Arbach Hannah, Butler Caley, McMenimen Kathryn A
Department of Chemistry, Mount Holyoke College, 50 College Street, South Hadley, MA, 01075, USA.
Cell Stress Chaperones. 2017 Jul;22(4):503-515. doi: 10.1007/s12192-017-0764-2. Epub 2017 Jan 27.
Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.
小分子热休克蛋白（sHsps）是细胞内维持蛋白质稳态机制中普遍存在的一部分，作为分子伴侣发挥作用。sHsps以不依赖ATP的方式结合并防止部分折叠的底物蛋白聚集。sHsps具有动态性，通过浓度依赖性平衡解离形成从二聚体到大型寡聚体的一系列结构。基于结构研究和诱变实验，有人提出二聚体是最小的活性伴侣单元，而较大的寡聚体可能作为sHsps的储存库，或在伴侣功能中发挥额外作用。其结构组织的复杂性和动态性质使得阐明其伴侣功能具有挑战性。HspB1和HspB5是两种典型的人类sHsps，它们的序列不同，在多种组织中表达。为了确定二聚体在伴侣活性中的作用，谷胱甘肽-S-转移酶（GST）通过基因连接作为融合蛋白与HspB1和HspB5（分别也称为Hsp27和αB-晶状体蛋白）的N端区域相连，以便通过使用GST来限制HspB1和HspB5的寡聚体形成，因为GST很容易形成二聚体结构。我们监测了这些融合蛋白的伴侣活性，结果表明它们主要形成二聚体和单体，并作为活性分子伴侣发挥作用。此外，这两种不同的融合蛋白对两种模型底物蛋白柠檬酸合酶（CS）和苹果酸脱氢酶（MDH）表现出不同的伴侣活性。与GST-HspB5和野生型HspB1相比，GST-HspB1能更有效地防止MDH聚集。然而，当CS作为底物时，GST-HspB1和GST-HspB5作为伴侣的效果相同。此外，野生型蛋白对底物的活性并不相同，这表明每种sHsp都表现出不同的底物特异性。因此，如本文所述，对于与MDH和CS结合的全长GST融合蛋白，底物特异性受到sHsp寡聚体构象和sHsp序列变异的共同调节。
