The overarching goals of this project are to define mechanisms of protein quality control in the mammalian endoplasmic reticulum (ER). The proposed research will utilize well-defined mammalian cell systems to determine how aberrant secretory cargo is selected and targeted for destruction and how organization of the quality control machinery within the ER coordinates protein transit and maturation. The quality control process is comprised of a series of interlocking steps that involve making critical choices on whether to transport a substrate or divert it for ER retention or degradation. The vast majority of proteins that traverse the mammalian secretory pathway receive N-linked glycans that are added shortly after a consensus N- glycosylation site emerges into the ER lumen. The composition of N-glycans is dynamic because they act as signals to recruit factors that assist the protein maturation and quality control processes. N-glycans support recruitment of the carbohydrate binding chaperones calnexin and calreticulin, which bind to monoglucosylated side chains. Persistent lectin chaperone binding is directed by UDP-glucose: glycoprotein glucosyltransferase (UGT1). Although UGT1 has been studied in reconstituted systems, the cellular role of this critical ER factor is poorly defined. Mannose trimming is proposed to be involved in sorting aberrant proteins to the ER-associated degradation (ERAD) pathway as inhibition of mannosidase activity stabilizes ERAD substrates. However, many questions still remain about the role of mannose trimming in quality control. While the over expression of the ER mannosidase-like protein EDEM1 accelerates the turnover of glycosylated ERAD substrates, its role in the quality control and sorting processes is unclear. Our results indicate that EDEM1 serves as a central link to deliver defective cargo to an ERAD dislocation complex by possessing bipartite binding properties. Finally, the exquisite spatial organization of the ER contributes to the efficiencies by which the ER temporally coordinates processes including protein maturation, quality control and ERAD. Tetratricopeptide repeat (TPR) domains are universal adaptor motifs that support protein-protein interactions involved in post-translational translocation and the formation of chaperone complexes within the cell. We have recently discovered a family of ER resident TPR-rich proteins that we propose to play a role in ER organization. This research proposal has three aims: (1) to define how UGT1 participates in ER quality control in a cellular context;(2) to elucidate the role and mechanism of action for EDEM1 in the ERAD process;and (3) to determine how novel TPR-containing proteins function in ER organization and homeostasis. A deeper knowledge of the processes in the early secretory pathway has widespread implications for understanding basic cell biology and the etiology of a range of human maladies as a growing number of diseases states are caused by defects in protein maturation and ER homeostasis.

Public Health Relevance

Insights gained into the cellular life of a protein will shed light on pathologies caused by deficiencies in protein maturation and quality control. Diseases associated with maturation and quality control defects include albinism, liver cirrhosis, emphysema, cystic fibrosis, Krabbe, Gaucher, and some forms of heart disease to name a few. Furthermore, the accumulation of misfolded proteins results in the activation of stress responses that is associated with obesity and diabetes. Understanding the quality control process for proteins that traverse the secretory pathway will aid in the development of methodologies or therapeutic agents that serve as protein folding correctors control the stringency of the quality control process or modulate the stress response caused by the accumulation of misfolded proteins. 1

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM086874-14
Application #
8486450
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Gindhart, Joseph G
Project Start
1999-04-01
Project End
2016-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
14
Fiscal Year
2013
Total Cost
$274,774
Indirect Cost
$96,249
Name
University of Massachusetts Amherst
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Guiliano, David B; Fussell, Helen; Lenart, Izabela et al. (2014) Endoplasmic reticulum degradation-enhancing ?-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation. Arthritis Rheumatol 66:2976-88
Sunryd, Johan C; Cheon, Banyoon; Graham, Jill B et al. (2014) TMTC1 and TMTC2 are novel endoplasmic reticulum tetratricopeptide repeat-containing adapter proteins involved in calcium homeostasis. J Biol Chem 289:16085-99
Hebert, Daniel N; Lamriben, Lydia; Powers, Evan T et al. (2014) The intrinsic and extrinsic effects of N-linked glycans on glycoproteostasis. Nat Chem Biol 10:902-10
Raghava, Smita; Giorda, Kristina M; Romano, Fabian B et al. (2013) SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release. Biochemistry 52:3939-48
Braakman, Ineke; Hebert, Daniel N (2013) Protein folding in the endoplasmic reticulum. Cold Spring Harb Perspect Biol 5:a013201
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
Hebert, Daniel N; Bernasconi, Riccardo; Molinari, Maurizio (2010) ERAD substrates: which way out? Semin Cell Dev Biol 21:526-32
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