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PubMed2006

Gene expression profiling of acute myeloid leukemia with translocation t(8;16)(p11;p13) and MYST3-CREBBP rearrangement reveals a distinctive signature with a specific pattern of HOX gene expression.

Acute myeloid leukemia (AML) with translocation t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinicobiological features. This translocation leads to fusion of MYST3 (MOZ) and CREBBP (CBP) genes, probably resulting in a disturbed transcriptional program of a myelomonocytic precursor. Nonetheless, its gene expression profile is unknown. We have analyzed the gene expression profile of 23 AML patients, including three with molecularly confirmed MYST3-CREBBP fusion gene, using oligonucleotide U133A arrays (Affymetrix). MYST3-CREBBP cases clustered together and clearly differentiated from samples with PML-RARalpha, RUNX1-RUNX1T1, and CBFbeta-MYH11 rearrangements. The relative expression of 46 genes, selected according to their differential expression in the high-density array study, was analyzed by low-density arrays in an additional series of 40 patients, which included 7 MYST3-CREBBP AML cases. Thus, genes such as prolactin (PRL) and proto-oncogene RET were confirmed to be specifically overexpressed in MYST3-CREBBP samples whereas genes such as CCND2, STAT5A, and STAT5B were differentially underexpressed in this AML category. Interestingly, MYST3-CREBBP AML exhibited a characteristic pattern of HOX expression, with up-regulation of HOXA9, HOXA10, and cofactor MEIS1 and marked down-regulation of other homeobox genes. This profile, with overexpression of FLT3, HOXA9, MEIS1, AKR7A2, CHD3, and APBA2, partially resembles that of AML with MLL rearrangement. In summary, this study shows the distinctive gene expression profile of MYST3-CREBBP AML, with overexpression of RET and PRL and a specific pattern of HOX gene expression.

Acute Disease↗
PubMed2004

Identification and characterization of several new members of the ZIP family of metal ion transporters in Medicago truncatula.

To broaden our understanding of micronutrient metal transport in plants, we have identified cDNAs for six new metal transporters in the model legume Medicago truncatula. All of the predicted proteins have high similarity to the ZIP protein family, and have been designated MtZIP1, MtZIP3, MtZIP4, MtZIP5, MtZIP6, and MtZIP7. The six predicted proteins ranged from 350 to 372 amino acids in length; sequence analysis revealed that all proteins contained eight transmembrane domains and the highly conserved ZIP signature motif. Most of the proteins also exhibited a histidine-rich region in the variable sequence between transmembrane domains III and IV. When MtZIPs were transformed into appropriate metal-uptake defective yeast mutants and grown on metal-limited media, MtZIP1, MtZIP5, and MtZIP6 proteins restored yeast growth on Zn-limited media, MtZIP4 and MtZIP7 proteins restored yeast growth on Mn-limited media, and MtZIP3, MtZIP5, and MtZIP6 proteins restored yeast growth on Fe-limited media. Therefore, we conclude that these proteins function as metal transporters in Medicago truncatula. The expression pattern for each gene was studied by semi-quantitative RT-PCR in roots and leaves from plants grown under various metal supplies. MtZIP1 transcripts were only detected in Zn-deficient roots and leaves. MtZIP3 and MtZIP4 expression was down regulated in leaves from Mn- and Fe-deficient plants and appeared to be upregulated under Zn-deficient conditions in both roots and leaves. MtZIP5 was upregulated in leaves under Zn and Mn deficiency. The expression of MtZIP6 and MtZIP7 was unaffected by the metal supply, at least in root and leaf tissues. Characterizing these proteins in a single organism will allow us to understand the interplay between various ZIP genes, and the role they play in the regulation/execution of plant metal homeostasis.

Amino Acid Sequence↗
PubMed2026

Evolutionary dynamics of HIV-1 recombinants: analysis of contemporary and historical viral populations in East Africa.

