Conjugation of the 15-kDa interferon-induced ubiquitin homolog is distinct from that of ubiquitin.
The biological effect of type 1 interferons is proposed to arise in part from the conjugation of ubiquitin cross-reactive protein (UCRP), the ISG15 gene product, to intracellular target proteins in a process analogous to that of its sequence homolog ubiquitin, a highly conserved 8.6-kDa polypeptide whose ligation marks proteins for degradation via the 26 S proteasome. Inclusion of CoCl2 during the purification of recombinant UCRP blocks the proteolytic inactivation of the polypeptide occurring by cleavage of the carboxyl-terminal glycine dipeptide required for activation and subsequent ligation. Intact UCRP supports a low rate of ubiquitin-activating enzyme (E1)-dependent ATP:PPi exchange but fails to form a stoichiometric E1-UCRP thiol ester or undergo transfer to ubiquitin carrier protein (E2). The binding affinity of E1 for UCRP is significantly diminished relative to that of ubiquitin. These results suggest that UCRP conjugation proceeds through an enzyme pathway distinct from that of ubiquitin, at least with respect to the step of activation. This was confirmed for an in vitro conjugation assay in which 125I-UCRP could be ligated in an ATP-dependent reaction to proteins present within an A549 human lung carcinoma cell extract and could be competitively inhibited by excess unlabeled UCRP but not ubiquitin. Other results demonstrate that 125I-UCRP conjugation is significantly increased in cell extracts after 24 h of incubation in the presence of interferon-beta, consistent with the late induction of UCRP conjugating activity. Thus, interferon-responsive cells contain a pathway for UCRP ligation that is parallel but distinct from that of ubiquitin.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
J
J
Narasimhan
J L
JL
Potter
A L
AL
Haas
D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D004952:Esters / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007372:Interferons / Q000502:physiology*; D007700:Kinetics; D011755:Pyrophosphatases / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D004952:Esters; D013438:Sulfhydryl Compounds; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D007372:Interferons; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase