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A-Kinase Anchoring Protein Dysregulation during Alcohol-Associated Liver Disease

酒精相关性肝病期间 A 激酶锚定蛋白失调

基本信息

批准号：
10705602
负责人：
Komal Ramani
金额：
$ 33.4万
依托单位：
CEDARS-SINAI MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-16 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10705602
关键词：
A kinase anchoring protein Adipose tissue Adrenergic Receptor Affect Alcoholic Liver Diseases Alcohols Binding Cell membrane Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclin D1 Data Development Disease Progression Enzymes Event Exhibits Fatty Liver Fatty acid glycerol esters Functional disorder Gravin Hepatic Stellate Cell Hepatocyte Homeostasis Hsp47 protein Human Impairment Lipase Lipids Lipolysis Literature Liver Liver diseases Location Maps Mediating MicroRNAs Mus Mutate Mutation Nature Outcome Pathway interactions Pattern Phosphopeptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Process Protein Kinase Proteins Proteomics Publishing Rationalization Recycling Regulation Role Scaffolding Protein Signal Transduction Site Testing Therapeutic Triglycerides Work alcohol effect alcohol exposure comparison control crosslink desensitization fatty acid oxidation lipid biosynthesis liver injury mRNA Expression mRNA Stability mouse model novel preservation prevent protein expression rab GTP-Binding Proteins recruit scaffold spatiotemporal trafficking

项目摘要

PROJECT SUMMARY Fatty liver potentiates alcohol-associated liver disease (ALD) development. AKAP12 (A-kinase anchoring protein 12) is a scaffolding protein in signal transduction by anchoring protein kinases (PKA, PKC), cyclin-D1 and heat shock protein 47. Our recent proteomics studies showed that AKAP12 exhibited decreased interaction with kinases and increased interaction with phosphatases by alcohol in mouse models and human ALD. This led us to examine whether alcohol regulated AKAP12 phosphorylation. By phospho-peptide mapping we determined that alcohol strongly inhibited AKAP12 phosphorylation in mouse liver. AKAP12’s alcohol-sensitive phospho- sites were mainly PKA-predicted substrates. Mutating these phospho-sites reduced the stability of AKAP12 protein, suggesting that phosphorylation stabilized AKAP12. The AKAP12 interactome revealed its interactions with proteins known to regulate lipid homeostasis such as PKA, 2-adrenergic receptor (2-AR) and lipolytic enzymes, adipose-triglyceride lipase (ATGL). We therefore assessed the functional role of this AKAP12 interactome in hepatocytes. It was shown previously that AKAP12-mediated PKA scaffolding recruits it to 2- AR. The signalosome of AKAP12, PKA and 2-AR regulates cAMP levels through the process of 2-AR de- sensitization/re-sensitization and may involve AKAP12 phosphorylation. 2-AR/PKA/cAMP signaling limits lipid accumulation and enhances lipolysis. Our novel preliminary data indicates that alcohol exposure dysregulates several components of the AKAP12/PKA/2-AR signalosome. Alcohol inhibited the interaction between PKA and 2-AR. AKAP12 was highly phosphorylated at a S696-S698 PKA-phospho-site that was previously published to facilitate AKAP12-2-AR binding. This site exhibited decreased phosphorylation upon alcohol exposure and mutating this AKAP12 site reduced its scaffolding towards both PKA and 2-AR. This AKAP12 mutation also reduced 2-AR protein stability. Alcohol interfered with AKAP12-2-AR interaction and facilitated AKAP12 and 2-AR lysosomal degradation. Based on these data, we hypothesize that decreased AKAP12 phosphorylation following alcohol exposure might interfere with AKAP12/PKA/2-AR signalosome, causing alterations in cAMP signaling, which in turn could dysregulate lipid homeostasis. Preliminary data that support this hypothesis are: 1) Enhancing endogenous AKAP12 levels reverses the inhibitory effect of alcohol on PKA-2-AR interaction; 2) AKAP12 induction inhibits alcohol-mediated increase of triglyceride levels; 3) AKAP12 induction reverses alcohol’s suppressive effect on fatty acid oxidation. Induction of AKAP12 also reverses alcohol’s suppressive effect on the activity of lipolytic enzyme, ATGL. Whether the AKAP12/PKA/2-AR scaffold controls lipid homeostasis is unknown. We hypothesize that dysregulation of the AKAP12 signalosome favors lipid accumulation. This proposal will evaluate how alcohol-sensitive AKAP12 phosphorylation destabilizes AKAP12, impairs its scaffolding activities and contributes to dysregulation of lipid homeostasis. The therapeutic benefit of AKAP12 phospho-stabilization in ameliorating lipid dysfunction will be tested in mouse models of ALD.

