Final Report Summary - SCHISTOSOMA PROTEASE (BIOACTIVE SERINE PROTEASES FROM HUMAN PARASITE SCHISTOSOMA MANSONI)
The main aim of the project was to characterize biological role and analyze structure-activity relationship of neglected serine proteases of Schistosoma mansoni.Schistosomiasis caused by parasites Schistosoma blood flukes, represents one of the most serious chronic parasitic infections in the developing world with more than 200 million people. Current therapy of schistosomiasis relies on a single drug, praziquantel. Therefore, a prophylactic vaccine, or new drug treatment is needed. Schistosomes are actively interacting with the host physiological processes. Their evasive and immuno-modulatory strategies are crucial in host-parasite interactions. Disrupting of these mechanisms by specific drug/vaccine targeting of modulating parasite molecules may possibly lead in diseases treatment. In addition to medical treatment, because of the ability of schistosomes to influence host physiological processes, their molecules involved subject of interest for biomedicine where can be used as instrumental molecules. Our research project is focused on most human schistosome, Schistosoma mansoni. The project is focused on serine proteases (serine proteolytic enzymes) which were except few cases largely neglected. These enzymes share apparent similarity on amino acid level to mammalian proteases involved as regulatory factors. It is hypothesize that it arose due to co-existence with the mammalian hosts and parasite needs to actively interact with host physiological processes involving proteolysis (such as anticoagulation, vasodilatation, immunity). We hypothesize that these proteases participate in the host-parasite interaction or are crucial for parasite survival within the mammalian vasculature.
This fellowship had tentative work plan for past 4 years as follows:
Year 1-2: Establishing the laboratory; Introducing parasite life cycle, RT-PCR and qPCR studies; Cloning and expression of proteases and their selected domains in E. coli; Antibody production and immunolocalization by confocal/electron microscopy. Expression of active recombinant proteases employing various systems and refolding if necessary; RNAi treatments (in vitro and in vivo tests); Protease activity assays, active site labeling if active protease available; Specific design of substrates and probes for labeling.
Year 3-4: Protease activity assays, active site labeling and positional scanning-synthetic combinatorial libraries. Large scale production and purification of recombinant proteases for crystallography will start. Project continuation with plans mentioned for previous years; Relevant native substrate testing; 2D protein gel mapping of relevant substrates; biological assays will start.
Work over the past 4-year period was focused on all objectives mentioned above. Original plan was expanded on additional parasite proteases and modified as response to emerged difficulties during the period of project. All genes were characterized, annotated and deposited to online databases. RNAi knockdowns were refined and optimized with output published. Characterization of expression profiles, activities in parasites and phylogenetic and structural studies of studied trypsin and chymotrypsin-like enzymes was recently submitted to the scientific journal. In detail characterization of the non-trypsin surface protease- prolyl endopeptidase including biological and biochemical aspects will be submitted by the end of the year 2013. Recombinantly expressed proteases were used for antibodies production. Challenging immulocalization studies together with RNAi specific knockdowns and RNA in situ localizations will be published during the following year 2014. Refolding and /or production of active recombinant proteases are still under progress. Some of these results will be published next year as well, as a part of the study utilizing unique peptide positional synthetic library. Student participation on every single goal of the project was positive aspect. In conclusion; this project enabled study of interesting topic, helped to the applicant to re-establish and establish relevant work contacts back in Europe, several scientific publications are and will be published and the research topic will be expanded further.
This fellowship had tentative work plan for past 4 years as follows:
Year 1-2: Establishing the laboratory; Introducing parasite life cycle, RT-PCR and qPCR studies; Cloning and expression of proteases and their selected domains in E. coli; Antibody production and immunolocalization by confocal/electron microscopy. Expression of active recombinant proteases employing various systems and refolding if necessary; RNAi treatments (in vitro and in vivo tests); Protease activity assays, active site labeling if active protease available; Specific design of substrates and probes for labeling.
Year 3-4: Protease activity assays, active site labeling and positional scanning-synthetic combinatorial libraries. Large scale production and purification of recombinant proteases for crystallography will start. Project continuation with plans mentioned for previous years; Relevant native substrate testing; 2D protein gel mapping of relevant substrates; biological assays will start.
Work over the past 4-year period was focused on all objectives mentioned above. Original plan was expanded on additional parasite proteases and modified as response to emerged difficulties during the period of project. All genes were characterized, annotated and deposited to online databases. RNAi knockdowns were refined and optimized with output published. Characterization of expression profiles, activities in parasites and phylogenetic and structural studies of studied trypsin and chymotrypsin-like enzymes was recently submitted to the scientific journal. In detail characterization of the non-trypsin surface protease- prolyl endopeptidase including biological and biochemical aspects will be submitted by the end of the year 2013. Recombinantly expressed proteases were used for antibodies production. Challenging immulocalization studies together with RNAi specific knockdowns and RNA in situ localizations will be published during the following year 2014. Refolding and /or production of active recombinant proteases are still under progress. Some of these results will be published next year as well, as a part of the study utilizing unique peptide positional synthetic library. Student participation on every single goal of the project was positive aspect. In conclusion; this project enabled study of interesting topic, helped to the applicant to re-establish and establish relevant work contacts back in Europe, several scientific publications are and will be published and the research topic will be expanded further.