Abstract：
J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.
摘要：
J域蛋白（JDPs）形成了一个非常大的分子伴侣家族，参与蛋白质稳定过程，比如蛋白质折叠，通过膜和降解/分解贩运。JDP是能够刺激ATP酶活性以及选择和向Hsp70呈递客户蛋白的Hsp70共伴侣。在线粒体中，人DjC20/HscB（III型JDP，仅在蛋白质的某些区域具有保守的J结构域）参与[FeS]蛋白质的生物发生，并协助人线粒体Hsp70（HSPA9）。人DjC20在其N端具有锌指结构域，它与J域紧密接触，似乎对其功能至关重要。这里,我们研究了hDjC20在溶液中的结构以及Zn2对其稳定性的重要性。重组hDjC20是纯的，折叠并能够刺激HSPA9ATP酶活性。它表现得像一个略微拉长的单体，小角度X射线散射和SEC-MALS证明。验证了hDjC20样品中Zn2+的存在，观察到1：1的化学计量，用高浓度的EDTA和DTPA去除是不可行的。然而,在EDTA存在下的热和化学变性导致蛋白质稳定性降低，表明螯合剂和变性剂之间的协同作用，促进蛋白质展开取决于金属去除。这些数据表明，Zn+2对蛋白质的亲和力非常高，证明其对hDjC20结构的重要性。
