Alterations In Lipid Metabolism In The Nervous System By Ethanol

乙醇改变神经系统脂质代谢

基本信息

批准号：
7732113
负责人：
Hee-Yong Kim
金额：
$ 233.31万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
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/7732113
关键词：
Alanine Animals Binding Biochemical Biological Cell Membrane Structures Cell Survival Cell membrane Cells Chromosome Pairing Coupled Data Development Diet Digestion Docosahexaenoic Acids Electrostatics Embryo Ethanol Exhibits Glutamate Receptor Goals Hippocampus (Brain)In Vitro Investigation Label Learning Ligands Link Lipids Long-Term Potentiation Lysine Mass Spectrum Analysis Mediating Mediator of activation protein Membrane Membrane Structure and Function Memory Metabolic Metabolism Methods Minor Modification Molecular Molecular Conformation Monitor Mus Mutate Nervous system structure Neurites Neurons Omega-3 Fatty Acids PH Domain Peptides Phosphopeptides Phosphoproteins Phosphorylation Phosphotransferases Physiological Polyunsaturated Fatty Acids Pregnancy Process Production Proteins RXR Reporting Research Role Signal Transduction Slice Somatomedins Staging Structure Supplementation Surface Plasmon Resonance Synapses Synapsins Techniques Water analog base cognitive function crosslink feeding fetal improved in vitro Model in vivo inhibitor/antagonist interest lipid mediator lipid metabolism mutant neurite growth neurodevelopment neuronal survival novel novel strategies prevent protein expression synaptogenesis titanium dioxide

项目摘要

During this period we continued to investigate the biological implication of membrane phosphotidylserine (PS) on Akt signaling that regulates cell survival, using various cellualr and in vitro models where PS levels were altered. Mass spectrometric analysis of conformational changes coupled with biomolecular interaction analysis based on surface plasmon resonance revelaed that PS promotes Akt-membrane binding and conformational changes for activation through electrostatic interaction. We found that both PH and regulatory domains of Akt interact with PS. When Neuro 2A cells were expressed with Akt mutated at the potential PS-binding residues (R15A or K20A) in the PH domain of Akt, in vitro Akt-PS binding as well as in vivo membrane translocation triggered by IGF was greatly diminished. Furthermore, IGF-induced phosphorylation of both T308 and S473 was abolished in these mutants, strongly indicating that PS is necessary for Akt activation in vivo. When basic residues in the regulatory domain were mutated with alanine, T308 phosphorylation significantly decreased and S-473 phosphorylation was abolished, indicating that PS-regulatory interaction is also important for phosphorylation of Akt, especially for S473 for optimal activation. We have also established during this period a novel strategy to study Akt-inhibitor interaction by probing Akt conformation altered by inhibitors. We have previously identified two inter-domain cross-linked lysine pairs, K30-K389 (PH-kinase) and K426-K284 (regulatory-kinase), in inactive Akt molecules, indicating a folded structure with the PH and regulatory domains covering the kinase domain. These cross-linked pairs were not observed within 24 angstrom spatial distance constraint upon interaction with membranes containing PIP3 and PS, suggesting that changes to an open conformation occurred after the membrane interaction. Using this strategy, we demonstrated that when Akt interacted with a PI analog, an open inter-domain conformation necessary for Akt activation occurred, even before Akt interacted with membranes. The Akt-PI analog interaction presumably prevented the Akt-PIP3 binding and hence blocked Akt membrane translocation and activation. In contrast, the folded inter-domain conformation was unchanged by interacting with Inhibitor VI, a peptide supposedly binding to the PH domain. Upon subsequent interaction with the membrane, the extent of PH-kinase cross-linking in comparison to the case without the inhibitor VI increased, indicating that the inhibitor impaired the opening of the PH domain for exposing T308 for phosphorylation at the plasma membrane. Our results demonstrated that molecular mechanisms for Akt inhibition can be deduced by monitoring the Akt conformational changes probed by mass spectrometry. During this report period we have also established quantitative mass spectrometric analysis of phosphoproteins. Proteins were first labeled with O-18 by tryptic digestion in either regular or O-18 labeled water, differentially labeled peptides were mixed together and phosphopeptides were enriched using titanium oxide prior to mass spectrometric analysis. This approach enabled us to unveil minor phosphopeptides generated during stimulation of the cells. This approach is now being applied to investigate the Akt interacting phosphoproteins during stimulation. Our previous findings suggested that DHA-induced neurite outgrowth may not be mediated by direct activation of RXR, suggesting other mediators are involved. During this period, we have also established an HPLC/ESI-MS/MS method for quantitative profiling of lipid mediators derived from DHA. Using this method, we found that DHA metabolizes to N-docosahexaenoylethanolamide (DEA) in developing hippocampi. We subsequently demonstrated that DEA is a novel and effective ligand to RXR and promotes neurite development, synaptogenesis and synaptic protein expression through activating RXR. We found in mouse embryonic hippocampal neuronal cultures that DHA at low micromolar concentrations uniquely promotes not only neurite development but also the expression of synaptic proteins such as synapsins and glutamate receptors and improves excitatory synaptic activity. DHA supplementation of E-18 hippocampal neuronal culture increased DEA content. Indeed, DEA at submicromolar concentrations activated RXR and promoted neurite growth and synaptic protein expression in an RXR-dependent manner. Conversely, depleting DHA by feeding mice with n-3 fatty acid deficient diet during pregnancy resulted in a marked reduction of DEA in E-18 fetal hippocampi as well as significant inhibition of neurite development and synaptogenesis in E-18 hippocampal cultures. Furthermore, hippocampal slices obtained from the deficient animals at a later stage of development exhibited retarded long-term potentiation (LTP) while the expression of hippocampal synapsins and glutamate receptors was significantly decreased, supporting a significant role of DHA in promoting synapse development and learning and memory. As the DEA production is directly linked to the DHA level, DEA- and RXR-dependent hippocampal neurite growth and synaptic protein expression provide a novel mechanism for the role of DHA in hippocampal development and hippocampus-related cognitive function. Based on these data, we suggest that DHA metabolism to DEA and RXR activation emerge as potential new targets for regulating physiological and pathophysiological processes of neurodevelopment and function.

