Role and Mechanism of Bypass Pathway Trafficking

描述(由申请人提供)：本提案的重点是在三种与人类疾病相关的情况下，对一种知之甚少的膜运输途径--内小体到高尔基体“旁路”途径--的基本细胞生物学机制。第一个是细菌毒素对细胞的入侵，细菌毒素绕过了导致人类疾病的旁路。第二种是锰毒性，它导致旁路途径的一个关键组成部分下调，并导致人类神经退行性疾病。第三个是由于肝细胞癌旁路通路转运的改变，产生了一个有希望的肝癌早期血清标记物。 起源于细胞表面或高尔基体的蛋白质和脂肪货物通过移动到早期/分选内小体而进入该途径。然后，货物“绕过”内吞途径中的降解分支，进入晚期的内小体，直接输送到高尔基体。我们是为数不多的几个阐明这条途径上人口贩运机制的实验室之一。我们的方法是识别在旁路途径中运输的蛋白质，绘制它们的靶向信号和相互作用图，并结合siRNA介导的敲除和基因替换使用生化和形态分析来测试它们的功能作用。我们发表的文章(Bachert等人，2001；Lstedt等人，1997；Natarajan和Lstedt，2004；Puri等人，2002；Puthenveedu et al.，2003)和未发表的工作表明，a)高尔基蛋白GPP130和GP73在旁路途径中循环，其对pH敏感的高尔基体靶向依赖于这种循环，b)GPP130和GP73旁路通路循环涉及存在于其管腔螺旋线圈结构域中的高尔基体和内体决定因素的组合使用，c)GPP130显示对pH敏感的结合到一个大的管腔复合体，d)类似的管腔决定因素介导的GPP130的分选发生在极化细胞中，并且受到碱基的限制，e)GPP130选择性地被用于旁路途径运输志贺毒素和其他旁路标记，这一功能取决于GP130的管腔分选决定因素和细胞质结构域，F)COG对接复合体也是必需的，并作用于GPP130的下游，g)响应低浓度的锰，GPP130的运输被迅速和选择性地改变，导致GPP130下调，以及h)在肝细胞癌的旁路途径中的GP73的运输被改变，使得它被切割，其胞外结构域从产生疾病的有希望的生物标志物的细胞中释放。 因此，我们的主要目的是检验这样一种假设，即GPP130和类似结构的旁路组件使用管腔、pH敏感的相互作用，将其整合到一个大型高尔基体修复复合体中，该复合体可以从分类和回收内小体的过程中萌芽。通过与这种复合体相互作用，志贺样毒素进入旁路途径。在其形成过程中，检索复合体还与有效的旁路运输所必需的运输组件相互作用--包括COG对接复合体--而GPP130胞质结构域在这种招募中起着关键作用。此外，我们还将确定Mn+2暴露和细胞转化分别改变旁路转运导致GPP130下调和GP73胞外结构域脱落的机制。 公共卫生相关性：膜转运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正在为未来有效的治疗铺平道路。一个重要但研究不足的例子是从细胞表面到内部隔室的旁路膜运输途径。我们对旁路运输机制的研究揭示了基于检索的高尔基蛋白靶向、志贺毒素感染人类、锰神经毒性(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记物(全球第五大癌症死亡原因)的新见解，并具有潜在的临床意义。因此，这项工作为将药物输送到胞液间隔、治疗涉及细菌毒素的传染病、治疗锰致运动障碍综合征以及无创早期发现肝癌提供了新的模式。

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.