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C Southan

Publications and source records attributed to C Southan.

At least 19 recordsLinked to original sources
PubMed2001

A genomic perspective on human proteases as drug targets.

Of the approximately 400 known human proteases, approximately 14% are under investigation as drug targets. Although the total is certain to rise during the finishing phase of the human genome project, the initial annotation of the approximately 30,000 human proteome set includes approximately 500 proteases. Bioinformatic analysis can now be performed on complete human protease families and will soon include comparisons with mice and fish. New sequences will require evaluation of their function in normal physiology and human disease. By revealing details such as splice variants and population polymorphisms, genomic sequence information will have a central role in the validation of protease drug targets.

Journal Article↗
PubMed2001

A genomic perspective on human proteases.

Over 400 human proteases documented in secondary databases can already be delineated in genomic sequence. A Genome Ontology annotation of 30585 sequences in the provisional human proteome set recognises 498 proteases, i.e. 1.6%. Homology searches against finished sequence and comparisons between mouse and zebrafish are likely to increase this total. However, the data already indicate that the mechanistic class, sequence family and domain distribution of the genomic complement of proteases is unlikely to shift significantly from that already observed in the transcript data. Genomically derived novel sequences will require bioinformatic analysis and biochemical verification. The increasing availability of annotated genomic data will enable studies of splice variants, transcriptional control, polymorphisms, pseudogenes, inactive homologues and evolution. Comparative work on complete human protease families should produce a more integrated picture of their biochemistry and physiology. Genomic data will also lead to the identification of new protease involvement in disease processes and their evaluation as drug targets.

Alternative Splicing↗
PubMed2001

Identification, genomic organization, and mRNA expression of LACTB, encoding a serine beta-lactamase-like protein with an amino-terminal transmembrane domain.

Database searching with bacterial serine beta-lactamases identified mouse expressed sequence tags (ESTs) with significant similarity scores.The cloned mouse cDNA encodes a novel 551-amino-acid protein, LACTB, with a predicted amino-terminal transmembrane domain but no signal peptide. It contains an active site motif related to C-class beta-lactamases. Homologues were detected in sequence data from human, rat, cow, rabbit, pig, toad, zebrafish, and Caenorhabditis elegans, but not in Saccharomyces cerevisiae or Drosophila melanogaster. The genes were mapped to human chromosome 15q22.1 and mouse chromosome 9. Sequencing of a 14.7-kb fragment of mouse genomic DNA defined six exons. A virtual human cDNA and a 549-residue protein, predicted from unfinished genomic sequence, showed the same intron/exon structure. Northern blot analysis showed expression of the 2.3-kb mRNA predominantly in mouse liver and human skeletal muscle. This is the first reported vertebrate example of this microbial peptidase family.

Amino Acid Sequence↗
PubMed2000

Sequencing, tissue distribution and chromosomal assignment of a novel ubiquitin-specific protease USP23.

We have identified human and mouse cDNAs encoding a novel ubiquitin-specific protease designated USP23. Both cDNAs encode a 62-kDa protein containing the highly conserved His and Cys domains characteristic of the C19 cysteine protease family of ubiquitin-specific processing proteases (UCH-2). Human tissue Northern blots revealed USP23 to be ubiquitously expressed, whereas USP12, its closest human paralogue, displayed a more restricted expression pattern. The human USP23 gene mapped to chromosome 1q22.

Amino Acid Sequence↗
PubMed2000

Website review: interPro (the integrated resource of protein domains and functional sites).

The family and motif databases, PROSITE, PRINTS, Pfam and ProDom, have been integrated into a powerful resource for protein secondary annotation. As of June 2000, InterPro had processed 384 572 proteins in SWISS-PROT and TrEMBL. Because the contributing databases have different clustering principles and scoring sensitivities, the combined assignments compliment each other for grouping protein families and delineating domains. The graphic displays of all matches above the scoring thresholds enables judgements to be made on the concordances or differences between the assignments. The website links can be used to analyse novel sequences and for queries across the proteomes of 32 organisms, including the partial human set, by domain and/or protein family. An analysis of selected HtrA/DegQ proteases demonstrates the utility of this website for detailed comparative genomics. Further information on the project can be found at the European Bioinformatics Institute at http://www.ebi.ac.uk/interpro/

Amino Acid Motifs↗
PubMed2000

Assessing the protease and protease inhibitor content of the human genome.

The revealing of the entire complement of protease and protease inhibitor sequences by the Human Genome Project will be of great importance to both academic and pharmaceutical research. Although the finishing phase is not yet complete, a selection of secondary annotation sources and comparisons with completed model organism genomes already allow useful estimates to be made. Conservative extrapolation suggests a total of approximately 1.8% for human proteases. This is close to the figures for yeast (1.7%) and worm (1.8%) but lower than the fly (3.4%) which has a large trypsin-like protease content. Using estimates for the human proteome of between 40,000 and 60,000 genes would extrapolate to 700-1,100 proteases, compared with approximately 360 currently represented as GenBank mRNAs. Preliminary comparisons between domain annotations for predicted human gene products and completed proteins suggest the genomic protease family and mechanistic class distributions will broadly reflect those in the current transcript data. The protease:inhibitor ratio at the mRNA level is currently approximately 9:1, but genome annotation data indicate that inhibitory domains are more widespread than this ratio would indicate.

Databases, Factual↗
PubMed2000

ASP1 (BACE2) cleaves the amyloid precursor protein at the beta-secretase site.

