Functional cooperativity between the trigger factor chaperone and the ClpXP proteolytic complex

A functional association is uncovered between the ribosome-associated trigger factor (TF) chaperone and the ClpXP degradation complex. Bioinformatic analyses demonstrate conservation of the close proximity of tig, the gene coding for TF, and genes coding for ClpXP, suggesting a functional interaction. The effect of TF on ClpXP-dependent degradation varies based on the nature of substrate. While degradation of some substrates are slowed down or are unaffected by TF, surprisingly, TF increases the degradation rate of a third class of substrates. These include λ phage replication protein λO, master regulator of stationary phase RpoS, and SsrA-tagged proteins. Globally, TF acts to enhance the degradation of about 2% of newly synthesized proteins. TF is found to interact through multiple sites with ClpX in a highly dynamic fashion to promote protein degradation. This chaperone–protease cooperation constitutes a unique and likely ancestral aspect of cellular protein homeostasis in which TF acts as an adaptor for ClpXP

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia co ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation2CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP306943/2015-8; 420567/2016-099999.004913/2015-092015/15822-1; 2012/01953-9; 2016/05019-0; 2012/50161-8Precision Medicine Initiative (PRiME) at the University of Toronto internal fellowship [PMRF2019-007]; Canadian Institutes of Health Research (CIHR) postdoctoral fellowshipCanadian Institutes of Health Research (CIHR); CNPq-Brazil fellowship [202192/2015-6]; Saskatchewan Health Research Foundation postdoctoral fellowship; Ontario Graduate Scholarship (OGS)Ontario Graduate Scholarship; Department of Biochemistry at the University of Toronto; Centre for Pharmaceutical Oncology (University of Toronto); CIHR Training Program in Protein Folding and Interaction Dynamics: Principles and Diseases fellowshipCanadian Institutes of Health Research (CIHR) [TGF-53910]; University of Toronto Fellowship from the Department of Biochemistry; OGS fellowship; NSERC PGS-D2 fellowship; CIHR Emerging Team Grants from the Institute of Infection and ImmunityCanadian Institutes of Health Research (CIHR) [XNE-86945]; CIHR Project grantCanadian Institutes of Health Research (CIHR) [PJT-148564]; Global Affairs Canada (Canada); CAPES (Brazil)CAPES [99999.004913/2015-09]; NSERCNatural Sciences and Engineering Research Council of Canada [RGPIN-2015-04877, DG-20234]; Canada Research Chairs ProgramCanada Research Chairs; CIHR new investigator programCanadian Institutes of Health Research (CIHR); FAPESPFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2015/15822-1, 2012/01953-9, 2016/05019-0, 2012/50161-8]; CNPqNational Council for Scientific and Technological Development (CNPq) [306943/2015-8, 420567/2016-0]; AbbVie [1097737]; BayerBayer AG [1097737]; Boehringer IngelheimBoehringer Ingelheim [1097737]; Genome Canada through Ontario Genomics Institute GrantGenome Canada [1097737, OGI-055]; GlaxoSmithKlineGlaxoSmithKline [1097737]; JanssenJohnson & Johnson USAJanssen Biotech Inc [1097737]; Lilly CanadaEli Lilly [1097737]; MerckMerck & Company [1097737]; Novartis Research Foundation [1097737]; Ontario Ministry of Economic Development and Innovation [1097737]; PfizerPfizer [1097737]; TakedaTakeda Pharmaceutical Company Ltd [1097737]; Wellcome Trust GrantWellcome Trust [1097737, 092809/Z/10/Z]; Canada Foundation for InnovationCanada Foundation for Innovation; NSERCNatural Sciences and Engineering Research Council of Canada; University of Saskatchewan; Government of Saskatchewan; Western Economic Diversification Canada; National Research Council Canada; CIHRCanadian Institutes of Health Research (CIHR

