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

我们分离了内质网膜结合（ERb）形式的蛋白酶体，我们将其称为“ERb蛋白酶体”，并研究了其结构特征，如何与内质网膜结合，以及它作为ERAD的新型蛋白酶的可能功能。HPLC法分离ERb亚基，并与20S蛋白酶体亚基进行详细分析，发现20S蛋白酶体的两个α5亚基中有一个在ERb中被修饰，我们称之为α5’亚基。α5’亚基的n端氨基酸序列为n -acetyl- met - ph - leu -Thr-Arg-Ser-， α5亚基的n端氨基酸序列为Thr-Arg-Ser-。由于Met- ph - leu序列对应于α5亚基的前肽，因此α5'亚基很可能是由α5亚基前体形式的n端Met的n -乙酰化而产生的。为了研究α5’亚基是否参与了ERb的膜结合，我们在大肠杆菌中制备了重组α5型和α5’型突变亚基，并研究了重组亚基与ERb的磷脂结合能力和特异性。α5型、α5′型突变亚基或ERb均未与PC、PE、PS、PI等主要膜组分结合，但与磷脂酰肌醇多磷酸（PIP、PIP2、PIP3）特异性结合，表明α5′亚基与ERb具有独特的膜结合特征。我们还利用293细胞检测了ERb及其衍生物在错误折叠糖蛋白（α1-antitrypsin null Hong和抗凝血酶Pro429Stop）的ERAD中的功能。脉冲追踪实验表明，α5′型亚基共转染后，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。

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.