Regulation and mechanistic basis of macrolide resistance by the ABC-F ATPase MsrD

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors currently proliferating among human pathogens that provide resistance against clinically important ribosome-targeting antibiotics. Here, we combine genetic and structural approaches to determine the activity of the streptococcal ARE ABC-F protein MsrD on the ribosome and its regulation in response to macrolide exposure. We show that cladinose-containing macrolides lead to insertion of MsrDL leader peptide into a conserved crevice of the ribosomal exit tunnel, which remained thus far undocumented, concomitantly with 23S rRNA rearrangements that preclude proper accommodation of release factors and inhibits termination. The stalled ribosome obstructs formation of a Rho-independent terminator which prevents msrD transcriptional attenuation. This stalled ribosome is rescued by MsrD powered by its two functionally asymmetric ATPase sites, but not by MsrD mutants which do not provide antibiotic resistance, showing evidence of equivalence between MsrD function in antibiotic resistance and its action on this complex.

Discovery proteomics defines androgen-regulated glycoprotein networks in prostate cancer cells, as well as putative biomarkers of prostatic diseases

Supraphysiologic androgen (SPA) inhibits cell proliferation in prostate cancer (PCa) cells by transcriptional repression of DNA replication and cell-cycle genes. In this study, quantitative glycoprotein profiling identified androgen-regulated glycoprotein networks associated with SPA-mediated inhibition of PCa cell proliferation, and androgen-regulated glycoproteins in clinical prostate tissues. SPA-regulated glycoprotein networks were enriched for translation factors and ribosomal proteins, proteins that are known to be O-GlcNAcylated in response to various cellular stresses. Thus, androgen-regulated glycoproteins are likely to be targeted for O-GlcNAcylation. Comparative analysis of glycosylated proteins in PCa cells and clinical prostate tissue identified androgen-regulated glycoproteins that are differentially expressed prostate tissues at various stages of cancer. Notably, the enzyme ectonucleoside triphosphate diphosphohydrolase 5 was found to be an androgen-regulated glycoprotein in PCa cells, with higher expression in cancerous versus non-cancerous prostate tissue. Our glycoproteomics study provides an experimental framework for characterizing androgen-regulated proteins and glycoprotein networks, toward better understanding how this subproteome leads to physiologic and supraphysiologic proliferation responses in PCa cells, and their potential use as druggable biomarkers of dysregulated AR-dependent signaling in PCa cells.

Dysregulation of Translation Factors EIF2S1, EIF5A and EIF6 in Intestinal-Type Adenocarcinoma (ITAC)

Intestinal-type adenocarcinoma (ITAC) is a rare cancer of the nasal cavity and paranasal sinuses that occurs sporadically or secondary to exposure to occupational hazards, such as wood dust and leather. Eukaryotic translation initiation factors have been described as promising targets for novel cancer treatments in many cancers, but hardly anything is known about these factors in ITAC. Here we performed in silico analyses, evaluated the protein levels of EIF2S1, EIF5A and EIF6 in tumour samples and non-neoplastic tissue controls obtained from 145 patients, and correlated these results with clinical outcome data, including tumour site, stage, adjuvant radiotherapy and survival. In silico analyses revealed significant upregulation of the translation factors EIF6 (ITGB4BP), EIF5, EIF2S1 and EIF2S2 (p < 0.05) with a higher arithmetic mean expression in ITAC compared to non-neoplastic tissue (NNT). Immunohistochemical analyses using antibodies against EIF2S1 and EIF6 confirmed a significantly different expression at the protein level (p < 0.05). In conclusion, this work identifies the eukaryotic translation initiation factors EIF2S1 and EIF6 to be significantly upregulated in ITAC. As these factors have been described as promising therapeutic targets in other cancers, this work identifies candidate therapeutic targets in this rare but often deadly cancer.

First-principles model of optimal translation factors stoichiometry

Enzymatic pathways have evolved uniquely preferred protein expression stoichiometry in living cells, but our ability to predict the optimal abundances from basic properties remains underdeveloped. Here we report a biophysical, first-principles model of growth optimization for core mRNA translation, a multi-enzyme system that involves proteins with a broadly conserved stoichiometry spanning two orders of magnitude. We show that predictions from maximization of ribosome usage in a parsimonious flux model constrained by proteome allocation agree with the conserved ratios of translation factors. The analytical solutions, without free parameters, provide an interpretable framework for the observed hierarchy of expression levels based on simple biophysical properties, such as diffusion constants and protein sizes. Our results provide an intuitive and quantitative understanding for the construction of a central process of life, as well as a path toward rational design of pathway-specific enzyme expression stoichiometry.

Inhibition of Mul1-mediated ubiquitination promotes mitochondria-associated translation

G-Protein Pathway Suppressor 2 (GPS2) was recently identified as an endogenous inhibitor of non-proteolytic ubiquitination mediated by the E2 ubiquitin-conjugating enzyme Ubc13. GPS2-mediated restriction of K63 ubiquitination is associated with the regulation of insulin signaling, inflammation and mitochondria-nuclear communication, however a detailed understanding of the targets of GPS2/Ubc13 activity is currently lacking, Here, we have dissected the GPS2-regulated K63 ubiquitome in mouse embryonic fibroblasts and human breast cancer cells, unexpectedly finding an enrichment for proteins involved in RNA binding and translation. Characterization of putative targets, including the RNA-binding protein PABPC1 and translation factor eiF3m, revealed a strategy for regulating the mitochondria-associated translation of selected mRNAs via Mul1-mediated ubiquitination. Our data indicate that removal of GPS2-mediated inhibition, either via genetic deletion or stress-induced nuclear translocation, promotes the ubiquitination of mitochondria-associated translation factors leading to increased expression of an adaptive antioxidant program. In light of GPS2 role in nuclear-mitochondria communication, these findings reveal an exquisite regulatory network for modulating mitochondrial gene expression through spatially coordinated transcription and translation.

