Deorphanizing the Peptidome

人类基因组的成功测序带来了21世纪的下一个科学机遇：蛋白质组的功能注释。大约五分之一的基因组编码分泌蛋白，其中一小部分代表g蛋白偶联受体（gpcr）的信号分子。我们将在本提案中探讨的主要挑战是对参与神经元GPCR信号传导的肽进行全面的“去孤儿化”（从而进行注释）。目前大约有100种非嗅觉孤儿gpcr，其中许多有望使用肽配体；然而，在过去的8年里，只有不到12个新的肽被发现，而在过去的几年里，一个也没有。生物信息学分析表明，基因组包含约150个未经测试的分泌蛋白，它们与已知的肽前体具有生化相似性。我们假设这些蛋白质含有孤儿gpcr缺失的肽配体。然而，为了使这些新的孤儿受体-肽匹配，迫切需要新的肽配体鉴定方法。为了鉴定新的多肽神经递质，我们建议采用一种创新的方法，将生物活性肽合成（Lindberg实验室）的专业知识与GPCR筛选（Roth实验室）的专业知识结合起来。我们将通过实验确认在生物信息学衍生的假定前体列表中存在激素原转化酶可切割位点。生物活性肽将通过生理酶的大规模体外翻译后修饰反应从所有经过验证的前体生成（Lindberg实验室）。然后，我们将通过使用简易筛选技术对已知和孤儿肽受体的整个基因组补体进行功能筛选，发现这些肽的同源受体（Roth实验室）。我们的研究结果将使我们能够将孤儿受体与新的肽配体相匹配，从而提供新的神经肽-受体信号对。由于神经肽信号通路对大脑功能至关重要，包括涉及情绪和认知、精神障碍和药物奖励的通路，我们的研究结果将显著推进我们对精神健康和疾病的理解，也可能产生新的药物靶点。虽然我们将专注于获取和测试神经元表达的前体/配体和受体，但我们的研究也可能发现其他配体-受体匹配；因此，对许多其他由肽- GPCR受体信号通路控制的生理过程的重大影响也是预期的。

The successful sequencing of the human genome has given rise to the next scientific opportunity of the twenty-first century: functional annotation of the proteome. About one-fifth of the genome encodes secretory proteins, a small number of which represent signaling molecules for G-protein coupled receptors (GPCRs). The major challenge that we will explore in this proposal is the comprehensive 'de-orphanization' (and thereby annotation) of the universe of peptides involved in neuronal GPCR signaling. There are currently about 100 non-olfactory orphan GPCRs, many of which are expected to use peptide ligands; however, fewer than a dozen novel peptides have been identified within the last eight years- and none at all within the last few years. Bioinformatics analyses indicate that the genome contains about 150 untested secretory proteins which possess biochemical similarities to known peptide precursors. We postulate that these proteins contain the missing peptide ligands for orphan GPCRs. However, in order to make these new orphan receptor- peptide matches, fresh approaches to peptide ligand identification are urgently needed. To identify novel peptide neurotransmitters we propose to take an innovative approach integrating expertise in bioactive peptide synthesis (Lindberg laboratory) with expertise in GPCR screening (Roth laboratory). We will experimentally confirm the presence of prohormone convertase-cleavable sites in a bioinformatically-derived list of putative precursors. Bioactive peptides will be generated from all validated precursors through large-scale in vitro posttranslational modification reactions using physiological enzymes (Lindberg laboratory). We will then discover cognate receptors to these peptides via functional screening against the entire genomic complement of known and orphan peptide receptors using facile screening technologies (Roth laboratory). Our results will enable us to match orphan receptors with novel peptide ligands, thus providing new neuropeptide-receptor signaling pairs. Since neuropeptide signaling pathways are critical to brain function and include pathways involved in mood and cognition, mental disorders, and drug reward, our results will significantly advance our understanding of mental health and disease, and may also generate new drug targets. While we will focus on obtaining and testing neuronally-expressed precursors/ligands and receptors, our research is also likely to uncover other ligand-receptor matches; thus a major impact on the many other physiological processes controlled by peptide- GPCR receptor signaling pathways is also anticipated.