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会：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(GPCRs)的信号分子。在这项提案中，我们将探索的主要挑战是对参与神经元GPCR信号的多肽进行全面的“去孤立化”(从而进行注释)。目前大约有100个非嗅觉孤儿GPCR，其中许多有望使用多肽配体；然而，在过去八年中发现的新多肽不到12个，在过去几年中根本没有发现任何一个。生物信息学分析表明，该基因组包含约150个未检测的分泌蛋白，它们与已知的多肽前体具有生物化学相似性。我们推测这些蛋白含有孤儿GPCRs缺失的多肽配体。然而，为了使这些新的孤儿受体-多肽匹配，迫切需要新的多肽配体鉴定方法。为了确定新的多肽神经递质，我们建议采取一种创新的方法，将生物活性多肽合成方面的专业知识(Lindberg实验室)与GPCR筛选专业知识(Roth实验室)相结合。我们将在实验上确认在生物信息学推导的假定前体列表中存在激素前体转换酶裂解位点。生物活性多肽将通过使用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中产生(林德伯格实验室)。然后，我们将通过使用简易筛选技术(Roth实验室)针对已知和孤儿多肽受体的整个基因组进行功能筛选，发现这些多肽的同源受体(Roth实验室)。我们的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，我们的结果将显著促进我们对心理健康和疾病的理解，并可能产生新的药物靶点。虽然我们将专注于获得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，也有望对由肽-GPCR受体信号通路控制的许多其他生理过程产生重大影响。

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.