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At least 253 records · Page 14Linked to original sources
PubMed1999

A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.

The t(1;19) chromosomal translocation of pediatric pre-B cell leukemia produces chimeric oncoprotein E2a-Pbx1, which contains the N-terminal transactivation domain of the basic helix-loop-helix (bHLH) transcription factor, E2a, joined to the majority of the homeodomain protein, Pbx1. There are three Pbx family members, which bind DNA as heterodimers with both broadly expressed Meis/Prep1 homeo-domain proteins and specifically expressed Hox homeodomain proteins. These Pbx heterodimers can augment the function of transcriptional activators bound to adjacent elements. In heterodimers, a conserved tryptophan motif in Hox proteins binds a pocket on the surface of the Pbx homeodomain, while Meis/Prep1 proteins bind an N-terminal Pbx domain, raising the possibility that the tryptophan-interaction pocket of the Pbx component of a Pbx-Meis/Prep1 complex is still available to bind trypto-phan motifs of other transcription factors bound to flanking elements. Here, we report that Pbx-Meis1/Prep1 binds DNA cooperatively with heterodimers of E2a and MyoD, myogenin, Mrf-4 or Myf-5. As with Hox proteins, a highly conserved tryptophan motif N-terminal to the DNA-binding domains of each myogenic bHLH family protein is required for cooperative DNA binding with Pbx-Meis1/Prep1. In vivo, MyoD requires this tryptophan motif to evoke chromatin remodeling in the Myogenin promoter and to activate Myogenin transcription. Pbx-Meis/Prep1 complexes, therefore, have the potential to cooperate with the myogenic bHLH proteins in regulating gene transcription.

3T3 Cells↗
PubMed1991

Modulation of the IgH enhancer's cell type specificity through a genetic switch.

Using defined regions of the immunoglobulin heavy-chain enhancer linked to minimal promoters and cDNAs that encode the two helix-loop-helix transcription factors ITF-1 and TFE3, we demonstrate that activity of an otherwise repressed enhancer can be stimulated in nonlymphoid cells. Repression in non-B cells is mediated by the microE5 motif. Derepression occurs at two levels. First, overexpression of ITF-1, and E12/E47-related protein that binds the microE5 motif, leads to transcriptional activation itself. Second, binding of ITF-1 physically displaces a repressor that normally blocks the stimulatory activity of TFE3, which binds the neighboring microE3 motif. TFE3 can only stimulate enhancer activity in the presence of ITF-1 or in the absence of a microE5 motif. Hence, one component of the enhancer's cell type specificity can be artificially modulated through a "genetic switch" in which activity is dictated by the relative levels of ITF-1 and a competing repressor.

Base Sequence↗
PubMed1990

Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif.

Activity of the immunoglobulin heavy and kappa light chain gene enhancers depends on a complex interplay of ubiquitous and developmentally regulated proteins. Two complementary DNAs were isolated that encode proteins, denoted ITF-1 and ITF-2, that are expressed in a variety of cell types and bind the microE5/kappa 2 motif found in both heavy and kappa light chain enhancers. The complementary DNAs are the products of distinct genes, yet both ITF-1 and ITF-2 are structurally and functionally similar. The two proteins interact with one another through their putative helix-loop-helix motifs and each possesses a distinct domain that dictates transcription activation.

Animals↗
PubMed2002

Molecular dynamics study of Ca(2+) binding loop variants of silver hake parvalbumin with aspartic acid at the "gateway" position.

The helix-loop-helix (i.e., EF-hand) Ca(2+) ion binding motif is characteristic of a large family of high-affinity calcium ion binding proteins, including the parvalbumins, oncomodulins and calmodulins. In this work we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B) and on functional fragments of this protein, consisting of the first four helical regions (the ABCD fragment), and the internal helix-loop- helix region (the CD fragment). In both whole protein and protein fragments (i.e., ABCD and CD fragments), the 9th loop residue in the calcium ion binding site in the CD helix-loop-helix region (the so-called "gateway" position) has been mutated from glutamic acid to aspartic acid. Aspartic acid is one of the most common residues found at the gateway position in other (non-parvalbumin) EF- hand proteins, but has never been found at the gateway position of any parvalbumin. (Interestingly, aspartic acid does occur at the gateway position in the closely related rat and human oncomodulins.) Consistent with experimental observations, the results of our molecular dynamics simulations show that incorporation of aspartic acid at the gateway position is very disruptive to the structural integrity of the calcium ion coordination site in the whole protein. The aspartic acid mutation is somewhat less disruptive to the calcium ion coordination sites in the two parvalbumin fragments (i.e., the ABCD and CD fragments), presumably due to the higher degree of motional freedom allowable in these protein fragments. One problem associated with the E59D whole protein variant is a prohibitively close approach of the aspartate carboxyl group to the CD calcium ion observed in the energy-minimized (pre-molecular dynamics) structure. This steric situation does not emerge during energy-minimization of the wild-type protein. The damage to the structural integrity of the calcium ion coordination site in the whole protein E59D variant is not relieved during the molecular dynamics simulation. In fact, during the course of the 300 picosecond simulation, all of the calcium ion ligands leave the primary coordination sphere. In addition, the conserved hydrogen- bonds (in the short beta-sheet structure) that links the CD site to the symmetry-related EF site (in the non-mutated whole protein) is also somewhat disrupted in the E59D whole protein variant. These results suggest that the Ca(2+) ion binding deficiencies in the CD loop are related, at least in part, to the unique interaction that exists between the paired CD and EF hands in the whole protein. Our theoretical results correlate well with previous studies on engineered EF-hand proteins and with all of our experimental evidence on whole silver hake parvalbumin and enzymatically-generated parvalbumin fragments.

