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 of protein structures, 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 for proteins of interest to measure the protein fold destabilization and changes in catalytic activity caused by the disease-related mutations. Finally, we correlate these findings with clinical phenotypes from inherited eye disease. In addition, in collaboration with the National Center for Advancing Translational Sciences (NCATS) we search for drug activators of catalytic activity of mutant protein affected by genetic mutation. Mutations in the tyrosinase gene are linked to oculocutaneous albinism type 1 (OCA1), an autosomal recessive disorder. Human tyrosinase is a Type 1 membrane bound glycoenzyme that catalyzes the initial and rate-limiting steps of melanin production in the melanosome. Full-length human tyrosinase (residues 1-529) recently was purified and characterized in the Lab (Kus et al.; Young et al, PLOS One, 2018). The human tyrosinase was overexpressed in Trichoplusia ni larvae infected with a baculovirus, solubilized with detergent, and purified using chromatography. Michaelis-Menten kinetics, enzymatic specific activity, and analytical ultracentrifugation were used to compare the tyrosinase in detergent with the soluble recombinant intra-melanosomal tyrosinase domain (residues 19-469). The tyrosinase domain analyzed using atomic force microscopy to show that dimensions of the domain are similar to that of derived from molecular modeling. Active tyrosinase is monomeric in detergent micelles suggesting no stable interactions between protein molecules. Both, tyrosinase and intra-melanosomal domain, exhibited similar enzymatic activity and ligand affinity in L-DOPA and L-Tyrosine reactions. In addition, expression in larvae is a scalable process that will allow high yield protein production. Thus, larval production of enzymatically active human full-length tyrosinase is a significant step in understanding the tyrosinase function and protein reactome responsible for melanin production in melanosome. This study has a potential to open a door for a future cure of OCA1 using the enzyme replacement therapy. Recently, we performed protein expression, purification, and in vitro analysis of de-glycosylated human tyrosinases using multiple site-directed mutagenesis to understand a significance of glycosylated asparagines, which were previously not accessible to mass-spectroscopy analysis (Dolinska & Sergeev, Biol. Chemistry, 2017). In the de-glycosylated in six N-glycosylation positions mutant the residue N371 was intact and tyrosinase kept a residual catalytic activity. The additional glycosylation of residue N371 seems to be the critical for a loss of tyrosinase function. In general, a total amount of pure protein and enzymatic activity for the intra-melanosomal domain and de-glycosylated mutants were steadily reduced due to a loss of protein stability followed by protein degradation. In addition, we demonstrated that the replacement of N-glycosylation residues decreases tyrosinase enzymatic activity more effectively compared to the effect of Endonuclease F1. A complete de-glycosylation of tyrosinase in vitro causes a decrease of functionally active enzyme and agrees with a loss of melanin in OCA1 patients. To understand experimental data and likely clinical impact of mutations we performed molecular modeling and computer simulations of protein atomic structures related to the inherited eye disease. Previously we applied unfolding mutation screen for the in-silico evaluation of the severity of genetic perturbations at the atomic level of protein structure (McCafferty & Sergeev, Scientific Reports, 2016). One of important conclusions from our work was that the method allows to determine residues critical for protein stability. Indeed, protein's amino acid sequence dictates the folds and final structure the macromolecule will form. We proposed that by identifying critical residues in a protein's atomic structure, we can select a critical stability framework within the protein structure essential to proper protein folding (McCafferty & Sergeev, PLOS One, 2017,2018). We use global computational mutagenesis based on the unfolding mutation screen to test the effect of every possible missense mutation on the protein structure to identify the residues that cannot tolerate a substitution without causing protein misfolding. This method was tested using molecular dynamics to simulate the stability effects of mutating critical residues in proteins involved in inherited disease, such as myoglobin, p53, and the 15th sushi domain of complement factor H. In addition, we prove that when the critical residues are in place, other residues may be changed within the structure without a stability loss. We validate that critical residues are conserved using myoglobin to show that critical residues are the same for crystal structures of 6 different species and comparing conservation indices to critical residues in 9 eye disease-related proteins. Our studies demonstrate that by using a selection of critical elements in a protein structure we can identify a critical protein stability framework. The frame of critical residues can be used in genetic engineering to improve small molecule binding for drug studies, identify loss-of-function disease-causing missense mutations in genetics studies, and aide in identifying templates for homology modeling. The computational studies were creating a foundation for creation of the web-site containing information on severities of missense mutations from inherited eye disease, which was published by us earlier (McCafferty & Sergeev, Scientific Data, 2016). At present, ocular proteome web site (https://neicommons.nei.nih.gov/#/proteomeData) contains in-silico predictions for 1,342,160 missense mutations associated with 102 protein structures from 150 inherited eye diseases. These diseases are albinism, retina degenerations, glaucoma, cataracts, and many others from eye research. This is a first database strictly oriented on analysis of genetic changes in proteins from inherited eye disease. The web-site contains information for each protein including disease phenotype, internet links to the databases such as Genetic Home Reference, UniProt, HGMD and LOVD. In addition, the search mechanism provides information on protein stability change and risk of protein destabilization due to genetic mutation, which were obtained in-silico from the atomic level of protein structure. All protein structures as well as the structures colored with risk evaluations are available for download. This site could be used as a tool for the express analysis of novel mutations in experimental biochemistry with a potential to use these data in clinical practice, next generation sequencing, and genotype-to-phenotype relationships in inherited eye disease.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAEY000476-10
Application #
9796708
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2018
Total Cost
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
McCafferty, Caitlyn L; Sergeev, Yuri V (2018) Correction: Global computational mutagenesis provides a critical stability framework in protein structures. PLoS One 13:e0191881
Kus, Nicole J; Dolinska, Monika B; Young 2nd, Kenneth L et al. (2018) Membrane-associated human tyrosinase is an enzymatically active monomeric glycoprotein. PLoS One 13:e0198247
Veleri, Shobi; Nellissery, Jacob; Mishra, Bibhudatta et al. (2017) REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival. Hum Mol Genet 26:2218-2230
McCafferty, Caitlyn L; Sergeev, Yuri V (2017) Global computational mutagenesis provides a critical stability framework in protein structures. PLoS One 12:e0189064
Dolinska, Monika B; Wingfield, Paul T; Sergeev, Yuri V (2017) Purification of Recombinant Human Tyrosinase from Insect Larvae Infected with the Baculovirus Vector. Curr Protoc Protein Sci 89:6.15.1-6.15.12
Dolinska, Monika B; Kus, Nicole J; Farney, S Katie et al. (2017) Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity. Pigment Cell Melanoma Res 30:41-52
Shang, Peng; Valapala, Mallika; Grebe, Rhonda et al. (2017) The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling. Aging Cell 16:349-359
Zernant, Jana; Lee, Winston; Collison, Frederick T et al. (2017) Frequent hypomorphic alleles account for a significant fraction of ABCA4 disease and distinguish it from age-related macular degeneration. J Med Genet 54:404-412
Dolinska, Monika B; Sergeev, Yuri V (2017) The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase. Biol Chem 399:73-77
George, Aman; Zand, Dina J; Hufnagel, Robert B et al. (2016) Biallelic Mutations in MITF Cause Coloboma, Osteopetrosis, Microphthalmia, Macrocephaly, Albinism, and Deafness. Am J Hum Genet 99:1388-1394

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