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: 9978891
• 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9978891
• 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组织在膜蛋白周围，以调节重要的生物活性。 功能，包括信号转导和分子转运。许多脂质和膜蛋白 形成称为脂筏的平台，脂筏与周围的膜相分离。动态 这些中等尺寸（5-200 nm）非结晶组装体的结构和功能重要性 很难描述。许多病原菌组织脂筏，这可以增加毒力， 抗生素耐药性在人类中，筏的形成是为了促进多种信号传导过程。这些过程，在 反过来，参与疾病的发病机制，包括阿尔茨海默氏症，帕金森氏症和心脏病。原子- 这些组件的分辨率动态结构细节将拓宽我们对信号的理解 过程和告知疾病病因。我们将使用固态NMR（SSNMR）来解决这个问题， 蛋白脂质体和生物膜中的功能测定。我们的研究项目围绕三个方面展开 主要研究方向：（1）了解脂质环境如何对膜蛋白进行位点特异性调控。 (2)以确定膜蛋白如何依次排列它们的环境。(3)确定…的程度 这些膜组件的长程有序和动态时标。我们的第一个目标是KirBac1.1 原核细胞内向整流钾离子（Kir）通道和一系列功能性脂质，包括合成脂质和 生物脂质提取物，已知与木筏有关。KirBac1.1与真核生物Kir有许多共同的行为 渠道它被阴离子脂质（特别是心磷脂）激活，对饱和脂质具有高亲和力， 胆固醇和其他脂质微区形成组分（包括来自天然生物体的类霍帕酸 类鼻疽伯克霍尔德氏菌）。KirBac1.1与真核生物共有的调控和结构特征 Kir通道激发了几个感兴趣的主题：（a）脂质心磷脂如何最大限度地激活 KirBac1.1和触发跨膜变构？心磷脂是自然界中一种重要的功能性脂质， 了解细胞膜的变构性不仅有助于了解钾离子传导的机制， 跨膜通讯的能力。(b)胆固醇/hopanoid诱导的位点和机制是什么 通道激活？理解这一点是确定固醇如何调节蛋白质以及它们如何 有助于形成双层结构。(c)功能性脂质结合位点如何使筏成核？心磷脂， 胆固醇和hopanoids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产生蛋白质-脂质和脂质-脂质相互作用。(d)如何 环状/非环状脂质壳的组织作为膜蛋白的二级调节因子？Kir 通道被胆固醇灭活，但对筏具有高亲和力。细胞膜是如何组织的 这样基尔通道就可以在木筏上，但仍然保持活动？(e)这些的长程有序度和寿命是多少 集会？目前尚不清楚这些组装是否在信号传导过程的时间尺度上持续存在。