POMC PROCESSING & BETA-ENDORPHIN RELATED OPIOID PEPTIDE

POMC加工

基本信息

批准号：
6175553
负责人：
Vivian Y. H Hook
金额：
$ 1.37万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-06-01 至 2000-10-14
项目状态：
已结题

项目摘要

Proteolytic processing of proopiomelanocortin (POMC) is required to produce active endogenous beta-endorphin-related opioid peptides that stimulate analgesia. The main goal is to assess the 70 kDa, PC1/3, and PC2 enzymes as candidate POMC processing enzymes by comparing POMC cleavage products, kinetics, enzyme colocalization with beta-endorphin, and effects of antisense expression on POMC processing in pituitary cells. The second goal is to assess whether POMC and proenkephalin (PE) are processed by different enzymes in antisense experiments. These in vitro and cellular studies of POMC processing are feasible because of our recent purification of endogenous POMC cleaving enzymes -- the 70 kDa aspartic proteinase, PC1/3, and PC2. Furthermore, functional rat anterior and intermediate pituitary cells in primary cultures allows examination of the role of each enzyme (in antisense studies) in cells from in vivo tissue that naturally express POMC. Firstly, in vitro processing of POMC will be compared for the 70 kDa aspartic proteinase, PC 1/3, and PC2 with respect to kinetics, cleavage sites, and beta-endorphin/ACTH products. Purified enzymes will be obtained from chromaffin granules of bovine adrenal medulla which are highly enriched in these proteases, and recombinant POMC will be expressed and purified from E. coli. POMC cleavage products will be identified by peptide microsequencing. Secondly, the secretory vesicle localization and neuroendocrine tissue distribution of these enzymes will be compared with that of beta-endorphin and ACTH by immunoelectron microscopy, immunofluorescence histochemistry, and radioimmunoassay. Thirdly, molecular cloning will isolate the 70 kDa aspartic proteinase cDNA by RT- PCR and RACE approaches based on peptide microsequencing of the enzyme; cDNA libraries will be screened with PCR generated probes, complementary oligonucleotides, and anti-aspartic proteinase antibodies. Expression in E. coli or mammalian cells (BSC40) will confirm the cDNA as the 70 kDa aspartic proteinase. Rat and mouse cDNAs will also be isolated for antisense studies in pituitary cells from these species. Lastly, the effects of enzyme antisense expression on POMC processing in anterior and intermediate pituitary cells will be compared. Results may implicate particular proteases in tissue-specific processing of POMC. Comparison of enzyme antisense studies in POMC processing cells with proenkephalin processing in chromaffin cells may indicate whether POMC and PE are processed by different proteases. These studies will provide a comprehensive evaluation of primary POMC processing enzyme(s).

阿黑皮质素原 (POMC) 需要进行蛋白水解加工产生活性内源性 β-内啡肽相关阿片肽刺激镇痛。主要目标是评估 70 kDa、PC1/3 和通过比较 POMC，PC2 酶作为候选 POMC 加工酶裂解产物、动力学、酶与 β-内啡肽的共定位，以及反义表达对垂体细胞 POMC 加工的影响。第二个目标是评估 POMC 和脑啡肽原 (PE) 是否在反义实验中由不同的酶加工。这些在体外 POMC 处理的细胞研究是可行的，因为我们最近内源性 POMC 裂解酶——70 kDa 天冬氨酸的纯化蛋白酶、PC1/3 和 PC2。此外，功能性大鼠前部和原代培养物中的中间垂体细胞可以检查每种酶（在反义研究中）在体内组织细胞中的作用自然表达 POMC。首先，将比较 70 kDa 的 POMC 体外处理天冬氨酸蛋白酶、PC 1/3 和 PC2 的动力学、切割网站和 β-内啡肽/ACTH 产品。将获得纯化的酶来自牛肾上腺髓质的嗜铬颗粒，具有高度富含这些蛋白酶，重组 POMC 将被表达并从大肠杆菌中纯化。 POMC 裂解产物将通过以下方式鉴定肽微测序。其次，分泌囊泡的定位和这些酶的神经内分泌组织分布将与通过免疫电子显微镜观察β-内啡肽和ACTH，免疫荧光组织化学和放射免疫分析。第三，分子克隆将通过 RT-分离 70 kDa 天冬氨酸蛋白酶 cDNA 基于酶肽微测序的 PCR 和 RACE 方法； cDNA 文库将使用 PCR 生成的探针进行筛选，互补寡核苷酸和抗天冬氨酸蛋白酶抗体。表达于大肠杆菌或哺乳动物细胞 (BSC40) 将确认 cDNA 为 70 kDa 天冬氨酸蛋白酶。大鼠和小鼠的 cDNA 也将被分离出来对这些物种的垂体细胞进行反义研究。最后，酶反义表达对前部和后部 POMC 加工的影响将比较中间垂体细胞。结果可能暗示 POMC 组织特异性加工中的特殊蛋白酶。比较脑啡肽原在 POMC 处理细胞中的酶反义研究嗜铬细胞中的加工可能表明 POMC 和 PE 是否经过不同的蛋白酶处理。这些研究将提供主要 POMC 加工酶的综合评价。