Abstract
Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates.
Highlights
It has long been known and well documented that pigment production within mammalian melanocytes is largely driven by the catalytic activity of tyrosinase (Tyr), a binuclear multifunctional enzyme whose primary role centers on the initial and rate-limiting reactions in melanin biosynthesis
Tyr’s catalytic function tends to be the focus of melanogenesis, there are two other tyrosinase-related proteins, tyrosinase-related protein 1 (Tyrp1) and Tyrp2, which have roles that have yet to be clearly defined in humans
RIneseualrtlsier work, we have shown that Tyrp1tr exists as a monomeric protein with a weight-average molecuIlnarewareliigerhtwoof r5k9,.2w±e2h.3avkeDsah, aoswsunmthinatg T10y%rp1catrrbeoxhisytsdraasteas mmaosnsodmueeritco pprrootteeiinn gwlyitchosaywlaetiiognht[-14]
Summary
It has long been known and well documented that pigment production within mammalian melanocytes is largely driven by the catalytic activity of tyrosinase (Tyr), a binuclear multifunctional enzyme whose primary role centers on the initial and rate-limiting reactions in melanin biosynthesis. All three proteins may form a hetero-oligomeric complex, which can play a role in the stabilization of Tyr and regulation of its enzymatic activity [2,3,4]. Affected individuals usually present one of two phenotypes: rufous OCA (ROCA), characterized by red-bronze skin, blue or brown irises, and ginger-red hair; or brown OCA (BOCA), characterized by light to brown or tan skin and light to brown hair In both cases, skin pigmentation can increase with age. Tyrp, the most abundant protein of the melanosome, is involved in maintenance of melanosome structure, can affect melanocyte proliferation as well as cell death, and may regulate or influence the type of melanin synthesized [2,16,17,18,19]. Ra(wPadnaetlaBa).reRashwodwantabayreopsheonwcnircblyeso;paelninceirrcelepsr;easelinntes rtehperebseesnttfistt.hTehbeesint fsiet.rtTshsehionwserthtsesShVowanthaleySsVis of expanearilmyseisnotsf ceoxnpedruimctendtsatco4n0,d0u0c0terdpmat,4200,0◦0C0.rpm, 20 °C
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