Monitor Protein Synthesis Research Articles

Deep Proteome Coverage Based on Ribosome Profiling Aids Mass Spectrometry-based Protein and Peptide Discovery and Provides Evidence of Alternative Translation Products and Near-cognate Translation Initiation Events*

An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing and highly sensitive mass spectrometry (MS) instrumentation. Recently, a strategy, termed ribosome profiling (or RIBO-seq), based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both Swiss-Prot- and RIBO-seq-derived translation products, applicable for MS/MS spectrum identification. To record the impact of using the constructed deep proteome database, we performed two alternative MS-based proteomic strategies as follows: (i) a regular shotgun proteomic and (ii) an N-terminal combined fractional diagonal chromatography (COFRADIC) approach. Although the former technique gives an overall assessment on the protein and peptide level, the latter technique, specifically enabling the isolation of N-terminal peptides, is very appropriate in validating the RIBO-seq-derived (alternative) translation initiation site profile. We demonstrate that this proteogenomic approach increases the overall protein identification rate 2.5% (e.g. new protein products, new protein splice variants, single nucleotide polymorphism variant proteins, and N-terminally extended forms of known proteins) as compared with only searching UniProtKB-SwissProt. Furthermore, using this custom database, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated upstream ORFs. Notably, the characterization of these new translation products revealed the use of multiple near-cognate (non-AUG) start codons. As deep sequencing techniques are becoming more standard, less expensive, and widespread, we anticipate that mRNA sequencing and especially custom-tailored RIBO-seq will become indispensable in the MS-based protein or peptide identification process. The underlying mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000124.

Monitoring Protein Synthesis One Codon at a Time through Ribosome Profiling

The ability to sequence genomes has far outstripped approaches for deciphering the information they encode. We have developed a suite of techniques based on ribosome profiling (deep sequencing of ribosome protected fragments) that dramatically expand our ability to follow translation in vivo. I will present recent applications of our ribosome profiling approach including the following: (1) Development of ribosome profiling protocols for a wide variety of eukaryotic and prokaryotic organisms. (2) Uses of ribosome profiling to globally monitor when chaperones, targeting factors or processing enzymes engage nascent chains. (3) Deciphering the driving force and biological consequences underlying the choice of synonymous codons.

Viewing folding of nascent polypeptide chains from ribosomes

Regulation of gene expression occurs at multiple stages and ultimately determines the protein levels in cells. The last decade has witnessed impressive progress in the development of approaches to measuring changes in cellular transcription and protein complement. However, techniques monitoring protein synthesis have lagged far behind. Current proteomic studies gauge the steady state levels of intracellular protein and rely on mass spectrometry, which has a low dynamic range and rarely samples more than 10% of the proteome [1]. RNA-seq is a sensitive approach for assessing transcriptional changes but the transcriptome correlates poorly with changes in protein levels [2,3]. Recent data suggest that the regulation of gene expression within cells occurs predominantly at the level of translation and that protein abundance correlates closely with translation rates [3]. Therefore, monitoring protein synthesis is crucial to understanding cellular homeostasis. Additionally, regulation of protein synthesis at the level of translation permits the cell to respond swiftly to adverse conditions [4]. Therefore, translational control is an integral part of cellular stress response. An intricate process associated with protein synthesis is folding. Proteins must achieve proper tertiary structure to acquire their designated properties and functions. Our current understanding of protein folding is predominantly based on in vitro refolding of denatured full-length proteins [5]. However, under native intracellular conditions, protein folding could occur concurrently with the synthesis of primary polypeptide chains on the ribosomes [6,7]. Cotranslational folding of partially synthesized nascent chains differs from refolding of full length polypeptides owing to the vectorial nature and the relatively slow elongation speed of the translation process. However, it is challenging to capture cotranslational folding events of nascent chains attached to the translating ribosome. Several approaches have been developed to dissect this process but most of them rely on in vitro systems with limited resolution [7,8]. There is thus an urgent need to establish new approaches to monitor cotranslational folding processes in vivo with high resolution. One solution is to look at the folding status of nascent polypeptide chains from the perspective of translating ribosomes.

