Characterisation of plasmodium falciparum pfnek3, an atypical MAP kinase activator

CHARACTERIZATION OF PLASMODIUM FALCIPARUM PFNEK3, AN ATYPICAL ACTIVATOR OF A MAP KINASE LYE YU MIN (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements ACKNOWLEDGEMENTS Most importantly, I would like to express my deepest gratitude to my supervisor, A/Prof Sim Tiow Suan for the opportunity to be a student in the lab starting in AY2002/3 to learn molecular biological techniques. This subsequently led to participation in an UROPS project followed by Honours year work and now, graduate studies. The lab is engaging, challenging and most importantly, it nurtured and guided me for a few important years in my life. Prof Sim has been encouraging and constructively critical to me, without whom, there would not have been two journal publications with a position for my authorship. I am indebted to Maurice Chan, my mentor. Maurice has always graciously taken extra steps to assist and advise me. I am also grateful to Dr. Doreen Tan for invaluable assistance during immunofluorescence analyses. Appreciation goes to Jasmine and Madam Seah for many fruitful discussions and technical help. It has been enriching working with Jason and Wenjie. They have assisted me on countless occasions. Huiyu is an ardent supporter of this work and a fantastic listener too. Chun Song has the ability to remain helpful and calm in the most testing situations for which I am thankful. I am indebted to Prof Christian Doerig (Univ. of Glasgow) for providing the plasmids carrying Pfmap1 and Pfmap2. The pXJ40 plasmid was a gift from Prof E. Manser (Centre for Molecular Medicine, Singapore). Technical expertise for analytical gel filtration was sought from Mr. Lam Kin Wai. MR4 provided the Plasmodium falciparum 3D7 parasites contributed by Dr D.J. Carucci. Mr Robin Philp, Ms Ee Kim Huey and Mr Li Rong from Protein Analytics Lab (Biopolis Shared Facilities) were extremely accommodating with the interpretation of mass spectra. I would like to thank my family and friends for their understanding, encouragement, support and advice. i Contents CONTENTS Acknowledgements………………………………………………………………………………………………….……..i Contents………………………………………………………………………………………………………………………….ii Abstract………………………………………………………………………………………………………………………… vi List of publications………………………………………………………………………………………………………. viii List of tables…………………………………………………………………………………………………………………..ix List of figures…………………………………………………………………………………………………………………..x Abbreviations………………………………………………………………………………………………………………..xii Symbols………………………………………………………………………………………………………………………..xiii 1 Introduction ............................................................................................................. 1 2 Literature review...................................................................................................... 5 2.1 The canonical MAPK cascade ............................................................................ 5 2.2 The unusual kinome of the malaria parasite..................................................... 6 2.3 The P. falciparum genome encodes two MAPKs............................................... 9 2.4 Rodent Map2 is maleͲspecific and necessary for the sexual cycle ................. 10 2.5 MAPKKs upstream of Pfmap1 and Ͳ2 remain elusive...................................... 11 2.6 Current thoughts ............................................................................................. 12 3 Materials and Methods.......................................................................................... 14 3.1 BioͲcomputation.............................................................................................. 14 3.2 Use of Escherichia coli for cloning and expression.......................................... 16 3.2.1 Growth and maintenance of E. coli ................................................................. 16 3.2.2 Preparation of electrocompetent E. coli ......................................................... 16 3.2.3 Transformation of E. coli.................................................................................. 17 3.3 Use of Saccharomyces cerevisiae for yeastͲtwoͲhybrid studies ..................... 18 3.3.1 Growth and maintenance of S. cerevisiae ....................................................... 18 3.3.2 Preparation of competent S. cerevisiae AH109............................................... 18 3.3.3 Yeast transformation ....................................................................................... 19 3.4 Mammalian cell culture................................................................................... 20 ii Contents 3.4.1 Growth and maintenance of mammalian cell lines......................................... 20 3.4.2 Mammalian cell transfection ........................................................................... 21 3.5 Manipulation of the Pfnek3 gene.................................................................... 21 3.5.1 Pfnek3 gene isolation and amplification.......................................................... 21 3.5.2 Obtaining deletionͲconstructs for the purpose of Y2H screens ...................... 26 3.5.3 Generating mammalianͲtwoͲhybrid (M2H) fusion plasmid constructs .......... 27 3.6 Conventional DNA ligation .............................................................................. 28 3.7 Gateway™Ͳbased cloning ................................................................................ 28 3.8 SiteͲdirected mutagenesis of Pfnek3 and Pfmap2 .......................................... 30 3.9 DNA sequencing .............................................................................................. 35 3.10 Recombinant protein production.................................................................... 36 3.10.1 Harvesting cellͲfree extracts ............................................................................ 36 3.10.2 Affinity purification of GSTͲ and HisͲtagged proteins...................................... 37 3.11 Estimation of protein concentration by Bradford assay ................................. 37 3.12 SDSͲPAGE ......................................................................................................... 38 3.13 Mass spectrometric identification of proteins................................................ 39 3.14 Analytical gel filtration..................................................................................... 40 3.15 Protein adsorption........................................................................................... 41 3.16 Protein kinase assay ........................................................................................ 41 3.16.1 Validation of the ELISAͲbased kinase assay..................................................... 42 3.17 Western blot.................................................................................................... 43 3.18 Antibody production ....................................................................................... 43 3.19 Immunofluorescence microscopy ................................................................... 45 3.20 YeastͲtwoͲhybrid protein interaction studies ................................................. 46 3.20.1 Principles.......................................................................................................... 46 3.20.2 Testing the bait fusion protein for autoͲactivation and toxicity ..................... 47 3.20.3 Total cDNA library synthesis, amplification and fractionation........................ 48 3.20.4 Constructing and screening a twoͲhybrid library ............................................ 52 3.20.5 Prey plasmid rescue and identification by DNA sequencing ........................... 53 iii Contents 3.20.6 ReͲtesting the interaction by smallͲscale transformation............................... 54 3.20.7 ɲͲgalactosidase reporter assay........................................................................ 54 3.21 MammalianͲtwoͲhybrid protein interaction studies ...................................... 55 3.21.1 Principles.......................................................................................................... 55 3.21.2 M2H cell transfection....................................................................................... 57 3.21.3 Normalized SEAP activity assay ....................................................................... 59 4 Results and Discussion........................................................................................... 62 4.1 BioͲcomputational identification of Pfnek3 .................................................... 62 4.2 Molecular cloning of FLͲ and TRͲPfnek3.......................................................... 67 4.3 Construction of plasmids for bacterial expression.......................................... 69 4.4 Construction of plasmids for yeastͲtwoͲhybrid studies .................................. 70 4.5 Construction of plasmids for mammalianͲtwoͲhybrid studies ....................... 72 4.6 Generating kinaseͲinactive mutants of Pfnek3 and Pfmap2........................... 74 4.7 Bacterial expression of recombinant proteins ................................................ 75 4.8 Ensuring the expression of GSTͲPfmap2 ......................................................... 76 4.9 Kinase activity of recombinant Pfnek3............................................................ 79 4.10 Pfnek3 directionally phosphorylates Pfmap2 ................................................. 82 4.11 Pfnek3 stimulates Pfmap2 kinase activity....................................................... 84 4.12 Pfnek3 autophosphorylates in the presence of Pfmap2, active or inactive ... 86 4.13 Pfnek3 stimulates Pfmap2 but not Pfmap1 or hMAPK1 ................................. 86 4.14 Determining Pfnek3 localization using a surrogate assay............................... 88 4.15 Expression of endogenous Pfnek3 .................................................................. 91 4.16 Total cDNA library construction for yeastͲtwoͲhybrid studies ....................... 93 4.17 Protein partners of Pfnek3 identified from yeastͲtwoͲhybrid ........................ 94 4.17.1 The advantages and limitations of Y2H assays .............................................. 100 4.18 MammalianͲtwoͲhybrid confirmation of protein interactions ..................... 106 4.19 Future challenges: unraveling the malarial MAPK cascade .......................... 109 4.19.1 Current strategies for kinase substrate identification................................... 109 iv Contents 4.19.2 A dualͲcomponent strategy to decipher the phosphoproteome network of the malaria parasite ............................................................................................................... 115 4.20 5 4.19.2.1 The in vitro component ...................................................................... 115 4.19.2.2 The in vivo component ....................................................................... 119 Outlook .......................................................................................................... 124 References ........................................................................................................... 126 Appendices Appendix I: Publications Appendix II: Media, buffers and reagents Appendix III: Vector maps v Abstract ABSTRACT The canonical mitogenͲactivated protein kinase (MAPK) signal cascade was previously suggested to be atypical in the malaria parasite. Two copies of Plasmodium falciparum MAPKs (Pfmap1 and Pfmap2) have been identified and are believed to influence parasite proliferation. However, the regulators and substrates of malarial MAPKs have remained elusive. Hence, the presence of alternative upstream MAPK kinases and substrates in the malaria parasite is a tantalizing research question. To address this issue, available transcriptome datasets were scrutinized to identify candidate Plasmodium MAPK regulators by comparing the transcriptional profiles of numerous protein kinases. As a result, a candidate kinase was identified to possess transcriptional activities similar to both Pfmap1 and Pfmap2. This geneͲofͲ interest, named Pfnek3 (P. falciparum NIMAͲlike kinase 3), is a homologue of the NIMA (Never in Mitosis, Aspergillus) mitotic kinase family. Immunofluorescence data indicated that endogenous Pfnek3 was expressed during late asexual to gametocyte stages. Sequence analyses unveiled unusual kinase sequence motifs. For instance, the lack of an ATPͲbinding glycine triad, a common feature of many eukaryotic kinases, might have contributed to the absence of a strong in vitro kinase activity. Moreover, the phylogenetic distance of Pfnek3 from mammalian NIMAͲkinases concurs with its preference for manganese, rather than magnesium, as a cofactor. Recombinant Pfnek3 exists primarily as monomers, unlike the majority of NIMA kinases, which are dimeric. vi Abstract Recombinant Pfnek3 was able to phosphorylate and stimulate Pfmap2, a malarial MAPK known to be necessary for the completion of the male sexual cycle. Contrastingly, this was not observed with two other MAPKs, Pfmap1 and human MAPK1, suggesting that the Pfnek3ͲPfmap2 interaction may be specific for Pfmap2 regulation and possibly playing a role in gametocyte maturation, during which both genes have been reported to be highly upregulated. Because protein interaction data is currently unavailable for Pfnek3, yeastͲtwoͲ hybrid experiments were performed using Pfnek3 as bait to screen a P. falciparum total cDNA library. Out of about a hundred clones, five yielded potential interaction data. However, only one interaction pair activated the ɲͲgalactosidase reporter gene when the interactions were reͲtested. As an attempt to confirm the interactions observed in the yeast system, mammalianͲtwoͲhybrid assays were employed. Reporter gene activation was not detected, suggesting that one of the two systems, or the interaction, could be artifactual. To pursue the role of Pfmap2 and Pfnek3 in the sexual development of the malaria parasite, a dualͲcomponent phosphoproteomics strategy is discussed. It would be useful to first attempt an in vitro component to determine the substrates of Pfmap2 and Pfnek3, followed by an in vivo study to determine the total phosphoproteome of the malaria parasite. Future studies could involve in vivo experiments that could illuminate the functions and substrates of a kinaseͲofͲinterest, but only when geneͲdisrupted parasites or kinaseͲspecific inhibitors become available. vii List of Publications LIST OF PUBLICATIONS Journal publications: [1] Low H, Lye YM, Sim TS. (2007) Pfnek3 functions as an atypical MAPKK in Plasmodium falciparum. Biochem Biophys Res Commun. 361(2):439Ͳ444. [2] Lye YM, Chan M and Sim TS (2006) Pfnek3: an atypical activator of a MAP kinase in Plasmodium falciparum. FEBS Letters 580: 6083Ͳ6092. [3] Lye YM, Chan M, Sim TS (2005) Endorsing functionality of Burkholderia pseudomallei glyoxylate cycle genes as antiͲpersistence drug screens. J. Mol. Cat. B: Enzymatic. 33: 51Ͳ 56. Poster abstracts: [4] Lye YM, Lin W, Tay J, Low H, Chan M, Sim TS (2007) A survey of genes coding for NͲ terminal regions in Plasmodium falciparum proteins affecting their heterologous bacterial expression. Research abstract in: Keystone symposia meeting: Cell signaling and Proteomics, Steamboat Springs, Colorado, USA. Mar 22 – 27, 2007. Poster 214, p129. [5] Lye YM, Tan D, Chan M, Sim TS (2007) Tracking Plasmodium falciparum’s protein network through heterologous interacting systems. Research abstract in: Keystone symposia meeting: Cell signaling and Proteomics, Steamboat Springs, Colorado, USA. Mar 22 – 27, 2007. Poster 312, p137. [6] Low H, Lye YM, Chan M, Sim TS (2006) Data mining for malarial kinases with bipartite localization signals. Research abstract in: 18th Annual Meeting of the Thai Society for Biotechnology, Bangkok, Thailand. Nov 2 – 3, 2006. Poster VIIͲPͲ8, p214. [7] Lye YM, Chan M, Sim TS (2006) A proposed textͲbased dataͲmining tool for malaria. Research abstract in: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Japan. Jun 18 – 23, 2006. Poster 3PͲBͲ522, p21. [8] Chan M, Lye YM, Sim TS (2006) Validity of datamining the transcriptome data of Plasmodium falciparum for procuring genes of its glycolytic and TCA pathways. Research abstract in: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress, Kyoto, Japan. Jun 18 – 23, 2006. Poster 3PͲBͲ525, p22. [9] Lye YM, Chan M, Sim TS (2004) NIMAͲrelated protein kinases in Plasmodium falciparum. Research abstract in: 4th Combined Scientific Meeting, Singapore. 8th NUSͲNUH Annual Scientific Meeting, Singapore, Oct 7Ͳ8, 2004. Poster PͲ62, p. 109. [10] Lye YM, Chan M, Sim TS (2004) Cloning and characterization of glyoxylate cycle genes from Burkholderia pseudomallei. Research abstract in: 1st PacificͲRim International Conference on Protein Science, Yokohama, Japan. April 14 – 18, 2004. Poster P15/16Ͳ 181, p. 154. viii List of Tables LIST OF TABLES Table 2Ͳ1: Annotated NIMA family protein kinases from the malaria parasite.........................8 Table 3Ͳ1: Cell lines and organisms used in this study. ...........................................................15 Table 3Ͳ2: List of primers used in this study............................................................................ 24 Table 3Ͳ3: Recipe for PCR amplification of Pfnek3 (Protocol Pf1.1).........................................26 Table 3Ͳ4: T4 ligation recipe ..................................................................................................... 29 Table 3Ͳ5: LR clonase™ recombination recipe using Gateway™ technology ...........................29 Table 3Ͳ6: Recipe for siteͲdirected mutagenesis......................................................................30 Table 3Ͳ7: Plasmids used in this study...................................................................................... 