BACKGROUND: Understanding the genetic evolution of HIV-1 Transmitted/Founder (T/F) virus is crucial for developing effective treatment and prevention strategies due to its rapid mutation and recombination rates. METHODS: This study compared the genetic diversity of 24 contemporary T/F viruses collected between 2016 and 2021 in Uganda and Kenya with 29 historical T/F sequences sampled between 2006 and 2011. RESULTS: Subtype analysis based on near-full-length (NFL) HIV-1 T/F genomes revealed that 57.1% (12/21) of contemporary viruses were recombinants, predominantly involving Subtype A1, D, and increasing Subtype C, with 33.3% (7/21) being A1D recombinants (A1 > D) and 19% (4/21) classified as complex recombinants involving three or more subtypes. Historical viruses showed a similar overall proportion (69%) but were mainly A1D mosaics (D > A1) with recombination confined primarily to the envelope region. In contrast, contemporary viruses shifted towards more complex recombinant patterns affecting additional genomic regions, including pol and accessory genes. Phylogenetic analysis demonstrated that contemporary viruses clustered into distinct, well-supported (98% bootstrap) sub-branches, suggesting divergency attributed to an imbalance in their proportions of subtype A1 and D sequences as well as a different content of A1 and D segments in the A1/D mosaic recombinants. CONCLUSIONS: These findings underscore the dynamic and shifting nature of HIV-1 genetic diversity in East Africa, highlighting the need for continuous molecular surveillance and region-specific treatment guidelines.

HIV-1↗
PubMed2002

Dominant-negative suppression of HCN1- and HCN2-encoded pacemaker currents by an engineered HCN1 construct: insights into structure-function relationships and multimerization.

I(f), a diastolic depolarizing current activated by hyperpolarization, is a key player in cardiac pacing. Despite the fact that I(f) has been known for over 20 years, the encoding genes, namely HCN1 to 4, have only recently been identified. Functional data imply that different HCN isoforms may coassemble to form heteromeric channel complexes, but little direct evidence is available. Subunit stoichiometry is also unknown. Although the pore region of HCN channels contains the glycine-tyrosine-glycine (GYG) signature motif found in K+-selective channels, they permeate both Na+ and K+. In the present study, we probed the functional importance of the GYG selectivity motif in pacemaker channels by replacing this triplet in HCN1 with alanines (GYG(349-351)AAA or HCN1-AAA). HCN1-AAA did not yield functional currents; coexpression of HCN1-AAA with wild-type (WT) HCN1 suppressed normal channel activity in a dominant-negative manner (55.2+/-3.2%, 68.3+/-4.3%, 78.7+/-1.6%, 91.7+/-0.8%, and 97.9+/-0.2% current reduction at -140 mV for WT:AAA cRNA ratios of 4:1, 3:1, 2:1, 1:1, and 1:2, respectively) without affecting gating (steady-state activation, activation and deactivation kinetics) or permeation (reversal potential) properties. HCN1-AAA coexpression, however, did not alter the expressed current amplitudes of Kv1.4 and Kv2.1 channels, indicating that its suppressive effect was channel-specific. Statistical analysis reveals that a single HCN channel is composed of 4 monomeric subunits. Interestingly, HCN1-AAA also inhibited HCN2 in a dominant-negative manner with the same efficacy. We conclude that the GYG motif is a critical determinant of ion permeation for HCN channels, and that HCN1 and HCN2 readily coassemble to form heterotetrameric complexes.

Amino Acid Motifs↗
PubMed2001

Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Amino Acid Sequence↗
PubMed2002

The Retinoic acid receptor alpha (RARalpha) chimeric proteins PML-, PLZF-, NPM-, and NuMA-RARalpha have distinct intracellular localization patterns.

Retinoic acid receptor alpha (RARalpha) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acute promyelocytic leukemia (APL). Disruption of RARalpha function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RARalpha expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RARalpha chimeric proteins, we engineered epitope-tagged versions of PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, and NuMA-RARalpha (X-RARalphaV5) and generated a panel of stable COS cell lines expressing X-RARalphaV5. Protein fractionation and Western analysis of these COS lines reveal that X-RARalpha proteins localize to both the cytoplasm and nucleus. NPM-RARalpha is predominantly nuclear whereas NuMA-RARalpha is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PML-RARalpha and PLZF-RARalpha share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RARalpha is also diffuse in the nucleus but, in contrast to PML-RARalpha and PLZF-RARalpha, is strongly associated with the nucleolus. Strikingly, NuMA-RARalpha predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RARalpha and NuMA-RARalpha, respectively. The distinct intracellular localization patterns and the shared ability of X-RARalpha to interact with their respective RARalpha partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis.

Animals↗
PubMed2003

Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing.

ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs) is a multidomain metalloproteinase belonging to the reprolysin family. The enzyme cleaves aggrecan core protein at several sites. Here we report that the non-catalytic ancillary domains of the enzyme play a major role in regulating aggrecanase activity, with the C-terminal spacer domain masking the general proteolytic activity. Expressing a series of domain deletion mutants in mammalian cells and examining their aggrecan-degrading and general proteolytic activities, we found that full-length ADAMTS-4 of 70 kDa was the most effective aggrecanase, but it exhibited little activity against the Glu(373)-Ala(374) bond, the site originally characterized as a signature of aggrecanase activity. Little activity was detected against reduced and carboxymethylated transferrin (Cm-Tf), a general proteinase substrate. However, it readily cleaved the Glu(1480)-Gly(1481) bond in the chondroitin sulfate-rich region of aggrecan. Of the constructed mutants, the C-terminal spacer domain deletion mutant more effectively hydrolyzed both the Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds. It also revealed new activities against Cm-Tf, fibromodulin, and decorin. Further deletion of the cysteine-rich domain reduced the aggrecanase activity by 80% but did not alter the activity against Cm-Tf or fibromodulin. Further removal of the thrombospondin type I domain drastically reduced all tested proteolytic activities, and very limited enzymatic activity was detected with the catalytic domain. Full-length ADAMTS-4 binds to pericellular and extracellular matrix, but deletion of the spacer domain releases the enzyme. ADAMTS-4 lacking the spacer domain has promiscuous substrate specificity considerably different from that previously reported for aggrecan core protein. Finding of ADAMTS-4 in the interleukin-1alpha-treated porcine articular cartilage primarily as a 46-kDa form suggests that it exhibits a broader substrate spectrum in the tissue than originally considered.

ADAM Proteins↗
PubMed2000

The 'KMSKS' motif in tyrosyl-tRNA synthetase participates in the initial binding of tRNA(Tyr).

Variants at each position of the 'KMSKS' signature motif in tyrosyl-tRNA synthetase have been analyzed to test the hypothesis that this motif is involved in catalysis of the second step of the aminoacylation reaction (i.e., the transfer of tyrosine from the enzyme-bound tyrosyl-adenylate intermediate to the tRNA(Tyr) substrate). Pre-steady-state kinetic studies show that while the rate constants for tyrosine transfer (k(4)) are similar to the wild-type value for all of the mobile loop variants, the K230A and K233A variants have increased dissociation constants (K(d)(tRNA)( )()= 2.4 and 1.7 microM, respectively) relative to the wild-type enzyme (K(d)(tRNA)( )()= 0.39 microM). In contrast, the K(d)(tRNA) values for the F231L, G232A, and T234A variants are similar to that of the wild-type enzyme. The K(d)(tRNA) value for a loop deletion variant, Delta(227-234), is similar to that for the K230A/K233A double mutant variant (3.4 and 3.0 microM, respectively). Double mutant free energy cycle analysis indicates there is a synergistic interaction between the side chains of K230 and K233 during the initial binding of tRNA(Tyr) (DeltaDeltaG(int) = -0.74 kcal/mol). These results suggest that while the 'KMSKS' motif is important for the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase, it does not play a catalytic role in the second step of the reaction. These studies provide the first kinetic evidence that the 'KMSKS' motif plays a role in the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1995

Investigating the structural determinants of the p21-like triphosphate and Mg2+ binding site.

Amongst the superfamily of nucleotide binding proteins, the classical mononucleotide binding fold (CMBF), is the one that has been best characterized structurally. The common denominator of all the members is the triphosphate/Mg2+ binding site, whose signature has been recognized as two structurally conserved stretches of residues: the Kinase 1 and 2 motifs that participate in triphosphate and Mg2+ binding, respectively. The Kinase 1 motif is borne by a loop (the P-loop), whose structure is conserved throughout the whole CMBF family. The low sequence similarity between the different members raises questions about which interactions are responsible for the active structure of the P-loop. What are the minimal requirements for the active structure of the P-loop? Why is the P-loop structure conserved despite the diverse environments in which it is found? To address this question, we have engineered the Kinase 1 and 2 motifs into a protein that has the CMBF and no nucleotide binding activity, the chemotactic protein from Escherichia coli, CheY. The mutant does not exhibit any triphosphate/Mg2+ binding activity. The crystal structure of the mutant reveals that the engineered P-loop is in a different conformation than that found in the CMBF. This demonstrates that the native structure of the P-loop requires external interactions with the rest of the protein. On the basis of an analysis of the conserved tertiary contacts of the P-loop in the mononucleotide binding superfamily, we propose a set of residues that could play an important role in the acquisition of the active structure of the P-loop.

