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PubMed1995

A novel enhancer, the pro-B enhancer, regulates Id1 gene expression in progenitor B cells.

The helix-loop-helix (HLH) Id proteins have been reported to function as inhibitors of various differentiation programs. The HLH motif mediates dimer formation between Id and the basic HLH transcription factors. Since Id proteins lack the basic region responsible for DNA binding, the heterodimers cannot bind to DNA. Id proteins have also been found to be involved in early B-cell differentiation. They are expressed at high levels in progenitor B cells (pro-B cells), and the expression is diminished in pre-B cells and mature B cells. This expression pattern correlates inversely with basic HLH protein activity and immunoglobulin enhancer function in B-cell development. Regulation of Id expression may play an important role in transcriptional control of immunoglobulin genes and therefore in B-cell differentiation. We have characterized the regulatory elements of the Id1 gene. Using stable transfectants, transient transfection, and mobility shift assays, we have identified an 8-bp element designated PBE (pro-B enhancer) downstream of the Id1 gene that is responsible for a pro-B-cell-specific enhancer activity. A pro-B-cell-specific protein complex was found to bind to the 8-bp PBE element. Substitution mutagenesis at this binding site showed that it is indeed of functional importance in regulating the pro-B-cell-specific expression of the Id1 gene.

Animals↗
PubMed2005

The role of extra-membranous inter-helical loops in helix-helix interactions.

The effect of a short loop connecting two transmembrane alpha-helices was studied using molecular dynamics simulations. Helices F and G from bacteriorhodopsin and two corresponding polyalanine helices were embedded in octane and POPC membranes in a transmembrane configuration both with and without the inter-helical loop. The results indicate that the membrane environment and the sequence of the loop are more influential on the dynamics and structure of the motif than the presence of a loop as such, at least for the time-scales investigated. The four residues in the FG loop are stabilized by four hydrogen bonds. These hydrogen bonds are not present in the polyalanine loop, causing it to be more flexible than the FG loop. This effect was observed independently of the protein environment, stressing the importance of the sequence. The structural analysis indicates that the loop has weak stabilizing properties in all environments. The stabilization due to the presence of the loop was strongest in a simulation of the FG fragment in a membrane-mimetic octane slab. In the simulations of the helix-loop-helix motif embedded in an explicit lipid bilayer model, the lipid bilayer interface compensates to a large extent for the absence of the loop.

Amino Acid Motifs↗
PubMed2004

The cross-clade neutralizing activity of a human monoclonal antibody is determined by the GPGR V3 motif of HIV type 1.

Both polyclonal and monoclonal human antibodies (Abs) to the V3 domain of HIV-1 gp120 display cross-clade neutralizing activity against primary isolates and T cell-adapted virus strains. The most broadly neutralizing of the human anti-V3 monoclonal Abs (mAbs), 447-52D, recognizes 14 amino acids, including the GPxR core epitope at the tip of the V3 loop. Monoclonal Ab 447-52D neutralized 92% of 38 primary isolates carrying the GPGR V3 motif regardless of whether the viruses belonged to clades A, B, F, or H; in contrast, none of 19 viruses with the GPGQ and other non-GPGR/Q sequences at the tip of the V3 loop was sensitive to mAb 447-52D. These data are consistent with the crystallographic resolution of a complex of the Fab fragment of mAb 447-52D with a V3 peptide that shows that the binding specificity of the mAb is due to recognition of the GPGR motif at the tip of the loop. The critical role of the Arg residue in this motif was determined using viruses pseudotyped with the envelope of primary isolate CA1 containing the GPGR motif or with a mutated envelope with a Gln (Q) replacing the Arg (R) at the tip of the loop. While the wild-type pseudovirus was neutralized by mAb 447-52D, the pseudovirus carrying the point mutation was resistant to neutralization. These data illuminate the structural basis for both the breadth and specificity of a broadly neutralizing human mAb and contribute to our understanding of the epitopes recognized by Abs that protect against infection with HIV-1.

