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PubMed2004

PspG, a new member of the Yersinia enterocolitica phage shock protein regulon.

The Yersinia enterocolitica phage shock protein (Psp) system is induced when the Ysc type III secretion system is produced or when only the YscC secretin component is synthesized. Some psp null mutants have a growth defect when YscC is produced and a severe virulence defect in animals. The Y. enterocolitica psp locus is made up of two divergently transcribed cistrons, pspF and pspABCDycjXF. pspA operon expression is dependent on RpoN (sigma(54)) and the enhancer-binding protein PspF. Previous data indicated that PspF also controls at least one gene that is not part of the psp locus. In this study we describe the identification of pspG, a new member of the PspF regulon. Predicted RpoN-binding sites upstream of the pspA genes from different bacteria have a common divergence from the consensus sequence, which may be a signature of PspF-dependent promoters. The Y. enterocolitica pspG gene was identified because its promoter also has this signature. Like the pspA operon, pspG is positively regulated by PspF, negatively regulated by PspA, and induced in response to the production of secretins. Purified His(6)-PspF protein specifically interacts with the pspA and pspG control regions. A pspA operon deletion mutant has a growth defect when the YscC secretin is produced and a virulence defect in a mouse model of infection. These phenotypes were exacerbated by a pspG null mutation. Therefore, PspG is the missing component of the Y. enterocolitica Psp regulon that was previously predicted to exist.

Animals↗

PubMed1999

A novel 14-base-pair regulatory element is essential for in vivo expression of murine beta4-galactosyltransferase-I in late pachytene spermatocytes and round spermatids.

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.

Animals↗

PubMed1996

Complete sequence of the mitochondrial DNA in the sea urchin Arbacia lixula: conserved features of the echinoid mitochondrial genome.

The complete nucleotide sequence (15,719 nucleotides) of the mitochondrial DNA (mtDNA) from the sea urchin Arbacia lixula is presented. The comparison of gene arrangement between different echinoderm orders of the same class provides evidence that the gene organization is conserved within the same echinoderm class. The peculiarities of sea urchin mtDNA features, already described, are confirmed by the A. lixula mtDNA sequence. The comparison of the entire sequences of mtDNA among A. lixula, Paracentrotus lividus, and Strongylocentrotus purpuratus allowed us to detect peculiar features, common to the three sea urchin species, that can represent the molecular signature of the mt genome in the sea urchin group. Analysis of the nucleotide composition indicates that A. lixula mtDNA, in contrast with the mtDNA of other sea urchins, shows a bias in the use of T and tends to avoid the use of C, most evident in the neutral part of the molecule, such as the third codon positions. This observation indicates that the three sea urchin mtDNAs evolve under different mutation pressure. Analysis of the sequence evolution allowed us to confirm the phylogenetic tree. However, the absolute divergence time, calculated on the basis of paleontological estimates, largely diverged from the expected one.

Amino Acid Sequence↗

PubMed2006

Ancient polymorphism and functional variation in the primate MHC-DQA1 5' cis-regulatory region.

Precise regulation of MHC gene expression is critical to vertebrate immune surveillance and response. Polymorphisms in the 5' proximal promoter region of the human class II gene HLA-DQA1 have been shown to influence its transcriptional regulation and may contribute to the pathogenesis of autoimmune diseases. We investigated the evolutionary history of this cis-regulatory region by sequencing the DQA1 5' proximal promoter region in eight nonhuman primate species. We observed unexpectedly high levels of sequence variation and multiple strong signatures of balancing selection in this region. Specifically, the considerable DQA1 promoter region diversity was characterized by abundant shared (or trans-species) polymorphism and a pronounced lack of fixed differences between species. The majority of transcription factor binding sites in the DQA1 promoter region were polymorphic within species, and these binding site polymorphisms were commonly shared among multiple species despite evidence for negative selection eliminating a significant fraction of binding site mutations. We assessed the functional consequences of intraspecific promoter region diversity using a cell line-based reporter assay and detected significant differences among baboon DQA1 promoter haplotypes in their ability to drive transcription in vitro. The functional differentiation of baboon promoter haplotypes, together with the significant deviations from neutral sequence evolution, suggests a role for balancing selection in the evolution of DQA1 transcriptional regulation in primates.

