Co-translational Process Research Articles

Methionine aminopeptidase 2 and its autoproteolysis product have different binding sites on the ribosome

Excision of the initiator methionine is among the first co-translational processes that occur at the ribosome. While this crucial step in protein maturation is executed by two types of methionine aminopeptidases in eukaryotes (MAP1 and MAP2), additional roles in disease and translational regulation have drawn more attention to MAP2. Here, we report several cryo-EM structures of human and fungal MAP2 at the 80S ribosome. Irrespective of nascent chains, MAP2 can occupy the tunnel exit. On nascent chain displaying ribosomes, the MAP2-80S interaction is highly dynamic and the MAP2-specific N-terminal extension engages in stabilizing interactions with the long rRNA expansion segment ES27L. Loss of this extension by autoproteolytic cleavage impedes interactions at the tunnel, while promoting MAP2 to enter the ribosomal A-site, where it engages with crucial functional centers of translation. These findings reveal that proteolytic remodeling of MAP2 severely affects ribosome binding, and set the stage for targeted functional studies.

Roles of the CCR4-Not complex in translation and dynamics of co-translation events.

The Ccr4-Not complex is a global regulator of mRNA metabolism in eukaryotic cells that is most well-known to repress gene expression. Delivery of the complex to mRNAs through a multitude of distinct mechanisms accelerates their decay, yet Ccr4-Not also plays an important role in co-translational processes, such as co-translational association of proteins and delivery of translating mRNAs to organelles. The recent structure of Not5 interacting with the translated ribosome has brought to light that embedded information within the codon sequence can be monitored by recruitment of the Ccr4-Not complex to elongating ribosomes. Thereby, the Ccr4-Not complex is empowered with regulatory decisions determining the fate of proteins being synthesized and their encoding mRNAs. This review will focus on the roles of the complex in translation and dynamics of co-translation events. This article is categorized under: Translation > Mechanisms Translation > Regulation.

NMR-Based Characterization of the Interaction between Yeast Oxa1-CTD and Ribosomes.

In mitochondria, the major subunits of oxidative phosphorylation complexes are translated by the mitochondrial ribosome (mito-ribosome). The correct insertion and assembly of these subunits into the inner mitochondrial membrane (IMM) are facilitated by mitochondrial oxidase assembly protein 1 (Oxa1) during the translation process. This co-translational insertion process involves an association between the mito-ribosome and the C-terminus of Oxa1 (Oxa1-CTD) Nuclear magnetic resonance (NMR) methods were mainly used to investigate the structural characterization of yeast Oxa1-CTD and its mode of interaction with the E. coli 70S ribosome. Oxa1-CTD forms a transient α-helical structure within the residues P342-Q385, which were reported to form an α-helix when combining with the ribosome. Two conserved contact sites that could interact with the ribosome were further identified. The first site was located on the very end of the N-terminus (V321-I327), and the second one encompassed a stretch of amino acid residues I348-Q370. Based on our discoveries and previous reports, a model has been proposed in which Oxa1-CTD interacts with ribosomes, accompanied by transient-to-stable transitions at the second contact site. These observations may enhance our understanding of the potential role of Oxa1-CTD in facilitating the assembly of oxidative phosphorylation complexes and provide insight into the structural characteristics of Oxa1-CTD.

Biological and Catalytic Properties of Selenoproteins.

Selenocysteine is a catalytic residue at the active site of all selenoenzymes in bacteria and mammals, and it is incorporated into the polypeptide backbone by a co-translational process that relies on the recoding of a UGA termination codon into a serine/selenocysteine codon. The best-characterized selenoproteins from mammalian species and bacteria are discussed with emphasis on their biological function and catalytic mechanisms. A total of 25 genes coding for selenoproteins have been identified in the genome of mammals. Unlike the selenoenzymes of anaerobic bacteria, most mammalian selenoenzymes work as antioxidants and as redox regulators of cell metabolism and functions. Selenoprotein P contains several selenocysteine residues and serves as a selenocysteine reservoir for other selenoproteins in mammals. Although extensively studied, glutathione peroxidases are incompletely understood in terms of local and time-dependent distribution, and regulatory functions. Selenoenzymes take advantage of the nucleophilic reactivity of the selenolate form of selenocysteine. It is used with peroxides and their by-products such as disulfides and sulfoxides, but also with iodine in iodinated phenolic substrates. This results in the formation of Se-X bonds (X = O, S, N, or I) from which a selenenylsulfide intermediate is invariably produced. The initial selenolate group is then recycled by thiol addition. In bacterial glycine reductase and D-proline reductase, an unusual catalytic rupture of selenium-carbon bonds is observed. The exchange of selenium for sulfur in selenoproteins, and information obtained from model reactions, suggest that a generic advantage of selenium compared with sulfur relies on faster kinetics and better reversibility of its oxidation reactions.

