Thông tin tài liệu
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
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