RESUMO
ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Células HEK293 , Humanos , Mutação , NAD/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 signaling is relatively well understood; and, at present, only mammalian TORC1 is a validated drug target, pursued in immunosuppression and oncology. However, the knowledge void surrounding TORC2 is dissipating. Acute inhibition of TORC2 with small molecules is now possible and structural studies of both TORC1 and TORC2 have recently been reported. Here we review these recent advances as well as observations made from tissue-specific mTORC2 knockout mice. Together these studies help define TORC2 structure-function relationships and suggest that mammalian TORC2 may one day also become a bona fide clinical target.