The Evolutionarily Conserved Obg‐like ATPase‐1 (OLA1) Acts as a Molecular Chaperone to Regulate Cellular Stress Responses

Abstract

BackgroundMolecular chaperones are highly regulated important mediators of cell stress responses. Chaperones, through direct interactions prevent aggregation of client proteins, critical for cell survival in conditions of stress. Since most chaperones that are fundamental to cell survival are prone to aggregation themselves, suggest the importance of other stress proteins in regulating proteostasis. OLA1, an Obg‐like ATPase is ubiquitously expressed and evolutionarily conserved from bacteria to humans. Among the GTPase family, OLA1 is a unique member with an exceptional ability to hydrolyze ATP rather than GTP. OLA1 is involved in diverse cellular processes including subcellular localization, and apoptosis. However, these functions cannot be explained by a single mode of action and the detailed mechanism by which OLA1 regulates cellular stress responses is unknown. Given recent implications of OLA1 in several disease pathogenesis there is a need to define the mechanism that enables OLA1 to function as a stress response protein.Methods and ResultsWe first assessed the response of OLA1 to unfavorable conditions by quantifying OLA1 expression after subjecting bovine pulmonary artery endothelial cells (PAECs) to heat stress (HS, 42°C) using quantitative PCR. We observed an increase in the expression level of OLA1 mRNA in a time‐dependent manner following HS. Further, HS triggers re‐localization of OLA1 proteins to the cytosol, which is in good agreement with proteins involved in stress regulation. To determine the functional relevance of this finding, we tested if OLA1 is capable of suppressing aggregation of proteins using thermolabile model substrates (Citrate synthase, CS, and Firefly Luciferase, Luc). We expressed OLA1 in E. coli and purified it to homogeneity. We initially ensured that recombinant OLA1 protein is properly folded and functional by monitoring its secondary structure and stability against thermal unfolding using CD spectroscopy. OLA1 showed CD spectra typical of an α‐sheet protein and was maintained in its native folded state at higher temperatures (Tm‐75°C). Chaperone assays revealed that OLA1 decreased aggregations of both CS and Luc in a concentration‐dependent manner. The presence of OLA1 substantially increased the soluble fraction of CS after heat treatment. Also, OLA1 inhibited aggregation of chemically denatured CS and Luc in temperature‐dependent manner. Interestingly, OLA1 forms large complex oligomers at higher temperatures, as assessed by size exclusion chromatography (SEC), which displayed a single peak with an apparent molecular size of 1350 kDa (50°C) vs 1235kDa (25°C). The heat‐induced change in OLA1 oligomeric states correlates with higher ATP hydrolyses.ConclusionsOur findings specify OLA1 as a molecular chaperone and its chaperone activity constitutes a key feature of its action in cell‐stress protection. The stability of OLA1 at higher temperature allows OLA1 to prevent HS‐induced damage to client‐proteins including Hsp70 and Hsp90.Support or Funding InformationNIH (K08‐AJA080271), Children's Research Institute (CRI) and the Department of Pediatrics, Medical College of Wisconsin. Recombinant Citrate synthase protein was subjected to 55°C for 10 minutes in the absence or presence of OLA1 at varying concentrations and the absorbance was measured at 360 nm continuosly for 60 minutes using spectrophotometry.imageRecombinant Citrate synthase protein was subjected to 55°C for 10 minutes in the absence or presence of OLA1 at varying concentrations and the absorbance was measured at 360 nm continuosly for 60 minutes using spectrophotometry. Recpmbinant Citrate synthase protein was incubated with increasing concentration of guanidine hydrochloride (GuHcl) at room temperature for 2 hours, in the presence or absence of 0.6uM OLA1 and the absorbance at 280 nm was measured over 20 minutes at varying temperatures using spectrophotometry.imageRecpmbinant Citrate synthase protein was incubated with increasing concentration of guanidine hydrochloride (GuHcl) at room temperature for 2 hours, in the presence or absence of 0.6uM OLA1 and the absorbance at 280 nm was measured over 20 minutes at varying temperatures using spectrophotometry. Quaternary structure of OLA1 was assessed by SEC‐HPLC using a superdex 200 (30/200) column. OLA1 at a concentration of 3 mg/mL was separated at 25°C (black) and at 55°C (red).imageQuaternary structure of OLA1 was assessed by SEC‐HPLC using a superdex 200 (30/200) column. OLA1 at a concentration of 3 mg/mL was separated at 25°C (black) and at 55°C (red).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.