Research – Highlights

Highlights of the Overall Laboratory Research

The Overall Laboratory has helped shape the current view of MMPs as key regulators of multiple signaling pathways that are integral to innate immunity rather than just dowdy degraders of the extracellular matrix (Butler & Overall 09 Nature Rev Drug Disc). We were at the forefront in this revision of in vivo roles for MMP with the first use of yeast 2-hybrid substrate screens for protease substrate discovery, that identified chemokines and CCN cytokines as novel MMP substrates (McQuibban et al 00 Science). Next we adapted proteomics for substrate discovery, but soon recognized inadequacies for the specialized tasks of substrate and cleavage site identification (López-Otín & Overall 02 Nature Rev Mol Cell Biol; Overall & Blobel 07 Nature Rev Mol Cell Biol). So, we initiated the new field of degradomics in 2000 to describe all genomic and proteomic investigations of proteases, their inhibitors and substrates. Following this paper in Science (McQuibban et al 00 Science) Dr. Carlos López-Otín (Chair of the 2007 MMP Gordon Research Conference) and Dr. Chris Overall wrote an invited review in Nature Reviews Molecular Cell Biology formally introducing the term degradomics and describing approaches to study proteolysis on a system-wide scale (López-Otín & Overall 02 Nature Rev Mol Cell Biol). This was updated by another paper in Nature Reviews Molecular Cell Biology, co-authored with Dr. Carl Blobel (Chair of the 2009 MMP Gordon Research Conference), describing proteomic and other innovative techniques to link proteases with substrates (Overall & Blobel 07 Nature Rev Mol Cell Biol). In Nature Reviews Genetics we annotated the complete human and mouse protease and inhibitor degradomes (Puente et al 03 Nature Rev Genetics).

We are leading the development of degradomics and its application to protease substrates. Initially, our quantitative proteomics experiments used isotope-coded affinity tags (ICAT) for substrate discovery (Tam et al 04 PNAS; Dean et al 07 Mol Cell Biol) and to analyze effects of MMP inhibitor drugs on cancer cells (Butler et al 07, 08 Mol Cell Biol). We then identified MMP substrates in cell culture (Dean & Overall 07 MCP; Prudova et al 10 MCP; Morrison et al 11 JBC; a.d.-Keller et al 12) using 4- or 8-plex iTRAQ labels. We developed PICS (proteomic identification of protease cleavage sites) (Schilling & Overall 08 Nature Biotech; Schilling et al 11a, b Nature Protocols) to profile the cleavage site specificity of 16 MMPs, most astacins (Becker-Pauly et al 11 MCP) and proteases of 4 classes using our new powerful web-based data processing site, WebPICS (Schilling et al 11), contributing >8000 cleavage sites to MEROPS, the protease database.

For identification of the substrate and the precise cleavage site, we developed the effective Terminal Amine Isotopic Labeling of Substrates (TAILS) for proteomics analysis of protease-generated (neo)-N-termini of natural substrates (Kleifeld et al 10 Nature Biotech; Doucet et al 10; Kleifeld et al 11 Nature Protocols). The tryptic peptides of proteomes are selectively removed leaving isotopic-labelled natural and neo-N-termini for MS/MS identification. To do so we patented a new class of polymer that we continue to enhance for proteomics (Beaudette et al 11). To complement N-TAILS we developed C-TAILS to identify the carboxy-termini of substrates and the C-terminome (Schilling et al 11, 10 Nature Methods). TAILS was improved using iTRAQ labels thereby enabling simultaneous analysis of up to 8 samples (Prudova et al 10 MCP). And now 10 samples by 10plex TMT Tags (Klein et al 14). We developed bioinformatics software (CLIPPER) for statistically valid identification of cleavage sites (a.d.-Keller et al 10, 12) and integrated our data into a KnowledgeBase of all protein termini and cleavage sites, TopFIND (Lange & Overall 12, 11 Nature Methods), and mathematically modeled the network of protease cascades and pathways (Fortelny et al 14, PLoS Biology) that interconnect to form the ‘protease web’ (Overall & Kleifeld 06a, Nature Rev Cancer). In validating the new protease substrates, limitations in Edman sequencing of cleavage products in gels triggered the refinement of MALDI-TOF methods for cleavage site identification (Starr & Overall 09) and a new sequencing method for proteins in solution: Amino Terminal Orientated Mass spectrometry of Substrates (ATOMS) (Doucet & Overall 11a,b).

