A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness

The autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants

Calcium binding, structural stability and guanylate cyclase activation in GCAP1 variants associated with human cone dystrophy

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal Ca2+ sensor (NCS) that regulates the activation of rod outer segment guanylate cyclases (ROS-GCs) in photoreceptors. In this study, we investigated the Ca2+-induced effects on the conformation and the thermal stability of four GCAP1 variants associated with hereditary human cone dystrophies. Ca2+ binding stabilized the conformation of all the GCAP1 variants independent of myristoylation. The myristoylated wild-type GCAP1 was found to have the highest Ca2+ affinity and thermal stability, whereas all the mutants showed decreased Ca2+ affinity and significantly lower thermal stability in both apo and Ca2+-loaded forms. No apparent cooperativity of Ca2+ binding was detected for any variant. Finally, the nonmyristoylated mutants were still capable of activating ROS-GC1, but the measured cyclase activity was shifted toward high, nonphysiological Ca2+ concentrations. Thus, we conclude that distorted Ca2+-sensor properties could lead to cone dysfunction

Integrated in silico and in vitro characterization of Rhodopsin mutations causing RP4

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were discovered in the rhodopsin gene (RHO). RHO belongs to the G protein Coupled Receptor superfamily of seven-transmembrane proteins. The vast majority of the rhodopsin mutations cause the Autosomal Dominant form (ADRP) of the disease. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants are obscure, which hampers rational drug design. Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. In order to define levels of expression and differences in post-translational modifications of the mutants compared to the wild type, the proteins were analyzed by Western blotting. Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. We could define a number of computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO. We also defined different abilities of the mutated proteins to be affected by 9-cis retinal. Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. This knowledge will be our starting point for the choice of a number of mutations to be used to reveal therapeutic effect of chaperone molecules

Classification of Rhodopsin mutations by integrated in silico and in vitro analyses for screening of chaperon molecules to rescue misfolding.

Purpose: About 140 point mutations were identified in the rhodopsin gene (RHO) as cause of Autosomal Dominant Retinitis Pigmentosa (ADRP), a genetic degenerative disease causing blindness in later life. A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Characterization of the structural and molecular features of such mutants will support the development of rational drug design. Methods: Wild type RHO and 33 different RHO mutations were analyzed in silico either in the rhodopsin form bound to retinal or in the opsin form by thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. In parallel, the same mutants were cloned in expression vectors and in vitro expressed in COS-7 cells either in the absence or presence of 9-cis retinal in the culture medium. The subcellular localization was analyzed with two monoclonal antibodies recognizing either the extracellular N-terminal or the intracellular C-terminal of RHO. Retention in the endoplasmic reticulum (ER) was assessed by analysis of co-localization with calnexin and calculation of the Pearson Correlation Coefficient (PCC) of co-localization. Results: In silico studies revealed that the selected ADRP RHO mutations share marked abilities to impair highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates to the stability of the protein. We defined computational indices whose combination led to a structural classification of the mutants. The in vitro level of analysis revealed increased ER retention and reduction of plasma membrane localization of most of the mutants compared to wild type RHO. We found a strong correlation of the perturbation indexes calculated by in silico analyses with PCC calculated by in vitro analyses. We could also characterize different abilities of the mutated proteins to be affected by treatment with 9-cis retinal. Conclusions: These two levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. We also developed a PCC index to evaluate the effect of retinal on protein folding and protein localization at the plasma membrane. This knowledge will be our starting point for in silico screening of compounds able to bind the retinal site and act as chaperones. The in vitro studies have developed a quantitative analysis to assess therapeutic effects of chaperone molecules

Integrated in silico and in vitro characterization of Rhodopsin mutations and molecular mechanisms activated in photoreceptor cell death

Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing blindness in later life. Despite the high genetic heterogeneity of RP, ~140 point mutations were identified in the rhodopsin gene (RHO) as cause of the Autosomal Dominant form of the disease (ADRP). A recent analysis indicates that 89% of the biochemically characterized RHO mutants are misfolded, supporting the protein-misfolding disease model suitable for treatments with pharmacological chaperones. Yet, the structural and molecular features of such mutants and the cell death pathways activated by mutant RHO are obscure, hampering rational drug design. Methods: In silico experiments on wild type RHO and 36 different mutations consisted in thermal unfolding simulations combined with the graph-based Protein Structure Network analysis. Subcellular localization and effects of retinal as chaperone in the same mutants were characterized in vitro in COS-7 cells. Molecular death pathways activated by RHO mutation were studied in vivo in transgenic and knock-in mice bearing the P23H mutation. Results: In silico studies revealed that ADRP RHO mutations share marked abilities to impair selected highly connected nodes in the protein structure network, i.e. hubs, essentially located in the retinal binding site, which participates in the stability core of the protein. The in vitro level of analysis revealed reduction in expression levels and plasma membrane localization of some of the mutants compared to wild type RHO as well as different abilities of the mutated proteins to be affected by 9-cis retinal. We characterized ER-stress pathways and calpain pathway activation in P23H mutant retinas. We defined the different contributions of these pathways by in vivo treatments with specific drugs blocking either ER-stress or calpains. Conclusions: The in silico and in vitro levels of analysis allowed a novel characterization of the different mutants to generate the first classification of ADRP RHO mutants based on a multiscale approach, i.e. at the cellular and atomic levels of detail. We also characterized cell death pathways activated by RHO mutants. This knowledge will be our starting point for an in silico screen of chaperone molecules to be tested in vivo

Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes.

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes

Mutations in the GUCA1A gene involved in hereditary cone dystrophies impair calcium-mediated regulation of guanylate cyclase

The GUCA1A gene encodes the guanylate cyclase activating protein 1 (GCAP1) of mammalian rod and cone photoreceptor cells, which is involved in the Ca 2+ -dependent negative feedback regulation of membrane bound guanylate cyclases in the retina. Mutations in the GUCA1A gene have been associated with different forms of cone dystrophies leading to impaired cone vision and retinal degeneration. Here we report the identification of three novel and one previously detected GUCA1A mutations: c.265G>A (p.Glu89Lys), c.300T>A (p.Asp100Glu), c.476G>T (p.Gly159Val) and c.451C>T (p.Leu151Phe). The clinical data of the patients carrying these mutations were compared with the functional consequences of the mutant GCAP1 forms. For this purpose we purified the heterologously expressed GCAP1 forms and investigated whether the mutations affected the Ca 2+ -triggered conformational changes and the apparent interaction affinity with the membrane bound guanylate cyclase. Furthermore, we analyzed Ca 2+ -dependent regulatory modes of wildtype and mutant GCAP1 forms. Although all novel mutants were able to act as a Ca 2+ -sensor protein, they differed in their Ca 2+ -dependent activation profiles leading to a persistent stimulation of guanylate cyclase activities at physiological intracellular Ca 2+ concentration. © 2009 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63544/1/21055_ftp.pd

Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca²⁺ plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca²⁺-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca²⁺-dependent manner. No binding is observed in buffer with 6 mM Mg²⁺ and 1 mM EGTA that chelates Ca²⁺, suggesting high specificity of the CaM–ribosome interaction dependent on the Ca²⁺ induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free <i>in vitro</i> translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes