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

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing (MBPP)
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Gindhart, Joseph G
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University of Massachusetts Amherst
Schools of Arts and Sciences
United States
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Lamriben, Lydia; Oster, Michela E; Tamura, Taku et al. (2018) EDEM1's mannosidase-like domain binds ERAD client proteins in a redox-sensitive manner and possesses catalytic activity. J Biol Chem 293:13932-13945
Krishnan, Beena; Hedstrom, Lizbeth; Hebert, Daniel N et al. (2017) Expression and Purification of Active Recombinant Human Alpha-1 Antitrypsin (AAT) from Escherichia coli. Methods Mol Biol 1639:195-209
Braakman, Ineke; Lamriben, Lydia; van Zadelhoff, Guus et al. (2017) Analysis of Disulfide Bond Formation. Curr Protoc Protein Sci 90:14.1.1-14.1.21
Lamriben, Lydia; Graham, Jill B; Adams, Benjamin M et al. (2016) N-Glycan-based ER Molecular Chaperone and Protein Quality Control System: The Calnexin Binding Cycle. Traffic 17:308-26
Hebert, Daniel N; Clerico, Eugenia M; Gierasch, Lila M (2016) Division of Labor: ER-Resident BiP Co-Chaperones Match Substrates to Fates Based on Specific Binding Sequences. Mol Cell 63:721-3
Chandrasekhar, Kshama; Ke, Haiping; Wang, Ning et al. (2016) Cellular folding pathway of a metastable serpin. Proc Natl Acad Sci U S A 113:6484-9
Tannous, Abla; Pisoni, Giorgia Brambilla; Hebert, Daniel N et al. (2015) N-linked sugar-regulated protein folding and quality control in the ER. Semin Cell Dev Biol 41:79-89
Tannous, Abla; Patel, Nishant; Tamura, Taku et al. (2015) Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation. Mol Biol Cell 26:390-405
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
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

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