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, provide a quantitative analysis of their impact on protein structure and stability, and corellate these findings with disease phenotype. Currently we use X-linked retinoschisis (XLRS), oculocutaneous albinism, and macular degeneration as our disease models. 1.XLRS is a vitreo-retinal degeneration caused by mutations in the protein retinoschisin (RS1), required for the structural and functional integrity of the retina. In our work the structural effect of mutational changes was analyzed in silico on the basis of atomic protein structure, molecular dynamics, and Gibbs free energy calculations. Previously, in 60 XLRS patients from NEI who share 27 missense mutations, the molecular models were correlated with retinal function as determined by the ERG and b-waves (Sergeev et al., 2010, HMG). To test the validity of this approach, we have now compared the molecular models with the electrophysiological features in a cohort of 38 XLRS patients from Moorfields Eye Hospital (London, UK) having one of 18 RS1 missense variants (Sergeev et al., 2013, HMG). Patients were grouped based on mutation severity predicted by molecular modeling: mild (class I), moderate (intermediate) and severe (class II). Most patients had an electronegative scotopic bright flash electroretinogram (ERG) (reduced b/a-wave ratio) in keeping with predominant inner retinal dysfunction. An association between the type of structural RS1 alterations and the severity of b/a-wave reduction was found in all but the oldest group of patients, significant in patients aged 15V30 years. Severe RS1 missense changes were associated with a lower ERG b/a ratio than were mild changes, suggesting that the extent of inner retinal dysfunction is influenced by the effect of the mutations on protein structure. The majority of class I mutations showed no changes involving cysteine residues. Class II mutations caused severe perturbations due to the removal or insertion of cysteine residues or due to changes in the hydrophobic core. The ERG b/a ratio in intermediate cases was abnormal but showed significant variability, possibly related to the role of proline or arginine residues. The grouping in classes helps predict the severity of ERG abnormalities relating to global inner retinal dysfunction and may influence patient management and the selection of candidates for possible future therapeutic interventions. The knowledge of a functional phenotype (b/a-ratios) from the patient genotype potentially could be useful in clinical trials or other clinical studies to identify patient groups with severe and less severe phenotypes and might provide some rationale to choose medical treatment for each group. Indeed, the small molecule drug treatment might be more appropriate for patients with mild missense changes. In contrast, gene therapy could be suggested as a treatment to compensate the effect of the null protein caused by the severe missense change. Finally, this work confirms the findings in our previous study (Sergeev et al, 2010, HMG), makes additional observations with a finer grading scale, and contains a comprehensive statistical analysis of in silico predictions for the completely independent patient cohort. For the first time this work does clearly show that the specific mutation makes a difference in the XLRS clinical severity and quite likely in the clinical course. In general, the ability to establish genotype-to-phenotype relationships could be used to assess disease risk using atomic models of proteins for a broad spectrum of inherited eye disorders. 2.Oculocutaneous albinism (OCA) 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 is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B;some OCA1B mutations are temperature-sensitive (Simeonov et al., 2013, Human Mutation). Previous studies have shown that temperature sensitive mutants exit the endoplasmic reticulum and enter the endosomal compartment at 31oC, but not 37oC, suggesting that intracellular targeting is an important mechanism for loss of enzymatic activity. To test these predictions on tyrosinase activity the intra-melanosomal domain of human tyrosinase (residues 19 - 469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae (Dolinska et al., PLOS One, 2013, resubmitted). The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1mg per 10g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37oC and neutral pH. Glycosylation occurred at several Asn residues, and the enzymes kinetic and pharmacologic properties were similar to the authentic enzyme described in the literature. We furthermore characterized the enzymology of two temperature-sensitive enzymes. The intra-melanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Indeed, compared to WT enzyme, the R422Q mutant at 37oC had lowered helical and increased beta-sheet content. This conformational change is equivalent to partial (localized) protein unfolding with concomitant reduction in the enzymes function. Hence, temperature dependent changes in catalytic efficiency of R422Q appear to be associated with structural change. The temperature sensitivity of the R422W mutant is not clearly detected by far-UV CD. This suggests that, at least in part, the temperature sensitive behavior is intrinsic to the tyrosinase structure. In conclusion, we have described methods which result in the production of pure wild-type and mutant tyrosinase proteins in quantities sufficient for targeted or panel drug screening. Pure recombinant enzyme would also have the potential for direct therapeutic treatment, if delivered to the retinal pigment epithelium of the eye prior to foveal development. Purified proteins can also be used for crystal screening and X-ray structure determination. High resolution structural information could be used, for example, in rational drug designs. Also, structural determinations could help understand at a molecular level how individual mutations contribute to the diverse phenotype in patients with albinism. 3.Age-related macular degeneration. Recently we have implied molecular modeling to investigate possibly risks of genetic mutations associated with macular degeneration and the potential functional consequences of the K155Q variant (Zhan X. et al., 2013, Nature Genetics, accepted).Our analysis identified K155Q as a rare C3 variant associated with a 2.91-fold increased risk of macular degeneration. Molecular modeling suggests that together with rare CFH variant R1210C and previously described common C3 variant R102G, K155Q may reduce binding of CFH to C3b, inhibiting the ability of Factor H to inactivate the alternative complement pathway.
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