Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

• 批准号: 7593549
• 负责人: James Hurley
• 金额: $ 19.75万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 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/7593549
• 关键词: Acquired Immunodeficiency Syndrome; Adaptor Signaling Protein; Address; Affinity; Binding; Binding Sites; Cations; Cell membrane; Cell physiology; Cells; Clathrin; Clathrin Adaptors; Clathrin-Coated Vesicles; Complex; Cytoplasmic Tail; Down-Regulation; Drosophila melanogaster; Ear; Endocytosis; Engineering; Eukaryotic Cell; Glutathione S-Transferase; Goals; HIV-1; Hela Cells; Human; Human Cell Line; Hybrids; IGF Type 2 Receptor; Infection; Integral Membrane Protein; Laboratories; Ligands; Link; Localized; Measures; Pathogenesis; Peptides; Personal Satisfaction; Play; Proteins; Purpose; RNA Interference; Recombinants; Recording of previous events; Role; Signal Transduction; Sorting - Cell Movement; Structure; System; T-Lymphocyte; Tail; Trafficking Protein Gene; Transcription Factor AP-1; Transcription Factor AP-2 Alpha; Tyrosine; Variant; Vesicle; Viral Proteins; Work; Yeasts; base; golgi associated, gamma adaptin homologous, ADP-ribosylation factor interacting protein; intracellular protein transport; macrophage; molecular recognition; novel; protein transport; receptor; simian human immunodeficiency virus

Specific sorting signals direct transmembrane proteins to the compartments of the endosomal-lysosomal system. The tyrosine-based sorting signal binds to the ear domains of the large subunits of AP complexes (AP1, 2, 3, and 4), and has been well characterized. Acidic-cluster-dileucine signals of the form DXXLL present within the cytoplasmic tails of sorting receptors, such as the cation-independent and cation-dependent mannose 6-phosphate receptors, are recognized by the VHS domain of the GGA adaptor proteins. In previous work, our laboratory determined the structure of the GGA VHS domain bound to DXXLL-motif peptides. We went on to characterize all of the domains of the GGA proteins in complex with relevant protein or peptide ligands. The DXXXLL motif exists in medically important host proteins such as CD4 and viral proteins such as HIV-1 Nef. Unlike the DXXLL motif, it binds to AP complexes, not GGAs. Despite its importance and substantial history, and years of effort by multiple laboratories, the structural basis for the DXXXLL motif is unknown. To address this problem, our lab engineered and expressed a variant AP2 complex that is competent to bind to DXXXLL motif peptides. Nef is an accessory protein of human and simian immunodeficiency viruses. It is a critical determinant of pathogenesis that promotes the progression from infection to AIDS. The pathogenic effects of Nef are in large part dependent on its ability to downregulate the macrophage and T-cell coreceptor, CD4. It has been proposed that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein trafficking machinery. To identify these components, Juan Bonifacinos laboratory, with whom we collaborate, developed a novel Nef-CD4 downregulation system in Drosophila melanogaster S2 cells. They found that human immunodeficiency virus type 1 (HIV-1) Nef downregulates human CD4 in S2 cells and that this process is subject to the same sequence requirements as in human cells. An RNA interference screen targeting protein trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP2 complex in the downregulation of CD4. The requirement for AP2 was confirmed in the human cell line HeLa. A yeast three-hybrid system and glutathione S-transferase pull-down analyses using the recombinant AP2 complex variant produced in our laboratory were used to demonstrate a robust, direct interaction between HIV-1 Nef and AP2. This interaction requires a dileucine motif in Nef that is also essential for downregulation of CD4.

特异性分选信号将跨膜蛋白引导至内体-溶酶体系统的区室。基于酪氨酸的分选信号结合到AP复合物（AP 1、2、3和4）的大亚基的耳域，并且已经被很好地表征。存在于分选受体（例如阳离子非依赖性和阳离子依赖性甘露糖6-磷酸受体）的细胞质尾内的DXXLL形式的酸性簇双亮氨酸信号被GGA衔接子蛋白的VHS结构域识别。在以前的工作中，我们的实验室确定了与DXXLL基序肽结合的GGA VHS结构域的结构。我们继续表征与相关蛋白质或肽配体复合的GGA蛋白的所有结构域。DXXXLL基序存在于医学上重要的宿主蛋白如CD 4和病毒蛋白如HIV-1 Nef中。与DXXLL基序不同，它结合AP复合物，而不是GGA。尽管它的重要性和实质性的历史，以及多个实验室多年的努力，DXXXLL基序的结构基础是未知的。为了解决这个问题，我们的实验室设计并表达了一种能够与DXXXLL基序肽结合的变体AP 2复合物。 Nef是人类和猿猴免疫缺陷病毒的辅助蛋白。它是促进从感染发展为艾滋病的发病机制的关键决定因素。Nef的致病作用在很大程度上取决于其下调巨噬细胞和T细胞辅助受体CD 4的能力。已经提出Nef通过将CD 4的胞质尾部连接到宿主细胞蛋白运输机制的组分来诱导下调。为了鉴定这些成分，我们与Juan Bonifacinos实验室合作，在果蝇S2细胞中开发了一种新的Nef-CD 4下调系统。他们发现，人类免疫缺陷病毒1型（HIV-1）Nef下调S2细胞中的人类CD 4，并且这一过程与人类细胞中的序列要求相同。靶向S2细胞中蛋白质运输基因的RNA干扰筛选揭示了在CD 4下调中对网格蛋白和网格蛋白相关的质膜定位的AP 2复合物的需求。在人细胞系HeLa中证实了对AP 2的需求。酵母三杂交系统和谷胱甘肽S-转移酶下拉分析，使用我们实验室生产的重组AP 2复合物变体，用于证明HIV-1 Nef和AP 2之间的强大的，直接的相互作用。这种相互作用需要Nef中的双亮氨酸基序，这也是下调CD 4所必需的。