Targeted Proteomic Approaches for Natural Product Biosynthetic Pathway Discovery

天然产物生物合成途径发现的靶向蛋白质组学方法

• 批准号: 9203060
• 负责人: KRISTINA HAKANSSON
• 金额: $ 28.1万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9203060
• 关键词: 4' -phosphopantetheine; Acyl Carrier Protein; Alkylation; Amino Acids; Anabolism; Antibiotics; Antineoplastic Agents; Antitoxins; Asthma; Bacteria; Biochemical; Bioinformatics; Biological Models; Biosynthetic Proteins; Brevenal; Carbohydrates; Carbon Dioxide; Carrier Proteins; Cells; Cessation of life; Chemicals; Clinical; Clinical Treatment; Clinical Trials; Collaborations; Complex; Crosslinker; Cystic Fibrosis; DNA; Data; Data Set; Dehydration; Detection; Development; Dimensions; Dinophyceae; Dissociation; Ecteinascidin 743; Electron Transport; Electrons; Engineering; Environment; Enzymes; Florida; Fourier transform ion cyclotron resonance; Gases; Gene Cluster; Generations; Genome; Health; Human; Human Genome; Hybrids; In Vitro; Ions; Irritants; Knowledge; Lasers; Link; Marine Invertebrates; Marines; Mass Spectrum Analysis; Methods; Mining; Modification; Molecular Weight; Multienzyme Complexes; Natural Products; Organism; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Phosphopeptides; Plankton; Plants; Positioning Attribute; Post-Translational Protein Processing; Process; Prosthesis; Proteins; Proteome; Proteomics; Reaction; Research; Research Personnel; Sampling; Serine; Shotguns; Source; Streptomyces; Structure; Sulfhydryl Compounds; System; Therapeutic; United States; Urochordata; absorption; base; bioactive natural products; biosynthetic product; brevetoxin; drug discovery; drug production; enzyme mechanism; experience; genome sequencing; human disease; improved; innovation; inorganic phosphate; interest; link protein; metaproteomics; microbial; novel; novel strategies; overexpression; peptide drug; peptide synthase; picromycin; polyketide synthase; polypeptide; protein expression; public health relevance; reconstitution; red tide; respiratory; whole genome

DESCRIPTION (provided by applicant): Natural products from organisms as diverse as bacteria, plants, and marine invertebrates constitute a rich source of molecules with wide-ranging bioactivities related to human disease, including antibiotics and anti- cancer agents. A plentitude of these structurally complex secondary metabolites are synthesized by large enzyme complexes, polyketide synthases (PKSs) and/or nonribosomal peptide synthetases (NRPSs), in a linear "assembly-line" manner. PKSs/NRPSs consist of multiple polypeptides (modules), each with multiple functional domains that covalently load appropriate building blocks (e.g., malonyl groups for PKSs and activated amino acids for NRPSs) and sequentially condense them onto the growing natural product chain. Often, additional enzymes are involved for further processing, such as attachment of carbohydrates. There is also enzymatic variety within each module such that, e.g., dehydration, reduction, and alkylation reactions may occur at any position in the growing natural product chain for increased structural diversity. Tremendous advances in our understanding of natural product biosynthetic pathways are beginning to allow pathway engineering for generation of compounds with new or improved bioactivities. However, in many cases, valuable natural products are known but the corresponding biosynthetic pathways remain undiscovered due to, e.g., challenges in genome sequencing. For such systems, pathway discovery at the protein rather than DNA level is emerging as an attractive approach that also verifies biosynthetic protein expression. However, due to the complexity of collected metaproteomic samples, targeted methods are needed. This proposal describes the development of innovative methods for targeted PKS/NRPS proteomics, as well as their application for pathway discovery in the dinoflagellate Karenia brevis. This marine plankton produces the highly structurally complex brevetoxins, responsible for the deaths and illnesses associated with the Florida red tide, as well as the antitoxin, brevenal, currently in clinical trils for treatment of asthma and cystic fibrosis. We will harness the high infrared absorption of phosphopantetheine (Ppant) prosthetic groups on PKSs/NRPSs to selectively detect Ppant-containing peptides in proteolytic digests with mass spectrometry in a parallel rather than the conventional sequential manner. This innovative strategy will be validated in highly complex metaproteomic samples such as the tunicate/microbial symbiont producer of the approved anti-cancer agent ET-743, for which we recently demonstrated feasibility of biosynthetic protein detection. We will also develop suitable bioinformatic approaches for automated mining of such complex datasets. For increased selectivity, we will develop IR-active chemical probes, resembling secondary metabolite biosynthetic intermediates, for loading onto PKSs/NRPSs. These approaches will be applied for PKS discovery in collected K. brevis samples. Biosynthetic pathway identification will allow characterization of the corresponding undoubtedly highly intricate biosynthetic mechanisms, and provide a gateway to sustainable drug production.

描述（由申请人提供）：来自细菌、植物和海洋无脊椎动物等多种生物的天然产物构成了丰富的分子来源，具有与人类疾病相关的广泛生物活性，包括抗生素和抗癌剂。大量这些结构复杂的次级代谢物是由大型酶复合物，聚酮合成酶（pks）和/或非核糖体肽合成酶（NRPSs）以线性“装配线”方式合成的。PKSs/NRPSs由多个多肽（模块）组成，每个多肽都有多个功能域，共价加载适当的构建块（例如PKSs的丙二酰基和NRPSs的活化氨基酸），并依次将它们凝聚到不断增长的天然产物链上。通常，还需要额外的酶来进行进一步的加工，比如碳水化合物的附着。在每个模块中也存在酶的多样性，例如，脱水、还原和烷基化反应可以在不断增长的天然产物链的任何位置发生，以增加结构多样性。我们对天然产物生物合成途径的理解取得了巨大的进步，这使得途径工程能够产生具有新的或改进的生物活性的化合物。然而，在许多情况下，已知有价值的天然产物，但由于基因组测序方面的挑战，相应的生物合成途径仍未被发现。对于这样的系统，在蛋白质而不是DNA水平上发现途径正在成为一种有吸引力的方法，也可以验证生物合成蛋白的表达。然而，由于收集的元蛋白质组学样本的复杂性，需要有针对性的方法。本提案描述了靶向PKS/NRPS蛋白质组学的创新方法的发展，以及它们在鞭毛藻短凯伦氏菌（Karenia brevis）中途径发现的应用。这种海洋浮游生物产生结构高度复杂的短藻毒素，造成与佛罗里达赤潮有关的死亡和疾病，以及抗毒素，短藻毒素，目前用于治疗哮喘和囊性纤维化的临床试验。我们将利用pks /NRPSs上磷蚁氨酸（Ppant）假体基的高红外吸收，用质谱法平行而不是传统的顺序方式选择性地检测蛋白水解消化中含有Ppant的肽。这一创新策略将在高度复杂的元蛋白质组学样品中得到验证，例如被批准的抗癌药物ET-743的被囊动物/微生物共生体生产者，我们最近证明了生物合成蛋白质检测的可行性。我们还将开发合适的生物信息学方法来自动挖掘这些复杂的数据集。为了提高选择性，我们将开发红外活性化学探针，类似于次生代谢物生物合成中间体，用于装载到pks /NRPSs上。这些方法将应用于在收集的短链镰刀菌样本中发现PKS。生物合成途径的识别将允许对相应的高度复杂的生物合成机制进行表征，并为可持续的药物生产提供门户。