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：
10416315
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/10416315
关键词：
A kinase anchoring protein Adipose tissue Adrenergic Receptor Affect 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 Mediating MicroRNAs Mus Mutate Mutation Nature Outcome Pathway interactions Pattern Peptide Mapping Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Process Protein Kinase Proteins Proteomics Publishing Recycling Regulation Role Scaffolding Protein Signal Transduction Site Testing Therapeutic Triglycerides Work alcohol effect alcohol exposure base crosslink 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.

项目概要脂肪肝会促进酒精相关性肝病 (ALD) 的发展。AKAP12（A 激酶锚定蛋白） 12) 是通过锚定蛋白激酶（PKA、PKC）、细胞周期蛋白-D1 和热进行信号转导的支架蛋白休克蛋白 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 中，激酶以及酒精与磷酸酶的相互作用增加。通过磷酸肽图谱我们确定了酒精是否调节 AKAP12 磷酸化。酒精强烈抑制小鼠肝脏中 AKAP12 的酒精敏感磷酸化。位点主要是 PKA 预测的底物，突变这些磷酸位点会降低 AKAP12 的稳定性。蛋白，表明磷酸化稳定了 AKAP12 AKAP12 相互作用组揭示了其相互作用。含有已知调节脂质稳态的蛋白质，如 PKA、2-肾上腺素受体 (2-AR) 和脂肪分解蛋白因此，我们评估了该 AKAP12 的功能作用。先前表明 AKAP12 介导的 PKA 支架将其招募到 2- AR。AKAP12、PKA 和 2-AR 的信号体通过 2-AR 脱去过程调节 cAMP 水平。敏化/再敏化，可能涉及 AKAP12 磷酸化，限制脂质。我们的新初步数据表明，酒精暴露会失调。 AKAP12/PKA/2-AR 信号体的多种成分酒精抑制 PKA 和 β2-AR 之间的相互作用。 2-AR。AKAP12 在 S696-S698 PKA 磷酸位点处高度磷酸化，该位点先前发表于促进 AKAP12-2-AR 结合。该位点在暴露于酒精和突变该 AKAP12 位点也减少了其对 PKA 和 2-AR 的支架。降低 2-AR 蛋白稳定性。酒精会干扰 AKAP12-2-AR 相互作用并促进 AKAP12 和根据这些数据，我们发现 2-AR 溶酶体降解会降低 AKAP12 磷酸化。酒精暴露后可能会干扰 AKAP12/PKA/2-AR 信号体，导致 cAMP 发生变化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 2) AKAP12 诱导抑制酒精介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也可以逆转酒精的抑制作用。 AKAP12/PKA/2-AR 支架是否控制脂质对脂肪分解酶 ATGL 活性的影响。我们勇敢地承认 AKAP12 信号体的失调有利于脂质。该提案将评估酒精敏感的 AKAP12 磷酸化如何破坏 AKAP12 的稳定性。损害其支架活性并导致脂质稳态失调。 AKAP12 磷酸稳定性改善脂质功能障碍的作用将在 ALD 小鼠模型中进行测试。