Amino Acid Motifs↗
PubMed2004

Receptor editing and marginal zone B cell development are regulated by the helix-loop-helix protein, E2A.

Previous studies have indicated that the E2A gene products are required to initiate B lineage development. Here, we demonstrate that E2A(+/-) B cells that express an autoreactive B cell receptor fail to mature due in part to an inability to activate secondary immunoglobulin (Ig) light chain gene rearrangement. Both RAG1/2 gene expression and RS deletion are severely defective in E2A(+/-) mice. Additionally, we demonstrate that E2A(+/-) mice show an increase in the proportion of marginal zone B cells with a concomitant decrease in the proportion of follicular B cells. In contrast, Id3-deficient splenocytes show a decline in the proportion of marginal zone B cells. Based on these observations, we propose that E-protein activity regulates secondary Ig gene rearrangement at the immature B cell stage and contributes to cell fate determination of marginal zone B cells. Additionally, we propose a model in which E-proteins enforce the developmental checkpoint at the immature B cell stage.

Animals↗
PubMed1993

The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs.

The multisubunit yeast transcription factor IIIC (TFIIIC; also called tau) can undergo considerable conformational changes upon binding to the A and B blocks of tRNA genes. After binding to DNA encoding tRNA (tDNA), TFIIIC acts as an assembly factor to recruit an initiation factor, TFIIIB, via its tau 131 subunit. We have cloned the gene encoding the tau 131 subunit and named it TFC4. This gene is unique, essential for cell viability, and encodes a M(r) 120,153 protein. Epitope-tagging and mobility-shift assays indicated the presence of a single tau 131 subunit in TFIIIC-tDNA complexes. tau 131 contains two sequence motifs, accounting for nearly one-half of the protein mass, that may provide a molecular explanation for the properties of TFIIIC-tDNA complex. A series of 11 copies of the tetratricopeptide repeat motif may account for the flexibility and interaction properties of TFIIIC. A motif akin to the basic helix-loop-helix motif of MyoD suggests the direct involvement of tau 131 in promoting DNA binding of TFIIIB.

Amino Acid Sequence↗
PubMed1998

The V3 loop of human immunodeficiency virus type-1 envelope protein is a high-affinity ligand for immunophilins present in human blood.

Human immunodeficiency virus type-1 (HIV-1) infection requires binding of the envelope protein gp120 to host CD4 receptors and the action of the chemokine receptors CXCR4 or CCR5, which define cell tropism. The proline-containing V3 loop of gp120 determines the selection of the chemokine receptor and participates in conformational changes on binding of gp120 to CD4. In this study, we show that macrophage-tropic and T-cell-tropic V3 loop peptides bind specifically to the active site of the immunophilins FK506-binding protein (FKBP12), and cyclophilins A and B. Macrophage-tropic and T-cell-tropic V3 loop peptides inhibited the peptidyl-prolyl cis-trans isomerase (PPIase) activities of the immunophilins. Kd values in the range 0.036-4.1 microM were determined with V3 loop peptides labeled with an environmentally sensitive fluorophore. The observed binding properties of the V3 loop peptides reveal structural motifs of linear water-soluble peptidic substrates for tight interaction with immunophilins. FKBP12, and cyclophilins A and B were found to be present in normal human blood in the ranges 0.8-1.7, 1.4-2.3 and 2.4-3.1 nM, respectively, as demonstrated by PPIase activity measurements and western blot analysis. Cyclophilins A and B levels in serum of HIV-1-infected individuals were increased 3.6-fold and 1.6-fold. Due to the interaction of immunophilins with V3 loop peptides and with the envelope protein gp120, a role of immunophilins in HIV pathogenesis as conformases or docking mediators seems possible, since immunophilin receptors on cell membranes and immunophilin-related virulence factors of pathogens have been identified.

Amino Acid Isomerases↗
PubMed1997

Maid: a maternally transcribed novel gene encoding a potential negative regulator of bHLH proteins in the mouse egg and zygote.