项目总结脂肪肝加剧了酒精相关性肝病(ALD)的发展。AKAP12(A-激酶锚定蛋白 12)是一种通过锚定蛋白激酶(PKA、PKC)、细胞周期蛋白-D1和热进行信号转导的脚手架蛋白休克蛋白47。我们最近的蛋白质组学研究表明，AKAP12与在小鼠模型和人类ALD模型中，乙醇与磷酸酶的相互作用增加。这让我们检测酒精是否调节AKAP12的磷酸化。通过磷酸肽图确定了酒精对小鼠肝脏AKAP12的磷酸化有强烈的抑制作用。AKAP12‘S酒精敏感磷酸盐位点主要是PKA预测的底物。突变这些磷酸基降低了AKAP12的稳定性蛋白质，这表明磷酸化稳定了AKAP12。AKAP12相互作用组揭示了其相互作用具有调节脂质稳态的已知蛋白质，如PKA、2-肾上腺素能受体(2-AR)和脂解酶，脂肪甘油三酯脂肪酶(ATGL)。因此，我们评估了该AKAP12的功能作用肝细胞中的相互作用体。以往的研究表明，AKAP12介导的PKA支架将其招募到2- 阿。AKAP12、PKA和2-AR信号小体通过2-AR去激活调节cAMP水平。敏化/再敏化，可能涉及AKAP12的磷酸化。2-AR/PKA/cAMP信号通路限制血脂积聚并增强脂肪分解作用。我们新的初步数据表明，酒精暴露会导致 AKAP12/PKA/2-AR信号体的几个组分。酒精可抑制PKA和PKA之间的相互作用 2-AR。AKAP12在S696-S698的PKA磷酸化位点高度磷酸化，该位点之前发表于促进AKAP12-2-AR结合。在酒精暴露下，这个部位的磷酸化水平降低，突变这个AKAP12位点减少了它对PKA和2-AR的支架作用。这个AKAP12突变也降低2-AR蛋白的稳定性。酒精干扰AKAP12-2-AR相互作用促进AKAP12和 2-AR溶酶体降解。基于这些数据，我们假设AKAP12的磷酸化水平降低酒精暴露可能干扰AKAP12/PKA/2-AR信号体，导致cAMP改变信号，这反过来可能会扰乱脂类平衡。支持这一假设的初步数据是： 1)提高内源性AKAP12水平可逆转酒精对PKA--2-AR相互作用的抑制作用； AKAP12诱导抑制酒精诱导的甘油三酯水平升高；3)AKAP12诱导逆转酒精对脂肪酸氧化的抑制作用。AKAP12的诱导也可逆转酒精的抑制作用对脂解酶ATGL活性的影响。AKAP12/PKA/2-AR支架是否控制血脂动态平衡是未知的。我们假设AKAP12信号体的失调有利于脂质积累。这项提案将评估酒精敏感的AKAP12磷酸化如何破坏AKAP12的稳定，损害其支架活性，并导致脂质平衡失调。治疗方面的好处 AKAP12在改善脂质紊乱方面的磷酸化稳定性将在ALD的小鼠模型中进行测试。