Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2.

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.

Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3alpha) of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S. cerevisiae. We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.

RGS4 is arginylated and degraded by the N-end rule pathway in vitro.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.

The N-end rule pathway in Xenopus egg extracts.

Ubiquitin-dependent degradation of intracellular proteins underlies a multitude of biological processes, including the cell cycle, cell differentiation, and responses to stress. One ubiquitin-dependent proteolytic system is the N-end rule pathway, whose targets include proteins that bear destabilizing N-terminal residues. This pathway, which has been characterized only in somatic cells, is shown here to be present also in germ line cells such as the eggs of the amphibian Xenopus laevis. We demonstrate that the set of destabilizing residues in the N-end rule pathway of Xenopus eggs is similar, if not identical, to that of somatic cells such as mammalian reticulocytes and fibroblasts. It is also shown that the degradation of engineered N-end rule substrates in egg extracts can be strongly and selectively inhibited by dipeptides bearing destabilizing N-terminal residues. This result allowed us to ask whether selective inhibition of the N-end rule pathway in egg extracts influences the apoptosis-like changes that are observed in these extracts. A dipeptide bearing a bulky hydrophobic (type 2) destabilizing N-terminal residue was found to delay the apoptotic changes in egg extracts, whereas dipeptides bearing basic (type 1) destabilizing N-terminal residues had no effect. High activity of the N-end rule pathway in egg extracts provides an alternative to reticulocyte extracts for the in vitro analyses of this pathway.

Th2-specific protein/DNA interactions at the proximal nuclear factor-AT site contribute to the functional activity of the human IL-4 promoter.

IL-4 is a pleiotropic immunoregulatory cytokine secreted by activated Th2, but not Th1, cells. The proximal IL-4 promoter contains MARE, C/EBP, P0, octamer-like, P1, and activating protein-1 elements. The half c-Maf binding site (MARE), P0, and P1 sites were previously shown to be involved in Th2-specific transcriptional activity. Except the MARE and P1 site, the molecular basis for Th2 specificity of the P0 site has not been analyzed. Here, we provide the first detailed analysis of the P0 binding factors and show that in Th2, but not in Th1, cells, NF-AT and proteins of the activating protein-1 family are involved in cooperative binding to the P0 and the adjacent octamer-like site. In the mouse Th2 D10 cells, Oct-1/Oct-2 are also found to participate in formation of the P0-binding complexes. Mutation, deletion, and methylation interference analysis demonstrate that both the P0 and the octamer-like sequence are required for inducible binding. Furthermore, we provide the first report of the functional relevance of each site in the human IL-4 promoter by mutagenesis/transfection analysis and demonstrate that the octamer-like, P0 and P1 sites are important for the biologic function of the IL-4 promoter. The MARE site, although it was shown to be critical for the function of the murine IL-4 promoter, does not appear essential for human IL-4 promoter activity in Jurkat T cells. These findings suggest that besides c-Maf, another Th2-specific factor(s) may be involved in tissue-specific expression of the IL-4 gene.

Differential interaction of nuclear factors with the PRE-I enhancer element of the human IL-4 promoter in different T cell subsets.

The immunomodulatory cytokine IL-4 affects cells of most hemopoietic lineages. IL-4 is secreted by activated Th2 but not Th1 cells and plays a major role in the immune response by modulating the differentiation of naive Th cells toward the Th2 phenotype. We have previously identified an enhancer element, PRE-I, that is essential for the function of the human IL-4 promoter. To investigate the mechanisms responsible for tissue-specific expression of the IL-4 gene, we analyzed nuclear factors binding to the PRE-I site and compared the binding activities of these factors to the IL-4 promoter of Th1 and Th2 cells. We show that PRE-I interacts with PMA- and PMA/ionomycin-inducible, cyclosporin A-sensitive nuclear factors. Using anti-C/EBPbeta (NF-IL6), anti-C/EBPdelta (NF-IL6beta), anti-NF-ATc, anti-NF-ATp, anti-Fos, and anti-Jun Abs we demonstrate that the previously identified PRE-I binding factor POS-1 is composed of different transcription factors in different Th cell subsets. In the IL-4-producing Th0-like human Jurkat and mouse EL-4 cells, POS-1 (designated POS-1a) contains NF-IL6beta and Jun. In the mouse Th2 D10 cells and in the human Th2 clones, POS-1 (designated POS-1b) contains NF-IL6beta, Jun, and NF-ATc/p. In contrast, POS-1 was not found in nuclear extracts of human Th1 clones. These findings suggest that PRE-I may play a role in the differential regulation of IL-4 gene expression levels.

Nuclear factor-IL6 activates the human IL-4 promoter in T cells.

Positive regulatory element I (PRE-I) is a strong enhancer element essential for expression of the human IL-4 gene. To identify transcription factors binding to PRE-I, we screened a cDNA expression library from Jurkat T cells and isolated a cDNA encoding nuclear factor (NF)-IL6 (also known as C/EBP beta). NF-IL6 mRNA was found in human Jurkat T cells and in the mouse Th2 clone D10, but not in Th1 clone 29. rNF-IL6 expressed in bacteria was shown to specifically bind to PRE-I. PRE-I forms multiple DNA-protein complexes with nuclear extracts from Jurkat cells. Some of these complexes were demonstrated to contain NF-IL6 by using anti-C/EBP beta Abs. Overexpression of NF-IL6 enhanced expression of the chloramphenicol acetyl transferase reporter gene linked to the PRE-I-thymidine kinase or the human IL-4 promoter more than 10-fold in Jurkat cells. Promoter deletion studies revealed two additional NF-IL6 binding sites located at positions -44 to -36 (C/EBP proximal) and -87 to -79 (C/EBP medial), respectively. Our results demonstrate that NF-IL6 is involved in transcriptional activation of the human IL-4 promoter in T cells.