31 Table 3Ͳ8: Fusion constructs used in this study. ......................................................................33 Table 3Ͳ9: Recipe for cDNA library preparation .......................................................................51 Table 3Ͳ10: cDNA library thermal cycling parameters .............................................................51 Table 3Ͳ11: M2H transfection setͲup ....................................................................................... 60 Table 3Ͳ12: Summary of kits used in the study ........................................................................61 Table 4Ͳ1: Typical protein kinase domain features ..................................................................65 Table 4Ͳ2: List of prey genes (identified from Y2H) subͲcloned into pVP16 ............................74 Table 4Ͳ3 : List of peptide ions detected in a LCͲMS/MS experiment identifying Pfmap2. ....78 Table 4Ͳ4: Representative raw data for Figure 4.12(C) Directional phosphorylation of Pfmap2 by Pfnek3 ..................................................................................................................................84 Table 4Ͳ5: Representative raw data for Figure 4.12(D) Kinase activity after coͲincubation of kinases ......................................................................................................................................84 Table 4Ͳ6: Representative raw data for Fig. 4.14 .....................................................................88 Table 4Ͳ7: Representative raw data for Figure 4.16(A)............................................................93 Table 4Ͳ8: Prey genes subͲcloned for M2H studies..................................................................99 Table 4Ͳ9: Advantages and disadvantages of yeastͲtwoͲhybrid screens ...............................105 Table 4Ͳ10: Comparison of twoͲhybrid technology to other methods ..................................105 Table 4Ͳ11: Representative raw data for Figure 4.20(A) MammalianͲtwoͲhybrid protein interaction assay..................................................................................................................... 109 Table 4Ͳ12: A list of phosphorylation site prediction web servers.........................................119 ix List of Figures LIST OF FIGURES Figure 2.1: A typical eukaryotic MAP kinase pathway......................................................... 6 Figure 3.1: Proposed strategy to study Pfnek3 as a potential, functional MAPKK........... 14 Figure 3.2: Schematic diagram depicting five deletionͲconstructs and the regions of the Pfnek3 gene that was PCR amplified for cloning into yeast bait vectors. ......................... 27 Figure 3.4: Screening for proteinͲprotein interactions with the Matchmaker¥ TwoͲHybrid System................................................................................................................................ 46 Figure 3.5: A process flow chart for twoͲhybrid protein interaction screening................ 48 Figure 3.6: Schematic diagram of the SMART III system to generate cDNA with the SMART III and CDS III anchors............................................................................................ 49 Figure 3.7: Reporter gene activation during a stable protein interaction event. ............. 57 Figure 3.8: Plasmids required for a M2H assay. ................................................................ 58 Figure 4.1: Sequence alignment of Pfnek3 with other NEKs and comparison of domain architecture with the closest homologue.......................................................................... 64 Figure 4.2: Phylogenetic analysis....................................................................................... 66 Figure 4.3: Structural models of Pfnek3 and Pfnek1. ........................................................ 66 Figure 4.4: Gel visualization of PCR products. ................................................................... 68 Figure 4.5: Restriction digestion screening for FLͲ and TRͲPfnek3ͲpGEX recombinant constructs........................................................................................................................... 70 Figure 4.6: Construction of yeast bait vectors fused with coding sequences derived from Pfnek3. ............................................................................................................................... 71 Figure 4.7: Plasmid construction for M2H protein interaction studies............................. 73 Figure 4.8: Generation of siteͲdirected kinaseͲinactive mutants, GSTͲȴPfnek3 and GSTͲ ȴPfmap2............................................................................................................................. 75 Figure 4.9: Recombinant expression of proteins............................................................... 76 Figure 4.10: Pfmap2 amino acid sequence covered by mass spectrometry (~35%)......... 77 Figure 4.12: Pfnek3 phosphorylates Pfmap2 and stimulates its kinase activity. .............. 83 x List of Figures Figure 4.13: Confirmatory experiments demonstrating that increased MBP phosphorylation was due to Pfmap2 preͲactivated with Pfnek3...................................... 85 Figure 4.14: Pfnek3 activates Pfmap2 but not hMAPK1.................................................... 88 Figure 4.15: Cytoplasmic localization of Pfnek3 in HepG2 cells. ....................................... 91 Figure 4.16: Endogenous expression of Pfnek3 via immunofluorescence microscopy. ... 92 Figure 4.18: Protein interactions of Pfnek3....................................................................... 97 Figure 4.19: Translation of prey fusion plasmid DNA sequences to determine the correctness of reading frame for interactions showing ɲͲgalactosidase activation. ........ 98 Figure 4.20: MammalianͲtwoͲhybrid interactions. ......................................................... 108 Figure 4.22: The KESTREL approach................................................................................ 112 Figure 4.23: Some phosphate sources used for the identification of kinase substrates. 114 Figure 4.24: Schematic diagram of a protein chipͲbased global phosphoproteome analysis............................................................................................................................. 115 Figure 4.26: The principles of SILAC................................................................................. 120 Figure 4.27: An outline for an organism level, proteomeͲscale in vivo identification of protein kinase substrates when a specific inhibitor is unavailable. ................................ 122 Figure 4.28: An integrated approach to study the phosphoproteome of the malaria parasite. ........................................................................................................................... 123 xi Abbreviations ABBREVIATIONS APS BSA CMV Ctrl DMSO DNA dNTP DTT FITC FL GFP GST IPTG LB M2H MAPK MAPKK MAPKKK MBP mRNA Nek NIMA NTR OD OL ORF PBS PCR Pfmap Pfnek PfPK7 RCC RT SD SDS SDSͲPAGE TDE TEMED TR Tris tRNA UV Y2H YPD Ammonium persulfate Bovine serum albumin Cytomegalovirus Control Dimethyl sulfoxide Deoxyribonucleic acid Deoxyribonucleotide triphosphate Dithiolthreitol Fluorescein isothiocyanate FullͲlength Green fluorescent protein GlutathioneͲSͲtransferase IsopropylͲɴͲDͲthiogalactopyranoside LuriaͲBertani Mammalian two hybrid MitogenͲactivated protein kinase MAPK kinase MAPKK kinase Myelin basic protein Messenger ribonucleic acid NIMAͲlike kinase Never in mitosis, Aspergillus NͲterminal region Optical density Oligonucleotide Open reading frame Phosphate buffered saline Polymerase chain reaction Plasmodium falciparum MAP kinase Plasmodium falciparum NIMAͲrelated kinase Plasmodium falciparum protein kinase 7 Regulator of chromosome condensation Reverse transcription/transcriptase Synthetic defined Sodium dodecyl sulfate Sodium dodecyl sulfate polyacrylamide gel electrophoresis TrisͲDTTͲEDTA N, N, N’, N’ͲtetramethylͲethylenediamine Truncated TrisͲ(hydroxymethyl)Ͳaminomethane Transfer ribonucleic acid Ultraviolet Yeast two hybrid Yeast extractͲPeptoneͲDextrose xii Symbols SYMBOLS % (v/v) % (w/v) °C µg µl µm µmol bp d Da g h kb kD kV L or l M mg min ml mm mM mth ng nm nmol pmol RCF RPM sec Milliliter per 100 milliliters Gram per 100 milliliters Degree Celsius Microgram Microliter Micrometer Micromole Base pairs Day Dalton Gram Hour Kilobases KiloͲDalton KiloͲVolt Liter Mole per liter (Molar) Milligram Minute Milliliter Millimeter Millimolar Month Nanogram Nanometer Nanomole Picomole Relative centrifugal force Revolution per minute Second xiii Chapter 1 Introduction 1 Introduction Plasmodium falciparum is responsible for the most lethal form of human malaria. Currently, reports of widespread reͲemergence of drug resistance exacerbate the problem (Martens and Hall, 2000). The development of effective vaccines has yet to produce significant success. Therefore, a more detailed understanding of parasite development and growth may be vital to fortifying our molecular arsenal against the disease. The malaria life cycle is appreciably complex (Leete and Rubin, 1996). Briefly, the sporozoites target the liver upon inoculation by the mosquito vector and develop into asexual forms that specifically infect red blood cells. During the asexual intraerythrocytic stage, some parasites develop into gametocytes which are picked up by another feeding mosquito. In the mosquito midgut, the gametocytes fuse and form zygotes that escape the midgut and transform into sporozoites that migrate to the vector’s salivary glands, ready to infect a new human host. Surprisingly, the signaling mechanisms throughout the entire developmental process are poorly understood. It has been established in other eukaryotic cells that molecular signaling is a key to a cell’s fate. Therefore, it is reasonable to suggest that signal transduction control is essential to parasite growth. A methodical approach to unveil these pathways may therefore lead to the identification of important signaling mediators. There is a growing interest in understanding the role of protein kinase pathways in the malaria parasite. Among eukaryotes, protein kinases belonging to the mitogenͲ activated protein kinase superfamily (MAPK, also called ERK, extracellularlyͲregulated 1 Chapter 1 Introduction kinase) are currently among the best understood. MAPKs are believed to be highly conserved among eukaryotes and are central to the transduction of extracellular mitogenic stimuli down a cascade of ATPͲdependent protein kinases. Two copies of P. falciparum MAPKs (Pfmap1 and Pfmap2) have been identified so far (Graeser et al., 1997; Dorin et al., 1999). Both MAPKs share a peptide sequence identity of 41% in their catalytic domain. Phosphorylation of a threonineͲtyrosine (TXY) sequence motif by an upstream kinase is usually needed for the activation of classical MAPKs. The TXY motif is completely conserved in Pfmap1 as TDY (PlasmoDB identifier: PF14_0294), and is altered to TSH in Pfmap2 (PlasmoDB identifier: PF11_0147). Pbmap2, the P. berghei counterpart of Pfmap2 was demonstrated to regulate male gametogenesis via geneͲdisruption studies as well as in sexͲspecific proteomic analyses (Khan et al., 2005; Rangarajan et al., 2005). Numerous attempts have been made to identify candidate kinases upstream of Pfmap1 and Pfmap2. For example, it has previously been suggested that Pfnek1, a NIMAͲ family kinase is an upstream regulator (i.e. MAPKK, also called MEK, MAPK/ERK Kinase) of Pfmap2 (Dorin et al., 2001). Unfortunately, in vivo activity of Pfnek1 was not established and recombinant Pfnek1 did not stimulate Pfmap1. A second protein kinase, P. falciparum Protein Kinase 7 (PfPK7), with a MEKͲlike motif was reported (Dorin et al., 2005). Interestingly, its sequence similarity to MEKs is limited to the CͲterminal domain while its NͲterminal region bears similarity to PKA (protein kinase A). In view of the above, and the fact that the malaria ‘kinome’ (genomeͲscale analyses of kinaseͲencoding sequences) reveals the lack of other MEKͲcoding DNA sequences (Ward et al., 2004), 2 Chapter 1 Introduction Dorin et al. (2005) suggested that a regular threeͲstep MAP kinase cascade is possibly nonͲexistent in the malaria parasite. Thus, the existence of unusual signaling mediators that can regulate plasmodial MAPKs remains an intriguing question of Plasmodium biology. The advent of the PlasmoDB repository (www.PlasmoDB.org) has eased our search for potential signaling mediators. Capitalizing on the transcriptome data offered in PlasmoDB (Bozdech et al., 2003; Le Roch et al., 2004), it was possible to identify a range of annotated gene products with expression profiles wellͲcorrelated with Pfmap1 and/or Pfmap2. The result of dataͲmining revealed five Plasmodium genes clustered as sequences with homology to the NIMA (Never in Mitosis, Aspergillus) protein kinase family (Ward et al., 2004). Of these five genes, three were revealed by microarray data to be expressed predominantly in gametocytes (Bozdech et al., 2003; Le Roch et al., 2004). NIMA, the founding member of the NEK kinase family was described in Aspergillus nidulans and shown to be required for G2/M transition (Osmani et al., 1988). Thus far, other NIMA homologues have been identified in many eukaryotes, and there is growing evidence that NIMAͲfamily kinases (NEKs) play the role of cell cycle regulators (O’Connell et al., 2003). We were intrigued by a sequence identified as PFL0080c (hereafter referred to as Pfnek3 following Ward et al., 2004), because it has an asexual intraerythrocytic expression profile akin to that of Pfmap1 whilst also being highly upͲregulated during the gametocyte stage, where Pfmap2 is specifically expressed (Dorin et al., 1999). Preliminary studies suggested that Pfmap2 and Pfnek3 act synergistically to 3 Chapter 1 Introduction phosphorylate substrate proteins in vitro (Lye et al., 2006). To understand the relationship between the kinases, the objectives of this study are as follows: 1. To identify the mode of interaction between Pfnek3 and Pfmap2. 2. To verify the directionality of phosphorylation between Pfnek3 and Pfmap2. 3. To determine the localization pattern of endogenous Pfnek3. 4. To reveal the protein interaction partners of Pfnek3. 4 Chapter 2 Literature Review 2 Literature review 2.1 The canonical MAPK cascade All living cells face the need for stimuli perception and mitotic signal transduction during cell cycle progression. Strategies for resolving this necessity have therefore evolved very early, as evident by the high level of conservedness of classical signal transduction pathways in all eukaryotic cells. One wellͲconserved pathway is the MAPK (mitogenͲactivated protein kinase) pathway and MAPK family members have been identified in all eukaryotes investigated so far, from unicellular organisms to mammals and plants (Figure 2.1). Reviewed in Garrington and Johnson (1999), the MAPKs, also called ERKs (extracellularly regulated kinases), are central to the adaptive responses of eukaryotic cells to a wide range of stimuli. Phosphorylation at both the Thr and Tyr residues of the conserved MAPK activation motif (TXY) by a specific MAPK kinase (MAPKK; also called MEK, for MAPK/ERK kinase) is necessary for MAPK activation. Correspondingly, another upstream kinase, MAPKKK or MEKK, often associated with membrane receptor tyrosine kinases, are in turn responsible for the phosphorylation and activation of MEKs. 5 Chapter 2 Literature Review Figure 2.1: A typical eukaryotic MAP kinase pathway. The pathway involves the sensing of extracellular stimuli which are transduced through a series of phosphotransfer cascades via GTP exchangers and GTPases which culminate in a kinase cascade resulting in the activation of MAPK1/2 which in turn phosphorylate its target proteins, many of which are phosphorylationͲactivated (or –deactivated) transcription factors. Figure constructed from textual description in Garrington and Johnson (1999). 