Effect of N-terminal region of nuclear Drosophila melanogaster small heat shock protein DmHsp27 on function and quaternary structure

PloS One
Mohamed Taha MoutaoufikRobert M Tanguay

Abstract

The importance of the N-terminal region (NTR) in the oligomerization and chaperone-like activity of the Drosophila melanogaster small nuclear heat shock protein DmHsp27 was investigated by mutagenesis using size exclusion chromatography and native gel electrophoresis. Mutation of two sites of phosphorylation in the N-terminal region, S58 and S75, did not affect the oligomerization equilibrium or the intracellular localization of DmHsp27 when transfected into mammalian cells. Deletion or mutation of specific residues within the NTR region delineated a motif (FGFG) important for the oligomeric structure and chaperone-like activity of this sHsp. While deletion of the full N-terminal region, resulted in total loss of chaperone-like activity, removal of the (FGFG) at position 29 to 32 or single mutation of F29A/Y, G30R and G32R enhanced oligomerization and chaperoning capacity under non-heat shock conditions in the insulin assay suggesting the importance of this site for chaperone activity. Unlike mammalian sHsps DmHsp27 heat activation leads to enhanced association of oligomers to form large structures of approximately 1100 kDa. A new mechanism of thermal activation for DmHsp27 is presented.

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Citations

Sep 6, 2020·Cell Stress & Chaperones·Mohamed Taha Moutaoufik, Robert M Tanguay
Nov 10, 2020·Infection, Genetics and Evolution : Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases·Joaquim Ferreira do Nascimento NetoWanderli Pedro Tadei

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Methods Mentioned

BETA
electrophoresis
transfection
gel filtration
PCR
size exclusion chromatography
fluorescence microscopy

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