Conjugation of the 15-kDa Interferon-induced Ubiquitin Homolog Is Distinct from That of Ubiquitin
Journal of Biological Chemistry
The biological effect of type 1 interferons is proposed to arise in part from the conjugation of ubiquitin crossreactive 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 CoCl 2 during the purification of recombinant UCRP blocks the proteolytic inactivation of the polypeptide occurring
... ypeptide 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:PP i 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 125 I-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 125 I-UCRP conjugation is significantly increased in cell extracts after 24 h of incubation in the presence of interferon-␤, 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. The interferons exert their biological effects through induction of a subset of cellular genes whose patterns of expression define the cell-and tissue-specific responses characteristic of these cytokines. Several of these interferon-induced genes have been the subject of considerable study (reviewed in Ref. 1); however, the mechanisms by which many of these proteins contribute to the interferon response remain poorly understood. One such protein within the latter group is the 15-kDa polypeptide originally identified by Farrell et al. (2) and later characterized by Knight and co-workers (3, 4). Interferon-induced expression of the 15-kDa protein is regulated by an upstream interferon-stimulated response element typical of all early genes induced by type 1 interferons, IFN-␣ 1 and IFN-␤ (5). Subsequently, the sequence of the 15-kDa protein was noted to possess significant homology to a tandem diubiquitin sequence, accounting for its cross-reaction with affinity-purified anti-ubiquitin antibodies (6). The carboxyl-terminal LRL-RGG sequence of ubiquitin essential for its conjugation to cellular proteins is conserved within the carboxyl terminus of the ubiquitin cross-reactive protein (UCRP), leading to the proposal that UCRP contributes to the type 1 interferon response through an analogous conjugation reaction (6). More recently, high molecular weight UCRP conjugates have been detected constitutively and within interferon-induced cultured cell lines using anti-UCRP-specific antibodies (7, 8) . Ubiquitin is one of the most highly conserved proteins found widely distributed among eucaryotes. The best studied function of ubiquitin is to target cellular proteins for degradation through a post-translational modification wherein the carboxyl terminus of ubiquitin is covalently linked via isopeptide bond to primary amines on target proteins (9). The resulting conjugates are degraded by a multienzyme ATP-dependent pathway requiring the 26 S multicatalytic protease complex (proteasome) (10). Conjugation of ubiquitin to cellular proteins proceeds through a three-step pathway, reviewed in Hershko and Ciechanover (10) and Pickart (11). The ubiquitin-activating enzyme (E1) catalyzes an ATP-coupled activation of the carboxyl-terminal glycine of ubiquitin to generate an enzyme-bound ubiquitin adenylate intermediate and free PP i (12). Transfer of activated ubiquitin to an active site cysteine of E1 releases AMP and generates a covalent E1-ubiquitin thiol ester (12). In the second step, ubiquitin is transferred by transacylation to a cysteine residue conserved among all members of a family of ubiquitin carrier proteins, E2 (13, 14). The third step involves aminolysis of these E2 thiol esters to form isopeptide bonds between the carboxyl-terminal glycine of ubiquitin and ⑀-amino groups of lysine residues on target proteins in both E3 (ubiquitin:protein ligase) -dependent and -independent mechanisms (15). A persistent question has been whether the regulatory pathway of ubiquitin ligation is unique within the cell or if other similar pathways exist. Discovery of UCRP and its conjugates suggests such ligation events represent a general regulatory strategy within cells. However, a remaining unresolved question is whether ubiquitin and UCRP ligation mechanisms share a common set of enzymes or proceed through parallel but distinct pathways. In order to address the latter question, we have examined the ability of recombinant human UCRP to support the reaction catalyzed by ubiquitin-activating enzyme.