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Chemical tools selectively target components of the PKA system

机译：化学工具有选择地针对PKA系统的组件

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Background: In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme insignal transduction and represents the main target of the second messenger cAMP. Here wedescribe the design, synthesis and characterisation of specifically tailored cAMP analogs which canbe utilised as a tool for affinity enrichment and purification as well as for proteomics based analysesof cAMP binding proteins.Results: Two sets of chemical binders were developed based on the phosphorothioate derivativesof cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively. Thesecompounds were tested via direct surface plasmon resonance (SPR) analyses for their bindingproperties to PKA R-subunits and holoenzyme. Furthermore, these analogs were used in an affinitypurification approach to analyse their binding and elution properties for the enrichment andimprovement of cAMP binding proteins exemplified by the PKA R-subunits. As determined by SPR,all tested Sp-analogs provide valuable tools for affinity chromatography. However, Sp-8-AEA-cAMPS displayed (i) superior enrichment properties while maintaining low unspecific binding toother proteins in crude cell lysates, (ii) allowing mild elution conditions and (iii) providing thecapability to efficiently purify all four isoforms of active PKA R-subunit in milligram quantities within8 h. In a chemical proteomics approach both sets of binders, Rp- and Sp-cAMPS derivatives, can beemployed. Whereas Sp-8-AEA-cAMPS preferentially binds free R-subunit, Rp-AHDAA-cAMPS,displaying antagonist properties, not only binds to the free PKA R-subunits but also to the intactPKA holoenzyme both from recombinant and endogenous sources.Conclusion: In summary, all tested cAMP analogs were useful for their respective application asan affinity reagent which can enhance purification of cAMP binding proteins. Sp-8-AEA-cAMPS wasconsidered the most efficient analog since Sp-8-AHA-cAMPS and Sp-2-AHA-cAMPS, demonstratedincomplete elution from the matrix, as well as retaining notable amounts of bound proteincontaminants. Furthermore it could be demonstrated that an affinity resin based on Rp-8-AHDAA-cAMPS provides a valuable tool for chemical proteomics approaches.
机译：背景：在真核细胞中，依赖于cAMP的蛋白激酶（PKA）是关键的酶信号转导，并且代表第二个信使cAMP的主​​要靶标。本文描述了专门定制的cAMP类似物的设计，合成和表征，可将其用作亲和力富集和纯化以及基于蛋白质组学的cAMP结合蛋白分析的工具。结果：基于cAMP的硫代磷酸酯衍生物开发了两组化学结合剂，Sp-cAMPS和Rp-cAMPS分别充当cAMP激动剂和-拮抗剂。通过直接表面等离子体共振（SPR）分析测试了这些化合物与PKA R亚基和全酶的结合特性。此外，将这些类似物用于亲和纯化方法中，以分析其结合和洗脱特性，以丰富和改善以PKA R亚基为代表的cAMP结合蛋白。由SPR确定，所有经过测试的Sp-analogs为亲和色谱提供了有价值的工具。然而，Sp-8-AEA-cAMPS表现出（i）优异的富集特性，同时保持与粗细胞裂解液中其他蛋白质的低非特异性结合，（ii）允许温和的洗脱条件，以及（iii）提供有效纯化活性PKA的所有四种同工型的能力8小时内以毫克为单位的R亚基。在化学蛋白质组学方法中，可以使用两组结合剂Rp-和Sp-cAMPS衍生物。 Sp-8-AEA-cAMPS优先结合游离R-亚基，而Rp-AHDAA-cAMPS表现出拮抗特性，不仅与游离PKA R-亚基结合，而且与重组和内源来源的完整PKA全酶结合。总之，所有测试的cAMP类似物均可用作亲和试剂，可增强cAMP结合蛋白的纯化。 Sp-8-AEA-cAMPS被认为是最有效的类似物，因为Sp-8-AHA-cAMPS和Sp-2-AHA-cAMPS表现出从基质中洗脱不完全，并保留了大量的结合蛋白污染物。此外，可以证明基于Rp-8-AHDAA-cAMPS的亲和树脂为化学蛋白质组学方法提供了有价值的工具。

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