SUnSET, a nonradioactive method to monitor protein synthesis

We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.

Cell-Specific Monitoring of Protein Synthesis In Vivo

Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

Monitoring protein synthesis by fluorescence recovery after photobleaching (FRAP) in vivo

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.

Single molecule fluorescence detection of BODIPY-FL molecules for monitoring protein synthesis

We have observed single DNA strands labeled with a small fluorescent molecule, BODIPY-FL, using objective based total internal fluorescence microscopy. In spite of its photobleaching, this small fluorophore is potentially useful for monitoring biochemical processes like protein synthesis because it can be efficiently incorporated into proteins by the ribosome through a charged epsilon-labeled fluorescent lysine tRNA. Using evanescent wave laser excitation at 488 nm, we have been able to observe single Bodipy molecules using integration times as low as 20 ms with a good signal to noise ratio, and for several images before photobleaching. We have measured the fluorescence decay due to photobleaching and have been able to lengthen it by a factor of 5 using oxygen scavenger systems.

Histone H2A.z Is Essential for Cardiac Myocyte Hypertrophy but Opposed by Silent Information Regulator 2α

In this study we have shown that the histone variant H2A.z is up-regulated during cardiac hypertrophy. Upon its knock-down with RNA interference, hypertrophy and the underlying increase in growth-related genes, protein synthesis, and cell size were down-regulated. During attempts to understand the mode of regulation of H2A.z, we found that overexpression of silent information regulator 2alpha (Sir2alpha) specifically induced down-regulation of H2A.z via NAD-dependent activity. This effect was reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2alpha-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. An increase in Sir2alpha also resulted in a dose-dependent reduction of the response to hypertrophic stimuli, whereas its inhibition resulted in enhanced hypertrophy and apoptosis. We have shown that Sir2alpha directly deacetylates H2A.z. Mutagenesis proved that lysines 4, 7, 11, and 13 do not play a role in the stability of H2A.z, whereas Lys-15 was indispensable. Meanwhile, Lys-115 and conserved, ubiquitinatable Lys-121 are critical for Sir2alpha-mediated degradation. Fusion of the C terminus of H2A.z (amino acids 115-127) to H2A.x or green fluorescence protein conferred Sir2alpha-inducible degradation to the former protein only. Because H2A.x and H2A.z have conserved N-tails, this implied that both the C and N termini are critical for mediating the effect of Sir2alpha. In short, the results suggest that H2A.z is required for cardiac hypertrophy, where its stability and the extent of cell growth and apoptosis are moderated by Sir2alpha. We also propose that Sir2alpha is involved in deacetylation of H2A.z, which results in ubiquitination of Lys-115 and Lys-121 and its degradation via a ubiquitin/proteasome-dependent pathway.

Saccharomyces cerevisiae Ub-Conjugating Enzyme Ubc4 Binds the Proteasome in the Presence of Translationally Damaged Proteins

Surveillance mechanisms that monitor protein synthesis can promote rapid elimination of misfolded nascent proteins. We showed that the translation elongation factor eEF1A and the proteasome subunit Rpt1 play a central role in the translocation of nascent-damaged proteins to the proteasome. We show here that multiubiquitinated proteins, and the ubiquitin-conjugating (E2) enzyme Ubc4, are rapidly detected in the proteasome following translational damage. However, Ubc4 levels in the proteasome were reduced significantly in a strain that expressed a mutant Rpt1 subunit. Ubc4 and Ubc5 are functionally redundant E2 enzymes that represent ideal candidates for ubiquitinating damaged nascent proteins because they lack significant substrate specificity, are required for the degradation of bulk, damaged proteins, and contribute to cellular stress-tolerance mechanisms. In agreement with this hypothesis, we determined that ubc4Delta ubc5Delta is exceedingly sensitive to protein translation inhibitors. Collectively, these studies suggest a specific role for Ubc4 and Ubc5 in the degradation of cotranslationally damaged proteins that are targeted to the proteasome.

Induced synthesis of albumin-like protein in damaged rat reticulocytes.

