The cGMP system in normal and degenerating mouse neuroretina: New proteins with cGMP interaction potential identified by a proteomics approach

Abstract

The hereditary disease Retinitis pigmentosa results in severe vision loss due to photoreceptor degeneration by unclear mechanisms. In several disease models, the second messenger cGMP accumulates in the degenerating photoreceptors, where it may over‐activate specific cGMP‐interacting proteins, like cGMP‐dependent protein kinase. Moreover, interventions that counteract the activity of these proteins lead to reduced photoreceptor cell death. Yet there is little or no information whether other than such regular cGMP‐interactors are present in the retina, which we, therefore, investigated in wild‐type and retinal degeneration (rd1, rd10, and rd2) mouse models. An affinity chromatography based proteomics approach that utilized immobilized cGMP analogs was applied to enrich and select for regular and potentially new cGMP‐interacting proteins as identified by mass spectrometry. This approach revealed 12 regular and 10 potentially new retinal cGMP‐interacting proteins (e.g., EPAC2 and CaMKIIα). Several of the latter were found to be expressed in the photoreceptors and to have proximity to cGMP and may thus be of interest when defining prospective therapeutic targets or biomarkers for retinal degeneration.

The stereospecific interaction sites and target specificity of cGMP analogs in mouse cortex

“…This method was optimized in a previous study (Rasmussen et al, 2020). In brief, a sample of the soluble cortex fraction [1.5 mg] was added to 60 µl of following cGMP agaroses: 8-AET-cGMP, 2-AH-cGMP, 2′-AHC-cGMP, Sp-8-AET-cGMPS, Sp-2′-AHC-cGMPS, Rp-8-AET-cGMPS, Rp-2′-AHC-cGMPS, or EtOH-NH, respectively, with 500 µl PBS [0.137 m NaCl, 2.7 mm KCl, 10 mm Na 2 HPO 4 , and 1.8 mm KH 2 PO 4 ].…”

“…The cGMP's effector proteins elicit a broad variety of cellular functions (Schlossmann & Schinner, 2012) and include PKG and PKA (Huang et al, 2014;Kim, Zhong, et al, 2016;Reed et al, 1996) (which phosphorylate downstream target proteins), cyclic nucleotidegated channels (Altenhofen et al, 1991) (CNGs, that upon stimulation allow cation fluxes), and the regulatory PDEs (Yan et al, 1996) (responsible for abrogating the cGMP signaling). However, also other proteins may bind to cGMP (Rasmussen et al, 2020).…”

“…Studies have identified essential stereospecificbinding sites and affinities between interactors like PDEs or PKG and cGMP analogs modified for a specific target and which possess inhibitory or activatory substitutions (Beltman et al, 1995;Butt et al, 1995;Hanke et al, 2011;Jeong J Kim et al, 2011;Poppe et al, 2008;Scholten et al, 2006Scholten et al, , 2007. Further, cGMP interactors (PKGs, PKA regulatory subunits, and catalytic subunits, PDEs) have been identified by affinity-based proteomic approaches in various tissues (Hanke et al, 2011; E. Kim & Park, 2003;Kovanich et al, 2010;Margarucci et al, 2011;Rasmussen et al, 2020;Scholten et al, 2006Scholten et al, , 2007, utilizing different cAMP-and/or cGMP-analogcarrying agaroses. These studies (E. Kim & Park, 2003;Scholten et al, 2006) tested the ability of agaroses with immobilized general cGMP analogs with cyclic phosphate (i.e., 8-AET-cGMP, 2-AHA-cGMP, 2-AH-cGMP, and 2′-AHC-cGMP agarose) to pull down cGMP-binding proteins.…”

Kinase activity profiling identifies putative downstream targets of cGMP/PKG signaling in inherited retinal neurodegeneration

“…Recent studies identified additional cGMP-interacting proteins such as PKAs, PKCs, and CaMKs as upregulated in IRD retinas, which might be interesting in developing new therapeutic targets (49,50). These recent findings corresponded well with our study where we also identified these same kinases as activated in rd1 diseased retinal explants and inhibited by the PKG inhibitor, CN03.…”

“…Recent studies identified additional cGMP-interacting proteins such as PKAs, PKCs, and CaMKs as upregulated in IRD retinas, which might be interesting in developing new therapeutic targets (49,50).…”