Post-translational modifications of Hsp90

我们已经报道了酵母中在多细胞真核生物中不保守的Hsp90共伴侣蛋白在人类细胞中被位点特异性翻译后修饰在功能上取代。具体来说，人类Hsp90 α中高度保守的酪氨酸627的磷酸化导致伴侣蛋白的远程构象改变，导致Hsp90 n结构域二聚化的丧失。虽然酵母Hsp90中的酪氨酸627没有被磷酸化，但其拟磷突变对酵母Hsp90构象的影响与非保守的酵母共伴侣蛋白Hch1的过表达和人Hsp90 α中酪氨酸627的磷酸化相似。我们的数据与这种翻译后修饰在高等真核生物中出现的可能性是一致的，这代表了Hch1蛋白的进化替代。(2)我们发现磷酸化诱导的Hsp90共同伴侣Cdc37的展开促进了对Hsp90的激酶募集和客户类特异性的Hsp90磷酸化。在Hsp90介导的蛋白激酶的伴侣过程中，该机制的核心成分Hsp90和伴侣蛋白Cdc37在不同的磷酸化状态之间循环，从而调节伴侣蛋白循环的进展。我们发现Cdc37在Y298位点的磷酸化导致c末端结构域的部分展开和折叠中间体的分布。通过揭开一个磷酸化肽序列，展开促进了Hsp90 Y197位点的磷酸化，该磷酸化肽序列作为对接位点，通过其SH2结构域将非受体酪氨酸激酶招募到伴侣蛋白复合体中。反过来，Hsp90在Y197的磷酸化特异性调节其与Cdc37的相互作用，从而仅影响蛋白激酶客户的陪伴。总之，我们发现通过提供客户类特异性，Cdc37等Hsp90伴侣蛋白不仅有助于客户招募，而且还以客户类特异性的方式塑造了Hsp90的翻译后修饰格局。最近，我们发表了Hsp90中间区域特定酪氨酸残基的磷酸化，通过诱导Hsp90独特且可逆的构象，显著增强了刺激共伴侣Aha1的关联。最近，我们发现Hsp90赖氨酸乳酸化是一种新的糖酵解依赖性PTM。线粒体抑制增加了这种修饰，LDH抑制减少了这种修饰。我们目前正在使用质谱法鉴定以这种方式修饰的人类HSP90上的特定赖氨酸残基。最近，我们发现当线粒体复合体I被特异性抑制时，内质Hsp70 BiP被乳酸化。我们将确定所涉及的赖氨酸残基，我们将突变这些赖氨酸并研究对HSP70功能的影响。

We have reported that an Hsp90 co-chaperone in yeast that is not conserved in multi-cellular eukaryotes is functionally replaced by site-specific postranslational modification in human cells. Specifically, phosphorylation of the highly conserved tyrosine 627 in human Hsp90 alpha causes a long-range conformational change in the chaperone resulting in loss of dimerization of the Hsp90 N-domains. Although tyrosine 627 in yeast Hsp90 is not phosphorylated its phosphomimetic mutation has a similar effect on yeast Hsp90 conformation as does over-expression of the non-conserved yeast co-chaperone Hch1 and phosphorylation of tyrosine 627 in human Hsp90 alpha. Our data are consistent with the possibility that appearance of this posttranslational modification in higher eukaryotes represents an evolutionary substitution for Hch1 protein. (2) We showed that phosphorylation-induced unfolding of the Hsp90 co-chaperone Cdc37 promotes kinase recruitment to Hsp90 and client class-specific Hsp90 phosphorylation. During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner. Most recently, we have published that phosphorylation of a specific tyrosine residue in the middle domain of Hsp90 dramatically enhances association of the stimulatory co-chaperone Aha1 by inducing a unique and reversible conformation of Hsp90. Most recently, we identified Hsp90 lysine lactylation as a novel, glycolysis-dependent PTM. This modification is increased by mitochondrial inhibition and decreased by LDH inhibition. We are currently using mass spectrometry to identify specific lysine residues on human HSP90 that are modified in this manner. Most recently, we have identified the endoplasmic Hsp70 BiP as being lactylated when mitochonddrial complex I is specifically inhibited. We will identify the lysine residues involved ,and we will mutate these lysines and study impact on HSP70 function.