Animals↗

PubMed2005

Evolutionary and experimental analyses of inorganic phosphate transporter PiT family reveals two related signature sequences harboring highly conserved aspartic acids critical for sodium-dependent phosphate transport function of human PiT2.

The mammalian members of the inorganic phosphate (P(i)) transporter (PiT) family, the type III sodium-dependent phosphate (NaP(i)) transporters PiT1 and PiT2, have been assigned housekeeping P(i) transport functions and are suggested to be involved in chondroblastic and osteoblastic mineralization and ectopic calcification. The PiT family members are conserved throughout all kingdoms and use either sodium (Na+) or proton (H+) gradients to transport P(i). Sequence logo analyses revealed that independent of their cation dependency these proteins harbor conserved signature sequences in their N- and C-terminal ends with the common core consensus sequence GANDVANA. With the exception of 10 proteins from extremophiles all 109 proteins analyzed carry an aspartic acid in one or both of the signature sequences. We changed either of the highly conserved aspartates, Asp28 and Asp506, in the N- and C-terminal signature sequences, respectively, of human PiT2 to asparagine and analyzed P(i) uptake function in Xenopus laevis oocytes. Both mutant proteins were expressed at the cell surface of the oocytes but exhibited knocked out NaP(i) transport function. Human PiT2 is also a retroviral receptor and we have previously shown that this function can be exploited as a control for proper processing and folding of mutant proteins. Both mutant transporters displayed wild-type receptor functions implying that their overall architecture is undisturbed. Thus the presence of an aspartic acid in either of the PiT family signature sequences is critical for the Na+-dependent P(i) transport function of human PiT2. The conservation of the aspartates among proteins using either Na+- or H+-gradients for P(i) transport suggests that they are involved in H+-dependent P(i) transport as well. Current results favor a membrane topology model in which the N- and C-terminal PiT family signature sequences are positioned in intra- and extracellular loops, respectively, suggesting that they are involved in related functions on either side of the membrane. The present data are in agreement with a possible role of the signature sequences in translocation of cations.

Amino Acid Sequence↗

PubMed2006

Dichloroacetate causes toxic neuropathy in MELAS: a randomized, controlled clinical trial.

OBJECTIVE: To evaluate the efficacy of dichloroacetate (DCA) in the treatment of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). BACKGROUND: High levels of ventricular lactate, the brain spectroscopic signature of MELAS, correlate with more severe neurologic impairment. The authors hypothesized that chronic cerebral lactic acidosis exacerbates neuronal injury in MELAS and therefore, investigated DCA, a potent lactate-lowering agent, as potential treatment for MELAS. METHODS: The authors conducted a double-blind, placebo-controlled, randomized, 3-year cross-over trial of DCA (25 mg/kg/day) in 30 patients (aged 10 to 60 years) with MELAS and the A3243G mutation. Primary outcome measure was a Global Assessment of Treatment Efficacy (GATE) score based on a health-related event inventory, and on neurologic, neuropsychological, and daily living functioning. Biologic outcome measures included venous, CSF, and 1H MRSI-estimated brain lactate. Blood tests and nerve conduction studies were performed to monitor safety. RESULTS: During the initial 24-month treatment period, 15 of 15 patients randomized to DCA were taken off study medication, compared to 4 of 15 patients randomized to placebo. Study medication was discontinued in 17 of 19 patients because of onset or worsening of peripheral neuropathy. The clinical trial was terminated early because of peripheral nerve toxicity. The mean GATE score was not significantly different between treatment arms. CONCLUSION: DCA at 25 mg/kg/day is associated with peripheral nerve toxicity resulting in a high rate of medication discontinuation and early study termination. Under these experimental conditions, the authors were unable to detect any beneficial effect. The findings show that DCA-associated neuropathy overshadows the assessment of any potential benefit in MELAS.

