Hsp40 and Hsp70 in Membrane Protein Triage

项目摘要： 膜蛋白（MP）的生物合成始于内质网（ER），是一个复杂的过程， ER膜两侧和膜双层内的结构域必须折叠和组装。MPs 正在研究的包括ABC转运蛋白、P型ATP酶、G蛋白偶联受体和BRICHOS蛋白。 导致MP错误折叠和过早降解的错义突变引起疾病，如 囊性纤维化、低促性腺激素性腺功能减退症、色素性视网膜炎和特发性肺纤维化。为了 防止毒性蓄积，错误折叠的MP被E3泛素靶向用于ER相关降解（ERAD） 连接酶复合物。ER跨膜Hsp 40 DNAJB 12（JB 12）将胞浆Hsp 70募集到细胞内， 在ER的胞质面，这些分子伴侣一起将错误折叠的MP递送到ERAD机器。在 然而，为了使错误折叠的蛋白质成为ERAD的候选物，它必须能够从细胞中提取。 限制ER膜并递送至胞质蛋白酶体。结构错误的蛋白质 阻止进入蛋白酶体的限制需要另一种质量控制机制， 确保降解。错误折叠的MP暴露了ER腔、膜和胞质溶胶中的表面，因此， 不同地点的ERQC因素的协调行动应对了这一挑战。MP蛋白的错误 管理是致命的，当流氓客户端采用有毒的形状，使流氓破坏膜， 主要毒害细胞的基本功能。Hsp 70和Hsp 40与折叠和降解机器一起作用， 分诊宪兵它们通过抑制聚集，重折叠客户端，选择性地 通过蛋白质生物合成系统降解和调节通量的客户。ERQC的一个主要问题 系统是在化学稳定的中间状态中积累的MP，这些中间状态是非天然的， 自缔合形成低聚物、无定形聚集体和淀粉样原纤维。这种错误折叠的构象 埋面通常由ERQC因素识别，其热力学稳定性阻碍了 展开事件所需的从膜中提取和降解的狭窄的中心腔， 蛋白酶体热力学稳定的MP中间体对ERAD具有抗性，我们发现， 它们通过JB 12依赖性ER-吞噬机制降解。实验的总体目标包括 在这个提议中，定义了Hsp 70分子伴侣系统、ERAD和ER-噬菌体之间的串扰节点 对细胞活力和蛋白质组的维持至关重要。

Project Summary: Membrane protein (MP) biogenesis begins in the endoplasmic reticulum (ER) and is a complex process, as domains on both sides of the ER membrane, and within the membrane bilayer, must fold and assemble. MPs understudy include ABC-Transporters, P-Type ATPases, G-protein coupled receptors, and BRICHOS proteins. Missense mutations that cause misfolding and premature degradation of MPs give rise to diseases such as cystic fibrosis, hypogonadotropic hypogonadism, retinitis pigmentosa and idiopathic lung fibrosis. In order to prevent toxic accumulation, misfolded MPs are targeted for ER-associated degradation (ERAD) by E3 ubiquitin ligase complexes. The ER transmembrane Hsp40 DNAJB12 (JB12) recruits cytosolic Hsp70 to the cytoplasmic face of the ER and together these chaperones deliver the misfolded MPs to ERAD machinery. In order for a misfolded protein to be a candidate for ERAD however, it must be competent for extraction from the confines of the ER membrane and delivered to the cytosolic proteasome. Misfolded proteins with structural restraints that prevent entrance into the proteasome necessitate an alternative quality control mechanism to ensure degradation. Misfolded MPs expose surfaces in the ER lumen, membrane, and the cytosol, so the coordinated action of ERQC factors in different locations manage this challenge. Mistakes in MP protein management are fatal when rogue clients adopt a toxic shape that enable rogues to damage membranes and dominantly poison essential cell functions. Hsp70 and Hsp40s act with folding and degradation machines to triage MPs. They shield against proteotoxicity through suppressing aggregation, refolding clients, selection of clients for degradation and regulating flux though protein biosynthetic systems. A major problem to ERQC systems are the MPs that accumulate in thermodynamically stable intermediate states that are non-native and self-associate to form oligomers, amorphous aggregates, and amyloid-like fibrils. Such misfolded conformers bury surfaces that are normally recognized by ERQC factors, and their thermodynamic stability hinders the unfolding events required for their extraction from membranes and degradation in the narrow central cavity of the proteasome. Thermodynamically stable MP intermediates are resistant to ERAD and we discovered that they are degraded by a JB12 dependent ER-phagy mechanism. The overall goal of the experiments included in this proposal is to define nodes of cross-talk between the Hsp70 chaperone system, ERAD and ER-phagy that are essential for cell viability and proteome maintenance.