2.2 The unusual kinome of the malaria parasite The sequencing of the P. falciparum genome has led to a plethora of opportunities for genomeͲwide analyses of broad gene groupings, and this has been done with protein kinases and termed the kinome (Ward et al., 2004). The malarial kinome has been reported to be evolutionarilyͲdivergent from most eukaryotes and this provides new opportunities for the identification of novel drug targets that are parasiteͲspecific and 6 Chapter 2 Literature Review thus less likely to present host toxicity issues. With the aim of identifying and classifying all protein kinases in the malaria parasite, Ward et al. (2004) used a variety of bioinformatics tools to identify 65 malarial kinase sequences and constructed a phylogenetic tree to position these sequences relative to the seven established eukaryotic protein kinase groups. The main features of the tree were: (1) several malarial sequences did not cluster within any of the known protein kinase groups; (2) the highest number of malarial protein kinases fall within the CMGC group, which is a collective term for cyclinͲdependent kinases (CDKs), mitogen activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDKͲlike kinases (CLKs), whose members are usually involved in the control of cell proliferation; and (3) no malarial protein kinases clustered with the tyrosine kinase group, pointing to the possible absence of a typical MAPK cascade in the parasite. Figure 2.2: The export of a GFPͲ tagged FIKK kinase from transgenic parasites into the host erythrocyte membrane. Parasite nuclei were confirmed with DAPI staining (blue). Figure source: Nunes et al. (2007). A novel family of 20 kinase sequences was identified and called the “FIKK” group, on the basis of a conserved “FIKK” amino acid motif. The FIKK family seems restricted to the Apicomplexan protozoan, with 20 members in P. falciparum. Many of the malarial FIKK kinases are believed to contain protein export signals that transport the FIKK 7 Chapter 2 Literature Review protein kinase to the parasitized human cell (Schneider and MercereauͲPuijalon, 2005). Recently, transgenic parasites expressing GFPͲtagged FIKK kinases allowed the detection of exported FIKK kinases at the erythrocyte cytoskeleton (Figure 2.2). Moreover, the FIKK kinases coͲimmunoprecipitated from red cells were enzymatically active, suggesting yet unknown roles of these kinases in the remodeling of the erythrocyte during an infection. Hence, these findings emphasize the need to study the function and localization of malarial protein kinases so as to validate them as candidates of antiͲplasmodial screens. Table 2Ͳ1: Annotated NIMA family protein kinases from the malaria parasite Literature Name1 www.PlasmoDB.org annotation Gene ID Predicted localization Reference Pfnek1 NIMAͲrelated protein kinase (PfnekͲ1) PFL1370w Ͳ Dorin et al. (2001) PFE1290w Ͳ PlasmoDB.org PFL0080c Apicoplast Lye et al. (2006) Pfnek2 Pfnek3 Serine/threonineͲprotein kinase Nek1, putative Serine/threonineͲprotein kinase Nek1, putative (this study) Pfnek4 Serine/threonine protein kinase 2, putative MAL7P1.100 Apicoplast Khan et al. (2005); Reininger et al. (2005) Pfnek5 Serine/Threonine kinase, putative PFF0260w Ͳ PlasmoDB.org Note: 1 Nomenclature follows Ward et al. (2004) 8 Chapter 2 2.3 Literature Review The P. falciparum genome encodes two MAPKs During investigations of molecular mechanisms possibly regulating parasite multiplication and development, two plasmodial MAPKs (Pfmap1 and Pfmap2) have been identified (Doerig et al., 1996; Dorin et al., 1999). Both malarial MAPKs were originally described as members of the MAPK1/2 subfamily, whose members are normally involved in the regulation of cell proliferation in response to external stimuli. Pfmap1 is expressed during erythrocytic schizogony and in gametocytes (Graeser et al., 1997), while Pfmap2 mRNA and protein are detectable only in the latter stage (Dorin et al., 1999). Pfmap1 contains the conserved peptide motif comprising the amino acids, TXY, as an activation site. Mammalian MAPK1 is activated by phosphorylation on the threonine and tyrosine residues of the TXY motif by an upstream protein kinase (i.e. MAPKK). Although the TXY motif is similarly present on the Plasmodium MAPK1 (Pfmap1), it is not yet clear which Plasmodium kinase is capable of activating Pfmap1. Intriguingly, the requirement of a MAPK1 homologue for parasite survival, development and proliferation has been demonstrated in Trypanosoma brucei (Muller et al., 2002). The case for Plasmodium MAPKs remains to be tested. Surprisingly, in Pfmap2, the TXY activation site is substituted by an atypical TSH motif. SiteͲdirected mutagenesis showed that both the Thr (T290) and the His (H292) residues in this motif are important for kinase activity of recombinant Pfmap2 (Dorin et al., 1999). Recent mass spectrometric studies indicate that the phosphorylation on T290 is crucial for the activation of Pfmap2 (Low et al., 2007) suggesting that the regulation of 9 Chapter 2 Literature Review Pfmap2 activity may differ from that of typical MAPKs. The only other example of such a divergent MAPK activation site is found in the freeͲliving protozoan Tetrahymena sp. (Nakashima et al., 1999). 2.4 Rodent Map2 is maleͲspecific and necessary for the sexual cycle In a pioneering study to understand the sexͲspecific proteomes of Plasmodium parasites, P. berghei, the rodent malaria parasite, was transfected with GFP reporter constructs under the control sexͲspecific promoters which enabled the differentiation and sorting of male and female gametocytes (Khan et al., 2005). A comparative proteomic analysis revealed the presence of sexͲspecific proteins, among which, Pbmap2, the rodent homologue of Pfmap2 was demonstrated to be specific to, and necessary for, male gametocytogenesis. In another independent study, Pbmap2Ͳdeficient parasites were impaired in sexual cycle completion (Rangarajan et al., 2005). These studies suggest the necessity of Pbmap2 to the malaria parasite and it follows that the use of a rodent malaria model for searching upstream kinases and candidate drugs capable of disrupting their phosphorylation appears relevant for human malaria. Figure 2.3: Pbmap2 knockͲout male gametocytes of the rodent malaria parasite, P. berghei, were disrupted in the ability to exflagellate. Abbreviations: M, male gametocytes; F, female gametocytes. (Image: Khan et al., 2005) 10 Chapter 2 Literature Review 2.5 MAPKKs upstream of Pfmap1 and Ͳ2 remain elusive Before the advent of the Plasmodium genome resource (www.PlasmoDB.org), P. falciparum MAPKK homologues were identified using PCR with degenerate primers, an approach which led to the identification of genes from various families of eukaryotic protein kinases (Dorin et al., 1999). When genome data became available, Dorin et al., (2001) isolated Pfnek1 (P. falciparum NIMAͲlike kinase 1), a protein kinase exhibiting maximal homology to members of the NIMA family of protein kinases (also called NIMAͲ like kinases, NEKs), but possessing a putative protein motif for activation (SMAHS) reminiscent of the conserved SMANS activation site found in mammalian MAPKK1/MAPKK2 enzymes. BacteriallyͲexpressed Pfnek1 can phosphorylate recombinant Pfmap2 in vitro. Unfortunately, in vivo functionality of Pfnek1 still awaits formal demonstration. Pfnek1 has no in vitro effect on Pfmap1 or on mammalian ERK2 (also called MAPK2). PfPK7 is another plasmodial protein kinase that encodes the ‘most MAPKKͲlike enzyme’ in the Plasmodium genome based on sequence similarity (Dorin et al., 2005). However, PfPK7 was unable to phosphorylate the two P. falciparum MAPK homologues in vitro, and was insensitive to PKA and MAPKK inhibitors. Together with the absence of a typical MAPKK activation site, this suggests that PfPK7 is not a MAPKK orthologue, although this enzyme is the most ‘MAPKKͲlike’ enzyme encoded in the P. falciparum genome. Consequently, Dorin et al. (2005) suggested that the classical threeͲcomponent MAPK pathway may be absent in the malaria parasite. 11 Chapter 2 Literature Review Figure 2.4: Transcription levels of Pfmap2 and Pfnek3 at various intraerythrocytic stages. Both kinases are predominantly expressed in the schizont and gametocyte stages (Bozdech et al., 2003; Le Roch et al., 2004). However, in proteomic studies, native Pfmap2 could only be specifically detected in male gametocytes (Khan et al., 2005). 2.6 Current thoughts Plasmodium falciparum causes 350 to 500 million clinical episodes of malaria occur every year (World Malaria Report, 2005). The impact of this parasite on the socioͲ economic development of affected countries is considerable as a result of the spread of drug resistance. Although the malaria parasite possesses only approximately 6000 genes, its life cycle is surprisingly complex, consisting of a succession of developmental stages staggering both the human and mosquito hosts. In order to complete its life cycle, the malaria parasite needs to sense changes in its environment and to provide rapid and adequate adaptive responses, such as stimulation or inhibition of the cell division machinery. In contrast, the genome of Trichomonas vaginalis, a human vaginal pathogen, encodes nearly 10 times more genes but displays a much simpler life cycle 12 Chapter 2 Literature Review (Carlton et al., 2007), suggesting that the malaria parasite possesses: (1) a lowͲ redundancy genome and/or (2) atypical gene products that contain multiple domains or singular domains with multiple functions, thereby enabling a single gene to take part in multiple physiological pathways. The existence of a large number of unusual malarial genes, that encode protein domains that do not possess homology to other eukaryotic genomes, points in the direction of such a possibility. The cell signaling pathways of the malaria parasite is currently believed to deviate from classical eukaryotic models. Therefore, the evolutionarilyͲdivergent protein kinases that possibly regulate the Plasmodium cell cycle progression become attractive drug targets. In particular, the MAPKs are believed to play central roles in the adaptive response of eukaryotic cells to a wide range of stimuli. However, the current understanding of molecular regulatory mechanisms on Pfmap1 and Pfmap2 is insufficient to explain the complexity of the parasite’s elusive reproductive pathways. Intriguingly, the classical eukaryotic MAPK pathway appears to be absent in the malaria parasite. To this day, the in vivo upstream regulators of Plasmodium MAPKs have remained enigmatic. With this in mind, there is a compelling imperative to characterize potential regulators of the previously identified plasmodial MAP kinases. As part of an earlier study, the synergistic kinase activity of Pfmap2 and Pfnek3 has been demonstrated. To take this lead further, the relationship between the kinases would be further dissected and the interaction partners of Pfnek3 identified in an attempt to decipher the malarial MAPK signaling pathway. 13 Chapter 3 Materials and Methods 3 Materials and Methods 3.1 BioͲcomputation Multiple sequence alignments were carried out using the ClustalW program (Thompson et al., 1994) Analysis of the protein sequences was performed using the software packages at the ExPASy molecular biology server (www.expasy.org). PlasmoDB (www.PlasmoDB.org) was the major source of sequence annotation and transcriptome data. Alignment dendrograms were viewed with the TreeView program (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) Databank (PlasmoDB) mining Parasitic mRNA and DNA extraction RT-PCR and PCR amplification to obtain Pfnek3 (full-length and various truncations) Site-direct mutagenesis Recombinant protein production expression Activity assays (ELISA) Kinase activation assays Ligate into cloning vectors Immunofluorescence microscopy Sub-clone into destination vectors (GST/6xHis/Twohybrid bait) Two-hybrid protein interaction studies Figure 3.1: Proposed strategy to study Pfnek3 as a potential, functional MAPKK. 14 Chapter 3 Materials and Methods Table 3Ͳ1: Cell lines and organisms used in this study. Strain Characteristics E. coli BL21 (DE3) Expression host with T7 RNA polymerase Novagen, gene placed under lacUV5 promoter USA control E. coli BL21 (DE3) CodonPlus™ As above. Contains the RIL plasmid to Stratagene, mitigate AT codon bias USA E. coli TOP10¥ A general purpose cloning host E. coli ccdͲsurvival P. falciparum 3D7 strain Source Invitrogen, A lethalityͲresistant strain required for USA the maintenance of native pDEST¥ series Gateway¥ destination vectors Genome sequencing strain MR4, USA P. falciparum Tan strain A clinical isolate from Singapore National University Hospital, Singapore S. cerevisiae AH109 strain For screening protein interactions in Clontech, yeastͲtwoͲhybrid experiments USA HepG2 Human liver cell line ATCC, USA MCFͲ7 Human breast cell line ATCC, USA 15 Chapter 3 3.2 Materials and Methods Use of Escherichia coli for cloning and expression 3.2.1 Growth and maintenance of E. coli Escherichia coli cultures were grown in autoclaved LuriaͲBertani (LB) medium as well as LB agar Petri dishes containing an addition of 1.5% (w/v) agar. Cultures were grown at 37oC with shaking at 250 RPM. When necessary, antibiotics such as kanamycin (50µg/ml), chloramphenicol (40µg/ml) and/or ampicillin (100µg/ml) were added to culture media. Stocks of E. coli strains were preserved at Ͳ80oC in 10% (w/v) glycerol. The strains of E. coli used in this study are listed in Table 3Ͳ1. 3.2.2 Preparation of electrocompetent E. coli The preparation of electrocompetent cells with the highest competence requires the cells to be in the early to midͲlogarithmic growth phase. A single wellͲisolated E. coli colony was inoculated into 5 ml of sterile LB broth and incubated overnight at 37oC with shaking at 250 RPM. Two ml of the overnight culture was then added to 100 ml of sterile LB broth. This is incubated at 37oC with shaking at 250 RPM until midͲlog phase (A600 0.6Ͳ 0.8). Following that, the culture was pelleted by centrifuging for 10 min at 7000 RPM at 4°C. Cells are washed once in 50 ml of iceͲcold 10% (w/v) glycerol, pelleted again, followed by supernatant removal and reͲsuspension by short vortexing. The reͲ suspended cells were then divided into 50 Pl aliquots. The cells can now be used for electroͲtransformation or frozen at Ͳ80°C for subsequent use. The minimum acceptable competency was 108 CFU/µg of pUC19 DNA. 16 Chapter 3 Materials and Methods 3.2.3 Transformation of E. coli The ligation reaction (10 out of a 20 µl reaction) was diluted with 100 µl of sterile water and the mixture added to 50 µl of competent cells in an iceͲchilled electroporation cuvette with an electrode gap of 0.2 cm (BioͲRad). The BioͲRad Gene Pulser¥ was adjusted to 2.5 kV and an electric pulse rendered across the cuvette. Immediately, one ml of sterile LB broth was added to the mixture to recover the cells at 37°C for one hour with shaking at 250 RPM. For heat shock transformation of commercial E. coli TOP10 competent cells, regular ligation mixes or TOPO™ ligaseͲfree cloning reactions were mixed with iceͲ thawed cells for 2 min on ice. Transformation was achieved by heat shock for 30 sec in a 42°C water bath followed by icing for 2 min. Sterile SOC medium (250 µl) was immediately added to the mixture followed by one hour of recovery at 37°C with shaking at 250 RPM. Selection for transformed colonies was achieved by spreading the transformation mixtures onto LB agar containing relevant antibiotics, depending on the antibiotic resistance marker encoded on the plasmid. The plates were incubated overnight at 37oC and wellͲisolated colonies were picked the following day. 17 Chapter 3 3.3 Materials and Methods Use of Saccharomyces cerevisiae for yeastͲtwoͲhybrid studies 3.3.1 Growth and maintenance of S. cerevisiae Yeast strains were stored in YPD or an auxotrophicͲselection SD medium with 25% (w/v) glycerol at –80°C. To prepare glycerol stocks, isolated colonies from an agar plate were reͲsuspended in 200–500 µl of YPD or the appropriate SD medium in a sterile 1.5 ml microfuge tube. Sterile 50% (w/v) glycerol was added to a final concentration of 25%. Frozen stocks were revived by streaking onto YPD or a selective SD dropͲout agar and incubated at 30°C. On YPD agar, colonies would generally take at least 4Ͳ5 days to appear. To prepare liquid overnight cultures, fresh (< 2 mth old, 2Ͳ4 mm diameter) colonies from a working stock plate were inoculated at one colony per 5 ml of broth. 3.3.2 Preparation of competent S. cerevisiae AH109 A flask containing 30 ml of YPD was inoculated with several young (< 1 mth) colonies that are 2–3 mm in diameter. Care was taken to disperse cell clumps by smearing the wire loop carrying the inoculums against the wall of the flask in a circular motion. The flask was incubated at 30°C overnight for 16–18 h with shaking at 250 RPM to stationary phase (OD600 > 1.5). The culture was transferred to another flask containing 300 ml of sterile YPD and incubated at 30°C for 3 h with shaking (250 RPM) achieving an OD600 of 0.4–0.6. Cell cultures were transferred to six 50 ml Falcon tubes and centrifuged at 1,000 gravitationalͲforces (RCF) for 5 min at room temperature (20–22°C). The supernatants were discarded and the cell pellets thoroughly reͲsuspended in sterile water. The cells were pooled into one tube (final volume 25–50 ml) and centrifuged at 18 Chapter 3 Materials and Methods 1,000 RCF for 5 min at room temperature. The supernatant was removed and the pellet reͲsuspended in 1.5 ml of freshly prepared, sterile 1x TE/lithium acetate (LiAc) mix. 3.3.3 Yeast transformation The desired plasmid (0.1 µg) was freshly mixed with 0.1 mg of herring testes carrier DNA. FreshlyͲmade yeast competent cells (0.1 ml) were added to each tube and vortexed. 0.6 ml of sterile PEG/LiAc solution was added to each tube and vortexed for 10 sec. Cells were incubated at 30°C for 30 min with shaking at 200 RPM, after which, 70 µl of DMSO was added. Cells were then mixed by gentle inversion and heat shocked for 15 min in a 42°C water bath. Cells were chilled on ice for 1–2 min for recovery followed by centrifugation for 5 sec at 14,000 RPM at room temperature. After removal of the supernatant, cells were reͲsuspended in 0.5 ml of sterile 1X TE buffer and 100 µl from each sample were spread on the relevant SDͲdropout agar plates that selected for cells expressing the corresponding auxotrophic markers. For coͲtransformation samples, three controls plates (ͲLeu, ͲTrp and ͲLeu/ͲTrp) were needed to ensure comparable transformation efficiencies of both bait and prey plasmids. Plates were incubated at 30°C until colonies appear (at least 2–4 days). 19 Chapter 3 Materials and Methods 3.4 Mammalian cell culture 3.4.1 Growth and maintenance of mammalian cell lines HepG2, a human liver cell line, was purchased from ATCC (USA). MCF7, a human breast cell line, was a gift from Prof Bay BH (National University of Singapore). Both cell lines were maintained in T25 flasks (Nunc, Denmark) using Dulbecco's Modified Eagle's Medium (DMEM; Sigma, USA) supplemented with 10% (v/v) fetal calf serum (FCS; Hyclone, USA). For maintaining HepG2 cells, 1X cellͲculture grade penͲstrep (Sigma, USA) was added to the medium. Flask caps were loosened for ventilation while incubated at 37°C and 5% CO2. Approximately 0.5Ͳ1.0 million cells were split into a fresh flask each time 80Ͳ100% confluence was achieved. To prepare frozen stocks, cells in 2Ͳ4 day old flasks (with 80Ͳ100% confluence) were dislodged from the flask walls using 1.5 ml of 1X cellͲculture grade trypsin (Sigma, USA) with 3Ͳ5 min of incubation at ambient temperatures. Thereafter, fresh culture medium was added to quench the trypsin, followed by vigorous pipeting and transferred to sterile 50 ml Falcon tubes. The cells were pelleted by centrifugation at 1500 RPM for 5 min at 20°C. The supernatant was decanted and the pellet resuspended with 1.5 ml of freezing medium, consisting of DMEM supplemented with 10% (v/v) FCS and 10% (v/v) DMSO (dimethyl sulfoxide; Sigma, USA). Thereafter, the cells were transferred to cryoͲ vials (Nunc, Denmark), sealed in Parafilm™, and floated in an isopropanol bath (~25°C), which was subsequently transferred to a Ͳ80°C freezer. 