Action Potentials↗

PubMed2025

Deciphering Pseudoendocrine Sarcoma: A Clinicopathological, Molecular, and Epigenetic Study Suggesting Biological Links With Solid Pseudopapillary Neoplasm of the Pancreas.

Pseudoendocrine sarcoma (PS) is a recently described neoplasm of uncertain differentiation, characterized by recurrent CTNNB1 mutations, frequent paravertebral location, and a neuroendocrine-like histomorphology. In this study, we report the clinicopathologic, immunohistochemical, transcriptomic, and epigenetic findings of 12 PS cases. The tumors affected 7 men and 5 women with a median age of 66 years and were located in the paraspinal/paravertebral region (n = 11) and the thigh (n = 1). Median tumor size was 82 mm (range, 32-170 mm). Histologically, the tumors comprised sheets and nests of epithelioid-to-ovoid cells with uniform nuclei and speckled chromatin, frequently associated with extracellular hyaline globules and fibrovascular cords/septa. Uncommon findings included microcalcifications, myxoid stroma, pseudopapillary, pseudoglandular, microcystic or corded architecture, and lumen and rosette-like structures. Necrosis was absent, and mitotic activity was low. On immunohistochemistry, the tumors showed aberrant nuclear staining for beta-catenin (8/8) and expression of CD56 (7/7), S100 (8/8), desmin (2/6), and androgen receptor (1/4). Pankeratin (AE1/AE3), progesterone receptor, synaptophysin, chromogranin, and INSM1 were negative. All tested cases harbored CTNNB1 mutations. Using a customized cohort, methylation profiling revealed that PS formed a common cluster with solid pseudopapillary neoplasm of the pancreas (SPNP), distinct from all methylation classes from the Heidelberg sarcoma classifier and a subset of paragangliomas. Transcriptomic analysis showed that PS formed an independent cluster from a control group of tumors (including SPNP). Differential gene expression analysis showed enrichment in genes of the Wnt signaling pathway (HALLMARK gene sets) and biological processes related to sensory perception, among others (gene ontology - biological process [GO-BP]). Additionally, upregulated genes were related to various fetal cell types from the cell type signature data set (MSigDB), particularly of neuronal and epithelial lineage. Immunohistochemical assessment of potential markers identified through gene expression analysis revealed focal-to-diffuse expression of GLUT1 (6/6) and focal/multifocal expression of Brachyury (4/9) and HuD (3/7). Follow-up information, available for 10 cases (median duration of 18 months; range, 7-69 months), showed local recurrences and metastatic spread in 2 patients each. Evidence of response to radiotherapy was documented in one tumor. Altogether, this study expands knowledge on PS and suggests biological links with SPNP, including a potential shared cell of origin.

Humans↗

PubMed2006

Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.

Amyloid Precursor Protein Secretases↗

PubMed2004

Signature of the oligomeric behaviour of nuclear receptors at the sequence and structural level.

Nuclear receptors (NRs) are ligand-dependent transcription factors that control a large number of physiological events through the regulation of gene transcription. NRs function either as homodimers or as heterodimers with retinoid X receptor/ultraspiracle protein (RXR/USP). A structure-based sequence analysis aimed at discovering the molecular mechanism that controls the dimeric association of the ligand-binding domain reveals two sets of differentially conserved residues, which partition the entire NR superfamily into two classes related to their oligomeric behaviour. Site-directed mutagenesis confirms the functional importance of these residues for the dimerization process and/or transcriptional activity. All homodimers belong to class I, in which the related residues contribute a communication pathway of two salt bridges linking helix 1 on the cofactor-binding site to the dimer interface. A salt bridge involving a differentially conserved arginine residue in loop H8-H9 defines the signature motif of heterodimers. RXR/USP and all Caenorhabditis elegans NRs belong to class I, supporting the hypothesis of an earlier emergence of this class.

