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

Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP

As cancer cells traverse collagen-rich extracellular matrix (ECM) barriers and intravasate, they adopt a fibroblast-like phenotype and engage undefined proteolytic cascades that mediate invasive activity. Herein, we find that fibroblasts and cancer cells express an indistinguishable pericellular collagenolytic activity that allows them to traverse the ECM. Using fibroblasts isolated from gene-targeted mice, a matrix metalloproteinase (MMP)–dependent activity is identified that drives invasion independently of plasminogen, the gelatinase A/TIMP-2 axis, gelatinase B, collagenase-3, collagenase-2, or stromelysin-1. In contrast, deleting or suppressing expression of the membrane-tethered MMP, MT1-MMP, in fibroblasts or tumor cells results in a loss of collagenolytic and invasive activity in vitro or in vivo. Thus, MT1-MMP serves as the major cell-associated proteinase necessary to confer normal or neoplastic cells with invasive activity

Rate of telomere shortening and cardiovascular damage: a longitudinal study in the 1946 British Birth Cohort.

AIM: Cross-sectional studies reported associations between short leucocyte telomere length (LTL) and measures of vascular and cardiac damage. However, the contribution of LTL dynamics to the age-related process of cardiovascular (CV) remodelling remains unknown. In this study, we explored whether the rate of LTL shortening can predict CV phenotypes over 10-year follow-up and the influence of established CV risk factors on this relationship. METHODS AND RESULTS: All the participants from the MRC National Survey of Health and Development (NSHD) with measures of LTL and traditional CV risk factors at 53 and 60-64 years and common carotid intima-media thickness (cIMT), cardiac mass and left ventricular function at 60-64 years were included. LTL was measured by real-time polymerase chain reaction and available at both time points in 1033 individuals. While LTL at 53 years was not linked with any CV phenotype at 60-64 years, a negative association was found between LTL and cIMT at 60-64 years (β = -0.017, P = 0.015). However, the strongest association was found between rate of telomere shortening between 53 and 60-64 years and values of cIMT at 60-64 years (β = -0.020, P = 0.006). This association was not affected by adjustment for traditional CV risk factors. Cardiac measurements were not associated with cross-sectional or longitudinal measures of LTL. CONCLUSION: These findings suggest that the rate of progression of cellular ageing in late midlife (reflected by the rate of LTL attrition) relates to vascular damage, independently from contribution of CV risk factor exposure

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

Stable isotopic labeling in proteomics

Labeling of proteins and peptides with stable heavy isotopes (deuterium, carbon-13, nitrogen-15, and oxygen-18) is widely used in quantitative proteomics. These are either incorporated metabolically in cells and small organisms, or postmetabolically in proteins and peptides by chemical or enzymatic reactions. Only upon measurement with mass spectrometers holding sufficient resolution, light, and heavy labeled peptide ions or reporter peptide fragment ions segregate and their intensity values are subsequently used for quantification. Targeted use of these labels or mass tags further leads to specific monitoring of diverse aspects of dynamic proteomes. In this review article, commonly used isotope labeling strategies are described, both for quantitative differential protein profiling and for targeted analysis of protein modifications

Systemic MMP inhibition for periodontal wound repair: results of a multi-centre randomized-controlled clinical trial

Aim : This multi-centre, prospective, controlled trial was designed to examine the biological response of the matrix metalloproteinase(MMP) inhibitor subantimicrobial dose doxycycline (SDD) combined with access flap surgery on periodontal wound repair in patients with chronic severe periodontitis. Material and Methods : Seventy subjects were enrolled into a 12-month, randomized, placebo-controlled, double-masked trial to evaluate disease response to 6 months therapy and “wash-out” of either placebo+surgery or SDD (20 mg b.i.d.)+surgery. Primary outcome measure included clinical attachment levels (CAL) and secondary outcomes included probing depth (PD), bleeding on probing (BOP), as well as gingival crevicular fluid bone marker assessment [collagen telopeptides (ICTP)]. These measurements were taken at baseline through 12 months post-surgery and drug administration. Results : Patients treated with SDD and surgery demonstrated stronger reductions in PD in surgically-treated sites of ges;7 mm as well as gains in CAL ( p <0.004). Furthermore, SDD+surgery resulted in short-term reductions in ICTP levels compared with placebo. Rebounds in ICTP levels and clinical parameters occurred when SDD was withdrawn. Conclusions : The results from this multi-centre study suggests that SDD in combination with surgery improves the short-term response of periodontal therapy by reducing PD, increasing CAL gain and inhibiting early stage bone resorption.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75470/1/j.1600-051X.2008.01351.x.pd

Reducción en el índice de peroxidizabilidad de la membrana mitocondrial y niveles de lipoxidación proteícos en el corazón de la rata después de la interrupción de la señalización del receptor betaadrenérgico con el betabloqueante atenolol

A new mammalian longevity model based on ß-adrenergic signaling interruption at the level of adenylyl cyclase has reported decreased bone and heart aging and mean and maximum longevity increases in AC5KO mice (1). We decided to mimic this model in male Wistar rats treated with the ß-blocker atenolol in the drinking water and to check if an oxidative stress decrease could be involved. Atenolol treatment did not modify heart mitROS generation rate and mitDNA oxidative damage but significantly decreased global peroxidizability index of mitochondrial membranes, as well as protein lipoxidation, probably mediated by changes in elongases and desaturases activities.Un nuevo modelo de longevidad en mamíferos basado en la interrupción de la vía de señalización beta-adrenérgica a nivel de la adenilato ciclasa ha revelado una ralentización del envejecimiento del corazón y el hueso de ratones AC5KO y un incremento de su longevidad media y máxima [1]. Decidimos mimetizar este modelo en ratas Wistar utilizando atenolol en el agua de bebida para comprobar si un descenso de estrés oxidativo podría estar implicado. El tratamiento no modificó la tasa de generación de radicales y el daño oxidativo al ADN del corazón, pero si redujo el índice de peroxidizabilidad y la lipoxidación proteica de las membranas mitocondriales, probablemente debido a cambios en las actividades elongasas y desaturasas