In order to understand how a pathogenic change in a gene causes disease, it is necessary to recognize how pathogenic mutations could affect a protein structure-function, protein-protein interactions in protein networks and how these changes could be associated with clinical parameters describing the disease phenotype. We imply molecular modeling to build protein structure, simulate the effect of pathogenic missense changes, and provide a quantitative analysis of their impact on protein structure and stability. Here we use oculocutaneous albinism, autosomal dominant maculopathy, and X-linked retinoschisis (XLRS) as our disease models. 1. Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are currently four types of OCA. For the first time, three full-length protein atomic structures, TYR (OCA1), TYRP1 (OCA3), and SLC45A2 (OCA4), were successfully modeled by homology and in silico analysis of missence changes from the NEI/NHGRI molecular diagnostic study has been performed. TYR is a type I trans-membrane monooxygenase. The 4-helix bundle is structurally conserved in different species to carry CuA and CuB ions essential for the catalytic reaction. The active site is formed by 6 His residues structurally coordinating the copper positions. Several missense changes from the collaborative NEI/NHGRI molecular diagnostic study were analyzed. S50L is found in the Cys-rich motif 1 of tyrosinase, whereas R298W is located within the Cys-rich motif 2. The mutations N364H, P384A, D394N, D437N and R403V disrupt the coordination of the copper ion center. A490D affects conformation of helix located in melanosomal membrane. TYRP1 is a type 1 membrane protein. This protein also has 2 Cys-rich motifs and an active site with 6 His residues coordinating 2 metal ions of unknown nature that could be either copper or zinc. A heterozygous missense mutation, A24T, was found in the border between a signal peptide and Cys-rich motif 1 of TYRP1. The structure of SLC45A2 is predicted as a multi-pass trans-membrane protein. The missense mutation L60R is predicted to be deleterious. To test our predictions on tyrosinase activity we engineered a construct of human C-terminal truncated tyrosinase, hTyrCtr. The expression of hTyrCtr in E. Coli was confirmed by Western blot analysis. Metal affinity chromatography shown poor binding to the column which suggests that C-terminal His-tag peptides have decreased binding capacity to the IMACS resin. In addition, L-Dopa enzymatic assay demonstrated that hTyrCtr is expressed as a non-active enzyme which might be due to either the loss of Cu2+ ions in a catalytic site or protein partial misfolding. In contrast, similar protein construct was implied for the protein expression in larvae. We shown that the hTyrCtr, and 2 mutant variants, R422Q and R422W, are active soluble proteins which catalyzes the rate-limiting conversions of tyrosine to DOPA and DOPA to DOPA-quinone. In perspective, a detailed understanding of protein structure and the mechanisms controlling tyrosine-modified tyrosinase interactions would allow to establish molecular chaperone screening for a future medical treatment of patients with the OCA-1B albinism. 2. Assembly of elastic fibers is critical for structural development as well as proper functioning of the extracellular matrix. Elastin and 10-nm fibrillin containing microfibrils form the major components of elastic fibers, which form integral part of extracellular matrices including Bruchs membrane. One of fibrillins, fibrillin-2 or FBN2, is a 2,912 amino acid polypeptide which consists of one amino-terminal trans-membrane domain, 4 epidermal growth factor-like (EGF) domains, 43 calcium-binding consensus sequences (Ca_EGF domains), and 9 transforming growth factor 1 binding protein-like (TB) domains. FBN-2 has 363 cysteine residues. The amino acid sequence of fibrillin-2 been used to generate a native and mutant variant structures for the Ca-EGF motifs 12-19 by homology modeling. Protein fold of Ca_EGF domain is maintained by 6 conserved cysteines which form 3 SS-bridges. In addition, negatively charged conserved residues are either involved in direct ligation to calcium or involved in stabilizing the calcium-binding site. Calcium ion improves the fold stability, help to fix a relative orientation of two neighbor Ca_EGF domains, and stabilize a spatial orientation of FBN-2. Disease-causing mutation E1144K introduces a positive charge into the negatively charged cavity and decreases the Ca-binding affinity. The interaction of K1144 and E1178 change a relative orientation of a neighbor domain. E1438K mutation is expected to have a similar structural effect. Both mutations are associated with a severe phenotype of disease and could change of microfiber packing and elasticity. The M1247T change is affecting the hydrophobic surface loop. The SS-bridge C1246-C1257 stabilizes the native fold of a protein by lowering entropy of the polypeptide chain and by condensing hydrophobic residues from the surface loop into local hydrophobic core using hydrophobic interactions. Thus, the mutation M1247T might affect the SS-bond stability and/or intermolecular interactions. Other mutations are mild changes. Mutations with severe phenotype are likely to cause a change in the fiber flexibility, packaging, and pathogenicity. This might cause the loss of elastic fibers, thickening and calcification of Bruchs membrane which are associated with dominant maculopathy and AMD pathophysiology. 3. Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. Molecular modeling predicted an association between the type of structural RS1 alterations and the severity of full-field ERG phenotype in all but the oldest group of patients. This is now a second study (Hum Mol Genet 19:1302, 2010) that indicates a genotype-ERGphenotype correlation, and it was done with a totally separate and independent cohort. There was a significant association between the predicted severity of RS1 perturbation and both photopic and scotopic ERG b/a-ratios, but only for one age group (15-30 years). Severe RS1 missense changes were associated with a lower ERG b/a ratio than for mild and moderate missense changes, suggesting a quantitatively distinct ERG phenotype. Age-related differences in dark-adapted ERG parameters are consistent with those reported previously in the RS1 knockout mouse. 4. One of possible clinical implications in a human eye disease is using chaperones for the stabilization of native protein structure in mutant variants affected by genetic mutations. This stabilization could be performed in controllable fashion by using small heat shock proteins (sHSPs) with genetically engineered structure. Our recent study have indicated a role for changing of protein hydrophobicity in the thermal adaptation of alpha-crystallin A and suggested ways to produce sHSP variants with altered chaperone-like activity. In this work we use molecular modeling, computational biology, and side-directed mutagenesis to evaluate the effect of mutations and to establish a link between sHSPs hydrophobicity and physiological temperatures. sHSPs maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. In addition, our work provided an evidence for an evolutionary mechanism that has adapted chaperone activity to different environmental temperatures though the alteration of hydrophobicity at crucial locations in the protein structure. This combination of experimental and computational design potentially could be used to create a new generation of artificial chaperones with a purpose to improve stability of mutant variants in inherited eye disease.
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