Protein Trafficking In The Endosomal-Lysosomal System

内体-溶酶体系统中的蛋白质运输

• 批准号: 7594182
• 负责人: JUAN BONIFACINO
• 金额: $ 195.93万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594182
• 关键词: Acids; Active Sites; Adaptor Signaling Protein; Arrestin; Arrestins; Bacterial Toxins; Binding; Binding Sites; Biochemical; Biogenesis; Blood Platelets; C-terminal; Capsid Proteins; Cells; Class; Collaborations; Complex; Defect; Disease; Ear; Early Endosome; Electron Microscopy; Endosomes; Environment; Enzymes; Extracellular Space; Goals; HIV-1; Helix (Snails); Hermanski-Pudlak Syndrome; Hydrolase; IGF Type 2 Receptor; Institutes; Integral Membrane Protein; Knowledge; Length; Lipids; Liquid substance; Lumen of the Lysosome; Lysosomes; Mammalian Cell; Mediating; Melanosomes; Membrane Proteins; Molecular; Movement; Multivesicular Body; Mutation; Names; National Institute of Diabetes and Digestive and Kidney Diseases; Organelles; Paris, France; Pathogenesis; Pathway interactions; Phase; Phenotype; Physiological; Physiological Processes; Play; Protein Sortings; Proteins; RNA Interference; Reaction; Recycling; Regulation; Role; Shiga Toxin; Signal Transduction; Sorting - Cell Movement; Structure; System; Time; Transcription Factor AP-1; Transcription Factor AP-2 Alpha; Transfection; Transport Vesicles; Tubular formation; Tyrosine; Ubiquitination; Vacuole; Work; X-Ray Crystallography; Yeasts; base; cell type; ear helix; human disease; intracellular protein transport; mutant; pathogen; poly(L-glutamic acid(60)-L-alanine(30)-L-tyrosine(10)); protein transport; receptor; retinal rods; retrograde transport; scaffold; sorting nexins; trafficking; ubiquitin ligase

We investigate the molecular mechanisms by which transmembrane proteins are sorted to intracellular compartments such as endosomes, lysosomes, and a group of cell-type-specific organelles known as lysosome-related organelles, which includes melanosomes and platelet dense bodies. Sorting to these compartments is mediated by recognition of signals in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of protein coats. Among these adaptor proteins are the heterotetrameric AP1, AP2, AP3 and AP4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (i.e., the GGAs) and the heteropentameric retromer complex. Current work is aimed at elucidating the structure, regulation and physiological roles of recognition proteins, as well as investigating human diseases that result from genetic defects in these proteins (i.e., the Hermansky-Pudlak syndrome) or from their exploitation by pathogens (i.e., HIV-1). The AP complexes and GGAs have ear domains that bind accessory proteins. We previously demonstrated that these interactions are mediated by a canonical sequence motif shared by the accessory proteins. The physiological relevance of these interactions, however, had not been established. This past year, we have examined the significance of interactions of the ear domains of the AP and GGA proteins with two accessory proteins, GAK and p56. We found that depletion of both GAK and p56 by RNA interference (RNAi) resulted in missorting of mannose 6-phosphate receptors (MPRs) and their cargo lysosomal hydrolases, resulting in a phenotype similar to that of certain lysosomal storage