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Query: UNIPROT:P06889 (Mol)
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The Drosophila CAD gene, also known as rudimentary, encodes a protein that carries out the first three enzymatic steps of de novo pyrimidine biosynthesis. The sequence for this gene, as previously published, appears to contain several errors. The correction of six bases in a 250 bp stretch encoding the aspartate transcarbamylase domain leads to changes of frame in two areas of the predicted amino acid sequence, consisting of lengths of 30 and 15 amino acid residues, respectively. The revised sequence shows significantly improved positional identity with both Syrian hamster and Escherichia coli aspartate transcarbamylases.
J Mol Biol 1994 Oct 21
PMID:Revision in sequence of CAD aspartate transcarbamylase domain of Drosophila. 793 64

Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation.
Somat Cell Mol Genet 1997 Jan
PMID:Site-directed substitution of Ser1406 of hamster CAD with glutamic acid alters allosteric regulation of carbamyl phosphate synthetase II. 921

The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases, while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory, NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change that characterize the AAA proteins.
J Mol Evol 1997 Nov
PMID:The evolution of the conserved ATPase domain (CAD): reconstructing the history of an ancient protein module. 934 2

Annexins are a unique family of membrane-associated, Ca2+ and phospholipid-binding proteins found in various tissues. Among the 12 isoforms, Annexin II, V and VI exist in heart tissue in the highest amounts. Annexin VI has been shown to affect intracellular Ca2+ cycling and contractility in isolated cardiomyocytes. Annexin V is present in both cardiomyocytes and non-myocyte cell types in the heart and may play a role in the regulation of cellular ion fluxes, organization and secretion, while the cardiac effects of annexin II are unclear. To identify changes in annexin II, V and VI isoforms that might occur in human heart failure, we measured mRNA and protein levels of these three annexins in transplanted left ventricular tissue of 12 patients with end-stage congestive heart failure due to coronary artery disease (CAD, n=6) or idiopathic dilated cardiomyopathy (DCM, n=6) who underwent cardiac transplantation. Normal heart tissue (C, n=6) was used as a control. Northern blot analyses showed a significant decrease (61%) in annexin VI mRNA levels in heart failure patients compared with controls (1.08+/-0.16 v 2.79+/-0.20 A.U.C. unit, determined by laser densitometry, mean+/-s.e.). In contrast, we found a 67% increase (2. 32+/-0.27 v 3.88+/-0.29) in annexin II mRNA levels and a two-fold increase (1.00+/-0.24 v 2.21+/-0.29) in annexin V mRNA levels in cardiomyopathic hearts as compared to normal hearts. Western blot analyses demonstrated a corresponding decrease (46.1%) in annexin VI protein levels in the heart failure group as compared to controls (2. 63+/-0.22 v 4.88+/-0.52), while annexin II protein levels showed a significant 40.7% increase in patients with heart failure compared to those in normal hearts (5.08+/-0.67 v 3.61+/-0.32). Annexin V protein levels were also significantly increased (45%) in heart failure patients compared with normal (2.14+/-0.19 v 1.48+/-0.11). No difference in either annexins II, V or VI mRNA and protein levels were found between CAD and DCM patients. We conclude that human end-stage heart failure is associated with a down regulation of annexin VI and up regulation of annexin II and V proteins. Coordinate changes were observed in steady-state mRNA levels. These results suggest that these annexin isoforms may contribute to the regulation of intracellular Ca2+ homeostasis in the cardiomyopathic heart.
J Mol Cell Cardiol 1998 Mar
PMID:Altered cardiac annexin mRNA and protein levels in the left ventricle of patients with end-stage heart failure. 951 22

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.
Plant Mol Biol 1998 Mar
PMID:A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression. 952 8

Amplification of genes involved in signal transduction and cell cycle control occurs in a significant fraction of human cancers. Loss of p53 function has been proposed to enable cells with gene amplification to arise spontaneously during growth in vitro. However, this conclusion derives from studies employing the UMP synthesis inhibitor N-phosphonacetyl-L-aspartate (PALA), which, in addition to selecting for cells containing extra copies of the CAD locus, enables p53-deficient cells to enter S phase and acquire the DNA breaks that initiate the amplification process. Thus, it has not been possible to determine if gene amplification occurs spontaneously or results from the inductive effects of the selective agent. The studies reported here assess whether p53 deficiency leads to spontaneous genetic instability by comparing cell cycle responses and amplification frequencies of the human fibrosarcoma cell line HT1080 when treated with PALA or with methotrexate, an antifolate that, under the conditions used, should not generate DNA breaks. p53-deficient HT1080 cells generated PALA-resistant variants containing amplified CAD genes at a frequency of >10(-5). By contrast, methotrexate selection did not result in resistant cells at a detectable frequency (<10(-9)). However, growth of HT1080 cells under conditions that induced DNA breakage prior to selection generated methotrexate-resistant clones containing amplified dihydrofolate reductase sequences at a high frequency. These data demonstrate that, under standard growth conditions, p53 loss is not sufficient to enable cells to produce the DNA breaks that initiate amplification. We propose that p53-deficient cells must proceed through S phase under conditions that induce DNA breakage for genetic instability to occur.
Mol Cell Biol 1998 May
PMID:Gene amplification in a p53-deficient cell line requires cell cycle progression under conditions that generate DNA breakage. 956 27

