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PubMed1996

Involvement of the E2A basic helix-loop-helix protein in immunoglobulin heavy chain class switching.

The basic helix-loop-helix (bHLH) transcriptional factor E2A has previously been shown to play a critical role in early B cell development, with E2A knockout mice and Id1 transgenic mice showing an arrest at the pro-B cell stage of development. More recent data suggest that E2A, through an interaction with the immunoglobulin heavy chain 3' enhancer, might also regulate later events in B cell development such as heavy chain class switching. The patterns of E2A protein expression in secondary lymphoid tissues support a role in later stages of B cell maturation. In particular, immunostaining reveals upregulation of E2A protein in cells of the dark zone of the germinal center, the site of immunoglobulin heavy chain class switching. To examine the role of E2A in class switching, the inhibitory HLH protein Id1 was expressed in B cell lines which normally undergo spontaneous and inducible switching from IgM to IgA. The forced expression of Id1 in these cell lines effectively blocked class switching. This Id1 blockade of class switching did not occur via downregulation of immunoglobulin heavy chain germline transcription or through inhibition of cell cycling. Furthermore, Id1 inhibited spontaneous and, to a lesser degree, cytokine inducible class switching. From these data, we conclude that E2A plays an important role in the class switching process.

Animals↗
PubMed2000

E2A proteins: essential regulators at multiple stages of B-cell development.

The development of mature B lymphocytes from multipotent progenitor cells requires the co-ordinated activities of a number of transcriptional regulatory proteins. The transcription factors encoded by the E2A gene are required for the development of committed B-lineage cells and regulate the expression of essential B-lineage genes at multiple stages of differentiation and activation. In this review we discuss the evidence that the E2A gene products function in the regulation of 1) transcription factors required for B-lineage determination, 2) essential proteins involved in pro-B and pre-B-cell development, 3) accessibilty and recombination of the IgH and IgL chain loci, and 4) isotype switching in activated, mature B lymphocytes.

Animals↗
PubMed1997

[New structural motifs in alpha-helical proteins].

Four novel structural motifs were found and characterized in alpha-helical proteins. One of them consists of three alpha helices and can be represented as a combination of an alpha-alpha corner and an L-shaped structure. Its second alpha helix is a part of both one of the two alpha helices of the alpha-alpha corner and one of the two L-structure helices. The second structural motif, named the ABCD unit, consists of four alpha helices A, B, C, and D. These are consecutive in sequence and arranged in space in such a manner that the helices B, C, and D form a left-handed superhelix and the helix A is located between the helices B and D and is approximately antiparallel to them. The third motif, alpha helix-loop-alpha helix, consists of two alpha helices and a long connection. Its alpha helices are arranged in an approximately parallel manner and together with the long connection, form a left-handed alpha-1-alpha superhelix in space. The fourth structural motif considered is formed by four consecutive alpha helices. It is named the phi motif, because its overall shape is reminiscent of the Greek letter phi. Various variants of these motifs are analyzed on numerous examples of proteins with the known spatial structures.

Protein Conformation↗
PubMed1993

Basal promoter and enhancer element of yeast U6 snRNA gene.

The yeast U6 snRNA gene, SNR6, transcribed by RNA polymerase III or C, is shown to have a mixed promoter with upstream, intragenic and downstream elements. The distant downstream B block behaves as a typical enhancer element. Required in vivo, and for transcription of chromatin templates in vitro, it was also active in reversed orientation. As shown by footprinting and electron microscopy, the factor TFIIIC, or tau, bound the B block in an oriented manner and was able to induce DNA looping. The factor TFIIIC appeared to act via a weak A block located at position +21. This A block-related motif was essential in vivo and with chromatin templates. When changed into a consensus A block it favored DNA looping by TFIIIC firmly anchored on the B block, and activated a B block lacking gene in vivo and in vitro. The role of the TATA box at -30 was most apparent using a purified transcription system. With the A block, it appeared to contribute to start site selection. The results suggest a model where three weak promoter elements collaborate to assemble the transcription complex by DNA looping and synergistic protein-DNA interactions.

Base Sequence↗
PubMed2004

Design and synthesis of de novo cytochromes c.

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.

Amino Acid Sequence↗
PubMed2001

Tertiary structure stability of the hairpin ribozyme in its natural and minimal forms: different energetic contributions from a ribose zipper motif.

