Monoclonal antibodies against pools of mono- and polyacetylated peptides selectively recognize acetylated lysines within the context of the original antigen(270 visite)(PDF privato182 visite) Sandomenico A, Focà A, Sanguigno L, Caporale A, Focà G, Pignalosa A, Corvino G, Caragnano A, Beltrami AP, Antoniali G, Tell G, Leonardi A, Ruvo M
*** IBB - CNR *** Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche (IBB-CNR), Napoli, Italy; Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), Napoli, Italy; Bioker Multimedica, Napoli, Italy;
Post-translational modifications (PTMs) strongly influence the structure<br> and function of proteins. Lysine side chain acetylation is one of the <br>most widespread PTMs, and it plays a major role in several physiological<br> and pathological mechanisms. Protein acetylation may be detected by <br>mass spectrometry (MS), but the use of monoclonal antibodies (mAbs) is a<br> useful and cheaper option. Here, we explored the feasibility of <br>generating mAbs against single or <span class="" "\\\"highlight\\\""""="" style="" "\\\"background-color:\\\""""="">multiple</span><br> acetylations within the context of a specific sequence. As a model, we <br>used the unstructured N-terminal domain of APE1, which is acetylated on <br>Lys27, Lys31, Lys32 and Lys35. As immunogen, we used a peptide mixture <br>containing all combinations of single or multi-acetylated variants <br>encompassing the 24-39 protein region. Targeted screening of the <br>resulting clones yielded mAbs that bind with high affinity to only the <br>acetylated APE1 peptides and the acetylated protein. No binding was seen<br> with the non-acetylated variant or unrelated acetylated peptides and <br>proteins, suggesting a high specificity for the APE1 acetylated <br>molecules. MAbs could not finely discriminate between the differently <br>acetylated variants; however, they specifically bound the acetylated <br>protein in mammalian cell extracts and in intact cells and tissue slices<br> from both breast cancers and from a patient affected by idiopathic <br>dilated cardiomyopathy. The data suggest that our approach is a rapid <br>and cost-effective method to generate mAbs against specific proteins <br>modified by <span class="" "\\\"highlight\\\""""="" style="" "\\\"background-color:\\\""""="">multiple</span> acetylations or other PTMs.
Petraglia F, Singh AA, Carafa V, Nebbioso A, Conte M, Scisciola L, Valente S, Baldi A, Mandoli A, Petrizzi VB, Ingenito C, De Falco S, Cicatiello V, Apicella I, Janssen-megens EM, Kim B, Yi G, Logie C, Heath S, Ruvo M, Wierenga ATJ, Flicek P, Yaspo ML, Della Valle V, Bernard O, Tomassi S, Novellino E, Feoli A, Sbardella G, Gut I, Vellenga E, Stunnenberg HG, Mai A, Martens JHA, Altucci L * Combined HAT/EZH2 modulation leads to cancer-selective cell death(145 visite) Oncotarget (ISSN: 1949-2553electronic, 1949-2553linking), 2018 May 22; 9(39): 25630-25646. Impact Factor:5.008 DettagliEsporta in BibTeXEsporta in EndNote
31 Records (31 escludendo Abstract e Conferenze). Impact factor totale: 147.939 (147.939 escludendo Abstract e Conferenze). Impact factor a 5 anni totale: 152.733 (152.733 escludendo Abstract e Conferenze).