Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome

Oxidatively modified proteins that accumulate in aging and many diseases can form large aggregates because of covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic, and threaten cell viability. Most oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the proteasome. Previous work from our laboratory has shown that purified 20 S proteasome degrades oxidized proteins without ATP or ubiquitin in vitro, but there have been no studies to test this mechanism in vivo. The aim of this study was to determine whether ubiquitin conjugation is necessary for the degradation of oxidized proteins in intact cells. We now show that cells with compromised ubiquitin-conjugating activity still preferentially degrade oxidized intracellular proteins, at near normal rates, and this degradation is still inhibited by proteasome inhibitors. We also show that progressive oxidation of proteins such as lysozyme and ferritin does not increase their ubiquitinylation, yet the oxidized forms of both proteins are preferentially degraded by proteasome. Furthermore, rates of oxidized protein degradation by cell lysates are not significantly altered by addition of ATP, excluding the possibility of an energy requirement for this pathway. Contrary to earlier popular belief that most proteasomal degradation is conducted by the 26 S proteasome with ubiquitinylated substrates, our work suggests that oxidized proteins are degraded without ubiquitin conjugation (or ATP hydrolysis) possibly by the 20 S proteasome, or the immunoproteasome, or both.     

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.     

The reactive site of alpha 1-antitrypsin is C-terminal, not N-terminal

alpha 1-Antitrypsin recovered from trypsin-alpha 1-antitrypsin complexes was shown to be a mixture of two peptides which remained associated in 6 M guanidine and in 1% acetic acid, but were separated by SDS-polyacrylamide gel electrophoresis. The larger peptide had an Mr of 47 000 and gave low yields on end-group analysis; the smaller had an Mr of 4000 and was the C-terminal 36-residue fragment of alpha 1-antitrypsin. These results explain the consistent but erroneous finding of a reactive site near the N-terminus of alpha 1-antitrypsin, and confirm that the reactive site is 36 residues from the C-terminus.     

Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome

Under homeostatic conditions, Nrf2 activity is constitutively repressed. This process is dependent on Keap1, to which Nrf2 binds through the Neh2 domain. Since the N-terminal subdomain of Neh2 (Neh2-NT) contains evolutionarily conserved motifs, we examined the roles they play in the degradation of Nrf2. In Neh2-NT, we defined a novel motif that is distinct from the previously characterized DIDLID motif and designated it DLG motif. Deletion of Neh2-NT or mutation of the DLG motif largely abolished the Keap1-mediated degradation of Nrf2. These mutations were found to enfeeble the binding affinity of Nrf2 to Keap1. The Neh2-NT subdomain directed DLG-dependent, Keap1-independent, degradation of a reporter protein in the nucleus. By contrast, mutation of DLG did not affect the half-life of native Nrf2 protein in the nucleus under oxidative stress conditions. These results thus demonstrate that DLG motif plays essential roles in the Keap1-mediated proteasomal degradation of Nrf2 in the cytoplasm.     

Cleavage of Shigella surface protein VirG occurs at a specific site, but the secretion is not essential for intracellular spreading.

The large plasmid-encoded outer membrane protein VirG (IcsA) of Shigella flexneri is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that VirG is located at one pole on the surface of the bacterium and secreted into the culture supernatant and that in host cells it is localized along the length of the F-actin tail. The roles of these VirG phenotypes in bacterial spreading still remain to be elucidated. In this study, we examined the surface-exposed portion of the VirG protein by limited trypsin digestion of S. flexneri YSH6000 and determined the sites for VirG processing during secretion into the culture supernatant. Our results indicated that the 85-kDa amino-terminal portion of VirG is located on the external side of the outer membrane, while the 37-kDa carboxy-terminal portion is embedded in it. The VirG cleavage required for release of the 85-kDa protein into the culture supernatant occurred at the Arg-Arg bond at positions 758 to 759. VirG-specific cleavage was observed in Shigella species and enteroinvasive Escherichia coli, which requires an as yet unidentified protease activity governed by the virB gene on the large plasmid. To investigate whether the VirG-specific cleavage occurring in extracellular and intracellular bacteria is essential for VirG function in bacterial spreading, the Arg-Arg cleavage site was modified to an Arg-Asp or Asp-Asp bond. The virG mutants thus constructed were capable of unipolar deposition of VirG on the bacterial surface but were unable to cleave VirG under in vitro or in vivo conditions. However, these mutants were still capable of eliciting aggregation of F-actin at one pole, spreading into adjacent cells, and giving rise to a positive Sereny test. Therefore, the ability to cleave and secrete VirG in Shigella species is not a prerequisite for intracellular spreading.     

The functional site of placental anticoagulant protein: essential histidine residue of placental anticoagulant protein

Placental anticoagulant protein (PAP) rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH 7.9 and 8 degrees C. Photooxidized PAP failed to bind the phospholipid vesicle. It seemed unlikely that the protein underwent a change in molecular size during the photooxidation on the basis of its behavior in electrophoresis and gel filtration. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged. The peptide, SHLRKV, was included in the functional site of PAP and still showed an anticoagulant activity. On the other hand, the peptide which substituted histidine by alanine, SALRKV, no longer showed the activity. It was shown that the histidine residue is involved in Ca2+ or the phospholipid binding site of the protein.     

Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase.

Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site. In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells. The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis. The binding of [3H]ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi. The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM. The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480. The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM). These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate. It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase.     

A novel DEAD box helicase Has1p from Plasmodium falciparum: N-terminal is essential for activity

Helicases catalyze the opening of nucleic acid duplexes and are implicated in many nucleic acid metabolic cellular processes that require single stranded DNA or reorganization of RNA structure. Previously we have reported that Plasmodium falciparum genome contains a number of DEAD box helicases. In the present study we report the cloning, expression and characterization of one of the novel members of DEAD box family from P. falciparum. Our results indicate that it is a homologue of Has1p from yeast and it contains DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities. This enzyme can utilize all the nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs) for its unwinding activity. Using a truncated derivative of this protein we further report that the N-terminal region of the protein is essentially required for its activity. These studies suggest that besides the conserved helicase domain the highly variable N-terminal region also contributes in the activity of the protein.     

Accelerated dephosphorylation of the beta2-adrenergic receptor by mutation of the C-terminal lysines: effects on ubiquitination, intracellular trafficking, and degradation

Agonist-mediated ubiquitination regulates some G protein-coupled receptors by targeting them to lysosomes for degradation. Phosphorylation also regulates receptor endocytosis and trafficking to lysosomes. To explore the roles of the two post-translational modifications, we mutated the three C-terminal lysines to arginines in the human beta 2-adrenergic receptor (beta 2AR) (K348/372/375R). The level of agonist-mediated ubiquitination of the mutant (3K/R) was greatly reduced compared to that of wild-type (WT) beta 2AR in whole cells and in cell-free assays. Downregulation of 3K/R also was attenuated compared to that of the WT, whereas internalization and recycling were more similar. During endocytosis, WT and 3K/R appeared in different vesicles and WT, but not 3K/R, was transported to lysosomes. Both were rapidly phosphorylated in agonist-stimulated cells, but upon agonist removal, the rate of dephosphorylation of 3K/R initially was approximately 5 times faster than that of WT. The increased rate also was observed in a cell-free, soluble assay and, thus, was not due to differences in receptor trafficking. Okadaic acid, a potent phosphatase inhibitor, reduced the level of dephosphorylation and increased the levels of lysosomal targeting and degradation of 3K/R. The reduced level of ubiquitination and rapid dephosphorylation of 3K/R appear to prevent it from being sorted to lysosomes in contrast to the phosphorylated and ubiquitinated WT beta 2AR. Our findings indicate that both phosphorylation and ubiquitination are involved in the intracellular sorting of beta 2AR between pathways of recycling to the plasma membrane and degradation in lysosomes, and that the rate of dephosphorylation may be another mechanism of regulating the sorting.     

The N-terminal half of EBNA2, except for seven prolines, is not essential for primary B-lymphocyte growth transformation.

Previous molecular genetic analyses of Epstein-Barr virus nuclear protein 2 (EBNA2) identified a negative effect of deletion of codons 19 to 33 on transformation and gene transactivation, while deletion of codons 19 to 110 was a null mutation for transformation and gene transactivation. We here report the surprising finding that codons 2 to 88, which encode the highly conserved unique N terminus (amino acids 1 to 58) and most of the polyproline repeat (amino acids 59 to 95), can be deleted with only minimal effects on transformation. Codons 97 to 122 can also be deleted with only minimal effects on transformation. However, deletion of 35 of the 37 prolines (amino acids 59 to 93) or deletion of codons 2 to 95 results in a null transforming phenotype. Although EBNA2 from which codons 59 to 93 were deleted was a null mutation for transformation, it was similar to some transforming mutants of EBNA2 in abundance, in interaction with RBPJK, and in transactivation of the LMP1 promoter in transient transfection assays. These data indicate that between three and seven prolines are critical for EBNA2 structure or for intermolecular interaction. Aside from these seven prolines, codons encoding the rest of the N-terminal half (amino acids 2 to 230) of EBNA2 are nonessential for primary B-lymphocyte growth transformation.     

A novel role for ATM in regulating proteasome-mediated protein degradation through suppression of the ISG15 conjugation pathway

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.     

Evidence for one essential tryptophan residue at the active site of relaxin.

The hormone relaxin, which is responsible for the rapid widening of the birth channel in mammals prior to parturition, was purified from hog ovarian extracts and shown to be homogeneous by exclusion chromatography in 6 M guanidine hydrochloride and SDS gel electrophoresis. Of the two disulfide-linked chains that comprise relaxin, the larger chain was shown to contain two tryptophan residues, one of which could be completely oxidized in native relaxin without measurable effect on its biological activity. Oxidation of the second residue completely inactivated the hormone. Modifications of lysine side chains or carboxymethylation of a single methionine residue at low pH did not impair the effectiveness of relaxin.