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

2021 ◽  
Vol 22 (19) ◽  
pp. 10809
Author(s):  
Amedeo Biasi ◽  
Valerio Marino ◽  
Giuditta Dal Cortivo ◽  
Paolo Enrico Maltese ◽  
Antonio Mattia Modarelli ◽  
...  
Keyword(s):  
Guanylate Cyclase ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Cone Dystrophy ◽  
Photoreceptor Outer Segment ◽  
Enzymatic Assays ◽  
Cgmp Signaling ◽  
Novel Variant ◽  
Neuronal Calcium Sensor Protein ◽  
Dynamics Simulations

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.

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

2021 ◽  
Vol 22 (8) ◽  
pp. 4030
Author(s):  
Valerio Marino ◽  
Giuditta Dal Cortivo ◽  
Paolo Enrico Maltese ◽  
Giorgio Placidi ◽  
Elisa De Siena ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Guanylate Cyclase ◽  
Molecular Mechanisms ◽  
Second Messengers ◽  
Bipolar Cells ◽  
Cone Dystrophy ◽  
Photoreceptor Outer Segment ◽  
Double Amino Acid ◽  
Dynamics Simulations ◽  
Unusual Phenotype

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.

scholarly journals Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology

1979 ◽  
Vol 183 (3) ◽  
pp. 615-622 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Sequence Homology ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Terminal Sequence ◽  
Complement Components ◽  
Factor B ◽  
Terminal Residue ◽  
Covalently Linked

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

scholarly journals CERKL gene knockout disturbs photoreceptor outer segment phagocytosis and causes rod-cone dystrophy in zebrafish

2017 ◽  
Vol 26 (12) ◽  
pp. 2335-2345 ◽  
Author(s):  
Shanshan Yu ◽  
Chang Li ◽  
Lincoln Biswas ◽  
Xuebin Hu ◽  
Fei Liu ◽  
...  
Keyword(s):  
Outer Segment ◽  
Gene Knockout ◽  
Cone Dystrophy ◽  
Photoreceptor Outer Segment

A novel variant of DHH in a familial case of 46,XY disorder of sex development: Insights from molecular dynamics simulations

2017 ◽  
Vol 87 (5) ◽  
pp. 539-544 ◽  
Author(s):  
Francoise Paris ◽  
Delphine Flatters ◽  
Sandrine Caburet ◽  
Bérangère Legois ◽  
Nadège Servant ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Familial Case ◽  
Disorder Of Sex Development ◽  
Sex Development ◽  
Novel Variant ◽  
Dynamics Simulations

scholarly journals The release of collagenase as an inactive proenzyme by bone explants in culture

1972 ◽  
Vol 126 (2) ◽  
pp. 275-289 ◽  
Author(s):  
G. Vaes
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Culture Media ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Inhibitor Complex ◽  
Ph Values ◽  
Liver Lysosomes ◽  
Skin Explants ◽  
Extracellular Activity

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme–inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a `procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.

scholarly journals Activation of a phospholipase Cβ2 deletion mutant by limited proteolysis

1998 ◽  
Vol 330 (1) ◽  
pp. 461-468 ◽  
Author(s):  
Petra SCHNABEL ◽  
Montserrat CAMPS
Keyword(s):  
Deletion Mutant ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Cell System ◽  
Heterotrimeric G Proteins ◽  
Sds Page ◽  
Catalytically Active ◽  
Heterodimeric Complex ◽  
High Degree ◽  
Hydrolysis Of

All phosphoinositide-specific phospholipases C (PLC) identified until today exhibit a high degree of similarity within two regions of 170 and 260 residues, respectively, which are designated regions X and Y. The PLCβ family, including four members designated PLCβ1, PLCβ2, PLCβ3 and PLCβ4, is regulated by heterotrimeric G proteins. In order to investigate structure-function relationships of PLCβ2, we expressed PLCβ2Δ, a deletion mutant of PLCβ2 which lacks most of the sequence downstream of region Y, in the baculovirus/insect cell system. The mutant was present in both soluble and particulate fractions of Sf9 cells and was demonstrated to be catalytically active and sensitive to βγ-subunits. Sf9 cytosol containing this mutant was subjected to limited proteolysis by trypsin and S. aureus protease V8, respectively. Immunochemical analysis revealed that both proteases cleaved the enzyme between the regions X and Y. Most interestingly, proteolytic cleavage at this site by both proteases stimulated the catalytic activity of PLC2β2Δ. The proteolytically activated enzyme was still sensitive to βγ-subunits and showed an unchanged concentration dependence on Ca2+. Gel filtration chromatography indicated that the fragments generated by cleavage between the regions X and Y were still connected and formed a functional heterodimeric complex. In order to visualize all fragments generated by protease V8, PLCβ2Δ was purified to homogeneity from Sf9 cytosol. Limited proteolysis of the purified enzyme by S. aureus protease V8 and subsequent SDS/PAGE and silver staining revealed that several cuts take place between the regions X and Y and that the resulting fragments remain intact. We hypothesize that the activating proteolytic cut induces a conformational change of the enzyme which might facilitate hydrolysis of the phospholipid substrate.