Amino Acid Motifs↗
PubMed1999

Diversity of conformational states and changes within the EF-hand protein superfamily.

The EF-hand motif, which assumes a helix-loop-helix structure normally responsible for Ca2+ binding, is found in a large number of functionally diverse Ca2+ binding proteins collectively known as the EF-hand protein superfamily. In many superfamily members, Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins. In calmodulin and troponin C, this is described as a change from the closed conformational state in the absence of Ca2+ to the open conformational state in its presence. It is now clear from structures of other EF-hand proteins that this "closed-to-open" conformational transition is not the sole model for EF-hand protein structural response to Ca2+. More complex modes of conformational change are observed in EF-hand proteins that interact with a covalently attached acyl group (e.g., recoverin) and in those that dimerize (e.g., S100B, calpain). In fact, EF-hand proteins display a multitude of unique conformational states, together constituting a conformational continuum. Using a quantitative 3D approach termed vector geometry mapping (VGM), we discuss this tertiary structural diversity of EF-hand proteins and its correlation with target recognition.

Amino Acid Motifs↗
PubMed2006

Genetic characteristics of the V3 region associated with CXCR4 usage in HIV-1 subtype C isolates.

CXCR4 coreceptor usage appears to occur less frequently among HIV-1 subtype C viruses. The aim of this study was to investigate the genetic determinants within the V3 region of subtype C isolates able to use CXCR4. Thirty-two subtype C isolates with known phenotypes (16 R5, 8 R5X4 and 8 X4 isolates) were assessed. A subtype C-specific V3 heteroduplex tracking assay (HTA) was used to determine sample complexity, and nucleotide sequencing analysis was used to compare characteristics associated with CCR5 and CXCR4-using isolates. There were sufficient genetic differences to discriminate between R5 viruses and those able to use CXCR4. In general, R5 isolates had an HTA mobility ratio >0.9 whereas CXCR4-using isolates were usually <0.9. Multiple bands were more frequently seen among the dualtropic isolates. Sequence analysis of the V3 region showed that CXCR4-using viruses were often associated with an increased positive amino acid charge, insertions and loss of a glycosylation site, similar to HIV-1 subtype B. In contrast, where subtype B consensus V3 has a GPGR crown motif irrespective of coreceptor usage, all 16 subtype C R5 viruses had a conserved GPGQ sequence at the tip of the loop, while 12 of the 16 (75%) CXCR4-using viruses had substitutions in this motif, most commonly arginine (R). These findings were confirmed using a larger published data set. We therefore suggest that changes within the crown motif of subtype C viruses might be an additional pathway to utilise CXCR4 and thus GPGQ may limit the potential for the development of X4 viruses.

Amino Acid Sequence↗
PubMed1993

A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells.

Previous studies demonstrated that the amino-terminal portions of E2A and E2-2 are crucial for transactivation. Subsequent findings showed that the same amino-terminal region of E2A is involved in two different translocation events contributing to the induction of a pre-B-cell acute lymphoblastic leukemia and a pro-B-cell acute lymphoblastic leukemia. These results led us to focus on the amino-terminal region of E2A to better understand its normal role in transcriptional regulation and its aberrant involvement in the two leukemias. We report here the identification of two conserved boxes in the E2A amino-terminal domain that show extensive homology within the transactivation domains of E12, E47, E2-2, HEB, and daughterless, all members of the same class of helix-loop-helix proteins. Together, both boxes are crucial for transcriptional activation and have the potential to form a new activation motif, that of a loop adjacent to an amphipathic alpha-helix, designated the loop-helix (LH) motif. A minimal region containing the LH motif is sufficient for transcriptional activation. Point mutations in the amphipathic helix of the minimal region reduce its transactivation capabilities dramatically. The same constructs expressed in yeast cells show identical patterns of activation, suggesting that the LH motif and its target proteins are functionally conserved in yeast cells. We propose that the LH motif represents a novel transactivation domain that is distinct from the previously characterized acidic blob, proline-rich, and glutamine-rich activation motifs. In addition, the LH motif is the first activation motif restricted to one class of DNA binding proteins.