Amino Acid Motifs↗

PubMed1996

Cloning and sequencing of the LYS1 gene encoding homocitrate synthase in the yeast Yarrowia lipolytica.

The alpha-aminoadipate pathway for the biosynthesis of lysine is present only in fungi and euglena. The first step in the pathway is the condensation of acetyl-CoA and alpha-ketoglutarate into homocitrate, and this step is carried out by the enzyme homocitrate synthase (EC 4.1.3.21). In spite of extensive genetic analysis, no mutation affecting this step has been isolated until now in model organisms such as Saccharomyces cerevisiae or Neurospora crassa, although identification of mutations affecting the structural gene (LYS1) for homocitrate synthase was reported in the yeast Yarrowia lipolytica several years ago. Here we used these mutants for the cloning and sequencing of the Yarrowia LYS1 gene. The LYS1 gene encodes a predicted 446 amino acid polypeptide, with a molecular mass of 48442 Da. The Lys1p sequence displays two regions, one near the N-terminal section and the other in the central region, that contain conserved signatures found in prokaryotic homocitrate synthases (nifV genes of Azotobacter vinelandii and Klebsiella pneumoniae), as well as in all alpha-isopropyl malate synthases so far described. A putative mitochondrial targeting signal of 41-45 amino acids is predicted at the N-terminus. The Lys1p sequence shows 84% identity at the amino acid level with the putative product of open reading frame D1298 of S. cerevisiae. Northern blot hybridizations revealed a LYS1 transcript of approximately 1.7 kb in Y. lipolytica. Deletion of the LYS1 gene resulted in a Lys- phenotype. Our results indicate that we cloned the structural gene for homocitrate synthase in Y. lipolytica, and that the enzyme is encoded by a single gene in this yeast.

Amino Acid Sequence↗

PubMed2026

The machine-learning classifier ALLCatchR2 identifies 20 T-ALL subtypes across cohorts and age groups.

T-cell acute lymphoblastic leukemia (T-ALL) comprises molecularly diverse subtypes, but robust cross-cohort validations and operational gene-expression definitions are lacking. To establish a gene-expression-anchored framework for T-ALL subtyping, we aggregated 2314 transcriptomes (15 cohorts, age: 0.8-90.8 years). An extended unsupervised approach defined 17 main clusters and 3 subclusters in samples with high blast fractions. Supervised analyses added an overarching immature T-ALL (early T cell precursor [ETP]-like) definition and resolved the LMO2 γδ-like subtype. All clusters contained samples from at least two cohorts. Characteristic genomic driver enrichments were consistent across cohorts, while gene-expression clusters did not correspond exclusively to single driver events but also reflected developmental origins. A machine-learning classifier based on ALLCatchR, our B-cell acute lymphoblastic leukemia (B-ALL) classifier, identified these 20 transcriptomic subtypes and the immature T-ALL (ETP-like) signature with 0.995-1.0 accuracy in a validation set (n = 203). Testing the classifier on a second hold-out data set (n = 265 samples) showed that 92.7% of predictions matched with corresponding driver alterations. Across all samples, 83.2% of cases received high-confidence predictions, 7.3% candidate predictions, and 9.5% remained unclassified, largely because of low blast fractions. We identified a novel gene-expression cluster markedly enriched (P < 0.001) for clonal hematopoiesis mutations (IDH2 R140Q, DNMT3A) and a stem-/progenitor cell-like gene expression. This novel clonal hematopoiesis-related T-ALL subtype was observed in six cohorts and accounted for 8.9% of adults and 39.5% of patients aged >50 years. We extended ALLCatchR into ALLCatchR2, a free R package that now enables B-/T-lineage separation, gene-expression subtyping, blast estimation, and developmental annotation to harmonize T-ALL classification across studies and clinical contexts.

Journal Article↗

PubMed2002

Cloning and characterization of a human novel gene C9orf19 encoding a conserved putative protein with an SCP-like extracellular protein domain.

