7 results on '"Guanylate Cyclase-Activating Proteins"'
Search Results
2. NMR and EPR-DEER Structure of a Dimeric Guanylate Cyclase Activator Protein‑5 from Zebrafish Photoreceptors
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Cudia, Diana, Roseman, Graham P, Assafa, Tufa E, Shahu, Manisha Kumari, Scholten, Alexander, Menke-Sell, Sarah-Karina, Yamada, Hiroaki, Koch, Karl-W, Milhauser, Glenn, and Ames, James B
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Biochemistry and Cell Biology ,Chemical Sciences ,Biological Sciences ,Rare Diseases ,Amino Acid Sequence ,Animals ,Cysteine ,Electron Spin Resonance Spectroscopy ,Guanylate Cyclase-Activating Proteins ,Magnesium ,Molecular Docking Simulation ,Mutation ,Nuclear Magnetic Resonance ,Biomolecular ,Protein Binding ,Protein Multimerization ,Protein Structure ,Quaternary ,Zebrafish ,Zebrafish Proteins ,Medicinal and Biomolecular Chemistry ,Medical Biochemistry and Metabolomics ,Biochemistry & Molecular Biology ,Biochemistry and cell biology ,Medical biochemistry and metabolomics ,Medicinal and biomolecular chemistry - Abstract
Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1-7). GCAPs form dimers that bind to Ca2+ and confer Ca2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg2+-bound, Ca2+-free, Fe2+-free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca2+-bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe2+ binding. Nitroxide spin-label probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe2+-dependent regulation of cyclase activity in zebrafish photoreceptors.
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- 2021
3. Prediction and validation of GUCA2B as the hub-gene in colorectal cancer based on co-expression network analysis: In-silico and in-vivo study
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Samira Nomiri, Reyhane Hoshyar, Elham Chamani, Zohreh Rezaei, Fatemeh Salmani, Pegah Larki, Tahmine Tavakoli, Faranak gholipour, Neda Jalili Tabrizi, Afshin Derakhshani, Mariacarmela Santarpia, Tindara Franchina, Oronzo Brunetti, Nicola Silvestris, and Hossein Safarpour
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Pharmacology ,Neoplastic ,Tumor ,WGCNA ,Colorectal cancer ,GUCA2B ,Molecular pathogenicity ,Transcriptome analysis ,Apoptosis ,Biomarkers, Tumor ,Colorectal Neoplasms ,Gene Expression Regulation, Neoplastic ,Gene Regulatory Networks ,Guanylate Cyclase-Activating Proteins ,Humans ,Intestinal Mucosa ,MicroRNAs ,Natriuretic Peptides ,Transcriptome ,General Medicine ,RM1-950 ,Gene Expression Regulation ,Therapeutics. Pharmacology ,Biomarkers - Abstract
Background: Several serious attempts to treat colorectal cancer have been made in recent decades. However, no effective treatment has yet been discovered due to the complexities of its etiology. Methods: we used Weighted Gene Co-expression Network Analysis (WGCNA) to identify key modules, hub-genes, and mRNA-miRNA regulatory networks associated with CRC. Next, enrichment analysis of modules has been performed using Cluepedia. Next, quantitative real-time PCR (RT-qPCR) was used to validate the expression of selected hub-genes in CRC tissues. Results: Based on the WGCNA results, the brown module had a significant positive correlation (r = 0.98, p-value=9e-07) with CRC. Using the survival and DEGs analyses, 22 genes were identified as hub-genes. Next, three candidate hub-genes were selected for RT-qPCR validation, and 22 pairs of cancerous and non-cancerous tissues were collected from CRC patients referred to the Gastroenterology and Liver Clinic. The RT-qPCR results revealed that the expression of GUCA2B was significantly reduced in CRC tissues, which is consistent with the results of differential expression analysis. Finally, top miRNAs correlated with GUCA2B were identified, and ROC analyses revealed that GUCA2B has a high diagnostic performance for CRC. Conclusions: The current study discovered key modules and GUCA2B as a hub-gene associated with CRC, providing references to understand the pathogenesis and be considered a novel candidate to CRC target therapy.
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- 2022
4. The Transition of Photoreceptor Guanylate Cyclase Type 1 to the Active State
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Manisha Kumari, Shahu, Fabian, Schuhmann, Alexander, Scholten, Ilia A, Solov'yov, and Karl-Wilhelm, Koch
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Alanine ,Guanylate Cyclase ,Retinal Cone Photoreceptor Cells ,Humans ,Guanylate Cyclase-Activating Proteins ,Hormones - Abstract
Membrane-bound guanylate cyclases (GCs), which synthesize the second messenger guanosine-3', 5'-cyclic monophosphate, differ in their activation modes to reach the active state. Hormone peptides bind to the extracellular domain in hormone-receptor-type GCs and trigger a conformational change in the intracellular, cytoplasmic part of the enzyme. Sensory GCs that are present in rod and cone photoreceptor cells have intracellular binding sites for regulatory Ca
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- 2022
5. Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa
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Anna Avesani, Laura Bielefeld, Nicole Weisschuh, Valerio Marino, Pascale Mazzola, Katarina Stingl, Tobias B. Haack, Karl-Wilhelm Koch, and Daniele Dell’Orco
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retina ,vision ,phototransduction ,guanylate cyclase (guanylyl cyclase) ,cyclic GMP ,calcium-binding proteins ,retinitis pigmentosa ,neurodegenerative disease ,GUCA1C ,GCAP ,Catalysis ,Inorganic Chemistry ,Animals ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy ,Organic Chemistry ,Calcium-Binding Proteins ,General Medicine ,Guanylate Cyclase-Activating Proteins ,Computer Science Applications ,Guanylate Cyclase ,Retinal Cone Photoreceptor Cells ,Calcium ,Cattle ,Retinitis Pigmentosa - Abstract
The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G>C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca2+-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 ms molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca2+ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.