We isolated an abundant novel cDNA SSEC-8 from a subtraction cDNA library enriched for maternal transcripts that are still present in the mouse 2 cell stage embryo. This gene is evolutionarily conserved and maps to the distal region of mouse chromosome 2. The deduced polypeptide sequence of the encoded protein contains a conserved helix-loop-helix (HLH) motif without a basic DNA binding domain, suggesting that it functions as a negative regulator of basic (b) HLH transcription factors. Gel mobility shift assays show that in vitro translated protein prevents the E12/MyoD bHLH dimer from binding to DNA. Also, transient overexpression of this protein in C2C12 cells reduced the transcription of a CAT-reporter regulated by an E12/MyoD driven enhancer. The 3'-UTR contains consensus sequences of cytoplasmic polyadenylation elements (CPE's), and the length of its poly (A) tail changes during oocyte maturation, indicating that its expression is controlled by timely activation of translation. This new gene, Maid, models the translational and transcriptional regulation of gene expression during the transition from gamete to embryo.

Amino Acid Sequence↗
PubMed1990

Insulin enhancer binding protein has helix-loop-helix structure.

Insulin gene expression is restricted to pancreatic B cells and the 5' flanking region is responsible for the tissue specificity. The GCCATCTG motif in this region of the rat insulin 1 gene functions as an enhancer for insulin transcription. A cDNA coding for a GCCATCTG motif-binding protein (IEBP1) was isolated from a rat pancreatic B cell tumor lambda gt11 library. The IEBP1 protein was found to be the rat counterpart of the immunoglobulin (Ig) enhancer binding protein E12/47 having a helix-loop-helix domain. This result indicates that the Ig gene and insulin gene employ the same (or a similar) binding protein as a part of their transcriptional apparatus.

Amino Acid Sequence↗
PubMed1999

Activation of constitutive 5-hydroxytryptamine(1B) receptor by a series of mutations in the BBXXB motif: positioning of the third intracellular loop distal junction and its G(o)alpha protein interactions.

Constitutive activity of the recombinant human 5-hydroxytryptamine(1B) (5-HT(1B)) receptor (RC code 2.1.5HT.01.B) was investigated by mutagenesis of the BBXXB motif (in which B represents a basic residue and X a non-basic residue) located in the C-terminal portion of the third intracellular loop. In contrast with wild-type 5-HT(1B) receptors, three receptor mutants (Thr(313)-->Lys, Thr(313)-->Arg and Thr(313)-->Gln) increased their agonist-independent guanosine 5'-[gamma-[(35)S]thio]triphosphate binding response by 26-41%. This activity represented approx. 30% of the maximal response induced by 5-HT and could be reversed by the inverse agonists methiothepin and 3-(3-dimethylaminopropyl)-4-hydroxy-N-(4-pyridin-4-yl phenyl)-benzenamide (GR 55562). Enhanced agonist-independent and agonist-dependent 5-HT(1B) receptor activation was provided by co-expression of a pertussis toxin-resistant rat G(o)alpha Cys(351)-->Ile protein. The wild-type 5-HT(1B) receptor displayed a doubling in basal activity, whereas a spectrum of enhanced basal activities (313-571%) was observed with a series of diverse amino acid substitutions (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, glutamine and arginine) at the 5-HT(1B) receptor position 313 in the presence of pertussis toxin (100 ng/ml). Consequently, the constitutive 5-HT(1B) receptor activity can be modulated by the mutation of Thr(313), and displays a graded range between 11% and 59% of maximal 5-HT(1B) receptor activation by 5-HT. No clear pattern is apparent in the framework of traditionally cited amino acid characteristics (i.e. residue size, charge or hydrophobicity) to explain the observed constitutive activities. The various amino acid substitutions that yielded enhanced activity are unlikely to make similar intramolecular interactions within the 5-HT(1B) receptor. It is hypothesized that the positioning of the junction between the third intracellular loop and transmembrane domain VI is altered by mutation of Thr(313) in the BBXXB motif and thereby unmasks G(alpha)-protein interaction points.