Sequential proteolytic processing of the Amyloid Precursor Protein (APP) by beta- and gamma-secretases generates the 4-kDa amyloid (A beta) peptide, a key component of the amyloid plaques seen in Alzheimer's disease (AD). We and others have recently reported the identification and characterisation of an aspartic proteinase, Asp2 (BACE), as beta-secretase. Here we describe the characterization of a second highly related aspartic proteinase, Asp1 as a second beta-secretase candidate. Asp1 is expressed in brain as detected at the mRNA level and at the protein level. Transient expression of Asp1 in APP-expressing cells results in an increase in the level of beta-secretase-derived soluble APP and the corresponding carboxy-terminal fragment. Paradoxically there is a decrease in the level of soluble A beta secreted from the cells. Asp1 colocalizes with APP in the Golgi/endoplasmic reticulum compartments of cultured cells. Asp1, when expressed as an Fc fusion protein (Asp1-Fc), has the N-terminal sequence ALEP..., indicating that it has lost the prodomain. Asp1-Fc exhibits beta-secretase activity by cleaving both wild-type and Swedish variant (KM/NL) APP peptides at the beta-secretase site.

Amyloid Precursor Protein Secretases↗
PubMed2000

Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response.

Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures. HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts. HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment. Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis. This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis. In human cell lines, it is present as two polypeptides of 38 and 40 kDa. HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E. coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C. The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

Alanine↗
PubMed1999

Disposable microbore high-pressure liquid chromatography columns for protein and peptide separations.

A range of high-performance liquid chromatography (HPLC) columns with internal diameters of 0.25 to 1.8 mm have been constructed by securing glass or plastic tubing into standard HPLC fittings. These were packed with chromatographic materials chosen for operation at moderate pressures with high flow rates. These columns were shown to be effective in a conventional HPLC instrument for peptide and protein separations in reverse-phase mode and for proteins in ion-exchange and size-exclusion modes. The simple construction and low cost of these microbore columns allow them to be considered as disposable. Using only small amounts of any type of packing material, they have the flexibility to be adapted to a wide range of analytical and micropreparative separations.

Chromatography, Gel↗
PubMed1998

Expression, purification and characterization of a human serine-dependent phospholipase A2 with high specificity for oxidized phospholipids and platelet activating factor.

Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.

Amino Acid Sequence↗
PubMed1996

Purification, properties, sequencing, and cloning of a lipoprotein-associated, serine-dependent phospholipase involved in the oxidative modification of low-density lipoproteins.

A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.

Amino Acid Sequence↗
PubMed1991

Separation by capillary electrophoresis followed by dynamic elution.

In this study we have explored the behaviour of peptides after capillary electrophoresis (CE) followed by elution under pressure. The use of D2O- rather than H2O-based buffer solutions appears to restrict the diffusion of peptides after CE, resulting in little loss of resolution when peptides are eluted by dynamic flow. In this paper we present results showing that a simple two-step process, involving CE at a low voltage, switching off the power supply, and connecting the fused capillary at the anode end to a syringe pump for dynamic flow, can retain separation characteristics and can be used for the isolation of picomole quantities of peptides for sequence determination.

Chromatography, High Pressure Liquid↗
PubMed1991

Analytical and micropreparative capillary electrophoresis of the peptides from calcitonin.

The capillary electrophoresis (CE) of peptide fragments from the tryptic digest of salmon calcitonin and elcatonin has been carried out in H2O- and D2O-based buffer solutions. Analysis in heavy water was found to be superior to that in H2O especially at a pH or pD of 7.93. From a single CE run on elcatonin digest we were also able to harvest three pure cleavage peptides in sufficient quantity to determine each amino acid residue by protein sequencing. The order of elution from CE agreed with that predicted on the basis of net charge calculated for each peptide.

Amino Acid Sequence↗
PubMed1990

Bovine dopamine beta-hydroxylase, primary structure determined by cDNA cloning and amino acid sequencing.

A cDNA clone encoding bovine dopamine beta-hydroxylase (DBH) has been isolated from bovine adrenal glands. The clone hybridizes to two oligonucleotide probes, one based on a previously reported active site peptide [DeWolf, W. E., Jr., et al. (1988) Biochemistry 27, 9093-9101] and the other based on the human DBH sequence [Lamouroux, A., et al. (1987) EMBO J. 6, 3931-3937]. The clone contains a 1.9-kb open reading frame that codes for the soluble form of bovine DBH, with the exception of the first six amino acids. Direct confirmation of 93% of the cDNA-derived sequence was obtained from cleavage peptides by protein sequencing and mass spectrometry. Differences were found between these two sequences at only two positions. Of the four potential N-linked carbohydrate attachment sites, two, Asn-170 and Asn-552, were shown to be partially and fully glycosylated, respectively. Within the 69% of the protein sequence confirmed by mass spectrometry, no other covalent modifications were detected.

Adrenal Glands↗
PubMed1990

Inactivation of dopamine beta-hydroxylase by p-cresol: evidence for a second, minor site of covalent modification at tyrosine 357.

p-Cresol is a mechanism-based inhibitor of bovine dopamine beta-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate: oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) (DBH) which covalently modifies a tyrosine at position 216 during inactivation (DeWolf, W.E., Jr., Carr, S.A., Varrichio, A., Goodhart, P.J., Mentzer, M.A., Roberts, G.D., Southan, C., Dolle, R.E. and Kruse, L.I. (1988) Biochemistry 27, 9093-9101). Here we report the recovery and characterization of additional minor peptides that are produced during the inactivation of DBH with p-[3H]cresol. Sequence and structural analysis of these peptides indicates tyrosine 357 as a second, minor site of modification.

Amino Acid Sequence↗
PubMed1990

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age.

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.

ATP Citrate (pro-S)-Lyase↗
PubMed1990

Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.

Amino Acid Sequence↗