The Degradome database: mammalian proteases and diseases of proteolysis

The degradome is defined as the complete set of proteases present in an organism. The recent availability of whole genomic sequences from multiple organisms has led us to predict the contents of the degradomes of several mammalian species. To ensure the fidelity of these predictions, our methods have included manual curation of individual sequences and, when necessary, direct cloning and sequencing experiments. The results of these studies in human, chimpanzee, mouse and rat have been incorporated into the Degradome database, which can be accessed through a web interface at http://degradome.uniovi.es. The annotations about each individual protease can be retrieved by browsing catalytic classes and families or by searching specific terms. This web site also provides detailed information about genetic diseases of proteolysis, a growing field of great importance for multiple users. Finally, the user can find additional information about protease structures, protease inhibitors, ancillary domains of proteases and differences between mammalian degradomes

Regulation of TMPRSS6 by BMP6 and iron in human cells and mice.

Mutations in transmembrane protease, serine 6 (TMPRSS6), encoding matriptase-2, are responsible for the familial anemia disorder iron-refractory iron deficiency anemia (IRIDA). Patients with IRIDA have inappropriately elevated levels of the iron regulatory hormone hepcidin, suggesting that TMPRSS6 is involved in negatively regulating hepcidin expression. Hepcidin is positively regulated by iron via the bone morphogenetic protein (BMP)-SMAD signaling pathway. In this study, we investigated whether BMP6 and iron also regulate TMPRSS6 expression. Here we demonstrate that, in vitro, treatment with BMP6 stimulates TMPRSS6 expression at the mRNA and protein levels and leads to an increase in matriptase-2 activity. Moreover, we identify that inhibitor of DNA binding 1 is the key element of the BMP-SMAD pathway to regulate TMPRSS6 expression in response to BMP6 treatment. Finally, we show that, in mice, Tmprss6 mRNA expression is stimulated by chronic iron treatment or BMP6 injection and is blocked by injection of neutralizing antibody against BMP6. Our results indicate that BMP6 and iron not only induce hepcidin expression but also induce TMPRSS6, a negative regulator of hepcidin expression. Modulation of TMPRSS6 expression could serve as a negative feedback inhibitor to avoid excessive hepcidin increases by iron to help maintain tight homeostatic balance of systemic iron levels

Earlier onset of tumoral anglogenesis in matrix metalloproteinase-19-deficient mice

Among matrix metalloproteinases (MMP), MMP-19 displays unique structural features and tissue distribution. In contrast to most MMPs, MMP-19 is expressed in normal human epidermis and down-regulated during malignant transformation and dedifferentiation. The contribution of MMP-19 during tumor angiogenesis is presently unknown. In an attempt to give new insights into MMP-19 in vivo functions, angiogenic response of mutant mice lacking MMP-19 was analyzed after transplantation of murine malignant PDVA keratinocytes and after injection of Matrigel supplemented with basic fibroblast growth factor. In situ hybridization and immunohistochemical analysis revealed that MMP-19 is produced by host mesenchymal cells but not by endothelial capillary cells or CD11b-positive inflammatory cells. Based on a new computer-assisted method of quantification, we provide evidence that host MMP-19 deficiency was associated with an increased early angiogenic response. In addition, increased tumor invasion was observed in MMP-19-/- mice. We conclude that, in contrast to most MMPs that promote tumor progression, MMP-19 is a negative regulator of early steps of tumor angiogenesis and invasion. These data highlight the requirement to understand the individual functions of each MMP to improve anticancer strategies

A Comprehensive Peptidome Profiling Technology for the Identification of Early Detection Biomarkers for Lung Adenocarcinoma

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000–5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273–283, FIBA 5–16, and LBN 306–313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens

High-carotenoid maize: development of plant biotechnology prototypes for human and animal health and nutrition

Carolight (R) is a transgenic maize variety that accumulates extraordinary levels of carotenoids, including those with vitamin A activity. The development of Carolight (R) maize involved the technical implementation of a novel combinatorial transformation method, followed by rigorous testing for transgene expression and the accumulation of different carotenoid molecules. Carolight (R) was envisaged as a way to improve the nutritional health of human populations that cannot access a diverse diet, but this ultimate humanitarian application can only be achieved after extensive testing for safety, agronomic performance and nutritional sufficiency. In this article, we chart the history of Carolight (R) maize focusing on its development, extensive field testing for agronomic performance and resistance to pests and pathogens, and feeding trials to analyze its impact on farm animals (and their meat/dairy products) as well as animal models of human diseases. We also describe more advanced versions of Carolight (R) endowed with pest-resistance traits, and other carotenoid-enhanced maize varieties originating from the same series of initial transformation experiments. Finally we discuss the further steps required before Carolight (R) can fulfil its humanitarian objectives, including the intellectual property and regulatory constraints that lie in its path

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) family

The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future

Combination of KIR2DS4 and FcγRIIa polymorphisms predicts the response to cetuximab in KRAS mutant metastatic colorectal cancer

Cetuximab is a standard-of-care treatment for RAS wild-type metastatic colorectal cancer (mCRC) but not for those harbor a KRAS mutation since MAPK pathway is constitutively activated. Nevertheless, cetuximab also exerts its effect by its immunomodulatory activity despite the presence of RAS mutation. The aim of this study was to determine the impact of polymorphism FcγRIIIa V158F and killer immunoglobulin-like receptor (KIR) genes on the outcome of mCRC patients with KRAS mutations treated with cetuximab. This multicenter Phase II clinical trial included 70 mCRC patients with KRAS mutated. We found KIR2DS4 gene was significantly associated with OS (HR 2.27; 95% CI, 1.08–4.77; P = 0.03). In non-functional receptor homozygotes the median OS was 2.6 months longer than in carriers of one copy of full receptor. Multivariate analysis confirmed KIR2DS4 as a favorable prognostic marker for OS (HR 6.71) in mCRC patients with KRAS mutation treated with cetuximab. These data support the potential therapeutic of cetuximab in KRAS mutated mCRC carrying non-functional receptor KIR2DS4 since these patients significantly prolong their OS even after heavily treatment. KIR2DS4 typing could be used as predictive marker for identifying RAS mutated patients that could benefit from combination approaches of anti-EGFR monoclonal antibodies and other immunotherapies to overcome the resistance mediated by mutation in RAS.This clinical trial was approved and supported by Merck S.L., an affiliate of Merck KGaA, Darmstadt. Germany [research project number 2010-023580-18, date: 05-06-2014