Molecular Recognition by Clathrin Adaptors

特定的分选信号将跨膜蛋白定向到内体-溶酶体系统的隔室。基于酪氨酸的分选信号与AP复合体(AP1、2、3和4)的大亚基的EAR结构域结合，并已得到很好的表征。存在于分选受体细胞质尾部的DXXLL形式的酸性簇二亮氨酸信号，如阳离子非依赖性和阳离子依赖型甘露糖6-磷酸受体，可被GGA接头蛋白的VHS结构域识别。在以前的工作中，我们实验室确定了与DXXLL基序多肽结合的GGA VHS结构域的结构。我们继续研究了GGA蛋白与相关蛋白质或多肽配体形成的复合体中的所有结构域。DXXXLL基序存在于重要的医学宿主蛋白中，如CD4和病毒蛋白，如HIV-1 Nef。与DXXLL基序不同，它与AP复合体结合，而不是GGA。尽管它的重要性和悠久的历史，以及多个实验室多年来的努力，DXXXLL基序的结构基础尚不清楚。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基序多肽结合的AP2变异体。 NEF是人类和猿类免疫缺陷病毒的辅助蛋白。它是促进从感染到艾滋病进展的致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它下调巨噬细胞和T细胞辅助受体CD4的能力。有人认为，Nef通过将CD4的胞浆尾巴与宿主细胞蛋白运输机制的组件联系起来而诱导下调。为了确定这些成分，与我们合作的Juan Bonifacinos实验室开发了一种新的Nef-CD4下调系统，用于果蝇黑腹果蝇S2细胞。他们发现，人类免疫缺陷病毒1型(HIV-1)Nef下调了S2细胞中人类CD4的表达，这一过程受到与人类细胞相同的序列要求的影响。针对S2细胞中蛋白质运输基因的RNA干扰筛选显示，在CD4下调过程中需要笼蛋白和与笼蛋白相关的、定位于质膜的AP2复合体。在人类细胞系HeLa中证实了对AP2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本实验室生产的重组AP2复合变异体证明了HIV-1Nef和AP2之间强大的直接相互作用。这种相互作用需要Nef中的一个二亮氨酸基序，该基序对下调CD4也是必不可少的。

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.