IDENTIFICATION OF CELLULAR PROTEINS THAT BIND TO THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF GENE-PRODUCT IN-VITRO - A ROLE FOR MYRISTYLATION
IDENTIFICATION OF CELLULAR PROTEINS THAT BIND TO THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF GENE-PRODUCT IN-VITRO - A ROLE FOR MYRISTYLATION
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DOI：
10.1099/0022-1317-74-8-1581
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发表时间：
1993-08-01
影响因子：
3.8
通讯作者：
COATES, K
中科院分区：
文献类型：
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作者：
HARRIS, M;COATES, K
人免疫缺陷病毒（HIV）1型nef基因产物在杆状病毒系统中表达为N-末端与谷胱甘肽-S-转移酶（GST）的融合蛋白。发现所得的nefGST融合蛋白在N末端被真正地肉豆蔻酰化，并且可以通过固定化谷胱甘肽上的一步亲和层析纯化至均一。利用nefGST对谷胱甘肽的高亲和力来开发鉴定能够与nef相互作用的细胞蛋白的测定。在Jurkat人T细胞系的提取物中鉴定了几种这样的蛋白质。nef结合蛋白和固定化的nefGST之间的相互作用可以通过添加可溶性nef特异性竞争。细胞分级显示nef结合蛋白存在于胞浆和膜相关组分中。非肉豆蔻基化衍生物未能结合到膜相关蛋白，但能够结合到胞质组。尽管亲和力降低。此外，一个单一的蛋白质存在于可溶性和膜相关的馏分表现出肉豆蔻基化独立的结合nef。通过与其他豆蔻酰化蛋白质如MARCKS（豆蔻酰化富含丙氨酸的C激酶底物）和劳斯肉瘤病毒转化蛋白src类比，仅与豆蔻酰化nef结合的膜相关蛋白可能代表nef的特异性膜靶点。与nef相互作用的胞质蛋白可能构成一个尚未确定的信号转导途径的可溶性组分，该途径是HIV-1感染细胞中nef作用的靶点。
The human immunodeficiency virus (HIV) type 1 nef gene product was expressed as an N-terminal fusion protein with glutathione-S-transferase (GST) in the baculovirus system. The resulting nefGST fusion protein was found to be authentically myristylated at the N terminus and could be purified to homogeneity by one-step affinity chromatography on immobilized glutathione. The high affinity of nefGST for glutathione was exploited to develop an assay to identify cellular proteins capable of interacting with nef. Several such proteins were identified in extracts from the Jurkat human T cell line. The interaction between nef-binding proteins and immobilized nefGST could be specifically competed by the addition of soluble nef. Cell fractionation showed that nef-binding proteins were present in both cytosolic and membrane-associated fractions. A non-myristylated derivative failed to bind to the membrane-associated proteins but was able to bind to the cytosolic group. albeit with reduced affinity. In addition, a single protein present in both soluble and membrane-associated fractions exhibited myristylation-independent binding to nef. By analogy with other myristylated proteins such as MARCKS (myristylated alanine-rich C kinase substrate) and the Rous sarcoma virus transforming protein, src, the membrane-associated proteins that bind only to myristylated nef may represent a specific membrane target for nef. The cytosolic proteins that interact with nef may constitute soluble components of an as yet unidentified signal transduction pathway which is the target of nef action in the HIV-1-infected cell.