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PubMed2006

Derivation of primary sequences and secondary structures of rev responsive element from HIV-1 infected mothers and infants following vertical transmission.

We have characterized the primary RRE sequences of HIV-1, including in vivo genetic variation and functional motifs required for Rev-RRE interactions as well as evaluated the RNA secondary structures of RRE derived from five mother-infant pairs following vertical transmission. Multiple (157) RRE sequences derived from mother-infant pairs showed that primary nucleotide sequences of RRE were highly conserved with a low degree of viral heterogeneity following vertical transmission. We found that the RRE sequences from mothers and infants folded and retained all the essential stem-loop formation required for Rev-RRE interactions. More importantly, a primary 9-nucleotide (5'-CACTATGGG-3') RRE sequence in the stem-loop B that is required for optimal Rev recognition and must be presented as a stem-bulge-stem structure was highly conserved in most of the sequences. The domains required for RRE-host protein interactions were also conserved in most of the RRE sequences. Taken together, the primary RRE sequences in the context of secondary structures were maintained and the Rev-RRE interaction domains were conserved following vertical transmission, which is consistent with a crucial role of RRE in HIV-1 pathogenesis.

Adult↗
PubMed2003

Identification of residues and domains of Raf important for function in vivo and in vitro.

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Alleles↗
PubMed1995

Down syndrome-critical region contains a gene homologous to Drosophila sim expressed during rat and human central nervous system development.

Many features of Down syndrome might result from the overdosage of only a few genes located in a critical region of chromosome 21. To search for these genes, cosmids mapping in this region were isolated and used for trapping exons. One of the trapped exons obtained has a sequence very similar to part of the Drosophila single-minded (sim) gene, a master regulator of the early development of the fly central nervous system midline. Mapping data indicated that this exonic sequence is only present in the Down syndrome-critical region in the human genome. Hybridization of this exonic sequence with human fetal kidney poly(A)+ RNA revealed two transcripts of 6 and 4.3 kb. In situ hybridization of a probe derived from this exon with human and rat fetuses showed that the corresponding gene is expressed during early fetal life in the central nervous system and in other tissues, including the facial, skull, palate, and vertebra primordia. The expression pattern of this gene suggests that it might be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome.

Amino Acid Sequence↗
PubMed1998

Expression of Id1 results in apoptosis of cardiac myocytes through a redox-dependent mechanism.

We have constructed a recombinant adenovirus (Ad.Id1) that allows for efficient expression of the helix-loop-helix protein Id1. After infection with Ad.Id1, neonatal cardiac myocytes display a significant reduction in viability, which was proportional to the level of Id1 expression. A similar effect was observed in adult myocytes. Morphological and biochemical assays demonstrated that Id1 expression resulted in myocyte apoptosis. In contrast, expression of Id1 in endothelial cells, vascular smooth muscle cells, or fibroblasts did not affect the viability of these cells. Along with the induction of apoptosis, the expression of Id1 in neonatal cardiac myocytes resulted in an increase in the level of intracellular reactive oxygen species. The source of these reactive oxygen species appears to be the mitochondria. Reducing the ambient oxygen concentration or treatment with a cell-permeant H2O2 scavenger prevented Id1-stimulated apoptosis in cardiac myocytes. These results suggest that the expression of Id1 leads to the induction of apoptosis in cardiac myocytes through a redox-dependent mechanism.

Adenoviridae↗
PubMed2000

The basic helix-loop-helix-PAS protein MOP9 is a brain-specific heterodimeric partner of circadian and hypoxia factors.

PAS (PER, ARNT, SIM) proteins play important roles in adaptation to low atmospheric and cellular oxygen levels, exposure to certain environmental pollutants, and diurnal oscillations in light and temperature. In an attempt to better understand how organisms sense environmental changes, we have characterized a novel member of the PAS superfamily, MOP9 (member of PAS superfamily), that maps to human chromosome 12p11.22-11.23. This protein displays significant homology to the Drosophila circadian factor CYCLE and its putative mammalian ortholog MOP3/bMAL1. Like its homologs, MOP9 forms a transcriptionally active heterodimer with the circadian CLOCK protein, the structurally related MOP4, and hypoxia-inducible factors, such as HIF1alpha. In a manner consistent with its role as a biologically relevant partner of these proteins, MOP9 is coexpressed in regions of the brain such as the thalamus, hypothalamus, and amygdala. Importantly, MOP9 is coexpressed with CLOCK in the suprachiasmatic nucleus, the site of the master circadian oscillator in mammals.

