Domain bridging interactions. A necessary contribution to the function and structure of Escherichia coli aspartate transcarbamoylase.
(33/437)
Aspartate transcarbamoylase undergoes a domain closure in the catalytic chains upon binding of the substrates that initiates the allosteric transition. Interdomain bridging interactions between Glu(50) and both Arg(167) and Arg(234) have been shown to be critical for stabilization of the R state. A hybrid version of the enzyme has been generated in vitro containing one wild-type catalytic subunit, one catalytic subunit in which Glu(50) in each catalytic chain has been replaced by Ala (E50A), and wild-type regulatory subunits. Thus, the hybrid enzyme has one catalytic subunit capable of domain closure and one catalytic subunit incapable of domain closure. The hybrid does not behave as a simple mixture of the constituent subunits; it exhibits lower catalytic activity and higher aspartate affinity than would be expected. As opposed to the wild-type enzyme, the hybrid is inhibited allosterically by CTP at saturating substrate concentrations. As opposed to the E50A holoenzyme, the hybrid is not allosterically activated by ATP at saturating substrate concentrations. Small angle x-ray scattering showed that three of the six interdomain bridging interactions in the hybrid is sufficient to cause the global structural change to the R state, establishing the critical nature of these interactions for the allosteric transition of aspartate transcarbamoylase. (+info)
Intracellular distribution of various enzymes concerned with DNA synthesis from normal and regenerating rat liver, and Yoshida sarcoma.
(34/437)
During the fractionation of various enzymes concerned with DNA synthesis from the postmicrosomal supernatant fraction of various tissues, DNA polymerace [EC 2.7.7.7], thymidine kinase [EC 2.7.1.75], dTMP kinase [EC 2.7.4.9], deoxycytidine kinase [EC 2.7.1.74], and deoxycytidine monophosphokinase (dCMP kinase) [EC 2.7.4.14] were found in the pellet fraction of postmicrosomal supernatant. Further, the uridine kinase [EC 2.7.1.48] and aspartate transcarbamylase [EC 2.1.3.2] activities of postmicrosomal supernatant from various tissues were also present in this pellet fraction. The activities of DNA polymerase, thymidine kinase, uridine kinase, and aspartate transcarbamylase from normal and regenerating rat liver, and Yoshida sarcoma were higher in the pellet fraction than in the supernatant. On the other hand, the activities of dTMP kinase, dCMP kinase, and orotidine-5'-phosphate decarboxylase [EC 4.1.1.23] were lower in the pellet fraction than in the supernatant. The pellet fractions of regenerating rat liver and Yoshida sarcoma showed a remarkable incorporation of various precursors (thymidine, dTMP, deoxycytidine, and dCMP) into DNA in the presence of a suitable DNA template, ATP and all four deoxynucleoside 5'-triphosphates for DNA synthesis. Normal adult rat liver catalyzed a much smaller incorporation of all these precursors, except for dCMP. (+info)
Increased gene amplification in immortal rodent cells deficient for the DNA-dependent protein kinase catalytic subunit.
(35/437)
Gene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In rodent cell lines, N-(phosphonacetyl)-L-aspartate (PALA) resistance is considered a measure of gene amplification because it is mainly attributable to amplification of the carbamyl-P-synthetase aspartate transcarbamylase dihydro-orotase (CAD) gene. In this paper we show that the radiosensitive hamster cell line V3, which is defective in DSB repair because of a mutation in the DNA-PKcs gene, displays also an increased frequency of gene amplification. In these cells, we found that the amplification of the CAD gene occurs with a frequency and a rate more than one order of magnitude higher than in control cell lines, although it relies on the same mechanisms. When the same analysis was performed in mouse embryo fibroblasts (MEFs) obtained from animals in which the DNA-PKcs gene was ablated by homologous recombination, a higher frequency of amplification compared with the controls was found only after cellular immortalization. In primary DNA-PKcs(-/-) MEFs, PALA treatment induced a block in the cell cycle, and no PALA-resistant clones were found. Our results indicate that the lack of DNA-PKcs increases the probability that gene amplification occurs in a genetic background already permissive, like that of immortalized cells, although it is not sufficient to make normal cells able to amplify. (+info)
Allosteric regulation of catalytic activity: Escherichia coli aspartate transcarbamoylase versus yeast chorismate mutase.
(36/437)
Allosteric regulation of key metabolic enzymes is a fascinating field to study the structure-function relationship of induced conformational changes of proteins. In this review we compare the principles of allosteric transitions of the complex classical model aspartate transcarbamoylase (ATCase) from Escherichia coli, consisting of 12 polypeptides, and the less complicated chorismate mutase derived from baker's yeast, which functions as a homodimer. Chorismate mutase presumably represents the minimal oligomerization state of a cooperative enzyme which still can be either activated or inhibited by different heterotropic effectors. Detailed knowledge of the number of possible quaternary states and a description of molecular triggers for conformational changes of model enzymes such as ATCase and chorismate mutase shed more and more light on allostery as an important regulatory mechanism of any living cell. The comparison of wild-type and engineered mutant enzymes reveals that current textbook models for regulation do not cover the entire picture needed to describe the function of these enzymes in detail. (+info)
c-Myc mediates activation of the cad promoter via a post-RNA polymerase II recruitment mechanism.
