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

BROLL, V.; LOPES, F.C.; MOYETTA, N.R.; MARTINELLI, A.H.S.; FRUTTERO, L.L.; SULIS, D.B.; DEMARTINI, D.R.; ZAMBELLI, B.; CIURLI, S.L.; CARLINI, C.R.

Porto Alegre

Encuentro; 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 to catalyze the hydrolysis of urea producing ammonium and carbon dioxide. However this protein is classified as moonlighting, producing effects independently of its enzymatic activity, such as neurotoxicity to mammals, exocytose induction, and pro-inflammatory activity with production of oxygen reactive species and cytokines. The mapping of structure-activity relationships of these non-enzymatic properties of ureases is still poorly understood. The bacterium Proteus mirabilis is an opportunistic pathogen that causes severe urinary infections. Urease produced by these bacteria acts as an important virulence factor. Urease from Proteus mirabilis (PMU) is a trimeric protein in which each monomer is composed by three structural subunits, PmUre alpha (66.0 kDa), PmUre beta (12.2 kDa) and PmUre gamma (11 kDa) expressed by the structural genes ureC, ureB and ureA, respectively. In that work we look for PMU moonlighting activities and evaluate which subunit of PMU could be related to those non-enzymatic biological activities of PMU. In order to do that, different plasmids were produced to obtain each subunit separately. PMU was purified from ionic exchange chromatography and a gel filtration as final step. All constructs where produced and each protein expressed with a Strep-tag at the N-terminal portion. PmUre gamma is a soluble protein purified with high purity from an affinity chromatography with the StrepTactin Sepharose HP column. PmUre beta and PmUre alpha were purified from inclusion bodies, following the same chromatographic protocol as PmUre gamma. PMU act as platelet agonist promoting aggregation and showed to be active against different yeasts causing morphological alterations. PmUre gamma and beta subunits showed to be toxic against insects, being both able to activate the immune system of Rhodnius prolixus, but when orally administrated in Dysdercus peruvianus PmUre beta was more toxic than alpha and gamma subunits. Both beta and alpha PMU subunits interact with platelet membranes but only PmUre beta is able to activate platelet aggregation. PmUre beta 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 beta carries some activities described for PMU. Although the contribution of the other two PMU´s subunits in each of its biological activities still needs clarification, we can speculate that PmUre beta conveys most of the urease´s moonlighting activities.