Research – Interests

Summary of the Overall Laboratory Research Interests

The Overall Lab founded the field of ‘degradomics’. Degradomics is the application of genomic and proteomic techniques to determine protease and protease-substrate repertoires—or ‘degradomes’—on an cell, tissue and organism-wide scale. The Overall Lab has developed world-leading innovative quantitative proteomic techniques to enrich and identify proteolysis products in vivo. This awareness and understanding derived from application of “omics” technologies to complex biological systems has revolutionized the understanding of the roles of proteases and matrix metalloproteinases (MMP) in particular, in physiological and pathological processes in vivo.

For technical reasons protein cleavage is opaque to conventional proteomics, which limits functional insight. Therefore, to specifically enrich for mature protein N-termini and neo-N-termini of proteins we utilize 4-8plex iTRAQ and 10plex TMT TAILS (Terminal Amine Isotopic Labeling of Substrates) (Nature Biotech 28, 281-288 (2010); Nature Protocols 6, 1578-1611 (2011). Using quantitative proteomics information determined through TAILS, our lab’s signature approach, the protein substrate and the exact cleavage site are determined in the same analysis. By analyzing all N-termini, the original mature N terminus of a protein together with protease generated neo-N termini, the N-terminome of the tissue is elucidated. By then analyzing all quantifiable peptides that change in abundance significantly between samples, protein expression and new protease substrates are identified. On a global scale this generates the proteolytic signature of the tissue. The proteolytic signature can be used to identify protease networks in vivo that are operative in the system under study and to identify new disease biomarkers, with the advantage of being mechanistically informative. Resultant new drug targets and new clinical tests for early, accurate patient diagnosis can thereby by translated.

We now aim to develop clinically relevant system-wide knowledge of inflammation in humans in vivo e.g., in response to bacterial infection in placental chorioamnionitis and periodontal diseases, in viral infection, in chronic inflammatory diseases, e.g., arthritis and periodontitis, and in tumor-associated inflammation.

In analyzing the N-terminome of normal human tissues we have revealed the unexpected finding that 50-75% of proteins in vivo start at N-termini that are not as annotated in UniProt. Rather, the N-termini of protein chains in vivo can commence at many points C terminal to the predicted start site and result from proteolytic processing to generate stable protein chains: Proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions and location.

To identify and understand the function of all proteins in the human body and to map the human proteome on a disease-centric and chromosome-centric basis, the international HUPO consortium was formed. By knowing all the proteins and their interactions and roles in pathways, knowledge will be gained about the fundamental ways cells and tissues function. Thus, our studies of human disease have the added value of contributing highly desired and unique knowledge of human protein N-termini to the Human Proteome Project (HPP) of HUPO.

Proteolysis alters the protein sequence and results in neo-N termini and hence novel semi-tryptic N-terminal peptides upon tryptic digestion proteomics. We hypothesized that some N terminal semi-tryptic peptides will exhibit beneficial m/z, ionization and fragmentation properties over their fully tryptic counterparts, rendering these peptides and cognate proteins identifiable. Hence, identifying in vivo cleavage sites in the proteome by all active proteases present in the tissue identifies protein speciation and also obtains proof for the expression of ‘missing proteins’, particularly if rare tissues and cells are analyzed. In our HPP project, “Termini Orientated Proteomics-Human Amino Terminome” (TOP-HAT), we have identified >350 missing proteins in less commonly studied cells e.g. erythrocytes, platelets, placenta, gingiva, dental pulp and lymphocytes.

Recently we found that an extracellular protease (MMP12) secreted from macrophages targets virus-infected target cells where it segues to the nucleus (Nature Medicine 20, 493-502 2014). Here MMP12 acts as a transcription factor and cleaves intracellular substrates that are essential for IFN-alpha secretion and antiviral immunity. Hence, by proteomically identifying proteolytic networks by TAILS and their actions on signaling and cytokine pathways in cancer, inflammation, and infection we have uncovered remarkable roles for proteases inside, outside and outside-to-inside the cell.