CONNEXIN32 MUTATIONS IN CHARCOT-MARIE-TOOTH-X DISEASE

描述（来自申请人的摘要）：I型的X-连锁形式 腓骨肌萎缩症（Charcot-Marie-Tooth disease，CMTX）是第二常见的遗传性 人类周围神经病变。CMTX是由差距突变引起的 连接通道蛋白连接蛋白32（Cx 32）被认为影响其 通过许旺细胞介导物质径向扩散的能力 髓鞘CMTX在遗传和表型上都是异质的： Cx 32基因编码区有超过200种不同的缺陷， 在CMTX患者中发现，其临床症状范围从非常 轻微/无症状，最终成为轮椅。研究 转染的组织培养细胞和转基因小鼠表明， 突变通过不同的机制干扰Cx 32功能：一些突变体 翻译效率低下和/或降解非常快;其他人似乎影响 在细胞表面的通道功能;和许多只检测到 在细胞内并作为野生型连接蛋白的显性负抑制剂。 不同的Cx 32突变如何导致这些不同的表型尚不清楚。我们 三个CMTX连锁Cx 32点突变体在PCI 2细胞中初步鉴定 转染子显示，每个突变体在折叠和寡聚方面都有缺陷， 组装并经历了不同的细胞内命运：E208 K Cx 32在细胞内积累。 内质网，而E186 K和R142 W突变体都是 运输到高尔基体区域，从那里它们要么运输到溶酶体， （RI 42 W Cx 32）或返回ER（EI 86 K Cx 32）。拟议研究的目标 是阐明CMTX连锁突变影响 Cx 32的组装、胞内转运和降解。这些研究 将在转染的组织培养细胞以及外周血中进行 来自表达Cx 32突变体的小鼠的神经组织。具体来说，我们将使用 分子和细胞生物学技术：（1）确定细胞内 CMTX连接的Cx 32的转运途径和分子伴侣相互作用 突变体;（2）鉴定和测试构象缺陷的可纠正性 在不同CMTX连锁突变诱导的Cx 32中;（3）阐明 Cx 32突变体进入细胞溶质并被细胞溶质降解的机制 蛋白酶体;（4）确定显性阴性的分子基础 活性的一些CMTX连锁突变体，并确定在体内的目标，这 髓鞘形成的雪旺细胞中的活性。这些研究将阐明 CMTX的表型多样性，并提供了第一个分子见解 连接蛋白的质量控制机制 组装成间隙连接通道。

DESCRIPTION (From the Applicant's Abstract): The X-linked form of type I Charcot-Marie-Tooth disease (CMTX) is the second most common hereditary peripheral neuropathy in humans. CMTX is caused by mutations in the gap junction channel protein connexin32 (Cx32) that are thought to affect its ability to mediate the radial diffusion of substances through the Schwann cell myelin sheath. CMTX is genetically as well as phenotypically heterogeneous: over 200 different defects in the coding region of the Cx32 gene have been found in CMTX patients, whose clinical symptoms range from very mild/asymptomatic to eventually becoming wheelchair-bound. Studies in transfected tissue culture cells and transgenic mice indicate that different mutations interfere with Cx32 function by distinct mechanisms: some mutants are translated inefficiently and/or degraded very quickly; others appear to affect channel function at the cell surface; and many are detected only intracellularly and act as dominant negative inhibitors of wild-type connexins. How different Cx32 mutations result in these diverse phenotypes is unknown. Our initial characterization of three CMTX-linked Cx32 point mutants in PCI2 cell transfectants revealed that each mutant was defective in folding and oligomeric assembly and underwent a distinct intracellular fate: E208K Cx32 accumulated in the endoplasmic reticulum whereas both the E186K and R142W mutants were transported to the Golgi region from which they trafficked either to lysosomes (RI42W Cx32) or back to the ER (EI86K Cx32). The goal of the proposed studies is to elucidate the molecular mechanisms by which CMTX-linked mutations affect the assembly, intracellular transport, and degradation of Cx32. These studies will be conducted in transfected tissue culture cells as well as in peripheral nerve tissue from mice expressing Cx32 mutants. Specifically, we will use molecular and cellular biological techniques to: (1) define the intracellular trafficking routes and molecular chaperone interactions of CMTX-linked Cx32 mutants; (2) identify, and test the correctability of, conformational defects in Cx32 induced by different CMTX-linked mutations; (3) elucidate the mechanisms whereby Cx32 mutants gain access to, and are degraded by, cytosolic proteasomes; and (4) determine the molecular basis for the dominant-negative activity of some CMTX-linked mutants and identify the in vivo target of this activity in myelinating Schwann cells. These studies will elucidate the causes of phenotypic diversity in CMTX as well as provide the first molecular insights into the quality control mechanisms that govern the fidelity of connexin assembly into gap junctional channels.