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

基本信息

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
项目类别：
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&apos -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

项目摘要

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体的天然产物构成了分子的丰富来源，这些分子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、聚酮合酶（PKS）和/或非核糖体肽合成酶（NRPS）以线性“装配线”方式合成的。 PKS/NRPS 由多个多肽（模块）组成，每个多肽（模块）具有多个功能域，这些功能域共价加载适当的构建块（例如，PKS 的丙二酰基和 NRPS 的活化氨基酸），并依次将它们凝结到不断增长的天然产物链上。通常，需要额外的酶进行进一步加工，例如附着碳水化合物。每个模块内还存在酶促多样性，例如脱水、还原和烷基化反应可以发生在不断增长的天然产物链中的任何位置，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进展，开始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，有价值的天然产物是已知的，但由于基因组测序方面的挑战等原因，相应的生物合成途径仍未被发现。对于此类系统，蛋白质而非 DNA 水平的途径发现正在成为一种有吸引力的方法，该方法还可以验证生物合成蛋白质的表达。然而，由于收集的宏蛋白质组样本的复杂性，需要有针对性的方法。该提案描述了靶向 PKS/NRPS 蛋白质组学创新方法的开发，以及它们在甲藻短卡伦虫通路发现中的应用。这种海洋浮游生物产生结构高度复杂的短尾毒素，导致与佛罗里达赤潮相关的死亡和疾病，以及抗毒素短尾醛，目前正在临床试验中用于治疗哮喘和囊性纤维化。我们将利用 PKS/NRPS 上磷酸泛硫氨酸 (Ppant) 辅基的高红外吸收，通过质谱法以平行而不是传统的顺序方式选择性检测蛋白水解消化物中含有 Ppant 的肽。这一创新策略将在高度复杂的宏蛋白质组样品中得到验证，例如已批准的抗癌剂 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此类复杂的数据集。为了提高选择性，我们将开发类似于次级代谢物生物合成中间体的红外活性化学探针，用于加载到 PKS/NRPS 上。这些方法将应用于在收集的短短杆菌样本中发现 PKS。生物合成途径的鉴定将允许表征相应的无疑高度复杂的生物合成机制，并为可持续药物生产提供途径。