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Made available in DSpace on 2021-06-25T10:37:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Objectives: The aim of this study was to evaluate the effect of specific inhibition of MMP-13 on inflammation and inflammatory bone resorption in a murine model of lipopolysaccharide (LPS)-induced periodontitis. Materials and methods: Periodontitis was induced in mice by micro-injections of LPS into the gingival tissues adjacent to the palatal surfaces of maxillary molars twice a week for 15 days. Matrix metalloproteinase-13 (Mmp-13) shRNA or a specific biochemical inhibitor were also injected into the same sites in alternating days with the LPS injections. Efficacy of shRNA-mediated silencing of Mmp-13 was verified by quantitative real-time polymerase chain reaction (qPCR) and immunoblot. Bone resorption was assessed by microcomputed tomography (uCT). Histological sections stained with hematoxylin/eosin (H/E) were used in the stereometric analysis of the inflammatory infiltrate. Gingival tissues were used to evaluate expression of Mmp-13, Il-6, Tnf-α, Ptgs2, and Rankl (qPCR). Protein levels of TGF-β and IL-10 in the tissues were determined by enzyme-linked immunosorbent assays (ELISA) or by MMP-13 and p38 immunoblot. Results: Silencing Mmp-13 expression reduced bone resorption significantly. Expression of Mmp-13, Il-6, and Tnf-α, as well as the protein levels of IL-6 and TNF-α, was reduced in the animals treated with adenovirus-delivered shRNA; however, these effects were not associated with modulation of p38 MAPK signaling. Interestingly, inhibition Mmp-13 did not affect the severity of inflammatory infiltrate. Conclusions: Site-specific inhibition of MMP-13 reduced bone resorption and production of inflammatory mediators associated with periodontal disease. Clinical relevance: The results suggest that site-specific inhibition of MMP-13 may be an interesting strategy to modulate inflammation and reduce bone resorption in osteolytic inflammatory diseases. Department of Diagnosis and Surgery School of Dentistry at Araraquara-State University of São Paulo UNESP, Rua Humaita, 1680, Centro Department of Biological Sciences School of Pharmaceutical Sciences UNESP Department of Physiology and Pathology School of Dentistry at Araraquara UNESP Department of Oral Biology University at Buffalo State University of New York Department of Diagnosis and Surgery School of Dentistry at Araraquara-State University of São Paulo UNESP, Rua Humaita, 1680, Centro Department of Biological Sciences School of Pharmaceutical Sciences UNESP Department of Physiology and Pathology School of Dentistry at Araraquara UNESP FAPESP: 2007/05583-3

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

Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation.

eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A(C39Y/G118D)) shows a sicker phenotype when combined with the dominant negative mutant eft2 ( H699K ) of the elongation factor eEF2. In this study, we used the eIF5A(K56A) mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A(K56A) mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A(K56A) mutant is synthetic sick with the mutant eft2 ( H699K ) of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A(K56A) mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A(K56A) mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A(K56A) mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.

Translational control is extremely important in all organisms, and some of its aspects are highly conserved among all primary kingdoms, such as those related to the translation elongation step. The previously classified translation initiation factor 5A (eIF5A) and its bacterial homologue elongation factor P (EF-P) were discovered in the late 70's and have recently been the object of many studies. eIF5A and EF-P are the only cellular proteins that undergo hypusination and lysinylation, respectively, both of which are unique posttranslational modifications. Herein, we review all the important discoveries related to the biochemical and functional characterization of these factors, highlighting the implication of eIF5A in translation elongation instead of initiation. The findings that eIF5A and EF-P are important for specific cellular processes and play a role in the relief of ribosome stalling caused by specific amino acid sequences, such as those containing prolines reinforce the hypothesis that these factors are involved in specialized translation. Although there are some divergences between these unique factors, recent studies have clarified that they act similarly during protein synthesis. Further studies may reveal their precise mechanism of ribosome activity modulation as well as the mRNA targets that require eIF5A and EF-P for their proper translation.

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved and essential protein present in all organisms except bacteria. To be activated, eIF5A requires the conversion of a specific residue of lysine into hypusine. This hypusine modification occurs posttranslationally in two enzymatic steps, and the polyamine spermidine is the substrate. Despite having an essential function in translation elongation, the critical role played by eIF5A remains unclear. In addition to demonstrating genetic interactions with translation factors, eIF5A mutants genetically interact with mutations in YPT1, which encodes an essential protein involved in endoplasmic reticulum (ER)-to-Golgi vesicle transport. In this study, we investigated the correlation between the function of eIF5A in translation and secretion in yeast. The results of in vivo translocation assays and genetic interaction analyses suggest a specific role for eIF5A in the cotranslational translocation of proteins into the ER, but not in the posttranslational pathway. Additionally, we observed that a block in eIF5A activation up-regulates stress-induced chaperones, which also occurs when SRP function is lost. Finally, loss of eIF5A function affects binding of the ribosome-nascent chain complex to SRP. These results link eIF5A function in translation with a role of SRP in the cell and may help explain the dual effects of eIF5A in differential and general translation.

Inflammation is part of an important mechanism triggered by the innate immune response that rapidly responds to invading microorganisms and tissue injury. One important elicitor of the inflammatory response is the Gram-negative bacteria component lipopolysaccharide (LPS), which induces the activation of innate immune response cells, the release of proinflammatory cytokines, such as interleukin 1 and tumor necrosis factor α(TNF-α), and the cellular generation of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS). Although essential to the immune response, uncontrolled inflammatory responses can lead to pathological conditions, such as sepsis and rheumatoid arthritis. Therefore, identifying cellular targets for new anti-inflammatory treatments is crucial to improving therapeutic control of inflammation-related diseases. More recently, the translation factor eIF5A has been demonstrated to have a proinflammatory role in the release of cytokines and the production of NO. As eIF5A requires and essential and unique modification of a specific residue of lysine, changing it to hypusine, eIF5A is an interesting cellular target for anti-inflammatory treatment. The present study reviews the literature concerning the anti-inflammatory effects of inhibiting eIF5A function. We also present new data showing that the inhibition of eIF5A function by the small molecule GC7 significantly decreases TNF-α release without affecting TNF-α mRNA levels. We discuss the mechanisms by which eIF5A may interfere with TNF-α mRNA translation by binding to and regulating the function of ribosomes during protein synthesis.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:28:48Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:32:23Z : No. of bitstreams: 1 2-s2.0-84875686470.pdf: 1096527 bytes, checksum: 8faf38291a907524aacb14c06e2c94f8 (MD5) Made available in DSpace on 2014-05-27T11:28:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-01 The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al. Department of Biological Sciences Univ Estadual Paulista - UNESP, Araraquara-Saõ Paulo Department of Biological Sciences Univ Estadual Paulista - UNESP, Araraquara-Saõ Paulo