Structural and functional characterization of the mammalian chaperone Asna1 (TRC40)

• Grant number: 21/13550-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Effective date (Start): March 04, 2022
• Effective date (End): March 03, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Flavia Carla Meotti
• Grantee: Bianca Dempsey Pinto
• Supervisor: Ursula Jakob
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: University of Michigan, United States
• Associated to the scholarship: 19/16224-1 - Proteomic analysis of endothelial cells secretome: study about uric acid and Atherosclerosis development, BP.DD

Abstract

Reactive oxygen species (ROS) are well known for their role in mediating physiological cellular processes. In certain conditions, ROS levels may exceed the capacity of the cellular antioxidant system, which can cause damage to proteins. To alleviate the risk of irreversible protein aggregation, cells have developed a molecular chaperone system that binds unfolding proteins under stress conditions. Some of these chaperone holdases can be specifically activated by ROS-mediated reversible thiol oxidation, such as bacterial Hsp33 or its eukaryotic counterpart Get3. Get3 is a dual function protein, targeting tail-anchored membrane proteins to the endoplasmic reticulum under non-stress conditions and acting as an ATP-independent chaperone upon oxidative stress. Asna1 is the conserved mammalian homolog of yeast Get3 but so far, it is still uncertain whether Asna1 can also act as a chaperone beyond its function as a targeting factor for ER membrane proteins. Preliminary structural and functional analyses of the recombinant protein revealed that Asna1 indeed gains chaperone activity upon oxidation. Interestingly, the proteomic analysis of our project in Brazil revealed that Ansa1 is exclusively expressed by endothelial HUVEC cells exposed to uric acid, which is known to cause inflammation and oxidative stress. In this project, we will further elucidate the functional switch in vivo and investigate the redox status of Asna1 after uric acid treatment in superoxide or peroxide-generating systems to monitor reversible oxidative modification. Knockdown experiments using Asna1-targeting siRNAs will further show the role of Asna1 under various stress conditions. This research will allow comprehending the relationship between uric acid and Asna1 together with a better understanding of the role of Asna1 in mammals. (AU)