Role and Mechanism of Bypass Pathway Trafficking

描述（由申请人提供）：该提案侧重于在三种人类疾病相关背景下，对膜运输途径-内体至高尔基体“旁路”途径-知之甚少的基本细胞生物学机制。第一种是细菌毒素侵入细胞，它们占据了导致人类疾病的旁路途径。第二个是锰毒性，它诱导旁路途径和人类神经退行性疾病的一个关键组成部分下调。第三个是由于肝细胞癌中旁路途径运输的改变而产生的有希望的肝癌早发血清标志物。 起源于细胞表面或高尔基体的蛋白质和脂质货物通过移动到早期/分选内体而进入该途径。货物然后“绕过”涉及晚期内体的内吞途径的降解分支，而是直接运输到高尔基体。我们是少数几个阐明这一途径中管理贩运机制的实验室之一。我们的方法是识别旁路途径中的蛋白质运输，绘制它们的靶向信号和相互作用，并使用生物化学和形态学测定结合siRNA介导的敲除和基因置换来测试它们的功能作用。我们发表的（Bachert等人，2001; Linstedt等人，1997; Natarajan和Linstedt，2004; Puri等人，2002; Puthenveedu等人，2003）和未发表的工作表明a）高尔基体蛋白GPP 130和GP 73在旁路途径中循环，并且它们的pH敏感性高尔基体靶向依赖于这种循环，B）GPP 130和GP 73旁路途径循环涉及高尔基体和存在于它们的内腔卷曲螺旋结构域中的内体决定簇的组合使用，c）GPP 130显示与大内腔复合物的pH敏感性结合，e）GPP 130是滋贺毒素和其它旁路标志物的旁路途径运输所选择性需要的，并且这种功能依赖于GPP 130内腔分选决定簇和胞质结构域，g）响应于低浓度的锰，GPP 130的运输被快速和选择性地改变，导致GPP 130下调，和h）旁路途径中的GP 73运输在HCC中被改变，使得其被切割并且其胞外域从细胞中释放，产生该疾病的有希望的生物标志物。 因此，我们的主要目的是测试的假设，GPP 130和类似结构的旁路途径组件使用内腔，pH敏感的相互作用，以纳入一个大的高尔基体检索复合物，芽从排序和回收内体。通过与这种复合物相互作用，志贺样毒素进入旁路途径。在其形成过程中，回收复合物还与有效旁路途径运输所必需的转运组分相互作用-包括COG对接复合物-并且GPP 130胞质结构域在这种募集中起关键作用。此外，我们将确定Mn+2暴露和细胞转化分别改变旁路运输引起GPP 130下调和GP 73胞外域脱落的机制。 公共卫生相关性：膜运输缺陷是许多人类疾病的原因，我们对这些缺陷的分子基础的理解为未来有效的治疗铺平了道路。一个重要但研究不足的例子是从细胞表面到其内部隔室的旁路膜运输途径。我们对旁路途径运输机制的研究揭示了新的和潜在的临床相关见解，包括基于检索的高尔基蛋白靶向，志贺样毒素对人类的感染，锰神经毒性（由于不发达国家受污染的井水造成的主要问题），以及人类肝癌早期血清生物标志物的产生（全球癌症死亡的第五大原因）。因此，这项工作提供了新的模式，药物输送到胞质隔室，治疗感染性疾病的细菌毒素参与，治疗锰诱导的运动功能障碍综合征，和非侵入性早期检测肝癌的可能性。

DESCRIPTION (provided by applicant): This proposal is focused on basic cell biological mechanisms of a poorly understood membrane trafficking pathway- the endosome-to-Golgi "bypass" pathway- in three human disease-related contexts. The first is cell invasion by bacterial toxins, which co-opt the bypass pathway causing human disease. The second is manganese toxicity, which induces downregulation of a key component of the bypass pathway and neurodegenerative disorders in humans. The third is the generation of a promising early onset serum marker for liver cancer due to alterations in bypass pathway trafficking in hepatocellular carcinoma. Protein and lipid cargo originating at the cell surface or Golgi apparatus gain access to the pathway by moving to early/sorting endosomes. The cargo then "bypasses" the degradative branch of the endocytic pathway involving late endosomes and instead traffics directly to the Golgi apparatus. We are one of the few labs elucidating the mechanisms that govern trafficking in this pathway. Our approach is to identify proteins trafficking in the bypass pathway, map their targeting signals and interactions, and test their functional role using biochemical and morphological assays in conjunction with siRNA-mediated knockdown and gene replacement. Our published (Bachert et al., 2001; Linstedt et al., 1997; Natarajan and Linstedt, 2004; Puri et al., 2002; Puthenveedu et al., 2003) and unpublished work indicates that a) Golgi proteins GPP130 and GP73 cycle in the bypass pathway and their pH-sensitive Golgi targeting depends on this cycling, b) GPP130 and GP73 bypass pathway cycling involves the combined use of Golgi and endosomal determinants present in their lumenal coiled-coil domains, c) GPP130 shows pH-sensitive binding to a large lumenal complex, d) analogous lumenal determinant-mediated sorting of GPP130 occurs in polarized cells and is basolaterally restricted, e) GPP130 is selectively required for bypass pathway trafficking of Shiga toxin and other bypass markers and this functioning depends on both the GPP130 lumenal sorting determinants and the cytoplasmic domain, f) the COG docking complex is also required and acts downstream of GPP130, g) in response to low concentrations of manganese GPP130 trafficking is rapidly and selectively altered leading to GPP130 downregulation, and h) GP73 trafficking in the bypass pathway is altered in HCC such that it is cleaved and its ectodomain is released from cells producing a promising biomarker of the disease. Thus, our major aim is to test the hypothesis that GPP130 and similarly structured bypass pathway components use lumenal, pH-sensitive interactions to be incorporated into a large Golgi retrieval complex that buds from sorting and recycling endosomes. By interacting with this complex, Shiga-like toxins access the bypass pathway. During its formation, the retrieval complex also interacts with transport components essential for efficient bypass pathway trafficking- including the COG docking complex- and the GPP130 cytoplasmic domain plays a critical role in this recruitment. In addition, we will determine the mechanism by which Mn+2 exposure and cellular transformation alter bypass trafficking causing GPP130 downregulation and GP73 ectodomain shedding, respectively. PUBLIC HEALTH RELEVANCE: Defects in membrane trafficking are responsible for many human diseases and our understanding of the molecular basis of these defects is paving the way to future effective therapeutics. An important, but understudied, example is the bypass membrane trafficking pathway from the cell surface to its interior compartments. Our investigation into the mechanism of bypass pathway trafficking is revealing novel and potentially clinically relevant insights into retrieval-based targeting of Golgi proteins, infection of humans by Shiga-like toxins, manganese neurotoxicity (a major problem due to contaminated well water in underdeveloped nations), and production of an early serum biomarker of human liver cancer (the fifth-leading cause of cancer death worldwide). Thus, the work offers possibilities for new modes for drug delivery to the cytosolic compartment, treatment of infectious diseases where bacterial toxins are involved, therapies against manganese-induced motor dysfunction syndrome, and non-invasive early detection of liver cancer.