Ribosomes and Protein Synthesis

cAMP response element-binding protein (CREB) is imported into mitochondria and promotes protein synthesis Domenico De Rasmo 1 , Anna Signorile 1 , Emilio Roca 1 and Sergio Papa 1,2 1 Department of Medical Biochemistry, Biology and Physics (DIBIFIM), University of Bari, Italy 2 Institute of Biomembranes and Bioenergetics (IBBE), Consiglio Nazionale delle Ricerche, Bari, Italy Introduction The cAMP response element-binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that recognizes the DNA consensus sequence TGACGTCA, the cAMP response element (CRE) in gene promoters [1,2]. Phosphorylation of CREB by cAMP-dependent pro- tein kinase (protein kinase A; PKA), as well as by Ca 2+ -dependent and other protein kinases, in response to different cellular signals, promotes tran- scription of CRE-regulated genes [1–4]. Activation of the expression of nuclear CRE-regulated genes has been shown to be involved in a variety of cellular pro- cesses, including apoptosis [5,6], oxidative stress [7], neuronal growth, and plasticity [5,8]. In yeast, cAMP was found to reverse the glucose repression of mito- chondriogenesis [9] and to activate the expression of mitochondrial genes [10,11] and nuclear genes [12,13] of respiratory chain proteins. In Saccharomyces cerere- visiae, where the RAS ⁄ cAMP ⁄ PKA system appears to be involved in regulation of the biogenesis of the oxidative phosphorylation system [13], a probable cis- regulatory element on mtDNA, responsible for cAMP- mediated transcription, was identified [11]. In yeast and mammalian cells, the cAMP cascade is involved in the regulation of mitochondrial dynamics [14] and bioenergetics [15–17]. In 1999, findings were presented [18] indicating that CREB is localized in the inner mitochondrial compart- ment as well as in the nucleus. These observations, based on the use of CREB and phospho-CREB anti- Keywords cAMP cascade; complex I; CREB; mitochondrial protein synthesis; PKA Correspondence S. Papa, Department of Medical Biochemistry, Biology and Physics, University of Bari, Policlinico, P.zza G. Cesare, 70124 Bari, Italy Fax: +39 080 5448538 Tel: +39 080 5448540 E-mail: papabchm@cimedoc.uniba.it (Received 9 March 2009, revised 26 May 2009, accepted 4 June 2009) doi:10.1111/j.1742-4658.2009.07133.x The cAMP response element-binding protein (CREB) is a ubiquitous transcription factor in the higher eukaryotes that, once phosphorylated, promotes transcription of cAMP response element-regulated genes. We have studied the mitochondrial import of CREB and its effect on the expression of mtDNA-encoded proteins. [ 35 S]Methionine-labelled CREB, synthesized in vitro in the Rabbit Reticulocyte Lysate system using a con- struct of the human cDNA, was imported into the matrix of isolated rat liver mitochondria by a membrane potential and TOM complex-dependent process. The imported CREB caused cAMP-dependent promotion of the synthesis of mitochondrially encoded subunits of oxidative phosphorylation enzyme complexes. Thus, CREB moves from the cytosol to mitochondria, in addition to the nucleus, and, when phosphorylated by cAMP-dependent protein kinase, promotes the expression of mitochondrial genes. Abbreviations ADU, arbitrary densitometric units; AKAP, A kinase anchoring protein; CAP, chloramphenicol; cPKA, catalytic subunit of cAMP dependent protein kinase; CRE, cAMP response element; CREB, cAMP response element-binding protein; db-cAMP, dibutyryl cAMP; IBMX, isobutylmethylxanthine; PKA, cAMP-dependent protein kinase (protein kinase A); RRL, Rabbit Reticulocyte Lysate. FEBS Journal 276 (2009) 4325–4333 ª 2009 Ribosomes and Protein Synthesis Ribosomes and Protein Synthesis Bởi: OpenStaxCollege The synthesis of proteins consumes more of a cell’s energy than any other metabolic process In turn, proteins account for more mass than any other component of living organisms (with the exception of water), and proteins perform virtually every function of a cell The process of translation, or protein synthesis, involves the decoding of an mRNA message into a polypeptide product Amino acids are covalently strung together by interlinking peptide bonds in lengths ranging from approximately 50 amino acid residues to more than 1,000 Each individual amino acid has an amino group (NH2) and a carboxyl (COOH) group Polypeptides are formed when the amino group of one amino acid forms an amide (i.e., peptide) bond with the carboxyl group of another amino acid ([link]) This reaction is catalyzed by ribosomes and generates one water molecule A peptide bond links the carboxyl end of one amino acid with the amino end of another, expelling one water molecule For simplicity in this image, only the functional groups involved in the peptide bond are shown The R and R' designations refer to the rest of each amino acid structure The Protein Synthesis Machinery In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation The composition of each component may vary across species; for instance, ribosomes may consist of different numbers of rRNAs and polypeptides 1/9 Ribosomes and Protein Synthesis depending on the organism However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors Link to Learning Click