In spite of the major structural and signaling roles of collagen in the extracellular matrix, and its involvement in many diseases, there are gaps in our understanding of basic triple-helix features and how they are affected by pathogenic mutations. The absence of an accessible recombinant collagen system limits mutagenesis-based exploration of structure, biologically active sites, pathogenic mutations, and drug screening. At this time, no small molecule drugs are known to bind to collagen for clinical applications. The long-term goals of this work are to establish a recombinant system to produce collagen-based scaffolds for tissue engineering and to discover drugs that will interact with collagen to inhibit pathogenic processes.
Aim #1 proposes to acquire fundamental knowledge about charged pair interactions and triple-helix bending. Motivated by the high content of charged residues and their involvement at every level of collagen function, a combined experimental and computational approach will define intra vs. interchain contributions and compare transposed charged pairs. Data suggest triple-helical molecules do not always have a linear structure, and a recently developed integrated solution structure approach, using x-ray scattering, analytical ultracentrifugation and constrained modeling, will be applied to define bending of the triple-helix and its sequence dependence. The objective of Aim #2 is to markedly improve an existing recombinant bacterial collagen system as a model for human collagen by introducing appropriate proline hydroxylation and by establishing the capacity to form heterotrimers using a coiled coil domain for chain selection. The structural and biological effectiveness of these enhancements will be tested by inserting within the bacterial collagen domain a human collagen platelet binding site requiring hydroxylation for platelet aggregation activity and a collagenase cleavage site requiring heterotrimers for activity.
Aim #3 is directed towards elucidating the mechanism through which Gly missense mutations in collagen lead to hereditable connective tissue disorders. The misfolding of mutant collagens appears to lead to degradation, through endoplasmic reticulum (ER) stress, UPR or autophagy, while mutation interference with collagen binding to cell receptors may represent an alternate mechanism in select cases. The direct effect of Gly missense mutations on triple-helix folding and on integrin binding will be determined on a bacterial system, and the detailed structural effects defined in model peptides, providing quantitative data to clarify the disease mechanism. The proposed research is significant because it will move the field forward in terms of foundational knowledge about basic triple-helix properties and will provide tools for finding drugs that can correct collagen defects. The innovative establishment of a recombinant collagen system with the capacity for hydroxylation of proline and heterotrimer formation creates a substantive new capacity to model and modify biologically important sites from human collagens and is well suited for initial screening of small molecules that can accelerate collagen folding, promote receptor binding, or inhibit degradation.

Public Health Relevance

The proposed research is relevant to public health because of the key role of collagen in many common diseases, in addition to the hereditary connective tissue disorders due directly to mutations in collagen. Fundamental knowledge about the collagen triple-helix structure, the effect of collagen mutations on folding and binding, and the development of a recombinant collagen system will promote discovery of drugs and advance efforts in tissue regeneration.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Wehrle, Janna P
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Tufts University
Engineering (All Types)
Schools of Engineering
United States
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