- Glycolaldehyde is an endogenous source of lysine N-pyrrolation
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Lysine N-pyrrolation converts lysine residues to Nα-pyrrole-L-lysine (pyrK) in a covalent modification reaction that significantly affects the chemical properties of proteins, causing them to mimic DNA. pyrK in proteins has been detected in vivo, indicating that pyrrolation occurs as an endogenous reaction. However, the source of pyrK remains unknown. In this study, on the basis of our observation in vitro that pyrK is present in oxidized low-density lipoprotein and in modified proteins with oxidized polyunsaturated fatty acids, we used LC– electrospray ionization–MS/MS coupled with a stable isotope dilution method to perform activity-guided separation of active molecules in oxidized lipids and identified glycolaldehyde (GA) as a pyrK source. The results from mechanistic experiments to study GA-mediated lysine N-pyrrolation suggested that the reactions might include GA oxidation, generating the dialdehyde glyoxal, followed by condensation reactions of lysine amino groups with GA and glyoxal. We also studied the functional significance of GA-mediated lysine N-pyrrolation in proteins and found that GA-modified proteins are recognized by apolipoprotein E, a binding target of pyrrolated proteins. Moreover, GA-modified proteins triggered an immune response to pyrrolated proteins, and monoclonal antibodies generated from mice immunized with GA-modified proteins specifically recognized pyrrolated proteins. These findings reveal that GA is an endogenous source of DNA-mimicking pyrrolated proteins and may provide mechanistic insights relevant for innate and autoimmune responses associated with glucose metabolism and oxidative stress.
- Chikazawa, Miho,Iwata, Shiori,Lim, Sei-Young,Negishi, Lumi,Shibata, Takahiro,Uchida, Koji,Yoshitake, Jun
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p. 7697 - 7709
(2020/06/09)
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- Development and validation of a docking-based virtual screening platform for the identification of new lactate dehydrogenase inhibitors
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The human muscle isoform of lactate dehydrogenase (hLDH5) is one of the key enzymes of the glycolytic process. It is overexpressed in metastatic cancer cells and is linked to the vitality of tumors in hypoxic conditions. With the aim of identifying new hLDH5 inhibitors, a fully automated docking-based virtual screening platform was developed by considering different protein conformations and the consensus docking strategy. In order to verify the reliability of the reported platform, a small database of about 10,000 compounds was filtered by using this method, and the top-ranked compounds were tested for their hLDH5 inhibition activity. Enzymatic assays revealed that, among the ten selected compounds, two proved to efficiently inhibit enzyme activity with IC50 values in the micromolar range. These results demonstrate the validity of the methodologies we followed, encouraging the application of larger virtual screening studies and further refinements of the platform. Furthermore, the two active compounds herein described may be considered as interesting leads for the development of new and more efficient LDH inhibitors.
- Granchi, Carlotta,Capecchi, Alice,Del Frate, Gianluca,Martinelli, Adriano,Macchia, Marco,Minutolo, Filippo,Tuccinardi, Tiziano
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p. 8772 - 8790
(2015/09/01)
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