through the steps of this PBS interactive to see protein synthesis in action Ribosomes Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes In E coli, there are between 10,000 and 70,000 ribosomes present in each cell at any given time A ribosome is a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs Ribosomes exist in the cytoplasm in prokaryotes and in the cytoplasm and rough endoplasmic reticulum in eukaryotes Mitochondria and chloroplasts also have their own ribosomes in the matrix and stroma, which look more similar to prokaryotic ribosomes (and have similar drug sensitivities) than the ribosomes just outside their outer membranes in the cytoplasm Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation In E coli, the small subunit is described as 30S, and the large subunit is 50S, for a total of 70S (recall that Svedberg units are not additive) Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus The complete mRNA/poly-ribosome structure is called a polysome tRNAs The tRNAs are structural RNA molecules that were transcribed from genes by RNA polymerase III Depending on the species, 40 to 60 types of tRNAs exist in the cytoplasm Serving as adaptors, specific tRNAs bind to sequences on the mRNA template and add the corresponding amino acid to the polypeptide chain Therefore, 2/9 Ribosomes and Protein Synthesis tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins Of the 64 possible mRNA codons—or triplet combinations of A, U, G, and C—three specify the termination of protein synthesis and 61 specify the addition of amino acids to the polypeptide chain Of these 61, one codon (AUG) also encodes the initiation of translation Each tRNA anticodon can base pair with one of the mRNA codons and add an amino acid or terminate translation, according to the genetic code For instance, if the sequence CUA occurred on an mRNA template in the proper reading frame, it would bind a tRNA expressing the complementary sequence, GAU, which would be linked to the amino acid leucine As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package Consider that tRNAs need to interact with three factors: 1) they must be recognized by the correct aminoacyl synthetase (see below); 2) they must be recognized by ribosomes; and 3) they must bind to the correct sequence in mRNA Aminoacyl tRNA Synthetases The process of pre-tRNA synthesis by RNA polymerase III only creates the RNA portion of the adaptor molecule The corresponding amino ...MINIREVIEW 15 N-Labelled proteins by cell-free protein synthesis Strategies for high-throughput NMR studies of proteins and protein–ligand complexes Kiyoshi Ozawa, Peter S. C. Wu, Nicholas E. Dixon and Gottfried Otting Research School of Chemistry, Australian National University, Canberra, ACT, Australia Introduction Cell-free protein synthesis in both the Escherichia coli coupled transcription-translation system and the wheat germ translation system has been remarkably improved so that milligram quantities of protein can routinely be prepared [1–6]. Compared to conventional recombin- ant protein production in vivo, cell-free protein synthe- sis offers a number of decisive advantages for the preparation of stable isotope labelled protein samples for analysis by NMR spectroscopy. (a) The target protein is the only protein synthesized and labelled during the reaction. Consequently the iso- tope-labelled amino acids are used very efficiently, and because no new metabolic enzymes are expressed in the medium, isotope scrambling is kept to a minimum. Moreover, isotope-filtered NMR experiments allow the selective observation of the isotope-labelled proteins without chromatographic purification. (b) The reaction is fast. This is advantageous for the synthesis of proteins that are sensitive to proteolytic degradation and for high-throughput applications. (c) The reaction can be carried out in small volumes. Therefore, isotope-labelled starting materials are used more efficiently and economically than for conven- tional in vivo labelling methods [7]. (d) The reaction is independent of cell growth. Therefore, toxic proteins and proteins containing non- natural amino acids can be made efficiently [8–10]. With the advent of cryogenic probe heads, hetero- nuclear single quantum coherence (HSQC) spectra of proteins made by cell-free expression can be recorded quickly at the concentration delivered by the reaction mixture. Keywords cell-free protein synthesis; combinatorial labelling; 15 N-HSQC; 15 N-labelled amino acids; protein–ligand interactions Correspondence G. Otting, Research School of Chemistry, Australian National University, Canberra, ACT, Australia Fax: +61 261250750 Tel: +61 261256507 E-mail: gottfried.otting@anu.edu.au Website: http://rsc.anu.edu.au/go/ (Received 9 May 2006, accepted 23 June 2006) doi:10.1111/j.1742-4658.2006.05433.x [ 15 N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [ 15 N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [ 15 N]-HSQC spectra by amino acid type selective [ 15 N]-labelling. The samples can be measured without chro- matographic protein purification, dilution of isotopes by transaminase activities are suppressed, and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum num- ber of samples for the identification of all [ 15 N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [ 15 N]-HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular, combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h, including protein production and NMR measurement. Abbreviations HSQC, heteronuclear single quantum DNA, RNA, and Protein Synthesis The Beginning…. All living things are made of-  Water (an inorganic compound)  Other inorganic compounds (mostly salts)  Organic Compounds: {contain carbon and hydrogen}  Carbohydrates  Lipids(fats)  Proteins*  Nucleic Acids*  *These are what we’ll talk about today When we look at a living thing,  What we see is mostly PROTEIN-  So, how does an organism produce its particular protein?  