Barbiroli A.

D’Incecco P.

Fessas D.

Mapelli C.

Motta P.

Musatti A.

Pellegrino S.

Rollini M.

Saitta F.

English

AIR Universita degli studi di Milano

of scFv, specific to hIL7, from combinatorial antibody libraries.The fusion protein, consisting of hIL7 for scFv binding and bac-terial alkaline phosphatase with enhanced catalytic activity (BAP-mut) for immune complex detection, was developed. The DNAsequences encoding human IL-7 and BAPmut were subclonedinto pET24a(+) plasmid vector under control of T7 promoter.E. coli BL21(DE3) cells were transformed with pET24-IL7-BAP-mut expression vector and protein synthesis was induced withauto-induction protocol. Target protein was accumulated in theform of bacterial inclusion bodies. IMAX was used for purifica-tion of solubilized proteins with subsequent optimization ofrenaturation method. Bifunctional activity of IL-7-BAPmutfusion protein after renaturation was confirmed in ELISA andWestern blot. It was shown that application of hIL7-BAPmutallows at least three times shortening the time of the screening ofimmune combinatory libraries of variable genes of IgG and doesnot require using of specific primary and secondary antibodies.P-41-074Analysis of butyrylcholinesterase interactionswith old inhibitors and new reactivatorsZ. Kovarik, T. Cadez, N. Macek HrvatInstitute for Medical Research and Occupational Health, Zagreb,CroatiaInhibition of the enzyme butyrylcholinesterase (BChE) in humantissues by binding of compounds to its active site serine is impor-tant for the detoxification and scavenging of xenobiotics such asorganophosphates (OP). Although BChE is generally consideredas having no physiological function, a growing body of evidenceindicates that BChE plays a central role in the development ofthe symptomatology of Alzheimer’s disease and related demen-tias. The most likely function for BChE is as a backup for acetyl-cholinesterase (AChE) and protection of synaptic AChE fromman-made and naturally occurring poisons. Newly consideredstrategies in medical protection against nerve agents focus on theuse of exogenously administered BChE. The overall idea is toadminister such an enzyme in combination with a specific oxime,to scavenge an OP before it can reach and inhibit native AChE,thus helping organism detoxification from the excess OP. Startingwith a directed library of pyridinium aldoximes, we identified effi-cient reactivators of sarin, cyclosarin, VX, and tabun-BChE con-jugates and kinetically characterized their interactions in detail.Moreover, for several oximes BChE reactivation potency wasshowed to be promising when compared to the standard oximesused in medical practice. However, an absence of universality ofreactivators underlies comprehensiveness of the reactivationmechanism and importance of the stabilization of the oximegroup in vicinity of the phosphorus conjugated at the catalyticserine. Its convenient position for the nucleophilic attack is themajor criteria for efficient reactivation of OP-BChE conjugates.Notwithstanding, we identified several efficient reactivators ofphosphorylated BChE that, due to a cumulative capacity to reac-tivate both AChE and BChE, possess the potential for bioscav-enging of OP. Acknowledgments: This work was supported bythe Croatian Science Foundation (IP-2018-01-7683).P-41-075Insights on the mechanism of action of classIIa bacteriocinsP. Motta1, P. D’Incecco1, F. Saitta1, D. Fessas1, A. Musatti1,C. Mapelli1, S. Pellegrino2, L. Pellegrino1, A. Barbiroli11Department of Food, Environmental and Nutritional Sciences,University of Milan, Milan, Italy, 2Department of PharmaceuticalSciences, University of Milan, Milan, ItalyBacteriocins are ribosomally synthesized peptides with antimicro-bial activity against pathogenic Gram positive bacteria. Thesepeptides have high potency and low toxicity, and are usuallyspecific for one or a few target microorganisms. Sakacin-A is aclass IIa bacteriocin, produced by Lactobacillus sakei, with aspecific anti- Listeria activity. For this reason, it has the potentialto be employed to reduce the risk of L. monocytogenes poisoningof ready-to-eat food products. Class IIa bacteriocins exert theiranti-Listeria activity by binding to a transmembrane receptor onthe target cells, but the exact underlying mechanism has yet to befully elucidated. The binding properties of the N- and the C-terminal domains of sakacin-A were tested against differentmicroorganisms, by using confocal laser scanning microscopyand peptides conjugated with 5(6)-carboxyfluorescein, to investi-gate the molecular mechanism responsible for the specific recog-nition of Listeria. In addition, the full length peptide wasinserted into liposomes of appropriate composition – where theC-terminal domain is hidden into the lipid bilayer, whereas theN-terminal half is exposed to the solvent – to perform pulldownexperiments on bacterial lysates. Analysis of the liposome-boundproteins confirms a specific interaction between sakacin-A andtransmembrane receptors on Listeria cells. Our data point to ageneral role of the N-terminal domain in the binding to differentGram positive bacteria, although this region is not “per se” suffi-cient for the specific recognition of Listeria. In conclusion, a bet-ter understanding of the mechanisms underlying the specificityand anti-microbial activity of sakacin-A will pave the way for amore efficient exploitation of class IIa bacteriocins as foodpreservative or as an alternative to non-protein antibiotics.Posters General topics – various431FEBS Open Bio 9 (Suppl. 1) (2019) 65–431 DOI: 10.1002/2211-5463.12675ª 2019 The Authors. FEBS Open Bio ª 2019 FEBS

Insights on the mechanism of action of class IIa bacteriocins

https://air.unimi.it/bitstream/2434/906345/2/FEBS%20Open%20Bio%20-%202019%20-%20-%20Posters%20%281%29.pdf

Insights on the mechanism of action of class IIa bacteriocins

Abstract

Similar works

Full text

Available Versions

AIR Universita degli studi di Milano