Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rtg1p is a basic helix-loop-helix transcription factor in the yeast Saccharomyces cerevisiae that is required for basal and regulated expression of CIT2, the gene encoding a peroxisomal isoform of citrate synthase. In respiratory incompetent rho degree petite cells, CIT2 transcription is elevated as much as 30-fold compared with respiratory competent rho + cells. Here we provide evidence that Rtg1p interacts directly with a CIT2 upstream activation site (UASr) and that the rho degree/rho + regulation is not due to a change in the levels of Rtg1p. A fusion protein consisting of the DNA binding domain of Gal4p fused to the NH2 terminus of the full-length wild-type Rtg1p was able to transactivate an integrated LacZ reporter under control of the Gal4p-responsive GAL1 UASG in a rho degree/rho(+)-dependent manner. Other Gal4p fusions to deletions or mutations of Rtg1p indicate that the helix-loop-helix domain is essential for transactivation. Regulated expression of CIT2 also requires the RTG2 gene product. The Gal4-Rtg1p fusion was unable to transactivate the LacZ reporter gene in a strain deleted for RTG2, suggesting that the RTG2 product does not act independently of Rtg1p in the rho degree/rho + transcriptional response.
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PMID:Transactivation by Rtg1p, a basic helix-loop-helix protein that functions in communication between mitochondria and the nucleus in yeast. 749 87

We used the minitransposon TnhlyAs [Gentschev, I., Maier, G., Kranig, A. and Goebel, W. (1996) Mol. Gen. Genet. 252, 266-274] for random insertion of the secretion signal (HlyAs) of Escherichia coli hemolysin (HlyA) into chromosomal genes. Four mini-TnhlyAs derivatives bearing the gltA (citrate synthase), deoC (2 deoxyribose-5 phosphate aldolase), tig (trigger factor) genes and an unknown ORF fused to hlyAs were identified and characterized. Our data suggest that TnhlyAs-generated hemolysin fusion proteins are secreted efficiently by the HlyB/HlyD/TolC hemolysin secretion machinery and that this can be useful for studies of gene expression or function.
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PMID:Construction of chromosomally encoded secreted hemolysin fusion proteins by use of mini-TnhlyAs transposon. 971 56

To examine functions of two small heat shock proteins of Escherichia coli, IbpA and IbpB, we constructed His-IbpA and His-IbpB, in which a polyhistidine tag was fused to the N-terminals. Both purified His-IbpA and His-IbpB formed multimers, which have molecular masses of about 2.0-3.0 MDa and consist of about 100-150 subunits. They suppressed the inactivation of several enzymes including citrate synthase and 6-phosphogluconate dehydrogenase by heat, potassium superoxide, hydrogen peroxide and freeze-thawing, but not the inactivation of glyceraldehyde-3-phosphate dehydrogenase by hydrogen peroxide. Both His-IbpA and His-IbpB suppressed enzyme inactivation by various treatments and were also found to be associated with their non-native forms. However, both His-IbpA and His-IbpB were not able to reactivate enzymes inactivated by heat, oxidants or guanidine hydrochloride. When heated to 50 degrees C, each multimeric form of His-IbpA or His-IbpB was dissociated to form a monomer for His-IbpA, and an oligomer of about one-quarter size for His-IbpB. These structural changes were reversible, as both heated proteins regained the multimeric structures after incubation at 25 degrees C. However, when exposed to hydrogen peroxide or potassium superoxide, the large multimeric forms of His-IbpA and His-IbpB were maintained. The results suggest that His-IbpA and His-IbpB suppress the inactivation of enzymes and bind non-native proteins to protect their structures from heat and oxidants.
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PMID:Escherichia coli small heat shock proteins, IbpA and IbpB, protect enzymes from inactivation by heat and oxidants. 1207 54

It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-microm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, approximately 45% of each chimera was mobile and had a diffusion coefficient of 4 x 10(-8) cm(2)/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 x 10(-8) cm(2)/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.
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PMID:Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex. 1219 36

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.
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PMID:On molecular taxonomy: what is in a name? 1517 35

We report here a development of the MultiSite Gateway(TM)-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.
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PMID:Development of a versatile expression plasmid construction system for Aspergillus oryzae and its application to visualization of mitochondria. 1688 May 96