As in, people protein vs tree protein? The answer is DNA!  The species-particular DNA sequences produce the species-particular proteins  GENES code for proteins  GENES are long strands of DNA on chromosomes What is DNA?  DNA is the genetic code,  Instructions for heredity,  Components of genes,  Director of protein synthesis  AND- DNA is also  A type of nucleic acid  A type of organic compound  A polymer {a compound made of repeating subunits}  WHAT DOES “DNA” STAND FOR? DNA’s proper name is-  Deoxyribonucleic acid!  Consists of a ribose SUGAR with a “missing oxygen” (that’s the de-oxy part)  And it’s found in the nucleus of eukaryotic organisms How does DNA code for protein synthesis?  First-what is “protein synthesis”?  It’s building, or assembling, a protein molecule from amino acids  Amino acids are smaller molecules found in the food we eat or produced in our cells  It happens at the ribosomes DNA and protein synthesis, then, happens this way:  1. DNA sequence codes (how letters are put together) produce messenger RNA sequence codes [...]... up! Structure of DNA A nucleotide of DNA is the base unit  A nucleotide consists of a phosphate, a sugar, and a nitrogen base  DNA is in a double strand  The nitrogen bases have compliment partners  Adenine-Thymine  Cytosine-Guanine Just a note about RNARNA is single-stranded and acts as a code for protein synthesis  RNA is still made of nucleotides that have a phosphate, a sugar, and a nitrogen... 2 The messenger RNA leaves the nucleus and attaches to a ribosome in the cytoplasm  3 Transfer RNA brings amino acids to the ribosome to match the sequence codes At the ribosome, Amino acids bond together and form polypeptides,  Which bond together to make proteins  Some examples of proteins are:    Melanin, the pigment that gives our skin color... Cytosine-Guanine Just a note about RNARNA is single-stranded and acts as a code for protein synthesis  RNA is still made of nucleotides that have a phosphate, a sugar, and a nitrogen base  The sugar is different and the basepairing is also different  Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors Jashmin Patel 1 , Laura E. McLeod 2 , Robert G. J. Vries 1 , Andrea Flynn 1 , Xuemin Wang 1,2 and Christopher G. Proud 1,2 1 Department of Biosciences, University of Kent at Canterbury, Canterbury, UK; 2 Division of Molecular Physiology, School of Life Sciences, University of Dundee, UK We have examined the effects of widely used stress-inducing agents on protein synthesis and on regulatory components of the translational machinery. The three stresses chosen, arsenite, hydrogen peroxide and sorbitol, exert their effects in quite different ways. Nonetheless, all three rapidly ( 30 min) caused a profound inhibition of protein syn- thesis. In each case this was accompanied by dephosphory- lation of the eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) and increased binding of this repressor protein to eIF4E. Binding of 4E-BP1 to eIF4E correlated with loss of eIF4F complexes. Sorbitol and hydrogen per- oxide each caused inhibition of the 70-kDa ribosomal pro- tein S6 kinase, while arsenite activated it. The effects of stresses on the phosphorylation of eukaryotic elongation factor 2 also differed: oxidative stress elicited a marked increase in eEF2 phosphorylation, which is expected to contribute to inhibition of translation, while the other stresses did not have this effect. Although all three proteins (4E-BP1, p70 S6 kinase and eEF2) can be regulated through the mammalian target of rapamycin (mTOR), our data imply that stresses do not interfere with mTOR function but act in different ways on these three proteins. All three stresses activate the p38 MAP kinase pathway but we were able to exclude a role for this in their effects on 4E-BP1. Our data reveal that these stress-inducing agents, which are widely used to study stress-signalling in mammalian cells, exert multiple and complex inhibitory effects on the translational machinery. Keywords: stress; initiation; elongation factor; mRNA translation; S6 kinase. The control of mRNA translation in mammalian cells involves the regulation of a range of components of the translational machinery, principally by changes in their phosphorylation, leading to modulation of their activities or their abilities to interact with one another [1,2]. Initiation factor 4E (eIF4E) plays a key role in mRNA translation and its control in eukaryotic cells. eIF4E binds to the 5¢ cap