4.6 Article

In vitro characterization of the intramelanosomal domain of human recombinant TYRP1 and its oculocutaneous albinism type 3-related mutant variants

Journal

PROTEIN SCIENCE
Volume 32, Issue 1, Pages -

Publisher

WILEY
DOI: 10.1002/pro.4518

Keywords

OCA3-related genetic mutations; oculocutaneous albinism type 3; protein stability; protein structure; tyrosinase-related protein 1

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Tyrosinase related protein 1 (TYRP1) is crucial for melanin synthesis in melanocytes, and mutations to the TYRP1 gene can cause a rare disease called oculocutaneous albinism type 3 (OCA3). We investigated the impact of genetic mutations on TYRP1 structure and function by engineering mutant variants mimicking OCA3-related changes. Our results showed that some mutants maintained native conformation and enzymatic activity, while others misfolded or disrupted essential interactions, explaining the phenotype changes associated with OCA3 mutations.
Tyrosinase related protein 1 (TYRP1) is the most abundant melanosomal protein of the melanocyte, where plays an important role in the synthesis of eumelanin, possibly catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid to 5,6-quinone-2-carboxylic acid. Mutations to the TYRP1 gene can result in oculocutaneous albinism type 3 (OCA3), a rare disease characterized by reduced synthesis of melanin in skin, hair, and eyes. To investigate the effect of genetic mutations on the TYRP1 structure, function, and stability, we engineered the intramelanosomal domain of TYRP1 and its mutant variants mimicking either OCA3-related changes, C30R, H215Y, D308N, and R326H or R87G mutant variant, analogous to OCA1-related pathogenic effect in tyrosinase. Proteins were produced in Trichoplusia Ni larvae, then purified, and analyzed by biochemical methods. Data shows that D308N and R326H mutants keep the native conformations and demonstrate no change in their stability and enzymatic activity. In contrast, mutations C30R and R87G localized in the Cys-rich domain show the variants misfolding during the purification process. The H215Y variant disrupts the binding of Zn2+ in the active site and thus reduces the strength of the enzyme/substrate interactions. Our results, consistent with the clinical and in silico studies, show that mutations at the protein surface are expected to have a negligible phenotype change compared to that of TYRP1. For the mutations with severe phenotype changes, which were localized in the Cys-rich domain or the active site, we confirmed a complete or partial protein misfolding as the possible mechanism of protein malfunction caused by OCA3 inherited mutations.

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