Amino Acid Motifs↗
PubMed2001

An IgH enhancer that drives transcription through basic helix-loop-helix and Oct transcription factor binding motifs. Functional analysis of the E(mu)3' enhancer of the catfish.

The transcriptional enhancer (E(mu)3') of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian E(mu) enhancer in both location and structure. It occurs between the mu and delta genes and contains numerous transcription factor binding sites, predominantly octamer and muE5 motifs of consensus and variant sequences. It lacks the classical muA-muE3(CBF)-muB core array of binding motifs seen within mammalian IgH E(mu) enhancers. To determine the functionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of consensus and variant octamer motifs (individually or in multiples) decreased enhancer function, mutation of a single consensus muE5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus muE5 site, combined with mutations of certain octamer sites, destroyed function in catfish B cells. Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal E(mu)3' enhancer (a) contains a consensus muE5 site and two octamer sites, (b) is B cell-specific, and (c) is active across species. The dependence of an Ig enhancer on sites that bind basic helix-loop-helix and Oct transcription factors has not previously been observed and confirms large differences in structure and function between fish and mammalian IgH enhancers.

Amino Acid Motifs↗
PubMed2004

The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies.

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight a-helices, 12 b-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

Amino Acid Oxidoreductases↗
PubMed1998

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins.

Adenosine Triphosphate↗
PubMed1990

Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor.

The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.

Amino Acid Sequence↗
PubMed1996

Various modes of basic helix-loop-helix protein-mediated regulation of murine leukemia virus transcription in lymphoid cell lines.

The transcriptionally regulatory regions of the lymphomagenic Akv and SL3-3 murine leukemia retroviruses (MLVs) contain two types of E-box consensus motifs, CAGATG. One type, EA/S, is located in the upstream promoter region, and the other, E(gre), is located in a tandem repeat with enhancer properties. We have examined the requirements of the individual E-boxes in MLV transcriptional regulation. In lymphoid cell lines only, the E(gre)-binding protein complexes included ALF1 or HEB and E2A basic helix-loop-helix proteins. Ectopic ALF1 and E2A proteins required intact E(gre) motifs for mediating transcriptional activation. ALF1 transactivated transcription of Akv MLV through the two E(gre) motifs equally, whereas E2A protein required the promoter-proximal E(gre) motif. In T- and B-cell lines, the E(gre) motifs were of major importance for Akv MLV transcriptional activity, while the EA/S motif had some effect. In contrast, neither E(gre) nor EA/S motifs contributed pronouncedly to Akv MLV transcription in NIH 3T3 cells lacking DNA-binding ALF1 or HEB and E2A proteins. The Id1 protein was found to repress ALF1 activity in vitro and in vivo. Moreover, ectopic Id1 repressed E(gre)-directed but not EA/S-directed MLV transcription in lymphoid cell lines. In conclusion, E(gre) motifs and interacting basic helix-loop-helix proteins are important determinants for MLV transcriptional activity in lymphocytic cell lines.

3T3 Cells↗
PubMed1998

Effect of mutations in the small envelope protein of hepatitis B virus on assembly and secretion of hepatitis delta virus.

The gene coding for the small (S) envelope protein of hepatitis B virus was mutated to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. This study was focused on a domain of the S protein that is exposed in the cytoplasm during synthesis and thereby represented a good candidate for interaction with the viral nucleocapsid during virion assembly. The mutations consisted of deletion/insertions spanning the entire cytosolic domain of S between amino acid residues 24 and 80. Although the expression of mutants clustered between residues 59 and 80 could not be obtained, we demonstrated that a large part of the cytosolic loop, from residues 29-47 and 49-59, does not contain motifs essential for production of hepatitis B virus subviral particles or HDV virions. However, deletion of residues 24-28 led to the synthesis of S protein mutant, which was competent for secretion of subviral particles but deficient for production of HDV. We concluded that the sequence between Arg-24 and Ile-28 located at the carboxyl boundary of the transmembrane signal I for S contains residue or residues important for HDV particle assembly.

Amino Acid Sequence↗
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↗