Osteogenesis imperfecta (OI) is a rare disease that causes patients to suffer from brittle or broken bones and varying degrees of deformities. Currently, there is no cure, and patients must rely on situational treatment as their symptoms arise, 'healing' them only until another painful break or fracture occurs. The most common cases of OI are caused by missense mutations in one of the two types I collagen genes, producing a mutant protein that compromises the integrity of bone structure. In recent years, studies show that protein misfolding stress responses are activated in a select number of OI mouse models. Additionally, in rare forms of OI, collagen modifying enzymes are rendered inactive and result in the same brittle bones and deformities. Taken together, these studies strongly suggest that OI represents a failure of the protein folding machinery to selectively identify and degrade poorly folded collagen. Unfortunately, a cell model system for studying collagen using the high throughput molecular biology and biochemical assays available today does not exist. Cells used to study collagen production and secretion are slow growing, and difficult to manipulate, while common cell lines used in many other applications express their own, endogenous collagen-I. In the absence of a proper, malleable cell model to probe different protein folding pathways with the ability to isolate each wild type and mutant strand individually, progress in this area remains slow. I have overcome the aforementioned difficulties by creating wild type and mutant collagen-I vectors that have genetically encoded, orthogonal antibody epitope tags for easily distinguishing between each collagen strand population. Additionally, I have used these vectors to create a cell model system that inducibly expresses my collagen constructs in the absence of endogenous collagen that would convolute any data obtained. With my cell model as the main route to discovery, I am investigating the interactions required for wild type collagen-I folding and secretion using state of the art mass spectrometry and biochemical assays that are accessible only because of our cell model system. I am also performing similar biochemical experiments on the OI-causing mutant collagen strands, and carefully examining differences that the mutation may induce in the collagen folding pathway. Finally, I am performing a SILAC-assisted (stable isotope labeling of amino acids in cell culture) mass spectrometry experiment comparing wild type and mutant collagen, providing the first quantitative, comparative interactomics study of full length human collagen-I. Our approach has dual benefits of both increasing our understanding of how wild type collagen-I folds and also delving into the specifics of the molecular causes of OI, by providing the first hypothesis-driven, directed investigation of how different OI-causing collagen strands misfold and diverge from the normal intracellular homeostatic pathways.
Osteogenesis imperefecta (OI) is a genetic disease that manifests as a result of mutated type I collagen genes. Current treatments are only temporary solutions to ease the symptoms of the disease, with no hope of treating the underlying cause of OI. This project proposes to delve into the molecular underpinnings of the pathology caused by OI-causing collagen-I mutations as a novel way to gain insight into the disease and how to potentially treat it.
| 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 |
| 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 |
