In Vitro Enzymatic Studies Reveal pH and Temperature Sensitive Properties of the CLIC Proteins

Author:

Alghalayini Amani12ORCID,Hossain Khondker Rufaka12ORCID,Moghaddasi Saba1,Turkewitz Daniel R.1ORCID,D’Amario Claudia1,Wallach Michael1,Valenzuela Stella M.12ORCID

Affiliation:

1. School of Life Sciences, University of Technology Sydney, Sydney, NSW 2007, Australia

2. ARC Research Hub for Integrated Device for End-User Analysis at Low-Levels (IDEAL), Faculty of Science, University of Technology Sydney, Sydney, NSW 2007, Australia

Abstract

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins’ enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment.

Funder

Australian Government Research Training Program Scholarship

Australian Research Council (ARC) Industry Transformational Research Hub Scheme

Bod Australia Pty Ltd. Scholarship

School of Life Sciences at University of Technology Sydney

Publisher

MDPI AG

Subject

Molecular Biology,Biochemistry

Reference69 articles.

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