20 Chapter 3 Materials and Methods To revive stocks, cryoͲvials were thawed quickly in a 37°C water bath and their contents transferred into 50 ml Falcon tubes containing 5 ml of culture medium. The Falcon tubes were then centrifuged to pellet the cells. The supernatants were decanted and replaced with 7.5 ml fresh cell culture media. After resuspension of the cells, the contents were transferred to a sterile T25 flask and incubated at 37°C with 5% CO2. 3.4.2 Mammalian cell transfection Prior to transfection, 6Ͳwell plates were seeded with about 1 million cells per well using antibioticͲfree DMEM supplemented with 10% (v/v) FCS and allowed to grow overnight achieving approx. 75Ͳ90 % confluence. The cells were rinsed once with sterile PBS and immersed with 2 ml of the serumͲreduced medium, OptiMEM™ (Invitrogen, USA). The wells were topped up with 500 µl of a mixture containing 10 µl of the Lipofectamine 2000™ reagent (Invitrogen, USA) and 4Ͳ20 µg of the desired plasmid(s). 3.5 Manipulation of the Pfnek3 gene 3.5.1 Pfnek3 gene isolation and amplification Parasites were grown according to Haynes et al. (1976). The isolation and amplification of the Pfnek3 gene was described in Lye et al. (2006). DNA and RNA were extracted from erythrocyteͲstage parasites using the QIAamp™ DNA Blood Mini Kit and RNeasy™ Mini Kit, respectively, following manufacturer’s instructions (both from Qiagen, USA). PCR was performed using a pair of primers, OL771 and OL768. For easy reference, all primers used in this study are listed in Table 3Ͳ2. Pfu DNA polymerase 21 Chapter 3 Materials and Methods (Promega, USA) was used for the amplification of this and other malarial genes in this laboratory (Chan and Sim, 2004), utilizing thermal cycling parameters comprising one min of denaturation at 94°C, followed by 35 repeats of the following: 94°C for one min, 45°C for two min and 68°C for three min. An extension step for 10 min at 68°C was carried out, followed by a final hold at 4°C. The reaction recipe is tabulated in Table 3Ͳ3. As evident in other studies (Dyson et al., 2004), the presence of NͲterminal hydrophobic regions in a recombinant protein has a propensity to interfere with subsequent heterologous expression in E. coli. Therefore, in anticipation of this possibility, the truncated form of the Pfnek3 gene encoding a peptide lacking the first 59 amino acids was cloned using another forward primer, OL767 and the reverse primer OL768. In order to verify the transcription of Pfnek3 in P. falciparum, the Sensiscript™ twoͲstep reverse transcription kit (Qiagen, USA) or the Transcriptor™ First Strand cDNA Synthesis Kit (Roche, USA) was employed. PolyͲA+ mRNAͲenriched templates purified from erythrocyteͲstage parasites were subjected to 3 h of reverse transcription at 37°C with OL768 for first strand synthesis prior to thermal cycling as described above. Modern cloning techniques have been hastened with the introduction of Gateway¥ technology (Invitrogen, USA). Because this technology involves genetic recombination rather than DNA ligation for subͲcloning, it was no longer necessary to include restriction enzyme sites in PCR primers. To amplify the Pfnek3 gene for Gateway¥ cloning, the following PCR primers were used instead: OL1106 which was 22 Chapter 3 Materials and Methods engineered to contain the directional vector insertion sequence (CACCATGGGA) and OL1107, a reverse primer. All PCR and RTͲPCR products were separated on 0.8% (w/v) agarose gels and the relevant DNA bands (FLͲ and TRͲPfnek3 = 1044 and 867 bp, respectively) were excised using a scalpel and purified using the DNA Gel Extraction kit, following manufacturer’s instructions (Qiagen, USA). The bluntͲend Pfnek3 PCR products were cloned into two different cloning vectors, (a) pCRͲBlunt IIͲTOPO™ for conventional cloning or (b) the pENTR/DͲTOPO™ directional cloning vector compatible with Gateway¥ technology (both from Invitrogen, USA). Recombinant constructs were chemicallyͲtransformed into E. coli TOP10 cells (Invitrogen, USA). Successful transformants were selected on LB agar containing 50µg/ml of filterͲsterilized kanamycin (Sigma, USA). Positive transformants were verified by restriction digestion for the regular pCRͲTOPO™ vectors or DNA sequencing for the pENTR/DͲTOPO™ Gateway™ vectors. DNA inserts on both vector types were authenticated by sequencing. The recombinant fullͲlength and truncated Pfnek3 were thereafter named FLͲPfnek3 and TRͲPfnek3, respectively. 23 Chapter 3 Materials and Methods Table 3Ͳ2: List of primers used in this study Primers Sequences Description Purpose Source M13F 5’ͲGTAAAACGACGGCCAGͲ3’ M13 forward primer Invitrogen M13R 5’ͲCAGGAAACAGCTATGACͲ3’ M13 reverse primer DNA sequencing of TOPO™ and DͲ TOPO™ plasmid inserts OL48 5’ͲTAATACGACTCACTATAGGGͲ3’ Sequences plasmids with a T7 promoter DNA Sequencing SigmaͲProligo OL74 5’ͲTATGCTAGTTATTGCTCAGͲ3’ Sequences plasmids from the T7 terminator DNA Sequencing SigmaͲProligo DNA sequencing of pGEX™ plasmid inserts. SigmaͲProligo For sequencing pGBKT7 only SigmaͲProligo OL674 OL675 5'Ͳ GGGCTGGCAAGCCACGTTTGGTGͲ 3' 5'Ͳ CCGGGAGCTGCATGTGTCAGAGGͲ 3' pGEX forward primer pGEX reverse primer OL733 5’Ͳ AGGGGTTATGCTAGTTAͲ3’ T7 terminator primer OL771 5’Ͳ GGATCCGTTTGCATTTACTTGTTTTͲ 3’ FLͲPfnek3 forward primer (BamHI site underlined) OL767 5’Ͳ GGATCCTGTGAGAAGAAATACCAG GͲ3’ TRͲPfnek3 forward primer (BamHI site underlined) 5’Ͳ GAATTCTTATTGAACCGTTATACAT Ͳ3’ Pfnek3 reverse primer (EcoRI site underlined; reverse complemented termination signal in bold) OL782 5’Ͳ GTCGACTTATTGAACCGTTATACAT Ͳ3’ Pfnek3 reverse primer (SalI site underlined; reverse complemented termination signal in bold) OL875 5’Ͳ GGATCCCCTAAAGAAGATTGCAAG Ͳ3’ Pfmap1 reverse primer with BamHI site underlined Amplify Pfmap1 SigmaͲProligo OL876 5’Ͳ GTCGACTTACTCATTTGAAACACAͲ 3’ Pfmap1 reverse primer with XhoI site underlined Amplify Pfmap1 SigmaͲProligo OL877 5’Ͳ GTCGACTTAAACATATTTATGTTTͲ 3’ Pfmap1 reverse primer with XhoI site underlined (for first 312 aa) Amplify Pfmap1 SigmaͲProligo OL768 DNA and RTͲPCR amplification of FLͲ and TRͲ Pfnek3, respectively. SigmaͲProligo SigmaͲProligo SigmaͲProligo DNA and RTͲPCR amplification of Pfnek3, for pGEX cloning. SigmaͲProligo 24 Chapter 3 Materials and Methods OL979 5’Ͳ GAGATATATATATCTatgGTATATG ATATATATGGͲ3’ Primer for siteͲdirected mutagenesis of Pfnek3 (K102M) OL980 5’Ͳ CCATATATATCATATACCATAGATA TATATATCTCͲ3’ Reverse complement of the above primer OL1105 5’Ͳ CACCATGGGAGTTTGCATTTACTTG TTTTͲ3’ FLͲPfnek3 forward primer OL1106 5’Ͳ CACCATGGGATGTGAGAAGAAATA CCAGGͲ3’ TRͲPfnek3 forward primer OL1107 5’Ͳ TTGAACCGTTATACATGATͲ3’ Pfnek3 reverse primer (no stop codon) OL1138 5’CAAATAAAAATGTGGCTATAATG AAGGTTAATAGͲ3’ Primer for siteͲdirected mutagenesis of Pfmap2 (K135M) OL1139 5’CTATTAACCTTCATTATAGCCACA TTTTTATTTGͲ3’ Reverse complement of the above primer OL 1176 5’Ͳ GAATTCATGGTTTGCATTTACTTGͲ 3’ OL1177 5’Ͳ GAATTCTGTGAGAAGAAATACCAG GͲ3’ OL1178 5’Ͳ GGATCCATTTAAACCATTTAATATT TGͲ3’ OL1179 5’Ͳ GAATTCAATAAAATAAAGATTGGT GͲ3’ OL1180 5’Ͳ GGATCCTTATTGAACCGTTATACAT GͲ3’ Forward primer for FLͲ Pfnek3 (EcoRI site underlined) Forward primer for TRͲ Pfnek3 (EcoRI site underlined) Reverse primer for NͲ terminal region of Pfnek3 (reverseͲ complemented with BamHI site underlined) Forward primer for CͲ terminal region of Pfnek3 with EcoRI site underlined Pfnek3 reverse primer (reverse complemented with BamHI site underlined and stop codon in bold) OL1344 5'ͲAGACTGATATGCCTCTAͲ3' OL1345 OL1346 To inactive mutant create Pfnek3 1st base 1st base 1st base For cloning DͲTOPO 1st base 1st base To create inactive Pfmap2 mutant 1st base 1st base YeastͲtwoͲhybrid 1st base YeastͲtwoͲhybrid 1st base YeastͲtwoͲhybrid 1st base YeastͲtwoͲhybrid 1st base YeastͲtwoͲhybrid 1st base 5’ sequencing primer for bait vector, pM MammalianͲ twoͲhybrid 1st base 5'ͲGGGGAGGTGTGGGAGGTͲ3' 3’ sequencing primer for bait vector, pM MammalianͲ twoͲhybrid 1st base 5'ͲCCGGGATTTACCCCCCAͲ3' 5’ sequencing primer for prey vector, pVP16 MammalianͲ twoͲhybrid 1st base 25 Chapter 3 Materials and Methods Table 3Ͳ3: Recipe for PCR amplification of Pfnek3 (Protocol Pf1.1) Reaction composition Amount (µl) DNA template (double stranded DNA) 1.0 (approximately 0.5 Ͳ 0.8 µg) dNTPs (Applied Biosystems) 3.0 Forward primer (10 pmol/µl) 1.0 Reverse primer ( 10 pmol/µl) 1.0 Pfu DNA polymerase (Promega, USA) 0.5 10X polymerase buffer (Promega, USA) 2.5 NucleaseͲfree water 16.5 3.5.2 Obtaining deletionͲconstructs for the purpose of Y2H screens YeastͲtwoͲhybrid (Y2H) protein interaction screens make use of the expression of reporter genes, in this case, ɲͲ and ɴͲgalactosidase, under the control of transcriptional activators encoded by the bait and prey vectors. The theoretical relevance of this system is contested by the omnipresence of false positives and/or nonͲexpression of heterologous proteins. Excessive background yeast growths (false positives) are due to autoͲactivation of the reporter genes by the DNAͲBD (binding domain) and/or DNAͲAD (activation domain) fusion proteins. In order to mitigate these possibilities, deletion of certain portions of bait and/or prey proteins may be required to eliminate unwanted transcriptional activity before twoͲhybrid screen results could be meaningfully interpreted (Bartel et al., 1993). In this study, a total of five constructs of different lengths were amplified by PCR from a P. falciparum genomic DNA template (Figure 3.2). PCR products were gelͲpurified and cloned into the polylinker region of the yeast bait vector (pGBKT7) encoding a DNAͲBD fusion of the Pfnek3 protein (methods described in Section 3.6). This strategy was designed to manage attrition owing to autoͲactivation of 26 Chapter 3 Materials and Methods the reporter genes, resulting in a large number of false positives and also to mitigate the possibility of nonͲexpression or toxicity of foreign proteins in yeast. Fragment no. Truncation scheme Description Designation FLͲPfnek3 TRͲPfnek3 FLͲNTͲ Pfnek3 TRͲNTͲ Pfnek3 CTͲPfnek3 Figure 3.2: Schematic diagram depicting five deletionͲconstructs and the regions of the Pfnek3 gene that was PCR amplified for cloning into yeast bait vectors. Fragments 1Ͳ5, respectively, were amplified using the following primer pairs: OL1176/OL1180, OL1177/OL1180, OL1176/OL1178, OL1177/OL1178 and OL1179/OL1180. 3.5.3 Generating mammalianͲtwoͲhybrid (M2H) fusion plasmid constructs Yeast bait and prey insertͲcontaining plasmids as well as destination (native mammalian bait and prey) plasmids were doubleͲdigested with BamHI and EcoRI to generate compatible ends for directionally ligating the geneͲcoding sequences into the linearized mammalian vectors in the correct reading frame (methods described in Section 3.6). The ligation mixes were transformed into E. coli TOP10 and the correct recombinants were analyzed by restriction digestion with BamHI and EcoRI. The 27 Chapter 3 Materials and Methods correctness of reading frame was verified by DNA sequencing. The PureYield¥ plasmid MidiPrep kit (Promega, USA) was used to purify larger amounts of the correct constructs achieving plasmid concentrations of 200Ͳ900 ng/µl for mammalian cell transfection. 3.6 Conventional DNA ligation DNA fragments cloned in the pCRͲBlunt IIͲTOPO™ cloning vector were first released by double restriction digestion using relevant enzymes to create compatible ends for subsequent ligation into the polylinker region of the destination vectors. RestrictionͲdigested DNA fragments were excised from electrophoresis gels, purified and ligated into the relevant plasmids previously linearized with the same restriction enzymes. Ligation was carried out using T4 DNA ligase following manufacturer’s recommendations (Table 3Ͳ4; Invitrogen, USA). The ligation mixture was incubated at 14Ͳ 16°C overnight, after which a 10 µl aliquot of the 20 µl ligation mix was diluted 10Ͳfolds with sterile water and transformed into electroͲcompetent E. coli. The transformants were screened for the presence of correct recombinant plasmids based on restriction enzyme digestion analysis and confirmed with full DNA sequencing on both strands. 3.7 Gateway™Ͳbased cloning For the case of Gateway¥ compatible vectors, the pENTR/DͲTOPO™ vector containing the relevant gene insert was incubated with the desired pDEST™ series destination vector in the presence of the Clonase™ LR mix (Invitrogen, USA) that contains a recombinase capable of transferring the gene insert from the pENTR/DͲ 28 Chapter 3 Materials and Methods TOPO™ plasmid into a pDEST™ plasmid. The reaction was carried out at 25°C for 1 h and stopped by adding Proteinase K (included in kit; 2 µl) and incubating at 37°C for 20 min. The reaction mixture was then chemicallyͲtransformed into E. coli TOP10 or BL21 (DE3) CodonPlus¥ cells (if protein expression was needed) and the transformants selected on LB agar containing the relevant antibiotics (100 µg/ml of ampicillin and an additional 40 µg/ml of chloramphenicol when selecting for E. coli CodonPlus¥ cells). Table 3Ͳ4: T4 ligation recipe Reagent T4 ligase (Invitrogen High Conc. 5U/ʅl ) 5X ligase buffer Linearized vector DNA (~0.1 – 0.3 ʅg/ʅl) Insert DNA (~0.1 – 0.3 ʅg/ʅl) ROͲgrade water (sterile) Total Amount (ʅl) 0.5 4 2 6 7.5 20 Table 3Ͳ5: LR clonase™ recombination recipe using Gateway™ technology Reagent Amount (ʅl) pENTR/DͲTOPO entry vector (100Ͳ300 ng) 1Ͳ10 pDEST destination vector (150 ng/µl) 2 5X Clonase™ reaction buffer 4 TE buffer pH 8.0 Top up to 16 ʅl LR Clonase™ mix 4 Total 20 29 Chapter 3 3.8 Materials and Methods SiteͲdirected mutagenesis of Pfnek3 and Pfmap2 To introduce amino acid residue changes into Pfnek3 and Pfmap2, the Quikchange¥ protocol (Stratagene, USA) was adapted for use. The codon encoding the amino acid to be replaced was identified. A replacement codon in agreement with the codon usage bias of that of E. coli was selected and introduced into a synthetic PCR primer containing the native gene sequence (about 15Ͳ20 nucleotides) immediately before and after the codon to be replaced. The desired Tm of the primer was about 45Ͳ 55°C and the PCR thermal cycling parameters follow Table 3Ͳ3 with elongated extension phases depending on the parental plasmid size. The extension rate of Pfu DNA polymerase was assumed to be 2 min/kb. A restriction enzyme, DpnI (20 U, New England Biolabs, USA), was added to the PCR product to degrade methylated parental plasmids and thereafter, transformed into E. coli CodonPlus™ cells and selected on LB agar containing the relevant antibiotics. A few colonies were picked for plasmid extraction and DNA sequencing to verify that only the desired mutation was introduced. The kinaseͲinactive Pfmap2 was generated with the primer pair OL1138/OL1139 and kinaseͲ inactive Pfnek3 was generated with OL979/OL980 (primer sequences in Table 3Ͳ2). Table 3Ͳ6: Recipe for siteͲdirected mutagenesis Reagent Parental plasmid DNA (~ 100 ng/ʅl) 10x Pfu buffer (Promega) Forward primer (10 pmol/µl) Reverse primer(10 pmol/µl) dNTP (10 mM; Applied Biosystems) Pfu DNA polymerase (Promega) NucleaseͲfree water Total Amount (ʅl) 0.5 2.5 1 1 3 0.5 16.5 25 30 Chapter 3 Materials and Methods Table 3Ͳ7: Plasmids used in this study Vector Characteristics Source To test E. coli competency. Ampicillin resistance marker pUC19 Invitrogen, USA (AmpR). Cloning vector for bluntͲend PCR products, kanamycin pCRͲBlunt resistance marker (KanR) and T7 promoter preceding Invitrogen, USA IIͲTOPO™ polylinker pENTR/DͲ TOPO™ Cloning vector for bluntͲend PCR products, KanR and recombination sites for transferring DNA sequences into Invitrogen, USA Gateway™ destination vectors. pDESTͲ17 Contains a T7 promoter for highͲlevel expression of the gene of interest and an NͲterminal 6xHis tag for purification. Two recombination sites, attR1 and attR2, downstream of the T7 promoter for recombination cloning of the gene of interest Invitrogen, USA from DͲTOPO vectors. Chloramphenicol resistance gene located between the two attR sites for counterͲselection. The ccdB gene located between the attR sites for negative selection. AmpR. pDESTͲ47 As above, except for a constitutive CMV promoter for expression of gene of interest fused with a CͲterminal GFP Invitrogen, USA tag. pGBKT7 Express bait protein as a GAL4 DNAͲBD fusion protein. Encodes the cͲMyc epitope. Yeast TRP1 auxotrophic marker. Clontech, USA Bacterial KanR. pGADT7Ͳ Rec Express prey protein as GAL4 AD fusion protein. Encodes the hemoagglutinin epitope. Yeast LEU2 auxotrophic marker. Clontech, USA Bacterial AmpR. pGEXͲ6PͲ1 GSTͲencoding expression vector, AmpR, tac promoter preceding polylinker, Prescission™ sequenceͲspecific Amersham protease targeting an 8Ͳamino acid recognition sequences for Biosciences, Sweden the removal of GST moiety. pGEXͲ3X Gifts of C. Doerig As above, except that Factor Xa could be used to cleave off (Wellcome Centre for GST. Provided as recombinant vectors carrying the Pfmap1 Molecular Parasitology, and Pfmap2 sequences. Glasgow) 31 Chapter 3 pM pVP16 Materials and Methods Used to generate a fusion of the GAL4 DNAͲBD and a protein of interest in the mammalianͲtwoͲhybrid system. The hybrid protein is targeted