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.
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.
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