Molecular Recognition by Clathrin Adaptors

网格蛋白适配器的分子识别

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.

特定的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与 AP 复合物大亚基（AP1、2、3 和 4）的耳域结合，并且已得到充分表征。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）细胞质尾部内的 DXXLL 形式的酸性簇二亮氨酸信号可被 GGA 接头蛋白的 VHS 结构域识别。在之前的工作中，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 蛋白质的所有结构域。 DXXXLL 基序存在于医学上重要的宿主蛋白（例如 CD4）和病毒蛋白（例如 HIV-1 Nef）中。与 DXXLL 基序不同，它结合 AP 复合物，而不是 GGA。尽管 DXXXLL 基序很重要，历史悠久，并且经过多个实验室多年的努力，但其结构基础仍不清楚。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 AP2 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 CD4 的能力。有人提出，Nef 通过将 CD4 的胞质尾部与宿主细胞蛋白质运输机制的组件连接来诱导下调。为了识别这些成分，我们与 Juan Bonifacinos 实验室合作，在果蝇 S2 细胞中开发了一种新型 Nef-CD4 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 CD4，并且该过程与人类细胞中的序列要求相同。针对 S2 细胞中蛋白质运输基因的 RNA 干扰筛选揭示了 CD4 下调中需要网格蛋白和网格蛋白相关的质膜定位 AP2 复合物。 AP2 的需求在人类 HeLa 细胞系中得到证实。使用我们实验室生产的重组 AP2 复合物变体进行的酵母三杂交系统和谷胱甘肽 S-转移酶下拉分析被用来证明 HIV-1 Nef 和 AP2 之间强大、直接的相互作用。这种相互作用需要 Nef 中的双亮氨酸基序，这对于 CD4 的下调也是必需的。