Parker Benjamin L, Shepherd Nicholas E, Trefely Sophie, Hoffman Nolan J, White Melanie Y, Engholm-Keller Kasper, Hambly Brett D, Larsen Martin R, James David E, Cordwell Stuart J
From the Discipline of Pathology, School of Medical Sciences, University of Sydney, Sydney 2006, Australia, the Diabetes and Obesity Program, Biological Mass Spectrometry Unit, Garvan Institute of Medical Research, 2010 Australia.
the School of Molecular Bioscience and.
J Biol Chem. 2014 Sep 12;289(37):25890-906. doi: 10.1074/jbc.M114.556035. Epub 2014 Jul 9.
Myocardial ischemia and cardioprotection by ischemic pre-conditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, and PKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning- and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.
心肌缺血以及缺血预处理诱导的心脏保护作用会引发信号网络，若缺血时间延长，这些信号网络会导致存活或细胞死亡。这些途径由蛋白质翻译后修饰介导，据推测这些修饰会相互作用并相互调节。在有或没有使用裂霉素抑制赖氨酸脱乙酰化的情况下，对经历缺血或缺血预处理的离体大鼠心脏中的磷酸肽和赖氨酸乙酰化肽进行了定量分析。我们发现，在缺血期间，赖氨酸乙酰化（乙酰 - 赖氨酸）依赖的AMP活化蛋白激酶、AKT和PKA激酶会被激活。在超过50%的研究蛋白质中，映射到三级结构上的磷酸化位点和乙酰 - 赖氨酸位点是相邻的，但在50个含有KXXS嗜碱性蛋白激酶共有基序的缺血预处理和/或缺血相关肽中，它们是相互排斥的。在肌肉型肌酸激酶的C末端对该基序中的修饰进行了建模。乙酰 - 赖氨酸使近端去磷酸化增加了10倍。通过二维核磁共振对修饰后的肌肉型肌酸激酶肽变体进行结构分析，结果显示通过赖氨酸 - 磷酸盐盐桥实现了稳定，而乙酰 - 赖氨酸会破坏该盐桥，导致主链灵活性增加和磷酸酶可及性提高。
