CELL TYPE-SPECIFIC PROCESSING OF OPIOID PEPTIDES

阿片肽的细胞类型特异性加工

• 批准号: 2140056
• 负责人: Gary Thomas
• 金额: $ 21.67万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140056
• 关键词: affinity labeling; cell type; chemical cleavage; chemical kinetics; endopeptidases; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; high performance liquid chromatography; immunoelectron microscopy; ion exchange chromatography; peptide hormone biosynthesis; posttranslational modifications; proopiomelanocortin; protease inhibitor; protein sequence; secretory protein; site directed mutagenesis; synthetic peptide; tissue /cell culture; transfection

DESCRIPTION (adapted from the applicant's abstract): Key to under- standing the early events in prohormone processing is the identification of the enzymes involved. Recently, this group and others have shown that a group of putative mammalian endoproteases, furin, PC2 and PC3, identified by homology to the yeast mating hormone processing enzyme, kex2p, are able to enhance the cleavage of precursor proteins when co- expressed in heterologous cells. Furthermore, two of these enzymes, PC2 and PC3, not only enhance processing of proopiomelanocortin (POMC) and proinsulin at authentic paired basic sites, but also have a pattern of expression consistent with a role in the tissue specific processing of these substrates in vivo.These co-expression studies, however, fail to show a direct interaction of PC2 and PC3 with their putative substrates and also suggest that factors in addition to differential expression of enzymes modulate the cell-specific processing phenotype. The proposed studies will first examine cleavage site specificities and kinetic properties of purified PC2 and PC3 using intact POMC and synthetic peptides in vitro. This information will aid the development of specific inhibitors and affinity labeling reagents with which to dissect the functional properties of these enzymes in vivo. Second, the importance of proteolytic maturation of PC2 and PC3, including its possible contribution to cell type specific activation of the enzymes and to the regulation of prohormone processing will be determined. Third, the following hypothesis will be evaluated: that compartmentalization and/or membrane association of PC2 and PC3 within the regulated secretory pathway is determined by their amphipathic helices and/or a protein binding domain conserved in evolution.Finally, the possibility that tissue specific prohormone processing reflects a two-tiered system, combining differential expression of PC2 and PC3 with post-translational regulation will be tested. Cell type-specific maturation, activation and modulation of PC2 and PC3 in vivo will be sought.

描述（根据申请人的摘要改编）： 在激素加工中站立的早期事件是识别 涉及的酶。 最近，这个小组和其他人表明 一组推定的哺乳动物内药物，Furin，PC2和PC3， 通过与酵母交配激素加工酶同源性鉴定， Kex2p，能够增强前体蛋白的裂解 在异源细胞中表达。此外，其中两个酶，PC2 和PC3，不仅增强了蛋白酶素皮质（POMC）和 在真实配对的基本站点上的proinsulin，但也有一个模式 表达与在组织特异性处理中的作用一致的表达 这些共同表达的研究未能 显示PC2和PC3与推定的底物的直接相互作用 还建议除了差异表达的因素 酶调节细胞特异性加工表型。 提议 研究将首先检查裂解位点特异性和动力学 使用完整的POMC和合成的纯化PC2和PC3的属性 体外肽。这些信息将有助于开发特定 抑制剂和亲和力标记试剂剖析试剂 这些酶在体内的功能特性。 第二，重要性 PC2和PC3的蛋白水解成熟度，包括可能 对酶的细胞类型特异性激活的贡献和 将确定激素加工的调节。 第三， 将评估以下假设：分隔和/或 PC2和PC3的膜协会在受监管的分泌中 途径由它们的两亲螺旋和/或蛋白质确定 结合域在进化中保守。在最后，这种可能性 组织特异性激素加工反映了一个两层的系统， 将PC2和PC3的差异表达与翻译后的差异表达相结合 法规将进行测试。细胞类型特异性的成熟，激活和 将寻求PC2和PC3的调节。