PROHORMONE PROCESSING MODEL IN APLYSIA

神经肽和其他肽激素作为前体合成 然后通过有限的蛋白水解作用裂解蛋白质。最近的发现 表明激素原的加工可能是由于 新描述的激素原转化酶 (PC) 家族 在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受到强烈的影响 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，酵母和海兔无脊椎动物模型做出了贡献 对我们对激素原加工的整体理解具有重要意义。 然而，故事还远没有结束。进一步发展和研究 适当的模型激素系统对于回答关键问题是必要的 问题。尽管异源共转染研究表明 转化酶可以处理多种激素原，这些 转化酶实际上在体内进行这种水解吗？他们是什么 作用机制？处理结果是连续动作的结果吗 超过一种酶？为下一个资助期描述的研究 将继续在海兔中开发激素原处理模型 加州涉及产卵激素（ELH），其合成方式为 袋细胞神经元的前体。相关基因也表达于 心房腺，外分泌器官。我们的调查结果以及报告 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PC1、PC2、furin1和furin2） 它们是 ELH 样前体的候选加工酶。因此， 海兔模型独特地允许比较 两个不同组织中的同源基因产物，神经内分泌（袋 细胞）和外分泌（心房腺），有两个复杂程度 基本上相同的转化酶。下一个项目期间的研究 将进一步明确激素原转化酶在 ELH 样前体的加工。过度表达程序将是 使用细菌和真核系统开发，以提供 足够量的 ELH 样前体和激素原转化酶 生化和酶研究。生化研究调查 转化酶介导的激素原加工动力学将提供 上述问题的答案。定点诱变将 探究前体的那些对于化学反应至关重要的结构特征 处理。此外，我们建议调查主要监管 影响激素原转化酶生物合成、激活的分子， 稳定性、活性。从长远来看，这些调查将 实质性开发了激素原加工的海兔模型 为今后的学习打下坚实的基础。更好地理解 激素原处理应该为疾病提供重要的见解 涉及特发性激素功能减退的过程。

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.