ARNTL Transcription Factors↗
PubMed1997

Design of short external guide sequences (EGSs) for cleavage of target molecules with RNase P.

The minimal substrate for human RNase P consists of the 5' leader sequence, aminoacyl acceptor stem, T-stem and T-loop of tRNA. The sequences corresponding to the D-stem, anticodon stem and loop and variable loop are replaced by a bulge which can be as small as 1 nt, but requires > 4 nt for optimal cleavage by RNase P. We found that a trans construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA while the other strand serves as an External Guide Sequence (EGS). We were also able to delete the nucleotides corresponding to nt 58 to 60 in the T-loop without affecting cleavage of the substrate. We propose that the sequence UUCG or UUCA (nucleotide 55 to 57 in the T-loop) positioned 3' to a double helical region of 12 to 13 basepairs containing a bulge of > 4 nt can form a structure that is recognized by human RNase P. The four nucleotides UUCR probably form a structure that resembles the uridine turn in the Tloop of tRNA. Since recognition by RNase P seems to be independent of the helical sequence, we suggest that this motif can be used for targeting RNA molecules for EGS-directed cleavage by RNase P. Based on these results, several 13-mer EGSs targeted to the 2.1 Kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1 Kb HBV transcript. Some of these were capable of inducing cleavage of the HBV RNA by RNase P. The use of such small EGSs for the inactivation of various genes will be discussed.

Base Sequence↗
PubMed1994

A transcriptional regulatory element common to a large family of hepatic cytochrome P450 genes is a functional binding site of the orphan receptor HNF-4.

Hepatic cytochrome P450 (CYP) genes, including members of CYP1 to CYP4 families, comprise the majority of the CYP gene superfamily. Previous study has demonstrated that HepG2-specific transcriptional activation of two CYP2C genes was dependent on a common element that bound a HepG2 nuclear protein designated HPF-1 (Venepally, P., Chen, D., and Kemper, B. (1992) J. Biol. Chem. 267, 17333-17338). This cis-acting element is highly homologous to the hepatocyte nuclear factor 4 (HNF-4) binding motif and is present in the promoters of more than 20 other CYP2 genes. To investigate the relationship between HPF-1 and HNF-4, we have compared their tissue distribution, DNA binding, and immunochemical characteristics, as well as transcriptional activity of their recognition elements. DNase I footprint analyses and gel-shift assays indicated that HPF-1, like HNF-4, was present in liver and kidney, but not brain and spleen. Both factors bound to either the HPF-1 site in the CYP2C2 promoter or an HNF-4 site in the human apolipoprotein CIII promoter. These complexes could be "supershifted" by an antiserum specific for HNF-4. When the sequence of the HPF-1 site in the CYP2C2 promoter was changed to that of the apolipoprotein CIII HNF-4 site, comparable transcriptional activities were obtained with the wild-type promoter and the HNF-4 mutant in transfected HepG2 cells. Cotransfection of HNF-4 with CYP2C2 promoter-luciferase constructs in COS-1 cells indicated that HNF-4 could trans-activate the promoters containing the HPF-1 site. These results indicate that the HPF-1 motif is a functional HNF-4-binding site, and the common immunological properties indicate that HPF-1 and HNF-4 are closely related and possibly identical. HNF-4, therefore, may act as a common regulator for the liver-specific expression of many CYP2 genes.

Animals↗
PubMed1999

The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity.

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.

Amino Acid Sequence↗
PubMed1999

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.

Acinetobacter calcoaceticus↗
PubMed1998

CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage.

The cellular tropism of human immunodeficiency virus type 1 (HIV-1) is dependent on utilization of specific chemokine co-receptor: macrophage-tropic/non-syncytium-inducing (NSI) viruses use CCR5, whereas T-cell tropic/syncytium-inducing (SI) viruses preferentially use CXCR4. We have analyzed co-receptor usage of 24 phylogenetically distinct primary HIV-1 isolates representing group M (clades A-F) and group O with known SI and NSI phenotype, using lymphocytes from donor with nonfunctional CCR5 (CCR5-/-; homozygous 32-bp deletion). While all SI isolates infected CCR5-/- lymphocytes (and hence do not require CCR5 for viral entry), all NSI isolates, regardless of clade, did not infect CCR5-/- lymphocytes. Thus, CCR5 expression is required for infection with NSI isolates and the CCR5 usage is independent of viral genotype. To localize the viral determinant involved in CCR5 binding, the V3 sequences across the clades were aligned based on the CCR5 usage. There were conserved uncharged residues at position 11 of V3 (mostly serine/glycine) and negatively charged residues at residue 25 (mostly glutamic/aspartic acid) among all isolates that used CCR5, whereas substitution with arginine or glutamine at these two positions led to usage of a co-receptor other than CCR5. This analysis led us to identify a consensus motif S/GXXXGPGXXXXXXXE/D within the V3 loop that predicts CCR5 co-receptor usage. Most isolates, with exception of one isolate, containing the conserved motif and predicted to utilize CCR5 indeed had an absolute requirement of CCR5 expression for infectibility. Site-directed mutagenesis in the infectious molecular clone further confirmed these results. Taken together, these data provide evidence that sequences within the V3 loop provide important residues that might be directly or indirectly involved in binding to a CCR5 co-receptor.

