Synthesis and evaluation of resins bearing substrate-like inhibitor functions for capturing copper amine oxidases

none6In order to make new tools available for investigating interactions between enzyme and substrate-like inhibitors in copper amine oxidases (CAOs), a class of enzymes that controls important cellular processes such as the crosslinking of elastin and collagen, cell proliferation and the regulation of intracellular polyamines, starting from a previous work we synthesized the poly(N,N-dimethylacrylamide)-based resin R2 and the new TentaGel resins T3 and T4 obtained by immobilizing onto commercial TentaGel S-Br properly designed CAO substrate-like inhibitor moieties. The anchoring of CAO substrate-like inhibitor moieties was carried out through stable ether bonds onto commercial TentaGel S-Br which contains bromomethyl groups susceptible to nucleophilic substitution reactions. We resorted to polyacrylamide gel electrophoresis (PAGE) experiments to ascertain the capability of the prepared resins to capture plasma amine oxidase (PAO) and diamine oxidase (DAO), members of the CAO family. The poly(N,N-dimethylacrylamide)-based resin R2 resulted able of blocking PAO and DAO being the first insoluble polymeric materials able to capture enzymes of the CAO family.Pocci M; Alfei S; Castellaro S; Lucchesini F; Milanese M; Bertini V.Pocci, Marco; Alfei, Silvana; Castellaro, Sara; Lucchesini, Francesco; Milanese, Marco; Bertini, Vincenz

Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

Zaprasis A, Brill J, Thüring M, et al. Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides. Applied and environmental microbiology. 2013;79(2):576-587.Bacillus subtilis can attain cellular protection against the detrimental effects of high osmolarity through osmotically induced de novo synthesis and uptake of the compatible solute l-proline. We have now found that B. subtilis can also exploit exogenously provided proline-containing peptides of various lengths and compositions as osmoprotectants. Osmoprotection by these types of peptides is generally dependent on their import via the peptide transport systems (Dpp, Opp, App, and DtpT) operating in B. subtilis and relies on their hydrolysis to liberate proline. The effectiveness with which proline-containing peptides confer osmoprotection varies considerably, and this can be correlated with the amount of the liberated and subsequently accumulated free proline by the osmotically stressed cell. Through gene disruption experiments, growth studies, and the quantification of the intracellular proline pool, we have identified the PapA (YqhT) and PapB (YkvY) peptidases as responsible for the hydrolysis of various types of Xaa-Pro dipeptides and Xaa-Pro-Xaa tripeptides. The PapA and PapB peptidases possess overlapping substrate specificities. In contrast, osmoprotection by peptides of various lengths and compositions with a proline residue positioned at their N terminus was not affected by defects in the PapA and PapB peptidases. Taken together, our data provide new insight into the physiology of the osmotic stress response of B. subtilis. They illustrate the flexibility of this ubiquitously distributed microorganism to effectively exploit environmental resources in its acclimatization to sustained high-osmolarity surroundings through the accumulation of compatible solutes