Analysis of transglutaminase protein substrates by functional proteomics.
Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.
Ruoppolo
Margherita
M
Dipartimento di Biochimica e Biotecnologie Mediche, Universita' di Napoli Federico II, Napoli, Italy.
Orrù
Stefania
S
D'Amato
Alfonsina
A
Francese
Simona
S
Rovero
Paolo
P
Marino
Gennaro
G
Esposito
Carla
C
D000345:Affinity Labels; D000588:Amines; D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives*; D002846:Chromatography, Affinity; D004791:Enzyme Inhibitors / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D013997:Time Factors; D011503:Transglutaminases / Q000032:analysis* / Q000737:chemistry
D000345:Affinity Labels; D000588:Amines; D004791:Enzyme Inhibitors; D010455:Peptides; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D011503:Transglutaminases