disorders. This defect could be reversed by transfection with the wild-type GAK or p56, but not with mutant GAK or p56 with substitution of their ear-binding motifs. This demonstrated for the first time that the ability of AP complexes and GGAs to engage in canonical ear-motif interactions is critical to their function in protein sorting and lysosome biogenesis. Newly-made acid hydrolases are sorted by binding to MPRs at the TGN. The hydrolase-receptor complexes are recognized by the GGA proteins, which mediate packaging into transport vesicles bound for endosomes. The acidic environment of endosomes induces the release of the hydrolases from the MPRs, after which the hydrolases follow the fluid phase to lysosomes, while the MPRs return to the TGN to mediate further rounds of transport. In previous work, we showed that the proteins, Vps26, Vps29 and Vps35, which are subunits of a protein complex named retromer, played a role in this retrograde transport of MPRs from endosomes to the TGN. We have recently examined the requirement of two other putative subunits of retromer, the sorting nexins 1 and 2 (SNX1 and SNX2). Using RNA interference, we found that depletion of either SNX protein by RNAi had no effect on MPR trafficking, but combined depletion of both SNX proteins impaired the recycling of MPRs to the TGN and caused its missorting to lysosomes, where they were degraded. As a consequence, lysosomal enzymes were missorted into the extracellular space, a phenotype that is typical of lysosomal storage disorders. These findings demonstrated that SNX1 and SNX2 play interchangeable but essential roles, as part of the retromer complex, in the sorting of MPRs from endosomes to the TGN. To elucidate the structural bases for the role of retromer in MPR retrograde transport, we collaborated with James Hurley (NIDDK), Alasdair Steven (NIAMS), and their co-workers, to solve the structure of the retromer Vps26-Vps29-Vps35 subcomplex by X-ray crystallography and electron microscopy. We found that this complex is a 21nm-long rod having Vps26 at one end and Vps29 at the other. Vps26 is structurally similar to arrestins, whereas Vps29 resembles a type of metallophophoesterase. Vps35 consists of a long alpha-helical solenoid that spans the length of the rod and covers the putative metallophosphoesterase active site on Vps29. Vps35 also exposes multiple grooves that could be binding sites for sorting signals on cargo molecules such as the MPRs. The retromer complex is not only used to retrieve intracellular sorting receptors from endosomes to the TGN, but is also exploited by certain bacterial toxins to access their target compartments. An example is Shiga toxin, which we have shown, in collaboration with Ludger Johannes and Graa Raposo (Curie Institute, Paris), to require retromer for its movement from endosomes to the TGN. This transport begins at vacuolar early endosomes and proceeds through tubules that are part of what we refer as the tubular endosomal network. The sorting of integral membrane proteins into the lumen of lysosomes involves passage through an intermediate organelle known as the multivesicular body (MVB). For most proteins, this sorting involves recognition of ubiquitinated proteins by several