Functional Interplay of Lipid Membrane Components: Activation, Inhibition, and Raft Formation

脂膜成分的功能相互作用：激活、抑制和筏形成

• 批准号: 10220069
• 负责人: Benjamin James Wylie
• 金额: $ 34.99万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-10 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220069
• 关键词: Affinity; Alzheimer' s Disease; Antibiotic Resistance; Behavior; Binding Sites; Biological; Biological Assay; Biological Process; Burkholderia pseudomallei; Cardiolipins; Cellular Membrane; Cholesterol; Communication; Disease; Environment; Etiology; GEM gene; HIV; Health; Heart Diseases; Human; Lipid Bilayers; Lipid Binding; Lipids; Membrane; Membrane Lipids; Membrane Microdomains; Membrane Proteins; Nature; Organism; Parkinson Disease; Pathogenesis; Pharmacologic Substance; Phase; Process; Proteins; Research; Resolution; Signal Transduction; Site; Sterols; Structure; Therapeutic; Transport Process; Virulence; improved; interest; membrane assembly; novel; pathogenic bacteria; programs; proteoliposomes; saturated fat; solid state nuclear magnetic resonance

We will determine how the lipid bilayer organizes around membrane proteins to regulate vital biological functions, including signal transduction and molecular transport. Many lipids and membrane proteins associate to form platforms called lipid rafts, which are phase-separated from the surrounding membrane. The dynamic structure and functional importance of these intermediate-sized (5-200 nm), non-crystalline assemblies are difficult to characterize. Many pathogenic bacteria organize lipid rafts which can increase virulence and antibiotic resistance. In humans, rafts form to facilitate multiple signaling processes. These processes are, in turn, involved in the pathogenesis of diseases, including Alzheimer’s, Parkinson’s, and heart disease. Atomic- resolution dynamic structural details of these assemblies will broaden our understanding of signaling processes and inform disease etiology. We will confront this problem using solid-state NMR (SSNMR) and functional assays in proteoliposomes and biological membranes. Our research program is built around three thematic thrusts: (1) To understand how the lipid environment regulates membrane proteins site-specifically. (2) To determine how membrane proteins, in turn, order their environment. (3) To determine the degree of long-range order and dynamic timescales of these membrane assemblies. Our first target is the KirBac1.1 prokaryotic inward-rectifier K+ (Kir) channel and an array of functional lipids, including synthetic lipids and biological lipid extracts, known to associate with rafts. KirBac1.1 shares many behaviors with eukaryotic Kir channels. It is activated by anionic lipids (especially cardiolipin) and has a high affinity for saturated lipids, cholesterol, and other lipid microdomain-forming components (including hopanoids from the native organism Burkholderia Pseudomallei). The shared regulatory and structural features between KirBac1.1 and eukaryotic Kir channels have inspired several topics of interest: (a) How does the lipid cardiolipin maximally activate KirBac1.1 and trigger transmembrane allostery? Cardiolipin is an essential functional lipid throughout nature, and understanding membrane allostery will inform not only the mechanism of K+ conductance, but the means of transmembrane communications. (b) What is the locus and mechanism of cholesterol/hopanoid induced channel activation? Understanding this is key to determining both how sterols regulate proteins and how they contribute to bilayer organization. (c) How do functional lipid binding sites nucleate rafts? Cardiolipin, cholesterol, and hopanoids are all associated with modulating protein activity and membrane organization; our aim is to understand how they create protein-lipid and lipid-lipid interactions in this process. (d) How does the organization of the annular/nonannular lipid shell act as a secondary regulator of membrane proteins? Kir channels are inactivated by cholesterol, but have a high affinity for rafts. How do cellular membranes organize such that Kir channels can be in rafts, yet retain activity? (e) What is the long-range order and lifetime of these assemblies? It is still unknown if these assemblies persist on the timescale of signaling processes.

我们将确定脂质双层如何围绕膜蛋白组织，以调节重要的生物 功能，包括信号转导和分子运输。许多脂质和膜蛋白联系在一起 形成称为脂筏的平台，它与周围的膜相分离。动态感 这些中等尺寸(5-200 nm)的非晶态组件的结构和功能重要性是 很难描述。许多致病菌会组织脂筏，这种脂筏可以增加毒力和 抗生素耐药性。在人类中，木筏的形成是为了促进多种信号传递过程。这些过程在 反过来，他参与了各种疾病的发病机制，包括阿尔茨海默氏症、帕金森氏症和心脏病。原子- 解析这些组件的动态结构细节将拓宽我们对信号传递的理解 处理并告知疾病病因学。我们将使用固态核磁共振(SS核磁共振)和 蛋白质脂质体和生物膜中的功能分析。我们的研究计划是围绕三个方面建立的 主题性研究：(1)了解脂质环境如何调节膜蛋白的位点特异性。 (2)确定膜蛋白如何依次对其环境进行排序。(三)确定程度 这些膜组件的长程顺序和动态时间尺度。我们的第一个目标是KirBac1.1 原核生物内向整流钾离子(KIR)通道和一系列功能脂质，包括合成脂质和 生物脂类提取物，已知与木筏有关。KirBac1.1与真核生物KIR有许多共同的行为 频道。它被阴离子脂类(尤其是心磷脂)激活，对饱和脂类有很高的亲和力， 胆固醇和其他形成类脂微域的成分(包括来自天然生物体的类胡萝卜素 假腮腺伯克霍尔德氏菌)。KirBac1.1与真核生物的共同调控和结构特征 KIR通道激发了几个感兴趣的话题：(A)脂类心磷脂如何最大限度地激活 KirBac1.1和触发跨膜变构？心磷脂在自然界中是一种必不可少的功能性脂质， 而了解膜变构不仅能揭示K+电导的机制，而且还能揭示K+电导的途径。 跨膜通讯。(B)胆固醇/类胡萝卜素诱导的部位和机制是什么？ 频道激活？了解这一点是确定甾醇如何调节蛋白质以及它们如何调节蛋白质的关键 为双层组织做出贡献。(C)功能性脂结合部位是如何使木筏成核的？心磷脂， 胆固醇和类何首乌都与调节蛋白质活性和膜组织有关；我们的 目的是了解它们如何在这一过程中创造蛋白质-脂和脂-脂相互作用。(D)政府如何 环状/非环状脂壳的组织作为膜蛋白的二级调节器？基尔 通道被胆固醇灭活，但对木筏有很高的亲和力。细胞膜是如何组织的？ 这样KIR通道就可以在木筏上，同时保持活动？(E)这些材料的长期顺序和寿命是多少 集合？目前还不清楚这些程序集是否会在信令过程的时间尺度上持续存在。