Protein Trafficking In The Endosomal-Lysosomal System

内体-溶酶体系统中的蛋白质运输

• 批准号: 9150089
• 负责人: JUAN BONIFACINO
• 金额: $ 227.86万
• 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/9150089
• 关键词: Adaptor Protein Complex 1; Adaptor Protein Complex 3; Adaptor Protein Complex Subunits; Adaptor Signaling Protein; Angiopoietin-2; Area; Autophagosome; Binding; Biochemical; Biogenesis; Capsid Proteins; Cell Adhesion Molecules; Cell membrane; Cell surface; Cell-Cell Adhesion; Cells; Clathrin; Complex; Congenital Abnormality; Congenital Disorders; Copper; Coxsackie Viruses; Cytoplasm; Defect; Disease; Drosophila genus; Endocytosis; Endoplasmic Reticulum; Endosomes; Epithelial Cells; Eukaryotic Cell; Event; Exhibits; Eye Color; Glycoproteins; Goals; Golgi Apparatus; HIV-1; Hemorrhage; Hermanski-Pudlak Syndrome; Hippocampus (Brain); Human; Impairment; Integral Membrane Protein; Intracellular Transport; Kinesin; Knowledge; Laboratories; Light; Lysosomes; Malignant Neoplasms; Mediating; Melanosomes; Membrane; Microtubules; Molecular; Monomeric GTP-Binding Proteins; Motor; Movement; Mutation; Names; Neoplasm Metastasis; Neurocutaneous Syndromes; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Nipah Virus; Organelles; Pathogenesis; Pathway interactions; Peripheral; Physiological Processes; Pigmentation physiologic function; Population; Positioning Attribute; Process; Protein Isoforms; Proteins; Pustular psoriasis; Recruitment Activity; Recycling; Regulation; Research; Retrieval; Role; SNAP receptor; SNAPIN gene; Series; Set protein; Signal Transduction; Signaling Molecule; Site; Sorting - Cell Movement; Surface; Syndrome; System; Tail; Therapeutic; Transferrin Receptor; Tyrosine; Vesicle; Virus Receptors; Wilson disease protein; Work; X-linked mental retardation syndrome 5; adenovirus receptor; base; cell motility; cell type; cerebral atrophy; fundamental research; insight; late endosome; migration; mutant; novel; pathogen; polarized cell; protein complex; protein transport; receptor; research study; retrograde transport; trafficking; trans-Golgi Network; tumor growth

We investigate the molecular mechanisms by which transmembrane proteins (referred to as cargo) are sorted to different compartments of the endomembrane system in eukaryotic cells. This system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes), and different domains of the plasma membrane in polarized cells (e.g., epithelial cells and neurons). Transport of cargo between these compartments is mediated by carrier vesicles or tubules that bud from a donor compartment, translocate through the cytoplasm, and eventually fuse with an acceptor compartment. Work in our laboratory focuses on the molecular machineries that mediate these processes, including (1) sorting signals and adaptor proteins that select cargo proteins for packaging into the transport carriers, (2) microtubule motors that drive movement of the transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of the transport carriers to acceptor compartments. These machineries are studied in the context of different intracellular transport pathways, including endocytosis, recycling to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, and polarized sorting in epithelial cells and neurons. Knowledge gained from this research is applied to the elucidation of disease mechanisms, including congenital disorders of protein traffic such as the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) and the neurocutaneous disorder MEDNIK syndrome. In addition, we study how the molecular mechanisms of protein transport are exploited by intracellular pathogens such as HIV-1. An AP-1/clathrin pathway for the sorting of transmembrane receptors to the somatodendritic domain of hippocampal neurons - A major focus of research in our laboratory is on processes mediated by recognition of sorting signals in the cytosolic tails of