Endogenous TOM20 Proximity Labeling: A Swiss-Knife for the Study of Mitochondrial Proteins in Human Cells.

Biotin-based proximity labeling approaches, such as BioID, have demonstrated their use for the study of mitochondria proteomes in living cells. The use of genetically engineered BioID cell lines enables the detailed characterization of poorly characterized processes such as mitochondrial co-translational import. In this process, translation is coupled to the translocation of the mitochondrial proteins, alleviating the energy cost typically associated with the post-translational import relying on chaperone systems. However, the mechanisms are still unclear with only few actors identified but none that have been described in mammals yet. We thus profiled the TOM20 proxisome using BioID, assuming that some of the identified proteins could be molecular actors of the co-translational import in human cells. The obtained results showed a high enrichment of RNA binding proteins close to the TOM complex. However, for the few selected candidates, we could not demonstrate a role in the mitochondrial co-translational import process. Nonetheless, we were able to demonstrate additional uses of our BioID cell line. Indeed, the experimental approach used in this study is thus proposed for the identification of mitochondrial co-translational import effectors and for the monitoring of protein entry inside mitochondria with a potential application in the prediction of mitochondrial protein half-life.

N-Myristoyltransferase, a Potential Antifungal Candidate Drug-Target for Aspergillus flavus.

The filamentous fungus Aspergillus flavus causes devastating diseases not only to cash crops but also to humans by secreting a series of secondary metabolites called aflatoxins. In the cotranslational or posttranslational process, N-myristoyltransferase (Nmt) is a crucial enzyme that catalyzes the myristate group from myristoyl-coenzyme A (myristoyl-CoA) to the N terminus or internal glycine residue of a protein by forming a covalent bond. Members of the Nmt family execute a diverse range of biological functions across a broad range of fungi. However, the underlying mechanism of AflNmt action in the A. flavus life cycle is unclear, particularly during the growth, development, and secondary metabolic synthesis stages. In the present study, AlfNmt was found to be essential for the development of spore and sclerotia, based on the regulation of the xylose-inducible promoter. AflNmt, located in the cytoplasm of A. flavus, is also involved in modulating aflatoxin (AFB1) in A. flavus, which has not previously been reported in Aspergillus spp. In addition, we purified, characterized, and crystallized the recombinant AflNmt protein (rAflNmt) from the Escherichia coli expression system. Interestingly, the crystal structure of rAlfNmt is moderately different from the models predicted by AlphaFold2 in the N-terminal region, indicating the limitations of machine-learning prediction. In conclusion, these results provide a molecular basis for the functional role of AflNmt in A. flavus and structural insights concerning protein prediction. IMPORTANCE As an opportunistic pathogen, A. flavus causes crop loss due to fungal growth and mycotoxin contamination. Investigating the role of virulence factors during infection and searching for novel drug targets have been popular scientific topics in the field of fungal control. Nmt has become a potential target in some organisms. However, whether Nmt is involved in the developmental stages of A. flavus and aflatoxin synthesis, and whether AlfNmt is an ideal target for structure-based drug design, remains unclear. This study systematically explored and identified the role of AlfNmt in the development of spore and sclerotia, especially in aflatoxin biosynthesis. Moreover, although there is not much difference between the AflNmt model predicted using the AlphaFold2 technique and the structure determined using the X-ray method, current AI prediction models may not be suitable for structure-based drug development. There is still room for further improvements in protein prediction.

From Nucleus to Membrane: A Subcellular Map of the N-Acetylation Machinery in Plants.

N-terminal acetylation (NTA) is an ancient protein modification conserved throughout all domains of life. N-terminally acetylated proteins are present in the cytosol, the nucleus, the plastids, mitochondria and the plasma membrane of plants. The frequency of NTA differs greatly between these subcellular compartments. While up to 80% of cytosolic and 20-30% of plastidic proteins are subject to NTA, NTA of mitochondrial proteins is rare. NTA alters key characteristics of proteins such as their three-dimensional structure, binding properties and lifetime. Since the majority of proteins is acetylated by five ribosome-bound N-terminal acetyltransferases (Nats) in yeast and humans, NTA was long perceived as an exclusively co-translational process in eukaryotes. The recent characterization of post-translationally acting plant Nats, which localize to the plasma membrane and the plastids, has challenged this view. Moreover, findings in humans, yeast, green algae and higher plants uncover differences in the cytosolic Nat machinery of photosynthetic and non-photosynthetic eukaryotes. These distinctive features of the plant Nat machinery might constitute adaptations to the sessile lifestyle of plants. This review sheds light on the unique role of plant N-acetyltransferases in development and stress responses as well as their evolution-driven adaptation to function in different cellular compartments.

