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.

The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum.

gp78, also known as the tumor autocrine motility factor receptor, is a transmembrane protein whose expression is correlated with tumor metastasis. We establish that gp78 is a RING finger-dependent ubiquitin protein ligase (E3) of the endoplasmic reticulum (ER). Consistent with this, gp78 specifically recruits MmUBC7, a ubiquitin-conjugating enzyme (E2) implicated in ER-associated degradation (ERAD), through a region distinct from the RING finger. gp78 can target itself for proteasomal degradation in a RING finger- and MmUBC7-dependent manner. Importantly, gp78 can also mediate degradation of CD3-delta, a well-characterized ERAD substrate. In contrast, gp78 lacking an intact RING finger or its multiple membrane-spanning domains stabilizes CD3-delta. gp78 has thus been found to be an example of a mammalian cellular E3 intrinsic to the ER, suggesting a potential link between ubiquitylation, ERAD, and metastasis.

Heavy metal surveys in Nordic lakes; concentrations, geographic patterns and relation to critical limits.

In the autumn of 1995, coordinated national lake surveys were conducted in the Nordic countries, including Russian Kola. The 11 metals (Pb, Cd, As, Zn, Cu, Ni, Co, Fe, Mn, Cr, V) investigated in nearly 3000 lakes have generally low concentrations and distinct geographical patterns. Direct and indirect influence of long-range transported air pollution is the major important factor for distribution of Pb, Cd, Zn and to a certain degree Co. Total organic carbon (TOC) concentrations in lakes are important for Fe and Mn but also to a certain degree for As, Cr and V. Bedrock geology is the major controlling factor for Cu and Ni, with the exception of areas around the smelters in the Kola peninsula, where the Cu and Ni concentrations in lakes are very high due to local airborne pollution. Bedrock and surficial geology is also an important factor for controlling the concentrations of As, Co, Cr and V. The results indicate that heavy metal pollution in lakes is a minor ecological problem on a regional scale in the Nordic countries.

Functional ecology and palaeolimnology: using cladoceran remains to reconstruct anthropogenic impact.

The field of lake palaeoecology has undergone significant changes. Powerful quantitative techniques have been developed to investigate anthropogenic impacts on lakes. Inclusion of zooplankton and benthic chydorid cladocerans has provided previously unavailable information on the historical development of planktivorous fish populations, submerged macrophytes and lake production, and has been used to document exotic species introductions, rapid genetic evolution and human disturbance of lakes. In particular, new techniques now allow a more complete evaluation of changes in past and present trophic structure to be made, and provide insights on the rapid evolutionary responses of aquatic invertebrate communities to anthropogenic perturbation of lakes.

Ubiquitin protein ligase activity of IAPs and their degradation in proteasomes in response to apoptotic stimuli.

To determine why proteasome inhibitors prevent thymocyte death, we examined whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis were selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzed their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, was spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain was relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program.

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53.

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.

RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.

A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.

Elf-1 regulates basal expression from the T cell antigen receptor zeta-chain gene promoter.

In mature T cells, limited synthesis of the TCR-zeta subunit is primarily responsible for regulating surface expression of TCRs. Transcription of zeta is directed by a complex promoter that includes two potential binding sites for the Ets family of transcription factors at -52 (zEBS1) and -135 (zEBS2). Mutation of these two sites results in a marked reduction of transcription from this promoter. Using electrophoretic mobility shift analysis, Elf-1 was demonstrated to be the Ets family member that binds to these sites. One site, zEBS1, matches the optimal Elf-1 consensus sequence in eight of nine bases, making it the best match of any known mammalian Elf-1 binding site. A role for Elf-1 in TCR-zeta trans-activation was confirmed by ectopic expression of Elf-1 in COS-7 cells. This resulted in an increase in TCR-zeta promoter activity that mapped to zEBS1 and zEBS2. Additional support for the involvement of Elf-1 in TCR-zeta trans-activation derives from the finding that a GAL4-Elf-1 fusion protein trans-activated TCR-zeta promoter constructs that had been modified to contain GAL4 DNA binding sites. These results demonstrate that Elf-1 plays an essential role in the trans-activation of a constitutively expressed T cell-specific gene, and that trans-activation occurs in the context of the native promoter in both lymphoid and nonlymphoid cells. Taken together with the existing literature, these data also suggest that the requirement for inducible factors in Elf-1-mediated trans-activation may decrease as the affinity and number of Elf-1 sites increase.

