Studies on structural characteristics and physiological function of a novel membrane-bound proteasome

新型膜结合蛋白酶体的结构特征和生理功能研究

• 批准号: 14380297
• 负责人: KOIDE Takehiko
• 金额: $ 9.02万
• 依托单位: University of Hyogo (2004)Himeji Institute of Technology (2002-2003)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380297/
• 关键词: ERb proteasome; 20S proteasome; ERAD; Quality control; phosphatidylinositol polyphosphates; α1-antitrypsin null Hong Kong; プロテアソーム; 品質管理機構; 小胞体関連分解; タンパク質分解; 基質特異性; SKLP

We have isolated the ER membrane-bound (ERb) form of proteasome that we referred to as "ERb proteasome", and studied the structural characteristics, how it binds to the ER membrane, and its possible function as a novel protease for ERAD.Isolation of subunits of ERb by HPLC and detailed analyses of isolated subunits, in comparison with those of 20S proteasome, revealed that one of two α5 subunits in 20S proteasome has been modified in ERb, which we referred to as α5' subunit. The α5' subunit had the N-terminal amino acid sequence of N-acetyl-Met-Phe-Leu-Thr-Arg-Ser-, while that of α5 subunit was Thr-Arg-Ser-. Since Met-Phe-Leu sequence corresponded to the propeptide of α5 subunit, it is highly likely that α5' subunit was produced by N-acetylation of the N-terminal Met of the precursor form of α5 subunit. To examine if α5' subunit is contributing to the membrane binding of ERb, we prepared recombinant α5-type and α5'-type mutant subunits in E.coli, and investigated the capability and specificity of phospholipid binding of the recombinant subunits as well as ERb. None of α5-type, α5'-type mutant subunit, or ERb bound to the major components of membrane such as PC, PE, PS or PI, but they specifically bound to phosphatidylinositol polyphosphates (PIP, PIP2 and PIP3), suggesting a unique characteristics of membrane binding of α5' subunit and ERb. We also examined the function of ERb and its derivative in ERAD of misfolded glycoproteins (α1-antitrypsin null Hong Kong and antithrombin Pro429Stop) using 293 cells. Pulse-chase experiments showed that ERADs of these misfolded glycoproteins were significantly enhanced when α5'-type subunit was co-transfected, suggesting the involvement of ERb in the ERAD of misfolded glycoproteins.

我们分离了内质网膜结合(ERb)形式的蛋白酶体，并研究了它的结构特征，它是如何与ER膜结合的，以及它可能作为一种新的ERAD酶的功能。通过高效液相色谱分离Erb的亚基，并与20S蛋白酶体的亚基进行详细的分析，发现20S蛋白酶体中的两个α5亚基中的一个在Erb中发生了修饰，我们称之为α5‘亚基。α5‘亚基的N-末端氨基酸序列为N-乙酰-蛋氨酸-亮氨酸-苏氨酸-精氨酸-丝氨酸，而α5’亚基的N-末端氨基酸序列为苏氨酸-精氨酸-丝氨酸。由于Met-Phe-Leu序列对应于α5亚基的前肽，α5‘亚基很可能是通过α5亚基前体的N-末端Met的N-乙酰化而产生的。为了研究α5‘亚基是否有助于ERb的膜结合，我们在大肠杆菌中制备了重组的α5’型和α5‘型突变亚基，并研究了重组亚基和ERb的磷脂结合能力和特异性。α5-型、α5‘-型突变亚基或Erb均不与膜的主要成分PC、PE、PS或PI结合，而与磷脂酰肌醇多聚磷酸盐(PIP、PIP2和PIP3)特异结合，提示α5’亚基与Erb具有独特的膜结合特性。我们还利用293细胞检测了Erb及其衍生物在错误折叠的糖蛋白(α1-抗胰蛋白酶空香港和抗凝血酶Pro429Stop)ERAD中的功能。脉冲追逐实验表明，当α5‘-型亚单位共转染时，这些错误折叠糖蛋白的ERAD显著增强，提示ERb参与了错误折叠糖蛋白的ERAD。