PROHORMONE PROCESSING MODEL IN APLYSIA

海兔的激素原加工模型

• 批准号: 2267487
• 负责人: ALEXANDER none KUROSKY
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2267487
• 关键词: Aplysia; alternatives to animals in research; biological models; circular dichroism; endopeptidases; enzyme inhibitors; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; hormone regulation /control mechanism; immunocytochemistry; immunologic assay /test; in situ hybridization; invertebrate hormone; laboratory rabbit; neuropeptides; peptide hormone; peptide hormone biosynthesis; protein purification; protein sequence; site directed mutagenesis; tissue /cell culture; transfection

Neuropeptides and other peptide hormones are synthesized as precursor proteins that are then cleaved by limited proteolysis. Recent findings indicate that the processing of prohormones may be due to the action of a newly described family of prohormone convertases (PCs) that are evolutionarily related to bacterial subtilisin. The mode of action, localization, and tissue specificity of these proteases are under intense investigation. The known convertases, as well as prohormone processing in general, are remarkably conserved from yeast to mammals. Consequently, the yeast and Aplysia invertebrate models have contributed significantly to our overall understanding of prohormone processing. However, the story is far from complete; further development and study of appropriate model hormone systems are necessary to answer critical questions. Although heterologous co-transfection studies have indicated that the convertases can process a variety of prohormones, do these convertases in fact carry out such hydrolysis in vivo? What is their mechanism of action? Does processing result from the consecutive action of more than one enzyme? Research described for the next funding period will continue to exploit a prohormone processing model in Aplysia californica involving egg-laying hormone (ELH), which is synthesized as a precursor by bag cell neurons. Related genes are also expressed in the atrial gland, an exocrine organ. Our findings, as well as reports of others, have demonstrated that the atrial gland and the bag cell neurons express a variety of convertases (including PC1, PC2, furin1, and furin2) that are candidate processing enzymes of the ELH-like precursors. Thus, the Aplysia model uniquely allows comparison of the processing of homologous gene products in two different tissues, neuroendocrine (bag cells) and exocrine (atrial gland), at two levels of complexity by essentially identical convertases. Research in the next project period will further define the role of the prohormone convertases in the processing of ELH-like precursors. Overexpression procedures will be developed, using bacterial and eucaryotic systems, to make available sufficient amounts of ELH-like precursors and prohormone convertases for biochemical and enzymic studies. Biochemical studies investigating the kinetics of convertase-mediated prohormone processing will provide answers for the questions raised above. Site-directed mutagenesis will probe those structural features of the precursors that are-critical for processing. In addition, we propose to investigate major regulatory molecules that impact on prohormone convertase biosynthesis, activation, stability, and activity. In the longer term, these investigations will substantially develop the Aplysia model of prohormone processing and provide a solid foundation for future studies. Better understanding of prohormone processing should provide important insights into disease processes involving idiopathic hormone hypofunction.

神经肽和其他多肽激素是合成的前体 这些蛋白质随后被有限的蛋白分解所切割。最新发现 表明前激素的处理可能是由于 一个新描述的前激素转换酶(PC)家族，它们是 在进化上与细菌枯草杆菌毒素相关。行动模式， 这些蛋白水解酶的定位和组织特异性都很低。 调查。已知的转化酶，以及激素前体的处理 总体而言，从酵母到哺乳动物都非常保守。 因此，酵母菌和海兔无脊椎动物模型做出了贡献 对我们对激素前体过程的整体理解具有重要意义。 然而，故事还远远没有完成；进一步的发展和研究 适当的模型荷尔蒙系统是回答关键问题所必需的 问题。尽管异源共转染研究表明 转换酶可以处理各种激素原，做这些 转化酶实际上在体内进行这样的水解吗？他们的产品是什么 作用机制？处理是否由连续的操作产生 一种以上的酶？为下一个资助期描述的研究 将继续在海兔开发激素前体处理模式 含有产卵激素(ELH)的Calfornica，合成为 一个由袋状细胞神经元组成的前体。相关基因也在细胞中表达 心房腺，外分泌器官。我们的发现，以及关于 另一些则证明了心房腺和袋状细胞神经元 表达各种转换酶(包括PC1、PC2、Furin1和Furin2) 它们是ELH样前体的候选加工酶。因此， Aplysia模型唯一允许比较 两种不同组织中的同源基因产物，神经内分泌(BAG) 细胞)和外分泌(心房腺)，在两个复杂程度上 本质上完全相同的转化酶。在下一个项目阶段进行研究 将进一步定义前激素转换酶在 ELH类前体的加工。过度表达的程序将是 利用细菌和真核系统开发的，以提供 足够量的ELH样前体和前激素转换酶 生化和酶学研究。生物化学研究调查 转化酶介导的激素原加工的动力学将提供 回答上面提出的问题。定点突变将 探索前兆的那些结构特征，这些结构特征对 正在处理。此外，我们建议调查主要监管机构 影响前激素转换酶生物合成、激活的分子， 稳定性和活跃性。从长远来看，这些调查将 实质上发展海兔的激素前体处理模型和 为今后的研究奠定了坚实的基础。更好地了解 激素原的处理应该提供对疾病的重要见解 涉及特发性荷尔蒙功能低下的过程。