The role of hHep1/Hsp70s in protein quality control and the regulation of condensate dynamics upon proteotoxic stress

• Grant number: 21/12775-3
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: June 01, 2022
• End date: May 31, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Julio Cesar Borges
• Grantee: Milene Nóbrega de Oliveira Moritz
• Supervisor: Serena Carra
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Institution abroad: Università degli Studi di Modena e Reggio Emilia, Modena (UNIMORE), Italy
• Associated to the scholarship: 19/22422-0 - Cellular functional study of high molecular weight oligomers of mortaline and the effects of mitochondrial co-chaperones on their structure, BP.PD

Abstract

70 kDa heat shock proteins (Hsp70s) are critical molecular chaperones for proteostasis capable of assisting protein folding, transportation through membranes, targeting to degradation, as well as preventing protein aggregation. Hsp70s are thus critical players of the protein quality control (PQC) system which includes protein synthesis, molecular chaperones and degradation systems. Upon stress, misfolding-prone proteins accumulate in the cytosol, where they are compartmentalized in puncta that colocalize with PQC players, as well as in nucleus where aggregation-prone proteins, including newly synthesized defective ribosomal products (DRiPs) accumulate inside two types of biomolecular condensates: nucleoli and promyelocytic leukemia (PML) bodies. Hsp70s, such as HSPA1A, are recruited to nucleoli and PML nuclear bodies (PML-NBs) upon stress. There, they participate to the clearance of nuclear misfolded proteins, thus maintaining the liquid-like dynamic properties of the biomolecular condensates. We recently demonstrated that the co-chaperone hHep1 (human Hsp70-escort protein 1) is localized in nucleus besides its common mitochondrial localization. hHep1 assists HSPA9 and HSPA1A (preventing their thermal-induced aggregation and remodeling such particles as well as stimulating the ATPase activities of the monomeric and supramolecular assemblies), which also justify the relevance to understand better the role of hHep1 in vivo. Moreover, HSPA9 (human mitochondrial Hsp70) is also localized in the nucleus, besides mitochondria. Cells transfected with hHep1 and HSPA9, under thermal stress, had an increased expression and colocalization both inside nucleus (unpublished data). These data suggest that a fraction of HSPA9 and hHep1 could participate to specialized nuclear functions; upon translocation to nuclei. Therefore, we hypothesized that hHep1 and Hsp70s could be involved in PQC condensates dynamic in nucleoli and/or PML bodies upon proteotoxic stress. To test this hypothesis, we propose to evaluate the dynamics of hHep1 and Hsp70s (HSPA9 and HSPA1A) in nuclear condensates, using confocal microscopy and fluorescence recovery after photobleaching (FRAP) approaches under controlled stress conditions. Besides, we also intent to investigate the influence of hHep1 in proteasome-mediated degradation of condensates under proteotoxic stress, using the proteasome inhibitor MG-132 and conversely we will deplete hHep1 and/or Hsp70s and assess their impact on the cell ability to clear DRiPs and polyUb proteins from nucleoli and PML-NBs. (AU)