Serine phosphorylation-regulated ubiquitination and degradation of beta-catenin.

Several lines of evidence suggest that accumulation of cytoplasmic beta-catenin transduces an oncogenic signal. We show that beta-catenin is ubiquitinated and degraded by the proteosome and that beta-catenin stability is regulated by a diacylglycerol-independent protein kinase C-like kinase activity, which is required for beta-catenin ubiquitination. We also define a six-amino acid sequence found in both beta-catenin and the NF-kappaB regulatory protein IkappaBalpha, which, upon phosphorylation, targets both proteins for ubiquitination. Mutation of a single serine within the ubiquitination targeting sequence prevents ubiquitination of beta-catenin. Mutations within the ubiquitination targeting sequence of beta-catenin may be oncogenic.

Subcellular localization and ubiquitin-conjugating enzyme (E2) interactions of mammalian HECT family ubiquitin protein ligases.

In most instances, the transfer of ubiquitin to target proteins is catalyzed by the action of ubiquitin protein ligases (E3s). Full-length cDNAs encoding murine E6-associated protein (mE6-AP) as well as Nedd-4, a protein that is homologous to E6-AP in its C terminus, were cloned. Nedd-4 and mouse E6-AP are both enzymatically active E3s and function with members of the UbcH5 family of E2s. Mouse E6-AP, like its human counterpart, ubiquitinates p53 in the presence of human papilloma virus E6 protein, while Nedd-4 does not. Consistent with its role in p53 ubiquitination, mE6-AP was found both in the nucleus and cytosol, while Nedd-4 was found only in the cytosol. Binding studies implicate a 150-amino acid region that is 40% identical between mE6-AP and Nedd-4 as a binding site for the C-terminal portion of an E2 enzyme (UbcH5B). Nedd-4 was determined to have a second nonoverlapping E2 binding site that recognizes the first 67 amino acids of UbcH5B but not the more C-terminal portion of this E2. These findings provide the first demonstration of physical interactions between mammalian E2s and E3s and establish that these interactions occur independently of ubiquitin and an intact E3 catalytic domain. Furthermore, the presence of two E2 binding sites within Nedd-4 suggests models for ubiquitination involving multiple E2 enzymes associated with E3s.

Identification of a family of closely related human ubiquitin conjugating enzymes.

Two very closely related human E2 ubiquitin conjugating enzymes, UbfH5B and UbcH5C, have been identified. These enzymes are products of distinct genes and are 88-89% identical in amino acid sequence to the recently described human E2, UbcH5 (now designated UbcH5A), UbcH5A-C are homologous to a family of five ubiquitin conjugating enzymes from Arabidopsis thaliana, AtUBC8-12. They are also closely related to Saccharomyces cerevisiae ScUBC4 and ScUBC5, which are involved in the stress response, and play a central role in the targeting of short-lived regulatory proteins for degradation. mRNAs encoding UbcH5A-C were co-expressed in all cell lines and tissues evaluated, with UbcH5C transcripts generally expressed at the highest levels. Analysis of Southern blots suggests that there are likely to be other related members of this family. Both UbcH5B and UbcH5C form thiol ester adducts with ubiquitin, and have activities similar to UbcH5A and AtUBC8 in the conjugation of ubiquitin to target proteins in the presence of the human ubiquitin protein ligase E6-AP. These results establish the existence of a highly conserved, and widely expressed, family of human ubiquitin conjugating enzymes.

Altered aromatic amine metabolism in epileptic patients treated with phenobarbital.

The fate of carcinogens differs among individuals who have different activities of drug-metabolizing enzymes that are important in activating and detoxifying carcinogens. A drug that profoundly alters the metabolism of the drugs and carcinogens is the anticonvulsive agent phenobarbital. To investigate why epileptic patients appear to have a low risk of cancer of the urinary bladder, and on the basis of the observation that levels of aromatic amine-hemoglobin adducts are strongly associated with various risk factors for cancer at that site, we determined aromatic amine-hemoglobin adducts in 62 epileptic patients as a surrogate measure of the reaction of carcinogenic metabolites with DNA in target tissue. Although adducts were detected in all subjects, the levels were proportional to daily tobacco consumption. When the subjects were stratified into groups smoking 20 g tobacco/day or more, smoking <20 g/day, and not smoking, an effect of medication was detected. Epileptic patients treated chronically with phenobarbital or primidone, which is effectively metabolized to phenobarbital, were found to have lower levels of 4-aminobiphenyl adducts than patients on the other treatment (P = 0.02; ANOVA). In nonsmokers, no effect of medication could be demonstrated above background variation; however, an increasing effect was seen with tobacco consumption with only one-half the increase in adducts per g of tobacco smoked as epileptic patients on other treatment. The difference in the increases (slopes of regression lines) was highly significant statistically. This reduction in the level of hemoglobin-aromatic amine adducts is probably due to induction of detoxification enzymes in the patients treated with phenobarbital.

