Moutaoufik Mohamed Taha, Morrow Geneviève, Finet Stéphanie, Tanguay Robert M
Laboratoire de génétique cellulaire et développementale, Département de biologie moléculaire, biochimie médicale et pathologie, Institut de biologie intégrative et des systèmes (IBIS) and PROTEO, Université Laval, Québec, Canada.
IMPMC UMR7590, CNRS, UPMC Paris 6, 4 place Jussieu, Paris, France.
PLoS One. 2017 May 16;12(5):e0177821. doi: 10.1371/journal.pone.0177821. eCollection 2017.
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.
通过使用尺寸排阻色谱法和非变性凝胶电泳进行诱变，研究了果蝇小核热休克蛋白DmHsp27的N端区域（NTR）在寡聚化和伴侣样活性中的重要性。N端区域中两个磷酸化位点S58和S75的突变，在转染到哺乳动物细胞中时，不影响DmHsp27的寡聚化平衡或细胞内定位。NTR区域内特定残基的缺失或突变确定了一个对该小分子热休克蛋白（sHsp）的寡聚结构和伴侣样活性很重要的基序（FGFG）。虽然完整N端区域的缺失导致伴侣样活性完全丧失，但去除29至32位的（FGFG）或F29A/Y、G30R和G32R的单点突变在胰岛素试验中增强了非热休克条件下的寡聚化和伴侣功能，表明该位点对伴侣活性很重要。与哺乳动物的sHsps不同，DmHsp27的热激活导致寡聚体增强缔合，形成约1100 kDa的大结构。本文提出了一种DmHsp27热激活的新机制。
