Abstract：
Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.
摘要：
热休克同源蛋白70(Hsc70/HSPA8)属于分子伴侣的Hsp70家族。Hsp70家族分子伴侣的基本功能取决于疏水多肽底物的结合和释放的ATP依赖性变构调节。Hsc70还参与各种其他细胞功能，包括蛋白质降解的选择性途径：伴侣介导的自噬（CMA）和内体微自噬（eMI）。其中Hsc70将含有KFERQ样五肽基序的底物蛋白从细胞质募集到溶酶体和晚期内体，分别。然而,Hsc70与五肽基序之间的相互作用是直接的还是由其他分子介导的仍然未知。在本研究中,我们在CMA/eMI模型底物中的KFERQ基序附近引入了一种光交联剂，并成功检测了其与Hsc70的交联，首次揭示了Hsc70与KFERQ基序之间的直接相互作用。此外,我们证明，D10N突变导致的Hsc70ATPase活性丧失明显降低了交联效率。我们目前的结果表明，Hsc70的ATP变异型参与了Hsc70与KFERQ样五肽的直接相互作用。