The substrate specificity of the mitochondrial metallopeptidase proteinase 1 (MP1) was investigated and its mitochondrial targeting signal identified. The substrate specificity of MP1 was examined with physiological peptides as substrates. Although the enzyme exhibits broad substrate specificity, there is a trend for peptides containing 13 or more residues to exhibit K(m) values of 2 muM or less. Three of four peptides containing 11 or fewer residues exhibited K(m) values above 10 muM. Similarly, peptides containing 13 or more residues exhibited k(cat) values below 10 min(-1), while three of four peptides containing 11 or fewer residues exhibited k(cat) values above 30 min(-1). Many of the peptide cleavage sites of MP1 resemble that of the mitochondrial processing protease (MPP); however, MP1 does not process the precursor form of citrate synthase. The enzyme, however, does cleave the released prepeptide from precitrate synthase. A mitochondria localization was shown in MP1 transfected NT2 and HepG2 cells. Deletion of the N-terminal 15 amino acids caused MP1 to be mislocalized to the cytoplasm and nucleus. Furthermore, when fused to green flourescent protein, this 15-amino acid N-terminal sequence directed the fusion protein to the mitochondria.
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PMID:Mammalian pitrilysin: substrate specificity and mitochondrial targeting. 1919 55

Agrobacterium tumefaciens is a plant pathogen that utilizes a type IV secretion system (T4SS) to transfer DNA and effector proteins into host cells. In this study we discovered that an alpha-crystallin type small heat-shock protein (alpha-Hsp), HspL, is a molecular chaperone for VirB8, a T4SS assembly factor. HspL is a typical alpha-Hsp capable of protecting the heat-labile model substrate citrate synthase from thermal aggregation. It forms oligomers in a concentration-dependent manner in vitro. Biochemical fractionation revealed that HspL is mainly localized in the inner membrane and formed large complexes with certain VirB protein subassemblies. Protein-protein interaction studies indicated that HspL interacts with VirB8, a bitopic integral inner membrane protein that is essential for T4SS assembly. Most importantly, HspL is able to prevent the aggregation of VirB8 fused with glutathione S-transferase in vitro, suggesting that it plays a role as VirB8 chaperone. The chaperone activity of two HspL variants with amino acid substitutions (F98A and G118A) for both citrate synthase and glutathione S-transferase-VirB8 was reduced and correlated with HspL functions in T4SS-mediated DNA transfer and virulence. This study directly links in vitro and in vivo functions of an alpha-Hsp and reveals a novel alpha-Hsp function in T4SS stability and bacterial virulence.
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PMID:The small heat-shock protein HspL is a VirB8 chaperone promoting type IV secretion-mediated DNA transfer. 2042 70

Insect pathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have an increasing role in the control of agricultural insect pests and vectors of human diseases. Many of the virulence factors are well studied but less is known of the metabolism of these fungi during the course of insect infection or saprobic growth. Here, we assessed enzyme activity and gene expression in the central carbon metabolic pathway, including isocitrate dehydrogenase, aconitase, citrate synthase, malate synthase (MLS) and isocitrate lyase (ICL), with particular attention to the glyoxylate cycle when M. anisopliae and B. bassiana were grown under various conditions. We observed that ICL and MLS, glyoxylate cycle intermediates, were upregulated during growth on 2-carbon compounds (acetate and ethanol) as well as in insect haemolymph. We fused the promoter of the M. anisopliae ICL gene (Ma-icl) to a marker gene (mCherry) and showed that Ma-icl was upregulated when M. anisopliae was grown in the presence of acetate. Furthermore, Ma-icl was upregulated when fungi were engulfed by insect haemocytes as well as during appressorium formation. Addition of the ICL inhibitor 3-nitroproprionate delayed conidial germination and inhibited appressorium formation. These results show that these insect pathogenic fungi have a flexible metabolism that includes the glyoxylate cycle as an integral part of germination, pathogenesis and saprobic growth.
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PMID:Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis. 2092 53

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a metabolic enzyme that catalyzes the oxidation of various aldehydes. Certain types of epithelial tissues in mammals, especially those continually exposed to environmental stress (e.g., corneal epithelium), express ALDH3A1 at high levels and its abundance in such tissues is perceived to help to maintain cellular homeostasis under conditions of oxidative stress. Metabolic as well as non-metabolic roles for ALDH3A1 have been associated with its mediated resistance to cellular oxidative stress. In this study, we provide evidence that ALDH3A1 exhibits molecular chaperone-like activity further supporting its multifunctional role. Specifically, we expressed and purified the human ALDH3A1 in E. coli and used the recombinant protein to investigate its in vitro ability to protect SmaI and citrate synthase (from precipitation and/or deactivation) under thermal stress conditions. Our results indicate that recombinant ALDH3A1 exhibits significant chaperone function in vitro. Furthermore, over-expression of the fused histidine-tagged ALDH3A1 confers host E. coli cells with enhanced resistance to thermal shock, while ALDH3A1 over-expression in the human corneal cell line HCE-2 was sufficient for protecting them from the cytotoxic effects of both hydrogen peroxide and tert-butyl hydroperoxide. These results further support the chaperone-like function of human ALDH3A1. Taken together, ALDH3A1, in addition to its primary metabolic role in fundamental cellular detoxification processes, appears to play an essential role in protecting cellular proteins against aggregation under stress conditions.
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PMID:Human aldehyde dehydrogenase 3A1 (ALDH3A1) exhibits chaperone-like function. 2852 14


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