The biological roles of the matrix metalloproteinases (MMPs) have been traditionally associated with the degradation and turnover of most of the components of the extracellular matrix (ECM). This functional misconception has been used for years to explain the involvement of the MMP family in developmental processes, cell homeostasis and disease, and led to clinical trials of MMP inhibitors for the treatment of cancer that failed to meet their endpoints and cast a shadow on MMPs as druggable targets. Accumulated evidence from a great variety of post-trial MMP degradomics studies, ranging from transgenic models to recent state-of-the-art proteomics screens, is changing the dogma about MMP functions. MMPs regulate cell behavior through finely tuned and tightly controlled proteolytic processing of a large variety of signaling molecules that can also have beneficial effects in disease resolution. Moreover, net proteolytic activity relies upon direct interactions between the different protease and protease inhibitor families, interconnected in a complex protease web, with MMPs acting as key nodal components. Such complexity renders simple interpretation of Mmp knockout mice very difficult. Indeed, the phenotype of these models reveals the response of a complex system to the loss of one protease rather than necessarily a direct effect of the lack of functional activity of a protease. Such a shift in the MMP functional paradigm, together with the difficulties associated with current methods of studying proteases this highlights the need for new high content degradomics approaches to uncover and annotate MMP activities in vivo and identify novel interactions within the protease web. Integration of these techniques with specifically designed animal models for final validation should lay the foundations for the development of new inhibitors that specifically target disease-related MMPs and/or their upstream effectors that cause deleterious effects in disease, while sparing MMP functions that are protective.

Deubiquitinases (DUBs) have fundamental roles in the ubiquitin system through their ability to specifically deconjugate ubiquitin from targeted proteins. The human genome encodes at least 98 DUBs, which can be grouped into 6 families, reflecting the need for specificity in their function. The activity of these enzymes affects the turnover rate, activation, recycling and localization of multiple proteins, which in turn is essential for cell homeostasis, protein stability and a wide range of signaling pathways. Consistent with this, altered DUB function has been related to several diseases, including cancer. Thus, multiple DUBs have been classified as oncogenes or tumor suppressors because of their regulatory functions on the activity of other proteins involved in tumor development. Therefore, recent studies have focused on pharmacological intervention on DUB activity as a rationale to search for novel anticancer drugs. This strategy may benefit from our current knowledge of the physiological regulatory mechanisms of these enzymes and the fact that growth of several tumors depends on the normal activity of certain DUBs. Further understanding of these processes may provide answers to multiple remaining questions on DUB functions and lead to the development of DUB-targeting strategies to expand the repertoire of molecular therapies against cancer.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults. We have analyzed exome sequencing data from 127 individuals with CLL and Sanger sequencing data from 214 additional affected individuals, identifying recurrent somatic mutations in POT1 (encoding protection of telomeres 1) in 3.5% of the cases, with the frequency reaching 9% when only individuals without IGHV@ mutations were considered. POT1 encodes a component of the shelterin complex and is the first member of this telomeric structure found to be mutated in human cancer. Somatic mutation of POT1 primarily occurs in gene regions encoding the two oligonucleotide-/oligosaccharide-binding (OB) folds and affects key residues required to bind telomeric DNA. POT1-mutated CLL cells have numerous telomeric and chromosomal abnormalities that suggest that POT1 mutations favor the acquisition of the malignant features of CLL cells. The identification of POT1 as a new frequently mutated gene in CLL may facilitate novel approaches for the clinical management of this disease.

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Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.