complexes known as ESCRT. In collaboration with James Hurley (NIDDK) and colleagues, we have solved the crystal structure of the ESCRT Vps27-Hse1 complex. The core of this complex is constituted by two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. These domains are similar to the previously described GAT domain of the GGAs. The two Vps27-Hse1 GAT domains are connected by an antiparallel coiled-coil to form a 90 -long barbell-like structure. These studies explain how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions. Although most proteins require ubiquitination for targeting to the MVB pathway, the yeast transmembrane protein Sna3p is an exception. Despite the fact that Sna3p itself is not ubiquitinated, we have found that the ubiquitin ligase Rsp5 and the ESCRT complexes are nonetheless required for Sna3p targeting to the MVB pathway. This targeting is mediated by a direct interaction between a PPAY motif within the Sna3p C-terminal cytosolic domain and the WW domains of Rsp5p. Sna3p is thus an example of a new class of proteins that follow a ubiquitination-independent, but ubiquitin-ligase-mediated, sorting pathway to the vacuole.

我们研究跨膜蛋白被分类到细胞内区室的分子机制，例如内体、溶酶体和一组称为溶酶体相关细胞器的细胞类型特异性细胞器，其中包括黑素体和血小板致密体。对这些区室的分选是通过作为蛋白质外壳的组成部分的衔接蛋白对跨膜蛋白的胞质结构域中的信号的识别来介导的。这些接头蛋白包括异四聚体 AP1、AP2、AP3 和 AP4 复合物、单体 GGA1、GGA2 和 GGA3 蛋白（即 GGA）以及异五聚体逆转录体复合物。目前的工作旨在阐明识别蛋白的结构、调节和生理作用，以及研究由这些蛋白的遗传缺陷（即赫曼斯基-普德拉克综合征）或病原体利用它们（即 HIV-1）引起的人类疾病。 AP 复合物和 GGA 具有结合辅助蛋白的耳结构域。我们之前证明这些相互作用是由辅助蛋白共享的规范序列基序介导的。然而，这些相互作用的生理相关性尚未确定。去年，我们研究了 AP 和 GGA 蛋白的耳结构域与两种辅助蛋白 GAK 和 p56 相互作用的重要性。我们发现，RNA 干扰 (RNAi) 消耗 GAK 和 p56 会导致 6-磷酸甘露糖受体 (MPR) 及其货物溶酶体水解酶的错误分选，从而导致与某些溶酶体贮积症相似的表型。这种缺陷可以通过用野生型 GAK 或 p56 转染来逆转，但用突变型 GAK 或 p56 替换其耳结合基序则不能逆转。这首次证明 AP 复合物和 GGA 参与典型耳基序相互作用的能力对于它们在蛋白质分选和溶酶体生物合成中的功能至关重要。 新制造的酸性水解酶通过与 TGN 处的 MPR 结合进行分类。水解酶-受体复合物被 GGA 蛋白识别，GGA 蛋白介导包装成与内体结合的运输囊泡。内体的酸性环境诱导 MPR 释放水解酶，随后水解酶沿着液相进入溶酶体，而 MPR 返回 TGN 介导进一步的运输。在之前的工作中，我们表明，蛋白质 Vps26、Vps29 和 Vps35（称为逆转录体的蛋白质复合物的亚基）在 MPR 从内体到 TGN 的逆行转运中发挥了作用。我们最近研究了逆转录酶的另外两个假定亚基，即分选连接蛋白 1 和 2（SNX1 和 SNX2）的需求。使用RNA干扰，我们发现RNAi对任一SNX蛋白的消耗对MPR运输没有影响，但两种SNX蛋白的联合消耗会损害MPR向TGN的再循环，并导致其错误分选至溶酶体，并在溶酶体中被降解。结果，溶酶体酶被错误分类到细胞外空间，这是溶酶体贮积症的典型表型。这些发现表明，SNX1 和 SNX2 作为逆转录体复合体的一部分，在 MPR 从内体到 TGN 的分类中发挥着可互换但重要的作用。 为了阐明retromer在MPR逆行运输中作用的结构基础，我们与James Hurley (NIDDK)、Alasdair Steven (NIAMS)及其同事合作，通过X射线晶体学和电子显微镜解析了retromer Vps26-Vps29-Vps35亚复合物的结构。我们发现这个复合物是一根21nm长的棒，一端有Vps26，另一端有Vps29。 Vps26 在结构上类似于抑制蛋白，而 Vps29 类似于一种金属磷酸酯酶。 Vps35 由一个长的 α 螺旋螺线管组成，该螺线管横跨杆的长度并覆盖 Vps29 上假定的金属磷酸酯酶活性位点。 Vps35 还暴露出多个凹槽，这些凹槽可能是用于对 MPR 等货物分子上的信号进行分类的结合位点。 逆转录酶复合物不仅用于将细胞内分选受体从内体回收到 TGN，而且还被某些细菌毒素利用来进入其目标区室。一个例子是志贺毒素，我们与 Ludger Johannes 和 Graa Raposo（巴黎居里研究所）合作证明，它需要逆转录酶才能从内体移动到 TGN。这种运输从液泡早期内体开始，并通过小管进行，小管是我们所说的管状内体网络的一部分。 将整合膜蛋白分选到溶酶体腔中涉及通过称为多泡体（MVB）的中间细胞器。对于大多数蛋白质，这种分选涉及通过称为 ESCRT 的几种复合物识别泛素化蛋白质。我们与 James Hurley (NIDDK) 及其同事合作，解析了 ESCRT Vps27-Hse1 复合物的晶体结构。该复合物的核心由两个相互缠绕的 GAT 结构域构成，每个结构域由一个亚基的两个螺旋和另一个亚基的两个螺旋组成。这些结构域与之前描述的 GGA 的 GAT 结构域相似。两个Vps27-Hse1 GAT结构域通过反平行卷曲螺旋连接形成90°长的杠铃状结构。这些研究解释了该复合物如何与脂质和泛素化膜蛋白协同结合，并充当泛素化反应的支架。 尽管大多数蛋白质需要泛素化才能靶向 MVB 途径，但酵母跨膜蛋白 Sna3p 是一个例外。尽管 Sna3p 本身并未泛素化，但我们发现泛素连接酶 Rsp5 和 ESCRT 复合物仍然是 Sna3p 靶向 MVB 途径所必需的。这种靶向是通过 Sna3p C 端胞质结构域内的 PPAY 基序与 Rsp5p 的 WW 结构域之间的直接相互作用介导的。因此，Sna3p 是一类新型蛋白质的一个例子，它遵循不依赖于泛素化但由泛素连接酶介导的液泡分选途径。