Designing Tissue Inhibitors of Metalloproteinases to Target Specific Metalloproteinases
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Authors
Mosca, Ethan
Issue Date
2024
Type
Thesis
Language
Keywords
Metalloproteinases , MMP , Protein Synthesis , TIMP , TIMP Hybrid , Tissue Inhibitor Of Metalloproteinases
Alternative Title
Abstract
Metalloproteinases (MPs) are zinc-dependent enzymes critical to the regulation of extracellular matrix (ECM) components. Overexpression of specific MPs is associated with numerous pathological conditions, including cancer, inflammatory disorders, and cardiovascular diseases, where they facilitate abnormal tissue degradation and disease progression. This study aims to create hybrid enzymes with enhanced selectivity and therapeutic potential. By redesigning the natural inhibitors of MPs, tissue inhibitors of metalloproteinases (TIMPs), via techniques such as loop and domain swapping, developed hybrid protein inhibitors that specifically target certain metalloproteinases while sparing others can be discovered. Studying TIMP hybrid variants binding towards various targets of interest (matrix metalloproteinases (MMPs), vascular endothelial growth factor receptor 2 (VEGFR2), and a disintegrin and metalloproteinases 17 (ADAM-17) revealed significant motifs that drive binding to these specific ECM targets which showed the promise of using these techniques to develop more efficient and selective protein variants to target. The recombinant expression of MPs was also explored for the need to use pure MMPs for biochemical and other assays to evaluate potential therapeutics of designed hybrid TIMPs. I further used recombinant protein expression systems to express MMP-9 protein in bacterial hosts such as Escherichia coli ( E. coli .) Bacterial expression systems are cost-effective and usually yield high quantities of protein but lack the complex post-translational modification which might result in insolubility of recombinant human proteins expressed in E. coli hosts. Cell-free protein synthesis emerged as a promising alternative, offering the advantages of rapid protein production without the limitations of cell-based systems but was found to be cost inefficient and low in reaction yield. Overall, the production of metalloproteinases through innovative design and synthesis strategies holds significant promise for laboratory applications, providing a targeted approach to complete experimental research more efficiently.