Metadegradomics

Abstract

Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for proteasegenerated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics. Molecular & Cellular Proteomics 7: 1925-1951, 2008.

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

“…However, deeper biological insight requires identifying the protease responsible for generation of neo-termini that distinguish cleavage products from the original protein termini. Whereas low- and high-throughput methods to identify the in vitro substrate repertoire of proteases, also known as the substrate degradome [7], are well established, in vivo identification is problematic [16]. In vitro experiments can only indicate potential cleavage in vivo because of difficulties assigning precise parameters governing cleavage in the actual biological system, such as protease and substrate colocalization spatially and temporally, presence of inhibitors, zymogen activation, pH, ion concentrations, interaction with nonprotein compounds [17], as well as O-glycosylation or phosphorylation of the protease or substrate [18].…”

High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family

“…Conversely, identification of the multiple cleavage sequences is required to recognize the MMPs’ cleavage signature (Jabaiah and Daugherty, 2011). This cleavage signature, if known, may directly relate the individual MMPs to their respective protein substrates (auf dem Keller, et al, 2010; Butler, et al, 2010; Butler, et al, 2009; Butler, et al, 2008; Butler and Overall, 2007; Dean and Overall, 2007; Doucet, et al, 2008; Doucet, et al, 2011; Dufour and Overall, 2013; Overall and Kleifeld, 2006; Ratnikov, et al, 2014; Schlage and Auf dem Keller, 2015). …”

Activity-based Proteomics of Enzyme Superfamilies: Serine Hydrolases as a Case Study

“…First, when applied in isolation, ABPP offers only limited insights into enzyme function. Only through integrating ABPP with other methods, such as emerging proteomic (51)(52)(53), peptidomic (54), and metabolomic (55) strategies to discover enzyme substrates in native biological systems, can the actual physiological activities of enzymes be defined. In cases in which this integrated approach has been applied to uncharacterized mSHs, specific metabolic functions have been discovered (e.g.…”