Amino Acid Sequence↗
PubMed1994

Lethal mutations within the conserved stem-loop of African cassava mosaic virus DNA are rapidly corrected by genomic recombination.

The nonanucleotide motif TAATATTAC occurs in the intergenic region of all geminiviruses that have been examined to date. The motif is invariably located within the loop of a potential stem-loop structure that has been implicated in viral DNA replication. To investigate the contribution of these sequences to virus proliferation, African cassava mosaic virus (ACMV) DNA B mutants have been screened for their ability to infect Nicotiana benthamiana when co-inoculated with DNA A. Mutants in which the putative stem structure was altered by the introduction of single nucleotide mismatches remained as infectious as the wild-type virus and the mutations were retained in the progeny. Mutants containing nucleotide substitutions within the loop sequences were similarly infectious but analysis of progeny showed that in most cases wild-type sequences were restored by recombination with DNA A. Stem-loop deletion mutants of both genomic components were not infectious when co-inoculated, although they were once again efficiently rescued by recombination when inoculated with the wild-type components. Co-inoculation of genomic components containing the motif TAGTATTAC did not result in a systemic infection while mutants containing the motif TAATACTAC were infectious and the mutation was stable. The results demonstrate that ACMV will tolerate some modification to this highly conserved region of the genome that might allow more precise mapping of the position at which the viral DNA is nicked during replication.

Base Sequence↗
PubMed2002

Early B-cell factor, E2A, and Pax-5 cooperate to activate the early B cell-specific mb-1 promoter.

Previous studies have suggested that the early-B-cell-specific mb-1(Igalpha) promoter is regulated by EBF and Pax-5. Here, we used in vivo footprinting assays to detect occupation of binding sites in endogenous mb-1 promoters at various stages of B-cell differentiation. In addition to EBF and Pax-5 binding sites, we detected occupancy of a consensus binding site for E2A proteins (E box) in pre-B cells. EBF and E box sites are crucial for promoter function in transfected pre-B cells, and EBF and E2A proteins synergistically activated the promoter in transfected HeLa cells. Other data suggest that EBF and E box sites are less important for promoter function at later stages of differentiation, whereas binding sites for Pax-5 (and its Ets ternary complex partners) are required for promoter function in all mb-1-expressing cells. Using DNA microarrays, we found that expression of endogenous mb-1 transcripts correlates most closely with EBF expression and negatively with Id1, an inhibitor of E2A protein function, further linking regulation of the mb-1 gene with EBF and E2A. Together, our studies demonstrate the complexity of factors regulating tissue-specific transcription and support the concept that EBF, E2A, and Pax-5 cooperate to activate target genes in early B-cell development.

Animals↗
PubMed2006

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor.

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

Cholecystokinin↗
PubMed2005

Helix-loop-helix proteins in lymphocyte lineage determination.

The cells of the lymphoid system develop from multipotent hematopoietic stem cells through a series of intermediate progenitors with progressively restricted developmental options. Commitment to a given lymphoid lineage appears to be controlled by numerous transcriptional regulatory proteins that activate lineage-specific gene expression programs and extinguish expression of lineage-inappropriate genes. In this review I discuss the function of transcription factors belonging to the helix-loop-helix protein family in the control of lymphoid cell fate decisions. A model of lymphocyte lineage determination based on the antagonistic activity of transcriptional activating and repressing helix-loop-helix proteins is presented.

Animals↗
PubMed1993

Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action.

2-5A-dependent RNAase, an interferon-induced enzyme that is activated by 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A), is implicated in both the molecular mechanisms of interferon action and the fundamental control of RNA stability in mammalian cells. Here we report the expression cloning and analysis of murine and human 2-5A-dependent RNAases. The 2-5A binding properties and RNAse activities of recombinant and naturally occurring forms of 2-5A-dependent RNAase were identical. Interferon induction of 2-5A-dependent RNAse expression was demonstrated by measuring the mRNA levels in cells treated with interferon and cycloheximide. Analysis of aligned murine and human 2-5A-dependent RNAse sequences revealed several intriguing features, including similarity to RNAase E, which is implicated in the control of mRNA stability in E. coli. Interestingly, a duplicated phosphate-binding loop motif was determined by deletion analysis and site-directed mutagenesis to function in the binding of 2-5A.

Amino Acid Sequence↗
PubMed1995

Expression of Id2 and Id3 mRNA in human lymphocytes.