In this study, we present evidence that red blood cell (RBC) membrane p68 in the Belgrade (b/b) rat is similar if not identical to rat serum albumin (RSA). Structural homology between RSA and the RBC p68 has been determined by a variety of biochemical and immunological criteria. This albumin-like protein is a normal constituent of rat RBC and it is partially exported by exosomes during erythroid differentiation. The endogenous origin of rat RBC albumin-like protein was demonstrated by monitoring protein synthesis in reticulocytes and by identification of reticulocyte mRNA for albumin. Haemolytic anaemia, either hereditary (b/b rat) or drug-induced (phenylhydrazine-treated normal rat), results in increased accumulation of the albumin-like protein in rat RBCs as a result of its induced synthesis.

In vitro translation in a cell-free system from Trypanosoma brucei yields glycosylated and glycosylphosphatidylinositol-anchored proteins.

African trypanosomes escape many cellular and unspecific immune reactions by the expression of a protective barrier formed from a repertoire of several hundred genes encoding immunologically distinct variant surface glycoproteins (VSGs). All mature VSGs are glycosylphosphatidylionositol-anchored and N-glycosylated. To study trypanosome-specific post-translational modifications of VSG, a cell-free system capable of in vitro translation, translocation into the rough endoplasmic reticulum, N-glycosylation and glycosylphosphatidylinositol-anchor addition was established using lysates of the bloodstream form of Trypanosoma brucei. Monitoring protein synthesis by [35S]methionine incorporation, labeled protein bands were readily detected by fluorography following SDS/PAGE. Appearance of these bands increased during a time-course of 45 min and was sensitive to cycloheximide but not chloramphenicol treatment. Efficiency of this system, in terms of incorporation of radiolabeled amino acids into newly formed proteins, is similar to reticulocyte lysates. The system does not, however, allow initiation of protein synthesis. Depending on the clone used, immunoprecipitation revealed one or two newly formed VSG bands. Upon digestion with N-glycosidase F these bands resulted in a single band of a lower apparent molecular mass, indicating that newly synthesized VSG underwent translocation and glycosylation in the cell-free system. Biotinylation of VSG and a combination of precipitation with immobilized avidin and detection of VSG using antibodies specific for clones and cross-reacting determinants revealed that newly formed VSG contained the glycosylphosphatidylinositol anchor.

The 60 kDa Heat Shock Proteins in the Hyperthermophilic Archaeon Sulfolobus shibatae

ne of the most abundant proteins in the hyperthermophilic archaeon Sulfolobus shibataeis the 59 kDa heat shock protein (TF55) that is believed to form a homo-oligomeric double ring complex structurally similar to the bacterial chaperonins. We discovered a second protein subunit in the S. shibatae ring complex (referred to as α) that is stoichiometric with TF55 (renamed β). The gene and flanking regions of α were cloned and sequenced and its inferred amino acid sequence has 54.4% identity and 74.4% similarity to β. Transcription start sites for both α and β were mapped and three potential transcription regulatory regions were identified. Northern analyses of cultures shifted from normal growth temperatures (70 to 75°C) to heat shock temperatures (85 to 90°C) indicated that the levels of α and β mRNAs increased during heat shock, but at all temperatures their relative proportions remained constant. Monitoring protein synthesis by autoradiography of total proteins from cultures pulse labeled with L-[ 35c]methionine at normal and heat shock temperatures indicated significant increases in α and β synthesis during heat shock. Under extreme heat shock conditions (≥90°C) α and β appeared to be the only two proteins synthesized. The purified α and β subunits combined to form high molecular mass complexes with similar mobilities on native polyacrylamide gels to the complexes isolated directly from cells. Equal proportions of the two subunits gave the greatest yield of the complex, which we refer to as a "rosettasome". It is argued that the rosettasome consists of two homo-oligomeric rings; one of α and the other of β. Polyclonal antibodies against α and β from S. shibataecross-reacted with proteins of similar molecular mass in 10 out of the 17 archaeal species tested, suggesting that the two rosettasome proteins are highly conserved among the archaea. The archaeal sequences were aligned with bacterial and eukaryotic chaperonins to generate a phylogenetic tree. The tree reveals the close relationship between the archaeal rosettasomes and the eukaryotic TCP1 protein family and the distant relationship to the bacterial GroEL/HSP60 proteins.

