Molecular pathogenesis of SIMPLE in Charcot-Marie-Tooth disease

描述(由申请人提供)： Charcot-Marie-Tooth病(CMT)是最常见的周围神经病变，发病率为1/2500，但其分子机制尚不清楚。从人类患者和CMT亚型啮齿动物模型(CMT1A和CMT1B)获得的雪旺细胞的成像研究显示，周围髓鞘蛋白，即外周髓鞘蛋白22(PMP22)和髓鞘蛋白零(MPZ)，积聚到细胞质包涵体中。这些发现提示PMP22和MPZ的异常转运参与了CMT的发病过程。然而，人们对外周髓鞘蛋白在细胞内运输的步骤和机制知之甚少。最近，一种功能未知的普遍表达的蛋白质SIMPLE的突变被确定为导致脱髓鞘形式的CMT(CMT1C)的遗传缺陷。Simple包含一个PSAP基序，它与Tsg101结合，Tsg101是一种参与泛素化货物蛋白分类的蛋白质，它与溶酶体途径有关，用于降解。初步研究表明，SIMPLE与Tsg101在早期内吞体内共定位，暗示了它们在体内的生理相互作用。综上所述，这些证据表明，SIMPLE在调节内小体到溶酶体的运输中发挥了作用。此外，SIMPLE的序列分析显示了一个环指基序，这是E3泛素蛋白连接酶活性的预测决定因素。这一发现提出了SIMPLE作为E3泛素蛋白连接酶泛化Tsg101以调节其功能的可能性。尽管有这些可能性，SIMPLE的泛素化活性还没有得到测试，SIMPLE在调节内吞运输中的作用仍然未知。这项提议将检验这样的假设，即SIMPLE作为内体E3泛素蛋白连接酶来调节内体到溶酶体的运输，并且SIMPLE的CMT1C突变破坏了这一功能。其具体目的是：1)确定SIMPLE的亚细胞定位和CMT1C突变的影响；2)检测SIMPLE的E3泛素蛋白连接酶活性和CMT1C突变的影响；3)阐明SIMPLE在调节内体到溶酶体运输中的功能作用。 公共卫生相关性：总体而言，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，对逆转或阻止脱髓鞘和轴突变性几乎没有作用。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。

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.