Cloning of the cDNA encoding human C/EBP gamma, a protein binding to the PRE-I enhancer element of the human interleukin-4 promoter.

The positive regulatory element-I (PRE-I) is a strong enhancer element essential for expression of the human interleukin-4 (IL-4)-encoding gene. In order to identify transcription factors binding to PRE-I, we screened a cDNA expression library from human Jurkat T-cells. A cDNA encoding the human CCAAT/enhancer binding protein-gamma (hC/EBP gamma) was cloned. The deduced amino acid (aa) sequence of HC/EBP gamma contains 150 aa with high homology to mouse Ig/EBP-1 and rat C/EBP gamma. The mRNA of hC/EBP gamma is expressed at a high level in Jurkat T-cells in three forms generated via differential polyadenylation. DNA-binding experiments with recombinant protein produced in bacteria demonstrate that hC/EBP gamma binds to PRE-I, but not to unrelated DNA fragments. Our data also show that hC/EBP gamma may cooperate with Fos to bind PRE-I.

The role of NF-Y and IRF-2 in the regulation of human IL-4 gene expression.

Activity of the IL-4 promoter was shown to be regulated by multiple cis-acting elements. In this study, two additional regulatory elements, a CCAAT element and a 15-nucleotide element homologous to the IFN- and virus-stimulation response element (ISRE), were identified in the human promoter region at -195 to -172. The ISRE-homologous sequence was shown to interact with two nuclear proteins, IRF-2, a transcriptional repressor of the IFN genes, and an NF-1-like factor. Mutations of the ISRE site increased overall IL-4 promoter activity twofold, suggesting a possible negative role of IRF-2 in the regulation of IL-4 transcription. The CCAAT element was found to interact with NF-Y, a nuclear factor essential for expression of MHC class II genes. Mutations of the CCAAT element at -195 to -172 resulted in a substantial decrease of IL-4 promoter activity. Furthermore, NF-Y was also found to bind to the previously described NF-ATp binding site of the IL-4 promoter (-79 to -62, originally described as "P element"), and the previously described P-binding complex NF-P was shown to contain NF-Y. These findings suggest that NF-Y may play an important role in the regulation of IL-4 gene expression.

[Preparation and bacterial expression of a mutant gene for human lymphotoxin]. / Poluchenie i bakteral'naia ékspressiia mutantnogo gena limfotoksina cheloveka.

Using the oligonucleotide directed mutagenesis, a human lymphotoxin (TNF beta) mutant gene lacking 21 N-terminal codons has been obtained. Recombinant plasmid pLT21 for expression of the mutant gene has been constructed. The mutant gene in the plasmid was placed under control of a tandem of constitutive promoters from coliphage T7. A simple procedure for isolation of recombinant protein was developed. The procedure allows to obtain the highly purified biologically active mutant protein with a good yield. During biosynthesis the recombinant protein undergoes a posttranslational processing resulted in the cleavage of N-terminal methionine and leucine residues.

[Hybrid human lymphokine genes. I. Design of recombinant plasmids coding hybrids of immune interferon and human tumor necrosis factor]. / Geny gibridnykh limfokinov cheloveka. I. Konstruirovanie rekombinantnykh plazmid, kodiruiushchikh gibridy immunnogo interferona i faktorov nekroza opukholei cheloveka.

Recombinant plasmids coding for hybrid proteins between human interferon gamma and human tumour necrosis factor alpha or beta have been constructed using site-directed mutagenesis. The genes were fused via a synthetic oligonucleotide linker coding for tetrapeptide Pro-Val-Gly-Pro. The fused genes were expressed in Escherichia coli under control of early promoters of bacteriophage T7. E. coli cells harbouring the plasmids with the hybrid genes gave rather high level of the fused proteins biosynthesis. The hybrid recombinant proteins proved to be unstable in E. coli cells.

Construction and properties of fusion proteins between human interferon-gamma and human tumour necrosis factors alpha and beta.

A number of recombinant plasmids coding for fusion proteins between human interferon-gamma (IFN-gamma) and human tumour necrosis factor alpha (TNF alpha) or beta (TNF beta) were constructed by using site-directed mutagenesis and ligation of the respective genes. In these proteins the whole IFN-gamma sequence of the molecule is linked at the N terminus via a short polypeptide linker to the TNF alpha sequence lacking two N-terminal amino acid residues or to the whole TNF beta sequence. A series of mutants with deletions in the interferon part of the fusion proteins were also produced. All the fusion genes obtained were efficiently expressed in Escherichia coli under the control of early promoters of bacteriophage T7. The recombinant fusion proteins were found to be unstable inside bacterial cells. Bacterial cell lysates expressing these fusion genes or their deletion mutants showed both biological activities in vitro: the antiviral activity of IFN-gamma and the cytotoxic activity of TNF.