Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease.
Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease.
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发表时间：
1994-08
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通讯作者：
C. Realini;W. Dubiel;G. Pratt;K. Ferrell;M. Rechsteiner
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作者：
C. Realini;W. Dubiel;G. Pratt;K. Ferrell;M. Rechsteiner
多催化蛋白酶（MCP）可以被两种不同的多亚基复合物激活。一个是26 S蛋白酶的调节组分，其包含至少15个不同的亚基。另一种是由31-和29-kDa亚基组成的六聚体激活剂。一个较小的亚基的cDNA已被克隆和测序。该cDNA编码249个氨基酸的蛋白质。嵌入在球状蛋白结构域的典型序列之间的是一段28个“交替”赖氨酸和谷氨酸残基。类似的区域，我们称之为KEKE基序，也发现在两个MCP亚基，亚基12的26 S蛋白酶和各种伴侣蛋白，包括热休克蛋白90，热休克蛋白70，和钙连接蛋白。在大肠杆菌中表达的激活剂cDNA产生的功能性蛋白质几乎没有区别直接从红细胞纯化的MCP激活剂。重组蛋白在双向聚丙烯酰胺凝胶电泳上形成3个等电蛋白，并与抗红细胞激活剂抗体发生反应。重组激活剂还结合多催化蛋白酶并刺激疏水或带电残基的羧基末端的切割。通过γ干扰素处理HeLa细胞诱导激活子亚基的合成。这最后两个发现对I类主要组织相容性受体的抗原呈递有影响。
The multicatalytic protease (MCP) can be activated by two distinct multisubunit complexes. One is the regulatory component of the 26 S protease, which contains at least 15 distinct subunits. The other is a hexameric activator composed of 31- and 29-kDa subunits. A cDNA for the smaller subunit has been cloned and sequenced. The cDNA encodes a protein of 249 amino acids. Embedded between sequences typical of globular protein domains is a stretch of 28 "alternating" lysine and glutamic acid residues. Similar regions, which we call KEKE motifs, are also found in two MCP subunits, in subunit 12 of the 26 S protease and in a variety of chaperonins including hsp90, hsp70, and calnexin. Expression of the activator cDNA in Escherichia coli produced a functional protein virtually indistinguishable from MCP activator purified directly from red blood cells. The recombinant protein formed three isoelectric species on two-dimensional polyacrylamide gel electrophoresis, and it reacted with antibodies to red blood cell activator. Recombinant activator also bound the multicatalytic protease and stimulated cleavage at the carboxyl terminus of hydrophobic or charged residues. Synthesis of the activator subunit was induced by gamma interferon treatment of HeLa cells. These last two findings have implications for antigen presentation by class I major histocompatibility receptors.