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- 2022
6. A Novel
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Amedeo, Biasi, Valerio, Marino, Giuditta, Dal Cortivo, Paolo Enrico, Maltese, Antonio Mattia, Modarelli, Matteo, Bertelli, Leonardo, Colombo, and Daniele, Dell'Orco
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Male ,Light Signal Transduction ,Adolescent ,GUCA1A ,phototransduction ,Retina ,Article ,calcium binding proteins ,Retinal Rod Photoreceptor Cells ,neuronal calcium sensor ,Humans ,Cone Dystrophy ,Child ,Cyclic GMP ,cone dystrophy ,guanylyl cyclase ,photoreceptors ,Middle Aged ,Guanylate Cyclase-Activating Proteins ,Pedigree ,Guanylate Cyclase ,Mutation ,retinal degeneration ,Calcium ,Female ,Signal Transduction - Abstract
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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- 2021
7. Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase
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Elena V. Olshevskaya, Alexander M. Dizhoor, and Igor V. Peshenko
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Retinal degeneration ,genetic structures ,CORD6, cone–rod dystrophy 6 ,ONL, outer nuclear layer ,Biochemistry ,chemistry.chemical_compound ,Mice ,GCAP, guanylyl cyclase–activating protein ,RD3, retinal degeneration-3 protein ,Mice, Knockout ,medicine.diagnostic_test ,OCT, optical coherence tomography ,CNG, cyclic nucleotide–gated channel ,Nuclear Proteins ,Photoreceptor outer segment ,eye ,Cell biology ,Isoenzymes ,medicine.anatomical_structure ,calcium-binding proteins ,GUCY2D ,TTBS, Tris-buffered saline containing 0.5% Tween-20 ,RD3 ,Retinitis Pigmentosa ,Research Article ,Photoreceptor Cells, Vertebrate ,Receptors, Cell Surface ,HEK293, human embryonic kidney cells 293 ,LCA12, Leber's congenital amaurosis type 12 ,cyclic GMP ,RetGC ,medicine ,RetGC, the retinal membrane guanylyl cyclase ,Animals ,Humans ,ROS, rod outer segment ,guanylate cyclase (guanylyl cyclase) ,Outer nuclear layer ,Molecular Biology ,Retina ,ERG, electroretinography ,GCAP ,Dystrophy ,Retinal ,Cell Biology ,medicine.disease ,photoreceptor ,Guanylate Cyclase-Activating Proteins ,HEK293 Cells ,chemistry ,Guanylate Cyclase ,Mutation ,retinal degeneration ,sense organs ,Electroretinography - Abstract
Different forms of photoreceptor degeneration cause blindness. Retinal degeneration-3 protein (RD3) deficiency in photoreceptors leads to recessive congenital blindness. We proposed that aberrant activation of the retinal membrane guanylyl cyclase (RetGC) by its calcium-sensor proteins (guanylyl cyclase–activating protein [GCAP]) causes this retinal degeneration and that RD3 protects photoreceptors by preventing such activation. We here present in vivo evidence that RD3 protects photoreceptors by suppressing activation of both RetGC1 and RetGC2 isozymes. We further suggested that insufficient inhibition of RetGC by RD3 could contribute to some dominant forms of retinal degeneration. The R838S substitution in RetGC1 that causes autosomal-dominant cone–rod dystrophy 6, not only impedes deceleration of RetGC1 activity by Ca2+GCAPs but also elevates this isozyme's resistance to inhibition by RD3. We found that RD3 prolongs the survival of photoreceptors in transgenic mice harboring human R838S RetGC1 (R838S+). Overexpression of GFP-tagged human RD3 did not improve the calcium sensitivity of cGMP production in R838S+ retinas but slowed the progression of retinal blindness and photoreceptor degeneration. Fluorescence of the GFP-tagged RD3 in the retina only partially overlapped with immunofluorescence of RetGC1 or GCAP1, indicating that RD3 separates from the enzyme before the RetGC1:GCAP1 complex is formed in the photoreceptor outer segment. Most importantly, our in vivo results indicate that, in addition to the abnormal Ca2+ sensitivity of R838S RetGC1 in the outer segment, the mutated RetGC1 becomes resistant to inhibition by RD3 in a different cellular compartment(s) and suggest that RD3 overexpression could be utilized to reduce the severity of cone–rod dystrophy 6 pathology.
- Published
- 2021
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