PROHORMONE PROCESSING MODEL IN APLYSIA

海兔的激素原加工模型

• 批准号: 2431192
• 负责人: ALEXANDER none KUROSKY
• 金额: $ 23.34万
• 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/2431192
• 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; prohormone convertase; 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.

神经肽和其他肽类激素作为前体被合成 然后通过有限的蛋白质水解被切割的蛋白质。最近的调查结果 表明激素原的加工可能是由于 一个新描述的激素原转化酶家族， 进化上与细菌枯草杆菌蛋白酶有关。作用方式， 这些蛋白酶的定位和组织特异性在强烈的 调查已知的转化酶，以及激素原加工 一般来说，从酵母到哺乳动物都非常保守。 因此，酵母和无脊椎动物模型 这对我们对激素原加工的整体理解有很大的帮助。 然而，这个故事还远未完成;进一步的发展和研究 适当的模型激素系统是必要的，以回答关键 问题.尽管异源共转染研究表明 转化酶可以处理各种激素原， 转化酶实际上在体内进行这种水解？什么是他们 行动机制？处理是否是连续操作的结果 一种以上的酶？下一个供资期的研究 将继续在南极洲开发激素原加工模型 涉及产卵激素（ELH），其合成为 是囊细胞神经元的前体相关基因也表达于 心房腺，一个外分泌器官。我们的调查结果，以及 其他人已经证明心房腺和袋细胞神经元 表达多种转化酶（包括PC 1、PC 2、furin 1和furin 2） 它们是ELH样前体的候选加工酶。因此，在本发明中， 该模型唯一允许比较的处理， 两种不同组织中同源基因产物，神经内分泌（袋 细胞）和外分泌（心房腺），在两个层次的复杂性， 基本上相同的转化酶。下一个项目期间的研究 将进一步确定激素原转化酶在 ELH类前体的加工。过表达程序将 利用细菌和真核系统， 足够量的ELH样前体和激素原转化酶， 生化和酶学研究。生物化学研究调查了 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。定点诱变将 探索那些对我们的研究至关重要的前体的结构特征， 处理.此外，我们建议调查主要的监管机构， 影响激素原转化酶生物合成，活化， 稳定性和活性。从长远来看，这些调查将 实质性地发展激素原加工的失智症模型， 为今后的学习打下坚实的基础。更好地了解 激素原的加工过程将为疾病的研究提供重要的见解 涉及特发性激素功能减退的过程。