to the cell’s nucleus by the GAL4 nuclear localization sequence. Transcription is initiated from the Clontech, USA constitutive SV40 early promoter (PSV40e); transcription is terminated at the SV40 poly A transcription termination signal. AmpR. Used to generate a fusion of the VP16 AD and a protein of interest. The hybrid protein is targeted to the cell’s nucleus by the SV40 nuclear localization sequence. Transcription is Clontech, USA initiated from the constitutive SV40 early promoter (PSV40e); transcription is terminated at the SV40 poly A transcription termination signal. AmpR. pM3ͲVP16 A positive control plasmid that expresses a fusion of the GAL4 Clontech, USA DNAͲBD to the VP16 AD. AmpR. pMͲ53 A positive control plasmid that expresses a fusion of the GAL4 Clontech, USA DNAͲBD to the mouse p53 protein. AmpR. pVP16ͲT A positive control plasmid that expresses a fusion of the VP16 AD to the SV40 large TͲantigen, which is known to interact Clontech, USA with p53. AmpR. pG5SEAP pG5SEAP contains GAL4 binding sites and an adenovirus E1b minimal promoter upstream of the secreted alkaline phosphatase (SEAP) gene. The DNAͲBD portion of a hybrid protein expressed from a pMͲderived plasmid localizes to the Clontech, USA GAL4 binding sites in pG5SEAP. If the hybrid test protein X interacts with test protein Y (expressed as a hybrid protein from a pVP16Ͳderived plasmid), the SEAP gene will be transcribed. pXJ40 An NͲterminal GFPͲfusion cloning vector under the control of Gift of E. Manser a constitutive CMV promoter. Used for coͲtransfecting (Centre for Molecular mammalian cells in M2H studies as normalization control. Medicine, Singapore) 32 Chapter 3 Materials and Methods Table 3Ͳ8: Fusion constructs used in this study. Construct Description GST FLͲPfnek3 TRͲPfnek3 GST GST FLͲƦPfnek3 GST TRͲƦPfnek3 Purpose Plasmid GSTͲtagged full length Pfnek3 Bacterial expression pGEXͲ6PͲ1 GSTͲtagged truncated Pfnek3 (excluding residues 1Ͳ59) Bacterial expression pGEXͲ6PͲ1 GSTͲtagged full length kinaseͲ inactive Pfnek3 Bacterial expression pGEXͲ6PͲ1 GSTͲtagged truncated kinaseͲ inactive Pfnek3 Bacterial expression pGEXͲ6PͲ1 GST Pfmap2 GSTͲtagged Pfmap2 Bacterial expression pGEXͲ3X GST ƦPfmap2 GSTͲtagged kinaseͲinactive Pfmap2 Bacterial expression pGEXͲ3X 6xHis TRͲPfnek3 HisͲtagged truncated Pfnek3 Bacterial expression pDESTͲ17 6xHis TRͲƦPfnek3 HisͲtagged truncated kinaseͲ inactive Pfnek3 Bacterial expression pDESTͲ17 NͲterminal GFPͲtagged full length Pfnek3 Localization pXJ40 NͲterminal GFPͲtagged truncated Pfnek3 Localization pXJ40 NͲterminal GFPͲtagged full length kinaseͲinactive Pfnek3 Localization pXJ40 NͲterminal GFPͲtagged truncated kinaseͲinactive Pfnek3 Localization pXJ40 CͲterminal GFPͲtagged full length Pfnek3 Localization pDEST47 GFP CͲterminal GFPͲtagged truncated Pfnek3 Localization pDEST47 TRͲPfnek3 GAL4ͲDNA binding domain tagged truncated Pfnek3 YeastͲ2Ͳ hybrid pGBKT7 GAL4ͲDNA binding domain tagged NͲterminal Pfnek3 YeastͲ2Ͳ hybrid pGBKT7 FLͲPfnek3 GFP TRͲPfnek3 GFP GFP FLͲƦPfnek3 GFP TRͲƦPfnek3 GFP FLͲPfnek3 TRͲPfnek3 GAL4 DNAͲBD GAL4 DNAͲBD FL-NT 33 Chapter 3 Materials and Methods GAL4 DNAͲBD TR-NT GAL4ͲDNA binding domain tagged NͲterminal Pfnek3 (excluding residues 1Ͳ59) GAL4 DNAͲBD CT GAL4ͲDNA binding domain tagged CͲterminal Pfnek3 YeastͲ2Ͳ hybrid pGBKT7 GAL4ͲDNA binding domain tagged truncated Pfnek3 MammalianͲ 2Ͳhybrid pM GAL4ͲDNA binding domain tagged NͲterminal Pfnek3 MammalianͲ 2Ͳhybrid pM TRͲPfnek3 GAL4 DNAͲBD GAL4 DNAͲBD FL-NT YeastͲ2Ͳ hybrid pGBKT7 GAL4 DNAͲBD TR-NT GAL4ͲDNA binding domain tagged NͲterminal Pfnek3 (excluding residues 1Ͳ59) MammalianͲ 2Ͳhybrid pM GAL4 DNAͲBD CT GAL4ͲDNA binding domain tagged CͲterminal Pfnek3 MammalianͲ 2Ͳhybrid pM YeastͲ2Ͳ hybrid pGADT7 YeastͲ2Ͳ hybrid pGADT7 YeastͲ2Ͳ hybrid pGADT7 YeastͲ2Ͳ hybrid pGADT7 YeastͲ2Ͳ hybrid pGADT7 MammalianͲ 2Ͳhybrid pVP16 MammalianͲ 2Ͳhybrid pVP16 MammalianͲ 2Ͳhybrid pVP16 MammalianͲ 2Ͳhybrid pVP16 MammalianͲ 2Ͳhybrid pVP16 GAL4 AD Hypothetical protein 1 GAL4 AD Hypothetical protein 2 GAL4 AD Merozoite capping protein 1 GAL4 AD TyrosylͲtRNA synthetase GAL4 AD Chromatin assembly factor 1 WD40 domain VP16 AD Hypothetical protein 1 VP16 AD Hypothetical protein 2 VP16 AD Merozoite capping protein 1 VP16 AD TyrosylͲtRNA synthetase VP16 AD Chromatin assembly factor 1 WD40 domain GAL4Ͳactivation domain tagged Prey 3 (Gene ID: PFC0245c) GAL4Ͳactivation domain tagged Prey 6 (Gene ID: MAL13P1.237) GAL4Ͳactivation domain tagged Prey 10 (Gene ID: PF10_0268) GAL4Ͳactivation domain tagged Prey 22 (Gene ID: MAL8P1.125) GAL4Ͳactivation domain tagged Prey 24 (Gene ID: PFA0520c) VP16Ͳactivation domain tagged Prey 3 (Gene ID: PFC0245c) VP16Ͳactivation domain tagged Prey 6 (Gene ID: MAL13P1.237) VP16Ͳactivation domain tagged Prey 10 (Gene ID: PF10_0268) VP16Ͳactivation domain tagged Prey 22 (Gene ID: MAL8P1.125) VP16Ͳactivation domain tagged Prey 24 (Gene ID: PFA0520c) 34 Chapter 3 3.9 Materials and Methods DNA sequencing DNA sequencing using fluorophoreͲlabeled dideoxynucleotide terminators is a rapid and convenient method for the DNA sequencing of the Pfnek3 gene. About 250 – 500 ng of the doubleͲstranded DNA was mixed with 3 µl of BigDye™ Version 3.1 Terminator Ready Reaction Mix containing A, C, G and TͲ dRhodamine Dye Terminators, dITP, dATP, dCTP, dTTP, TrisͲHCl pH 9.0, MgCl2, thermostable pyrophosphatase, AmpliTaq™ DNA polymerase, FS. A relevant sequencing primer (~3.2 pmol), 5X sequencing buffer and deionized water were added to a final volume of 10 µl in a 0.2 ml tube. The thermal cycling program used was in accordance with the manufacturer’s instructions (PerkinͲElmer, USA). To purify the extension products, the entire 10 µl reaction content was transferred to a 1.5 ml microfuge tube containing 1.5 µl of 3 M sodium acetate (pH 4.6), 7.25 µl of deionized water and 31.25 µl of 95% (v/v) ethanol. The mixture was allowed to precipitate for 15 minutes, and then centrifuged at 12,000 RPM for at least 20 min. Ethanol in the supernatant was aspirated and the pellet was rinsed with 250µl of 70% (v/v) ethanol to remove salt content before drying in an incubator (50°C) for 10Ͳ15 min. The dehydrated extension products were then sent for sequencing by the Department of Microbiology Sequencing Team, National University of Singapore, using the ABI PRISM 3100 Gene Analyzer (PerkinͲElmer, USA). 35 Chapter 3 Materials and Methods 3.10 Recombinant protein production Bacterial expression plasmids (pGEXͲ3X, pGEXͲ6PͲ1 and pDEST17) carrying Pfnek3, Pfmap1, Pfmap2 or their mutants were electroͲtransformed into E. coli BL21 (DE3) CodonPlus™ (Stratagene, USA) and thereafter selected on LB agar supplemented with ampicillin (100 µg/ml) and chloramphenicol (40 µg/ml). Two ml of overnight culture was inoculated into 100 ml of antibioticͲsupplemented LB broth and subsequently induced at midͲlog phase (OD600 0.6Ͳ0.8) to express fusion proteins with the addition of 0.5 mM of IPTG (isopropylͲbetaͲDͲthiogalactopyranoside) and incubated overnight at 15Ͳ 20°C in a rotary incubator. 3.10.1 Harvesting cellͲfree extracts The IPTGͲinduced cultures were harvested by centrifugation at 7000 RPM for 15 min at 4°C and the supernatant was discarded. The cell pellet was washed twice with 25 ml of 1X PBS buffer and finally resuspended in 1Ͳ2 ml of 1X PBS buffer. The cell suspension was then transferred into a Bijou bottle prior to cell lysis by sonication on ice with a 1/8” probe (Sanyo MSE Soniprep, UK). The cells were disrupted at 10 µm amplitude for eight rounds of 10 sec pulses, with 20 sec intervals between pulses. The cell lysates were then centrifuged at 12000 RPM for 15 min at 4°C to remove cell debris. The clear supernatant obtained is hereafter referred to as the soluble protein fraction and transferred into clean microfuge tubes. 36 Chapter 3 Materials and Methods 3.10.2 Affinity purification of GSTͲ and HisͲtagged proteins Affinity purification was achieved by incubating the soluble protein fraction with glutathioneͲsepharose 4B (120 µl; Amersham, Sweden) or nickel resin (120 µl; Invitrogen, USA) preͲwashed twice with 1X PBS or 10 mM imidazole, respectively, in spin columns. Crude cellͲfree extracts (soluble protein fraction) were added to the spin column and allowed to incubate on ice with periodic mixing for 15 min. Before elution, the spin column was washed twice with 1X PBS or 10 mM imidazole by centrifugation at 2000 RPM. To elute, the glutathione and nickel resins were incubated with at least 200 µl of iceͲcold 10mM reduced glutathione in 50 mM TrisͲHCl, pH 8.0 (Sigma, USA) or 100Ͳ 200 mM imidazole, respectively, for at least 10 min and eluted into a fresh microfuge tube by centrifugation at 2000 RPM. In some experiments, recombinant proteins were cleaved of their GST moiety by incubating for at least 3 h at 4°C with the Precission™ siteͲ specific protease (Amersham, Sweden). 3.11 Estimation of protein concentration by Bradford assay Protein concentrations were determined using the BioͲRad Protein Assay Reagent (BioͲRad, USA). As a reference, protein standards were prepared using a range of concentrations of bovine serum albumin (BSA; 0 to 1 mg/ml) and test samples were prepared in duplicates using 1X PBS. The BioͲRad Protein Assay Reagent was diluted fiveͲ folds in deionized water and filtered through a Whatman™ No.1 filter paper. For each assay, 250 µl of diluted reagent was added to 10 µl of BSA standards or test sample using a clearͲbottom 96Ͳwell plate. After allowing the color to develop in about 5 min, the 37 Chapter 3 Materials and Methods samples were measured at OD595 using a plate reader fitted with relevant wavelength filters (Tecan Genios™, Austria). The protein concentration of each test sample was determined from a standard curve prepared for each lot of Bradford reagent. 3.12 SDSͲPAGE The SDSͲPAGE apparatus was assembled according to the manufacturer’s instructions (Hoefer, USA). The composition of the various buffers used for gel preparation is shown in Appendix II. In an Erlenmeyer flask, the 10% acrylamide solution for the resolving gel was mixed with 0.1% (w/v) SDS, TEMED and ammonium persulfate (APS). Without delay, the solution was mixed thoroughly and poured into the gap between the glass plates. The acrylamide was overlaid with 70% (v/v) denatured ethanol to obtain a levelͲloading surface. The gel was left to polymerize at room temperature for at least one hour. Thereafter, the ethanol was discarded and any remaining unͲpolymerized gel was washed away with deionized water. The 4% stacking gel solution was prepared similarly. The stacking gel solution was poured onto the polymerized resolving gel and a clean Teflon™ comb was immediately inserted to create protein loading wells. The gel was then left to polymerize at room temperature for at least one hour. When the acrylamide has polymerized, the Teflon™ comb was removed and the wells rinsed with deionized water to remove any unͲpolymerized acrylamide. After mounting the gel onto the electrophoresis apparatus (SE 260 Mighty Small II System, Hoefer, USA), TrisͲglycine electrophoresis buffer [25 mM TrisͲbase, 250 mM glycine, 0.1% (w/v) SDS] was added to 38 Chapter 3 Materials and Methods fill the top and bottom reservoirs. Each protein sample was mixed with 1X SDSͲPAGE gel loading buffer (50 mM TrisͲHCl pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) and denatured by boiling for 5 min before loading into the wells. A preͲ stained protein standard was used as size markers (BioͲRad, USA). Electrophoresis was carried out at 20 mA (constant current) per gel until the bromophenol blue dye front reached the bottom of the resolving gel. The gel was then removed from the electrophoresis setup and stained with Coomassie Blue (0.25% (w/v) Coomassie Brilliant Blue RͲ250, 45% (v/v) methanol, 10% (v/v) acetic acid) for 30 min. The gel was deͲstained by soaking in a deͲstaining solution (30% methanol, 10% acetic acid) until distinct protein bands appeared. Alternatively, the EZͲBlue¥ high sensitivity protein gel stain was used (Sigma, USA). The gel was photographed using the BioͲRad GelͲDoc¥ system. 3.13 Mass spectrometric identification of proteins Liquid chromatographyͲtandem mass spectrometry (LCͲMS/MS) was performed at the Biopolis Shared Facilities Protein Analytics Lab. Protein samples intended for mass spectrometry were gelͲpurified by SDSͲPAGE and the relevant CoomassieͲstained band excised, transferred to a clean microfuge tube and frozen in deionized water until ready for analysis. Prior to LCͲMS/MS, the protein samples were reduced with 10 µL 1M DTT (dithiothreitol) per mg of protein (1 mg/ml) and disulphide linkages disrupted with the addition of 20 µL 1M iodoacetic acid. Thereafter, a sequencingͲgrade trypsin (Promega, USA) was used to digest the protein sample. All MS/MS samples were analyzed using 39 Chapter 3 Materials and Methods Mascot (Matrix Science, UK; version 2.1.03). Mascot was set up to search the MSDB_20060831 database (3239079 entries) assuming digestion by trypsin. Mascot was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 1000 PPM. Oxidation of methionine, iodoacetamide derivative of cysteine and phosphorylation of serine, threonine and tyrosine were specified in Mascot as variable modifications. Scaffold (version 1.06.06, Proteome Software Inc., USA) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 50.0% probability as specified by the Peptide Prophet algorithm (Keller et al., 2002). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). 3.14 Analytical gel filtration Analytical gel filtration was carried out using a Superdex 200 HR 10/30 column run on the AKTA Purifier liquid chromatography system (Amersham, Sweden). The desired protein sample was loaded onto the Superdex 200 filtration column equilibrated in TrisͲHCl (pH 7.5) and the eluted protein was monitored by UV absorbance at 280 nm. The column was calibrated using thyroglobulin, catalase, albumin and chymotrypsinogen A as molecular weight standards (Sigma, USA). 40 Chapter 3 Materials and Methods 3.15 Protein adsorption To assay for kinase substrate preference, a panel of four polypeptides (preͲ diluted to 0.5 mg/ml) was adsorbed to proteinͲbinding 96Ͳwell plates (Costar, USA): (1) histone H1, (2) casein kinase I substrate (peptide sequence RRKDLHDDEEDEAMSITA), (3) casein kinase II substrate (peptide sequence RRADDSDDDDD; all from Calbiochem, USA) and (4) dephosphorylated myelin basic protein (MBP; Upstate, USA). Similarly, GSTͲ fusion kinases were also adsorbed for use as substrates for other kinases. Adsorption was achieved by overnight incubation of the proteins in microplate wells at 4°C in a freshlyͲmade carbonate buffer (0.15 M sodium carbonate and 0.35 M sodium bicarbonate, pH 9.6) or PBS. Before use, remaining adsorptive surfaces were blocked with PBS/1% BSA for one hour at room temperature. To control wellͲtoͲwell consistency of the coated microplates, MBPͲcoated wells were assayed with hMAPK1 to ensure uniform adsorption. To ascertain equal binding of different GSTͲtagged kinases to the microplate wells, antiͲGST primary antibodies (1:1000 dilution; Sigma, USA) were used to detect adsorbed enzymes, and processed with standard peroxidaseͲbased chemiluminescence to demonstrate uniform adsorption. 3.16 Protein kinase assay Each kinase reaction mix of 60 µl comprised 10 µg of GSTͲfusion kinase or the inactive mutant as control, TrisͲHCl (50 mM, pH 7.2), ATP (500 µM), MgCl2 and/or MnCl2 (20 mM and 5 mM, respectively). The kinase mixes were incubated for 30 min at 30°C. Any phosphorylation activity in the kinase mixes were stopped by adding Laemmli’s 41 Chapter 3 Materials and Methods buffer (Laemmli, 1970) and denatured at 95°C for 5 min. The denatured kinase mixes were resolved on 10% SDSͲPAGE gels and subjected to the ProͲQ™ phosphoprotein gel stain (Invitrogen, USA). The major advantage of the ELISA architecture lies in the use of the microplate which is amenable to highͲthroughput inhibitor screenings. In view of this, 30 µl fractions of the aforesaid kinase mixes were transferred into substrateͲcoated ELISA microplates immediately after the assembly of the mixtures, to assay their kinase activities. The microplates were simultaneously incubated at 30°C for 30 min and thereafter quenched by repeated washing with PBS. The quenched microplate kinase assays were blocked for 30 min with PBS/1% BSA and then incubated with mixed monoclonal antiͲphosphoserine/threonine antibodies (1:500 dilution; Upstate, USA). The microplate was then subjected to a peroxidaseͲconjugated secondary antibody (1:4000, Upstate, USA) and the SuperSignal ELISA Femto peroxidase substrate (Pierce, USA). Any resulting chemiluminescence due to the presence of phosphorylated serine and/or threonine residues was measured using the Genios¥ microplate reader (Tecan, Austria). In some experiments, two different GSTͲfusion kinases were coͲincubated in the kinase mixes to detect any in vitro difference in phosphotransfer activity. As controls, either one of the two GSTͲfusion kinases was replaced with its respective kinaseͲdead mutant. 3.16.1 Validation of the ELISAͲbased kinase assay Autoradiography is commonly used for protein kinase assays. However, to facilitate future highͲthroughput inhibitor screens, a chemiluminescent microplate assay was chosen in this study. The performance of the MBPͲcoated microplates prepared in 42 Chapter 3 Materials and Methods this study was benchmarked against commerciallyͲavailable ones by adapting the manufacturer’s quality control protocol using human MAPK1 (Upstate, USA cat. no. 30Ͳ 011). To optimize plate reader sensitivity, between 1 Ͳ 100 ng of human MAPK1 (per reaction of 30 µl) was tested with a range of ATP concentrations (100Ͳ500 µM) to determine whether maximal levels of MBP phosphorylation could be consistently achieved. 