structure (containing 7-methylguanosine triphosphate; m 7 GTP) which is present at the 5¢ end of all cellular cytoplasmic mRNAs [3,4]. eIF4E can be regulated by its own phosphorylation (which occurs at a single major site (Ser209) [5,6]; and by binding proteins (4E-BPs) that modulate its availability for initiation complex formation (reviewed in [7]). eIF4E forms a complex termed eIF4F, which also contains the translation factors eIF4G (formerly called p220) and eIF4A. eIF4A has ATP-dependent RNA helicase activity thought to be required to unwind regions of self-complementary secondary structure in the 5¢ UTRs of certain mRNAs [4,8]. Such secondary structure inhibits translation and therefore mRNAs with 5¢ UTRs that contain significant secondary structure are often poorly translated. In contrast to many other cellular mRNAs, translation of heat shock protein mRNAs appears to be relatively cap-independent (reviewed in [9–11]), and trans- lation of the mRNA for the stress-protein Open Access Available online http://arthritis-research.com/content/7/5/R1091 R1091 Vol 7 No 5 Research article The protective effect of licofelone on experimental osteoarthritis is correlated with the downregulation of gene expression and protein synthesis of several major cartilage catabolic factors: MMP-13, cathepsin K and aggrecanases Jean-Pierre Pelletier 1 , Christelle Boileau 1 , Martin Boily 1 , Julie Brunet 1 , François Mineau 1 , Changshen Geng 1 , Pascal Reboul 1 , Stefan Laufer 2 , Daniel Lajeunesse 1 and Johanne Martel- Pelletier 1 1 Osteoarthritis Research Unit, University of Montreal Hospital Centre, Notre-Dame Hospital, Montreal, Quebec, Canada 2 Department of Pharmaceutical Chemistry/Medicinal Chemistry, Eberhard-Karls-University Tübingen, Institute of Pharmacy, Tübingen, Germany Corresponding author: Jean-Pierre Pelletier, dr@jppelletier.ca Received: 22 Dec 2004 Revisions requested: 3 Feb 2005 Revisions received: 6 Jun 2005 Accepted: 17 Jun 2005 Published: 19 Jul 2005 Arthritis Research & Therapy 2005, 7:R1091-R1102 (DOI 10.1186/ar1788) This article is online at: http://arthritis-research.com/content/7/5/R1091 © 2005 Pelletier et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract This study sought to evaluate the levels of mRNA expression and protein synthesis of MMP-13, cathepsin K, aggrecanase-1 (ADAMTS-4), aggrecanase-2 (ADAMTS-5) and 5-lipoxygenase (5-LOX) in cartilage in the experimental anterior cruciate ligament (ACL) dog model of osteoarthritis (OA), and to examine the effects of treatment with licofelone, a 5-lipoxygenase (LOX)/ cyclooxygenase (COX) inhibitor, on the levels of these catabolic factors. Sectioning of the ACL of the right knee was performed in three experimental groups: group 1 received no active treatment (placebo group); and groups 2 and 3 received therapeutic concentrations of licofelone (2.5 or 5.0 mg/kg/day orally, respectively) for 8 weeks, beginning the day following surgery. A fourth group consisted of untreated dogs that were used as normal controls. Specimens of cartilage were selected from lesional areas of OA femoral condyles and tibial plateaus, and were processed for real-time quantitative PCR and immunohistochemical analyses. The levels of MMP-13, cathepsin K, ADAMTS-4, ADAMTS-5 and 5-LOX were found to be significantly increased in OA cartilage. Licofelone treatment decreased the levels of both mRNA expression and protein synthesis of the factors studied. Of note was the marked reduction in the level of 5-LOX gene expression. The effects of the drug were about the same at both tested dosages. In vivo treatment with therapeutic dosages of licofelone has been found to reduce the degradation of OA cartilage in experimental OA. This, coupled with the results of the present study, indicates that the effects of licofelone are mediated by the inhibition of the major cartilage catabolic pathways involved in the destruction of cartilage matrix macromolecules. Moreover, our findings also indicate the possible auto-regulation of 5-LOX gene expression by licofelone in OA cartilage. Introduction Along with the graying of the world's population, osteoarthritis (OA), the most common form of arthritis, is becoming an increasingly significant medical and financial burden. In this context, the clear need for a better understanding of the dis- ease process has rendered undeniable the importance of find- ing drugs that can reduce or stop its progression. Recent studies have revealed new and interesting information regarding the role played by eicosanoids in the pathophysiol- ogy of arthritic diseases, including OA [1-6]. For instance, leu- kotriene-B 4 (LTB 4 ) has proven to be an important regulating factor in the ... encountered, a release factor binds and dissociates the components and frees the new protein Folding of the protein occurs during and after translation 7/9 Ribosomes and Protein Synthesis Art Connections... transferred to the tRNA, and AMP is released The Mechanism of Protein Synthesis As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination The.. .Ribosomes and Protein Synthesis depending on the organism However, the general structures and functions of the protein synthesis machinery are comparable from