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Structural and chemical biology

结构和化学生物学

基本信息

批准号：
10916884
负责人：
Anirban Banerjee
金额：
$ 325.98万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10916884
关键词：
Acyl Coenzyme A Acylation Anabolism Anemia Area Ataxia Attention Binding Biochemical Biological Biological Assay Biology COVID-19 Carbon Catalysis Cell membrane Cell physiology Cells Chemicals Collaborations Cryoelectron Microscopy Cues Cysteine Disease Elements Engineering Enzymes Equilibrium Eukaryota Family Family member Genetic Goals Heart Heme Homeostasis Human Hydrophobicity In Vitro Individual Integral Membrane Protein Intervention Iron Iron Overload Knock-out Knowledge Lead Legionella Legionella pneumophila Legionnaires&apos Disease Link Lipids Literature Macrophage Maintenance Malignant Neoplasms Membrane Membrane Lipids Membrane Proteins Membrane Structure and Function Metabolic Metal Ion Binding Metals Mitochondria Molecular Neurodegenerative Disorders Organism Palmitoyl Coenzyme A Pharmacologic Substance Phosphorylation Physiological Play Pneumonia Post-Translational Protein Processing Process Proliferating Property Protein Family Protein S Proteins Reaction Recombinants Resolution Role Signal Transduction Starvation Structure Sulfur Surface Time Touch sensation Transition Elements Universities Vacuole Virus X-Ray Crystallography Zebrafish biophysical techniques cofactor design enzyme mechanism experimental study heme biosynthesis human disease in vitro Assay inhibitor insight interest knock-down member mutant neuropsychiatric disorder novel overexpression oxygen transport palmitoylation pathogen pathogenic bacteria proteoliposomes reconstitution response tool

项目摘要

1) Structure and function of eukaryotic integral membrane enzymes that catalyze protein lipidation - A. Structural and chemical biology of zDHHC palmitoylacyltransferases - Of the different forms of protein lipidation, protein S-acylation, commonly known as protein palmitoylation, is the most prevalent. Nearly five thousand cellular proteins are modified by posttranslational S-acylation of cysteines. Unlike other lipid attachments, which are thought to be permanent, S-acylation can be reversed by cellular thioesterases, thus enabling dynamic modulation of the local hydrophobicity of substrate proteins. In humans, S-acylation is catalyzed by 23 members of the zDHHC family of integral membrane enzymes, which contain a signature Asp-His-His-Cys (zDHHC) motif. zDHHC enzymes use fatty acyl coenzyme A (predominantly the 16 carbon palmitoyl-CoA) to generate an acyl-enzyme intermediate from which the acyl chain is subsequently transferred to a substrate. With 23 enzymes and thousands of substrates, the complexity of protein S-acylation approaches that of protein phosphorylation and ubiquitylation. Yet, fundamental aspects of zDHHC enzymes, including their mechanism of catalysis and acyl-CoA binding and recognition, have been challenging to analyze without detailed structural information. To obtain insights into the structural mechanism of zDHHC enzymes, we had earlier solved the crystal structures of two zDHHC family members: human zDHHC20 and a catalytically inactive mutant of zebrafish zDHHC15. We also solved the structure of human zDHHC20 conjugated to an irreversible inhibitor that mimics an intermediate in the enzymatic cycle. Using our structures we have now started investigating the question of what are the physiological substrates of each zDHHC enzyme. Each of the 23 zDHHC enzymes catalyzes the S-acylation of many substrates. However, using genetic knockout, knockdown or overexpression of individual zDHHC enzymes with one substrate at a time, it has been suggested in the literature that many substrates can be acted on by more than one zDHHC enzyme. Nevertheless, there is an urgent need to develop a strategy that enables asking this question in the context of the native levels of the zDHHC enzymes. We have designed orthogonal synthetic long chain fatty acyl CoAs that can pair with engineered versions of zDHHC enzymes. We have used these orthogonal acyl CoAs and engineered enzymes to discover the substrates of human zDHHC3 and human zDHHC20. Our results have revealed known substrates, novel substrates as well as proteins that were not known to be palmitoylated in the literature at all. 2) Molecular mechanism of transporters that move transition metals across membranes - A. Structure and function of the mitochondrial iron transporter, Mitoferrin - Mitochondria play a central role in the cellular utilization and balance of iron. Mitoferrin-1 and -2 are the only known major transporters of iron into mitochondria. Subsequently, the iron is utilized in the biosynthesis of heme and in the biosynthesis of iron-sulfur clusters, important cofactors involved in a wide range of cellular activities. Mitoferrin was proposed as an iron transporter from genetic and cell-based studies but the iron transport activity has never been demonstrated through an in vitro assay. To bridge this knowledge gap, we have purified recombinant Mitoferrin-1 and probed its metal ion-binding and transport functions. In order to do so, we had to set up a the first robust in vitro iron transport assay in the literature. With this assay, we demonstrated that Mitoferrin-1 is indeed an iron transporter. Currently we are pursuing high-resolution structural studies of Mitoferrin that will lead to an atomic level understanding of its mechanism. We are also pursuing biochemical studies of Mitoferrin-2 to understand its metal transport properties and how it is distinct from Mitoferrin-1. B. Molecular mechanism of MavN, an iron transporter at the host-pathogen interface of Legionella pneumophila - Legionella pneumophila is a bacterial pathogen that causes a potentially fatal form of pneumonia called Legionnaire's Disease by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. Vacuolar membrane-localized MavN is one amongst only a handful of "core" effectors that are highly conserved in Legionella and was hypothesized to support iron transport. In collaboration with Ralph Isberg (Tufts University), we had determined the topology of MavN and had demonstrated in a proteoliposome reconstituted in vitro transport assay that MavN is a robust transporter of Fe2+. Currently we are conducting further studies to investigate the molecular mechanism of Fe2+ transport by MavN.