3.17 Western blot As an alternative to the ProͲQ™ phosphoprotein gel stain, kinase reactions were performed as described above with the exception that 2.5 µg of MBP was included in the reaction mix as kinase substrate. The mixture was then subjected to SDSͲPAGE and transferred to PVDF (polyvinyldifluoride; Millipore, USA) membranes and probed with a monoclonal antiͲphosphoserine/threonine antibody mix (Upstate, USA) and visualized by chemiluminescence with a peroxidaseͲconjugated antibody (Pierce, USA) and the SuperSignal™ West Femto reagent (Pierce, USA). The image was captured on XͲray film (Fuji Film, Japan). 3.18 Antibody production An affinityͲpurified antiͲPfnek3 antibody was prepared by GeneScript Corp. (New Jersey, USA). Two New Zealand White rabbits were immunized with a synthetic peptide derived from the Pfnek3 amino acid sequence (NQGDLHSDILRKKLC, residues 154Ͳ167). The terminal cysteine residue was incorporated to allow coupling to keyhole limpet 43 Chapter 3 Materials and Methods hemocyanin. The antibody was tested using ELISA and Western blot prior to fluorescence microscopy. To verify the specificity of antiͲPfnek3, proteinͲadsorbing microplates were used, as described in Protein adsorption (Section 3.15), to bind GSTͲPfmap2, GSTͲPfnek3 or their kinaseͲinactive mutants. Microplates were probed using a 1:1000 dilution of antiͲPfnek3 and processed for chemiluminescence as described earlier under Protein kinase assay (Section 3.16). The uniformity of adsorption of GSTͲtagged kinases were assessed with antiͲGST antibodies and processed by secondary antibody peroxideͲbased chemiluminescence. To further verify the specificity of antiͲPfnek3, purified recombinant GSTͲPfmap2 and GSTͲPfnek3 were subjected to Western blot, as described above, and probed with a 1:1000 dilution of antiͲPfnek3, followed by a peroxidaseͲconjugated antiͲ rabbit secondary antibody. Visualization was achieved using a peroxidase substrate, TMB (3,3',5,5'Ͳtetramethylbenzidine; Sigma, USA). 44 Chapter 3 Materials and Methods Figure 3.3: Selection of peptide sequences for synthesis as antigens. The sequences (in red) were proposed, using a proprietary software (Genescript Corp, USA), based on the probability of immunogenicity in rabbits as well as the predicted exterior exposure of the peptide in the context of the tertiary structure of the protein. The NQGDLHSDILRKKL sequence was selected for synthesis and immunizing hosts. 3.19 Immunofluorescence microscopy GradientͲcentrifugation enriched parasites (both sexual and asexual stages) were smeared, airͲdried and fixed on polyͲLͲlysine slides using 4% paraformaldehyde/PBS. Fixed cells were permeabilized with 0.1% Triton XͲ100/PBS for 5 min, blocked with 2% (w/v) BSA/PBS for an hour and immunolabeled with the affinityͲpurified Pfnek3 antibody (diluted 1:100) for an hour. This was followed by a fluorescein isothiocyanate (FITC)Ͳ conjugated antiͲrabbit secondary antibody (45 min; 1:33 dilution; Acris antibodies, Germany). Slides were washed at least three times (10 min of shaking at 60 RPM each time) in PBS containing 0.1% Tween 20. DNA was stained with Hoechst 33342 (Invitrogen, USA) at a final concentration of 5 µg/ml. Cover slips were mounted with 90% glycerol in 50 mM Tris (pH 8.0) containing 2.5% (w/v) 1,4Ͳdiazabicyclo[2.2.2]octane 45 Chapter 3 Materials and Methods (DABCO; Sigma, USA) and viewed with an Olympus BX60 fluorescence microscope with the same microscope and camera settings for images stained with the same fluorophore. 3.20 YeastͲtwoͲhybrid protein interaction studies 3.20.1 Principles YeastͲtwoͲhybrid (Y2H) assays can be used to identify novel proteinͲprotein interactions, confirm suspected interactions, and define interacting domains. A bait gene is expressed as a fusion to the GAL4 DNAͲbinding domain (DNAͲBD), while another gene or cDNA is expressed as a fusion to the GAL4 activation domain (AD). When bait and library fusion proteins interact in a yeast reporter strain such as AH109, the DNAͲBD and AD are brought into proximity thereby activating the transcription of the reporter genes: ADE2, HIS3, lacZ, and MEL1. DNAͲBD and AD fusions were created by cloning cDNAs into the yeast expression vectors pGBKT7 and pGADT7ͲRec, respectively. pGBKT7 expresses proteins as fusions to the GAL4 DNAͲBD, while pGADT7ͲRec expresses proteins as fusions to the GAL4 AD. Pfnek3 Total cDNA Figure 3.4: Screening for proteinͲprotein interactions with the Matchmaker¥ TwoͲHybrid System. A positive interaction event switches on the transcription of the ADE2 and HIS3 auxotrophic selection genes as well as the reporters lacZ and MEL1, which encode assayable ɴͲgalactosidase and ɲͲ galactosidase, respectively. (Image adapted from Clontech, 2006) 46 Chapter 3 Materials and Methods 3.20.2 Testing the bait fusion protein for autoͲactivation and toxicity Yeast strain AH109 was transformed with the hybrid construct using the smallͲ scale transformation protocol according to Clontech’s instructions. Transformants were spread on the following media: (a) SD/–Trp, (b) SD/–His/–Trp and (c) SD/–Ade/–Trp. A negative control comprised of yeast cells transformed with an “empty” pGBKT7 bait vector. For the bait/pGBKT7 fusion to be suitable for use in further twoͲhybrid screens, the bait DNAͲBD fusion protein should not autoͲactivate the transcription of the reporter gene. Desirable transformant colonies were white and did not grow on SD/– His/–Trp or SD/–Ade/–Trp media. Bait constructs that gave rise to transformant colonies that grew on SD/–His/–Trp or SD/–Ade/–Trp were discontinued from use. Also, bait constructs that caused yeast colonies to appear noticeably later than the “empty” bait transformed controls were omitted from subsequent studies as these are likely to be a result of poor heterologous protein expression or toxicity to host cells. 47 Chapter 3 Materials and Methods Generate malaria cDNA library Construct a DNA-DB fusion of bait Enrich poly A+ RNA RT with oligo-dT primers Amplify cDNA by LD-PCR Purify (size-select) cDNA Clone bait gene into pGBKT7 vector in E. coli Prepare competent yeast cells Screen two-hybrid interaction by yeast cotransformation Co-transform yeast with: • • • cDNA pGADT7-Rec (native prey plasmid) pGBKT7/bait plasmid Transform yeast with bait plasmid. Test for autonomous reporter gene activation and cell toxicity. No autoactivation + viable cells Auxotrophic selection for yeasts expressing interacting proteins Auto-activation or cell toxicity Troubleshoot: E.g. truncate bait gene Prey plasmid rescue, DNA sequencing and re-testing phenotype with X-Į-gal assay Figure 3.5: A process flow chart for twoͲhybrid protein interaction screening. 3.20.3 Total cDNA library synthesis, amplification and fractionation In the firstͲstrand cDNA synthesis step, MMLV (Moloney Murine Leukemia Virus) reverse transcriptase (RT) was used to synthesize a total cDNA library from mRNA. To prime RNA for cDNA synthesis, a modified oligo(dT) primer (called CDS III primer) supplied by Clontech (USA) was used. When MMLV RT encounters a 5'Ͳterminus on the template, the enzyme’s terminal transferase activity adds a few additional nucleotides, 48 Chapter 3 Materials and Methods primarily deoxycytidine, to the 3' end of the product cDNA. The SMART III oligonucleotide (Clontech, USA), which has an oligo(G) sequence at its 3' end, baseͲpairs with the deoxycytidine stretch, creating an extended template. MMLV RT then switches templates and continues replicating to the end of the oligonucleotide (Figure 3.6). In the majority of syntheses, the resulting singleͲstrand cDNA contains the complete 5' end of the mRNA as well as the sequence complementary to the SMART III oligo, which then serves as a universal priming site (SMART anchor) in the subsequent amplification by longͲdistance PCR (LDͲPCR). Only those singleͲstrand cDNAs having a SMART anchor sequence at the 5' end can serve as a template and be exponentially amplified by LDͲ PCR. SMART III primer 5’ SMART anchor 3’ SMART anchor GGGGGGG 3’ CCCCCCCCC RT Template switch to SMART III primer 5’ RT CDS III primer cDNA TTTTTT 5’ mRNA template AAAAAA 3’ Reverse transcription using CDS III primer Figure 3.6: Schematic diagram of the SMART III system to generate cDNA with the SMART III and CDS III anchors. First, reverse transcription is performed on endogenous mRNA using the CDS III primer, a modified oligoͲdT primer (boxed with dotted lines). At the 3’ end of the cDNA product, an oligoͲ dC overhang is added by the reverse transcriptase. The SMART III primer contains an oligoͲdG sequence that could base pair to the oligoͲdC overhang. The reverse transcriptase switches to the SMART III primer as template to extend the 3’ end of the cDNA product with a SMART anchor (boxed in solid lines). The SMART III and CDS III sequences serve as the binding sites for the 5’ and 3’ PCR primers (in the following step) as well as the recombination sites during in vivo prey library construction. Abbreviation: RT, reverse transcriptase. 49 Chapter 3 Materials and Methods In the second step, singleͲstranded cDNA was amplified by LDͲPCR to produce a doubleͲstranded cDNA library. The Advantage 2 Polymerase Mix consists of TITANIUM™ nuclease activityͲdeficient Taq DNA polymerase and a proofͲreading Tth polymerase, and an antibody for hotͲstarting the thermal cycling. The recipe and protocol for cDNA synthesis is described in Table 3Ͳ9 and Table 3Ͳ10. After obtaining doubleͲstranded cDNA from the PCR step, CHROMA SPIN TEͲ400 columns (Clontech, USA), containing resins that fractionate molecules based on size, were used to obtain cDNA larger than 200 bp by size exclusion. These molecules quickly move through the gel bed when the column is centrifuged, while molecules smaller than the resin pore size are held back. 50 Chapter 3 Materials and Methods Table 3Ͳ9: Recipe for cDNA library preparation Step Reagent / description 1 Add poly A+ RNA (~0.025–1.0 µg) 2 Add CDS III primer 3 Mix contents in a 0.2 ml PCR tube and spin briefly 4 Incubate at 72°C for 2 min 5 IceͲchill for 2 min and spin down briefly 6 Add 5X FirstͲstrand buffer 7 Add DTT (20 mM) 8 Add dNTP Mix (10 mM) 9 Add MMLV reverse transcriptase 10 Mix gently by tapping and spin briefly 11 Incubate at 42°C for 10 min 12 Add SMART III oligonucleotide 13 Incubate at 42°C for 3 h in a hotͲlid thermal cycler Place the tube at 75°C for 10 min to terminate firstͲstrand 14 synthesis 15 Cool the tube to room temperature, then add RNase H 16 Incubate at 37°C for 20 min Proceed to LDͲPCR by taking a 2 µl aliquot from the firstͲ strand synthesis and place it in a clean, prechilled, 0.2Ͳml 17 PCR tube. Any firstͲstrand reaction mixture not used right away was stored at –20°C up to three mth 18 Preheat the PCR thermal cycler to 95°C 19 To 2 µl firstͲstrand ss cDNA, add nucleaseͲfree water 20 Add 10X Advantage 2 PCR buffer 21 Add 50X dNTP mix 22 Add 5' PCR primer 23 Add 3' PCR primer 24 Add 10X GCͲMelt solution 25 Add 50X Advantage 2 polymerase mix Total volume Table 3Ͳ10: cDNA library thermal cycling parameters Temperature (°C) Duration 95 30 sec 95 10 sec 68 6 min + 5 sec increment per successive cycle 68 5 min 4 Hold Volume (µl) 3 1 2 1 1 1 1 1 70 10 2 2 2 10 2 100 40 cycles 51 Chapter 3 Materials and Methods 3.20.4 Constructing and screening a twoͲhybrid library To screen the P. falciparum cDNA library prepared earlier, the pGBKT7 vector carrying the bait DNA sequence (5 µg), the SmaI–linearized native pGADT7ͲRec prey plasmid (6 µl of 0.5 µg/µl) and the fractionated P. falciparum cDNA library (20 µl), generated as described earlier, and 20 µl of herring testes carrier DNA was added to a sterile 15 ml Falcon tube. FreshlyͲmade competent cells (600 µl) were added to the tube. PEG/LiAc (2.5 ml) was added and vortexed. The tube was incubated at 30°C for 45 min with shaking at 200 RPM. DMSO (160 µl) was added to the tube which was then heatͲshocked in a 42°C water bath for 20 min with mixing every 10 min. The mixture was centrifuged at 700 RCF for 5 min and the supernatant decanted. YPD Plus liquid medium (3 ml; Clontech, USA) was added and the tube incubated at 30°C with shaking at 200 RPM for 90 min. The tube was then centrifuged at 700 RCF for 5 min, the supernatant discarded and reͲsuspend in 6 ml of sterile 0.9% (w/v) NaCl. To select for transformants expressing interacting proteins, the coͲ transformation mixture was spread on nutritionalͲselection QDO plates (Quadruple DropͲOut medium = SD/–Ade/–His/–Leu/–Trp; 150 µl/plate). To assess the transformation efficiency of both the pGBKT7 and pGADT7ͲRec plasmids, 150 µl of the coͲtransformation mixture was spread on (a) SD/–Trp and (b) SD/–Leu plates, (c) SD/– Leu/–Trp, which select for cells carrying pGBKT7/bait, pGADT7ͲRec/prey or both plasmids, respectively. 52 Chapter 3 Materials and Methods The plates were incubated at 30°C at least 4Ͳ8 days. The small, pink colonies that appeared after 2 days but never grew to >1 mm in diameter were ignored because true Ade+ and His+ colonies were robust and could grow to >2 mm. Large pearlͲwhite colonies were each picked and streaked on fresh QDO plates and offspring colonies were inoculated into 5 ml QDO broth and incubated at 30°C for 24Ͳ48 h to stationary phase before being considered for pGADT7Ͳprey plasmid rescue. 3.20.5 Prey plasmid rescue and identification by DNA sequencing A large (2–4 mm), fresh (2–4 days) yeast colony from a QDO selection plate was inoculated into 5 ml QDO broth and incubated for 24Ͳ48 h at 30°C with shaking at 250 RPM. An aliquot (2 ml) of the stationary phase culture was pelleted by centrifugation at 14,000 RPM for 5 min. The supernatant was discarded and the pellet reͲsuspended in the residual liquid. Twenty µl of lyticase solution (5 U/µl; Sigma, USA) was added to each tube and thoroughly reͲsuspended by vortexing. The tubes were incubated at 37°C for 2Ͳ 3 h with shaking at 250 RPM. Twenty µl of 25% (w/v) SDS was added to each tube and vortexed vigorously for 1 min. The samples were then frozen for 30 min at –80°C and thawed before being vortexed again to ensure complete lysis of the cells. The samples were then processed with the Wizard™ SV plasmid miniprep kit according the instructions provided (Promega, USA). The pGADT7ͲRec/prey recombinant plasmid was then transformed into E. coli TOP10 cells by electroporation and selected on LB plates containing ampicillin (100µg/ml). The plasmids were isolation from E. coli using the Wizard™ SV plasmid 53 Chapter 3 Materials and Methods miniprep kit to obtain sufficient amounts for DNA sequencing. Only prey inserts at least 400 bp long were considered relevant for reͲtesting of interaction phenotype using the small scale yeast transformation protocol. 3.20.6 ReͲtesting the interaction by smallͲscale transformation For the purpose of interaction confirmation, a mixture containing the pGBKT7Ͳ bait recombinant plasmid (0.1 µg), recombinant pGADT7 containing a putative prey DNA (0.1 µg) and herring testes carrier DNA (0.1 mg) was prepared. FreshlyͲmade yeast competent cells (0.1 ml) were added to each tube and vortexed. 0.6 ml of sterile PEG/LiAc solution was added to each tube and vortexed for 10 sec. Cells were incubated at 30°C for 30 min with shaking at 200 RPM, after which, 70 µl of DMSO was added. Cells were then mixed by gentle inversion and heatͲshocked for 15 min in a 42°C water bath. Cells were chilled on ice for 1–2 min for recovery followed by centrifugation for 5 sec at 14,000 RPM at room temperature. After removal of the supernatant, cells were reͲ suspended in 0.5 ml of sterile 1X TE buffer and 100 µl from each sample were spread on QDO agar plates that selected for cells expressing the auxotrophic markers. For coͲ transformation samples, three controls plates (ͲLeu, ͲTrp and ͲLeu/ͲTrp) were needed to ensure comparable transformation efficiencies of both bait and prey plasmids. Plates were incubated at 30°C until colonies appear (at least 2–4 days). 3.20.7 ɲͲgalactosidase reporter assay The XͲɲͲgal assay is a colorimetric method for the detection of yeast ɲͲ galactosidase activity resulting from the downstream expression of the MEL1 reporter 54 Chapter 3 Materials and Methods gene in GAL4Ͳbased Y2H systems when two proteins interact. Upon binding of the GAL4 tag of the protein of interest to the MEL1 upstream activating sequence, the MEL1 gene product, ɲͲgalactosidase, is upregulated and secreted to the periplasmic space and culture medium where it hydrolyses an artificial colorimetric substrate, XͲɲͲgal (Clontech, USA). To monitor the expression of MEL1 directly on nutritional selection plates, XͲɲͲGal (dissolved at 20 mg/ml in dimethylformamide just before use) was spread on top of the medium and allowed to dry partially before spreading transformed yeast cultures. As ɲͲgalactosidase accumulated in the medium, it hydrolyzed XͲɲͲGal causing yeast colonies to turn blue. 3.21 MammalianͲtwoͲhybrid protein interaction studies 3.21.1 Principles The mammalianͲtwoͲhybrid (M2H) system utilizes the secreted alkaline phosphatase (SEAP) reporter, for testing proteinͲprotein interactions first identified in Y2H screens, in mammalian cells. The assay is an important followͲup to yeast screens because it tests interactions under conditions that allow for postͲtranslational changes to hybrid proteins (i.e., phosphorylation, acetylation and proteolysis) that cannot be replicated in yeast. This assay is also useful for confirming the relevance of proteinͲ protein interactions identified via Y2H screens. Such confirmation eliminates the possibility of a false positive arising as an artifact from working in yeast cells (Clontech, 2005). 