Moonlighting urease of Proteus mirabilis: digging for the biologically active portion at the protein sequence

BROLL VALQUIRIA; CORTES LOPES FERNANDA; MOYETTA NATALIA; MARTINELLI ANNE HELENE; FRUTTERO LEONARDO; SULIS D; DEMARTINI DIOGO; ZAMBELLI BARBARA; CIURLI S.L

Porto Alegre

Jornada; XVII Encontro do Programa de Pós-Graduação em Biologia Celular e Molecular (PPGBCM); 2016

Universidade Federal do Rio Grande do Sul

Ureases are widely spread nickel-dependent enzymes. Their main function is tocatalyze the hydrolysis of urea producing ammonium and carbon dioxide. Ureases are moonlighting proteins, producing effects independently of enzymatic activity, such as neurotoxicity to mammals, exocytose induction, and pro-inflammatory activity with production of oxygen reactive species and cytokines. Jaburetox, a recombinant peptide corresponding to an internal sequence of the jack bean urease, is highly toxic to insect and fungi. The mapping of structure-activity relationships of these non-enzymatic properties of ureases is still poorly characterized. The bacterium Proteus mirabilis is an opportunistic pathogen that causes severe urinary infections. Urease from Proteus mirabilis (PMU) acts as an important virulence factor. PMU is a trimeric protein in whicheach monomer is composed by three structural subunits, PmUre(66.0 kDa),PmUre(12.2 kDa) and PmUre(11 kDa), expressed by the structural genes ureC,ureB and ureA, respectively. Considering its moonlighting activities, we aim to evaluate which subunit of PMU could be related to each of its non-enzymatic biological activities. In order to do that, different plasmids were produced to obtain each subunit separately. PMU was purified by ion exchange chromatography and a gel filtration as final step. The constructs were produced and each protein expressed with a Strep-tag at the Nterminal portion. PmUre is a soluble protein, obtained with high purity after an affinity chromatography on a StrepTactin Sepharose HP column. PmUre and PmUrewere purified from inclusion bodies, following the same chromatographic protocol as PmUre. PMU is an agonist promoting platelet aggregation and is fungitoxic against differentyeasts causing morphological alterations. PmUre and  subunits are toxic against insects, interfering with the immune system of Rhodnius prolixus. When administrated orally to Dysdercus peruvianus, PmUre was more toxic to the insect than the isolated or  subunits. Both  and  PMU subunits interacted with platelet membranes but only PmUre was able to activate platelet aggregation. PmUre displayed fungitoxic activity against yeasts at lower doses than the other two subunits. Our data agree with previous results (Postal et al., 2012) showing that other peptides, besides Jaburetox, formed from Jack bean urease by digestion with papain are able to inhibit yeast growth. We conclude that PmUre carries some activities described for PMU. Although the contribution of the other two PMU?s subunits in each of its biological activities still needsclarification, we can speculate that PmUre conveys most of the urease?s moonlighting activities