ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Owing to unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes.

We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.