The hairpin catalytic motif in tobacco ringspot virus satellite RNA consists of two helix-loop-helix elements on two adjacent arms of a four-way helical junction. The bases essential for catalytic activity are located in the loops that are brought into proximity by a conformational change as a prerequisite for catalysis. The two loops interact via a ribose zipper motif involving the 2'-hydroxyls of A10, G11, A24, and C25 [Rupert, P. B., and Ferre d'Amare, A. R. (2001) Nature 401, 780-786]. To quantify the energetic importance of the ribose zipper hydrogen bonds, we have incorporated deoxy modifications at these four positions and determined the resulting destabilization of the docked conformer by means of time-resolved fluorescence resonance energy transfer. In a minimal form of the ribozyme, in which the loops are placed on the arms of a two-way helical junction, all modifications lead to a significant loss in tertiary structure stability and altered Mg2+ binding. Surprisingly, no significant destabilization was seen with the natural four-way junction ribozyme, suggesting that hydrogen bonding interactions involving the 2'-hydroxyls do not contribute to the stability of the docked conformer. These results suggest that the energetic contributions of ribose zipper hydrogen bonds are highly context dependent and differ significantly for the minimal and natural forms of the ribozyme.

Energy Transfer↗
PubMed1986

Two RNA species co-purify with RNase P from the fission yeast Schizosaccharomyces pombe.

RNase P activity from Schizosaccharomyces pombe co-purifies with two RNA species. These RNAs are associated with enzyme activity as judged by titrated micrococcal nuclease inactivation experiments. The two RNAs, K1- and K2-RNA, are 285 and 270 nucleotides long, respectively. Both RNAs are transcribed from one gene, present in a single copy in the haploid genome. The primary and a secondary structure of K RNAs have been determined and compared with M1 RNA, their counterpart from Escherichia coli. Very limited sequence homology was observed, and this agrees with the finding that no cross-hybridization with M1 RNA can be detected in a Southern analysis with yeast genomic DNA. However, the secondary structures of K RNA and M1 RNA show the same basic organization and one conserved local motif, the sequence GUG--AGGPu in an exposed hairpin loop.

Base Sequence↗
PubMed1994

Related target enhancers for dorsal and NF-kappa B signaling pathways.

Drosophila dorsoventral (DV) patterning and mammalian hematopoiesis are regulated by related signaling pathways (Toll, interleukin-1) and transcription factors (dorsal, nuclear factor-kappa B). These factors interact with related enhancers, such as the rhomboid NEE and kappa light chain enhancer, that contain similar arrangements of activator and repressor binding sites. It is shown that the kappa enhancer can generate lateral stripes of gene expression in transgenic Drosophila embryos in a pattern similar to that directed by the rhomboid NEE. Drosophila DV determinants direct these stripes through the corresponding mammalian cis regulatory elements in the kappa enhancer, including the kappa B site and kappa E boxes. These results suggest that enhancers can couple conserved signaling pathways to divergent gene functions.

Animals↗
PubMed2003

Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm.

Phosphorylation plays a central role in regulating the activation and signalling lifetime of protein kinases A, B (also known as Akt) and C. These kinases share three conserved phosphorylation motifs: the activation loop segment, the turn motif and the hydrophobic motif. This review focuses on how phosphorylation at each of these sites regulates the maturation, signalling and down-regulation of PKC as a paradigm for how these sites control the function of the ABC kinases.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

The high specificities of Phaseolus vulgaris erythro- and leukoagglutinating lectins for bisecting GlcNAc or beta 1-6-linked branch structures, respectively, are attributable to loop B.

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E(4)- and L(4)-PHA) are unique in recognizing larger structures. E(4)- and L(4)-PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a beta1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E(4)- and L(4)-PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E(4)- and L(4)-PHA toward bisecting GlcNAc and beta1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E(4)- and L(4)-PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a beta1,6-linked branch.

Acetylglucosamine↗
PubMed2000

X-Ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B).

Parvalbumins constitute a class of calcium-binding proteins characterized by the presence of several helix-loop-helix (EF-hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7:2397-2408), we presented the sequence of the major parvalbumin isoform from the silver hake (Merluccius bilinearis) and presented spectroscopic and structural information on the excised "EF-hand" portion of the protein. In this study, the X-ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C2221, with a = 75.7 A, b = 80.7 A, and c = 42.1 A. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at -188 degrees C. The structure refined to a conventional R-value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 A [1 > 2sigma(I)]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a beta parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X-ray results in addressing the issue of mobility of key residues in the calcium-binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.

