Nascent Chains Research Articles

Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation.

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.

YidC from Escherichia coli Forms an Ion-Conducting Pore upon Activation by Ribosomes.

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.

Transmembrane Helices 7 and 8 Confer Aggregation Sensitivity to the Cystic Fibrosis Transmembrane Conductance Regulator.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up to the second transmembrane domain (TMD2), including the R region, largely resisted aggregation. Limited proteolysis identified a folded structure that was prone to aggregation and consisted of TMD2 and at least part of the Regulatory Region R. Only when both TM7 (TransMembrane helix 7) and TM8 were present, TMD2 fragments became as aggregation-sensitive as wild-type CFTR, in line with increased thermo-instability of late CFTR nascent chains and in silico prediction of aggregation propensity. In accord, isolated TMD2 was degraded faster in cells than isolated TMD1. We conclude that TMD2 extended at its N-terminus with part of the R region forms a protease-resistant structure that induces heat instability in CFTR and may be responsible for its limited intracellular stability.

Reducing High Temperature System Development Cycle Time and Cost with Additively Manufactured Connectors

Connectors are a fundamental part of Ozark IC’s high temperature modules from characterization and test packages to Ozark IC’s XNode® data acquisition and single-board computing modules. Development of rugged high temperature electronic systems requires the co-development of similarly rugged high temperature connectors. The design and development of connector solutions enable test and evaluation of high temperature systems which would otherwise require fulfillment of a commercial connector order. There is a nascent supply chain of rugged, high temperature-rated connector designs but manufacturer lead times for such commercial products can exceed an entire design, prototype, and test cycle. Such delays are incompatible with rapid design iterations and fail-fast strategies employed in the development of extreme environment electronics – especially when the design and test cycles can change the requirements or specifications of the connectors. Polymer and ceramic additive manufacturing techniques enable in-house connector fabrication and support a connector co-development cycle that complements the pace and interdependent requirements of high temperature system development. These solutions are simple and reliable under laboratory conditions, operating for thousands of hours at 200 °C [1] and hundreds of hours at 800 °C [2]. The use of proprietary connectors manufactured with additive techniques and digital tooling lends an extra degree of freedom to system design and enables the trade-off of connector with system design specifications that greatly accelerates system ruggedization. Electrical connectors represent the final interface between an electronics module and the outside world. A failure of a connector is a failure of packaging that makes the module opaque to the tester and renders the power and robustness of any electronic system moot.

Mutation of the PEBP-like domain of the mitoribosomal MrpL35/mL38 protein results in production of nascent chains with impaired capacity to assemble into OXPHOS complexes.

Located in the central protuberance region of the mitoribosome and mitospecific mL38 proteins display homology to PEBP (Phosphatidylethanolamine Binding Protein) proteins, a diverse family of proteins reported to bind anionic substrates/ligands and implicated in cellular signaling and differentiation pathways. In this study, we have performed a mutational analysis of the yeast mitoribosomal protein MrpL35/mL38 and demonstrate that mutation of the PEBP-invariant ligand binding residues Asp(D)232 and Arg(R)288 impacted MrpL35/mL38's ability to support OXPHOS-based growth of the cell. Furthermore, our data indicate these residues exist in a functionally important charged microenvironment, which also includes Asp(D)167 of MrpL35/mL38 and Arg(R)127 of the neighboring Mrp7/bL27m protein. We report that mutation of each of these charged residues resulted in a strong reduction in OXPHOS complex levels that was not attributed to a corresponding inhibition of the mitochondrial translation process. Rather, our findings indicate that a disconnect exists in these mutants between the processes of mitochondrial protein translation and the events required to ensure the competency and/or availability of the newly synthesized proteins to assemble into OXPHOS enzymes. Based on our findings, we postulate that the PEBP-homology domain of MrpL35/mL38, together with its partner Mrp7/bL27m, form a key regulatory region of the mitoribosome.

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C).

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

Phospholipid dependency of membrane protein insertion by the Sec translocon

Membrane protein insertion into and translocation across the bacterial cytoplasmic membrane are essential processes facilitated by the Sec translocon. Membrane insertion occurs co-translationally whereby the ribosome nascent chain is targeted to the translocon via signal recognition particle and its receptor FtsY. The phospholipid dependence of membrane protein insertion has remained mostly unknown. Here we assessed in vitro the dependence of the SecA independent insertion of the mannitol permease MtlA into the membrane on the main phospholipid species present in Escherichia coli. We observed that insertion depends on the presence of phosphatidylglycerol and is due to the anionic nature of the polar headgroup, while insertion is stimulated by the zwitterionic phosphatidylethanolamine. We found an optimal insertion efficiency at about 30 mol% DOPG and 50 mol% DOPE which approaches the bulk membrane phospholipid composition of E. coli.

Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-β-glucans.

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-β-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-β-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-β-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry

The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

Galidesivir Triphosphate Promotes Stalling of Dengue-2 Virus Polymerase Immediately Prior to Incorporation.

Millions of people are infected by the dengue and Zika viruses each year, resulting in significant morbidity and mortality. Galidesivir is an adenosine nucleoside analog that can attenuate flavivirus replication in cell-based assays and animal models of infection. Galidesivir is converted to the triphosphorylated form by host kinases and subsequently incorporated into viral RNA by viral RNA polymerases. This has been proposed to lead to the delayed termination of RNA synthesis. Here, we report direct in vitro testing of the effects of Galidesivir triphosphate on dengue-2 and Zika virus polymerase activity. Galidesivir triphosphate was chemically synthesized, and inhibition of RNA synthesis followed using a dinucleotide-primed assay with a homopolymeric poly(U) template. Galidesivir triphosphate was equipotent against dengue-2 and Zika polymerases, with IC50 values of 42 ± 12 μM and 47 ± 5 μM, respectively, at an ATP concentration of 20 μM. RNA primer extension assays show that the dengue-2 polymerase stalls while attempting to add a Galidesivir nucleotide to the nascent RNA chain, evidenced by the accumulation of RNA products truncated immediately upstream of Galidesivir incorporation sites. Nevertheless, Galidesivir is incorporated at isolated sites with low efficiency, leading to the subsequent synthesis of full-length RNA with no evidence of delayed chain termination. The incorporation of Galidesivir at consecutive sites is strongly disfavored, highlighting the potential for modulation of inhibitory effects of nucleoside analogs by the template sequence. Our results suggest that attenuation of dengue replication by Galidesivir may not derive from the early termination of RNA synthesis following Galidesivir incorporation.

Phase-separated ribosome-nascent chain complexes in genotoxic stress response.

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

Co-translational binding of importins to nascent proteins

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

Using mechanistic models and machine learning to design single-color multiplexed nascent chain tracking experiments.

mRNA translation is the ubiquitous cellular process of reading messenger-RNA strands into functional proteins. Over the past decade, large strides in microscopy techniques have allowed observation of mRNA translation at a single-molecule resolution for self-consistent time-series measurements in live cells. Dubbed Nascent chain tracking (NCT), these methods have explored many temporal dynamics in mRNA translation uncaptured by other experimental methods such as ribosomal profiling, smFISH, pSILAC, BONCAT, or FUNCAT-PLA. However, NCT is currently restricted to the observation of one or two mRNA species at a time due to limits in the number of resolvable fluorescent tags. In this work, we propose a hybrid computational pipeline, where detailed mechanistic simulations produce realistic NCT videos, and machine learning is used to assess potential experimental designs for their ability to resolve multiple mRNA species using a single fluorescent color for all species. Our simulation results show that with careful application this hybrid design strategy could in principle be used to extend the number of mRNA species that could be watched simultaneously within the same cell. We present a simulated example NCT experiment with seven different mRNA species within the same simulated cell and use our ML labeling to identify these spots with 90% accuracy using only two distinct fluorescent tags. We conclude that the proposed extension to the NCT color palette should allow experimentalists to access a plethora of new experimental design possibilities, especially for cell Signaling applications requiring simultaneous study of multiple mRNAs.

Entangled Motifs in Membrane Protein Structures.

Entangled motifs are found in one-third of protein domain structures, a reference set that contains mostly globular proteins. Their properties suggest a connection with co-translational folding. Here, we wish to investigate the presence and properties of entangled motifs in membrane protein structures. From existing databases, we build a non-redundant data set of membrane protein domains, annotated with the monotopic/transmembrane and peripheral/integral labels. We evaluate the presence of entangled motifs using the Gaussian entanglement indicator. We find that entangled motifs appear in one-fifth of transmembrane and one-fourth of monotopic proteins. Surprisingly, the main features of the distribution of the values of the entanglement indicator are similar to the reference case of general proteins. The distribution is conserved across different organisms. Differences with respect to the reference set emerge when considering the chirality of entangled motifs. Although the same chirality bias is found for single-winding motifs in both membrane and reference proteins, the bias is reversed, strikingly, for double-winding motifs only in the reference set. We speculate that these observations can be rationalized in terms of the constraints exerted on the nascent chain by the co-translational bio-genesis machinery, which is different for membrane and globular proteins.