PROTEIN BIOSYNTHESIS IN SALT‐SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

ABSTRACTWe have developed an in vivo14C‐amino acid labelling procedure for monitoring protein synthesis in salt‐shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, 14C‐phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short‐term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.

Formation of the Ca2+-activated photoprotein obelin from apo-obelin and mRNA inside human neutrophils

1. A method has been developed to incorporate the apoprotein of the Ca2+-activated photoprotein obelin, and mRNA purified from the hydroid Obelia, into the cytoplasm of intact human neutrophils. This was based on internal release from pH-sensitive immunoliposomes taken up initially by phagocytosis. 2. Addition of the prosthetic group of obelin, coelenterazine, to these cells containing apo-obelin or Obelia mRNA resulted in formation of active Ca2+-activated obelin. 3. The obelin formed within the neutrophils responded to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (1 microM) and to the membrane attack complex of complement (C5B6789n). 4. The formation of the apo-obelin from mRNA within neutrophils was inhibited by over 80% in the absence of added amino acids, and by over 90% by the protein-synthesis inhibitor puromycin (100 micrograms/ml). 5. The translation of Obelia mRNA inside cells provides a method for circumventing consumption of Ca2+-activated photoproteins during cell activation or injury, and for monitoring protein synthesis in living cells.

Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse.

Spermatogenic cells isolated from prepubertal and adult mice by unit gravity sedimentation have been used to examine proteins synthesized in a stage-specific manner throughout meiosis and early spermiogenesis. Preleptotene, leptotene/zygotene, and pachytene spermatocytes were isolated from 17-day-old mice. Adult pachytene spermatocytes and round spermatids were isolated from mature animals. These germ cells were then cultured in defined medium with [35S]methionine [( 35S]met) for 4-5 h. For each cell type, relative [35S]met incorporation was determined and labeled proteins were compared by two-dimensional (2D) polyacrylamide gel electrophoresis and autoradiography. Levels of [35S]met incorporation by isolated germ cells correlate closely with previous autoradiographic estimates of protein synthesis during spermatogenesis (Monesi, 1967). Pachytene spermatocytes from prepubertal mice incorporate the highest levels of [35S]met, when expressed either as cpm/-10(6) cells or cpm/mg protein. Comparisons of 2D autoradiograms indicated that many proteins, including actin and tubulins, are synthesized at approximately equal levels in all stages examined. Other proteins, including heat-shock proteins and multiple plasma membrane constituents, are synthesized in a stage-specific manner in leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. These studies define conditions for monitoring protein synthesis in isolated spermatogenic cells prior to the pachytene stage of meiosis, provide a 2D map of proteins synthesized at these earlier meiotic stages, and examine the synthesis of several proteins previously identified on 2D gels with biochemical and immunological methods.

RNA and Protein Synthesis During Meiotic Prophase in Tetrahymena thermophila1

Tetrahymena is one of the few organisms from which large amounts of precisely staged meiotic material can be obtained. We took advantage of this fact to monitor RNA and protein synthesis during meiosis. The rate of total protein synthesis as well as the synthesis of the majority of heavily labeled conjugation-specific polypeptides (monitored by high resolution two-dimensional gel electrophoresis) was maximal during meiotic prophase. We therefore cloned cDNAs corresponding to genes active during this time. The mRNA levels of three conjugation-specific genes (pC1, pC2, and pC7) and one conjugation-induced gene (pC3) were followed by using the corresponding labeled cDNAs to probe RNA isolated from different times during mating that was also followed cytologically. Synthesis of the conjugation-specific mRNAs was maximal just prior to maximum crescent stage (pachytene). Evidence is presented for transcription by the normally inactive micronucleus just prior to the maximum crescent stage, confirming an earlier report. The significance of these results is discussed.