Post-translational modification of glutamine and lysine residues of HIV-1 aspartyl protease by transglutaminase increases its catalytic activity.
The human immunodeficiency virus type 1 aspartyl protease (HIV-1 PR) is a homodimeric aspartyl endopeptidase that is required for virus replication. HIV-1 PR was shown to act invitro as acyl-donor and -acceptor for both guinea pig liver transglutaminase (TG, EC 2.3.2.13) and human Factor XIIIa. These preliminary evidences suggested that the HIV-1 PR contains at least three TG-reactive glutaminyl and one lysyl residues. We report here that the incubation of HIV-1 PR with TG increases its catalytic activity. This increase is dependent upon the time of incubation, the concentration of TG and the presence of Ca2+. Identification of epsilon-(gamma-glutamyl)lysine in the proteolytic digest of the TG-modified HIV-1 PR suggested intramolecular covalent cross-linking of this protease which may promote a non-covalent dimerization and subsequent activation of this enzyme via a conformational change. This hypothesis is supported by the observation that the TG-catalyzed activation of HIV-1 PR was completely abolished by spermidine (SPD) which acts as a competitive inhibitor of epsilon-(gamma-glutamyl)lysine formation. Indeed, in the presence of 1mM SPD the formation of the isopeptide was decreased of about 80%. The main products of the TG-catalyzed modification of HIV-1 PR in the presence of SPD were N(1)-mono(gamma-glutamyl)SPD and N(8)-mono(gamma-glutamyl)SPD. Negligible amount of N(1),N(8)-bis(gamma-glutamyl)SPD were found. The significance of these results is discussed with respect to the activation of the protease by post-translational modification and design of potential inhibitors.
Lentini
Alessandro
A
Department of Biology, University "Tor Vergata", Rome, Italy.
Tabolacci
Claudio
C
Melino
Sonia
S
Provenzano
Bruno
B
Beninati
Simone
S
D000818:Animals; D057055:Aspartic Acid Proteases / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D026122:Factor XIIIa / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006168:Guinea Pigs; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013095:Spermidine / Q000031:analogs & derivatives / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*
D005973:Glutamine; C055363:N(1)-(gamma-glutamyl)spermidine; D026122:Factor XIIIa; D011503:Transglutaminases; D057055:Aspartic Acid Proteases; D008239:Lysine; D013095:Spermidine