Pyruvate Kinase M (PKM) binds ribosomes in a poly-ADP ribosylation dependent manner to induce translational stalling.

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation.

Molecular chaperones in stroke-induced immunosuppression.

Stroke-induced immunosuppression is a process that leads to peripheral suppression of the immune system after a stroke and belongs to the central nervous system injury-induced immunosuppressive syndrome. Stroke-induced immunosuppression leads to increased susceptibility to post-stroke infections, such as urinary tract infections and stroke-associated pneumonia, worsening prognosis. Molecular chaperones are a large class of proteins that are able to maintain proteostasis by directing the folding of nascent polypeptide chains, refolding misfolded proteins, and targeting misfolded proteins for degradation. Various molecular chaperones have been shown to play roles in stroke-induced immunosuppression by modulating the activity of other molecular chaperones, cochaperones, and their associated pathways. This review summarizes the role of molecular chaperones in stroke-induced immunosuppression and discusses new approaches to restore host immune defense after stroke.

Molecular basis of the TRAP complex function in ER protein biogenesis

The translocon-associated protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the ribosome to facilitate biogenesis of secretory and membrane proteins. TRAP plays a key role in the secretion of many hormones, including insulin. Here we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position is further stabilized by a finger-like loop. This positions a cradle-like lumenal domain of TRAP below the translocon for interactions with translocated nascent chains. Our structure-guided TRAP mutations in Caenorhabditis elegans lead to growth deficits associated with increased ER stress and defects in protein hormone secretion. These findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

Structure of the actively translating plant 80S ribosome at 2.2 Å resolution

In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing.

The N-terminal region of yeast mitoribosomal Mrp7/bL27m protein serves to optimize translation of nascent chains competent for OXPHOS complex assembly.

The extreme N-terminal residues of the mitochondrial ribosomal bL27m proteins reside within the ribosomal peptidyl transferase center (PTC) and are conserved from their bacterial ancestors. Mutation or truncation of the N-terminal region of the yeast Mrp7/bL27m protein did not inhibit protein synthesis but significantly impacted the efficacy of the mitochondrial translational process with respect to yielding proteins competent to assemble into functional oxidative phosphorylation enzymes. The requirement for the N-terminal residues of Mrp7/bL27m to support normal mitotranslation wasmore apparent under respiratory growth. We demonstrate that the N-terminal region of Mrp7/bL27m impacts the environment of the PTC and speculate the bL27m proteins serve to fine-tune and optimize mitoribosomal activity with respect to the downstream fate of the nascent chain.

Rapid cell type-specific nascent proteome labeling in Drosophila.

Controlled protein synthesis is required to regulate gene expression and is often carried out in a cell type-specific manner. Protein synthesis is commonly measured by labeling the nascent proteome with amino acid analogs or isotope-containing amino acids. These methods have been difficult to implement in vivo as they require lengthy amino acid replacement procedures. O-propargyl-puromycin (OPP) is a puromycin analog that incorporates into nascent polypeptide chains. Through its terminal alkyne, OPP can be conjugated to a fluorophore-azide for directly visualizing nascent protein synthesis, or to a biotin-azide for capture and identification of newly-synthesized proteins. To achieve cell type-specific OPP incorporation, we developed phenylacetyl-OPP (PhAc-OPP), a puromycin analog harboring an enzyme-labile blocking group that can be removed by penicillin G acylase (PGA). Here, we show that cell type-specific PGA expression in Drosophila can be used to achieve OPP labeling of newly-synthesized proteins in targeted cell populations within the brain. Following a brief 2 hr incubation of intact brains with PhAc-OPP, we observe robust imaging and affinity purification of OPP-labeled nascent proteins in PGA-targeted cell populations. We apply this method to show a pronounced age-related decline in neuronal protein synthesis in the fly brain, demonstrating the capability of PhAc-OPP to quantitatively capture in vivo protein synthesis states. This method, which we call POPPi (PGA-dependent OPP incorporation), should be applicable for rapidly visualizing protein synthesis and identifying nascent proteins synthesized under diverse physiological and pathological conditions with cellular specificity in vivo.