Amino Acid Sequence↗
PubMed1998

Novel modified tripeptide inhibitors of alpha 4 beta 7 mediated lymphoid cell adhesion to MAdCAM-1.

MAdCAM-1 specifically binds the lymphocyte integrin alpha 4 beta 7 and participates in the homing of leukocytes to intestinal mucosal sites. The LDT sequence located on the CD loop of MAdCAM-1 is an important binding site for MAdCAM-1/alpha 4 beta 7 interactions. N-Terminus acylation of the LDT motif and modification of the C-terminus carboxamide with amines led to low micromolar MAdCAM-1 inhibitors.

Cell Adhesion↗
PubMed2000

The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis.

The release factor eRF1 terminates protein biosynthesis by recognizing stop codons at the A site of the ribosome and stimulating peptidyl-tRNA bond hydrolysis at the peptidyl transferase center. The crystal structure of human eRF1 to 2.8 A resolution, combined with mutagenesis analyses of the universal GGQ motif, reveals the molecular mechanism of release factor activity. The overall shape and dimensions of eRF1 resemble a tRNA molecule with domains 1, 2, and 3 of eRF1 corresponding to the anticodon loop, aminoacyl acceptor stem, and T stem of a tRNA molecule, respectively. The position of the essential GGQ motif at an exposed tip of domain 2 suggests that the Gln residue coordinates a water molecule to mediate the hydrolytic activity at the peptidyl transferase center. A conserved groove on domain 1, 80 A from the GGQ motif, is proposed to form the codon recognition site.

Amino Acid Sequence↗
PubMed1999

High-level variability in the ORF-K1 membrane protein gene at the left end of the Kaposi's sarcoma-associated herpesvirus genome defines four major virus subtypes and multiple variants or clades in different human populations.

Infection with Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is common in certain parts of Africa, the Middle East, and the Mediterranean, but is rare elsewhere, except in AIDS patients. Nevertheless, HHV8 DNA is found consistently in nearly all classical, endemic, transplant and AIDS-associated KS lesions as well as in some rare AIDS-associated lymphomas. The concept that HHV8 genomes fall into several distinct subgroups has been confirmed and refined by PCR DNA sequence analysis of the ORF-K1 gene encoding a highly variable glycoprotein related to the immunoglobulin receptor family that maps at the extreme left-hand end of the HHV-8 genome. Among more than 60 different tumor samples from the United States, central Africa, Saudi Arabia, Taiwan, and New Zealand, amino acid substitutions were found at a total of 62% of the 289 amino acid positions. These variations defined four major subtypes and 13 distinct variants or clades similar to those found for the HIV ENV protein. The B and D subtype ORF-K1 proteins differ from the A and C subtypes by 30 and 24%, respectively, whereas A and C differ from each other by 15%. In all cases tested, multiple samples from the same patient were identical. Examples of the B subtype were found almost exclusively in KS patients from Africa or of African heritage, whereas the rare D subtypes were found only in KS patients of Pacific Island heritage. In contrast, C subtypes were found predominantly in classic KS and in iatrogenic and AIDS KS in the Middle East and Asia, whereas U.S. AIDS KS samples were primarily A1, A4, and C3 variants. We conclude that this unusually high diversity, in which 85% of the nucleotide changes lead to amino acid changes, reflects some unknown powerful biological selection process that has been acting preferentially on this early lytic cycle membrane signalling protein. Two distinct levels of ORF-K1 variability are recognizable. Subtype-specific variability indicative of long-term evolutionary divergence is both spread throughout the protein as well as concentrated within two 40-amino-acid extracellular domain variable regions (VR1 and VR2), whereas intratypic variability localizes predominantly within a single 25-amino-acid hypervariable Cys bridge loop and apparently represents much more recent changes that have occurred even within specific clades. In contrast, numerous extracellular domain glycosylation sites and Cys bridge residues as well as the ITAM motif in the cytoplasmic domain are fully conserved. Overall, we suggest that rather than being a newly acquired human pathogen, HHV8 is an ancient human virus that is preferentially transmitted in a familial fashion and is difficult to transmit horizontally in the absence of immunosuppression. The division into the four major HHV8 subgroups is probably the result of isolation and founder effects associated with the history of migration of modern human populations out of Africa over the past 35,000 to 60,000 years.

