1,721,254 research outputs found
Detection and quantification of protein carbonyl-quenching activities by high-resolution mass spectrometry
No full textReactive carbonyl species (RCS) are highly electrophilic compounds generated in the organism upon oxidative stress. These molecules are implicated in the pathogenesis and progression of different oxidative-based disorders, such as diabetes, fibrosis and Alzheimer’s disease. Detoxification of RCS by nucleophilic compounds able to form unreactive adducts (carbonyl quenching agents) represents a promising strategy to reduce RCS concentration and to prevent their spontaneous and detrimental reaction with nucleophilic moieties of DNA, lipids and proteins1.
We analyzed and compared the quenching ability of known carbonyl quenching agents including aminoguanidine, hydralazine, pyridoxamine and carnosine. Their ability to prevent protein carbonylation was evaluated by testing different RCS such as 4-hydroxy-trans-2-nonenal, methylglyoxal, glyoxal and malondialdehyde. The quenching ability was quantified by using an innovative approach based on high-resolution mass spectrometry and on ubiquitin, as model protein2.
An approach based on mass spectrometry was applied to identify and characterize the reaction products between RCS and the nucleophilic residues of ubiquitin. Increasing amounts of carbonyl quenchers prevented the formation of protein adducts, as determined by calculating the UC50 values, that is the concentration required to inhibit ubiquitin carbonylation by 50%. Quantitative analyses showed different carbonyl quenching activities: carnosine efficiently quenched the 4-hydroxy-trans-2-nonenal, while aminoguanidine was more active on methylglyoxal and glyoxal. Hydralazine efficiently quenched all reactive carbonyl species, while pyridoxamine was particularly active on malondialdehyde. Selectivity was evaluated by testing the reactivity of the tested compounds towards pyridoxal (an endogenous aldehyde); the results indicated that only carnosine and pyridoxamine are highly selective.
The reaction products between RCS and the different carbonyl quenchers were fully characterized by high-resolution mass spectrometry, leading to the elucidation of the quenching reaction mechanisms.
We then tested the quenching ability of complex mixtures, such as natural extracts. This revealed the ability of green coffee bean extract and procyanidins from Vitis vinifera to prevent protein carbonylation, thus demonstrating that the proposed analytical strategy can be used to test the ability of pure compounds as well as of natural extracts as carbonyl quenching agents.
References
(1) Aldini, G.; Vistoli, G.; Stefek, M.; Chondrogianni, N.; Grune, T.; Sereikaite, J.; Sadowska-Bartosz, I.; Bartosz, G. Molecular Strategies to Prevent, Inhibit and Degrade Advanced Glycoxidation and Advanced Lipoxidation End Products. Free Radic. Res. 2013.
(2) Colzani, M.; Criscuolo, A.; De Maddis, D.; Garzon, D.; Yeum, K.-J.; Vistoli, G.; Carini, M.; Aldini, G. A Novel High Resolution MS Approach for the Screening of 4-Hydroxy-Trans-2-Nonenal Sequestering Agents. J. Pharm. Biomed. Anal. 2014, 91, 108–118
Protein modelling by fragmental approach : connecting global homologies with local peculiarities
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Carnosine in the context of histidine-containing dipeptides
No full textEven though histidine constitutes one of the least abundant residues, it often plays key biochemical roles in protein environments due to the peculiar reactivity of the imidazole ring that characterizes its side chain. Such a relevant chemical profile can also explain the remarkable (and not yet fully clarified) biological roles played by histidine containing dipeptides and in particular by carnosine and its derivatives. Hence, the chapter starts by analyzing the key reactivity of the imidazole ring to explain and rationalize the biological properties of histidine-containing dipeptides as grouped into proteinogenic and nonproteinogenic compounds
Modeling of human ghrelin receptor (hGHS-R1a) in its close state and validation by molecular docking
No full textThe objective of this study was to generate a reliable model of human ghrelin receptor (hGHS-R1a) in its close state by means of a hybrid fragmental approach in which the transmembrane bundle was modeled using the rhodopsin as the template to assure a marked closeness among the transmembrane helices, while the remaining segments (i.e., loops plus terminal domains) were modeled searching different templates to favor the local homologies. The reliability of this model was assessed docking both a tetrapeptide, which represents the ghrelin's active core, and a set of 50 peptidomimetic secretagogues taken from the literature. The analysis of obtained complexes unveils a set of stabilizing interactions with crucial hGHS-R1a residues in remarkable agreement with both mutational analyses and pharmacophore hypotheses. Also the significant correlation between docking scores and biological activities affords an encouraging validation for such hGHS-R1a model, suggesting that also the receptor in its close state (similarly to the hGHS-R1a in its open state which was modeled in our previous study, Pedretti A, Villa M, Pallavicini M, Valoti E, Vistoli G. J. Med. Chem.2006, 49, p 3077.) may be involved in ligand binding and could find fertile applications in ligand desig
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