Role of ubiquitin in proteasomal degradation of mutant α1-antitrypsin Z in the endoplasmic reticulum
Role of ubiquitin in proteasomal degradation of mutant α1-antitrypsin Z in the endoplasmic reticulum
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DOI：
10.1152/ajpgi.2000.278.1.g39
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发表时间：
2000-01-01
影响因子：
4.5
通讯作者：
Perlmutter, DH
中科院分区：
文献类型：
--
作者：
Teckman, JH;Gilmore, R;Perlmutter, DH
突变α 1-抗胰蛋白酶（α 1-AT）Z分子的细胞内降解延迟与α 1-AT缺乏症患者亚组中内质网（ER）内更大的滞留和对肝脏疾病的易感性相关。最近的研究表明，α（1）ATZ通常通过涉及蛋白酶体的机制在ER中降解，如在完整细胞中使用为表达α（1）ATZ而设计的人成纤维细胞系和在用纯化的α（1）ATZ mRNA编程的无细胞微粒体易位测定系统中所证明的。为了确定α（1）ATZ的蛋白酶体降解是否需要泛素系统，以及是否涉及泛素系统的特定组分，我们现在使用了两种方法。首先，我们通过瞬时转染在表达α（1）ATZ的人成纤维细胞系中过表达显性负性泛素突变体（UbK 48 R-G76 A）。结果表明，UbK 48 R-G76 A介导的α（1）ATZ降解有明显的、特异性的和选择性的抑制，表明泛素系统至少部分参与了α（1）ATZ的ER降解。其次，我们将网织红细胞裂解物进行DE 52层析，并在无细胞系统中测试所得的良好表征的级分。结果表明，α（1）ATZ的降解既有泛素依赖的蛋白酶体机制，也有非泛素依赖的蛋白酶体机制，泛素结合酶E2-F1可能在泛素依赖的蛋白酶体机制中发挥作用。
A delay in intracellular degradation of the mutant al-antitrypsin (alpha(1)AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineered for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha(1)ATZ and whether specific components of the ubiquitin system can be implicated, we have now used two approaches. First, we overexpressed a dominant-negative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that there was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected reticulocyte lysate to DE52 chromatography and tested the resulting well-characterized fractions in the cell-free system. The results showed that there were both ubiquitin-dependent and -independent proteasomal mechanisms for degradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.