Mapping and Exploring the Collagen‑I Proteostasis Network

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.