Matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and ADAM with thrombospondin motifs (ADAMTSs) belong to the zinc-dependent proteases superfamily, responsible for degradation and remodeling of the extracellular matrix (ECM). Overexpression of metalloproteinases (MPs) have been replicated in many fatal and severe health conditions such as cancers, cardiovascular diseases and neurological disorders. Therefore, MPs remain major therapeutic targets to investigate for decades. Many small molecule MP inhibitors have failed to advance to clinical trials because of their off-target side effects and toxicity to patients. Tissue inhibitors of metalloproteinases (TIMP), a protein family of four members, are endogenous inhibitors of MPs. Among all four TIMPs, TIMP-3 is the most unique with the broadest binding spectrum and affinity to various inhibitory substrates. TIMP-3 is the only TIMP able to effectively regulate a large pool of ADAMs, ADAMTSs and MMPs activities, such as ADAM-17. ADAM-17 is also known as tumor necrosis factor α (TNF-α) converting enzyme (TACE) for its role in ectodomains cleavage of TNF-α and almost 100 other membrane proteins including cytokines, growth factors and growth factor receptors. ADAM-17 is considered one of the primary drug targets, especially in regards to inflammatory diseases and cancer. Understanding which mechanism TIMP-3 employs to inhibit certain MPs, via sequence and structure, might provide means to design more efficient, protein-based MP inhibitors.
We used yeast surface display and protein engineering techniques such as directed evolution to identify regions of TIMP-3 interacting with target MPs. We first displayed the library of TIMP-3 mutants on the yeast surface and then analyzed variants’ expression level and binding affinity via flow cytometry. Wild type human full-length TIMP-3 displayed on yeast historically displayed low expression levels in our laboratory. We then screened the TIMP-3 mutagenesis library for improved protein expression on yeast surface and MP binding affinity via fluorescent-activated cell sorting (FACS). To characterize our library of mutant’s enrichment landscape, we analyzed our DNA sequences, acquired from next generation sequencing after each round of FACS. This study allowed us to explore possible TIMP-3 regions and motifs interacting with desired MPs with high affinity and selectivity.
