From the State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, Peking University Shenzhen Graduate School, Shenzhen, China, 518055 and.
Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu Province, China, 210093.
J Biol Chem. 2019 Mar 15;294(11):4247-4258. doi: 10.1074/jbc.RA118.005844. Epub 2019 Jan 22.
Cluster of differentiation 38 (CD38) is the best-studied enzyme catalyzing the synthesis of the Ca messenger cyclic ADP-ribose. It is a single-pass transmembrane protein, but possesses dual orientations. We have documented the natural existence of type III CD38 in cells and shown that it is regulated by a cytosolic activator, calcium- and integrin-binding 1 (CIB1). However, how type III CD38 can be folded correctly in the reductive cytosol has not been addressed. Using the yeast two-hybrid technique with CD38's catalytic domain (sCD38) as bait, here we identified a chaperone, Hsp70-interacting protein (Hip), that specifically interacts with both the type III CD38 and sCD38. Immunoprecipitation coupled with MS identified a chaperone complex associated specifically with sCD38. Pharmacological and siRNA-mediated knockdown of Hsp90 chaperones decreased the expression levels of both sCD38 and type III CD38, suggesting that these chaperones facilitate their folding. Moreover, knockdown of Hsc70 or DNAJA2 increased the levels of both CD38 types, consistent with the roles of these proteins in mediating CD38 degradation. Notably, Hip knockdown decreased type III CD38 substantially, but only marginally affected sCD38, indicating that Hip was selective for the former. More remarkably, DNAJA1 knockdown decreased sCD38 but increased type III CD38 levels. Mechanistically, we show that Hsc70 mediates lysosomal degradation of type III CD38, requiring the lysosomal receptor Lamp2A and the C19-motif in the C terminus of CD38. Our results indicate that folding and degradation of type III CD38 is effectively controlled in cells, providing further strong support of its physiological relevance.
CD38 是研究最为透彻的酶之一，它可以催化 Ca 信使环 ADP-核糖的合成。它是一种单次跨膜蛋白，但具有双重取向。我们已经证明了细胞中 III 型 CD38 的自然存在，并表明它受到细胞质激活剂钙和整合素结合蛋白 1（CIB1）的调节。然而，III 型 CD38 如何在还原细胞质中正确折叠尚未得到解决。使用以 CD38 的催化结构域（sCD38）为诱饵的酵母双杂交技术，我们鉴定了一种伴侣蛋白 Hsp70 相互作用蛋白（Hip），它与 III 型 CD38 和 sCD38 特异性相互作用。免疫沉淀结合 MS 鉴定出与 sCD38 特异性相关的伴侣复合物。Hsp90 伴侣蛋白的药理学和 siRNA 敲低降低了 sCD38 和 III 型 CD38 的表达水平，表明这些伴侣蛋白有助于它们的折叠。此外，Hsc70 或 DNAJA2 的敲低增加了两种 CD38 类型的水平，与这些蛋白在介导 CD38 降解中的作用一致。值得注意的是，Hip 敲低显著降低了 III 型 CD38 的水平，但对 sCD38 的影响很小，表明 Hip 对前者具有选择性。更显著的是，DNAJA1 的敲低降低了 sCD38 的水平，但增加了 III 型 CD38 的水平。从机制上讲，我们表明 Hsc70 介导 III 型 CD38 的溶酶体降解，需要溶酶体受体 Lamp2A 和 CD38 C 端的 C19 基序。我们的结果表明，III 型 CD38 的折叠和降解在细胞中得到有效控制，为其生理相关性提供了进一步的有力支持。