Africa↗
PubMed1993

Trp221 is involved in the protective effect of elongation factor eEF-2 on the ricin/alpha-sarcin site of the ribosome.

Elongation factor eEF-2 treated by N-bromosuccinimide under conditions which oxidize 2 Trp residues (Trp343 and Trp221) is inactivated in ribosome-dependent GTP hydrolysis and polyphenylalanine synthesis, and inactivation correlates with the specific oxidation of Trp221 (Guillot, D., Penin, F., Di Pietro, A., Sontag, B., Lavergne, J. P., and Reboud, J. P. (1993) J. Biol. Chem. 268, 20911-20916). It is shown here that this oxidation prevents neither GTP binding to eEF-2 nor the formation of the ribosome-eEF-2-GPP(NH)P complex, but that oxidized eEF-2 is no longer able to protect ribosomes against ricin inactivation. These observations suggest that Trp221 or an amino-acid sequence containing this residue interacts with the 28 S rRNA loop including the GAGA sequence, which is the target of ricin. Such a hypothesis is discussed in relation with data on RNA recognition motifs described in different proteins.

Animals↗
PubMed1997

Leishmania braziliensis, molecular characterization of an elongation factor 1alpha gene.

The elongation factor EF-1alpha is one of the most studied components of the translation machinery owing to its abundance and possible role in other cellular functions. EF-1alpha mediates the correct coupling of the aminoacyl-tRNA on the A site of the ribosome in a GTP-dependent reaction. We have previously described an EF-1alpha DNA sequence in Leishmania amazonensis, pLEF11 (accession No. M92653), using PCR. In this paper we describe the DNA sequence and genomic organization of L. braziliensis EF-1alpha gene. Southern blot analysis revealed that EF-1alpha is organized as a 2 kb tandem repeat. The pLEF11 probe recognized a 1.8 kb mRNA from promastigotes in Northern blots. A clone containing the first copy and a half of the EF-1alpha tandem repeat was isolated by screening a L. braziliensis genomic library. Southern blot analysis showed that the isolated clone (lambda2.2) presented the same hybridization profile as that of a genomic blot. The partial sequencing of clone lambda2.2 spans 2959 nucleotides in length, which has two open reading frames separated by a putative non-coding region. The nucleotide and the predicted peptide sequence of the first coding region presented approximately 80% identity with other eukaryotic EF-1alpha genes. The sequence also displayed the four consensus motifs corresponding to the GTP-binding site (G1, G2, G3 and G4). Computer analysis of the sequence of both coding regions revealed three divergent nucleotides, which generated two changes at the amino acid level. One was found to be located in the G2 domain. The non-coding region of the EF-1alpha gene sequence showed potential regulatory elements such as polypyrimidine tracks, chi-homologous sequences and stem-loop forming sequences.

Amino Acid Sequence↗
PubMed1993

1H-NMR resonance assignments, secondary structure, and global fold of the TR1C fragment of turkey skeletal troponin C in the calcium-free state.

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the 1H-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D 1H-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard "EF-hand" motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M.N.G. (1988) J. Mol. Biol. 203, 761-779].

Amino Acid Sequence↗
PubMed1998

Crystal structure of botulinum neurotoxin type A and implications for toxicity.

Botulinum neurotoxin type A (BoNT/A) is the potent disease agent in botulism, a potential biological weapon and an effective therapeutic drug for involuntary muscle disorders. The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined at 3.3 A resolution. The structure reveals that the translocation domain contains a central pair of alpha-helices 105 A long and a approximately 50 residue loop or belt that wraps around the catalytic domain. This belt partially occludes a large channel leading to a buried, negative active site--a feature that calls for radically different inhibitor design strategies from those currently used. The fold of the translocation domain suggests a mechanism of pore formation different from other toxins. Lastly, the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.

Binding Sites↗