55 Chapter 3 Materials and Methods In the M2H assay, fusions of Pfnek3 to the GAL4 DNAͲBD (binding domain) of the bait vector (pM) were constructed (Figure 3.7). Similarly, the pVP16 vector is used to construct fusions of prey sequences, identified from Y2H studies, to an activation domain (AD) derived from the VP16 protein of the herpes simplex virus. pG5SEAP is a reporter vector which encodes the SEAP reporter gene under the control of a GAL4Ͳresponsive element in the form of five consensus GAL4 binding sites and the minimal promoter of the adenovirus E1b gene (Figure 3.7). A fourth plasmid (pXJ40, gift of E. Manser, Centre for Molecular Medicine, Singapore) that encodes a GFP reporter under the control of a constitutive CMV promoter allowed for the wellͲtoͲwell normalization of transfection efficiencies by quantitating the amount of GFP fluorescence using a microplate fluorometer (Tecan Genios™, Austria). All four plasmids were coͲtransfected into mammalian host cell lines (HepG2 or MCF7) using a liposomeͲbased method. TwoͲthree days postͲtransfection, the cell culture media can then be assayed for SEAP activity using a chemiluminescent substrate as an indication of interaction between Pfnek3 and its putative partner proteins. 56 Chapter 3 Materials and Methods Pfnek3 Prey Figure 3.7: Reporter gene activation during a stable protein interaction event. The GAL4 tag encoded by the pM vector directs the binding of the bait fusion protein to the UAS (upstream activating sequence) of the pG5SEAP vector. During a stable interaction between the bait and the prey, which is fused with the viral transcription AD (activation domain), the expression of SEAP is upregulated and its activity could thus be detected via standard microplate assays (Image modified from Clontech, 2005). 3.21.2 M2H cell transfection Prior to transfection, 6Ͳwell plates were seeded with about 1 million cells/well using antibioticͲfree Dulbecco’s modified Eagle’s medium (DMEM; containing 10% (v/v) fetal calf serum) and allowed to grow overnight achieving 75Ͳ90 % confluence. The medium was replaced with serumͲreduced OptiMEM™ (Invitrogen, USA) mixed with 10 µl of Lipofectamine 2000™ reagent per 2.5 ml of OptiMEM™ per well containing the respective amounts of each plasmid described below. Mammalian fusion plasmids containing the bait Pfnek3 sequence and the prey sequences identified from Y2H screens (4 µg each) were coͲtransfected together with the pG5SEAP reporter plasmid (2 µg) and pXJ40 GFP reporter plasmid (2 µg) into either MCF7 or HepG2 cells using the Lipofectamine 2000™ reagent (Invitrogen, USA). 57 Chapter 3 Materials and Methods Pfnek3 Gene X GFP Plasmid Purpose pXJ40 “Bait” “Prey” Reporter Transfection normalization Figure 3.8: Plasmids required for a M2H assay. The bait plasmid (pM) was used to clone bait gene Pfnek3 (or its truncations) and the prey plasmid (pVP16) was used to subͲclone prey DNA identified from prior yeastͲtwoͲhybrid screens. pG5SEAP encodes a secreted alkaline phosphatase (SEAP), the expression of which, depends on stable interaction of the bait and prey proteins. SEAP activity could be assayed using a chemiluminescent substrate and read in a luminometer. A GFPͲexpressing plasmid (pXJ40; gift of E. Manser, Centre for Molecular Medicine), was coͲtransfected with all abovementioned plasmids as a normalization control for transfection efficiency (modified from Clontech, 2005). 58 Chapter 3 Materials and Methods 3.21.3 Normalized SEAP activity assay Any sustained protein interaction event could activate the expression of SEAP, which could subsequently be detected using a chemiluminescent assay. A small amount (150 µl) of postͲtransfection (48Ͳ72 h) sample media was transferred into clean microfuge tubes. To remove cell debris, the tubes were centrifuged at 12,000 RPM for 10 sec and the supernatant transferred into fresh tubes. The cell media were heated at 65°C for 30 min to inactivate endogenous phosphatase activities, since SEAP is heatͲstable. The presence of GFPͲexpressing cells was confirmed by direct observation with an Olympus IX70 inverted fluorescence microscope. To normalize the SEAP assay readouts, the 6Ͳwell plates containing the transfected cells were measured for average GFP expression levels at 16 spots per well using a plate fluorometer (Tecan Genios™, Austria) set to excite at 488 nm and detect emission at 535 nm. Using white, flat bottom 96Ͳwell plates (Costar, USA), heatͲinactivated cell media (40 µl) were mixed with the 1x dilution buffer (20 µl), assay buffer (60 µl) and a chemiluminescent substrate, CSPD™ (60 µl of diluted CSPD™; preͲdiluted 1:20 with enhancer solution). The resulting chemiluminescence was read with a plate luminometer (Tecan Genios™, Austria) at 1 min intervals for 30 min to determine signal maxima. GFPͲ normalized SEAP activity readouts were expressed as “folds over basal control”, which consisted of samples transfected with “empty” bait and prey vectors. 59 Chapter 3 Materials and Methods Table 3Ͳ11: M2H transfection setͲup Transfection Sample GAL4 DNAͲ BD plasmid (4 µg) VP16 AD plasmid (4 µg) SEAP reporter (2 µg) GFP normalization (2 µg) SEAP activity pMͲPfnek3 pVP16Ͳ prey pG5SEAP pXJ40 To be determined pMͲ53 pVP16ͲT pG5SEAP pXJ40 Typically about 100Ͳ folds over basal control Basal controlb pM pVP16 pG5SEAP pXJ40 Low Bait controlc pM pVP16Ͳ prey pG5SEAP pXJ40 To be determined Prey controlc pMͲPfnek3 pVP16 pG5SEAP pXJ40 To be determined Positive controla Notes a pMͲ53 encodes p53 which is known to interact strongly with the large T antigen encoded by pVP16ͲT, both provided by Clontech as positive controls. b A basal control that allows for the determination of endogenous alkaline phosphatase activities. c Critical controls that involve the replacement of either one of the sample bait or prey with their respective “empty” bait or prey vectors so as to eliminate the possibility of SEAP transcriptional autoͲactivation. 60 Chapter 3 Materials and Methods Table 3Ͳ12: Summary of kits used in the study Kit Purpose Source QIAamp¥ DNA Blood Mini Kit DNA extraction from P. falciparum Qiagen RNeasy¥ Mini Kit RNA extraction from P. falciparum Qiagen MinElute¥ DNA Gel Extraction Kit Recovery of DNA from agarose gel Qiagen Sensiscript¥ twoͲstep transcriptase kit Reverse transcription Qiagen Enrichment of PolyͲA+ mRNA Qiagen reverse OligoTEX¥ mRNA enrichment Wizard¥ Plus SV MINIprep DNA Small scale plasmid purification purification system Promega PureYield¥ MIDIprep purification system Medium scale plasmid purification Promega DNA sequencing PerkinͲ Elmer BigDye¥ Terminator Reaction mix DNA Ready Zero Blunt¥ TOPO¥ PCR cloning Cloning of bluntͲend PCR products kit Invitrogen directional Cloning of bluntͲend PCR products Invitrogen for Gateway¥ cloning pENTR™/DͲTOPO cloning kit LRͲClonase™ kit For Gateway¥Ͳbased transfer of DNA from DͲTOPO vectors to pDEST Invitrogen vectors 6xHis purification kit Purification of 6xHis tagged proteins Invitrogen Matchmaker¥ TwoͲHybrid Library cDNA library construction yeastͲtwoͲhybrid screening Construction & Screening Kit and Matchmaker¥ Mammalian TwoͲ Mammalian twoͲhybrid screening Hybrid assay kit Great EscAPe¥ detection kit SEAP activity Detection of expression in hybrid assays Clontech Clontech SEAP reporter mammalianͲtwoͲ Clontech 61 Chapter 4 Results and Discussion 4 Results and Discussion 4.1 BioͲcomputational identification of Pfnek3 The predicted ORF of Pfnek3 encodes a protein with limited homology of about 10% sequence identity at its kinase domain to its closest NEK family member, human NEK9, identified by BLASTp searches (Figure 4.1). This information contrasted against a phylogenetic tree plotted using ClustalW which placed Pfnek3 in a different clade from mammalian NEKs (Figure 4.2). In comparison to the first malarial NEK reported (Pfnek1), Pfnek3 is hardly similar in terms of residue composition (17% identity) and peptide length (1057 vs. 347 amino acids, respectively). Close scrutiny of the alignment data revealed a few peculiarities which may explain the uniqueness of Pfnek3 (Figure 4.1). Firstly, the major region of similarity with human NEK9 is embedded only in the kinase domain. Secondly, a putative NͲterminal signal sequence and localization signal or transmembrane region in Pfnek3 predicted by the PlasmoAP, PATS and TMHMM2 algorithms, respectively, may give an indication of an unknown cellular localization. Thirdly, Pfnek3 appears to be devoid of any kinase activation motif commonly located at subdomain VIII (Figure 4.1). Collaterally, Pfnek1 lacks the classical NIMA activation motif, FXXT, at subdomain VIII which was replaced by a SMAHS motif reminiscent of a MEK activation site (Dorin et al., 2001) (included in Figure 4.1 for comparison). Interestingly, Pfnek3 lacks both the FXXT and SMAHS motifs (Figure 4.1, subdomain VIII). The 11 subͲdomains of the protein kinase domain (Figure 4.1) encoded by the ORF of Pfnek3 were not completely in agreement with known NEKs (Hanks and Hunter, 62 Chapter 4 Results and Discussion 1995). SubͲdomain I does not contain the glycine triad (GXGXXG) principally involved in ATP binding. Instead, the corresponding motif is altered to FMTSDS in Pfnek3, with none of the Gs in the triad conserved (Figure 4.1). Reportedly, nearly a third of human protein kinases lacks the third G of the triad, and 11% lack the first one (Kostich et al., 2002). So far, a small collection of kinases, for example, Schizosaccharomyces pombe MIK1, are reportedly devoid of all three Gs but yet can possess protein kinase activity (Lee et al., 1994), and this may now include Pfnek3. Also, the highly conserved lysine residue in subͲ domain II that is critical to contacting, anchoring and orienting the ɲͲ and ɴͲphosphates of ATP could not be detected initially using PROSITE algorithms. However, based on multiple sequence alignments with other protein kinases, it was putatively identified as K102. Intriguingly, the residues in the vicinity of K102 in Pfnek3 (Figure 4.1) have deviated from consensus sequences reported in other NIMAͲrelated kinases (Hanks and Hunter, 1995). Furthermore, the HRDXXXXN baseͲacceptor motif of eukaryotic protein kinases in subͲdomain VIb is represented by a different motif, HGDLKSTN. Since the frequency of a glycine (underlined) appearing in this subͲdomain among eukaryotic protein kinases is believed to be low, this particular glycine (G199) serves as an interesting target, therefore, warranting further studies on its role. Finally, the aspartate residue conserved in the DFG motif of subͲdomain VII (D217), is thought to elicit the binding of Mg2+ or Mn2+ ion associated with the ɴͲ and ɶͲphosphates of ATP. 63 Chapter 4 Asp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 Results and Discussion --------------------MSVLG-EYERHCDSINSDFGSESGGCGDSS --------------------MSALG-RYDRHCDSINSDFGDSVRSCG-------------------------------------------------------------------------------------MPSKYDDG-------MVCIYLFCFLLFHLFVVNTRMRILCNLLIVICEYMFPFYSFILFKFVKML Putative signal peptide 29 26 8 50 Putative localization or transmembrane region sp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 PGPSASQGPRAGGGAAEQEELHYIPIRVLGRGAFGEATLYRRTEDDSLVV ---------------PEQEELHYIPIRVLGHGAYGEATLYRRTEDDSLVV -------------------MEKYVRLQKIGEGSFGKAILVKSTEDGRQYV ----------------ESRLNEYEVIKKIGNGRFGEVFLVKHKRTQEFFC SLLLPSILSCEKKYQVYKNNGYKFETVLDFMTSDSEIHLIRSIESDEIYI .: * : . 79 61 31 42 100 sp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 WKEVDLTRLSEKERRDALNEIVILALLQ-HDNIIAYYNHFM--DNTTLLI WKEVGLARLSEKERRDALNEIVILSLLQ-HDNIIAYYNHFL--DSNTLLI IKEINISRMSSKEREESRREVAVLANMK-HPNIVQYRESFE--ENGSLYI WKAISYRGLKEREKSQLVIEVNVMRELK-HKNIVRYIDRFLNKANQKLYI SKVYDIYGINEDDLNKYMNELYIMNKLRNCENIVNIIDYIK--ENDTLSF * . :.. : . *: :: :: **: : : . .* : 126 108 78 91 148 sp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 ELEYCNGGNLYDKILRQKD--KLFEEEMVVWYLFQIVSAVSCIHKAG--ELEYCNGGNLFDKIVHQKA--QLFQEETVLWYLFQIVSAVSCIHKAG--VMDYCEGGDLFKRINAQKG--VLFQEDQILDWFVQICLALKHVHDRK--LMEFCDAGDLSRNIQKCYKMFGKIEEHAIIDITRQLLHALAYCHNLKDGP ILEFCNQGDLHSDILRKKLNNEIYTESEIFNILHQILNGLNIIHQNG--:::*: *:* * * :. *: .: *. 171 153 123 141 195 sp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 ----ILHRDIKTLNIFLTK-----------------ANLIKLGDYGLAKK ----ILHRDIKTLNIFLTK-----------------ANLIKLGDYGLAKQ ----ILHRDIKSQNIFLTK-----------------DGTVQLGDFGIARV NGERVLHRDLKPQNIFLSTGIRHIGKISSQANNLNSRPIAKIGDFGLSKN ----IIHGDLKSTNIFIK------------------DNKIKIGDFGISSE ::* *:*. ***:. ::**:*:: 200 182 152 191 223 sp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 LNSEYSMAETLVGTPYYMSPELCQGV--KYNFKSDIWAVGCVIFELLTLK LSSEYSMAETCVGTLYYMSPEICQGV--KYSFKSDIWAVGCVLYELLTLT LNSTVELARTCIGTPYYLSPEICENK--PYNNKSDIWALGCVLYELCTLK IGIE-SMAHSCVGTPYYWSPELLLHETKSYDDKSDMWALGCIIYELCSGK QSSNN-----NLGTLNCLSYESIKFK--KTNKLSDLFQVGCILYELATLS . :** * * . **:: :**:::** : . 248 230 200 240 266 sp|Q8TD19|NEK9_HUMAN sp|Q7ZZC8|NEK9_XENOPUS sp|Q96PY6|NEK1_HUMAN PlasmoDB|PFL1370W|Pfnek-1 PlasmoDB|PFL0080c|Pfnek-3 RTFDATNPLNLCVKIVQGIRAMEVDSSQYSLELIQMVHSCLDQDPEQRPT RTFDATNPLNLCVKIVQGNWAVGLDNTVYTQELIEVVHACLEQDPEKRPT HAFEAGSMKNLVLKIISG--SFPPVSLHYSYDLRSLVSQLFKRNPRDRPS TPFHKANNFAQLISELKR--GPELPIKGKSKELNLLIKNLLNLSAKERPS SPFCATN-INDMISLFE--------DKNYKSYIIKNISSIYSQKLVN--.* . :. .. . : : . . . I II III IV V VI a VIb VIII X B NTR Kinase domain Kinase domain RCC RCC RCC RCC RCC RCC VII IX 298 280 248 288 304 XI Pfnek3 Human NEK9 ~10% amino acid sequence identity in the kinase domains Figure 4.1: Sequence alignment of Pfnek3 with other NEKs and comparison of domain architecture with the closest homologue. (A) An NͲterminal stretch of 59 primarily hydrophobic residues was predicted to be a signal peptide and organelleͲtargeting signal or transmembrane region using, respectively, the PlasmoAP, PATS (score: 0.662 of 1) and TMHMM2 algorithms (Foth et al., 2003; Zuegge et al., 2001 and Krogh et al., 2001). Pfnek3 does not exhibit complete agreement with the 11Ͳsubdomain model of eukaryotic protein kinases (RomanͲnumbered boxes). For example, Pfnek3 belongs to a minority of kinases completely devoid of all three Gs in the glycine triad (GXGXXG) in subdomain I. Although Pfnek3 lacks the glycine triad (residues 80–85), a lysine residue, potentially important for ATP binding is conserved at K102. Mutation at this residue obliterates the kinase activity of Pfnek3. (B) Schematic diagram of 64 Chapter 4 Results and Discussion the Pfnek3 domain architecture compared to human NEK9, its closest homologue identified by nonͲ redundant BLASTp searches. Both kinases share a sequence identity of about 10% at their respective kinase domains. The kinase domains were demarcated with ScanProsite (www.expasy.org; Pfnek3: residues 52–334; human NEK9: residues 52–308). A marked divergence of protein architectures is exemplified by Pfnek3 having limited accessory domains. Abbreviations: RCC, Regulator of chromosome condensation domain; NTR, NͲterminal region. Table 4Ͳ1: Typical protein kinase domain features Kinase subdomain Typical features (Hanks and Hunter, 1995) I The glycine triad, GxGxxG motif (absent in Pfnek3) II An invariant lysine residue, important for ATP binding. KїM mutations will inactivate the kinase. III An invariant glutamate residue that assists in the stabilization of the interaction with ATP. IV No invariant residues. Not critical for kinase activity. Usually a ɴͲstrand structure. V Connects the small lobe and large lobe of the kinase domain VI a An ɲͲhelical region acting mainly to support the enzyme structure VI b Kinase catalytic loop which is characterized by a conserved motif, HRDLKxxN, which is represented by HGDLKSTN in Pfnek3. The appearance of a G (underlined) in place of R is unusual. VII Characterized by a DFG triplet motif believed to be involved in chelating Mg2+ or Mn2+ VIII Typically represented by an ‘APE’ triplet motif in most kinases, but changed to an ‘SPE’ motif in NIMA family kinases. However, in Pfnek3, the motif has been changed to ‘SYE’. The glutamate residue (underlined) forms an ion bridge with an invariant arginine in subdomain XI (which is absent in Pfnek3), thereby stabilizing the large lobe. Subdomain VIII also contains phosphorylation sites in many kinases, e.g. the SMAHS motif in Pfnek1. But corresponding phosphorylation sites are not detected in Pfnek3. IX A primarily ɲͲhelical structure involving an invariant aspartate residue. X The most poorly conserved subdomain typically containing insertions of varying lengths and its function is therefore unknown. XI Typically contains an invariant arginine residue that forms in ion bridge with a glutamate in subdomain VIII. However, this arginine residue is not detected in Pfnek3. Location on kinase domain Small lobe Linker region Large lobe 65 Chapter 4 Results and Discussion Figure 4.2: Phylogenetic analysis. The fullͲlength Pfnek3 amino acid sequence was used to query the SwissͲProt and TrEMBL nonͲredundant databases at www.expasy.org. Incompletely annotated sequences were filtered out and the remaining sequences were extracted and submitted to the ClustalW sequence alignment package, which generated dendrograms viewable with the TreeView software available at http://taxonomy.zoology.gla.ac.uk/rod/treeview.html. The dendrogram detaches Pfnek3, as well as the rodent malarial homologue (Pbnek3) from mammalian NEKs. Abbreviations: PK, protein kinase; sp, SwissͲ Prot database sequence identifier; tr, TrEMBL database sequence identifier. Pfnek3 Pfnek1 Figure 4.3: Structural models of Pfnek3 and Pfnek1. Full protein sequences of Pfnek3 and Pfnek1 were submitted to SwissͲModel (www.expasy.org). Both resulting models were demarcated at their respective kinase domains after derivation from a solved protein structure (PDB id: 2javA) based on the highest percentages of amino acid identities. A major dissimilarity was identified at the CͲterminal of the kinase domain (blue ɴͲsheets), which corresponded to kinase subdomain XI. 66 Chapter 4 Results and Discussion 4.2 Molecular cloning of FLͲ and TRͲPfnek3 The Pfnek3 ORF was amplified using either cDNA or genomic DNA from P. falciparum Tan or 3D7 strains as template, and the amplified product size appeared identical (~ 1 kb) suggesting that the Pfnek3 gene is nonͲintronic, as was later confirmed by DNA sequencing. The Pfnek3 DNA sequence from the 3D7 strain varied from the Tan stain at a single base (AїC at nucleotide 975), which was a conserved strain variation. The Tan strain Pfnek3 DNA was retained for further work. The cloning of a truncated Pfnek3 (TRͲPfnek3; Figure 4.4) was necessitated by the fact that the 59Ͳresidue hydrophobic NͲterminal domain of the fullͲlength version (FLͲPfnek3), which was predicted to be a signal peptide or an organelleͲtargeting signal (indicated in Figure 4.1) and therefore may interfere with the solubility of the expressed recombinant protein. Pfu DNA polymerase is a high fidelity enzyme that generates bluntͲend PCR products. Hence, the vector, pCRͲBlunt IIͲTOPO™, was employed to clone the PCR and RTͲPCR products of Pfnek3. Vaccinia virus DNA topoisomerase I is exploited in TOPO™ cloning to efficiently ligate bluntͲend PCR products into the vector. This reaction occurs spontaneously in 5 minutes at room temperature. Moreover, the cloning kit enables direct selection of recombinants via disruption of the lethal E. coli gene, ccdB, which therefore obviated the need for blueͲwhite selection. Thereafter, the recombinant vectors were chemicallyͲtransformed into competent E. coli strain TOP10. The transformed cells were then selected for on kanamycinͲsupplemented (50 µg/ml) LB plates incubated at 37oC overnight. 