Enzymes involved in neuropeptide biosynthesis and regulation

描述（由申请人提供）：神经元、内分泌和免疫组织中的多种蛋白质经历蛋白水解加工。这些蛋白质和肽中的许多是细胞间信使。大多数神经内分泌肽由前体蛋白合成。这些前体的翻译后加工是产生生物活性肽的关键步骤。在大多数情况下，这是通过在涉及枯草杆菌蛋白酶家族的内肽酶的“经典”切割位点处的前体的蛋白水解而发生的。通过在“非经典”切割位点加工产生生物活性肽的子集;负责这些切割的蛋白酶尚未被充分探索。金属内切蛋白酶家族的成员被认为参与非经典加工。其中，内皮素转化酶-2（ECE-2）是一个很好的候选者，因为它表现出与其作为神经肽生物合成酶一致的功能特性。在本授权申请中，我们提出检验“ECE-2是通过非经典加工产生新型生物活性肽的神经肽加工酶”的假设。具体目标是：（i）表征ECE-2的生化和酶学性质，（ii）研究缺乏ECE-2的动物的行为表型，包括成瘾行为，以及（iii）使用差分同位素标记和质谱法检查缺乏ECE-2的小鼠中的肽谱。这些研究是协同作用的，并将通过提供有关新型神经肽的信息来扩展我们目前对神经肽加工的知识。此外，这些研究的结果将为未来探索这些新的神经肽在体内的功能的研究奠定基础。由于各种神经肽参与大脑的正常功能，因此了解调节其内源性水平的机制成为开发用于治疗各种病理学（包括神经退行性疾病和药物成瘾）的策略的先决条件。公共卫生相关性：该提案描述了对内皮素转换酶-2的研究，内皮素转换酶-2是一种肽加工酶，被证明与许多疾病有关，包括药物成瘾和阿尔茨海默病。拟议研究的结果将有助于开发治疗这些和其他神经内分泌疾病的新疗法。

DESCRIPTION (provided by applicant): A wide variety of proteins in neuronal, endocrine and immune tissues undergo proteolytic processing. Many of these proteins and peptides are intercellular messengers. Most neuroendocrine peptides are synthesized from precursor proteins. Post-translational processing of these precursors is a key step in the production of biologically active peptides. In the majority of cases this occurs by proteolysis of the precursors at 'classical' cleavage sites involving endopeptidases of the subtilisin family. A subset of bioactive peptides are generated by processing at 'non-classical' cleavage sites; the protease(s) responsible for these cleavages have not been well explored. Members of the metalloendoprotease family are thought to be involved in non- classical processing. Among them, endothelin converting enzyme-2 (ECE-2) is a good candidate since it exhibits functional properties that are consistent with it being a neuropeptide biosynthetic enzyme. In this grant application we propose to examine the hypothesis that "ECE-2 is neuropeptide processing enzyme that generates novel bioactive peptides by non-classical processing". The specific aims are, (i) to characterize the biochemical and enzymological properties of ECE-2, (ii) to study the behavioral phenotype of animals lacking ECE-2 including addictive behaviors, and (iii) to examine the peptide profile in mice lacking ECE-2 using differential isotopic labeling and mass spectrometry. These studies are synergistic and will expand our current knowledge of neuropeptide processing by providing information about novel neuropeptides. Furthermore, findings from these studies will form a basis for future research exploring the functions of these novel neuropeptides in vivo. Since a variety of neuropeptides are involved in normal functioning of the brain, understanding the mechanisms that regulate their endogenous levels becomes a prerequisite to the development of strategies for the treatment of a variety of pathologies including neurodegenerative disorders and drug addiction. PUBLIC HEALTH RELEVANCE: This proposal describes studies on endothelin converting enzyme-2 - a peptide processing enzyme shown to be involved in a number of disorders including drug addiction and Alzheimer's disease. The findings from the proposed studies would help in the development of novel therapeutics for the treatment of these and other neuroendocrine disorders.