(37/437)
The c-Myc protein is a site-specific DNA-binding transcription factor that is up-regulated in a number of different cancers. We have previously shown that binding of Myc correlates with increased transcription of the cad promoter. We have now further investigated the mechanism by which Myc mediates transcriptional activation of the cad gene. Using a chromatin immunoprecipitation assay, we found high levels of RNA polymerase II bound to the cad promoter in quiescent NIH 3T3 cells and in differentiated U937 cells, even though the promoter is inactive. However, chromatin immunoprecipitation with an antibody that recognizes the hyperphosphorylated form of the RNA polymerase II carboxyl-terminal domain (CTD) revealed that phosphorylation of the CTD does correlate with c-Myc binding and cad transcription. We have also found that the c-Myc transactivation domain interacts with cdk9 and cyclin T1, components of the CTD kinase P-TEFb. Furthermore, activator bypass experiments have shown that direct recruitment of cyclin T1 to the cad promoter can substitute for c-Myc to activate the promoter. In summary, our results suggest that c-Myc activates transcription of cad by stimulating promoter clearance and elongation, perhaps via recruitment of P-TEFb. (+info)
High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6.
(38/437)
MutS and MutL homologs have been implicated in multiple genetic stabilization pathways. The activities participate in the correction of DNA biosynthetic errors, are involved in cellular responses to certain types of DNA damage, and serve to ensure the fidelity of genetic recombination. We show here that the rate of CAD (carbamyl-P synthetase/aspartate transcarbamylase/dihydroorotase) gene amplification is elevated 50- to 100-fold in human cell lines deficient in MLH1 or MSH6, as compared with mismatch repair-proficient control cells. Fluorescence in situ hybridization indicates that these amplification events are the probable consequence of unequal sister chromatid exchanges involving chromosome 2, as well as translocation events involving other chromosomes. These results implicate MutS alpha and MutL alpha in the suppression of gene amplification and suggest that defects in this genetic stabilization function may contribute to the cancer predisposition associated with mismatch repair deficiency. (+info)
N-(phosphonacetyl)-L-aspartate and calcium leucovorin modulation of fluorouracil administered by constant rate and circadian pattern of infusion over 72 hours in metastatic gastrointestinal adenocarcinoma.
(39/437)
BACKGROUND: We have reported that N-(phosphonacetyl)-L-aspartic acid (PALA) 1266 mg/m2 can safely be given 24 hours prior to the start of a 72-hour infusion of fluorouracil (FUra) and leucovorin (LV) at doses of 2000 and 500 mg/m2/day. Since inhibition of aspartate carbamoyltransferase (ACTase) activity was evident 4 hours post PALA, we wished to evaluate PALA given 1 hour prior to FUra. Further, we studied the toxicity and pharmacokinetics with FUra given by either fixed- or variable-rate infusion. PATIENTS AND METHODS: Twenty-seven patients with gastrointestinal tract adenocarcinomas were treated with PALA 1266 mg/m2/15 min followed by a 72-hour infusion of FUra and LV (1750 & 500 mg/m2/day) given by fixed- or variable-rate (peak at 4:00 A.M.). RESULTS: Clinical toxicity was similar in two consecutive cycles in 17 patients receiving fixed- and variable-rate infusion at the same FUra dose. Overall, grade 3 stomatitis and hand-foot syndrome occurred in 12% and 4% patients receiving fixed- and in 16% and 10.5% of patients receiving variable-rate infusions. Six of 24 evaluable patients (25%) had a partial response. The profile of FUra plasma levels (Cp) over a 24-hour period during fixed- and variable-rate infusions were strikingly different, but the average Cp and area under the concentration-time curves were comparable. ACTase activity was significantly decreased at 4 and 24 hours after PALA (12% and 18% of baseline; P < 0.001), but enzyme activity had recovered to 40% by 72 hours. CONCLUSIONS: This regimen was active and well tolerated with similar toxicities with FUra given by either fixed- or variable rate infusion. PALA 1266 mg/m2 significantly inhibited ACTase activity for at least 24 hours. (+info)
Carboxyl-terminal transactivation activity of hypoxia-inducible factor 1 alpha is governed by a von Hippel-Lindau protein-independent, hydroxylation-regulated association with p300/CBP.
(40/437)
Hypoxia-inducible factor 1 complex (HIF-1) plays a pivotal role in oxygen homeostasis and adaptation to hypoxia. Its function is controlled by both the protein stability and the transactivation activity of its alpha subunit, HIF-1 alpha. Hydroxylation of at least two prolyl residues in the oxygen-dependent degradation domain of HIF-1 alpha regulates its interaction with the von Hippel-Lindau protein (VHL) that targets HIF-1 alpha for ubiquitination and proteasomal degradation. Several prolyl hydroxylases have been found to specifically hydroxylate HIF-1 alpha. In this report, we investigated possible roles of VHL and hydroxylases in the regulation of the transactivation activity of the C-terminal activating domain (CAD) of HIF-1 alpha. We demonstrate that regulation of the transactivation activity of HIF-1 alpha CAD also involves hydroxylase activity but does not require functional VHL. In addition, stimulation of the CAD activity by a hydroxylase inhibitor, hypoxia, and desferrioxamine was severely blocked by the adenoviral oncoprotein E1A but not by an E1A mutant defective in targeting p300/CBP. We further demonstrate that a hydroxylase inhibitor, hypoxia, and desferrioxamine promote the functional and physical interaction between HIF-1 alpha CAD and p300/CBP in vivo. Taken together, our data provide evidence that hypoxia-regulated stabilization and transcriptional stimulation of HIF-1 alpha function are regulated through partially overlapping but distinguishable pathways. (+info)