Protein Trafficking In The Endosomal-Lysosomal System

我们研究了跨膜蛋白被分选到内膜系统的不同部分的分子机制，如内小体、溶酶体和一组称为溶酶体相关细胞器的特定细胞类型的细胞器(如黑素小体和血小板致密小体)。分选是通过识别存在于跨膜蛋白胞液区域的信号，由作为膜涂层(例如，网状蛋白涂层)成分的接头蛋白来实现的。在这些接头蛋白中，有异四聚体AP-1、AP-2、AP-3和AP-4复合体，单体GGA蛋白和异五聚体逆转聚体复合体。正确的分选需要运输机械的其他部件的功能，这些部件负责调节小泡系留和融合。本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，并研究由这些蛋白的遗传缺陷引起的人类疾病(例如，Hermansky-Pudlak综合征；神经退行性和神经发育障碍)。 AP-1、AP-2和AP-3是网状蛋白相关的适配器复合体，它们识别两种类型的分类信号，称为基于酪氨酸的和基于二亮氨酸的。我们实验室以前的研究表明，基于酪氨酸的信号与MU1、MU2和Mu3亚基结合，而基于二亮氨酸的信号与相应AP复合体中的两个亚单位的组合(即半复合体)结合，即γ-sigma1、α-sigma2和Delta-sigma3。在过去的一年里，我们证明了淀粉样前体蛋白裂解酶1(BACE1)胞浆尾部基于二亮氨酸的分选信号与AP-2相互作用，介导蛋白质的快速内吞和溶酶体靶向。然而，BACE1的内吞作用对于APP的切割是必不可少的，这支持了致病的淀粉样β蛋白是在晚期分泌途径中产生的这一概念，而不是在内吞体内产生的。此外，我们与宾夕法尼亚大学医学院的Michael Marks小组合作，证明了AP-1和AP-3对眼皮肤白化病2型(OCA2)蛋白胞液尾部另一种基于二亮氨酸的分选信号的识别介导了OCA2对黑素体的分选。 在过去的一年里，我们实验室的一个主要努力是检查信号-适配器相互作用在神经元和上皮细胞极化分选中的作用。神经元被极化成树突、胞体和轴突。每个结构域的质膜上都有一组不同的跨膜蛋白，包括受体、通道、转运体和黏附分子。我们假设，对这些结构域的分选可能是通过分选信号与AP复合体的相互作用来调节的。我们的研究表明，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在内的各种跨膜受体的胞浆尾部都有导致这些蛋白被分选到海马神经体树突域的信息。在TFR和CAR的情况下，这种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，这些受体的尾巴与AP-1的mu1A亚基结合。显性-负干扰和RNAi方法表明，胞浆尾巴与AP-1的相互作用负责躯体树突状细胞的分选。分选包括在胞体水平上将受体蛋白从去往轴突区域的运输载体中排除。干扰AP-1依赖的躯体树突分选导致树突棘成熟缺陷并减少突触数量。最近，AP-1的sigma1A和sigma1B亚基的突变被证明是两种神经发育障碍的原因，分别被称为Mednik综合征和Fry综合征。我们的发现表明，这些障碍可能是由于未能将某些货物分类到特定神经元群体的躯体树突域而引起的。 上皮细胞也被极化成基底侧域和顶域。我们与Weill Cornell医学院的Enrique Rodriguez-Boulan团队合作，证明了基于酪氨酸的分选信号与AP-1的mu1A和mu1B亚基相互作用，介导了TFR和CAR等跨膜蛋白分选到极化上皮细胞的基底外侧区域。综上所述，对神经元和上皮细胞的研究表明，AP-1复合体是细胞极性的全球调节因子。

We investigate the molecular mechanisms by which transmembrane proteins are sorted to different compartments of the endomembrane system such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). 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. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and 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. 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. This past year we demonstrated that a dileucine-based sorting signal in the cytosolic tail of the β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) interacts with AP-2 to mediate rapid endocytosis and lysosomal targeting of the protein. BACE1 endocytosis, however, is dispensable for APP cleavage, supporting the notion that the pathogenic amyloid-beta peptide is generated in the late secretory pathway rather than in endosomes. In addition, in collaboration with the group of Michael Marks (University of Pennsylvannia School of Medicine), we showed that recognition of another dileucine-based sorting signal in the cytosolic tail of the oculocutaneous albinism type 2 (OCA2) protein by AP-1 and AP-3 mediates OCA2 sorting to melanosomes. A major effort of our laboratory this past year was to examine the role of signal-adaptor interactions in polarized sorting in neurons and epithelial cells. 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 could be 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 occurred 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 synapses. Recently, mutations in the sigma1A and sigma1B subunits of AP-1 were shown to be the cause of two neurodevelopmental disorders known as MEDNIK syndrome and Fried syndrome, respectively. Our findings suggest that these disorders may arise from failure to sort certain cargos to the somatodendritic domain of specific neuronal populations. Epithelial cells are also polarized into basolateral and apical domains. We collaborated with the group of Enrique Rodriguez-Boulan (Weill Cornell Medical College) to demonstrate that interaction of tyrosine-based sorting signals with the mu1A and mu1B subunits of AP-1 mediates sorting of transmembrane proteins such as TfR and CAR to the basolateral domain of polarized epithelial cells. Taken together, studies with neurons and epithelial cells indicate that the AP-1 complex is a global regulator of cell polarity.