Little is known of the key transcriptomic, proteomic, and proteolytic modification differences between the cells and extracellular signaling networks in human disease on a system-wide scale. We have now perfected these approaches for in vivo application and analyzed wild type vs. Mmp-/- mice in a variety of murine models of inflammation including skin (a.d.-Keller et al 13 Science Signaling) and arthritis in Mmp8-/- (Cox et al 10) and Mmp12-/- mice (Bellac et al 14). Thereby, we uncovered beneficial activities of MMPs that dampen inflammation, e.g., inactivation of most CCL monocyte chemokines by MMPs (Starr et al 12a, b JBC), including CCL7 in arthritis (McQuibban et al 00 Science), and of all CXCL PMN chemokines by MMP12 (Dean et al 08 Blood). This stirred our thinking to conceive drug anti-target (Overall & Kleifeld 06 Nature Rev Cancer; Dufour & Overall 13) vs. drug target concepts (Overall & López-Otín 02 Nature Rev Cancer). We also reported that the macrophage secreted MMP12 translocates to the nucleus of viral infected cells, where it binds the IκBa promoter, up-regulating its transcription. IκBa activation was essential for IFNa secretion and survival. MMP12 also clears systemic IFNa, forming a negative feedback loop. This is blocked by a drug we patented that does not cross the cell membrane and thus spares the anti-target activities of MMP12 and so is protective in vivo against viral infection (Marchant et al 14 Nature Medicine).