Helix-loop-helix (HLH) transcription factors are involved in cellular growth and differentiation. The Id (inhibitor of DNA binding and differentiation) HLH proteins, in a dominantly negative fashion, regulate transcriptional activities of basic HLH proteins. We examined by northern hybridization the expression of Id2 and Id3 mRNA in human leukemia/lymphoma lines and patient samples, as well as resting and activated normal human lymphocytes from peripheral blood (PBL). The Id2 mRNA was abundantly expressed in 5/12 T-cell and 3/4 B-cell lines, and Id3 mRNA was detected in 4/12 T-cell and 3/4 B-cell lines. Interestingly, Id2, but not Id3, mRNA was strongly expressed in 4/5 T-cell lines infected with human T-cell leukemia virus type I (HTLV-I) (ATL-1k, MT-2, S-LB1) and type II (Mo). Another unexpected finding was that T-cell leukemias and T-cell lines often expressed either Id2 or Id3 mRNA. In addition, resting PBL constitutively expressed prominent levels of Id2 mRNA, but not Id3 mRNA. Upon PHA-stimulation, Id2 expression decreased and Id3 levels increased with biphasic kinetics. Taken together, our studies revealed three unexpected findings which require further analysis: (1) expression of Id2 mRNA is often associated with lymphocytic transformation by HTLV-I or -II; (2) T-cells usually express either Id2 or Id3 mRNA, but B-cells often express both simultaneously; (3) non-dividing, normal PBL express high levels of Id2 and no Id3 mRNA; and with the onset of cellular proliferation, levels of Id2 mRNA decrease while levels of Id3 mRNA increase, suggesting that regulation of expression of these closely related genes is disparate.

B-Lymphocytes↗
PubMed1994

Transcriptional regulation of T-cell genes during T-cell development.

The expression of many T cell specific genes has been shown to be regulated at the transcriptional level. Recent studies of T-cell specific promoters and enhancers have allowed the identification of a number of transcription factors that appear to play distinct but complementary roles in regulating gene expression during T-cell development and activation.

Animals↗
PubMed2001

Brinker requires two corepressors for maximal and versatile repression in Dpp signalling.

decapentaplegic (dpp) encodes a Drosophila transforming growth factor-beta homologue that functions as a morphogen in the developing embryo and in adult appendage formation. In the wing imaginal disc, a Dpp gradient governs patterning along the anteroposterior axis by inducing regional expression of diverse genes in a concentration-dependent manner. Recent studies show that responses to graded Dpp activity also require an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Here we show that Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, we demonstrate that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. Our results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters.

Alcohol Oxidoreductases↗
PubMed1999

Evolution of bHLH transcription factors: modular evolution by domain shuffling?

Multidomain proteins usually contain several conserved and apparently independently evolved domains. As a result, classifications based on only a single small domain may obscure the true evolutionary relationships of the proteins. The current classification of basic helix-loop-helix (bHLH) domain-containing proteins is based on the conserved bHLH domain alone. Herein, we explore whether sequence homology and, therefore, evolutionary relationships can be detected among the flanking or non-bHLH components of the amino acid sequences of 122 bHLH proteins. These 122 proteins were the same proteins previously used to construct the existing classification of the bHLH-domain-containing proteins. Several possible scenarios are examined in order to explain the observed patterns of sequence divergence, including (1) monophyly, (2) convergent evolution, (3) addition of functional components to the bHLH domain, and (4) modular evolution with domain shuffling. Drawing on several lines of evidence, we suggest that modular evolution by domain shuffling may have played an important role in the evolution of this large group of transcriptional regulators.

DNA-Binding Proteins↗
PubMed1999

Degradation of Id proteins by the ubiquitin-proteasome pathway.

Id proteins act as negative regulators of bHLH transcription factors by forming transcriptionally inactive protein complexes. The proposed function of these proteins includes promotion of cell growth and cell cycle progression, induction of apoptosis, and inhibition of cellular differentiation. We investigated the role of the ubiquitin-mediated proteolytic pathway in the degradation of the Id3 protein. We found Id3 to be a short-lived protein and estimated the half-life to be approximately 20 min in 293 cells. Using specific inhibitors of the 26S proteasome and mutant fibroblast cells with a temperature-sensitive defect in the essential E1 ubiquitin-activating enzyme, we show that Id3 and the related Id1 and Id2 proteins are degraded through the ubiquitin-proteasome pathway. We found the Id4 protein to be much less sensitive to inhibitors of the 26S proteasome, but its degradation was dependent on the E1 enzyme. In addition, we observed that coexpression of the bHLH protein E47 with Id3 significantly reduced the rate of degradation of Id3, suggesting that Id3 is less susceptible to degradation by the 26S proteasome when complexed to a bHLH protein. -Bounpheng, M. A., Dimas, J. J., Dodds, S. G., Christy, B. A. Degradation of Id proteins by the ubiquitin-proteasome pathway.

Cells, Cultured↗