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The formation of protein complexes is crucial to most biological functions. The cellular mechanisms governing protein complex biogenesis are not yet well understood, but some principles of cotranslational and posttranslational assembly are beginning to emerge. In bacteria, this process is favored by operons encoding subunits of protein complexes. Eukaryotic cells do not have polycistronic mRNAs, raising the question of how they orchestrate the encounter of unassembled subunits. Here we review the constraints and mechanisms governing eukaryotic co- and posttranslational protein folding and assembly, including the influence of elongation rate on nascent chain targeting, folding, and chaperone interactions. Recent evidence shows that mRNAs encoding subunits of oligomeric assemblies can undergo localized translation and form cytoplasmic condensates that might facilitate the assembly of protein complexes. Understanding the interplay between localized mRNA translation and cotranslational proteostasis will be critical to defining protein complex assembly in vivo.

Made available in DSpace on 2018-12-11T17:04:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-10-01 The translation elongation factor eIF5A is conserved through evolution and is necessary to rescue the ribosome during translation elongation of polyproline-containing proteins. Although the site of eIF5A binding to the ribosome is known, no systematic analysis has been performed so far to determine the important residues on the surface of eIF5A required for ribosome binding. In this study, we used clustered charged-to-alanine mutagenesis and structural modeling to address this question. We generated four new mutants of yeast eIF5A: tif51A-4, tif51A-6, tif51A-7 and tif51A-11, and complementation analysis revealed that tif51A-4 and tif51A-7 could not sustain cell growth in a strain lacking wild-type eIF5A. Moreover, the allele tif51A-4 also displayed negative dominance over wild-type eIF5A. Both in vivo GST-pulldowns and in vitro fluorescence anisotropy demonstrated that eIF5A from mutant tif51A-7 exhibited an importantly reduced affinity for the ribosome, implicating the charged residues in cluster 7 as determinant features on the eIF5A surface for contacting the ribosome. Notably, modified eIF5A from mutant tif51A-4, despite exhibiting the most severe growth phenotype, did not abolish ribosome interactions as with mutant tif51A-7. Taking into account the modeling eIF5A + 80S + P-tRNA complex, our data suggest that interactions of eIF5A with ribosomal protein L1 are more important to stabilize the interaction with the ribosome as a whole than the contacts with P-tRNA. Finally, the ability of eIF5A from tif51A-4 to bind to the ribosome while potentially blocking physical interaction with P-tRNA could explain its dominant negative phenotype. Department of Biological Sciences School of Pharmaceutical Sciences São Paulo State University-UNESP, Rod Araraquara-Jaú Km01 Physics and Interdisciplinary Science Department Physics Institute of Sao Carlos University of Sao Paulo-USP Molecular and Cellular Biology Department University of California Department of Biological Sciences School of Pharmaceutical Sciences São Paulo State University-UNESP, Rod Araraquara-Jaú Km01

Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.

Made available in DSpace on 2020-12-12T01:46:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The genus Paracoccidioides consist of dimorphic fungi geographically limited to the subtropical regions of Latin America, which are responsible for causing deep systemic mycosis in humans. However, the molecular mechanisms by which Paracoccidioides spp. causes the disease remain poorly understood. Paracoccidioides spp. harbor genes that encode proteins involved in host cell interaction and mitochondrial function, which together are required for pathogenicity and mediate virulence. Previously, we identified TufM (previously known as EF-Tu) in Paracoccidioides brasiliensis (PbTufM) and suggested that it may be involved in the pathogenicity of this fungus. In this study, we examined the effects of downregulating PbTUFM using a silenced strain with a 55% reduction in PbTUFM expression obtained by antisense-RNA (aRNA) technology. Silencing PbTUFM yielded phenotypic differences, such as altered translation elongation, respiratory defects, increased sensitivity of yeast cells to reactive oxygen stress, survival after macrophage phagocytosis, and reduced interaction with pneumocytes. These results were associated with reduced virulence in Galleria mellonella and murine infection models, emphasizing the importance of PbTufM in the full virulence of P. brasiliensis and its potential as a target for antifungal agents against paracoccidioidomycosis. Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Análises Clínicas Laboratório de Micologia Clinica Faculdade de Odontologia UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Fisiologia e Patologia Unidad de Biología Celular y Molecular Corporación para Investigaciones Biológicas (CIB) Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Ciências Biológicas Laboratório de Biologia Molecular e Celular de Microrganismos Grupo de Investigación MICROBA -Escuela de Microbiología Universidad de Antioquia Facultad de Medicina Universidad de Antioquia Instituto Carlos Chagas Fundação Oswaldo Cruz (Fiocruz) Institute of Science and Technology São Paulo State University (UNESP) Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Análises Clínicas Laboratório de Micologia Clinica Faculdade de Odontologia UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Fisiologia e Patologia Faculdade de Ciências Farmacêuticas UNESP – Univ Estadual Paulista Campus Araraquara Departamento de Ciências Biológicas Laboratório de Biologia Molecular e Celular de Microrganismos Institute of Science and Technology São Paulo State University (UNESP)

Made available in DSpace on 2018-12-11T17:04:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-04-01 National Institute of General Medical Sciences eIF5A is the only protein known to contain the essential and unique amino acid residue hypusine. eIF5A functions in both translation initiation due to its stimulation of methionyl-puromycin synthesis and translation elongation, being highly required for peptide-bound formation of specific ribosome stalling sequences such as poly-proline. The functional interaction between eIF5A, tRNA, and eEF2 on the surface of the ribosome is further clarified herein. Fluorescence anisotropy assays were performed to determine the affinity of eIF5A to different ribosomal complexes and reveal its interaction exclusively and directly with the 60S ribosomal subunit in a hypusine-dependent manner 60S-eIF5A-Hypi 60S-eIF5A-Lysi = 385 nM). A 3-fold increase in eIF5A affinity to the 80S is observed upon Meti binding, indicating positive cooperativity between P-site tRNA binding and eIF5A binding to the ribosome. Previously identified conditional mutants of yeast eIF5A, eIF5AQ22H/L93F and eIF5AK56A ,display a significant decrease in ribosome binding affinity. Binding affinity between ribosome and eIF5A-wild type or mutants eIF5AK56A ,butnoteIF5AQ22H/L93F , is impaired in the presence of eEF2 by 4-fold, consistent with negative cooperativity between eEF2 and eIF5A binding to the ribosome. Interestingly, high-copy eEF2 is toxic only to eIF5AQ22H/L93F and causes translation elongation defects in this mutant. These results suggest that binding of eEF2 to the ribosome alters its conformation, resulting in a weakened affinity of eIF5A and impairment of this interplay compromises cell growth due to translation elongation defects. School of Pharmaceutical Sciences UNESP - Univ Estadual Paulista Department of Biological Sciences Department of Molecular and Cellular Biology University of California Davis School of Pharmaceutical Sciences UNESP - Univ Estadual Paulista Department of Biological Sciences National Institute of General Medical Sciences: R01GM092927