Melanopsin Signal Transduction Studied by FTIR Spectroscopy

通过 FTIR 光谱研究黑视蛋白信号转导

• 批准号: 8271423
• 负责人: KENNETH J ROTHSCHILD
• 金额: $ 31.41万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8271423
• 关键词: Adrenergic Receptor; Affect; Arrestins; Bacteriorhodopsins; Biological Clocks; Calcitonin Receptor; Cells; Circadian Rhythms; Collaborations; Color; Complex; Cyclic Nucleotides; Diet Habits; Disease; G Protein-Coupled Receptor Signaling; G alpha q Protein; Glaucoma; Histamine Receptor; Hormonal; Human; In Vitro; Inositol; Invertebrates; Isotope Labeling; Isotopes; Laboratories; Light; Measures; Medical; Membrane; Membrane Proteins; Methods; Microscopy; Modeling; Molecular; Muscarinic Acetylcholine Receptor; Optics; Pathway interactions; Pharmaceutical Preparations; Photons; Physiological; Physiological Processes; Process; Property; Proteins; Proton Pump; Psyche structure; Receptor Signaling; Recombinants; Research; Retina; Retinal Ganglion Cells; Rhodopsin; Sensory Rhodopsins; Sequence Homology; Serotonin; Signal Transduction; Site-Directed Mutagenesis; Sleep; Sleep Disorders; Spectroscopy, Fourier Transform Infrared; Squid; Techniques; Texas; Time; Transducers; United States National Institutes of Health; Universities; Visual; Water; Work; absorption; alertness; analog; arrestin 2; base; interfacial; melanopsin; method development; photoactivation; public health relevance; receptor; response; time use; tripolyphosphate; two-photon

DESCRIPTION (provided by applicant): The overall objective of this project is to investigate the molecular basis of signal transduction in human melanopsin (MO), the recently discovered light-receptor in photosensitive retinal ganglion cells, which underlies the control of circadian rhythms and pupillary response. Because melanopsin is involved in a variety of physiological functions including sleep, mental alertness, eating habits, and hormonal levels, as well potentially involved in a variety of disorders including sleep disorders, seasonal affected disorders and glaucoma, the NIH has highlighted melanopsin as a priority for future research. Remarkably, the properties of melanopsin strongly resemble invertebrate rhodopsin instead of the extensively studied vertebrate "visual" rhodopsins. Similarities include a close primary sequence homology and signaling through the Gq-protein (phospholipaseC/inositol triphosphate) pathway instead of the Gt-protein cyclic nucleotide pathway. Importantly, melanopsin and its analog invertebrate rhodopsins such as squid rhodopsin (sRh) serve as models for investigating the signal transduction mechanism in the hundreds of GPCRs in human cells. Such GPCRs signal through the Gq-protein pathway and are inhibited by 2-arrestin2 instead of the more specialized visual 2-arrestin. Prominent examples include serotonin, histamine, adrenergic, muscarinic and calcitonin receptors which are targets of current and potentially new drugs. A key feature of melanopsin and invertebrate rhodopsins but not vertebrate rhodopsins is their optical bistability. This property allows them to be "cycled" between two different stable states using two different colors of light. In this project, we will exploit this two-photon property in order to investigate the detailed structural changes occurring upon light activation in melanopsin, sRh and the complexes they formed with 2- arrestin2 and Gq-protein. This research will be facilitated by the application of several advanced FTIR difference techniques, many developed in our laboratory, in conjunction with site-directed mutagenesis and isotope labeling. Application of this approach has led previously to several milestones including the first detailed characterization of the conformational changes which occur during vertebrate rhodopsin photoactivation and the proton pumping mechanism of bacteriorhodopsin. We have recently demonstrated the ability of this approach to also detect and characterize structural changes in key residues and internal water molecules that lie in the interfacial contact region between membrane protein signaling receptors such as sensory rhodopsin II and its cognate transducer. HtrII In preliminary studies, we have measured static and time resolve FTIR difference spectra of squid rhodopsin and its 2-arrestin2 complex. By using isotope editing, we can characterize conformational changes separately in the receptor and 2-arrestin2 components. The proposed studies will also benefit from our recent development of methods to: i) measure sub-picosecond protein changes; ii) probe minute quantities of membrane proteins including single crystals using time-resolved FTIR microscopy and iii) rapidly in vitro express membrane proteins in nanolipoparticles (NLPs). This work will be facilitated by close collaborations with the laboratories of Dr. J. Navarro at the University of Texas Medical Branch, Galveston who will prepare sRho/2-arrestin2 crystals and perform parallel x-ray crystallographic studies; Dr. W. DeGrip at the University of Nijmegen whose laboratory has expressed and characterized functional recombinant melanopsin and Dr. M. Coleman at the LLNL and UC Davis whose laboratory has developed cell-free techniques to express GPCRs in NLPs. PUBLIC HEALTH RELEVANCE: The overall objective of this project is to investigate the mechanism by which human melanopsin, the recently discovered light-receptor in the retina, controls the body's internal clock as well as pupillary response. Understanding melanopsin is important because it is involved in key physiological processes including sleep, mental alertness, eating habits, and hormonal levels as well as disorders involving these processes. The application of advanced infrared spectroscopic methods developed in our laboratory will allow us to determine the detailed molecular response of melanopsin and the complexes it forms with other proteins to light on time scales as short as one trillionth of a second.