The initial steps of pyrimidine biosynthesis in yeast and mammals are catalyzed by large multifunctional proteins of similar size, sequence and domain structure, but appreciable functional differences. The mammalian protein, CAD, has carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities. The yeast protein, ura2, catalyzes the first two reactions and has a domain, called pDHO, which is homologous to mammalian DHOase, but is inactive. In CAD, only CPSase is regulated, whereas both CPSase and ATCase in the yeast protein are inhibited by UTP. These functional differences were explored by constructing a series of mammalian yeast chimeras. The isolated ATCase domain is catalytically active, but is not regulated. The inclusion of the yeast sequences homologous to the mammalian regulatory domain (B3) and the intervening pDHO domain did not confer regulation. Chimeric proteins in which the homologous regions of the mammalian protein were replaced by the corresponding domains of ura2 exhibited full catalytic activity, as well regulation of the CPSase, but not the ATCase, activities. The yeast B3 subdomain confers UTP sensitivity on the mammalian CPSase, suggesting that it is the locus of CPSase regulation in ura2. Taken together, these results indicate that there are regulatory site(s) in ura2. Channeling is impaired in all the chimeric complexes and completely abolished in the chimera in which the pDHO domain of yeast is replaced by the mammalian DHO domain.
J Mol Biol 1998 Aug 14
PMID:Allosteric regulation and substrate channeling in multifunctional pyrimidine biosynthetic complexes: analysis of isolated domains and yeast-mammalian chimeric proteins. 969 53

In animals, UTP feedback inhibition of carbamyl phosphate synthetase II (CPSase) controls pyrimidine biosynthesis. Suppressor of black (Su(b) or rSu(b)) mutants of Drosophila melanogaster have elevated pyrimidine pools, and this mutation has been mapped to the rudimentary locus. We report that rSu(b) is a missense mutation resulting in a glutamate to lysine substitution within the second ATP binding site (i.e. CPS.B2 domain) of CPSase. This residue corresponds to Glu780 in the Escherichia coli enzyme (Glu1153 in hamster CAD) and is universally conserved among CPSases. When a transgene expressing the Glu-->Lys substitution was introduced into Drosophila lines homozygous for the black mutation, the resulting flies exhibited the Su(b) phenotype. Partially purified CPSase from rSu(b) and transgenic flies carrying this substitution exhibited a dramatic reduction in UTP feedback inhibition. The slight UTP inhibition observed with the Su(b) enzyme in vitro was due mainly to chelation of Mg2+ by UTP. However, the Km values for glutamate, bicarbonate, and ATP obtained from the Su(b) enzyme were not significantly different from wild-type values. From these experiments, we conclude that this residue plays an essential role in the UTP allosteric response, probably in propagating the response between the effector binding site and the ATP binding site. This is the first CPSase mutation found to abolish feedback inhibition without significantly affecting other enzyme catalytic parameters.
J Mol Biol 1999 Mar 26
PMID:A mutation that uncouples allosteric regulation of carbamyl phosphate synthetase in Drosophila. 1008 Aug 91

Although the Myc family of transcription factors is upregulated in many human tumors, it is unclear which genes are targets for the deregulated Myc. Previous studies suggest that hamster and rat carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase Cad genes are regulated by c-Myc. In fact, of all putative target genes thought to be activated by c-Myc, only the Cad gene showed loss of growth regulation in rat cells nullizygous for c-Myc. However, it was unknown whether upregulation of CAD, which performs the first three rate-limiting steps of pyrimidine biosynthesis, contributes to c-Myc's role in human neoplasia. To explore this possibility, we cloned the human cad promoter. We found that c-Myc could bind to an E box in the human cad promoter in gel shift assays and that growth regulated transcription from the human cad promoter was dependent on this c-Myc binding site. However, the increased amount of c-Myc found in Burkitt's lymphoma cell lines did not lead to increased cad mRNA levels. Thus, we suggest that although c-Myc is clearly important for the normal transcriptional control of the cad promoter, it is unlikely that increased levels of CAD are important mediators of c-Myc-induced neoplasia. Therefore, an understanding of the mechanism by which overexpressed c-Myc contributes to the development of Burkitt's lymphoma requires the identification of additional c-Myc target genes.
Mol Carcinog 2000 Feb
PMID:CAD, a c-Myc target gene, is not deregulated in Burkitt's lymphoma cell lines. 1065 1

Early-onset torsion dystonia is a hereditary movement disorder thought to be caused by decreased release of dopamine into the basal ganglia, without apparent neuronal degeneration. Recent cloning of the gene responsible for this disease, TOR1A (DYT1), identified the encoded protein, torsinA, as a member of the AAA+ superfamily of chaperone proteins and revealed highest levels of expression in dopaminergic neurons in human brain. Most cases of this disease are caused by a deletion of one glutamic acid residue in the C-terminal region of the protein. Antibodies generated against torsinA revealed expression of a predominant immunoreactive protein species similar to the predicted size of 37.8 kDa in neural, glial and fibroblastic lines by western blot analysis. This protein is N-glycosylated with high mannose content and not, apparently, phosphoryl-ated. Overexpression of torsinA in mouse neural CAD cells followed by immunocytochemistry, revealed a dramatically different pattern of distribution for wild-type and mutant forms of the protein. The wild-type protein was found throughout the cytoplasm and neurites with a high degree of co-localization with the endoplasmic reticulum (ER) marker, protein disulfide isomerase. In contrast, the mutant protein accumulated in multiple, large inclusions in the cytoplasm around the nucleus. These inclusions were composed of membrane whorls, apparently derived from the ER. If disrupted processing of the mutant protein leads to its accumulation in multilayer membranous structures in vivo, these may interfere with membrane trafficking in neurons.
Hum Mol Genet 2000 May 22
PMID:Mutant torsinA, responsible for early-onset torsion dystonia, forms membrane inclusions in cultured neural cells. 1081 22


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