A novel human transcript, C9orf19, mapped to the genomic region involved in hereditary inclusion body myopathy (IBM2) at chromosome 9p12-p13, has been cloned and characterized. A single cDNA clone consisting of the full-length 1.9 kb transcript has been isolated from a human placenta cDNA library and further analyzed. Genomic characterization of the C9orf19 gene identified five exons extending over 27.2 kb of genomic DNA, located 12 kb centromeric to the tumor suppressor RECK gene. C9orf19 mRNA is expressed in a wide range of adult tissues as a single transcript, most abundantly in lung and peripheral blood leukocytes. The predicted protein contains the SCP-like extracellular protein signature classified to IPR001283, a family of evolutionary related proteins with extracellular domains, which includes the human glioma pathogenesis-related protein (GliPR), the human testis specific glycoprotein (TPX-1), and several other extracellular proteins from rodents (SCP), insects venom allergens (Ag5, Ag3), plants pathogenesis proteins (PR-1) and yeast hypothetical proteins. Homology searches with the deduced 154 amino acid protein sequence of C9orf19 revealed highly similar proteins in mouse, drosophila, nematode and yeast. Mutation analysis of C9orf19 in IBM2 patients excluded it as the disease causing gene and revealed four single nucleotide polymorphisms within and in the vicinity of the gene, which will certainly be useful tools to study its potential role in several human diseases mapped to chromosome 9p12-p13. Parallel to this study, the gene termed GNE, approximately 50 kb centromeric to C9orf19, was shown to be the disease causing gene in IBM2.

Amino Acid Sequence↗

PubMed2003

Evolution and variation of the SARS-CoV genome.

Knowledge of the evolution of pathogens is of great medical and biological significance to the prevention, diagnosis, and therapy of infectious diseases. In order to understand the origin and evolution of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus), we collected complete genome sequences of all viruses available in GenBank, and made comparative analyses with the SARS-CoV. Genomic signature analysis demonstrates that the coronaviruses all take the TGTT as their richest tetranucleotide except the SARS-CoV. A detailed analysis of the forty-two complete SARS-CoV genome sequences revealed the existence of two distinct genotypes, and showed that these isolates could be classified into four groups. Our manual analysis of the BLASTN results demonstrates that the HE (hemagglutinin-esterase) gene exists in the SARS-CoV, and many mutations made it unfamiliar to us.

Amino Acid Motifs↗

PubMed2006

A cadmium-transporting P1B-type ATPase in yeast Saccharomyces cerevisiae.

Detoxification and homeostatic acquisition of metal ions are vital for all living organisms. We have identified PCA1 in yeast Saccharomyces cerevisiae as an overexpression suppressor of copper toxicity. PCA1 possesses signatures of a P1B-type heavy metal-transporting ATPase that is widely distributed from bacteria to humans. Copper resistance conferred by PCA1 is not dependent on catalytic activity, but it appears that a cysteine-rich region located in the N terminus sequesters copper. Unexpectedly, when compared with two independent natural isolates and an industrial S. cerevisiae strain, the PCA1 allele of the common laboratory strains we have examined possesses a missense mutation in a predicted ATP-binding residue conserved in P1B-type ATPases. Consistent with a previous report that identifies an equivalent mutation in a copper-transporting P1B-type ATPase of a Wilson disease patient, the PCA1 allele found in laboratory yeast strains is nonfunctional. Overexpression or deletion of the functional allele in yeast demonstrates that PCA1 is a cadmium efflux pump. Cadmium as well as copper and silver, but not other metals examined, dramatically increase PCA1 protein expression through post-transcriptional regulation and promote subcellular localization to the plasma membrane. Our study has revealed a novel metal detoxification mechanism in yeast mediated by a P1B-type ATPase that is unique in structure, substrate specificity, and mode of regulation.

Adenosine Triphosphatases↗

PubMed1993

Structure and expression of the excision repair gene ERCC6, involved in the human disorder Cockayne's syndrome group B.