transmembrane proteins by adaptor proteins that are components of protein coats (e.g., clathrin coats). Two types of sorting signal referred to as tyrosine-based and dileucine-based participate in various sorting events, including endocytosis, transport to lysosomes and melanosomes, and sorting to the basolateral surface of polarized epithelial cells. In previous work, we found that tyrosine-based signals bind to a conserved site on the mu1, mu2 and mu3 subunits of three hetero-tetrameric adaptor protein (AP) complexes, AP-1, AP-2 and AP-3, respectively. Dileucine-based signals, on the other hand, bind to a different site that spans the surface of two subunits, gamma-sigma1, alpha-sigma2 and delta-sigma3, of the corresponding AP-1, AP-2 and AP-3 complexes. In recent years, we extended our studies to the role of signal-adaptor interactions in the process of polarized sorting in neurons. Neurons are highly polarized cells with distinct somatodendritic and axonal domains. The plasma membrane of each of these domains possesses a distinct set of transmembrane proteins, including receptors, channels, transporters and adhesion molecules. We found that several transmembrane proteins, including the transferrin receptor (TfR), the Coxsackie virus and adenovirus receptor (CAR), and the Nipah virus fusion glycoprotein (NiV-F), are sorted to the somatodendritic domain by interaction of tyrosine-based signals with the mu1A subunit of AP-1. More recently, we discovered that a different set of proteins, including the copper transporter ATP7B and the SNARE VAMP4, also undergo sorting to the somatodendritic domain, but in this case through recognition of dileucine-based signals by the gamma1-sigma1 subunits of AP-1. Together with previous work on epithelial cells, these findings establish the AP-1 complex as a global regulator of polarized sorting in different cell types. Defects in polarized sorting likely underlie the pathogenesis of several neurocutaneous disorders caused by mutation in sigma1 subunit isoforms, such as the MEDNIK syndrome (sigma1A), Fried/Pettigrew syndrome (sigma1B) and pustular psoriasis (sigma1C). BORC: a novel multisubunit complex that regulates lysosome positioning and motility - In another line of research, we recently obtained unexpected insights into the mechanisms of lysosome positioning and motility. This research was an outgrowth of our previous work on the biogenesis of LROs such as melanosomes. Years ago, we discovered that mutations in AP-3 cause eye color defects in Drosophila and the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) type 2 (HPS-2) in humans. Other types of HPS are caused by mutations in subunits of the hetero-octameric BLOC-1 and the heterodimeric BLOC-3 complexes. Whereas AP-3 mediates sorting of transmembrane proteins to melanosomes, the functions of BLOC-1 and BLOC-3 are less well understood. In experiments aimed at identifying proteins that interact with BLOC-1, we made the surprising discovery of a related hetero-octameric complex named BORC (for BLOC-one-related complex). Biochemical analyses showed that BORC comprises three subunits that are shared with BLOC-1 (named BLOS1, BLOS2 and Snapin) and five unique subunits (named KXD1, MEF2B, Myrlysin, Lyspersin and Diaskedin). Further studies revealed that BORC is associated with late endosomes and lysosomes, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that drives kinesin-dependent movement of lysosomes toward the peripheral cytoplasm. Mutations in BORC cause collapse of the lysosomal population into the pericentrosomal area. In addition, BORC-mutant cells exhibit defective autophagic flux, probably due to the inability of lysosomes to reach autophagosomes in the cell periphery. These cells also display reduced spreading and migration, likely caused by impaired lysosome-dependent delivery of adhesion and signaling molecules to the plasma