Cotranslational folding and assembly of the dimeric Escherichia coli inner membrane protein EmrE

In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally (i.e., while at least one of the two chains is still attached to the ribosome). Whether this is also possible for integral membrane proteins is, however, unknown. Here, we apply force profile analysis (FPA)-a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion-to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the Escherichia coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in transmembrane helix 1 (TMH1) and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins thus appear to be able to both start to fold and start to dimerize during the cotranslational membrane insertion process.

The ribosome stabilizes partially folded intermediates of a nascent multi-domain protein

Co-translational folding is crucial to ensure the production of biologically active proteins. The ribosome can alter the folding pathways of nascent polypeptide chains, yet a structural understanding remains largely inaccessible experimentally. We have developed site-specific labelling of nascent chains to detect and measure, using 19F nuclear magnetic resonance (NMR) spectroscopy, multiple states accessed by an immunoglobulin-like domain within a tandem repeat protein during biosynthesis. By examining ribosomes arrested at different stages during translation of this common structural motif, we observe highly broadened NMR resonances attributable to two previously unidentified intermediates, which are stably populated across a wide folding transition. Using molecular dynamics simulations and corroborated by cryo-electron microscopy, we obtain models of these partially folded states, enabling experimental verification of a ribosome-binding site that contributes to their high stabilities. We thus demonstrate a mechanism by which the ribosome could thermodynamically regulate folding and other co-translational processes.

From Alpha to Beta – a co-translational way to fold?

ABSTRACT Protein folding in the cell is largely a co-translational process occurring during protein synthesis on the ribosome. It has become evident that co-translational folding is characteristic to almost every protein in the cell of pro- and eukaryotic origin that are single and multidomain, single and multisubunit, cytosolic, secretory and membrane. Co-translational protein folding begins very early during the process of polypeptide chain synthesis on the ribosome, with some secondary structure elements forming inside the ribosomal tunnel and some tertiary structures forming inside the vestibule (lower/wider) region of the ribosomal exit tunnel. However, many details of co-translational folding remains incompletely understood. New data show that folding of a β-barrel protein begins with formation of an α-helix inside the ribosome that rearranges into a β-hairpin structure as the growing peptide reaches the wider/vestibule region of the exit tunnel. While it was previously suggested that such scenario can take place on the ribosome, the new data provide the first experimental evidence in support of this notion.

Cell-Free Expression to Probe Co-Translational Insertion of an Alpha Helical Membrane Protein.

The majority of alpha helical membrane proteins fold co-translationally during their synthesis on the ribosome. In contrast, most mechanistic folding studies address refolding of full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation of membrane proteins is emerging as a useful tool to address folding during translation by a ribosome. We summarise the benefits of this approach and show how it can be successfully extended to a membrane protein with a complex topology. The bacterial leucine transporter, LeuT can be synthesised and inserted into lipid membranes using a variety of in vitro transcription translation systems. Unlike major facilitator superfamily transporters, where changes in lipids can optimise the amount of correctly inserted protein, LeuT insertion yields are much less dependent on the lipid composition. The presence of a bacterial translocon either in native membrane extracts or in reconstituted membranes also has little influence on the yield of LeuT incorporated into the lipid membrane, except at high reconstitution concentrations. LeuT is considered a paradigm for neurotransmitter transporters and possesses a knotted structure that is characteristic of this transporter family. This work provides a method in which to probe the formation of a protein as the polypeptide chain is being synthesised on a ribosome and inserting into lipids. We show that in comparison with the simpler major facilitator transporter structures, LeuT inserts less efficiently into membranes when synthesised cell-free, suggesting that more of the protein aggregates, likely as a result of the challenging formation of the knotted topology in the membrane.

Monitoring protein folding on and off the ribosome using structural mass spectrometry approaches

All proteins are synthesized by the ribosome and the relatively slow nature of this process allows for nascent proteins to sample conformational space before synthesis is complete. This search differs significantly from re-folding: translation is vectorial and the folding environment near the ribosome is highly charged and sterically restricted. While much is known about in vitro refolding, to date, our understanding of the co-translational process has been hampered by the lack of tools to monitor biophysical properties in the complex non-equilibrium environment required for co-translational folding.

Protein Targeting Into the Thylakoid Membrane Through Different Pathways.

In higher plants, chloroplasts are essential semi-autonomous organelles with complex compartments. As part of these sub-organellar compartments, the sheet-like thylakoid membranes contain abundant light-absorbing chlorophylls bound to the light-harvesting proteins and to some of the reaction center proteins. About half of the thylakoid membrane proteins are encoded by nuclear genes and synthesized in the cytosol as precursors before being imported into the chloroplast. After translocation across the chloroplast envelope by the Toc/Tic system, these proteins are subsequently inserted into or translocated across the thylakoid membranes through distinct pathways. The other half of thylakoid proteins are encoded by the chloroplast genome, synthesized in the stroma and integrated into the thylakoid through a cotranslational process. Much progress has been made in identification and functional characterization of new factors involved in protein targeting into the thylakoids, and new insights into this process have been gained. In this review, we introduce the distinct transport systems mediating the translocation of substrate proteins from chloroplast stroma to the thylakoid membrane, and present the recent advances in the identification of novel components mediating these pathways. Finally, we raise some unanswered questions involved in the targeting of chloroplast proteins into the thylakoid membrane, along with perspectives for future research.

