In order to understand how a pathogenic mutation causes inherited eye disease, it is necessary recognize how pathogenic mutations could affect protein structure-function, metabolic pathways, and how these perturbations could be associated clinical parameters describing the disease phenotype. For this purpose we perform molecular modeling to build protein structure, evaluate the severity of genetic missense changes from the atomic level of protein, and provide a quantitative analysis of the mutation impact on protein structure, stability and catalytic activity. We also do experimental in-vitro studies to measure the protein folding destabilization and changes in catalytic activity caused by the disease-related mutations. Finally we correlate these findings with clinical data on inherited eye disease phenotype. Currently we were using oculocutaneous albinism,Stargardt macular degeneration, congenital achromatopsia,autosomal recessive retinitis pigmentosa and others as our disease models. Oculocutaneous albinism is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with no or reduced TYR activity are classified as OCA1A or OCA1B forms, respectively. This year we were doing large scale purifications, biochemical, biophysical, and in-silico studies of human recombinant tyrosinases including OCA1-causing mutant variants. We also studied a role of N-glycosylation in 5-, 6-, and 7-site deglycosylated recombinant variants. Surprisingly, OCA1A mutant variants, T373K and R77Q, and N-deglycosylated variants show low protein expression, protein purity, and very low or no catalytic activity. As well this year we performed experimental and in-silico study with a purpose to understand folding and stability of OCA1B causing missense variants R402Q, P406L, R422Q, and R422W. Autosomal recessive Stargardt disease is the most common form of juvenile macular dystrophy and results from mutations in the ABCA4 gene. Approximately 49% of pathogenic ABCA4 missense mutations occur in the trans-membrane or nucleotide binding domains.The atomic structure of these domains could be modeled using molecular modeling. This year we continued work on molecular modeling of disease causing mutations in Stargardts disease and testing our predictions with clinical and EyeGENE data. The large-scale mutational analysis and predictions of mutation severities from atomic structure could be useful tool for the functional annotation of genetic variants from next-generation sequencing data and establishing the genotype-to-phenotype relationships in genetic disease. In addition, we performed modeling of a CEP290 oligomeric structure and several mutant variants (Rachel R. et al., Human Molecular Genetics, 2015). CEP290 localized in the transition zone of connecting cilia, precisely to the region of Y-linkers between central microtubules and plasma membrane. Based on the requirement of CEP290 in photoreceptor and ventricular ependyma ciliogenesis, we sought to predict CEP290 tertiary structure to gain further insight into its possible mechanism of action at the transition zone. Protein sequence information on centrosomal protein of 290 kDa (CEP290) from human and mice was used to predict structurally disordered regions and structural coiled-coil domains. Domain prediction of both mouse and human CEP290 identified extensive coiled-coils throughout the protein, almost to the exclusion of other domains. Only a few other cilia proteins share such a domain structure. To explore the tertiary structure of CEP290, we identified six coiled coil domains covering the full length of the protein, interspersed with five small peptide regions exhibiting structural disorder, which indicate five nodes that might maintain molecular flexibility of an otherwise rigid, rod-like molecule. We demonstrated that CEP290 has the propensity to form oligomeric structures. The entire protein is predicted to have a maximum theoretical length of about 348 nm, thus forming a long, thin rod-like molecule with approximate diameter 1.4 nm. In contrast, the truncated CEP290 protein of 116 kDa had a relatively high propensity to form a dimeric coiled-coil structure compared with that of the full-length trimeric protein. The structures of full-length and truncated CEP290 are interrupted by the coiled-coil vimentin-like structure (residues 696 to 752). The rest of the truncated CEP290 protein revealed 13% sequence identity to tropomyosin with 12 coiled-coil heptad motifs (HxxHCxC) confirming the coiled-coil conformation. The 116 kDa protein is shortened by 58% and predicted to have a maximum length of 145 nm. This study resulted in a model of photoreceptor connecting cilium which agrees with electron microscopy data. Our work clarifies possible positions for rod-like coiled-coil domain proteins such as Cep290, which localize to the region of the Y-linkers between the plasma membrane and the microtubule ring. We also implied molecular modeling to investigate the potential structural and functional consequences as well as possible risks associated with genetic mutations causing inherited eye disease. Such an examples are proteins, PDE6C and CNGA1, involved in congenital achromatopsia, macular atrophy, and autosomal recessive retinitis pigmentosa, respectively (Katagari S et al, Ophthalmic Genetics, 2015; PLOS One, 2014). In the first case, the ophthalmic examination revealed achromatopsia and severe macular atrophy in the older female sibling at 30 years of age and mild macular atrophy in the brother at 26 years of age. The genetic analysis identified a novel homozygous PDE6C mutation E591K as the disease-causing allele in the siblings. Each parent was heterozygous for the mutation. Molecular modeling showed that the mutation could cause a conformational change in the PDE6C protein and result in reduced phosphodiesterase activity. The PDEase catalytic domain has a binding site for divalent Zn2+ cations. The E591K mutation replaced a negatively-charged glutamic acid with a positively-charged lysine residue. This alteration dramatically changes the interaction between two helices, H5 and H12, located within the catalytic domain. The results of this modeling analysis were consistent with results of a structural study of phosphodiesterase inhibition by the C-terminal region of the g-subunit. In our PDE6C model, the H5 and H12 helices were positioned close to the H- and M-loops, residues 610-632 and 748-770, respectively. These loops formed a distinct interface that contributed to the g-subunit binding site. The disruption of this interface causes retinal degeneration in atrd3 mice. Our findings indicated that the H5 and H12 helices might be involved in the stabilization of the g-subunit binding site. PDE6C plays an important role in cone photoreceptors by rapidly decreasing intracellular levels of the second messenger cGMP. Reportedly, known PDE6C gene mutations reduce PDE activity, based on data from a PDE5/PDE6 chimeric protein expressed in Sf9 insect cells. Therefore, the E591K mutation might reduce PDE activity and thereby disturb the closure of the cGMP-gated ion channel in the cone outer segment membrane, resulting in the loss of hyperpolarization in the cone photoreceptors and leading to achromatopsia.
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