描述（由申请人提供）：该项目的总体目的是研究人类黑色素蛋白（MO）信号转导的分子基础，这是光敏感的视网膜神经节细胞中最近发现的光受体，这是对昼夜节律和瞳孔控制的控制。回复。因为黑色素蛋白参与了各种生理功能，包括睡眠，精神警觉，饮食习惯和荷尔蒙水平，以及可能涉及多种疾病，包括睡眠障碍，季节性受影响的疾病和青光眼，NIH已重点介绍了黑色素蛋白作为优先事项未来的研究。值得注意的是，黑色素蛋白的特性强烈类似于无脊椎动物的视紫红质，而不是广泛研究的脊椎动物“视觉”视紫红蛋白。相似性包括通过GQ蛋白（磷脂/三磷酸磷酸肌醇）途径而不是GT蛋白蛋白环核苷酸途径的近距离序列同源性和信号传导。重要的是，黑色素蛋白及其类似的无脊椎动物动蛋白（如鱿鱼视紫红蛋白（SRH））是研究人类细胞中数百种GPCR中信号转导机制的模型。这种GPCR通过GQ蛋白途径信号，并被2-arrestin2抑制，而不是更专业的视觉2- arrestin。突出的例子包括5-羟色胺，组胺，肾上腺素能，毒蕈碱和降钙素受体，它们是当前和潜在的新药的靶标。黑色素和无脊椎动物视紫红蛋白而不是脊椎动物视紫红蛋白的关键特征是它们的光学双重性。此属性使它们可以使用两种不同的光线在两个不同的稳定状态之间“循环”。在这个项目中，我们将利用这一两光子特性，以研究黑色素蛋白，SRH的光激活以及用2- harstin2和gq蛋白形成的复合物发生的详细结构变化。这项研究将通过应用几种高级FTIR差异技术的应用，其中许多在我们的实验室中开发，并结合位置定向的诱变和同位素标记。这种方法的应用先前导致了几个里程碑，包括对脊椎动物紫红素光活化过程中发生的构象变化的首次详细表征和细菌近红淡天的质子泵浦机理。最近，我们证明了这种方法还检测和表征关键残基和内部水分子的结构变化，这些变化位于膜蛋白信号受体之间的界面接触区域（例如感觉视紫红质II及其同源透性）之间的界面接触区域。 HTRII在初步研究中，我们已经测量了鱿鱼视紫红质及其2-甲词复合物的静态和时间解决FTIR差异光谱。通过使用同位素编辑，我们可以分别表征受体和2-arrestin2成分中的构象变化。拟议的研究还将受益于我们最近的方法发展：i）测量亚皮秒蛋白的变化； ii）使用时间分辨的FTIR显微镜和iii探针量的膜蛋白，包括单晶和iii）纳米脂肪粒子中的体外表达膜蛋白迅速（NLP）。这项工作将通过与加尔维斯顿德克萨斯大学医学分公司的J. Navarro博士的实验室进行密切合作，他们将准备SRHO/2-arrestin2晶体并进行并行X射线晶体学研究； Nijmegen大学的W. Degrip博士的实验室表达并表征了功能性重组黑色素蛋白和LLNL和UC Davis的M. Coleman博士，其实验室已经开发了无细胞的技术来表达NLP中的GPCR。 公共卫生相关性：该项目的总体目的是调查人类黑色素蛋白（最近发现的视网膜受体受体，控制人体的内部时钟和瞳孔反应）的机制。了解黑色素蛋白很重要，因为它参与了关键的生理过程，包括睡眠，精神警觉，饮食习惯和荷尔蒙水平以及涉及这些过程的疾病。在我们的实验室中开发的先进红外光谱方法的应用将使我们能够确定黑色素蛋白的详细分子反应及其与其他蛋白质形成的复合物，以在时间尺度上点亮一秒钟的时间尺度。

项目成果

Retinal chromophore structure and Schiff base interactions in red-shifted channelrhodopsin-1 from Chlamydomonas augustae.

• DOI: 10.1021/bi500445c
• 发表时间: 2014-06-24
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Ogren JI;Mamaev S;Russano D;Li H;Spudich JL;Rothschild KJ
• 通讯作者: Rothschild KJ

Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

通过傅里叶变换红外 (FTIR) 差异光谱和定点诱变研究通道视紫红质-1 中的质子转移。

• DOI: 10.1074/jbc.m114.634840
• 发表时间: 2015
• 期刊: The Journal of biological chemistry
• 作者: Ogren,JohnI;Yi,Adrian;Mamaev,Sergey;Li,Hai;Spudich,JohnL;Rothschild,KennethJ
• 通讯作者: Rothschild,KennethJ

Comparison of the structural changes occurring during the primary phototransition of two different channelrhodopsins from Chlamydomonas algae.

• DOI: 10.1021/bi501243y
• 发表时间: 2015-01-20
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Ogren JI;Yi A;Mamaev S;Li H;Lugtenburg J;DeGrip WJ;Spudich JL;Rothschild KJ
• 通讯作者: Rothschild KJ