Interactions between nascent proteins and the ribosome surface inhibit co-translational folding

Most proteins begin to fold during biosynthesis on the ribosome. It has been suggested that interactions between the emerging polypeptide and the ribosome surface might allow the ribosome itself to modulate co-translational folding. Here we combine protein engineering and NMR spectroscopy to characterize a series of interactions between the ribosome surface and unfolded nascent chains of the immunoglobulin-like FLN5 filamin domain. The strongest interactions are found for a C-terminal segment that is essential for folding, and we demonstrate quantitative agreement between the strength of this interaction and the energetics of the co-translational folding process itself. Mutations in this region that reduce the extent of binding result in a shift in the co-translational folding equilibrium towards the native state. Our results therefore demonstrate that a competition between folding and binding provides a simple, dynamic mechanism for the modulation of co-translational folding by the ribosome.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation

C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

Cracking the "Sugar Code": A Snapshot of N- and O-Glycosylation Pathways and Functions in Plants Cells.

Glycosylation is a fundamental co-translational and/or post-translational modification process where an attachment of sugars onto either proteins or lipids can alter their biological function, subcellular location and modulate the development and physiology of an organism. Glycosylation is not a template driven process and as such produces a vastly larger array of glycan structures through combinatorial use of enzymes and of repeated common scaffolds and as a consequence it provides a huge expansion of both the proteome and lipidome. While the essential role of N- and O-glycan modifications on mammalian glycoproteins is already well documented, we are just starting to decode their biological functions in plants. Although significant advances have been made in plant glycobiology in the last decades, there are still key challenges impeding progress in the field and, as such, holistic modern high throughput approaches may help to address these conceptual gaps. In this snapshot, we present an update of the most common O- and N-glycan structures present on plant glycoproteins as well as (1) the plant glycosyltransferases (GTs) and glycosyl hydrolases (GHs) responsible for their biosynthesis; (2) a summary of microorganism-derived GHs characterized to cleave specific glycosidic linkages; (3) a summary of the available tools ranging from monoclonal antibodies (mAbs), lectins to chemical probes for the detection of specific sugar moieties within these complex macromolecules; (4) selected examples of N- and O-glycoproteins as well as in their related GTs to illustrate the complexity on their mode of action in plant cell growth and stress responses processes, and finally (5) we present the carbohydrate microarray approach that could revolutionize the way in which unknown plant GTs and GHs are identified and their specificities characterized.

Electrostatic Interactions between Nascent Chains and the Ribosome Exit Tunnel Generate Forces that Modify Translation Rates

Several co-translational processes have been shown to generate tensile forces that are transmitted to the catalytic core of the ribosome. These forces have the ability to modify translation rates, which can in turn affect the structure and function of the protein being synthesized, which can then modify the cellular phenotype. These processes all occur as the nascent chain emerges from the exit tunnel. It has also been shown that positively charged residues can stall translation while still in the exit tunnel, although the mechanism by which stalling occurs is still unknown.

Probing the Role of Chain Connectivity in the Co-Translational Folding Process of Halotag through Circular Permutation

Though in vitro protein folding studies have produced insight into mechanisms underlying protein folding and energy landscapes, we do not completely understand how cellular factors modulate folding. All proteins have the opportunity to fold when synthesized from N- to C-terminus by the ribosome, which builds polypeptide chains at a rate of about 10 residues per second. This is much slower than the average folding rate of most small proteins, giving the nascent chain time to sample different conformations throughout translation.

Synonymous but Not Silent: The Codon Usage Code for Gene Expression and Protein Folding.

Codon usage bias, the preference for certain synonymous codons, is found in all genomes. Although synonymous mutations were previously thought to be silent, a large body of evidence has demonstrated that codon usage can play major roles in determining gene expression levels and protein structures. Codon usage influences translation elongation speed and regulates translation efficiency and accuracy. Adaptation of codon usage to tRNA expression determines the proteome landscape. In addition, codon usage biases result in nonuniform ribosome decoding rates on mRNAs, which in turn influence the cotranslational protein folding process that is critical for protein function in diverse biological processes. Conserved genome-wide correlations have also been found between codon usage and protein structures. Furthermore, codon usage is a major determinant of mRNA levels through translation-dependent effects on mRNA decay and translation-independent effects on transcriptional and posttranscriptional processes. Here, we discuss the multifaceted roles and mechanisms of codon usage in different gene regulatory processes.