在此期间，我们继续研究膜磷酸苷（PS）对调节细胞存活的Akt信号的生物学意义，使用各种CellualR和PS水平改变PS水平的体外模型。构象变化的质谱分析以及基于表面等离子体共振的生物分子相互作用分析的质谱分析，ps促进了PS促进Akt-膜结合和构象变化，从而通过静电相互作用激活。我们发现AKT的pH和调节域都与PS相互作用。当神经2a细胞用Akt在Akt的pH结构域的潜在PS结合残基（R15A或K20A）处突变时，体外Akt-PS结合以及由IGF触发的体内膜易位的体外AKT-PS结合。此外，在这些突变体中废除了IGF诱导的T308和S473的磷酸化，这强烈表明PS对于体内Akt激活是必需的。当用丙氨酸突变调节结构域中的基本残基时，T308磷酸化显着降低并消除了S-473磷酸化，这表明PS调节的相互作用对于AKT的磷酸化也很重要，尤其是对于S473而言，对于最佳激活而言。在此期间，我们还建立了一种新的策略来研究Akt抑制剂相互作用，通过探测抑制剂改变的AKT构象。我们先前已经在非活性Akt分子中鉴定了两个域间交联的赖氨酸对K30-K389（pH-激酶）和K426-K284（调节 - 激酶），表明具有pH和调节域覆盖激酶域的折叠结构。在与含有PIP3和PS的膜相互作用时，在24个Angstrom空间距离约束中未观察到这些交联对，这表明在膜相互作用后发生了开放构象的变化。使用此策略，我们证明了当AKT与PI类似物相互作用时，即使在AKT与膜相互作用之前，也会发生AKT激活所需的开放域间构象。 AKT-PI模拟相互作用可能阻止了Akt-Pip3结合，因此阻断了Akt膜的易位和激活。相反，通过与抑制剂VI相互作用（据说肽与pH结构域结合的肽VI），折叠的域间构象没有变化。随后与膜相互作用后，与没有抑制剂VI的情况相比，pH-激酶交联的程度增加，这表明抑制剂损害了pH结构域在质膜膜上暴露于T308的pH结构域的开放。我们的结果表明，可以通过监测质谱探测的AKT构象变化来推断出AKT抑制的分子机制。在此报告期间，我们还建立了对磷蛋白的定量质谱分析。首先，通过胰蛋白酶消化在常规或O-18标记的水中，将蛋白质用O-18标记，将差异标记的肽混合在一起，并在进行质谱分析之前使用氧化钛富含磷酸肽。这种方法使我们能够在细胞刺激过程中揭示产生的小磷酸肽。现在，正在应用这种方法来研究刺激过程中AKT相互作用的磷蛋白。我们以前的发现表明，DHA诱导的神经突生长可能不会通过RXR的直接激活来介导，这表明涉及其他介体。在此期间，我们还建立了一种HPLC/ESI-MS/MS方法，用于对DHA衍生的脂质介质的定量分析。使用这种方法，我们发现DHA在发育中的海马中代谢为N-二甲苯甲酰乙醇酰胺（DEA）。随后，我们证明了DEA是一种新颖有效的配体RXR，并通过激活RXR促进神经突发育，突触发生和突触蛋白表达。我们在小鼠胚胎海马神经元培养物中发现，在低微摩尔浓度下DHA不仅可以独特地促进神经突发育，还可以促进突触蛋白（例如突触蛋白和谷氨酸受体）的表达，并改善兴奋性突触活性。 E-18海马神经元培养的DHA补充增加了DEA含量。实际上，亚摩尔摩尔浓度下的DEA激活了RXR，并以RXR依赖性方式促进了神经突的生长和突触蛋白表达。相反，通过在怀孕期间用N-3脂肪酸喂养小鼠来耗尽DHA导致E-18胎儿海马中DEA显着降低，以及对E-18海马培养物中神经突发育和突触发生的显着抑制。此外，在后来发育阶段，从缺陷动物获得的海马切片表现出迟缓的长期增强（LTP），而海马突触蛋白和谷氨酸受体的表达显着降低，从而支持DHA在促进突触的发展和学习和记忆中的重要作用。由于DEA产生与DHA水平直接相关，因此DEA和RXR依赖性海马神经突的生长和突触蛋白表达为DHA在海马发育和海马相关的认知功能中的作用提供了一种新颖的机制。基于这些数据，我们建议DHA代谢DEA和RXR激活作为调节神经发育和功能的生理和病理生理过程的潜在新靶标。