Schmidt Bela Z, Perlmutter David H
Department of Pediatrics, Univ. of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213-2583, USA.
Am J Physiol Gastrointest Liver Physiol. 2005 Sep;289(3):G444-55. doi: 10.1152/ajpgi.00237.2004. Epub 2005 Apr 21.
In alpha1-antitrypsin (alpha1-AT) deficiency, a mutant form of alpha1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant alpha1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The alpha1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which alpha1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant alpha1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of approximately 110-130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic alpha1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the alpha1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that approximately 85% of alpha1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and approximately 15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of alpha1-AT Z as well as on its residual secretion.
在α1-抗胰蛋白酶（α1-AT）缺乏症中，α1-AT的一种突变形式在肝细胞内质网（ER）中聚合，通过获得毒性功能机制导致慢性肝炎和肝细胞癌。尽管细胞对突变型α1-AT Z的反应的某些方面已得到部分表征，包括几种蛋白酶体和非蛋白酶体处理机制的参与，但细胞反应途径的其他部分，特别是与其相互作用的伴侣蛋白和被激活的信号转导途径，仍未完全阐明。已知α1-AT Z分子与钙连蛋白相互作用，但根据一项研究，它不与Grp78相互作用。为了系统地寻找α1-AT Z在内质网中相互作用的伴侣蛋白，我们使用了几种不同基因工程细胞系统的化学交联方法。突变型α1-AT Z与Grp78、Grp94、钙连蛋白、Grp170、UDP-葡萄糖糖蛋白：葡糖基转移酶以及两种约110 - 130 kDa的未知蛋白发生交联。顺序免疫沉淀/免疫印迹分析和共免疫沉淀技术在没有化学交联的情况下证实了每种相互作用。发现相同的伴侣蛋白与保留在内质网中的两种非聚合性α1-AT突变体相互作用，表明这些相互作用并非α1-AT Z突变体所特有。此外，蔗糖密度梯度离心研究表明，约85%的α1-AT Z存在于与多种伴侣蛋白形成的异质可溶性复合物中，约15%存在于不含伴侣蛋白的极大聚合物/聚集体中。干扰内质网伴侣蛋白合成和/或活性的试剂，如衣霉素和钙离子载体A23187，对α1-AT Z的溶解度、降解及其残余分泌有不同影响。
