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Title 

Reduced protein denaturation in thermotolerant cells by elevated levels of HSP70

 

열내성이 유도된 세포에서 HSP70 단백질 증가에 의한 단백질 변성 감소

Authors 

Mi Young HanYoung-Mee Park

Publisher 

Korean Society of Pharmacognosy

Issue Date 

1996

Citation 

Korean Journal of Pharmacognosy, vol. 32, no. 3, pp. 433-444

Keywords 

diamideheat shock proteins (HSPs)protein denaturationthermotoleranceheat shock protein 70proteinprotein antibodyanimal cellcell lineprotein aggregation

Abstract 

We describe a novel approach to evaluate quantitatively the amounts of denatured proteins in cells upon heat exposure. A thiol compound, diamide [azodicarboxylic acid bis (dimethylamide)] causes protein cross-linking with exposed sulfhydryl residues of denatured proteins. Since denatured proteins expose normally well-hidden sulfhydryl groups, these will be preferentially cross-linked by diamide. Thus diamide acts to 'trap' denatured proteins. We observed that protein aggregates (high molecular weight protein aggregates, HMA) appeared on SDS-polyacrylamide gels run under non-reducing conditions and that the amount of HMA can be quantified by scanning the gels using a gas flow counter. Heating cells followed by a fixed dose of diamide exposure resulted in HMA increases in a heat-dose dependent manner, demonstrating that the quantitation of HMA could ve as a measure of heat-denatured proteins. We compared thermotolerant and nontolerant cells and found decreased HMA in tolerant cells upon heat treatment. As an attempt to examine the kinetics of protein renaturation (or 'repair'), we measured the amounts of aggregates formed by the addition of diamide at various times after heat shock. Such experiments demonstrate an equally rapid disappearance of HMA in previously unheated and in thermotolerant cells. Levels of HMA in tolerant cells increased significantly after electroporation of HSP70 specific mAbs, suggesting an involvement of HSP70 in reducing HMA levels in thermotolerant cells upon heat exposure. Immunoprecipitation studies using anti-HSP70 antibody indicated an association of HSP70 with heat-denatured proteins. Our results suggest that heat induces protein denaturation, and that elevated level of HSP70 present in thermotolerant cells protects them by reducing the level of protein denaturation rather than by facilitating the 'repair' (or degradation) process.

ISSN 

0253-3073

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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