The overall goal of this proposal is to understand the biological processes involved in the protein folding, quality control and degradation of the membrane glycoprotein tyrosinase. Tyrosinase is a melanocyte-specific enzyme required for melanin synthesis. Mutations in tyrosinase is a melanocyte-specific enzyme required for melanin synthesis. Mutations in tyrosinase are the cause of tyrosinase-negative albinism. Many of these mutated forms contain amino acid substitution that have the potential to reduce the efficiency of proper folding and to cause retention in the endoplasmic reticulum (ER), the site of protein maturation for membrane glycoproteins. The key role of the ER in the regulation of tyrosine has been demonstrated in a melanotic melanoma cells in which ER-retention of wild type tyrosinase leads to subsequent degradation by the cytosolic ubiquitin-dependent proteasomal pathway. Understanding the mechanisms involved in tyrosinase biosynthesis and degradation will help us to address the following fundamental questions: Are the tyrosinase-mutants in albino melanocytes retained in the ER? What is the mechanism of retention? Can this retention be relieved to produce a correctly localized and functional protein? Can this retention by selectively implemented? The specific aims of this proposal are: 1) To analyze the protein maturation (folding, co- and post-translational modification, and chaperone binding) of wild type and mutant forms of tyrosinase in a cell-free maturation system. 2) To constitute the ER- retention/degradation pathway of tyrosinase in a cell-free system, identify and characterize the proteins involved in both the sorting and degradation processes. 3) To characterize the maturation, quality control and degradation of tyrosinase in normal and malignant melanocytes. These studies will provide insights into the maturation, quality control and degradation mechanisms of tyrosinase and the etiology of pigmentation-related diseases. In addition, a better understanding of the biosynthetic and degradation processes of the cell would assist in the design of therapeutic drugs targeted against genetic diseases caused by trafficking and ER maturation defects in general, and albinism and/or hypo- and hyperpigmentation in particular.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA079864-01
Application #
2736678
Study Section
Molecular Cytology Study Section (CTY)
Program Officer
Spalholz, Barbara A
Project Start
1999-04-01
Project End
2004-01-31
Budget Start
1999-04-01
Budget End
2000-01-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Massachusetts Amherst
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
153223151
City
Amherst
State
MA
Country
United States
Zip Code
01003
Hebert, Daniel N (2012) An MBoC favorite: Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation. Mol Biol Cell 23:2236
Tamura, Taku; Cormier, James H; Hebert, Daniel N (2011) Characterization of early EDEM1 protein maturation events and their functional implications. J Biol Chem 286:24906-15
Tamura, Taku; Sunryd, Johan C; Hebert, Daniel N (2010) Sorting things out through endoplasmic reticulum quality control. Mol Membr Biol 27:412-27
Pearse, Bradley R; Hebert, Daniel N (2010) Lectin chaperones help direct the maturation of glycoproteins in the endoplasmic reticulum. Biochim Biophys Acta 1803:684-93
Pearse, Bradley R; Tamura, Taku; Sunryd, Johan C et al. (2010) The role of UDP-Glc:glycoprotein glucosyltransferase 1 in the maturation of an obligate substrate prosaposin. J Cell Biol 189:829-41
Jejcic, Alenka; Daniels, Robert; Goobar-Larsson, Laura et al. (2009) Small molecule targets Env for endoplasmic reticulum-associated protein degradation and inhibits human immunodeficiency virus type 1 propagation. J Virol 83:10075-84
Cormier, James H; Tamura, Taku; Sunryd, Johan C et al. (2009) EDEM1 recognition and delivery of misfolded proteins to the SEL1L-containing ERAD complex. Mol Cell 34:627-33
Wang, Ning; Glidden, Emily J; Murphy, Stephanie R et al. (2008) The cotranslational maturation program for the type II membrane glycoprotein influenza neuraminidase. J Biol Chem 283:33826-37
Pearse, Bradley R; Gabriel, Luke; Wang, Ning et al. (2008) A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein. J Cell Biol 181:309-20
Tamura, Taku; Cormier, James H; Hebert, Daniel N (2008) Sweet bays of ERAD. Trends Biochem Sci 33:298-300

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