1)催化蛋白质脂化的真核生物膜酶的结构和功能 A. zDHHC棕榈酰酰基转移酶的结构和化学生物学-在不同形式的蛋白质脂化中，通常称为蛋白质棕榈酰化的蛋白质S-酰化是最普遍的。近五千种细胞蛋白质通过半胱氨酸的翻译后S-酰化修饰。与其他被认为是永久性的脂质附着不同，S-酰化可以被细胞硫酯酶逆转，从而能够动态调节底物蛋白的局部疏水性。在人类中，S-酰化由23个zDHHC家族的膜内酶催化，其包含特征性Asp-His-His-Cys（zDHHC）基序。zDHHC酶使用脂肪酰基辅酶A（主要是16碳棕榈酰辅酶A）来产生酰基-酶中间体，酰基链随后从该酰基-酶中间体转移到底物。蛋白质S-酰化的复杂性接近于蛋白质磷酸化和泛素化的复杂性，涉及23种酶和数千种底物。然而，zDHHC酶的基本方面，包括它们的催化机制和酰基辅酶A结合和识别，在没有详细结构信息的情况下进行分析是具有挑战性的。为了深入了解zDHHC酶的结构机制，我们之前已经解决了两个zDHHC家族成员的晶体结构：人zDHHC 20和斑马鱼zDHHC 15的催化失活突变体。我们还解决了与模拟酶循环中中间体的不可逆抑制剂缀合的人zDHHC 20的结构。使用我们的结构，我们现在已经开始研究每个zDHHC酶的生理底物是什么的问题。23种zDHHC酶中的每一种都催化许多底物的S-酰化。然而，使用基因敲除、敲低或每次用一种底物过表达单个zDHHC酶，文献中已经提出许多底物可以被多于一种zDHHC酶作用。然而，迫切需要开发一种策略，使zDHHC酶的天然水平的背景下提出这个问题。我们已经设计了正交合成的长链脂肪酰基CoA，其可以与zDHHC酶的工程化版本配对。我们已经使用这些正交酰基CoA和工程化酶来发现人zDHHC 3和人zDHHC 20的底物。我们的研究结果揭示了已知的底物，新的底物以及在文献中根本不知道被棕榈酰化的蛋白质。 2)使过渡金属跨膜移动的转运蛋白的分子机制- A.线粒体铁转运蛋白的结构和功能-线粒体在细胞铁的利用和平衡中起着核心作用。线粒体铁蛋白-1和-2是唯一已知的铁进入线粒体的主要转运蛋白。随后，铁被用于血红素的生物合成和铁硫簇的生物合成，铁硫簇是参与广泛细胞活动的重要辅因子。线粒体铁蛋白被认为是遗传和细胞基础研究中的铁转运蛋白，但其铁转运活性从未通过体外试验得到证实。为了弥补这一知识空白，我们纯化了重组Mitoferrin-1，并探讨了其金属离子结合和转运功能。为了做到这一点，我们必须在文献中建立第一个稳健的体外铁转运试验。通过该试验，我们证明了Mitoferrin-1确实是一种铁转运蛋白。目前，我们正在进行高分辨率的Mitoferrin的结构研究，这将导致其机制的原子水平的理解。我们还在进行Mitoferrin-2的生化研究，以了解其金属转运特性以及它与Mitoferrin-1的区别。 B。嗜肺军团菌是一种细菌病原体，通过在含军团菌的空泡（LCV）中的巨噬细胞内复制，引起一种称为军团菌病的潜在致命形式的肺炎。LCV内的细菌存活和增殖依赖于包含高功能冗余的数百种分泌的效应蛋白。嗜酸性细胞膜定位的MavN是少数在军团菌中高度保守的“核心”效应子之一，并被假设为支持铁转运。与Ralph Isberg（塔夫茨大学）合作，我们确定了MavN的拓扑结构，并在蛋白脂质体重构的体外转运试验中证明了MavN是Fe 2+的稳健转运蛋白。目前，我们正在进行进一步的研究，以探讨通过MavN的Fe 2+运输的分子机制。