67 Chapter 4 Results and Discussion In order to authenticate that the growing colonies were indeed transformants, the respective plasmids were extracted and restriction enzyme digestion carried out on the plasmids of at least four wellͲisolated colonies. Representative clones are depicted in Figure 4.4. Restriction enzyme digestion analysis using BamHI and EcoRI revealed that both the fullͲlength and truncated copies of the Pfnek3 gene were cloned. Verification of the gene sequence was performed by DNA sequencing. Moreover, by sequencing genomic PCR and RTͲPCR products, Pfnek3 is confirmed to be nonͲintronic. The verified fullͲlength and truncated Pfnek3 clones were hereafter named TOPOͲFLͲPfnek3 and TOPOͲTRͲPfnek3, respectively. B A Pfnek3 kb 23 9.4 6.5 4.4 2.0 0.5 M - RT FL TR kb 23 9.4 6.5 2.0 Pfnek3 M TR FL ~ 3.5 kb (Linearized pCRTOPO¥ cloning vector) 0.5 Figure 4.4: Gel visualization of PCR products. (A) The RTase negative control (ͲRT) ensured that the template was cDNA and not genomic DNA. Legend: M = ʄ/HindIII DNA marker; RT = reverse transcriptase; FL = fullͲlength; TR = truncated. (B) Restriction enzyme digestion of pCRͲTOPO™ͲPfnek3 recombinant vectors. 68 Chapter 4 4.3 Results and Discussion Construction of plasmids for bacterial expression To obtain sufficient amounts of the Pfnek3 protein for functional assays, attempts were made to subclone both FLͲ and TRͲPfnek3 into an expression vector. Respectively, TOPO™ͲFLͲPfnek3 and TOPO™ͲTRͲPfnek3 were doubleͲdigested with BamHI and EcoRI to release the coding fragments which were separated on, and purified from, agarose gels. Thereafter, the purified FLͲ and TRͲPfnek3 fragments were inserted inͲframe into the polylinker region of the pGEXͲ6PͲ1 expression vector in order to produce recombinant proteins with GST purification tags. T4 DNA ligase was employed for constructing recombinant vectors encoding FLͲ and TRͲPfnek3. The ligation products were then transformed into electrocompetent E. coli CodonPlus™ cells. Positive transformants were selected for on LB agar supplemented with ampicillin (100µg/ml) and chloramphenicol (50µg/ml). Primary screening for successful transformants were carried out by doubleͲdigesting the plasmid extracts of ampicillinͲresistant clones with BamHI and EcoRI (Figure 4.5). DNA sequencing authenticated the pGEX™ clones of FLͲ and TRͲPfnek3 and were thereafter named pGEX™ͲFLͲPfnek3 and pGEX™ͲTRͲPfnek3. 69 Chapter 4 Results and Discussion Pfnek3 kb M FL TR 23 9.4 6.5 4.4 2.0 ~ 4.9 kb Linearized pGEX-6P-1 0.5 Figure 4.5: Restriction digestion screening for FLͲ and TRͲPfnek3ͲpGEX recombinant constructs. All lanes are indicative of positive constructs and representative bands for FLͲPfnek3 and TRͲ Pfnek3 are indicated by arrowheads. Legend: M, ʄ/HindIII DNA marker; FL, fullͲlength; TR, truncated. 4.4 Construction of plasmids for yeastͲtwoͲhybrid studies In order to study the protein interactions of Pfnek3, it was first necessary to clone the gene into the bait vector (pGBKT7), which encodes a GAL4 DNAͲBD fusion of Pfnek3. Building on current knowledge on the degree of expressibility of malarial proteins in yeast (LaCount et al., 2005), five modular constructs (Figure 4.6) were created by PCRͲ amplification with relevant primers (listed in Table 3Ͳ2), all designed with an EcoRI and a BamHI site flanking the 5’ and 3’ ends of the gene, respectively, to facilitate insertion of the fragments into the polylinker region of pGBKT7. The insertion of the fragments was verified by double digestion with EcoRI and BamHI (Figure 4.6) and the correctness of the reading frame subsequently confirmed by DNA sequencing. 70 Chapter 4 A B Results and Discussion 1 Full length Kinase domain FLͲPfnek3 2 Truncated Kinase domain TRͲPfnek3 3 Full length N terminal Kinase FLͲNTͲPfnek3 4 Truncated N terminal Kinase TRͲNTͲPfnek3 5 C terminal domain kb M 1 4 3 2 CTͲPfnek3 5 Linearized pCR-TOPO¥ vector 1.0 0.75 0.5 0.25 C kb M 2 3 4 5 Linearized pGBKT7 vector 1.0 0.75 0.5 0.25 Figure 4.6: Construction of yeast bait vectors fused with coding sequences derived from Pfnek3. (A) Schematic diagram for Pfnek3 and various deletionͲconstructs amplified by PCR. (B) RestrictionͲdigestion of the TOPO™ cloning vector carrying Pfnek3 and its deletion mutants released Pfnek3 DNA, which was confirmed subsequently by DNA sequencing. (C) SubͲcloning of Pfnek3 and its deletion mutants into the yeast bait vector (pGBKT7). Restriction digestion of the recombinant vectors released Pfnek3 DNA, with the exception of construct 1. Constructs 2Ͳ5 were subsequently used for interaction studies. M: Fermentas GeneRuler™1kb DNA ladder. 71 Chapter 4 4.5 Results and Discussion Construction of plasmids for mammalianͲtwoͲhybrid studies The Y2H system is widely used to detect protein interactions. Typically, interactions identified in yeast are followed up with confirmatory studies using alternative methods before the results can be accepted (Osman, 2004). Some confirmatory techniques used are coͲIP, GST pullͲdown or coͲimmunofluorescence. The limitation of the first two techniques is the in vitro nature of the experiments whereas the last technique requires cells to express detectable amounts of endogenous proteins. There is also a heavy dependence on the specificity of the primary antibodies, the production of which requires a lengthy optimization process. In contrast, the mammalianͲtwoͲhybrid (M2H) assay is a more recent technology which allows for the overͲexpression of proteinsͲofͲinterest and also obliterates the use of antibodies. The chemiluminescenceͲbased readout could be amenable to the microplate format, rendering it compatible with highͲthroughput instrumentation. The mammalianͲtwoͲhybrid assay is an important followͲup to yeast screens because it tests interactions under physiological conditions that allow for posttranslational changes to hybrid proteins, i.e., phosphorylation, acetylation, proteolysis that cannot be replicated in yeast or other in vitro methods. In this study, Pfnek3 bait sequences were released from recombinant yeast bait vectors (pGBKT7) by restriction digest, gelͲpurified, and ligated into the mammalian bait vector (pM). Similarly, prey sequences identified from prior Y2H experiments were released from the yeast prey vector (pGADT7) by restriction digest and ligated into the mammalian prey vector (pVP16). The resultant recombinant vectors were first screened 72 Chapter 4 Results and Discussion by restriction digestion (Figure 4.7) and subsequently sequenced to confirm that the sequences were inserted in the correct reading frame. A kb M1 2 3 4 5 Linearized pM vector 1.0 0.75 0.5 M2: Fermentas GeneRuler™ 100 bp DNA ladder 0.25 B bp Legend: M1: Fermentas GeneRuler™1kb DNA ladder M2 P3 P6 P10 P22 P24 3000 2000 Linearized pVP16 vector 1500 500 Figure 4.7: Plasmid construction for M2H protein interaction studies. (A) M2H bait vectors (pM) were inserted with Pfnek3 truncations no. 2Ͳ5 as per Y2H studies (Figure 3.2). (B) Prey vector (pVP16) was inserted with gene sequences obtained from previous Y2H prey vectors that showed promising protein interaction in the yeast system (Table 4Ͳ2). 73 Chapter 4 Results and Discussion Table 4Ͳ2: List of prey genes (identified from Y2H) subͲcloned into pVP16 Prey number pGADT7 clone numbera Sequencing IDb PlasmoDB gene IDc Annotation P3 3Ͳ1 164 PFC0245c Hypothetical protein P6 6Ͳ1 170 MAL13P1.237 Hypothetical protein P10 10Ͳ1 176 PF10_0268 Merozoite capping protein 1 P22 22Ͳ1 229 MAL8P1.125 TyrosylͲtRNA synthetase P24 24Ͳ1 233 PFA0520c Chromatin assembly factor 1 protein WD40 domain a E. coli cloning host; b Sequencing identifier on log; c PlasmoDB gene identifier (www.plasmodb.org) 4.6 Generating kinaseͲinactive mutants of Pfnek3 and Pfmap2 To validate subsequent experiments, it was necessary to generate kinaseͲinactive constructs for use as controls. A conserved lysine residue located at kinase subͲdomain II is often associated with ATPͲbinding during a phosphotransfer reaction. The mutation of this lysine to methionine is known to abrogate kinase activity in Pfmap2 (Dorin et al., 1999). To similarly create an inactive Pfnek3, a K102M mutation was introduced using PCR (Figure 4.8). A Pfmap2ͲK135M mutation was also generated according to Dorin et al. (1999). Both constructs were transformed into E. coli CodonPlus™ and the introduction of the desired mutation was confirmed by DNA sequencing. 74 Chapter 4 A kb 23 9.4 6.5 2.0 0.5 Results and Discussion M1 ǻPfnek3 B kb M2 ǻPfmap2 10.0 8.0 6.0 5.0 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.75 0.5 0.25 Legend: M1: ʄ /HindIII markers M2: Fermentas GeneRuler™1kb DNA ladder Figure 4.8: Generation of siteͲdirected kinaseͲinactive mutants, GSTͲȴPfnek3 and GSTͲ ȴPfmap2. 4.7 Bacterial expression of recombinant proteins The conceptuallyͲtranslated FLͲPfnek3 is 347 amino acids long and SDSͲPAGE indicated that the GSTͲfusion protein was approximately 60 kD (Figure 4.9). Analytical gel filtration of FLͲ and TRͲPfnek3 revealed that recombinant Pfnek3 existed primarily as monomers of molecular weights similar to that on SDSͲPAGE gels (Figure 4.9). 75 Chapter 4 Results and Discussion A B Figure 4.9: Recombinant expression of proteins. (A) Expression of GSTͲtagged kinases. (B) Analysis of purified GSTͲFLͲPfnek3 and GSTͲTRͲ Pfnek3 via analytical gel filtration indicates enriched fractions of monomers of about 60 kD. 4.8 Ensuring the expression of GSTͲPfmap2 The expression levels of malarial proteins in E. coli are generally low (Mehlin et al., 2006). To perform downstream research (e.g. activity assays), it would be advantageous to obtain larger amounts of sufficient purity. For the purpose of this project, it was useful to increase the bacterial culture volume from 50 ml to 100 ml. 76 Chapter 4 Results and Discussion However, the increase of bacterial culture volume also implied that during GST purification, larger amounts of bacterial proteins were often coͲpurified (pGEX instruction manual; unmarked bands on Figure 4.9). This complicated the interpretation of SDSͲPAGE gels, particularly for Pfmap2. To confirm its identity and expression, liquid chromatography tandem mass spectrometry (LCͲMS/MS) was employed and the results compared against the MASCOT database (Figure 4.10 and Table 4Ͳ3). Figure 4.10: Pfmap2 amino acid sequence covered by mass spectrometry (~35%). Tryptic peptides detected by LCͲMS/MS (score ш 25; Table 4Ͳ3) are indicated in bold. 77 Chapter 4 Results and Discussion Table 4Ͳ3 : List of peptide ions detected in a LCͲMS/MS experiment identifying Pfmap2. Mr (experimental) 632.27 682.30 751.35 765.29 811.38 893.35 927.38 976.36 1039.41 1051.41 1167.53 1207.47 1207.58 1209.39 1290.33 1311.50 1311.64 1324.61 1337.51 1337.56 1397.48 1580.65 1580.77 1836.65 2583.99 Mr (calculated) 632.36 682.41 751.43 765.40 811.44 893.44 927.52 976.49 1039.58 1051.52 1167.67 1207.65 1207.65 1209.55 1290.52 1311.72 1311.72 1323.67 1337.65 1337.65 1397.65 1580.86 1580.86 1836.76 2584.20 Scorea 20 24 15 44 25 35 46 32 32 33 32 37 33 65 70 51 55 75 88 40 69 43 34 46 63 Expect Peptide 73 22 1.5e+02 0.24 22 2.7 0.25 6.9 9.3 7.5 14 3.9 10 0.0054 0.0016 0.16 0.059 0.00063 3.9eͲ05 2.1 0.0027 0.97 7.9 0.65 0.013 LFPTR YIFLK LNIHQK SDYIIR KYSSISK ICDFGLAR ALSHPYLK VPDNYEIK QLTSHVVTR SHINNPTNR KQLTSHVVTR FIHESGIIHR FIHESGIIHR ENLENFSTEK DEEEEDANVNK TPIFLTEQHVK TPIFLTEQHVK DIHIVNDLEEK KENLENFSTEK KENLENFSTEK GSYGYVYLAYDK EIQSFHADLIIPAK EIQSFHADLIIPAK ESSSRDEEEEDANVNK DIHIVNDLEEKEENEEPGPHNK a A probability based Mowse score such that an ion score is Ͳ10*Log(P), where P is the probability that the observed match is a random event (Pappin et al., 1993). Individual ions scores > 54 indicate identity or extensive homology (p[...]... proteomic analyses (Khan et al., 2005; Rangarajan et al., 2005) Numerous attempts have been made to identify candidate kinases upstream of Pfmap1 and Pfmap2 For example, it has previously been suggested that Pfnek1, a NIMAͲ family kinase is an upstream regulator (i.e MAPKK, also called MEK, MAPK/ERK Kinase) of Pfmap2 (Dorin et al., 2001) Unfortunately, in vivo activity of Pfnek1 was not established and recombinant... MitogenͲactivated protein kinase MAPK kinase MAPKK kinase Myelin basic protein Messenger ribonucleic acid NIMAͲlike kinase Never in mitosis, Aspergillus NͲterminal region Optical density Oligonucleotide Open reading frame Phosphate buffered saline Polymerase chain reaction Plasmodium falciparum MAP kinase Plasmodium falciparum NIMAͲrelated kinase Plasmodium falciparum protein kinase 7 Regulator of chromosome condensation... unicellular organisms to mammals and plants (Figure 2.1) Reviewed in Garrington and Johnson (1999), the MAPKs, also called ERKs (extracellularly regulated kinases), are central to the adaptive responses of eukaryotic cells to a wide range of stimuli Phosphorylation at both the Thr and Tyr residues of the conserved MAPK activation motif (TXY) by a specific MAPK kinase (MAPKK; also called MEK, for MAPK/ERK kinase) ... site Mammalian MAPK1 is activated by phosphorylation on the threonine and tyrosine residues of the TXY motif by an upstream protein kinase (i.e MAPKK) Although the TXY motif is similarly present on the Plasmodium MAPK1 (Pfmap1), it is not yet clear which Plasmodium kinase is capable of activating Pfmap1 Intriguingly, the requirement of a MAPK1 homologue for parasite survival, development and proliferation... for MAPK activation Correspondingly, another upstream kinase, MAPKKK or MEKK, often associated with membrane receptor tyrosine kinases, are in turn responsible for the phosphorylation and activation of MEKs 5 Chapter 2 Literature Review Figure 2.1: A typical eukaryotic MAP kinase pathway The pathway involves the sensing of extracellular stimuli which are transduced through a series of phosphotransfer... Introduction kinase) are currently among the best understood MAPKs are believed to be highly conserved among eukaryotes and are central to the transduction of extracellular mitogenic stimuli down a cascade of ATPͲdependent protein kinases Two copies of P falciparum MAPKs (Pfmap1 and Pfmap2) have been identified so far (Graeser et al., 1997; Dorin et al., 1999) Both MAPKs share a peptide sequence identity of. .. reminiscent of the conserved SMANS activation site found in mammalian MAPKK1/MAPKK2 enzymes BacteriallyͲexpressed Pfnek1 can phosphorylate recombinant Pfmap2 in vitro Unfortunately, in vivo functionality of Pfnek1 still awaits formal demonstration Pfnek1 has no in vitro effect on Pfmap1 or on mammalian ERK2 (also called MAPK2) PfPK7 is another plasmodial protein kinase that encodes the ‘most MAPKKͲlike... regulators of the previously identified plasmodial MAP kinases As part of an earlier study, the synergistic kinase activity of Pfmap2 and Pfnek3 has been demonstrated To take this lead further, the relationship between the kinases would be further dissected and the interaction partners of Pfnek3 identified in an attempt to decipher the malarial MAPK signaling pathway 13 Chapter 3 Materials and Methods... the Apicomplexan protozoan, with 20 members in P falciparum Many of the malarial FIKK kinases are believed to contain protein export signals that transport the FIKK 7 Chapter 2 Literature Review protein kinase to the parasitized human cell (Schneider and MercereauͲPuijalon, 2005) Recently, transgenic parasites expressing GFPͲtagged FIKK kinases allowed the detection of exported FIKK kinases at the... to, and necessary for, male gametocytogenesis In another independent study, Pbmap2Ͳdeficient parasites were impaired in sexual cycle completion (Rangarajan et al., 2005) These studies suggest the necessity of Pbmap2 to the malaria parasite and it follows that the use of a rodent malaria model for searching upstream kinases and candidate drugs capable of disrupting their phosphorylation appears relevant ... functions as an atypical MAPKK in Plasmodium falciparum Biochem Biophys Res Commun 361(2):439Ͳ444 [2] Lye YM, Chan M and Sim TS (2006) Pfnek3: an atypical activator of a MAP kinase in Plasmodium falciparum. .. reaction Plasmodium falciparum MAP kinase Plasmodium falciparum NIMAͲrelated kinase Plasmodium falciparum protein kinase Regulator of chromosome condensation Reverse transcription/transcriptase... copies of Plasmodium falciparum MAPKs (Pfmap1 and Pfmap2) have been identified and are believed to influence parasite proliferation However, the regulators and substrates of malarial MAPKs have