Transcriptional regulation of the T cell antigen receptor zeta subunit: identification of a tissue-restricted promoter.

The cell surface expression of T cell antigen receptors (TCR) is regulated in part by the limiting synthesis of the zeta subunit. Utilizing fragments from the 5' region of the human zeta gene, two discrete regions that promote transcription were characterized. Both of these elements are located within 125 bases of the most 3' site of transcription initiation. The more proximal (3') promoter exhibits activity in lymphoid as well as nonlymphoid cells. In contrast, the more distal (5') promoter element functions in a tissue-restricted fashion. The tissue-specific promoter is localized to a 29-base fragment. The sequence of this region is remarkable for a stretch of 11 consecutive purines that are required for activity. This element constitutes the only known tissue-specific promoter for an invariant TCR subunit. Consistent with the unique role served by the zeta subunit in assembly of the TCR, this study demonstrates that the expression of the zeta gene is regulated in a fashion distinct from other TCR components.

Activation-dependent ubiquitination of a T cell antigen receptor subunit on multiple intracellular lysines.

The T cell antigen receptor zeta chain and other T cell antigen receptor components are ubiquitinated on receptor occupancy. A systematic mutagenesis of the zeta subunit was undertaken to determine the sites of ubiquitination. Ubiquitination was found to occur in the cytoplasmic domain of zeta with multiple lysines serving as sites for mono- and polyubiquitination. The mutation of all potential sites of ubiquitination did not inhibit receptor tyrosine phosphorylation or the ubiquitination of other T cell antigen receptor subunits. Lysines introduced into nonnative positions in the zeta molecule were also able to serve as sites for ubiquitination. These findings demonstrate that once a T cell antigen receptor is targeted for ubiquitination, there is little specificity with regard to the lysine residues that are modified.

T cell antigen receptor-eta subunit. Low levels of expression and limited cross-species conservation.

The eta-subunit of the TCR is derived from an alternative splice product of the TCR-zeta gene. The eta-subunit has been extensively characterized in murine T cells, where up to 10% of TCR bear zeta-eta heterodimers rather than zeta-homodimers. In contrast to the significant levels of eta found in murine T cells, in human PBL an eta-like region is expressed spliced to upstream exons of the zeta-gene at no more than 0.25% the level of zeta-mRNA. Analysis of genomic DNA from five additional mammalian species demonstrated that eta-like sequences are highly conserved at a nucleic acid level. The protein sequences encoded in the eta-regions from various species ranged from 28 to 92 amino acids in length, with the first seven deduced amino acids of eta-being common to all species. Within three to five amino acids of this region, all species have five consecutive charged amino acids. Beyond this point, due to translation in different reading frames, there is no significant amino acid homology. The findings of low level of expression of eta-RNA, limited cross-species conservation on a protein level, and the lack of an established functional role for this alternative splice product raise questions as to the potential roles that this subunit may play in TCR function.

Organization of the human T cell receptor zeta/eta gene and its genetic linkage to the Fc gamma RII-Fc gamma RIII gene cluster.

The zeta-subunit is the most recently characterized stoichiometric human TCR component. In this study we describe the molecular organization of the human zeta-gene. The zeta transcript is generated as the spliced product of eight exons that are separated by distances of 0.7 kb to more than 8 kb. Ribonuclease protection studies revealed multiple transcription initiation sites distributed over a range of approximately 115 bases. A variable number tandem repeat restriction fragment polymorphism contained within the structural gene has allowed for the localization of zeta within the human genome. Additionally, a restriction fragment polymorphism within the Fc gamma RII-Fc gamma RIII gene cluster has allowed for its localization on the map of human chromosome 1q and for the establishment of its linkage to the zeta-gene locus. A region that is highly homologous on a nucleotide level with the eta-exon of the murine zeta-gene is localized to the 3' region of the human zeta-gene. Surprisingly, translation of this region into protein results in a structure that is markedly divergent from its murine counterpart. This finding has important implications regarding the potential role of eta in T cell function.