scholarly journals Normal GCAPs partly compensate for altered cGMP signaling in retinal dystrophies associated with mutations in GUCA1A

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Daniele Dell’Orco ◽  
Giuditta Dal Cortivo
Keyword(s):  
Guanylate Cyclase ◽  
Autosomal Dominant ◽  
Compensatory Mechanism ◽  
Missense Mutations ◽  
Wild Type ◽  
Gene Encoding ◽  
Retinal Dystrophies ◽  
Cgmp Signaling ◽  
Intracellular Ca ◽  
Phototransduction Cascade

AbstractMissense mutations in the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) are associated with autosomal dominant cone/cone-rod (CORD) dystrophies. The nature of the inheritance pattern implies that a pool of normal GCAP proteins is present in photoreceptors together with the mutated variant. To assess whether human GCAP1 and GCAP2 may similarly regulate the activity of the retinal membrane guanylate cyclase GC-1 (GC-E) in the presence of the recently discovered E111V-GCAP1 CORD-variant, we combined biochemical and in silico assays. Surprisingly, human GCAP2 does not activate GC1 over the physiological range of Ca2+ whereas wild-type GCAP1 significantly attenuates the dysregulation of GC1 induced by E111V-GCAP1. Simulation of the phototransduction cascade in a well-characterized murine system, where GCAP2 is able to activate the GC1, suggests that both GCAPs can act in a synergic manner to mitigate the effects of the CORD-mutation. We propose the existence of a species-dependent compensatory mechanism. In murine photoreceptors, slight increases of wild-type GCAPs levels may significantly attenuate the increase in intracellular Ca2+ and cGMP induced by E111V-GCAP1 in heterozygous conditions. In humans, however, the excess of wild-type GCAP1 may only partly attenuate the mutant-induced dysregulation of cGMP signaling due to the lack of GC1-regulation by GCAP2.

scholarly journals Comprehensive Geno- and Phenotyping in a Complex Pedigree Including Four Different Inherited Retinal Dystrophies

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 137
Author(s):  
Johannes Birtel ◽  
Martin Gliem ◽  
Kristina Hess ◽  
Theresa H. Birtel ◽  
Frank G. Holz ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Night Blindness ◽  
Molecular Genetic ◽  
Cone Dystrophy ◽  
Molecular Testing ◽  
Congenital Stationary Night Blindness ◽  
Molecular Genetic Testing ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
Novel Variant

Inherited retinal dystrophies (IRDs) are characterized by high clinical and genetic heterogeneity. A precise characterization is desirable for diagnosis and has impact on prognosis, patient counseling, and potential therapeutic options. Here, we demonstrate the effectiveness of the combination of in-depth retinal phenotyping and molecular genetic testing in complex pedigrees with different IRDs. Four affected Caucasians and two unaffected relatives were characterized including multimodal retinal imaging, functional testing, and targeted next-generation sequencing. A considerable intrafamilial phenotypic and genotypic heterogeneity was identified. While the parents of the index family presented with rod-cone dystrophy and ABCA4-related retinopathy, their two sons revealed characteristics in the spectrum of incomplete congenital stationary night blindness and ocular albinism, respectively. Molecular testing revealed previously described variants in RHO, ABCA4, and MITF as well as a novel variant in CACNA1F. Identified variants were verified by intrafamilial co-segregation, bioinformatic annotations, and in silico analysis. The coexistence of four independent IRDs caused by distinct mutations and inheritance modes in one pedigree is demonstrated. These findings highlight the complexity of IRDs and underscore the need for the combination of extensive molecular genetic testing and clinical characterization. In addition, a novel variant in the CACNA1F gene is reported associated with incomplete congenital stationary night blindness.

scholarly journals Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Giuditta Dal Cortivo ◽  
Valerio Marino ◽  
Claudio Iacobucci ◽  
Rosario Vallone ◽  
Christian Arlt ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Protein ◽  
Gel Filtration ◽  
Native Mass Spectrometry ◽  
Oligomeric State ◽  
Hydrodynamic Properties ◽  
Mechanism Of Interaction ◽  
Accessible Surface ◽  
Dynamics Simulations ◽  
Solvent Accessible Surface

Abstract Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the α7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with α7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an α7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule.

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