Defining Interactomes of Wild‐Type Versus Misfolding Type I Collagen Variants in Osteogenesis Imperfecta
Bibliographic record
Abstract
Osteogenesis imperfecta (OI), also known as “brittle bone disease”, is a clinically heterogeneous, heritable connective tissue disorder that occurs in approximately one out of every 15,000–20,000 births. Unfortunately, despite decades of research effort, there is currently no cure for OI. Treatment options are symptomatic and limited to orthopaedic care and the administration of antiresorptive bone drugs such as bisphosphonates that have deleterious side effects. Disrupted collagen‐I proteostasis caused by the production of mutant, misfolding collagen‐I strands is a key pathological mechanism underpinning OI. However, a detailed mechanistic understanding of the fundamental processes of wild‐type and mutant collagen‐I maturation is currently lacking. We created a new cellular platform, based on osteosarcoma Saos2 cells, for biochemical studies of collagen folding and misfolding. We prepared eight different Saos‐2 cell lines inducibly expressing either HA‐tagged wild‐type or mutant collagen‐I under control of the Tet‐On system . The collagen mutations introduced were Col1A1‐Gly247Ser, Col1A1‐Gly352Ser, Col1A1‐Gly883Ser, Col1A1‐Cys1299Trp, Col1A2‐Gly502Ser, and Col1A2‐Gly610Cys. The tetracycline‐inducible expression of HA‐tagged collagen‐I proteins was confirmed by Western blotting. Importantly, HA‐tagged collagen was found to be inserted into triple‐helical collagen‐I molecules. We next employed mass spectrometry‐based proteomics strategy to define the proteostasis network of wild‐type collagen‐I in Saos‐2 cells. Our proteomic data revealed about 120 different proteins, including all known collagen‐I interactors such as Hsp47, BIP, PDI, CRTAP and more that might play a role in collagen‐I proteostasis. We then used comparative and TMT‐assisted quantitative proteomics to establish the differential interactomes of wild‐type and OI‐causing collagen‐I variants. We identified several proteins that differently engage wild‐type and mutant collagen‐I, including proteins in the PDI and HSP40/70/90 families, and ER proteins involved in protein post‐translational modifications. Proteins identified as important factors are being knocked down with shRNA or overexpressed (both in the Saos‐2 system and in primary OI patient fibroblasts). Biochemical approaches, including Western blotting to evaluate both intra‐ and extra‐cellular levels of collagen‐I, radiolabeling to follow the extent of post‐translational modifications, and pulse‐chase experiments to determine the rates of secretion and degradation, are being used to validate their effects on mutant collagen‐I maturation. Our mechanistic findings in follow‐up studies are revealing new players in cellular collagen‐I folding and quality control that may play an important role in OI pathology. Support or Funding Information Funding from the Mallinckrodt Foundation, the Smith Family Foundation, the NIH/NIAMS (1R03AR067503), the Fonds de la Recherche en Santé du Québec (FRSQ), and the Canadian Institute of Health Research (CIHR).
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".