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浆结构域中的信号，由作为膜涂层成分的接头蛋白来完成的。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GGA1、GGA2和GGA3蛋白(GGAs)，以及异五聚体逆转录复合体。正确的分选需要运输机制中调节囊泡拴系和融合的额外组件的功能，例如异四聚体GARP复合体。本实验室目前的工作旨在阐明外壳蛋白和囊泡拴系因子的结构、调节和生理作用，并调查由遗传缺陷(Hermansky-Pudlak综合征；阿尔茨海默病；发育障碍)或病原体(HIV-1)利用这些蛋白而导致的人类疾病。 遗传模型系统使我们能够确定AP复合体在生物体生理学和发育中的功能。对果蝇的研究表明，GGAS和AP-1是通过受体将酸性水解酶分类为溶酶体所必需的。与埃里克·埃弗雷特(北卡罗来纳大学)合作进行的其他研究表明，编码AP-2的Beta2亚单位的基因缺失会导致非综合征性腭裂。这种表型与AP-2的MU2亚单位编码基因的消融所导致的表型相反，后者是胚胎致死的。这两种突变结果不同的原因是AP-1的β1亚基可以替代β2，而AP-1的MU1亚基不能替代AP-2的MU2亚基。在腭部发育过程中，β2的缺失可能会损害转化生长因子-β信号转导。这些观察结果揭示了这种常见出生缺陷的发病机制。 AP-4复合体是AP复合体家族中最新发现的，也是人们了解最少的。在最近的工作中，我们发现AP-4与阿尔茨海默病淀粉样前体蛋白(APP)的胞浆尾巴相互作用。AP-4-APP相互作用的中断将APP的分布从内小体转移到跨高尔基网络(TGN)，并增强了APP在伽马分泌酶催化下对致病淀粉样β蛋白的加工。这些发现将AP-4确立为APP处理和贩运的新调节因子，并成为阿尔茨海默病的潜在风险因素。 新合成的酸性水解酶的分选依赖于甘露糖6-磷酸受体(MPRs)，它与TGN上的水解酶结合，并将它们输送到内体。水解酶随后被输送到溶酶体，而受体则返回TGN进行额外的分选。在以前的工作中，我们证明了MPR的选择是由逆转录复合体介导的。我们最近发现了另一个名为GARP的复合体，它能够将含有MPR的载体拴系并融合到TGN。 我们之前已经证明，人类GARP复合体由三个亚基组成，命名为VPS52、VPS53和VPS54。我们现在发现，以前被命名为ANG2/Fat-Free的蛋白质是GARP复合体的第四个亚基。干扰任何GARP亚基都会阻止MPR和志贺毒素等货物从内体输送到TGN。其结果是，酸性水解酶被错误地分选到细胞外空间，溶酶体变得功能障碍，细胞表现出缺陷的脂质运输和自噬。 我们还阐明了VPS54突变Wobbler小鼠运动神经元变性的结构基础，这是一种肌萎缩侧索硬化症(ALS)的动物模型。在西班牙毕尔巴鄂的CIC-BioGUNE公司的合作下，我们解决了VPS54的C-末端结构域的晶体结构，该结构含有摇摆器突变(亮氨酸-967为谷氨酰胺)。结构分析表明，GARP与其他多亚基连接复合体有关，如胞外囊和COG。此外，我们观察到摇摆器突变破坏了VPS54的稳定性，导致其在小鼠所有组织中的降解增强和水平降低。因此，这只小鼠的运动神经元退化是由于GARP水平降低所致。 最后，在另一个项目中，我们确定了在某些形式的色素沉着和出血障碍，Hermansky-Pudlak综合征中存在缺陷的BLOC-3复合体由HPS1和HPS4两个亚基组成的1：1组装。此外，我们还证明了该复合体与GTP结合的(活性)形式的Rab9 GTP酶相互作用，提示它可能在黑素小体和血小板致密小体的生物发生中发挥Rab9效应。

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. Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (GGAs), 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 heterotetrameric GARP complex. 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 (Hermansky-Pudlak syndrome; Alzheimer's disease; developmental disorders) or pathogens' (HIV-1) exploitation of these proteins. Genetic model systems have allowed us to determine the functions of AP complexes in organismal physiology and development. Studies in Drosophila revealed that the GGAs and AP-1 are required for receptor-mediated sorting of acid hydrolases to lysosomes. Other studies performed in collaboration with Eric Everett (University of North Carolina) showed that disurption of the gene encoding the beta2 subunit of AP-2 results in non-syndromic cleft palate. This phenotype is in contrast to that resulting from ablation of the gene encoding the mu2 subunit of AP-2, which is embryonic lethal. The explanation for the different outcomes of these two mutations is that the beta1 subunit of AP-1 can substitute for beta2, whereas the mu1 subunit of AP-1 cannot substitute for the mu2 subunit of AP-2. The absence of beta2 could impair TGF-beta signaling during development of the palate. These observations shed light on the pathogenesis of this common birth defect. The AP-4 complex is the most-recently discovered and least understood of the family of AP complexes. In recent work, we have found that AP-4 interacts with the cytosolic tail of the Alzheimer's disease amyloid precursor protein (APP). Disruption of the AP-4-APP interaction shifts the distribution of APP from endosomes to the trans-Golgi network (TGN) and enhances gamma-secretase-catalyzed processing of APP to the pathogenic amyloid-beta peptide. These findings establish AP-4 as a novel regulator of APP processing and trafficking, and as a potential risk factor for Alzheimer's disease. The sorting of newly-synthesized acid hydrolases to lysosomes relies on mannose 6-phosphate receptors (MPRs), which bind the hydrolases at the TGN and transport them to endosomes. The hydrolases are subsequently delivered to lysosomes whereas the receptors return to the TGN for additional rounds of sorting. In previous work, we showed that selection of MPRs for retrieval to the TGN is mediated by the retromer complex. We have recently identified another complex named GARP, which enables tethering and fusion of MPR-containing carriers to the TGN. We had previously shown that the human GARP complex comprises three subunits named VPS52, VPS53 and VPS54. We have now discovered that a protein previously named ANG2/Fat-free is a fourth subunit of the GARP complex. Interference with any of the GARP subunits blocks the delivery of cargos such as MPRs and Shiga toxin from endosomes to the TGN. As a consequence, acid hydrolases are missorted to the extracellular space, lysosomes become dysfunctional, and cells exhibit defective lipid traffic and autophagy. We have also elucidated the structural basis for motor neuron degeneration in the VPS54-mutant wobbler mouse, an animal model for amyotrophic lateral sclerosis (ALS). In collaboration with Aitor Hierro (CIC-bioGUNE, Bilbao, Spain), we solved the crystal structure of the C-terminal domain of VPS54, which harbors the wobbler mutation (leucine-967 to glutamine). The structure revealed that GARP is related to other multisubunit tethering complexes such as the exocyst and COG. In addition, we observed that the wobbler mutation destabilizes VPS54, resulting in its enhanced degradation and decreased levels in all tissues of the mouse. Motor neuron degeneration in this mouse therefore results from decreased levels of GARP. Finally, in another project, we definitively established that the BLOC-3 complex that is defective in some forms of the pigmentation and bleeding disorder, Hermansky-Pudlak syndrome, consists of a 1:1 assembly of two subunits, HPS1 and HPS4. In addition, we demonstrated that this complex interacts with the GTP-bound (active) form of the Rab9 GTPase, suggesting that it might function as a Rab9 effector in the biogenesis of melanosomes and platelet dense bodies.