[Malignant schwannoma of the trigeminal nerve]. / Malignes Schwannom des Nervus trigeminus.

We report on diagnosis, treatment and clinical course of a solitary schwannoma of the trigeminal nerve with consecutive metastatic spread in a 45-year-old woman. The patient presented a temporal lobe syndrome with psychomotor seizures. Trigeminal nerve function was intact apart from a transient hypaesthesia in the mandibular branch. Assuming a meningioma the tumor was removed via a temporal approach. Secondary to the definitive histological diagnosis radiation therapy was performed. After that the patient was symptom-free. 4 months later a large recurrent tumor was found involving the cavernous sinus and the pterygopalatine fossa. In a second operation the tumor was resected intra- and extradurally through an infratemporal preauricular approach in cooperation with an oral and maxillo-facial surgeon. At this time multiple pulmonary metastases developed showing no response to polychemotherapy (EVI). The patient died 13 months after onset of the disease. Hitherto, only 5 cases of a primary malignant schwannoma of the trigeminal nerve have been published in the world literature.

Cancer among epileptic patients exposed to anticonvulsant drugs.

Cancer incidence among 8,004 patients hospitalized for epilepsy between 1933 and 1962 in the Filadelfia treatment community in Denmark was compared to that of the general population. Patients received powerful and prolonged treatment with phenobarbital, phenytoin, and other anticonvulsants. This new survey extends the follow-up from 1976 through 1984. Among 7,864 patients with epilepsy not known to have received radioactive Thorotrast, record linkage with national cancer incidence files identified 789 cancers, compared to 664 expected [relative risk (RR) = 1.19; 95% confidence interval = 1.11-1.27]. Significant risks were found for cancers of the brain and central nervous system (RR = 5.7; n = 118) and the lung (RR = 1.4; n = 106). The excess numbers of brain cancer were concentrated within 10 years of hospitalization (RR = 20.7; n = 80) and decreased significantly over time, which suggests that brain tumors account for the seizure disorder and are not due to phenobarbital exposure as suggested by some epidemiologic studies. No overall risk was apparent when brain cancers were excluded (RR = 1.03). Because bladder cancer was significantly decreased (RR = 0.6; n = 18), the excess risk of lung cancer may not have been related to the "anecdotal" heavy smoking reported among confined groups of epileptic patients in the early years of the study period. The incidence of malignant melanoma was also significantly low (RR = 0.5; n = 7), which suggested limited exposure to sunlight among confined patients. The risk of non-Hodgkin's lymphoma was increased, but not significantly (RR = 1.4; n = 16), which is interesting in view of previous reports suggesting an association with phenytoin. Overall, these data provide little evidence that phenobarbital and phenytoin are carcinogenic to humans, but the excess risks of lung cancer and non-Hodgkin's lymphoma among epileptic patients in our study deserve further evaluation.

Consequences of the t(14;18) chromosomal translocation in follicular lymphoma: deregulated expression of a chimeric and mutated BCL-2 gene.

The t(14;18) chromosomal translocation of human follicular lymphoma recombines the candidate transforming gene bcl-2, located at 18q21, with the immunoglobulin (Ig) H-chain joining region (JH) at 14q32. To elucidate the consequences of this translocation, we cloned bcl-2 cDNAs from a pre-B cell line (Nall-1) and a t(14;18) lymphoma cell line (SU-DHL-6) and compared these sequences with their genomic counterparts. These studies revealed the complexity of bcl-2 gene expression in which six potential polyadenylation signals in exon 3 and two different 5' exons (exons 1 and 2) and promoters are alternatively used to generate different sized bcl-2 mRNAs. A single open reading frame (ORF), at the junction of exons 2 and 3, predicts a 239 amino acid, 26 kD protein. Most chromosome 18 breakpoints cluster within a 150 bp region of exon 3. In SU-DHL-6 the t(14;18) translocation juxtaposes a truncated bcl-2 gene with J6 in a tail-to-head configuration, resulting in the deregulated expression of chimeric bcl-2/Ig transcripts. Importantly, the SU-DHL-6 bcl-2 cDNA also contained several point mutations in the ORF, two of which altered the primary amino acid sequence. The deregulated expression of an altered bcl-2 gene may play a critical role in the disordered growth and differentiation of follicular B cell lymphoma.