The human repair gene ERCC6--a presumed DNA (or RNA) helicase--has recently been found to function specifically in preferential nucleotide excision repair (NER). This NER subpathway is primarily directed towards repair of (the transcribed strand of) active genes. Mutations in the ERCC6 gene are responsible for the human hereditary repair disorder Cockayne's syndrome complementation group B, the most common form of the disease. In this report, the genomic organization and expression of this gene are described. It consists of at least 21 exons, together with the promoter covering a region of 82-90 kb on the genome. Postulated functional domains deduced from the predicted amino acid sequence, including 7 distinct helicase signatures, are--with one exception--encoded on separate exons. Consensus splice donor and acceptor sequences are present at all exon borders with the exception of the unusual splice donor at the end of exon VII. The 'invariable' GT dinucleotide in the consensus (C,A)AG/GTPuAGT is replaced by the exceptional GC. Based on 42 GC splice donor sequences identified by an extensive literature search we found a statistically highly significant better 'overall' match of the surrounding nucleotides to the consensus sequence compared to normal GT-sites. This confirms and extends the observation made recently by Jackson (Nucl. Acids Res., 19, 3795-3798 (1991)) derived from analysis of 26 cases. Analysis of ERCC6 cDNA clones revealed the occurrence of alternative polyadenylation, resulting in the (differential) expression of two mRNA molecules (which are barely detectable on Northern blots) of 5 and 7 kb in length.

Animals↗

PubMed2002

Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity.

In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins. One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine. Since production of this pigment is cryptic in the wild-type strain, E. chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant. These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM. Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis. No specific function could be assigned to IndA. In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS). The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS. These data suggest that glutamine is the precursor of indigoidine. We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated. Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis. DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions. The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E. chrysanthemi pathogenicity. Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha. Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.

Amino Acid Sequence↗

PubMed2002

Contribution of an active site cation-pi interaction to the spectroscopic properties and catalytic function of protein tyrosine kinase Csk.

Csk is a soluble protein tyrosine kinase that phosphorylates and negatively regulates protein tyrosine kinases of the Src family. The spectral properties of the intrinsic Trp fluorescence of Csk and their underlying structural features were investigated in combination with urea denaturation and site-specific mutagenesis. It was found that W352 contributed approximately 35% of the total Trp fluorescence of Csk even though seven other Trp residues were present. The enhanced contribution by W352 to Csk fluorescence was due to an interaction between its indole ring and the positively charged guanidino group of R318. W352 is located on the peptide substrate binding P+1 loop, and R318 is located on the catalytic loop. The W352-R318 interaction, called a cation-pi interaction, uniquely couples the two loops in the active site. Mutations that disrupted this coupling resulted in varying levels of decreases in Csk activity, and consistent and significant increases in K(m) values for its physiological substrate, Src protein tyrosine kinase. These results indicated that structural coupling between the two loops by the cation-pi interaction played an important role in Csk substrate binding. Since both R318 and W352 are highly conserved among protein tyrosine kinases, this cation-pi interaction is likely a signature structural feature of most, if not all, PTKs. These studies elucidated the roles of two conserved signature residues in Csk and formed a baseline for further structure-function studies of Csk and other PTKs.

Animals↗

PubMed1999

Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding.

Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of an N-methyl group from betaine to homocysteine to produce dimethylglycine and methionine, respectively. The enzyme is found in the pathway of choline oxidation and is abundantly expressed in liver and kidney. We have recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid purification of human BHMT for further physical and mechanistic studies, including characterizing its metal binding properties, we have overexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification by a self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system in conjunction with site-directed mutagenesis, we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of these Cys residues to Ala results in the complete loss of activity and a significant reduction in the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surrounding these Cys residues with similar amino acid sequences retrievable from protein databases, we have identified the following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be a signature for a family of zinc-dependent methyltransferases that utilize thiols or selenols as methyl acceptors. Some of the members of this family include the vitamin B(12)-dependent methionine synthases, E. coli S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.

Base Sequence↗