Autosomal dominant osteogenesis imperfecta (OI) is typically caused by mutations in collagen-I genes that engender brittle bones and other pathologic phenotypes. Severe OI pathology may be linked to the secretion of malformed, mutant strand-containing collagen-I triple helices or to cellular stress owing to misfolding collagen strands accumulating inside cells and ultimately causing apoptosis. Haploinsufficiency owing to reduced collagen-I secretion can also cause OI with moderate pathologic phenotypes. Targeting the cell's protein homeostasis (or proteostasis) network to resolve failures in collagen-I folding and quality control could one day lead to a new therapeutic paradigm for OI. Such a system-targeted therapeutic strategy could also prove valuable for other collagenopathies, such as Ehlers-Danlos Syndrome. However, we must first learn much more about how the cell solves the collagen-I folding problem and how the quality control machinery handles misfolding collagen-I. Here, we deploy quantitative mass spectrometry-based proteomics to identify the proteostasis network machinery responsible for (1) folding and secreting wild-type collagen-I strands, (2) folding and secreting the OI-causing, misfolding collagen-?1(I) Gly247Ser and Cys1299Trp variants, and (3) identifying and disposing of misfolding collagen-I strands. Interactomics studies have not been previously performed with collagen-I owing to the absence of a suitable collagen-I expressing cell model system. We recently overcame this critical roadblock by generating immortalized fibrosarcoma cells that inducibly express wild-type and OI-causing collagen-I tagged with distinct antibody epitopes. We can now selectively immunoprecipitate wild-type and misfolding collagens, along with their interacting partners, from these cells, making comparative interactomics studies possible for the first time. We shall carefully prioritize collagen-I interacting partners we identify on the basis of multiple parameter. Top hits will be validated using RNAi depletion and assays already established in our lab to elucidate how collagen-I homeostasis is influenced by those interacting partners. Our most important findings will eventually be validated in mutation-matched primary cell lines obtained from OI patients via the Coriell Cell Repository. In the longer term, we will extend these studies to other collagen-I variants, study the molecular mechanisms by which the cell solves the collagen-I folding and misfolding problem, develop high throughput assays for collagen folding and secretion, and establish new strategies that adapt the proteostasis network to enhance collagen-I homeostasis and/or prevent the secretion of misfolded collagen-I triple helices.

Public Health Relevance

Osteogenesis imperfecta, or brittle bone disease, is a prototypical collagenopathy caused primarily by mutations in collagen-I genes that, despite decades of research effort, still lacks effective therapies. This research will unveil molecular details of collagen-I biogenesis by identifying the cellular machinery involved in folding nascent collagen-I triple helices and responding to collagen-I misfolding caused by mutations. Findings could one day lead to the identification of new therapeutic targets applicable to OI and the other collagenopathies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Small Research Grants (R03)
Project #
5R03AR067503-02
Application #
9118077
Study Section
Special Emphasis Panel (ZAR1)
Program Officer
Chen, Faye H
Project Start
2015-08-01
Project End
2018-05-31
Budget Start
2016-06-09
Budget End
2017-05-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
DiChiara, Andrew S; Li, Rasia C; Suen, Patreece H et al. (2018) A cysteine-based molecular code informs collagen C-propeptide assembly. Nat Commun 9:4206
Wong, Madeline Y; Doan, Ngoc Duc; DiChiara, Andrew S et al. (2018) A High-Throughput Assay for Collagen Secretion Suggests an Unanticipated Role for Hsp90 in Collagen Production. Biochemistry 57:2814-2827
Phillips, Angela M; Shoulders, Matthew D (2016) The Path of Least Resistance: Mechanisms to Reduce Influenza's Sensitivity to Oseltamivir. J Mol Biol 428:533-537
DiChiara, Andrew S; Taylor, Rebecca J; Wong, Madeline Y et al. (2016) Mapping and Exploring the Collagen-I Proteostasis Network. ACS Chem Biol 11:1408-21
Dewal, Mahender B; DiChiara, Andrew S; Antonopoulos, Aristotelis et al. (2015) XBP1s Links the Unfolded Protein Response to the Molecular Architecture of Mature N-Glycans. Chem Biol 22:1301-12