Protein Trafficking In The Endosomal-Lysosomal System

内体-溶酶体系统中的蛋白质运输

• 批准号: 8736848
• 负责人: JUAN BONIFACINO
• 金额: $ 279.78万
• 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/8736848
• 关键词: Adaptor Signaling Protein; Angiopoietin-2; Axon; Binding; Binding Sites; Biochemical; Blood Platelets; Capsid Proteins; Cell Adhesion Molecules; Cell membrane; Cells; Clathrin; Clathrin Adaptors; Coated vesicle; Collaborations; Complex; Couples; Coxsackie Viruses; Dendrites; Dendritic Spines; Disease; Dominant-Negative Mutation; Endosomes; Epithelial Cells; Event; Excitatory Synapse; Exclusion; Failure; GTP Binding; Glutamate Receptor; Goals; Hemorrhage; Hermanski-Pudlak Syndrome; Hippocampus (Brain); Integral Membrane Protein; Knowledge; Laboratories; Light; Lysosomes; Mediating; Melanosomes; Membrane; Mental Retardation; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Movement Disorders; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Nerve Degeneration; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Oculocutaneous albinism type 2; Organelles; Pathogenesis; Pathway interactions; Pennsylvania; Physiological; Physiological Processes; Pigmentation physiologic function; Population; Proteins; RNA Interference; Recruitment Activity; Regulation; Roentgen Rays; Role; SNAP receptor; Signal Transduction; Site; Sorting - Cell Movement; Spain; Structure; Syndrome; System; TFAP2A gene; Tail; Transcription Factor AP-1; Transferrin Receptor; Transport Vesicles; Tyrosine; Ubiquitin; United States National Institutes of Health; Universities; Vesicle; Virus Receptors; Work; adenovirus receptor; base; cell type; human disease; insight; medical schools; neuronal cell body; polarized cell; protein transport; receptor; receptor binding; syntaxin 6; trafficking; trans-Golgi Network

We investigate the molecular mechanisms by which transmembrane proteins are sorted to different compartments of the endomembrane system such as endosomes, lysosomes, lysosome-related organelles (e.g., melanosomes and platelet dense bodies) and specific domains of the plasma membrane in polarized cells (e.g., epithelial cells and neurons). Sorting is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats (e.g., clathrin coats). Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA proteins, and the heteropentameric retromer complex. Proper sorting requires the function of additional components of the trafficking machinery that mediate vesicle tethering and fusion, such as the multisubunit tethering complex GARP. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, as well as investigating human diseases that result from genetic defects of these proteins (e.g., Hermansky-Pudlak syndrome; neurodegenerative and neurodevelopmental disorders). AP-1, AP-2, and AP-3 are clathrin-associated adaptor complexes that recognize two types of sorting signal referred to as tyrosine-based and dileucine-based signals. Previous studies from our laboratory showed that tyrosine-based signals bind to the mu1, mu2 and mu3 subunits, whereas dileucine-based signals bind to a combination (i.e., a hemicomplex) of two subunits, gamma-sigma1, alpha-sigma2 and delta-sigma3, from the corresponding AP complexes. A major goal of our work this past year was the analysis of the role of signal-adaptor interactions in polarized sorting in neurons. Neurons are polarized into dendrites, soma and axons. The plasma membrane of each of these domains possesses a distinct set of transmembrane proteins, including receptors, channels, transporters and adhesion molecules. We hypothesized that sorting to these domains was mediated by interaction of sorting signals with AP complexes. Our studies showed that the cytosolic tails of various transmembrane receptors, including the transferrin receptor (TfR), the Coxsackie virus and adenovirus receptor (CAR), and the glutamate receptor proteins mGluR1, NR2A and NR2B, all have information leading to the sorting of these proteins to the somatodendritic domain of hippocampal neurons. In the case of TfR and CAR, this information occurs in the form of tyrosine-based sorting signals. Protein interaction analyses showed that the tails of these receptors bind to the mu1A subunit of AP-1. Dominant-negative interference and RNAi approaches demonstrated that interaction of cytosolic tails with AP-1 was responsible for somatodendritic sorting. Sorting involved exclusion of the receptor proteins from transport carriers destined for the axonal domain at the level of the soma. Interference with AP-1-dependent somatodendritic sorting caused defective maturation of dendritic spines and decreased the number of excitatory synapses. Recently, mutations in the sigma1A and sigma1B subunits of AP-1 were shown to be the cause of two syndromic mental retardation disorders known as MEDNIK syndrome and Fried syndrome, respectively. Unlike mu1A, sigma1A and sigma1B recognize dileucine-based sorting signals. Our findings suggest that the MEDNIK and Fried syndromes may arise from failure to sort dileucine-containing cargos to the somatodendritic domain of specific neuronal populations. Together with previous findings in epithelial cells, our recent results establish AP-1 as a global regulator of polarized sorting in various cell types. Structural studies provided valuable insights into the mechanisms by which AP complexes recognize sorting signals. In collaboration with James Hurley (NIDDK, NIH), we elucidated the mechanism by which the small GTPase Arf1 recruits the AP-1 complex to membranes and induces a conformational change that allows AP-1 to bind both tyrosine-based and dileucine-based sorting signals. X-ray crystallographic analysis of Arf1 in complex with the AP-1 core showed that two molecules of Arf1-GTP bind to distinct sites on the gamma and beta1 subunits of AP-1. The AP-1 core in this complex is in the open conformation that exposes binding sites for tyrosine-based and dileucine based sorting signals. The structure reveals how Arf1-induced conformational activation couples membrane recruitment to sorting-signal recognition. In addition to AP-1, we also studied the mechanisms of signal recognition by the related AP-3 complex. In collaboration with the group of Michael Marks (University of Pennsylvania School of Medicine), we showed that recognition of a dileucine-based sorting signal in the cytosolic tail of the oculocutaneous albinism type 2 (OCA2) protein by both AP-1 and AP-3 mediates OCA2 sorting to melanosomes. In a separate study, we solved the crystal structure of the mu3A subunit of AP-3 in complex with a tyrosine-based sorting signal. This structure revealed that the binding site on mu3A is similar to that on the mu2 subunit of AP-2. These studies contributed to the elucidation of the mechanisms by which the AP-3 complex sorts transmembrane proteins to melanosomes, thus shedding light on the pathogenesis of the pigmentation and bleeding disorder Hermansky-Pudlak syndrome type 2. Finally, in collaboration with Aitor Hierro (CIC-bioGUNE, Bilbao, Spain), we uncovered the structural mechanism by which the tethering factor GARP interacts with its cognate vesicle fusion SNARE proteins. X-ray crystallographic analyses showed how the Ang2 subunit of GARP binds to the Habc domain of the Syntaxin 6 SNARE. These findings highlight a key event in the pathway by which transport vesicles emanating from endosomes fuse with the trans-Golgi network. The integrity of this pathway is essential for neuronal function and viability, and its perturbation leads to neurodegenerative movement disorders.

