The human thioesterase II protein binds to a site on HIV-1 Nef critical for CD4 down-regulation
The human thioesterase II protein binds to a site on HIV-1 Nef critical for CD4 down-regulation
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DOI：
10.1074/jbc.m000536200
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发表时间：
2000-07-28
影响因子：
4.8
通讯作者：
Baltimore, D
中科院分区：
文献类型：
--
作者：
Cohen, GB;Rangan, VS;Baltimore, D
使用HIV-I Nef亲和柱从T细胞裂解物中纯化35-kDa Nef相互作用蛋白。通过肽微序列分析将35 kDa蛋白鉴定为人硫酯酶II（hTE）酶，该酶先前在酵母双杂交筛选中鉴定为潜在的Nef相互作用蛋白。免疫荧光研究表明，hTE定位于过氧化物酶体，Nef和hTE的共表达导致Nef重新定位于过氧化物酶体。Nef和hTE的相互作用被Nef中残基Asp（108）、Leu（112）、Phe（121）、Pro（122）和Asp（123）处的点突变消除。所有这些突变也消除了Nef下调HIV感染细胞表面CD 4的能力。基于Nef的X射线和NMR结构，这些残基定义了Nef上对CD 4下调至关重要的表面。这些突变的子集也影响Nef下调I类主要组织相容性复合体的能力。这些结果，与以前的研究一起，确定了Nef的大部分已知功能的关键区域。然而，并非所有Nef等位基因都以高亲和力与hTE结合，因此hTE在HIV感染过程中的作用仍然不确定。
A HIV-I Nef affinity column was used to purify a 35-kDa Nef-interacting protein from T-cell lysates. The 35-kDa protein was identified by peptide microsequence analysis as the human thioesterase II (hTE) enzyme, an enzyme previously identified in a yeast two-hybrid screen as a potential Nef-interacting protein. Immunofluorescence studies showed that hTE localizes to peroxisomes and that coexpression of Nef and hTE leads to relocalization of Nef to peroxisomes. Interaction of Nef and hTE was abolished by point mutations in Nef at residues Asp(108), Leu(112), Phe(121), Pro(122), and Asp(123). All of these mutations also abrogated the ability of Nef to down-regulate CD4 from the surface of HIV-infected cells. Eased on the x-ray and NMR structures of Nef, these residues define a surface on Nef critical for CD4 down-regulation. A subset of these mutations also affected the ability of Nef to down-regulate major histocompatibility complex class I. These results, taken together with previous studies, identify a region on Nef critical for most of its known functions. However, not all Nef alleles bind to hTE with high affinity, so the role of hTE during HIV infection remains uncertain.