Bibliography

  • Beaudette, P., Rossi, N.A.A., Huesgen, P.F., Yu, X., Shenoi, R., Doucet, A., Overall, C.M., and Kizhakkedathu, J. 2011. Development of Soluble Ester-Linked Aldehydes and their Peptide Binding Characteristics. Analytical Chemistry 83, 6500-6510.
  • auf dem Keller, U., Prudova, A., Gioia, M., Butler, G.S., and Overall, C.M. 2010. A Statistics Based Platform for Quantitative N-Terminome Analysis and Identification of Protease Cleavage Products. Molecular Cellular Proteomics 9, 912-927.
  • auf dem Keller and Overall, C.M. 2012. CLIPPER—An Add-on to the Trans-Proteomic Pipeline for the Automated Analysis of TAILS N-terminomics Data. Biological Chemistry 39, 1477-1483.
  • auf dem Keller, U., Prudova, A., Eckhard, U., Fingleton, B., and Overall, C.M. 2013. Systems-Level Analysis of Proteolytic Events in Increased Vascular Permeability and Complement Activation in Skin Inflammation. Science Signaling 6, rs2, 1-15: DOI: 10.1126/scisignal.2003512.
  • Butler, G.S. and Overall, C.M. 2009. Proteomic Identification of Multitasking Proteins in Unexpected Locations Complicates Drug Targeting. Nature Reviews Drug Discovery 8, 935-948.
  • Cox, J.H., Starr, A.E., Kappelhoff, R., Yan, R., Roberts, C.R., Overall, C.M. 2010. Matrix Metalloproteinase-8 Deficiency Exacerbates Inflammatory Arthritis Through Delayed Neutrophil Apoptosis and Reduced Caspase-11 Expression. Arthritis and Rheumatism 62, 3,645-3,655. Editorial Highlighted.
  • Dean, R.A. and Overall, C.M. 2007. Proteomic Discovery of Metalloprotease Substrates in the Cellular Context by iTRAQ Labeling Reveals A Diverse MMP-2 Substrate Degradome. Molecular Cellular Proteomics 6, 611-623.
  • Dean, R.A., Cox, J.H., Bellac, C.L., Doucet, A., Starr, A.E., and Overall, C.M. (2008). Macrophage-specific Metalloelastase (MMP-12) Truncates and Inactivates ELR+ CXC Chemokines and Generates CCL2, 7, 8, and 13 Antagonists:  Potential Role of the Macrophage in Terminating PMN Influx. Blood 112, 3444-3453.
  • Doucet, A., and Overall, C.M. 2011. Broad Coverage Identification of Multiple Proteolytic Cleavage Sites in Complex High Molecular Weight Proteins Using Quantitative Proteomics as a Complement to Edman Sequencing. Molecular Cellular Proteomics 10, M110.003533 1-12.
  • Doucet, A., Kleifeld, O., Kizhakkedathu, J.N., and Overall, C.M. 2011. Identification of Proteolytic N-Termini by Terminal Amine Isotopic Labeling of Substrates (TAILS). Gel-Free Proteomics: Methods and Protocols, Edited by Gaevert, K. and Vandekerckhov, J., Methods in Molecular Biology 753, 273-287, Humana Press Inc., Totowa, N.J., USA.
  • Kleifeld, O., Doucet, A., auf dem Keller, U., Prudova, A., Schilling, O., Starr, A., Foster, L.J., Kizhakkedathu, J.N., and Overall, C.M. 2010. Isotopic labeling of Terminal Amines in Complex Samples Identifies Protein N-termini and Protease Cleavage Products. Nature Biotechnology 28,281-288.
  • Kleifeld, O., Doucet, A., Prudova, A., auf dem Keller, U., Gioia, M., Kizhakkedathu, J., and Overall, C.M. 2011. System-Wide Proteomic Identification of Protease Cleavage Products by Terminal Amine Isotopic Labeling of Substrates. Nature Protocols 6, 1578-1611.
  • Lange, P. and Overall, C.M. 2011. TopFIND, a Knowledgebase Linking Protein Termini with Function. Nature Methods 8, 703-704.
  • Lange, P., Huesgen, P. and Overall, C.M. 2012. TopFIND 2.0—Linking Protein Termini with Proteolytic Processing and Modifications Altering Protein Function. Nucleic Acids Research, 40 (Database Issue), D351-61.
  • Lange, P., Huesgen, P.F., Nguyen, K., and Overall, C.M. 2014. Annotating N termini for the Human Proteome Project: N termini and Na-acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome. J. Proteomics Res 13, 2028–2044.
  • López-Otín, C. and Overall, C.M. 2002. Protease Degradomics: A New Challenge for Proteomics. Invited Review. Nature Reviews Molecular Cell Biology 3, 509-519.
  • Marchant, D., Bellac, C., Wadsworth, S., Dufour, A., Butler, G.S., Cheung, C., Ng, J., Luo, Z., Garmaroudi, F.S., Heilbron, K., Karpov, A., Devel, L., Georgiadis, D., Hegele, R.G., Luo, H., Dive, V., McManus, B.M., Overall, C.M. 2014. Transcriptional Upregulation of IκBa by a Moonlighting Extracellular Metalloprotease is Essential for Antiviral Immunity. Nature Medicine 20, 493-502, doi 10.1038/nm.3508.
  • McQuibban, G.A., Gong, J.-H., Tam, E., McCulloch, C.A.G., Clark-Lewis, I., and Overall, C.M. 2000. Inflammation Dampened by Gelatinase A Cleavage of MCP-3. Science 289, 1202-1206.
  • Overall, C.M. and López-Otín, C. 2002. Strategies for MMP Inhibition in Cancer: Innovations for the Post-Trial Era. Invited Review. Nature Reviews Cancer 2, 657-672.
  • Overall, C.M. and Kleifeld, O. 2006. Validating MMPs as Drug Targets and Anti-targets for Cancer Therapy. Nature Reviews Cancer 6, 227-239.
  • Overall, C.M. and Blobel, C.P. 2007. In Search of Partners: Linking Extracellular Proteases to Substrates.  Nature Reviews Molecular Cell Biology 8,245-257.
  • Prudova, A., auf dem Keller, U., Butler, G.A., and Overall, C.M. 2010. Multiplex N-Terminome Analysis of MMP-2 and MMP-9 Degradomes by iTRAQ-TAILS Quantitative Proteomics. Molecular Cellular Proteomics 9, 894-911.
  • Puente, X.S., Sanchez, L.M., Overall, C.M., and López-Otín, C. 2003. Human and Mouse Proteases: A Comparative Genomic Approach. Nature Reviews Genetics 4, 544-558.
  • Schilling, O. and Overall, C.M. 2008. Proteome-derived Database Searchable Peptide Libraries for Identifying Protease Cleavage Sites. Nature Biotechnology 26, 685-694.
  • Schilling, O., Barré, O., Huesgen, P.F., and Overall, C.M. 2010. Proteome-wide Analysis of Protein Carboxy Termini: C Terminomics. Nature Methods 7, 508-511. Featured in C&EN.
  • Schilling, O., Huesgen, P.F., Barré, O., auf dem Keller, U., and OveralI, C.M. 2011. Characterization of the Prime and Non-Prime Active Site Specificities of Proteases by Proteome-derived Peptide Libraries and Tandem Mass Spectrometry. Nature Protocols 6, 111-120.
  • Starr, A.E, Bellac, C.L, Dufour, A., Goebeler, V., and Overall, C.M. 2012. Biochemical Characterization and N-Terminomics Analysis of Leukolysin, the Membrane-Type 6 Matrix Metalloproteinase (MMP25): Chemokine and Vimentin Cleavages Enhance Cell Migration and Macrophage Phagocytic Activities. Journal of Biological Chemistry 287, 13382-13395.
  • Starr, A.E., Dufour, A., Maier, J., and Overall, C.M. 2012. Biochemical Analysis of Matrix Metalloproteinase Activation of Chemokines CCL15 and CCL23 and Increased Glycosaminoglycan Binding of CCL16. Journal of Biological Chemistry 287, 5,848-5,860.
  • Tam, E.M., Morrison, C.M., Wu, Y., Stack, S., and Overall, C.M. 2004. Membrane Protease Proteomics: Isotope Coded Affinity Tag/Tandem Mass Spectrometry Identification of Undescribed MT1-MMP Substrates, Proceedings National Academy of Sciences U.S.A. 101, 6917-6922.