Investigation of macromolecular complexes containing HSP70 and HSP90 in human endothelial cells in response to hypoxia and heat shock

Author(s)

Luiza Bitencourt de Carvalho Terci Coimbra

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2023-10-11

Examining board members

Thaís Larissa Araujo de Oliveira Silva; Júlio César Borges

• Advisor: Thaís Larissa Araujo de Oliveira Silva

Abstract

The 70kDa heat shock protein (HSP70) is a molecular chaperone essential for proteostasis and conserved in all kingdoms of life. In endothelial cells it is essential for migration and angiogenesis. Although HSP70 and HSP90 perform their chaperone functions mostly in monomeric and dimeric forms, respectively, oligomerization of these HSPs has been described in tumor cells, fungi and bacteria under stress conditions. The oligomerization of HSP70 was increased by heat stress in eukaryotic cells and Escherichia coli. In human tumor cells, the formation of high molecular weight complexes nucleated by the constitutive form of HSP70 (HSC70) and HSP90, called epichaperoma, is dependent on proteotoxic stress mediated by Myc. In vascular cells it is unknown whether HSPs exist in macromolecular complexes and whether they are modulated by physiologically relevant stresses in the vasculature such as: heat shock and hypoxia. We investigated in primary human endothelial cells from different vascular cradles, such as umbilical vein (HUVEC) and coronary artery (HCAEC), the existence and dynamics of HSPs in basal, stress and recovery conditions. The native gel reveals the presence of HSP70 complexes and HSP90 dimers in the basal condition in HUVEC and HCAEC. HSC70 complexes are reduced by hypoxia, increased after heat shock (HS) and return to basal levels after recovery from HS. HSP40, co-chaperone of HSP70, forms high molecular weight complexes only after recovery, a similar phenomenon was observed for stress-inducible HSP70 complexes (HSPA1A/B). The total protein expression of HSC70 and HSP90 was not altered, while the levels of HSP70 and HSP40 were significantly increased in HS and recovery. There is no interaction between HSPs in the macromolecular complexes in primary endothelial cells, unlike the hyperconnected network present in tumor cells. HSP90 dimers are reduced by HS and hypoxia and enriched after recovery from HS. Heat shock promotes translocation of HSC70 to the nucleus, reduction of cell-cell adhesion proteins found on the endothelial surface, VEcadherin and CD31/PECAM-1, as visualized by immunofluorescence. In addition, the pharmacological inhibition of HSP70 on migration and tube formation in endothelial cells was not affected by prior exposure to heat shock. Our results reveal a new face of the human heat shock response: temporal modulation of HSPs in complexes in endothelial cells. In addition, the reduction of complexes after chronic hypoxia is potentially associated with its deleterious vascular effects. Therefore, the results corroborate the essential function of HSP70 in vascular proteostasis and advance our understanding of the dynamics of the formation and stability of macromolecular complexes containing HSPs in the endothelium. (AU)

• FAPESP's process: 20/11249-3 - The pathophysiology of endothelial cells isolated from patients with hypertensive pulmonary thromboembolism: mechanisms regulated by HSP70
• Grantee: Luiza Bitencourt de Carvalho Terci Coimbra
• Support Opportunities: Scholarships in Brazil - Master