Chaperone Systems in Model Organisms

Molecular chaperones are typically promiscuous interacting proteins that function globally in the cell for quality control and maintenance of protein homeostasis. They are highly conserved across all organisms. A comprehensive view of their cellular function was obtained through a systematic global integrative network approach. We deciphered interactions involving all core chaperones of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps, that is central to the topology of the proteome network. Chaperone interactor properties demonstrate preferential protein domains and folds such as the WD40 repeat domain. We further found that many chaperones interact with proteins that form condensates under stress conditions. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities. From an extensive genomic analysis in bacteria we demonstrate the conservation of the close proximity of the tig gene, coding for the ribosome-associated chaperone trigger factor (TF), and the genes coding for the ClpXP proteolytic complex suggesting a possible functional association between the protein folding and the protein degradation systems. The effect of TF on ClpXP-dependent degradation varied depending on the nature of the substrate. For a class of substrates, TF increased the degradation rate. The ClpXP-dependent degradation was enhanced for the λ phage replication protein λO, the master regulator of stationary phase RpoS, and SsrA-tagged proteins by TF in vitro and in vivo. Globally, it is estimated that TF enhances the degradation of about subset of newly synthesized E. coli proteins. Through extensive biochemical and structural analyses, it was found that TF interacts through multiple sites with the ClpX oligomer in a highly dynamic fashion. TF binds the ATPase-domain of ClpX and consequently promotes the movement of the protruding N-terminal zinc binding domain (ZBD) of the chaperone towards the ClpP cylinder. With this movement, TF enhances the degradation of ZBD-bound substrates. These results reveal a novel function of TF as a ClpXP adaptor protein and highlight its broad substrate specificity. This chaperone-protease cooperation constitutes a novel aspect of cellular protein homeostasis.Ph.D.2021-07-21 00:00:0

Genetic Dissection of Virulence and Immune-eliciting Functions and Characterization of the Immune Response of the Pseudomonas syringae HopZ1 Type III Effector Family

Successful pathogens like Pseudomonas syringae translocate type III effector proteins (T3SE) into host cells. Plant hosts react by specifically recognizing these effectors via R proteins that trigger defense responses. The T3SE family HopZ1 has evolved into three allelic forms as a result of diversifying selection. In this thesis, I investigated how virulence and immune-eliciting functions are determined in HopZ1a and HopZ1b in Arabidopsis. Mutational analysis of HopZ1a identified ten residues important for immune elicitation and at least three are involved in virulence functions. These results suggest that distinct key amino acid residues in HopZ1a mediate the two activities. The closely related HopZ1b T3SE elicits an inconsistent immune response in Arabidopsis. We found that HopZ1b-triggered immune response involves a TIR-type R protein and plastid-derived SA. Together, these results highlight an uncharacterized ETI response to the HopZ1 family of T3SEs.MAS

Novel function discovery with GeneMANIA::a new integrated resource for gene function prediction in Escherichia coli

Motivation: The model bacterium Escherichia coli is among the best studied prokaryotes, yet nearly half of its proteins are still of unknown biological function. This is despite a wealth of available large-scale physical and genetic interaction data. To address this, we extended the GeneMANIA function prediction web application developed for model eukaryotes to support E.coli. Results: We integrated 48 distinct E.coli functional interaction datasets and used the GeneMANIA algorithm to produce thousands of novel functional predictions and prioritize genes for further functional assays. Our analysis achieved cross-validation performance comparable to that reported for eukaryotic model organisms, and revealed new functions for previously uncharacterized genes in specific bioprocesses, including components required for cell adhesion, iron–sulphur complex assembly and ribosome biogenesis. The GeneMANIA approach for network-based function prediction provides an innovative new tool for probing mechanisms underlying bacterial bioprocesses. Contact: [email protected]; [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Acyldepsipeptide analogs dysregulate human mitochondrial ClpP protease activity and cause apoptotic cell death

Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.</p

Novel function discovery with GeneMANIA: A new integrated resource for gene function prediction in Escherichia coli

Motivation: The model bacterium Escherichia coli is among the best studied prokaryotes, yet nearly half of its proteins are still of unknown biological function. This is despite a wealth of available large-scale physical and genetic interaction data. To address this, we extended the GeneMANIA function prediction web application developed for model eukaryotes to support E.coli. Results: We integrated 48 distinct E.coli functional interaction datasets and used the GeneMANIA algorithm to produce thousands of novel functional predictions and prioritize genes for further functional assays. Our analysis achieved cross-validation performance comparable to that reported for eukaryotic model organisms, and revealed new functions for previously uncharacterized genes in specific bioprocesses, including components required for cell adhesion, iron-sulphur complex assembly and ribosome biogenesis. The GeneMANIA approach for network-based function prediction provides an innovative new tool for probing mechanisms underlying bacterial bioprocesses