Amino Acid Sequence↗
PubMed2004

Shared transcriptional signature in Caenorhabditis elegans Dauer larvae and long-lived daf-2 mutants implicates detoxification system in longevity assurance.

In the nematode Caenorhabditis elegans, formation of the long-lived dauer larva and adult aging are both controlled by insulin/insulin-like growth factor-1 signaling. Potentially, increased adult life span in daf-2 insulin/insulin-like growth factor-1 receptor mutants results from mis-expression in the adult of a dauer larva longevity program. By using oligonucleotide microarray analysis, we identified a dauer transcriptional signature in daf-2 mutant adults. By means of a nonbiased statistical approach, we identified gene classes whose expression is altered similarly in dauers and daf-2 mutants, which represent potential determinants of life span. These include known determinants of longevity; the small heat shock protein/alpha-crystallins are up-regulated in both milieus. The cytochrome P450, short-chain dehydrogenase/reductase, UDP-glucuronosyltransferase, and glutathione S-transferase (in daf-2 mutants) gene classes were also up-regulated. These four gene classes act together in metabolism and excretion of toxic endobiotic and xenobiotic metabolites. This suggests that diverse toxic lipophilic and electrophilic metabolites, disposed of by phase 1 and phase 2 drug metabolism, may be the major determinants of the molecular damage that causes aging. In addition, we observed down-regulation of genes linked to nutrient uptake, including nhx-2 and pep-2. These work together in the uptake of dipeptides in the intestine, implying dietary restriction in daf-2 mutants. Some gene groups up-regulated in dauers and/or daf-2 were enriched for certain promoter elements as follows: the daf-16-binding element, the heat shock-response element, the heat shock-associated sequence, or the hif-1-response element. By contrast, the daf-16-associated element was enriched in genes down-regulated in dauers and daf-2 mutants. Thus, particular promoter elements appear longevity-associated or aging associated.

Aging↗
PubMed2004

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.

Adaptation, Physiological↗
PubMed2000

Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature-tagged mutagenesis.

Group B streptococcal (GBS) infections are the most common cause of bacterial sepsis in the immediate newborn period. Apart from the capsule, the factors required for survival of GBS in the host are not well defined. In this study, signature-tagged transposon mutagenesis (STM) was used to identify genes required for growth and survival of GBS in a neonatal rat sepsis infection model. Approximately 1600 transposon mutants were screened in pools of 80 mutants, and approximately 120 mutants defective for survival in the animal host were identified. We successfully cloned and sequenced DNA flanking the transposon insertions from 92 of the mutants. Fifty per cent of the mutants had transposon insertions in genes with homologues in the public databases, whereas the remaining 50% had transposon insertions in genes with unknown function. A significant proportion of the avirulent mutants had transposon insertions in genes encoding transport-associated or regulatory proteins or in genes involved in cell surface metabolism, emphasizing the significance of these functions for in vivo survival of GBS. Overall, STM analysis revealed GBS genomic loci that encode a wide variety of functional gene classes, underscoring the diversity of bacterial processes required for the infection process. Currently, the function of the genes identified during the screening can only be inferred by homology to previously described genes. However, a number of the genes identified in this study have been shown to correlate with virulence in other pathogens. A virulence of a subset of mutants identified during the screening was confirmed by performing competitive index assays and lethal dose assays. This represents the first report of a genome-wide scan for virulence factors in GBS. The identified genes will further our understanding of the pathogenesis of GBS infections and may represent targets for intervention or lead to the development of novel therapies.

Animals↗
PubMed2005

Microarray-based detection of Salmonella enterica serovar Typhimurium transposon mutants that cannot survive in macrophages and mice.

DNA microarrays provide an opportunity to combine the principles of signature-tagged mutagenesis (STM) with microarray technology to identify potentially important bacterial virulence genes. The scope of DNA microarrays allows for less laborious screening on a much larger scale than possible by STM alone. We have adapted a microarray-based transposon tracking strategy for use with a Salmonella enterica serovar Typhimurium cDNA microarray in order to identify genes important for survival and replication in RAW 264.7 mouse macrophage-like cells or in the spleens of BALB/cJ mice. A 50,000-CFU transposon library of S. enterica serovar Typhimurium strain SL1344 was serially passaged in cultured macrophages or intraperitoneally inoculated into BALB/cJ mice. The bacterial genomic DNA was isolated and processed for analysis on the microarray. The novel application of this approach to identify mutants unable to survive in cultured cells resulted in the identification of components of Salmonella pathogenicity island 2 (SPI2), which is known to be critical for intracellular survival and replication. In addition, array results indicated that a number of SPI1-associated genes, currently not associated with intracellular survival, are negatively selected. However, of the SPI1-associated mutants individually tested for intracellular survival, only a sirA mutant exhibited reduced numbers relative to those of wild-type bacteria. Of the mutants unable to survive in mice, significant proportions are either components of the SPI2 pathogenicity island or involved in lipopolysaccharide synthesis. This observation is in agreement with results obtained in the original S. enterica serovar Typhimurium STM screen, illustrating the utility of this approach for the high-throughput identification of virulence factors important for survival in the host.

