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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >A complex between peptide : N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins
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A complex between peptide : N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins

机译：肽：N-聚糖酶和两种蛋白酶连接的蛋白之间的复合物提示了错误折叠的糖蛋白降解的机制

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N-glycanase (PNGase) has been proposed to participate in the proteasome-dependent glycoprotein degradation pathway. The finding that yeast PNGase interacts with the 19S proteasome subunit through the protein Rad23 supports this hypothesis. In this report, we have used immunofluorescence, subcellular fractionation, coimmunoprecipitation, and in vitro GST pull-down techniques for detecting intracellular localization and interactions of PNGase, HR23B, and S4 by using human (h) and mouse (m) homologs. Immunofluorescence studies revealed that hPNGase, hHR23B, and hS4 are present in close proximity to the endoplasmic reticulum (ER) when calnexin was used as an ER marker in HeLa cells. Subcellular fractionation suggests not only cytoplasmic but also ER association of hPNGase in HeLa cells. Immunoprecipitation analysis revealed the interaction of h/mPNGase with the 19S proteasome subunit, hS4, through hHR23B. Using an in vitro GST pull-down assay, we also have shown that recombinant mPNGase requires its N terminus and middle domain for interaction with mHR23B. Finally, using misfolded yeast carboxypeptidase Y and chicken ovalbumin as glycoprotein substrates, we have established that mHR23B acts as a receptor for deglycosylated proteins. Based on this finding, we propose that after cleglycosylation of misfolded glycoproteins by PNGase, the aglyco forms of these proteins are recognized by HR23B and targeted for degradation.
机译：N-聚糖酶（PNGase）已被提议参与蛋白酶体依赖性糖蛋白降解途径。酵母PNGase通过Rad23蛋白与19S蛋白酶体亚基相互作用的发现支持了这一假设。在本报告中，我们使用了免疫荧光，亚细胞分级分离，共免疫沉淀和体外GST下拉技术，通过使用人类（h）和小鼠（m）同源物检测PNGase，HR23B和S4的细胞内定位和相互作用。免疫荧光研究表明，当将钙连蛋白用作HeLa细胞中的ER标记时，hPNGase，hHR23B和hS4紧邻内质网（ER）存在。亚细胞分级表明不仅在HeLa细胞中hPNGase的细胞质而且与ER关联。免疫沉淀分析显示h / mPNGase通过hHR23B与19S蛋白酶体亚基hS4相互作用。使用体外GST下拉试验，我们还显示重组mPNGase需要其N末端和中间结构域才能与mHR23B相互作用。最后，使用错折叠的酵母羧肽酶Y和鸡卵清蛋白作为糖蛋白底物，我们已经确定mHR23B充当去糖基化蛋白的受体。基于此发现，我们建议在通过PNGase对错误折叠的糖蛋白进行糖基化之后，这些蛋白的无糖形式将被HR23B识别并靶向降解。

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