Effect of Structural Changes Induced by Deletion of 54FLRAPSWF61 Sequence in αB-crystallin on Chaperone Function and Anti-Apoptotic Activity.

Sundararajan Mahalingam,Krishna K Sharma,Srabani Karmakar,Puttur Santhoshkumar

doi:10.3390/ijms221910771

Sundararajan Mahalingam, Krishna K Sharma + Show 2 more

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https://doi.org/10.3390/ijms221910771

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Abstract

Previously, we showed that the removal of the 54–61 residues from αB-crystallin (αBΔ54–61) results in a fifty percent reduction in the oligomeric mass and a ten-fold increase in chaperone-like activity. In this study, we investigated the oligomeric organization changes in the deletion mutant contributing to the increased chaperone activity and evaluated the cytoprotection properties of the mutant protein using ARPE-19 cells. Trypsin digestion studies revealed that additional tryptic cleavage sites become susceptible in the deletion mutant than in the wild-type protein, suggesting a different subunit organization in the oligomer of the mutant protein. Static and dynamic light scattering analyses of chaperone–substrate complexes showed that the deletion mutant has more significant interaction with the substrates than wild-type protein, resulting in increased binding of the unfolding proteins. Cytotoxicity studies carried out with ARPE-19 cells showed an enhancement in anti-apoptotic activity in αBΔ54–61 as compared with the wild-type protein. The improved anti-apoptotic activity of the mutant is also supported by reduced caspase activation and normalization of the apoptotic cascade components level in cells treated with the deletion mutant. Our study suggests that altered oligomeric assembly with increased substrate affinity could be the basis for the enhanced chaperone function of the αBΔ54–61 protein.

Highlights

Published: 5 October 2021 α-Crystallin, a small heat shock protein family member, is the most abundant protein in vertebrate eye lenses and is responsible for maintaining lens transparency [1,2]
The Increase in Chaperone Activity of αB∆54–61 Mutant Varies with the Substrate Protein
N-terminal domain (NTD) from αA-crystallin leads to an extreme reduction in the oligomeric size, from 800 to

Summary

Introduction

Published: 5 October 2021 α-Crystallin, a small heat shock protein family member, is the most abundant protein in vertebrate eye lenses and is responsible for maintaining lens transparency [1,2]. It has two subunits, A and B, with a molecular mass of ~20 kD, and the sequence homology between the two subunits is about 56%. While the N-terminal domain is considered essential for subunit–subunit interactions and quaternary structural arrangement [8], the C-terminal extension with a higher polar amino acid content is responsible for the soluble nature of the protein [9].

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