Obtaining recombinant GFP fused to a C-terminal degron sequence for interaction tests with human Hsp70s

• Grant number: 24/14681-4
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2024
• End date: October 31, 2025
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Julio Cesar Borges
• Grantee: Martina Jusan Fernandes Dau
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 17/26131-5 - The chaperome: study of the relationship of the structure of its components and the maintenance of proteostasis, AP.TEM

Abstract

Molecular chaperones are proteins responsible for protein quality control, including the identification of misfolded proteins and directing them for degradation. Human heat shock proteins of 70 kDa (Hsp70 or HSPA in humans) comprise a family of chaperones that also participate in the proteasome system via ubiquitin through a complex mechanism involving cytoplasmic Hsp90 and co-chaperones, such as CHIP (C-terminus of Hsc70-interacting protein), an E3 ubiquitin ligase. To study this system, we propose the use of an unstable variant of the green fluorescent protein GFP with a CL1 degron sequence at its C-terminal (eGFPuv-dCL1). In a cellular model expressing eGFPuv-dCL1, we demonstrated that inhibition of Hsp70 through the specific inhibitor MKT-077 results in the accumulation of eGFPuv-dCL1 in HEK293 cells, as evaluated by the increase in green fluorescence in these cells. These results suggest that the targeting of eGFPuv-dCL1 to the proteasome is impaired. However, using the Hsp90 inhibitor geldanamycin (GA), no accumulation of eGFPuv-dCL1 was observed. These data suggest that cytoplasmic Hsp70s, and not Hsp90s, are involved in directing eGFPuv-dCL1 for degradation via the proteasome. In this context, the scientific initiation project aims to study the interdependence between Hsp70s and the degradation system through the physical interaction between eGFPuv-dCL1 and human cytoplasmic Hsp70s. To this end, the objective of this project is to produce, purify, and characterize recombinant eGFPuv-dCL1 for interaction tests with 2 of the main recombinant human cytoplasmic Hsp70s.