membrane. Because of the critical importance of cell adhesion and motility in tumor growth, invasion, and metastasis, the BORC pathway of lysosome dispersal could be an attractive target for pharmacologic inhibition in cancer therapeutics. GARP and EARP: multisubunit tethering complexes involved in endosomal retrieval pathways - Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, prior to our work, it was unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. In the course of studies on the Golgi-associated retrograde protein (GARP) complex, we discovered a related complex named endosome-associated recycling protein (EARP). The two complexes share the Ang2 (also known as Vps51), Vps52 and Vps53 subunits, but whereas GARP comprises a fourth subunit named Vps54, EARP contains a previously uncharacterized protein named Syndetin. This change determines differential localization of GARP to the TGN and EARP to recycling endosomes. Importantly, we found that EARP is involved in recycling of internalized proteins to the plasma membrane. These findings should contribute to the understanding of the pathogenesis of progressive cerebello-cerebral atrophy type 2, a neurodegenerative disorder caused by mutations in Vps53, which in light of our results could result from impairment of both GARP-mediated retrograde transport to the TGN and EARP-mediated recycling to the plasma membrane.

我们研究了跨膜蛋白（称为货物）被分选到真核细胞内膜系统不同区室的分子机制。该系统由一系列膜封闭细胞器组成，包括内质网（ER）、高尔基体、反式高尔基体网络（TGN）、内体、溶酶体、溶酶体相关细胞器（LRO）（例如黑素体）以及极化细胞质膜的不同结构域（例如上皮细胞） 细胞和神经元）。这些区室之间的货物运输是由载体囊泡或小管介导的，这些载体囊泡或小管从供体区室中出芽，通过细胞质易位，并最终与受体区室融合。我们实验室的工作重点是介导这些过程的分子机制，包括（1）分选信号和接头蛋白，用于选择货物蛋白包装到运输载体中，（2）驱动运输载体和其他细胞器通过细胞质运动的微管马达，以及（3）促进运输载体与受体室融合的束缚因子。这些机制在不同的细胞内转运途径的背景下进行研究，包括胞吞作用、循环到质膜、从内体到 TGN 的逆行转运、溶酶体和 LRO 的生物发生以及上皮细胞和神经元的极化分选。从这项研究中获得的知识应用于阐明疾病机制，包括先天性蛋白质运输疾病，例如色素沉着和出血性疾病赫曼斯基-普德拉克综合征（HPS）和神经皮肤疾病MEDNIK综合征。此外，我们还研究 HIV-1 等细胞内病原体如何利用蛋白质转运的分子机制。 用于将跨膜受体分选至海马神经元体树突结构域的 AP-1/网格蛋白途径 - 我们实验室的一个主要研究重点是通过作为蛋白质外壳（例如网格蛋白外壳）组成部分的接头蛋白识别跨膜蛋白胞浆尾部中的分选信号介导的过程。基于酪氨酸和基于双亮氨酸的两种类型的分选信号参与各种分选事件，包括胞吞作用、转运至溶酶体和黑素体以及分选至极化上皮细胞的基底外侧表面。在之前的工作中，我们发现基于酪氨酸的信号分别结合到三个异源四聚体衔接蛋白（AP）复合物AP-1、AP-2和AP-3的mu1、mu2和mu3亚基上的保守位点。另一方面，基于双亮氨酸的信号结​​合到跨越相应 AP-1、AP-2 和 AP-3 复合物的两个亚基（gamma-sigma1、alpha-sigma2 和 delta-sigma3）表面的不同位点。近年来，我们将研究扩展到信号适配器相互作用在神经元极化分选过程中的作用。神经元是高度极化的细胞，具有独特的体树突和轴突结构域。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。我们发现几种跨膜蛋白，包括转铁蛋白受体（TfR）、柯萨奇病毒和腺病毒受体（CAR）以及尼帕病毒融合糖蛋白（NiV-F），通过基于酪氨酸的信号与AP-1的mu1A亚基的相互作用被分类到体细胞树突结构域。最近，我们发现一组不同的蛋白质，包括铜转运蛋白 ATP7B 和 SNARE VAMP4，也经历了体细胞树突结构域的分类，但在这种情况下，是通过 AP-1 的 gamma1-sigma1 亚基识别基于双亮氨酸的信号。结合之前对上皮细胞的研究，这些发现确立了 AP-1 复合物作为不同细胞类型中极化分选的全局调节剂。极化分选的缺陷可能是由 sigma1 亚基亚型突变引起的几种神经皮肤疾病的发病机制，例如 MEDNIK 综合征 (sigma1A)、Fried/Pettigrew 综合征 (sigma1B) 和脓疱性银屑病 (sigma1C)。 BORC：一种调节溶酶体定位和运动的新型多亚基复合物 - 在另一项研究中，我们最近对溶酶体定位和运动机制获得了意想不到的见解。这项研究是我们之前关于黑色素体等 LRO 生物发生研究的成果。多年前，我们发现 AP-3 突变会导致果蝇眼睛颜色缺陷以及人类色素沉着和出血性疾病 Hermansky-Pudlak 综合征 (HPS) 2 型 (HPS-2)。其他类型的 HPS 是由异八聚体 BLOC-1 和异二聚体 BLOC-3 复合物亚基突变引起的。 AP-3 介导跨膜蛋白向黑素体的分选，而 BLOC-1 和 BLOC-3 的功能尚不清楚。在旨在鉴定与 BLOC-1 相互作用的蛋白质的实验中，我们令人惊讶地发现了一种相关的异八聚体复合物，名为 BORC（BLOC-one 相关复合物）。生化分析表明，BORC 包含三个与 BLOC-1 共有的亚基（名为 BLOS1、BLOS2 和 Snapin）和五个独特的亚基（名为 KXD1、MEF2B、Myrlysin、Lyspersin 和 Diaskedin）。进一步的研究表明，BORC 与晚期内体和溶酶体相关，其功能是招募小 GTP 酶 Arl8。这启动了一系列相互作用，驱动溶酶体依赖驱动蛋白向外周细胞质移动。 BORC 突变导致溶酶体群体崩溃到中心体周围区域。此外，BORC突变细胞表现出有缺陷的自噬流，可能是由于溶酶体无法到达细胞外围的自噬体。这些细胞还表现出扩散和迁移减少，这可能是由于溶酶体依赖性粘附和信号分子向质膜的传递受损所致。由于细胞粘附和运动在肿瘤生长、侵袭和转移中至关重要，溶酶体分散的 BORC 途径可能成为癌症治疗中药物抑制的一个有吸引力的靶点。 GARP 和 EARP：参与内体修复途径的多亚基束缚复合物 - 内吞受体再循环到细胞表面涉及通过一系列膜结合区室，其机制尚不清楚。特别是，在我们的工作之前，尚不清楚内吞回收是否需要多亚基束缚复合物的功能，就像其他细胞内运输途径的情况一样。在对高尔基体相关逆行蛋白（GARP）复合物的研究过程中，我们发现了一种相关的复合物，称为内体相关回收蛋白（EARP）。这两个复合物共享 Ang2（也称为 Vps51）、Vps52 和 Vps53 亚基，但 GARP 包含第四个亚基（名为 Vps54），而 EARP 包含一种先前未表征的蛋白质（名为 Syndetin）。这种变化决定了 GARP 到 TGN 的差异定位和 EARP 到回收内体的差异定位。重要的是，我们发现EARP参与内化蛋白质到质膜的再循环。这些发现应有助于理解进行性小脑脑萎缩 2 型的发病机制，这是一种由 Vps53 突变引起的神经退行性疾病，根据我们的结果，这可能是由于 GARP 介导的 TGN 逆行运输和 EARP 介导的质膜再循环受损所致。