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: 8630175
• 负责人: KRISTINA HAKANSSON
• 金额: $ 28万
• 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/8630175
• 关键词: 4' -phosphopantetheine; Acyl Carrier Protein; Alkylation; Amino Acids; Anabolism; Antibiotics; Antineoplastic Agents; Antitoxins; Asthma; Bacteria; Biochemical; Bioinformatics; Biological Factors; Biological Models; 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; 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; Organism; Pantetheine; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Phosphopeptides; Plankton; Plants; Positioning Attribute; Post-Translational Protein Processing; Process; Prosthesis; Proteins; Proteome; Proteomics; Reaction; Recombinant Proteins; Research; Research Personnel; Sampling; Serine; Shotguns; Source; Streptomyces; Structure; Sulfhydryl Compounds; System; Therapeutic; United States; Urochordata; absorption; base; brevetoxin; drug discovery; drug production; enzyme mechanism; experience; genome sequencing; human disease; improved; innovation; inorganic phosphate; interest; microbial; novel; novel strategies; overexpression; peptide synthase; picromycin; polyketide synthase; polypeptide; protein aminoacid sequence; protein expression; public health relevance; reconstitution; red tide; respiratory

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 trials 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.

天然产物来自各种生物，如细菌、植物和海洋无脊椎动物，构成了丰富的 与人类疾病有关的具有广泛生物活性的分子的来源，包括抗生素和抗肿瘤药物 抗癌药物。大量这些结构复杂的次生代谢物是通过大量的 酶复合体，聚酮合成酶(PKS)和/或非核糖体多肽合成酶(NRPS) 线性“流水线”方式。PKSS/NRPS由多个多肽(模块)组成，每个模块具有多个 共价负载适当构建块的功能结构域(例如，PKS的丙二酰基和 NRPS的活性氨基酸)，并将它们顺序凝聚到不断增长的天然产品链上。 通常，更多的酶被用于进一步的处理，例如碳水化合物的附着。的确有 在每个模块内也有酶的变化，例如，脱水、还原和烷基化反应可以 发生在不断增长的自然产品链中的任何位置，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 允许通过途径工程来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 在某些情况下，有价值的天然产物已知，但相应的生物合成途径仍未发现 例如，由于基因组测序中的挑战。对于这样的系统，在蛋白质上发现途径 DNA水平正在成为一种有吸引力的方法，也可以验证生物合成蛋白质的表达。 然而，由于收集的偏蛋白质组样本的复杂性，需要有针对性的方法。这 提案描述了靶向PKS/NRPS蛋白质组学的创新方法的发展，以及 它们在短甲藻途径发现中的应用。这种海洋浮游生物产生 结构高度复杂的短杆菌毒素，与佛罗里达红相关的死亡和疾病有关 该药目前正处于治疗哮喘和囊性纤维化的临床试验中。 我们将利用磷酸丙氨酸(PPANT)假体基团的高红外吸收 PKSS/NRPSs联用质谱法选择性检测小鼠蛋白水解液中的PANT多肽 并行而不是传统的顺序方式。这一创新战略将在 复杂的代谢蛋白质组样本，例如批准的抗癌药物的衣酸盐/微生物共生体生产者 试剂ET-743，我们最近证明了对其进行生物合成蛋白检测的可行性。我们还将 开发合适的生物信息学方法来自动挖掘这类复杂的数据集。对于增加的 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PKSS/NRPS上。这些方法将应用于收集到的PKS发现 K.Brivis样本。生物合成途径的鉴定将允许表征相应的 毫无疑问，生物合成机制非常复杂，并为可持续的药物生产提供了一扇大门。