PDF(Pubmed)
Abstract：
Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs.
摘要：
遗传性视网膜退行性疾病（IRD）是一组罕见的疾病，导致感光细胞的进行性丧失，最终,失明。cGMP依赖性蛋白激酶G（PKG）的过度激活，cGMP信号的关键效应之一，先前发现与感光细胞死亡有关，并在鼠IRD模型中进行了研究，以阐明视网膜变性的病理生理学。然而,PKG是一种丝氨酸/苏氨酸激酶（STK），具有数百个潜在的磷酸化靶标，到目前为止,关于PKG活化的靶相互作用和下游效应的特异性知之甚少。这里,我们对来自rd10小鼠IRD模型的器官型视网膜外植体培养物进行了kinome活性和磷酸化蛋白质组学分析。在用PKG抑制剂CN03处理外植体后,观察到肽磷酸化的总体降低,最显著的减少发生在七个肽中，包括那些来自已知的PKG底物循环AMP反应元件结合CREB的，以及Ca2/钙调蛋白依赖性激酶（CaMK）肽和TOP2A。磷酸蛋白质组数据，反过来,显示磷酸化降低的蛋白质，以及磷酸化增加的蛋白质。两个数据集的整合确定了由PKG抑制改变的常见生物网络，其中包括主要来自所谓的AGC和CaMK激酶家族的激酶(例如，PKG1,PKG2,PKA,CaMK,RSKs,和AKTs）。通路分析证实了CREB的作用，钙调素,丝裂原活化蛋白激酶（MAPK）和CREB调节。在显示磷酸化活性降低的肽和途径中，基板CREB，验证了CaMK2和CaMK4的视网膜定位和活性，在rd10视网膜中使用免疫染色和免疫印迹。总之,对kinome活性和磷酸化蛋白质组数据的综合分析揭示了鼠IRD模型中已知和新的PKG底物。该数据为更好地理解cGMP介导的光感受器变性中涉及的生物途径奠定了基础。此外，经过验证的PKG靶标如CREB和CaMK值得探索作为新的(替代)生物标志物,以确定IRD的临床PKG靶向治疗的效果.
