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Biochemical investigation of the enzyme PRMT7 function in Leishmania spp. parasite infectivity: screening targets for Leishmaniasis relevance

Grant number: 14/50954-3
Support type:Regular Research Grants
Duration: April 01, 2015 - June 30, 2016
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Cooperation agreement: CONFAP ; Newton Fund, with FAPESP as a partner institution in Brazil ; MRC, UKRI
Principal Investigator:Angela Kaysel Cruz
Grantee:Angela Kaysel Cruz
Principal investigator abroad: Pegine Walrad
Institution abroad: University of York, England
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The Leishmaniases are the ninth largest disease burden amongst infectious diseases and the second biggest killer of parasitic diseases, threatening one tenth of the world's population. Who estimates 0.7-1.2 million new cases of cutaneous Leishmaniases annually while visceral Leishmaniasis infects 200,000-400,000 killing 10-20% of them. Brazil is stricken by all pathologies of this disease with 30,000 new infections every year. No vaccine exists and there is a growing resistance to available treatments; which are expensive, highly toxic and unsuitable for children. Despite the urgent need for intervention, leishmaniases remains a neglected disease. Leishmania spp. parasites adapt to drastic changes in environments during transmission between insect vectors and human hosts. Lifecycle-specific adaptions enable parasite transmission between hosts and perpetuate parasitic infections and diseases. Molecular regulators that coordinate the fitness of these parasites to different hosts and promote parasite infection are critically important to our broader understanding of this disease. In a recent collaboration, Angela Cruz and Pegine Walrad laboratories initiated the investigation of the role of a relevant molecular regulator in Leishmania major, the arginine methyl transferase PRMT7, which resulted in a publication on the scientific journal Molecular Microbiology. The enzyme PRMT7 of the parasite is differentially expressed during L. major development and is a novel regulator of Leishmania virulence. We seek to combine the expertise of both groups and facilities to biochemically characterize PRMT7 function and explore how it and its target proteins enable parasite infectivity. PRMT7 is the first Leishmania methylating enzyme to be implicated in host-parasite interactions. Preliminary evidence suggests it may act via modification of regulatory RNA binding proteins, RBPs. We will initiate this study by crosslinking Leishmania cells and immune precipitating associating proteins. We will identify candidate targets by utilizing the foremost iTRAQ LC-MS and gel free 20 fractionation technologies of the Centre for Excellence in Mass Spectrometry in University of York. This strategy is up to fifty times more sensitive than standard methods. We will compare results with those previously isolated. Also, we will conduct in vitro methyltransferase assays using [3H] AdoMet, recombinant PRMT7 and target candidates coupled to high resolution cation exchange chromatography to verify monomethyl arginine formation by PRMT7. We will isolate associating transcripts of two confirmed, prioritized RNA binding protein targets of PRMT7 and test whether RNA interactions are changed in PRMT7 knockout parasites. Associating RNAs will be identified via RNA Hi-Seq at the MRC Centre for Genomic Research (UK).We will examine and discuss the data implications from this work and it will direct and inspire our joint grant proposal for BBSRC-FAPESP call. Understanding the function and activity of PRMT7 enzyme implicated in infectivity will provide useful insight into ways to combat Leishmaniasis. (AU)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
FERREIRA, TIAGO R.; DOWLE, ADAM A.; PARRY, EWAN; ALVES-FERREIRA, ELIZA V. C.; HOGG, KAREN; KOLOKOUSI, FOTEINI; LARSON, TONY R.; PLEVIN, MICHAEL J.; CRUZ, ANGELA K.; WALRAD, PEGINE B. PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites. Nucleic Acids Research, v. 48, n. 10, p. 5511-5526, JUN 4 2020. Web of Science Citations: 0.

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