我们研究跨膜蛋白被分类到内膜系统不同区室的分子机制，例如内体、溶酶体、溶酶体相关细胞器（例如黑素体和血小板致密体）和极化细胞质膜的特定域（例如上皮细胞和神经元）。分选是通过作为膜涂层（例如网格蛋白涂层）成分的衔接蛋白识别跨膜蛋白胞质结构域中存在的信号来介导的。这些接头蛋白包括异四聚体 AP-1、AP-2、AP-3 和 AP-4 复合物、单体 GGA 蛋白和异五聚体逆转录体复合物。正确的分选需要介导囊泡束缚和融合的运输机制的附加组件的功能，例如多亚基束缚复合体 GARP。我们实验室目前的工作旨在阐明外壳蛋白和囊泡束缚因子的结构、调节和生理作用，以及研究由这些蛋白质的遗传缺陷引起的人类疾病（例如赫曼斯基-普德拉克综合征；神经退行性和神经发育障碍）。 AP-1、AP-2 和 AP-3 是网格蛋白相关接头复合物，可识别两种类型的分选信号（称为基于酪氨酸和基于双亮氨酸的信号）。我们实验室之前的研究表明，基于酪氨酸的信号与 mu1、mu2 和 mu3 亚基结合，而基于双亮氨酸的信号与来自相应 AP 复合物的两个亚基（gamma-sigma1、alpha-sigma2 和 delta-sigma3）的组合（即半复合物）结合。去年我们工作的一个主要目标是分析信号适配器相互作用在神经元极化排序中的作用。神经元被极化为树突、胞体和轴突。每个结构域的质膜都拥有一组独特的跨膜蛋白，包括受体、通道、转运蛋白和粘附分子。我们假设对这些域的分选是通过分选信号与 AP 复合物的相互作用介导的。我们的研究表明，各种跨膜受体的胞质尾部，包括转铁蛋白受体（TfR）、柯萨奇病毒和腺病毒受体（CAR）以及谷氨酸受体蛋白mGluR1、NR2A和NR2B，都具有导致这些蛋白分类到海马神经元体树突结构域的信息。对于 TfR 和 CAR，该信息以基于酪氨酸的分选信号的形式出现。蛋白质相互作用分析表明这些受体的尾部与 AP-1 的 mu1A 亚基结合。显性失活干扰和 RNAi 方法表明，胞质尾部与 AP-1 的相互作用负责体树突分选。分选涉及将受体蛋白从运往体细胞水平轴突结构域的运输载体中排除。干扰 AP-1 依赖性体细胞树突分选会导致树突棘成熟缺陷并减少兴奋性突触的数量。最近，AP-1 sigma1A 和 sigma1B 亚基的突变被证明是两种综合征性精神发育迟滞疾病的原因，分别称为 MEDNIK 综合征和 Fried 综合征。与 mu1A 不同，sigma1A 和 sigma1B 识别基于双亮氨酸的分选信号。我们的研究结果表明，MEDNIK 和 Fried 综合征可能是由于未能将含有双亮氨酸的货物分类到特定神经元群体的体细胞树突域而引起的。结合之前在上皮细胞中的发现，我们最近的结果表明 AP-1 是各种细胞类型中极化分选的全局调节剂。 结构研究为 AP 复合物识别分选信号的机制提供了宝贵的见解。我们与 James Hurley（NIDDK、NIH）合作，阐明了小 GTPase Arf1 将 AP-1 复合物招募到膜上并诱导构象变化的机制，使 AP-1 能够结合基于酪氨酸和基于双亮氨酸的分选信号。 Arf1 与 AP-1 核心复合物的 X 射线晶体分析表明，两个 Arf1-GTP 分子与 AP-1 的 γ 和 β1 亚基上的不同位点结合。该复合物中的 AP-1 核心处于开放构象，暴露了基于酪氨酸和基于双亮氨酸的分选信号的结合位点。该结构揭示了 Arf1 诱导的构象激活如何将膜募集与分选信号识别结合起来。 除了AP-1之外，我们还研究了相关AP-3复合物的信号识别机制。与 Michael Marks 团队（宾夕法尼亚大学医学院）合作，我们发现 AP-1 和 AP-3 对眼皮肤白化病 2 型 (OCA2) 蛋白胞质尾部基于双亮氨酸的分选信号的识别可介导 OCA2 分选至黑素体。在另一项研究中，我们解析了 AP-3 mu3A 亚基与酪氨酸分选信号复合物的晶体结构。该结构表明 mu3A 上的结合位点与 AP-2 的 mu2 亚基上的结合位点相似。这些研究有助于阐明 AP-3 复合物将跨膜蛋白分类到黑素体的机制，从而揭示色素沉着和出血性疾病 Hermansky-Pudlak 综合征 2 型的发病机制。 最后，我们与 Aitor Hierro（CIC-bioGUNE，毕尔巴鄂，西班牙）合作，揭示了束缚因子 GARP 与其同源囊泡融合 SNARE 蛋白相互作用的结构机制。 X 射线晶体分析显示了 GARP 的 Ang2 亚基如何与 Syntaxin 6 SNARE 的 Habc 结构域结合。这些发现强调了内体发出的运输囊泡与跨高尔基体网络融合途径中的一个关键事件。该通路的完整性对于神经元功能和活力至关重要，其扰动会导致神经退行性运动障碍。