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

Financing watershed management in Nicaragua. 1. An analysis of the legal framework

3 ilus. 5 tab. 7 ref.Se analizaron los principales instrumentos del marco legal que permiten institucionalizar procesos relacionados con el financiamiento del manejo de cuencas en Nicaragua. Los mecanismos concesionarios sobresalientes fueron los fondos ambientales. Los mecanismos de desarrollo de mercado m?s importantes fueron el pago por servicios ambientales y el sistema de cobro de c?nones por uso y vertidos. Los instrumentos econ?micos de control directo m?s importantes fueron los relacionados con los reg?menes de concesiones. En el marco jur?dico nicarag?ense predominan los incentivos de orden fiscal basados en instrumentos espec?ficos, como la exoneraci?n, exenci?n y deducci?n de impuestos. Casi la mitad de los actores nacionales consideran que se deben incorporar instrumentos para mejorar la institucionalidad del financiamiento del manejo de cuencas. Entre las debilidades del marco legal resaltan el bajo presupuesto de las instituciones, desconocimiento, falta de implementaci?n y apropiaci?n de los mecanismos de financiamiento y de instrumentos econ?micos. Entre las fortalezas del marco legal se destacan la incorporaci?n de elementos de valoraci?n ambiental e institucionalizaci?n del enfoque de cuencas y recursos h?dricos. The main instruments in the Nicaraguan legal framework were analyzed in order to find the processes that encourage the institutionalization of financing watershed management. The most noteworthy concessionary funding mechanisms were the environmental funds. The mechanisms for the development of markets were payments for environmental services and the system of fees for use and disposal. The most important economic instruments of direct control were those related to concessions. Fiscal incentives are dominant in the Nicaraguan legal framework they rely on specific instruments such as tax exemptions, exonerations and deductions. Almost half of national stakeholders considered that new instruments need to be incorporated if institutionalization of financing watershed management is to be improved. Predominant weaknesses in the legal framework are low institutional budgets, lack of knowledge and lack of implementation and making use of financing mechanisms and economic instruments. Among the strengths of the legal framework are environmental assessment and adoption of watershed and water resource approaches

Proteome-wide identification of family member-specific natural substrate repertoire of caspases

Caspases are proteolytic enzymes that are essential for apoptosis. Understanding the many discrete and interacting signaling pathways mediated by caspases requires the identification of the natural substrate repertoire for each caspase of interest. Using an amplification-based protein selection technique called mRNA display, we developed a high-throughput screen platform for caspase family member specific substrates on a proteome-wide scale. A large number of both known and previously uncharacterized caspase-3 substrates were identified from the human proteome. The proteolytic features of these selected substrates, including their cleavage sites and specificities, were characterized. Substrates that were cleaved only by caspase-8 or granzyme B but not by caspase-3, were readily selected. The method can be widely applied for efficient and systematic identification of the family member specific natural substrate repertoire of any caspase in an organism of interest, in addition to that of numerous other proteases with high specificity

Polyserase-I, a human polyprotease with the ability to generate independent serine protease domains from a single translation product

We have identified and cloned a human liver cDNA encoding an unusual mosaic polyprotein, called polyserase-I (polyserine protease-I). This protein exhibits a complex domain organization including a type II transmembrane motif, a low-density lipoprotein receptor A module, and three tandem serine protease domains. This unusual modular architecture is also present in the sequences predicted for mouse and rat polyserase-I. Human polyserase-I gene maps to 19p13, and its last exon overlaps with that corresponding to the 3′ UTR of the gene encoding translocase of mitochondrial inner membrane 13. Northern blot analysis showed the presence of a major polyserase-I transcript of 5.4 kb in human fetal and adult tissues and in tumor cell lines. Analysis of processing mechanisms of polyserase-I revealed that it is synthesized as a membrane-associated polyprotein that is further processed to generate three independent serine protease units. Two of these domains are proteolytically active against synthetic peptides commonly used for assaying serine proteases. These proteolytic activities of the polyserase-I units are blocked by serine protease inhibitors. We show an example of generation of separate serine protease domains from a single translation product in human tissues and illustrate an additional mechanism for expanding the complexity of the human degradome, the entire protease complement of human cells and tissues

Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1–matrix metalloproteinase substrates

By proteolytic modification of low abundant signaling proteins and membrane receptors, proteases exert potent posttranslational control over cell behavior at the postsecretion level. Hence, substrate discovery is indispensable for understanding the biological role of proteases in vivo. Indeed, matrix metalloproteinases (MMPs), long associated with extracellular matrix degradation, are increasingly recognized as important processing enzymes of bioactive molecules. MS is now the primary proteomic technique for detecting, identifying, and quantitating proteins in cells or tissues. Here we used isotopecoded affinity tag labeling and multidimensional liquid chromatography inline with tandem MS to identify MDA-MB-231 breast carcinoma cell proteins shed from the cell surface or the pericellular matrix and extracellular proteins that were degraded or processed after transfection with human membrane type 1-MMP (MT1-MMP). Potential substrates were identified as those having altered protein levels compared with the E240A inactive MT1-MMP mutant or vector transfectants. New substrates were biochemically confirmed by matrix-assisted laser desorption ionization–time-of-flight MS and Edman sequencing of cleavage fragments after incubation with recombinant soluble MT1-MMP in vitro. We report many previously uncharacterized substrates of MT1-MMP, including the neutrophil chemokine IL-8, secretory leukocyte protease inhibitor, pro-tumor necrosis factor α, death receptor-6, and connective tissue growth factor, indicating that MT1-MMP is an important signaling protease in addition to its traditionally ascribed roles in pericellular matrix remodeling. Moreover, the high-throughput and quantitative nature of isotope-coded affinity tag labeling combined with tandem MS sequencing is a previously undescribed degradomic screen for protease substrate discovery that should be generally adaptable to other classes of protease for exploring proteolytic function in complex and dynamic biological contexts

A proteomic approach for the discovery of protease substrates

Standardized, comprehensive platforms for the discovery of protease substrates have been extremely difficult to create. Screens for protease specificity are now frequently based on the cleavage patterns of peptide substrates, which contain small recognition motifs that are required for the cleavage of the scissile bond within an active site. However, these studies do not identify in vivo substrates, nor can they lead to the definition of the macromolecular features that account for the biological specificity of proteases. To use properly folded proteins in a proteomic screen for protease substrates, we used 2D difference gel electrophoresis and tandem MS to identify substrates of an apoptosis-inducing protease, granzyme B. We confirmed the cleavage of procaspase-3, one of the key substrates of this enzyme, and identified several substrates that were previously unknown, as well as the cleavage site for one of these substrates. We were also able to observe the kinetics of substrate cleavage and cleavage product accumulation by using the 2D difference gel electrophoresis methodology. “Protease proteomics” may therefore represent an important tool for the discovery of the native substrates of a variety of proteases