title: Mapping and Exploring the Collagen-I Proteostasis Network.
abstract: Collagen-I is the most abundant protein in the human body, yet our understanding of how the endoplasmic reticulum regulates collagen-I proteostasis (folding, quality control, and secretion) remains immature. Of particular importance, interactomic studies to map the collagen-I proteostasis network have never been performed. Such studies would provide insight into mechanisms of collagen-I folding and misfolding in cells, an area that is particularly important owing to the prominence of the collagen misfolding-related diseases. Here, we overcome key roadblocks to progress in this area by generating stable fibrosarcoma cells that inducibly express properly folded and modified collagen-I strands tagged with distinctive antibody epitopes. Selective immunoprecipitation of collagen-I from these cells integrated with quantitative mass spectrometry-based proteomics permits the first mapping of the collagen-I proteostasis network. Biochemical validation of the resulting map leads to the assignment of numerous new players in collagen-I proteostasis, and the unanticipated discovery of apparent aspartyl-hydroxylation as a new post-translational modification in the N-propeptide of collagen-I. Furthermore, quantitative analyses reveal that Erp29, an abundant endoplasmic reticulum proteostasis machinery component with few known functions, plays a key role in collagen-I retention under ascorbate-deficient conditions. In summary, the work here provides fresh insights into the molecular mechanisms of collagen-I proteostasis, yielding a detailed roadmap for future investigations. Straightforward adaptations of the cellular platform developed will also enable hypothesis-driven, comparative research on the likely distinctive proteostasis mechanisms engaged by normal and disease-causing, misfolding collagen-I variants, potentially motivating new therapeutic strategies for currently incurable collagenopathies.
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Andrew S
authors: AS
authors: DiChiara
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Rebecca J
authors: RJ
authors: Taylor
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Madeline Y
authors: MY
authors: Wong
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Ngoc-Duc
authors: ND
authors: Doan
authors: Koch Institute for Cancer Research, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States.
authors: Amanda M Del
authors: AM
authors: Rosario
authors: Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
authors: Matthew D
authors: MD
authors: Shoulders
mesh_terms: D002460:Cell Line; D024042:Collagen Type I / Q000737:chemistry / Q000378:metabolism*; D000090602:Collagen Type I, alpha 1 Chain; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D057165:Proteostasis Deficiencies / Q000378:metabolism
chemical_list: C000601279:COL1A2 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain