Qiu Wei, Kohen-Avramoglu Rita, Rashid-Kolvear Fariborz, Au Crystal S, Chong Taryne M, Lewis Gary F, Trinh Denny K Y, Austin Richard C, Urade Reiko, Adeli Khosrow
Division of Clinical Biochemistry, Department of Laboratory Medicine and Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8.
Biochemistry. 2004 Apr 27;43(16):4819-31. doi: 10.1021/bi034862z.
Co- and posttranslational regulation of apolipoprotein B (apoB) has been postulated to involve degradation by both proteasomal and nonproteasomal pathways; however, nonproteasomal mechanisms of apoB degradation are currently unknown. We have previously demonstrated an intracellular association of newly synthesized apoB with endoplasmic reticulum (ER)-60, an ER-localized protein, possessing both proteolytic and chaperone activities. In the present paper, adenoviral expression vectors containing rat ER-60 cDNA were used to achieve dose- and time-dependent overexpression of ER-60 to investigate its role in apoB100 turnover. Overexpressed ER-60 accumulated in the microsomal lumen of HepG2 cells and was associated with apoB100 in dense lipoprotein particles. Overexpression of ER-60 in HepG2 cells significantly reduced both intracellular and secreted apoB100, with no effect on the secretion of a control protein, albumin. Similar results were obtained in McA-RH7777 rat hepatoma cells. ER-60-stimulated apoB100 degradation and inhibition of apoB100 secretion were sensitive to the protease inhibitor, p-chloromercuribenzoate (pCMB), in a dose-dependent manner but were unaffected by the proteasomal or lysosomal protease inhibitors, N-acetyl-leucinyl-leucinyl-nor-leucinal, E64, and leupeptin. Interestingly, enhanced expression of ER-60 induced apoB100 fragmentation in permeabilized HepG2 cells and resulted in detection of a unique 50 kDa degradation intermediate, a process that could be inhibited by pCMB. Intracellular stability and secretion of apoB100 in primary hamster hepatocytes were also found to be sensitive to pCMB. When taken together, the data suggest an important role for ER-60 in promoting apoB100 degradation via a pCMB-sensitive process in the ER. ER-60 may act directly as a protease or may be involved indirectly as a chaperone/protein factor targeting apoB100 to this nonproteasomal and pCMB-sensitive degradative pathway.
载脂蛋白B（apoB）的共翻译和翻译后调控被认为涉及蛋白酶体和非蛋白酶体途径的降解；然而，apoB降解的非蛋白酶体机制目前尚不清楚。我们之前已经证明新合成的apoB与内质网（ER）-60在细胞内存在关联，ER-60是一种内质网定位的蛋白质，具有蛋白水解和伴侣活性。在本文中，使用含有大鼠ER-60 cDNA的腺病毒表达载体来实现ER-60的剂量和时间依赖性过表达，以研究其在apoB100周转中的作用。过表达的ER-60积聚在HepG2细胞的微粒体腔中，并与致密脂蛋白颗粒中的apoB100相关联。HepG2细胞中ER-60的过表达显著降低了细胞内和分泌的apoB100，而对对照蛋白白蛋白的分泌没有影响。在McA-RH7777大鼠肝癌细胞中也获得了类似的结果。ER-60刺激的apoB100降解和对apoB100分泌的抑制对蛋白酶抑制剂对氯汞苯甲酸（pCMB）呈剂量依赖性敏感，但不受蛋白酶体或溶酶体蛋白酶抑制剂N-乙酰亮氨酰-亮氨酰-正亮氨酸、E64和亮抑酶肽的影响。有趣的是，ER-60表达增强在通透的HepG2细胞中诱导apoB100片段化，并导致检测到一种独特的50 kDa降解中间体，这一过程可被pCMB抑制。还发现原代仓鼠肝细胞中apoB100的细胞内稳定性和分泌对pCMB敏感。综合来看，数据表明ER-60在通过内质网中对pCMB敏感的过程促进apoB100降解中起重要作用。ER-60可能直接作为蛋白酶起作用，或者可能间接作为伴侣/蛋白质因子将apoB100靶向这种非蛋白酶体且对pCMB敏感的降解途径。