Animals↗
PubMed2002

Involvement of Lsp, a member of the LraI-lipoprotein family in Streptococcus pyogenes, in eukaryotic cell adhesion and internalization.

Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the mga core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of Streptococcus pyogenes) and ORF2 exhibited a high degree of homology to the lmb/ORF2 genes of S. agalactiae (B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic lsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase. lsp and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn(2+) and Cu(2+) ion concentrations decreased lsp operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The lsp mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the lsp mutant showed decreased transcription of prtF2 encoding the fibronectin-binding protein F2. Both the lsp and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the lsp mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of speB (cysteine protease) and/or the global regulators mga, csrRS, and nra.

Bacterial Proteins↗
PubMed2004

Cell cycle-mediated regulation of smooth muscle alpha-actin gene transcription in fibroblasts and vascular smooth muscle cells involves multiple adenovirus E1A-interacting cofactors.

Expression of smooth muscle alpha-actin in growth factor-induced myofibroblasts and in differentiated vascular smooth muscle cells is transcriptionally controlled by multiple positive or negative trans-acting factors interacting with distinct cis-elements in the 5'-flanking region of the gene. Because none of the transcriptional regulators reported to date is smooth muscle cell- or myofibroblast-specific per se, the dynamic interplay among many factors interacting at specific sites along the promoter appears to be a signature feature of smooth muscle alpha-actin gene regulation in these cell types. Herein, the ability of the adenovirus E1A 12 S protein to bind and functionally inactivate specific cell regulatory factors has been exploited to identify several previously unknown coactivators of the mouse smooth muscle alpha-actin promoter in rodent fibroblasts and vascular smooth muscle cells. In transient cotransfection assays, ectopic expression of wild type E1A suppressed promoter activity in a dose- and cis-element-dependent manner. In asynchronous cells, N-terminal E1A mutants defective in CREB-binding protein (CBP) and p300 binding capacity exhibited markedly reduced inhibitory activity toward a smooth muscle alpha-actin promoter driven by a composite TEF-1-, SRF-, and Sp1/3-regulated enhancer. In synchronized cells, however, a more complex mutant E1A inhibitory pattern indicated that collaboration between CBP/p300 and the retinoblastoma family of pocket proteins was required to produce a fully functional enhancer. Cotransfection experiments conducted with Rb(-/-) fibroblasts demonstrated the necessity of pRB in augmenting smooth muscle alpha-actin enhancer/promoter activity. Physical interaction studies with the use of purified wild type and mutant E1A proteins confirmed that CBP, p300, and pRB were targets of E1A binding in nuclear extracts of vascular smooth muscle cells and/or fibroblasts. Collectively, these results suggest that a repertoire of E1A-interacting proteins, namely CBP/p300 and pRB, serve to integrate the activities of multiple trans-acting factors to control smooth muscle alpha-actin gene transcription in a cell type- and cell cycle-dependent manner.

Actins↗
PubMed1997

Structure-function relationships within the peptide deformylase family. Evidence for a conserved architecture of the active site involving three conserved motifs and a metal ion.

