Hsp90-mediated Assembly of the 26 S Proteasome Is Involved in Major Histocompatibility Complex Class I Antigen Processing*
Hsp90-mediated Assembly of the 26 S Proteasome Is Involved in Major Histocompatibility Complex Class I Antigen Processing*
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DOI：
10.1074/jbc.m803077200
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发表时间：
2008-10
影响因子：
4.8
通讯作者：
T. Yamano;Shusaku Mizukami;S. Murata;T. Chiba;Keiji Tanaka;H. Udono
中科院分区：
文献类型：
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作者：
T. Yamano;Shusaku Mizukami;S. Murata;T. Chiba;Keiji Tanaka;H. Udono
热休克蛋白90（hsp90）和蛋白酶体激活剂PA28刺激主要组织相容性复合体（MHC）I类抗原加工。目前尚不清楚hsp90是否影响蛋白酶体活性以产生T细胞表位，尽管PA 28与20 S蛋白酶体的结合刺激了酶活性。在这里，我们表明，热休克蛋白90是必不可少的装配的26 S蛋白酶体，因此，参与表位的生产。通过凝胶内水解试验确定，向细胞裂解物中加入重组hsp90α以ATP依赖的方式增强26 S蛋白酶体的胰凝乳蛋白酶样活性。我们成功地将组氨酸标记的hsp90α和PA 28 α诱导的新组装的26 S蛋白酶体从细胞提取物中拉下来用于体外表位产生测定，我们发现这些结构对hsp90抑制剂格尔德霉素敏感。我们发现了一个被hsp90α和PA28α下拉的蛋白酶体特有的切割表位，而在hsp90α和PA28α下拉中分别鉴定了两个不同的表位。通过用于蛋白酶体下拉的蛋白质组合忠实地增强了这些相应肽在体内的加工。格尔德霉素在体内抑制热休克蛋白90部分破坏了26 S蛋白酶体结构，与下调的MHC I类表达一致。我们的研究结果表明，热休克蛋白90促进MHC I类抗原的处理，通过表位生产的26 S蛋白酶体的复合物。
Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90α to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90α- and PA28α-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90α and PA28α, whereas two different epitopes were identified in the hsp90α- and PA28α-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.