Preferential Modification of Nuclear Proteins by a Novel Ubiquitin-like Molecule

Abstract

Sentrin is a novel ubiquitin-like protein that protects cells against both anti-Fas and tumor necrosis factorinduced cell death. Antiserum recognizing the N terminus of sentrin revealed the presence of a 18-kDa sentrin monomer, a 90-kDa band (p90), and multiple high molecular mass bands. Because sentrin possesses the conserved Gly-Gly residues near the C terminus, it is likely that these additional bands represent conjugation of sentrin to other proteins in a manner that is similar to the ubiquitination pathway. Transient expression of hemagglutinin epitope-tagged sentrin mutants in COS cells demonstrated that the sentrin C terminus is cleaved, which allows it to be conjugated to other proteins via the conserved C-terminal Gly residue. Immunocytochemical staining and cell fractionation analysis demonstrated that sentrin monomer is localized predominantly to the cytosol. However, p90 and the majority of sentrinized proteins appeared to be localized to the nucleus. When the conserved Gly-Gly residues of sentrin were changed to Gly-Ala, only sentrin monomer and p90 but not the high molecular mass bands were observed. Thus, p90 generation appears to be required for the formation of high molecular mass bands in the nucleus. Taken together, sentrinization represents a novel pathway for nuclear protein modification, which is distinct from ubiquitination.Sentrin was originally isolated in a yeast two hybrid screen using the death domain of Fas as bait (1). It also interacts with tumor necrosis factor (TNF) 1 receptor 1 death domain but not with the death domain of FADD/MORT1 or CD40. When overexpressed in mammalian cells, sentrin protects cells against both anti-Fas and TNF-induced cell death. The mechanism of action of sentrin has not been clearly elucidated. Sentrin could block cell death signaling by blocking the assembly of the death-inducing signal complex. Alternatively, due to its homology to ubiquitin (18% identical and 48% similar), sentrin could exert its anti-death effect through modification of other proteins in a process similar to ubiquitination.Protein modification by ubiquitin is critical for targeting proteins to be degraded by proteasomes (2-4). Conjugation of ubiquitin to other proteins requires initial activation of the conserved C-terminal Gly residue catalyzed by a specific ubiquitin-activating enzyme, E1. An intermediate, ubiquitin adenylate, is formed by displacement of PPi from ATP. Ubiquitin adenylate is then transferred to a thiol site in E1 with release of AMP. Next, ubiquitin is transferred to a family of ubiquitin carrier proteins, E2, through transacylation. Finally, ubiquitin is transferred from E2 to its target protein through an isopeptide linkage with the ⑀-amino group of the Lys residue of the target protein. The transfer of ubiquitin from E2 to the target protein may require the participation of a ligase, E3. The internal Lys of ubiquitin, in particular Lys 48 , can also be modified by another ubiquitin to form multiubiquitin chains that may be crucial for proteosome recognition (5)....

Functional analysis and intracellular localization of p53 modified by SUMO-1

“…In fact, free SUMO-1 protein can be detected in Saos-2 cells (Figure 7a) making it questionable whether one could modulate the levels of sumoylation by co-transfection with a SUMO-1-expressing plasmid. To overcome this obstacle, we used a SUMO-1 construct (SUMO-4aa) with a deletion of the last four amino acids in a transactivation assay: it was shown that cleavage of the last four amino acids was required for the activation of SUMO-1 protein (Mahajan et al, 1997;Kamitani et al, 1997). As a negative control, we used another SUMO-1 construct (SUMO-6aa) with deletion of the last six amino acids, which was shown previously to be defective in sumoylation .…”

Regulation of Hypoxia-Inducible mRNAs by the von Hippel-Lindau Tumor Suppressor Protein Requires Binding to Complexes Containing Elongins B/C and Cul2

“…Thus, one model, which remains to be tested, is that complexes containing Cul2 and elongins B and C target certain proteins for ubiquitin-dependent proteolysis. A more speculative model, given that elongin B contains a ubiquitin-like domain, is that elongin B itself becomes covalently linked to certain proteins, such as has been observed for the ubiquitinlike protein named either GMP1, SUMO-1, or Sentrin (20,33). In either case, pVHL competes with elongin A in vitro for binding to elongins B and C (6).…”

SUMO-1 conjugation to topoisomerase I: A possible repair response to topoisomerase-mediated DNA damage

“…The notion that SUMO-1 may be involved in targeting proteins to the nuclear pore complex came from studies of SUMO-1 conjugated RanGAP1 which is known to bind to the nuclear pore complex (24,37,39,40). SUMO-1 conjugates also have been shown to be primarily located in the nucleus (54) and often targeted to the nuclear bodies as most evidently demonstrated in the case of PML (52). One could speculate that SUMO-1 conjugated hTOP1 molecules in cells treated with CPT are either targeted to the nuclear pore complex for nuclear export or to the nuclear bodies.…”