Protease Activity Profiling Using Molecular Indexing of Proteins By Self Assembly (MIPSA)

Background: Post-translational modifications (PTMs) are structural or chemical modifications to proteins that enable them to participate in various cell signaling processes. Their disregulation has been correlated with pathogenesis. For example, imbalances in proteolysis, a structural PTM, in the tumor microenvironment (TME) has been linked with many hallmarks of cancer like metastasis. Currently the substrates for most proteases are not known.

Methods: We propose to study the PTM proteolysis using Molecular Indexing of Proteins by Self Assembly (MIPSA). It is a display technology that produces libraries of full-length proteins covalently coupled to uniquely identifying DNA barcodes for analysis by next generation sequencing. Full-length proteins provide conformational structure that is important in many protein-protein interactions. The advantage of the covalent bond between the protein and barcode allows us to subject these proteins to denaturation assays without losing barcode information. To study proteolysis in cancer we aim to use a library of 16,700 human proteins that contain various classes of molecules like cytokines, chemokines, transcription factors, receptors, etc. We aim to incubate proteases of interest with the MIPSA library, separate or fractionate cleaved and un-cleaved proteins and subsequently sequence the fractions to identify non-canonical substrates. We propose two workflows to characterize protease activities: (1) An initial on-bead cleavage strategy to show proof-of-concept and (2) eventual in-solution cleavage strategy.

Results: Using the on-bead cleavage assay as a proof-of-concept, we show that MIPSA can be used to identify protease substrates from a mixture of proteins. But because the substrates are immobilized on beads, a certain degree of assay sensitivity might be lost. To overcome this, we plan to develop a version of the assay where the proteins are present in-solution and will have more access to the protease. We can fractionate the cleavage products based on their size, as cleavage products would experience a size shift thereby moving their attached DNA barcodes to other fractions. This could potentially increase the sensitivity of the assay owing to the many fractions we collect, each having better signal to noise ratios.

Conclusions: Protease activities can serve as diagnostic biomarkers and also as therapeutic agents for cancer, especially in the TME. MIPSA offers an unbiased, relatively low-cost and high-throughput way to profile protease activities. Identified substrates can be used to design diagnostic probes, therapeutic conjugates or further elucidate tumor pathway biology.