Proteins From Hereditary Eye Diseases: In-silico and Experimental Studies

National Eye Institute

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Abstract

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 X-linked retinoschisis (XLRS) and oculocutaneous albinism type 1 (OCA1) as our disease models. 1. Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. Last year we suggested a severity score for the protein structure-based classification of the pathogenic missense mutations impact in human XLRS. Conclusions from this work were recently refined using the human XLRS affected cohort of patients from the NEI clinic. This analysis was applied independently to ERG b/a-wave ratios as a function of average patient age versus severity of XLRS phenotype. As result, phenotypic b/a-wave ratios declined for younger individuals accompanied by a corresponding rise of computed impact score, a measure of the structural severity of the mutation. Although this change is statistically less significant for older patients, a common trend showing a slight decrease with computed impact seems to be similar to that of described for younger individuals. Differences in phenotypes were considered for 3 groups of mutations, low- and high-impact missense changes and severe non-missense mutations. Low-impact missense changes show average b/a-wave ratio close to 1.0 for a maximum with 15 XLRS patients of different ages. This ratio corresponds to a maximum of 10 patients with an average value of approximately 0.6, which is consistent with more severe XLRS phenotype as expected for high-impact missense variants. In addition, the severity of high impact missense changes is confirmed by identical correspondence to 15 patients with the severe non-missense variants. This study confirmed that pathogenic missense mutations could be separated in mild and severe groups of XLRS phenotype by using the molecular grading scale. Thus, low- and high-impact missense changes in protein structure are associated with mild and severe changes in the XLRS phenotype. The results for the expected XLRS phenotypes currently predicted for 115 missense changes. From our analysis, half of the mild mutations could be considered as mutations which were partially or fully exposed at the protein surface;another half are the mutations predicted to be buried in the hydrophobic core. Mutations from this latter group were variants related to insertion, exchange, or removal of charged residues or replacement with a homologous hydrophobic or polar residue. In contrast, the majority of missense variants were predicted to cause severe changes, related to insertion or removal of cysteine residues (23 mutations) and/or buried in the hydrophobic core. Finally, this study confirms results of our previous work that severe phenotype maximum structure perturbations are related to dramatic changes in a protein hydrophobic core or to the deletion or insertion of cysteine residues affecting in general the stability of protein fold. The kinetics of the disease progression might depend on the degree of the mutation impact on protein stability caused by the pathogenic missense change. In the future, we have to incorporate age, genotype, and molecular modeling into ERG analysis to understand a functional role of pathogenic mutations. 2. Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder characterized by absence of pigment (melanin) in eyes, hair and skin. Pathogenic mutations causing OCA1 decrease or abolish the melanogenic pathway by affecting tyrosinase enzymatic activity which is the rate limiting step in pigment production. An atomic structure of mammalian tyrosinase currently is not available. We imply molecular modeling to generate the mouse tyrosinase atomic structure by homology to the available crystal structures of prokaryotic and mushroom tyrosinase, invertebrate hemocyanin, and plant catechol oxidase. Molecular modeling has demonstrated that the copper-binding domain is conserved between bacterial, mushroom and mammalian tyrosinases. In addition, mammalian tyrosinase most likely contain EGF-like structural motif at the N-terminus. The effect of the c-2J (R77L) and c-h (H420R) tyrosinase mutants was modeled in silico. The c-2J mutation, R77L, occurs in an EGF/laminin-like motif. Although the precise function of this domain is not known, the R77L mutation is expected to have a severe effect on tyrosine binding, which is take place at the hydrophobic surface of the catalytic site. Thus, we would expect that elevating tyrosine concentrations would have little to no effect on mutant tyrosinase function, in agreement with in vivo results. The analysis of the Himalayan (c-h) mutation, H420R, proposes a weaker structural change. The model predicts a role in the coordination of copper near the active site where elevated tyrosine concentrations binding might stabilize the enzyme enough to help for less efficient copper coordination and residual enzymatic activity. These predictions are consistent with in vivo data. In silico analysis suggested that H420R but not R77L tyrosinase can effectively bind tyrosine. The ambient tyrosine might act as a small molecular chaperone and selectively stabilize the protein in Hymalayan mice. We tested this idea in vitro by expressing either wild-type, R77L, or H420R mutant tyrosinase proteins in Chinese-hamster ovary (CHO) cells and measuring tyrosinase protein stability using cycloheximide to inhibit new protein synthesis. The elevated tyrosine stabilizes H240R, but not R77L, tyrosinase. These results agree with the in vivo observations that pharmacological elevation of plasma tyrosine increases pigmentation in the Himalayan model of OCA-1B, but not in the Tyrc-2J/c-2J model of OCA-1A. We also investigated the enzymatic activity of R77L and H420R mutant proteins in vitro, compared to wild-type protein in albino mouse melanocytes, so-called Melan-c cells. A saturating concentration of tyrosine increased enzyme activity in Melan-c cells expressing the H420R mutant tyrosinase, but not R77L mutant tyrosinase, regardless of comparable levels of protein expression. This suggests that elevated tyrosine results in increased enzymatic activity of tyrosinase in Melan-c cells. The results are consistent with previous in silico analysis and in vivo data. Tyrosinase is a Type I membrane protein with a single trans-membrane fragment located at the C-terminus. We designed, expressed in larvae, developed a high-yield protein purification procedure, and purified the human tyrosinase with the truncated trans-membrane fragment. The modified human tyrosinase is an enzymaticaly active soluble protein which catalyzes the rate-limiting conversions of tyrosine to DOPA and DOPA to DOPA-quinone. We used the PNGase F endoglycosidase to show that the modified tyrosinase contains N-linked oligosaccharides. The tyrosinase is a monomeric protein (Mr 56 kDa) as demonstrated by size-exclusion chromatography and analytical ultracentrifugation. In perspective, a detailed understanding of protein structure and the mechanisms controlling tyrosine-modified tyrosinase interactions would allow in silico and large-scale drug scrinning for a future medical treatment of patients with the OCA-1B albinism.