Molecular pathogenesis of SIMPLE in Charcot-Marie-Tooth disease

描述（由申请人提供）： 腓骨肌萎缩症（CMT）是最常见的周围神经病变，患病率为1/2500，但这种疾病的分子机制仍然未知。从CMT亚型（CMT 1A和CMT 1B）的人类患者和啮齿动物疾病模型获得的许旺细胞的成像研究揭示了外周髓鞘蛋白，即外周髓鞘蛋白22（PMP 22）和髓鞘蛋白零（MPZ）向细胞质包涵体的积累。这些结果表明，异常贩运的PMP 22和MPZ参与CMT的发病机制。然而，很少有人知道的步骤和机制参与细胞内贩运的外周髓鞘蛋白。最近，SIMPLE突变，一种功能未知的泛在表达蛋白，被鉴定为导致脱髓鞘形式CMT（CMT 1C）的遗传缺陷。SIMPLE含有结合Tsg 101的PSAP基序，Tsg 101是一种参与将泛素化货物蛋白分选到溶酶体途径进行降解的蛋白质。初步研究表明，SIMPLE与Tsg 101共定位于早期内体，暗示它们在体内的生理相互作用。总之，这些证据表明SIMPLE在调节内体至溶酶体运输中的作用。此外，序列分析的SIMPLE揭示了一个环指基序，预测决定E3泛素蛋白连接酶活性。这一发现提出了SIMPLE作为E3泛素蛋白连接酶来泛素化Tsg 101以调节其功能的可能性。尽管有这些可能性，但SIMPLE的泛素化活性尚未得到测试，SIMPLE在内吞转运调节中的作用仍然未知。该提议将检验SIMPLE作为内体E3泛素蛋白连接酶调节内体至溶酶体运输的假设，以及SIMPLE的CMT 1C突变破坏该功能的假设。具体目标是：1）表征SIMPLE的亚细胞定位和CMT 1C突变的影响; 2）检查SIMPLE的E3泛素-蛋白连接酶活性和CMT 1C突变的影响; 3）阐明SIMPLE在调节内体至溶酶体运输中的功能作用。 公共卫生相关性：总的来说，这些研究将评估与显著降低生活质量的毁灭性疾病有关的分子途径。目前的治疗方法，如物理治疗和止痛药，不足以对抗日益恶化的运动和感觉症状，也不能逆转或阻止脱髓鞘和轴突变性。因此，本项目将研究CMT的分子基础，希望为这些患者提供更好的治疗方法。

DESCRIPTION (provided by applicant): Charcot-Marie-Tooth disease (CMT) is the most common peripheral neuropathy with a prevalence of 1 in 2,500, but the molecular mechanisms underlying this disease remain unknown. Imaging studies of Schwann cells acquired from human patients and rodent disease models of CMT subtypes (CMT1A and CMT1B) revealed accumulations of peripheral myelin proteins, namely, peripheral myelin protein 22 (PMP22) and myelin protein zero (MPZ), to cytoplasmic inclusion bodies. These findings suggest that aberrant trafficking of PMP22 and MPZ are involved in CMT pathogenesis. However, little is known about the steps and the machinery involved in the intracellular trafficking of peripheral myelin proteins. Recently, mutations in SIMPLE, an ubiquitiously expressed protein of unknown function, are identified as the genetic defects responsible for a demyelinating form of CMT (CMT1C). SIMPLE contains a PSAP motif that binds Tsg101, a protein involved in the sorting of ubiquitinated cargo proteins to the lysosomal pathway for degradation. Preliminary studies demonstrated that SIMPLE colocalizes with Tsg101 at the early endosomes, implicating their physiological interaction in vivo. Together, this evidence suggest a role of SIMPLE in the regulation of endosome-to-lysosome trafficking. Furthermore, sequence analysis of SIMPLE reveals a RING finger motif, a predictive determinant of E3 ubiquitin-protein ligase activity. This finding raises the possibility that SIMPLE act as an E3 ubiquitin-protein ligase to ubiquitinate Tsg101 in order to regulate its function. Despite these possibilities, the ubiquitinating activity of SIMPLE has not been tested, and the role of SIMPLE in the regulation of endocytic trafficking remains unknown. This proposal will test the hypothesis that SIMPLE acts as an endosomal E3 ubiquitin-protein ligase to regulate endosome-to- lysosome trafficking, and that CMT1C mutations of SIMPLE disrupt this function. The specific aims are to: 1) characterize the subcellular localization of SIMPLE and the impact of CMT1C mutations; 2) examine the E3 ubiquitin-protein ligase activity of SIMPLE and the effects of CMT1C mutations; 3) elucidate the functional role of SIMPLE in the regulation of endosome-to-lysosome trafficking. PUBLIC HEALTH RELEVANCE: Overall, these studies will assess the molecular pathways involved in a devastating disease that significantly decrease the quality of life. Current treatments such as physical therapy and pain medications are insufficient to battle worsening motor and sensory symptoms and do little to reverse or halt demyelination and axonal degeneration. Therefore, this project will examine the molecular basis of CMT in hopes of providing better treatments for these patients.