Mitochondrial Protein Translation: Emerging Roles and Clinical Significance in Disease

Mitochondria are one of the most important organelles in cells. Mitochondria are semi-autonomous organelles with their own genetic system, and can independently replicate, transcribe, and translate mitochondrial DNA. Translation initiation, elongation, termination, and recycling of the ribosome are four stages in the process of mitochondrial protein translation. In this process, mitochondrial protein translation factors and translation activators, mitochondrial RNA, and other regulatory factors regulate mitochondrial protein translation. Mitochondrial protein translation abnormalities are associated with a variety of diseases, including cancer, cardiovascular diseases, and nervous system diseases. Mutation or deletion of various mitochondrial protein translation factors and translation activators leads to abnormal mitochondrial protein translation. Mitochondrial tRNAs and mitochondrial ribosomal proteins are essential players during translation and mutations in genes encoding them represent a large fraction of mitochondrial diseases. Moreover, there is crosstalk between mitochondrial protein translation and cytoplasmic translation, and the imbalance between mitochondrial protein translation and cytoplasmic translation can affect some physiological and pathological processes. This review summarizes the regulation of mitochondrial protein translation factors, mitochondrial ribosomal proteins, mitochondrial tRNAs, and mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) in the mitochondrial protein translation process and its relationship with diseases. The regulation of mitochondrial protein translation and cytoplasmic translation in multiple diseases is also summarized.

Essential role of translation factor eIF5a in cytokine production and cell cycle regulation in primary CD8 T lymphocytes

Translational control adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Translation can be regulated through sequestration of mRNAs by regulatory proteins or RNAs, but also by the availability of ribosomes and translation factors which enable initiation and elongation of nascent polypeptides. Traditionally initiation of mRNA translation has been considered to be a major translational control point, however, control of peptide elongation can also play a role. Here we show that post-translational modification of the elongation factor, eIF5a, controls translation of subsets of proteins in naive T-cells upon activation. Sequencing of nascent polypeptides indicated that functional eIF5a was required for the production of proteins which regulate T-cell proliferation and effector function. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation can contribute to post-transcriptional regulons important for the control of inflammation.

Cap-dependent translation initiation monitored in living cells

Despite extensive biochemical, genetic, and structural studies, a complete understanding of mRNA translation initiation is still lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-molecule resolution are necessary to fully elucidate regulation of this paramount process. We fused tags suitable for live imaging to eIF4E, eIF4G1 and 4E-BP1 without affecting their function. We combined Fluorescence Correlation Spectroscopy (FCS) and Single-Particle Tracking (SPT) to interrogate the binding dynamics of initiation factors to the 5'cap. Both FCS and SPT were able to detect eIF4E:eIF4G1 binding to the mRNA in the cytoplasm of proliferating cells and neuronal processes. Upon inhibition of phosphorylation by mTOR, 4E-BP1:eIF4E complexes rapidly dissociated from the 5'cap followed by eIF4G1 dissociation. Imaging of the binding dynamics of individual translation factors in living cells revealed the temporal regulation of translation at unprecedented resolution.

mSWI/SNF interacts with the ribosome and its inhibition/mutations alter translation and sensitize to mTOR/PI3K inhibitors

The chromatin remodelers mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) subunits are mutated, deleted or amplified in more than 40% of cancers. Understanding their functions in normal cells and the consequences of cancers alterations will lead to path toward new targeted therapies. Canonically, mSWI/SNF complexes regulate the structure of chromatin, however they likely have additional functions which could be relevant in carcinogenesis. Here, we highlight the substantial alteration of mSWI/SNF subunits expression in both the nucleus and cytoplasm in breast cancer cases. We demonstrate mSWI/SNF cytoplasmic localization and interaction with the translation initiation machinery. Short-term inhibition and depletion of specific subunits alter protein synthesis, implicating a direct role for these factors in translation. Inhibition and depletion of specific subunits increase sensitivity to mTOR-PI3K inhibitors, suggesting a potential therapeutic opportunity for diseases harboring mutations in these complexes. Indeed, SMARCA4 pathogenic mutations decrease protein synthesis. Furthermore, taking advantage of the DepMap studies, we demonstrate cancer cells harboring mutations of specific mSWI/SNF subunits exhibit a genetic dependency on translation factors and are particularly sensitive to translation pathway inhibitors. In conclusion, we report an unexpected cytoplasmic role for mSWI/SNF in protein synthesis, suggesting potential new therapeutic opportunities for patients afflicted by cancers demonstrating alterations in its subunits.

First-principles model of optimal translation factors stoichiometry

Enzymatic pathways have evolved uniquely preferred protein expression stoichiometry in living cells, but our ability to predict the optimal abundances from basic properties remains underdeveloped. Here we report a biophysical, first-principles model of growth optimization for core mRNA translation, a multi-enzyme system that involves proteins with a broadly conserved stoichiometry spanning two orders of magnitude. We show that a parsimonious flux model constrained by proteome allocation is sufficient to predict the conserved ratios of translation factors through maximization of ribosome usage The analytical solutions, without free parameters, provide an interpretable framework for the observed hierarchy of expression levels based on simple biophysical properties, such as diffusion constants and protein sizes. Our results provide an intuitive and quantitative understanding for the construction of a central process of life, as well as a path toward rational design of pathway-specific enzyme expression stoichiometry.