Thermus thermophilus peptide deformylase was characterized. Its enzymatic properties as well as its organization in domains proved to share close resemblances with those of the Escherichia coli enzyme despite few sequence identities. In addition to the HEXXH signature sequence of the zinc metalloprotease family, a second short stretch of strictly conserved amino acids was noticed, EGCLS, the cysteine of which corresponds to the third zinc ligand. The study of site-directed mutants of the E. coli deformylase shows that the residues of this stretch are crucial for the structure and/or catalytic efficiency of the active enzyme. Both aforementioned sequences were used as markers of the peptide deformylase family in protein sequence databases. Seven sequences coming from Haemophilus influenzae, Lactococcus lactis, Bacillus stearothermophilus, Mycoplasma genitalium, Mycoplasma pneumoniae, Bacillus subtilus and Synechocystis sp. could be identified. The characterization of the product of the open reading frame from B. stearothermophilus confirmed that it actually corresponded to a peptide deformylase with properties similar to those of the E. coli enzyme. Alignment of the nine peptide deformylase sequences showed that, in addition to the two above sequences, only a third one, GXGXAAXQ, is strictly conserved. This motif is also located in the active site according to the three-dimensional structure of the E. coli enzyme. Site-directed variants of E. coli peptide deformylase showed the involvement of the corresponding residues for maintaining an active and stable enzyme. Altogether, these data allow us to propose that the three identified conserved motifs of peptide deformylases build up the active site around a metal ion. Finally, an analysis of the location of the other conserved residues, in particular of the hydrophobic ones, was performed using the three-dimensional model of the E. coli enzyme. This enables us to suggest that all bacterial peptide deformylases adopt a constant overall tertiary structure.

Amidohydrolases↗
PubMed2003

In vivo functional genomics of Pseudomonas aeruginosa for high-throughput screening of new virulence factors and antibacterial targets.

Pseudomonas aeruginosa is a model for studying opportunistic pathogens that are highly resistant to most classes of antibiotics and cause chronic pulmonary infections. We have developed and adapted a multiplex polymerase chain reaction-based signature-tagged mutagenesis (STM) for high-throughput screening of a collection of 7968 P. aeruginosa mutants in a rat model of chronic respiratory infection. After three rounds of screening, a total of 214 mutants, representing transposition events into 148 open reading frames, were shown to be attenuated in lung infection and were retained for further analysis. As proof of concept supporting this technology, we identified 11 insertions in typical virulence genes such as those coding for pili implicated in motility, attachment and swarming, alginate synthesis and its expression, a mucus transcription regulator, extracellular enzymes such as alkaline protease, esterase and amino peptidase, a rhamnosyl surfactant transferase and a lipopolysaccharide glycosyl transferase. Detailed analysis of the 148 STM mutants, including seven auxotrophs, revealed insertions in 21 of the 26 known gene classes used to characterize sequenced bacterial genomes. We noted that at least 46% of STM mutants identified had insertions in hypothetical proteins or proteins of unknown function and that approximately 40% of all STM mutants had insertions in surface proteins including the outer membrane, the periplasm and the inner membrane. Interestingly, 11 STM mutants attenuated for lung infection were also identified in microarray and transcriptome for quorum sensing and mucoidy production. The remaining 130 mutants were systematically analysed for their capability to express fully known virulence factors. In addition, testing the ability of these mutants to infect alternative model host Drosophila melanogaster revealed 36 STM mutants defective in protease, twitching motility, swimming and swarming. Finally, we identified many genes, the activity of which in respiratory infection was not fully appreciated.

Alkaline Phosphatase↗
PubMed2006

The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells.

Deregulation of the cell cycle is a common strategy employed by many DNA and RNA viruses to trap and exploit the host cell machinery toward their own benefit. In many coronaviruses, the nucleocapsid protein (N protein) has been shown to inhibit cell cycle progression although the mechanism behind this is poorly understood. The N protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) bears signature motifs for binding to cyclin and phosphorylation by cyclin-dependent kinase (CDK) and has recently been reported by us to get phosphorylated by the cyclin-CDK complex (Surjit, M., Kumar, R., Mishra, R. N., Reddy, M. K., Chow, V. T., and Lal, S. K. (2005) J. Virol. 79, 11476-11486). In the present study, we prove that the N protein of SARS-CoV can inhibit S phase progression in mammalian cell lines. N protein expression was found to directly inhibit the activity of the cyclin-CDK complex, resulting in hypophosphorylation of retinoblastoma protein with a concomitant down-regulation in E2F1-mediated transactivation. Coexpression of E2F1 under such conditions could restore the expression of S phase genes. Analysis of RXL and CDK phosphorylation mutant N protein identified the mechanism of inhibition of CDK4 and CDK2 activity to be different. Whereas N protein could directly bind to cyclin D and inhibit the activity of CDK4-cyclin D complex; inhibition of CDK2 activity appeared to be achieved in two different ways: indirectly by down-regulation of protein levels of CDK2, cyclin E, and cyclin A and by direct binding of N protein to CDK2-cyclin complex. Down-regulation of E2F1 targets was also observed in SARS-CoV-infected VeroE6 cells. These data suggest that the S phase inhibitory activity of the N protein may have major significance during viral pathogenesis.

Animals↗