Characterizing D-amino acid-containing peptides

含 D-氨基酸肽的表征

• 批准号: 8495426
• 负责人: Jonathan V. Sweedler
• 金额: $ 31.44万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8495426
• 关键词: Age; Amino Acids; Aminopeptidase; Animal Model; Animal Organ; Animals; Aplysia; Binding; Biochemical; Biochemical Pathway; Biological Markers; Biological Models; Brain; Capillary Electrophoresis; Carboxypeptidase; Cell model; Cells; Comparative Study; Complement; Complex; Computer Simulation; Data; Databases; Detection; Development; Digestion; Disease; Endocrine; Endocrine system; Enzymes; Fluorescence; Genomics; Goals; Health; Homeostasis; Hormones; House mice; Illicit Drugs; Immunohistochemistry; In Situ Hybridization; Invertebrates; Investigation; L Forms; Lasers; Link; Location; Mammals; Mass Spectrum Analysis; Measurement; Modeling; Modification; Molecular Structure; Mus; Nerve Tissue; Nervous System Physiology; Nervous system structure; Organism; Pancreas; Pattern; Peptide Mapping; Peptide Signal Sequences; Peptides; Pharmacologic Substance; Phase; Physiological; Physiology; Planarians; Post-Translational Protein Processing; Posterior Pituitary Gland; Prions; Process; Property; Protocols documentation; Rattus; Rattus norvegicus; Reagent; Reporting; Research; Resistance; Sampling; Shapes; Signal Pathway; Signal Transduction; Signaling Molecule; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Structure; System; Technology; Testing; Tissue Sample; Tissues; base; biological systems; enzyme activity; epimerization; genetic manipulation; improved; intercellular communication; mass spectrometer; molecular shape; multiple reaction monitoring; neurophysiology; novel; protein aminoacid sequence; receptor; regenerative; research study; tandem mass spectrometry; three dimensional structure; trend

DESCRIPTION (provided by applicant): The three-dimensional structure of a cell-cell signaling peptide determines how it interacts with its cognate receptor and with various degradation enzymes. An enigmatic, poorly understood peptide modification-the enzymatic epimerization of a single amino acid residue-results in the formation of a D-amino acid-containing peptide (DAACP). Despite significant progress in DAACP characterization, discovery efforts are hampered by shortfalls in current technology. Why do we think there are unknown DAACPs? More than 30 have been reported in a surprising range of animals and organs including the brain; in mammals an undetermined enzyme activity converts several all-L-amino acid-containing peptides into DAACPs, and a unique aminopeptidase in nervous tissue is capable of degrading DAACPs. Accordingly, we hypothesize that DAACPs are widely present in nervous and endocrine tissues throughout the Metazoa. The overarching goal is to characterize the DAACP peptidome and determine the function of the newly uncovered DAACPs. We will create a comprehensive three-stage DAACP discovery funnel (Aim 1): (1) putative DAACP candidates are identified without the need for peptide standards; (2) the presence of the D-amino acid in the putative DAACP is confirmed; (3) using appropriate peptide standards and semi-purified peptides, the DAACPs are sequenced for absolute confirmation. Specifically, the funnel consists of linked analytical approaches: separation and characterization of isobaric peptides, analyses of peptides resistant to enzymatic digestion, separation/digestion of the peptides into their component amino acids for characterization via multiple reaction monitoring and chiral amino acid capillary electrophoresis, and finally, chiral tandem mass spectrometric peptide sequencing hyphenated to in silico structure determination. After funnel optimization, several model organisms will be used in the discovery phase: the exceptional neurophysiological model Aplysia californica with its known and putative DAACPs, and the regenerative planarian model with its ease of genetic manipulations (Aim 2); followed by experiments in mice and rat endocrine and selected nervous tissues (Aim 3). Most signaling peptide systems are ancient and well conserved; it appears that DAACPs are also. These comparative studies allow common biochemical pathways and physiologies to guide subsequent peptide bioactivity tests. Characterizing unknown DAACPs, mapping them to specific locations, correlating their levels to an animal's physiological state, and even electrophysiological testing combines to provide unparalleled information on their function (Aim 4). These research efforts are timely given the wealth of genomic and peptidome information available for our selected animal models and the recent discovery of enzymatic and biochemical data suggesting the presence of DAACPs. The discovery of new signaling DAACPs will improve our understanding of the functioning of the nervous and endocrine systems, and the discovery funnel will have application to a range of fundamental and applied investigations.

描述（由申请人提供）：细胞-细胞信号肽的三维结构决定了它如何与其同源受体以及各种降解酶相互作用。一种神秘的、人们知之甚少的肽修饰——单个氨基酸残基的酶催化差向异构化——导致形成含有 D-氨基酸的肽 (DAACP)。尽管 DAACP 表征取得了重大进展，但当前技术的缺陷阻碍了发现工作。为什么我们认为存在未知的 DAACP？据报道，在一系列令人惊讶的动物和器官（包括大脑）中，有超过 30 种此类现象；在哺乳动物中，一种未确定的酶活性将几种含有全 L 氨基酸的肽转化为 DAACP，而神经组织中的一种独特的氨肽酶能够降解 DAACP。因此，我们假设 DAACP 广泛存在于整个后生动物的神经和内分泌组织中。总体目标是表征 DAACP 肽组并确定新发现的 DAACP 的功能。我们将创建一个全面的三阶段DAACP发现漏斗（目标1）：（1）无需肽标准品即可识别假定的DAACP候选者； (2) 确认推定的DAACP中存在D-氨基酸； (3) 使用适当的肽标准品和半纯化肽，对 DAACP 进行测序以进行绝对确认。具体来说，该漏斗由相关的分析方法组成：同量异位肽的分离和表征、对酶消化具有抗性的肽的分析、将肽分离/消化成其组成氨基酸，以便通过多反应监测和手性氨基酸毛细管电泳进行表征，以及最后，手性串联质谱肽测序与计算机结构测定相结合。漏斗优化后，几种模型生物将用于发现阶段：特殊的神经生理学模型加州海兔（Aplysia californica）及其已知和推定的 DAACP，以及再生涡虫模型及其易于遗传操作（目标 2）；随后在小鼠和大鼠内分泌和选定的神经组织中进行实验（目标 3）。大多数信号肽系统都是古老且保存完好的。看来 DAACP 也是如此。这些比较研究允许常见的生化途径和生理学来指导后续的肽生物活性测试。表征未知的 DAACP，将其映射到特定位置，将其水平与动物的生理状态相关联，甚至结合电生理测试来提供有关其功能的无与伦比的信息（目标 4）。鉴于我们选定的动物模型拥有丰富的基因组和肽组信息，以及最近发现的酶和生化数据表明 DAACP 的存在，这些研究工作是及时的。新信号 DAACP 的发现将提高我们对神经和内分泌系统功能的理解，并且发现漏斗将应用于一系列基础和应用研究。