Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+: Hints to Understand Protein Activity

Bonì, Francesco; Marino, Valerio; Bidoia, Carlo; Mastrangelo, Eloise; Barbiroli, Alberto; Dell’Orco, Daniele; Milani, Mario

doi:10.3390/biom10101408

Cited by 13 publications

(24 citation statements)

References 43 publications

(72 reference statements)

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“…Several lines of evidence in the last years have indicated that WT GCAP1 is a functional dimer under physiological conditions [ 36 , 37 ]; thus, we conducted analytical gel filtration and Dynamic Light Scattering (DLS) measurements to address the effects of the double point mutation on the quaternary structure of GCAP1, as well as on its aggregation propensity. Analytical Size exclusion Chromatography (SEC) chromatograms ( Figure 5 A) of the double mutant showed a prominent peak which shifted from 14.58 mL ( Figure 5 A, blue trace) in the presence of Mg 2+ (corresponding to a molecular mass (MM) of 38.2 kDa, Table 2 ) to 14.50 mL ( Figure 5 A, red trace) in the presence of Ca 2+ (corresponding to a MM of 40.2 kDa, Table 2 ), thus suggesting that the double mutant is predominantly a dimer upon cation binding.…”

Section: Resultsmentioning

confidence: 99%

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells

Marino

Cortivo

Maltese³

et al. 2021

IJMS

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Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1–cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers’ accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.

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Section: Resultsmentioning

confidence: 99%

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells

Marino

Cortivo

Maltese³

et al. 2021

IJMS

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“…This happens because the relatively subtle movement of the exiting helix in EF4 affects the GCAP1 fold on the other side of the molecule [ 63 ] (Fig. 3 ) and dimerization state of GCAP1 [ 12 , 64 ]. Extensive site-directed mutagenesis of GCAP2 [ 32 , 78 ] and especially GCAP1 [ 92 ] identified the cyclase binding interface of GCAP as a compact patch of the residues located on one side of the molecule and occupying large portions of EF1, -2, and a part of the entering helix of EF3 [ 92 ] (Fig.…”

Section: Metal Binding Properties Of the Photoreceptor-specific Ef-hand Sensor Proteinsmentioning

confidence: 99%

Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein

Dizhoor

Peshenko

2021

Pflugers Arch - Eur J Physiol

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This article presents a brief overview of the main biochemical and cellular processes involved in regulation of cyclic GMP production in photoreceptors. The main focus is on how the fluctuations of free calcium concentrations in photoreceptors between light and dark regulate the activity of retinal membrane guanylyl cyclase (RetGC) via calcium sensor proteins. The emphasis of the review is on the structure of RetGC and guanylyl cyclase activating proteins (GCAPs) in relation to their functional role in photoreceptors and congenital diseases of photoreceptors. In addition to that, the structure and function of retinal degeneration-3 protein (RD3), which regulates RetGC in a calcium-independent manner, is discussed in detail in connections with its role in photoreceptor biology and inherited retinal blindness.

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“…WT-GCAP1 forms functional dimers under physiological conditions [ 35 , 36 ]. We tested whether this was the case also for N104H-GCAP1 by running analytical gel filtration ( Figure 8 A) and DLS ( Figure 8 B) measurements.…”

Section: Resultsmentioning

confidence: 99%

“…In conclusion, we found that point mutations in the same position (Asn104) of the GUCA1A gene not only lead to clinically different phenotypes, but also generate distinct molecular phenotypes despite the absence of major structural alterations observed in any case (this study and previous ones [ 20 , 28 ]). Mutation-specific sensitivity toward cations, subtle alterations of protein stability in the presence of Mg 2+ and Ca 2+ , specific alteration of protein flexibility in distinct signaling states and the dependence of GCAP1 dimerization on the presence of specific cations [ 36 ] all point to a very complex molecular scenario, in which focusing on the effects of mutations on individual proteins might be of little use in advancing the molecular understanding of disease. Instead, unveiling the molecular details of the protein–protein and protein–ion interactions involved in the altered signaling cascade should be the final goal to achieve a molecular-level understanding of the extremely heterogenous retinal dystrophies, including COD, and would constitute a solid basis for designing effective therapeutic interventions.…”

Section: Discussionmentioning

confidence: 99%

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase

Biasi

Marino

Cortivo

et al. 2021

IJMS

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Guanylate cyclase-activating protein 1 (GCAP1), encoded by the GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.

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Modulation of Guanylate Cyclase Activating Protein 1 (GCAP1) Dimeric Assembly by Ca2+ or Mg2+: Hints to Understand Protein Activity

Cited by 13 publications

References 43 publications

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells

Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein

A Novel GUCA1A Variant Associated with Cone Dystrophy Alters cGMP Signaling in Photoreceptors by Strongly Interacting with and Hyperactivating Retinal Guanylate Cyclase

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