Animals↗
PubMed2006

Molecular determinants for differential membrane trafficking of PMCA1 and PMCA2 in mammalian hair cells.

The plasma membrane Ca2+-ATPase-2 (PMCA2) is expressed in stereocilia of hair cells of the inner ear, whereas PMCA1 is expressed in the basolateral plasma membrane of hair cells. Both extrude excess Ca2+ from the cytosol. They are predicted to contain ten membrane-spanning segments, two large cytoplasmic loops as well as cytosolic N- and C-termini. Several isoform variants are generated for both PMCA1 and PMCA2 by alternative splicing, affecting their first cytosolic loop (A-site) and their C-terminal tail. To understand how these isoforms are differentially targeted in hair cells, we investigated their targeting regions and expression in hair cells. Our results show that a Leu-Ile motif in 'b'-tail splice variants promotes PMCA1b and PMCA2b basolateral sorting in hair cells. Moreover, apical targeting of PMCA2 depends on the size of the A-site-spliced insert, suggesting that the conformation of the cytoplasmic loop plays a role in apical targeting.

Amino Acid Motifs↗
PubMed2005

TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V(H)) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V(H) mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells. MATERIALS AND METHODS: TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells. RESULTS: Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V(H) show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V(H) were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression. CONCLUSION: TWIST2 is differentially methylated in CLL cells relative to Ig V(H) mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.

Antimetabolites, Antineoplastic↗
PubMed2000

Puromycin-rRNA interaction sites at the peptidyl transferase center.

The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439, G2505, and G2553 for E. coli, and G2058, A2503, G2505, and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E. coli 23S rRNA. In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E. coli ribosomes. Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin, along with other peptidyl-transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region. This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs. In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.

Base Sequence↗
PubMed1995

Identification of functional domains of the aryl hydrocarbon receptor.

Functional domains of the mouse aryl hydrocarbon receptor (Ahr) were investigated by deletion analysis. Ligand binding was localized to a region encompassing the PAS B repeat. The ligand-mediated dissociation of Ahr from the 90-kDa heat shock protein (HSP90) does not require the aryl hydrocarbon receptor nuclear translocator (Arnt), but it is slightly enhanced by this protein. One HSP90 molecule appears to bind within the PAS region. The other molecule of HSP90 appears to require interaction at two sites: one over the basic helix-loop-helix region, and the other located within the PAS region. Each mutant was analyzed for dimerization with full-length mouse Arnt and subsequent binding of the dimer to the xenobiotic responsive element (XRE). In order to minimize any artificial steric hindrances to dimerization and XRE binding, each Ahr mutant was also tested with an equivalently deleted Arnt mutant. The basic region of Ahr is required for XRE binding but not for dimerization. Both the first and second helices of the basic helix-loop-helix motif and the PAS region are required for dimerization. These last results are analogous to those previously obtained for Arnt (Reisz-Porszasz, S., Probst, M.R., Fukunaga, B. N., and Hankinson, O. (1994) Mol. Cell. Biol. 14, 6075-6086) compatible with the notion that equivalent regions of Ahr and Arnt associate with each other. Deletion of the carboxyl-terminal half of Ahr does not affect dimerization or XRE binding but, in contrast to an equivalent deletion of Arnt, eliminates biological activity as assessed by an in vivo transcriptional activation assay, suggesting that this region of Ahr plays a more prominent role in transcriptional activation of the cyp1a1 gene than the corresponding region of Arnt.

Animals↗
PubMed2003

Unusual DNA duplex and hairpin motifs.

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G*A base pairs are separated by one or two pairs of purine*purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G*C or A.T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G.A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G*A, A*A, A*C or sheared-like G(anti)*C(syn) base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.

Base Pair Mismatch↗
PubMed1997

IKK-1 and IKK-2: cytokine-activated IkappaB kinases essential for NF-kappaB activation.

Activation of the transcription factor nuclear factor kappa B (NF-kappaB) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit IkappaB. A large multiprotein complex, the IkappaB kinase (IKK) signalsome, was purified from HeLa cells and found to contain a cytokine-inducible IkappaB kinase activity that phosphorylates IkappaB-alpha and IkappaB-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappaB-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappaB signaling pathway.

Cell Cycle Proteins↗