CONNEXIN32 MUTATIONS IN CHARCOT-MARIE-TOOTH-X DISEASE

描述(摘自申请人摘要)：第I类的X链接形式 Charcot-Marie-Tooth病(CMTX)是第二常见的遗传性疾病 人类的外周神经病。CMTX是由缝隙中的突变引起的 连接通道蛋白连接蛋白32(Cx32)被认为影响其 调节物质通过雪旺细胞的径向扩散的能力 髓鞘。CMTX在遗传和表型上都是异质性的： Cx32基因编码区有200多种不同的缺陷 在CMTX患者中发现，他们的临床症状从非常 轻微/无症状到最终不得不坐轮椅。研究项目： 转基因组织培养细胞和转基因小鼠表明不同 突变通过不同的机制干扰Cx32的功能：一些突变是 翻译效率低下和/或降级非常快；其他似乎影响 在细胞表面的通道功能；许多仅被检测到 并作为野生型连接蛋白的显性负抑制因子。 不同的Cx32突变如何导致这些不同的表型尚不清楚。我们的 三个CMTX连锁Cx32点突变在PCI2细胞中的初步鉴定 转染结果显示，每个突变体都存在折叠缺陷和寡聚体。 组装并经历了明显的细胞内命运：E208K Cx32在 而E186K和R142W突变体均为内质网 被运送到高尔基地区，从那里他们要么贩卖到溶酶体 (RI42W Cx32)或返回ER(EI86K Cx32)。拟议研究的目标是 是阐明CMTX连锁突变影响的分子机制 Cx32的组装、细胞内转运和降解。这些研究 将在转基因的组织培养细胞和外周血细胞中进行 表达Cx32突变体的小鼠神经组织。具体地说，我们将使用 分子和细胞生物学技术：(1)定义细胞内 CMTX连锁Cx32的转运途径和分子伴侣相互作用 突变体；(2)识别并测试构象缺陷的可修复性 Cx32由不同的CMTX连锁突变诱导；(3)阐明 Cx32突变体进入胞浆并被胞浆降解的机制 蛋白酶体；以及(4)确定显性负性的分子基础 某些CMTX连锁突变体的活性及其体内靶点的确定 髓鞘雪旺细胞的活性。这些研究将阐明原因。 CMTX的表型多样性，以及提供了第一个分子洞察力 进入管理连接蛋白保真度的质量控制机制 组装成缝隙连接通道。

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.