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隔室的分子机制，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（例如，黑素体和血小板致密体）。分选是通过作为膜衣组分的衔接蛋白（例如，网格蛋白涂层）。在这些衔接蛋白中有异四聚体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复合物。正确的分选需要额外的功能组件的运输机械介导囊泡拴系和融合。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，并研究由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是网格蛋白相关的衔接子复合物，其识别称为基于酪氨酸和基于二亮氨酸的两种类型的分选信号。先前的研究表明，基于酪氨酸的信号与mu 1、mu 2和mu 3亚基结合，而基于二亮氨酸的信号与组合（即，半复合物）的两个亚基，γ-σ 1、α-σ 2和δ-σ 3，来自相应的AP复合物。基于结构的突变分析使我们能够精确定位基于二亮氨酸的分选信号的结合位点的确切位置，该信号适合AP-1、AP-2和AP-3复合物上的DEXXXLLI共有基序。相应位点的位置和地形是相似的，尽管不同相互作用的强度和氨基酸需求取决于信号的确切序列和所涉及的特定AP复合物。我们还证明了由各种γ（即，γ 1和γ 2）和σ 1（即，sigma 1A、sigma 1B和sigma 1C）亚单位同种型。这些AP-1变体结合基于双亮氨酸的信号，对某些序列具有明显的偏好，这意味着它们在功能上不等同。事实上，sigma 1A和sigma 1B的突变最近被证明是两种严重的神经发育障碍的原因，分别被称为MEDNIK和Fried综合征。基于我们的工作，我们假设这些疾病中的缺陷是由具有基于双亮氨酸的信号的特定货物蛋白的异常分选引起的。 AP-4复合物与其他AP复合物的不同之处在于它不识别基于酪氨酸和基于二亮氨酸的典型信号。我们最近发现，AP-4的mu 4亚基与阿尔茨海默病淀粉样前体蛋白（APP）胞质尾部的YKFFE序列结合。生化和X射线晶体学分析表明，APP序列的属性和穆4上的结合位点的位置是不同的，从那些典型的基于酪氨酸的信号结合到其他AP复合物的穆亚基。两种APP样蛋白APLP 1和APLP 2具有也与mu 4相互作用的相关序列。AP-4-APP相互作用的破坏将APP的分布从内体转移到反式高尔基体网络，并增强APP到致病性淀粉样β肽的γ-分泌酶催化的加工。这些结果表明，APP和AP-4参与一种新型的信号-适配器相互作用，介导APP从trans-Golgi网络转运到内体，从而减少蛋白质的淀粉样蛋白加工。因此，AP-4应该被认为是APP加工和运输的新调节剂，以及阿尔茨海默病的潜在危险因素。 在以前的工作中，我们发现一个名为GARP的复合物是介导各种货物蛋白（包括分选受体、加工内肽酶、融合蛋白以及细菌和植物毒素）从内体到哺乳动物细胞中的trans-Golgi网络的逆行运输的分子机制的关键组成部分。我们发现，这种复合物的分子功能是促进束缚和融合的内体衍生的运输载体的trans-Golgi网络。生物化学分析表明，人GARP复合物包括四个亚基，命名为Vps 52，Vps 53，Vps 54和Ang 2。干扰任何GARP亚基都会阻断许多货物向高尔基体网络的传递，导致溶酶体功能、脂质运输和自噬的全面缺陷。与Aitor Hierro（CIC bioGUNE，毕尔巴鄂，西班牙）合作，我们解决了Vps 54的C-末端片段的晶体结构，这是第一个解决GARP复合物任何部分的原子结构。这一结构揭示了GARP与其他多亚基拴系复合物如外囊和COG有关。此外，我们发现，在肌萎缩侧索硬化症（ALS）的动物模型wobbler小鼠中鉴定的Vps 54中亮氨酸-967替换为谷氨酰胺，使蛋白质不稳定，导致所有组织中GARP复合物的水平降低。因此，这种突变小鼠特有的运动神经元变性是由于GARP水平降低和随之而来的逆行转运缺陷所致。

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 (e.g., Hermansky-Pudlak syndrome; neurodegenerative and neurodevelopmental disorders) of these proteins. 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 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. Structure-based mutational analyses allowed us to pinpoint the exact location of the binding sites for dileucine-based sorting signals fitting the DEXXXLLI consensus motif on the AP-1, AP-2 and AP-3 complexes. The location and topography of the corresponding sites are similar, although the strength and amino acid requirements of different interactions depend on the exact sequence of the signal and the particular AP complex involved. We also demonstrated the occurrence of multiple AP-1 complexes resulting from combinatorial assembly of various gamma (i.e., gamma1 and gamma2) and sigma1 (i.e., sigma1A, sigma1B and sigma1C) subunit isoforms encoded by different genes. These AP-1 variants bind dileucine-based signals with marked preferences for certain sequences, implying that they are not functionally equivalent. Indeed, mutations in sigma1A and sigma1B have recently been shown to be the cause of two severe neurodevelopmental disorders known as the MEDNIK and Fried syndromes, respectively. Based on our work, we hypothesize that defects in these diseases are caused by abnormal sorting of specific cargo proteins having dileucine-based signals. The AP-4 complex is distinct from the other AP complexes in that it does not recognize canonical tyrosine-based and dileucine-based signals. We recently found that, instead, the mu4 subunit of AP-4 binds an YKFFE sequence from the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP). Biochemical and X-ray crystallographic analyses revealed that the properties of the APP sequence and the location of the binding site on mu4 are distinct from those of canonical tyrosine-based signals binding to the mu subunits of other AP complexes. Two APP-like proteins, APLP1 and APLP2, have related sequences that also interact with mu4. Disruption of the AP-4-APP interaction shifts the distribution of APP from endosomes to the trans-Golgi network and enhances gamma-secretase-catalyzed processing of APP to the pathogenic amyloid-beta peptide. These results demonstrate that APP and AP-4 engage in a novel type of signal-adaptor interaction that mediates transport of APP from the trans-Golgi network to endosomes, thereby reducing amyloidogenic processing of the protein. AP-4 should thus be considered a novel regulator of APP processing and trafficking, and a potential risk factor for Alzheimer's disease. In previous work, we found that a complex named GARP is a critical component of the molecular machinery that mediates retrograde transport of various cargo proteins, including sorting receptors, processing endopeptidases, fusogenic proteins and bacterial and plant toxins, from endosomes to the trans-Golgi network in mammalian cells. We showed that the molecular function of this complex is to promote tethering and fusion of endosome-derived transport carriers to the trans-Golgi network. Biochemical analyses showed that the human GARP complex comprises four subunits named Vps52, Vps53, Vps54 and Ang2. Interference with any of the GARP subunits blocks the delivery of many cargos to the trans-Golgi network, leading to global defects in lysosomal function, lipid traffic and autophagy. In collaboration with Aitor Hierro (CIC-bioGUNE, Bilbao, Spain), we solved the crystal structure of a C-terminal fragment from Vps54, the first atomic structure to be solved for any part of the GARP complex. This structure revealed that that GARP is related to other multisubunit tethering complexes such as the exocyst and COG. In addition, we found that a leucine-967 to glutamine substitution in Vps54 identified in the wobbler mouse, an animal model for amyotrophic lateral sclerosis (ALS), destabilizes the protein, leading to lower levels of the GARP complex in all tissues. The motor neuron degeneration that is characteristic of this mutant mouse is therefore due to decreased levels of GARP and the ensuing defects in retrograde transport.