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At least 73 records · Page 4Linked to original sources
PubMed2001

Adenovirus-mediated gene therapy specific for small cell lung cancer cells using a Myc-Max binding motif.

Recent clinical trials of gene therapy for patients with thoracic cancers have shown that these treatments were well tolerated with minimal side effects and that we need to further enhance specificity as well as efficiency of gene transfer to target cancer cells. We previously reported that myc-overexpressing SCLC cell lines became selectively sensitive to ganciclovir (GCV) by transducing the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the Myc-Max response elements (a core nucleotide sequence, CACGTG) and that this construct (MycTK) could be utilized to develop a novel treatment against chemo-radio-resistant SCLC. We report here in vivo antitumor effects and safety of a replication-deficient adenoviral vector containing the Myc-Max binding motif (AdMycTK) on SCLC cells. In vitro infection with AdMycTK selectively rendered myc-overexpressing SCLC cell lines 63- to 307-fold more sensitive to GCV. In vivo injections with AdMycTK followed by GCV administration markedly suppressed the growth of myc-overexpressing tumors established in the subcutis or in the peritoneal cavity of athymic mice. On the other hand, infection with AdMycTK did not significantly affect either in vitro GCV sensitivity of the cells expressing very low levels of the myc genes or the growth of their subcutaneous tumors. Moreover, we observed no apparent side effects of this treatment including body weight loss or biochemical abnormalities in contrast to the treatment with AdCATK that conferred strong but nonspecific expression of the HSV-TK gene. These results suggested that AdMycTK/GCV therapy is effective on SCLC patients whose tumors overexpress myc family oncogenes.

Adenoviridae↗
PubMed2000

Molecular analysis of PTEN and MXI1 in primary bladder carcinoma.

Loss of heterozygosity (LOH) on 10q is associated with late-stage events in urothelial neoplastic progression. The tumor suppressor gene PTEN, which is mutated or homozygously deleted in numerous cancers, maps to a region of 10q within the reported region of minimal loss in bladder tumors. In two recent studies alterations in the PTEN gene occur at a low frequency in bladder tumors displaying 10q LOH. We have screened 35 late-stage bladder tumors for mutations in PTEN and MXI1, both genes mapping to chromosome 10q. Using single-strand conformation polymorphism analysis, we identified 6 tumors harboring mutations in PTEN and 2 additional tumors displaying homozygous deletion at this locus. No MXI1 mutations were identified within the same tumor panel. Of 16 bladder tumor cell lines analyzed, 2 showed homozygous deletion of PTEN and 3 harbored point mutations resulting in an amino acid change. Two cell lines harbored missense mutations in MXI1. We report a significantly higher frequency of PTEN alterations in bladder carcinoma (23%) than was previously recorded, with no accompanying mutations in the MXI1 gene.

Amino Acid Substitution↗
PubMed2000

Helix-loop-helix transcription factors in electrically active and inactive skeletal muscles.

The muscle-specific helix-loop-helix (HLH) transcription factors myoD, myogenin, MRF4, and myf-5 are called the muscle regulatory factor family (MRF). Levels of MRFs are strongly regulated by muscle electrical activity and are thought to control downstream genes that are important for muscle phenotype such as the acetylcholine receptor (AChR) and possibly genes connected to muscle metabolic properties. The MRFs interact with ubiquitously expressed HLH factors such as E-proteins and Id-proteins to form heterodimers. In the present paper, we report the effects of paralysis obtained by nerve impulse block with tetrodotoxin (TTX) and denervation on messenger ribonucleic acid (mRNA) levels for Id-1, E47, myogenin, AChR alpha-subunit and beta-actin. Both Id-1 and E47 showed twofold increases in absence of nerve evoked electrical activity. These changes in the ubiquitously expressed HLH factors might have important functional implications for downstream gene expression, but in comparison, myogenin mRNA was increased 10-fold. We conclude that myogenin and the other muscle-specific MRFs remain the transcription factors with the strongest activity dependence that has so far been described in muscle.

Animals↗
PubMed2002

Multifunctional folded polypeptides from peptide synthesis and site-selective self-functionalization--practical scaffolds in aqueous solution.

The site selectivity of His-mediated lysine and ornithine side-chain acylation in a designed four-helix bundle protein scaffold was mapped by reaction of several polypeptides with one equivalent of mono-p-nitrophenyl fumarate in aqueous solution at pH 5.9 and room temperature followed by an analysis of the degrees and sites of acylation. Integration of the HPLC chromatograms of the acylated polypeptides and trypsin cleavage followed by mass spectrometry analysis of the tryptic fragments provided the experimental evidence. Based on these and previously published results a strategy was developed for the site-selective and stepwise incorporation of three residues into a folded polypeptide in aqueous solution at room temperature. The first substituent was incorporated by reaction of a 1.7-fold excess of the corresponding active ester with the polypeptide at pH 5.9, the second substituent was introduced in a 3-fold excess after the pH value was raised to 8, and the third substituent was incorporated by reaction of a 10-fold excess with the polypeptide at pH 5.9. No intermediate steps of purification were taken and the overall yield was 30 % or more. Examples of the substituents included are carbohydrates, an enzyme inhibitor, a fumarate, and an acetate group. The introduction of different substituents into three individually addressable positions in a stepwise, efficient, and controllable reaction demonstrates that designed folded polypeptides are practically useful scaffolds that can be functionalized by using very simple chemistry in aqueous solution. Predicted applications include designed receptors, biosensors, and molecular devices.

Acylation↗
PubMed2002

Setting the stage for new catalytic functions in designed proteins--exploring the imine pathway in the efficient decarboxylation of oxaloacetate by an Arg-Lys site in a four-helix bundle protein scaffold.

Fourteen 42-residue polypeptides have been designed to identify reactive sites for the catalysis of the decarboxylation of oxaloacetate, a chemical transformation that proceeds through the formation of an imine intermediate. The sequences fold into helix-loop-helix motifs and dimerize to four-helix bundles. The catalytically active lysine residues were incorporated in several surface exposed positions, but also in positions characterised by hydrophobic properties to reduce their pKa values. The molecular environments of the Lys residues were systematically varied, to find which residues were able to stabilise and bind the imine intermediate in the decarboxylation reaction. A two-residue Arg-Lys site formed the main component of the reactive site of the helix-loop-helix dimer Decarb-K34_R33, which obeyed saturation kinetics in catalysing the reaction with a kcat/KM of 0.59 M-1S-1. The rate constant measured was nearly three orders of magnitude larger than the second-order rate constant of the butylamine-catalysed reaction (0.0011 M-1S-1), and four orders of magnitude larger than the pseudo first-order rate constant of the uncatalyzed reaction (1.3 x 10(-5) s(-1)). The sequence of Decarb-K34_R33 contained only a single lysine residue. It was flanked by an arginine in the preceding position in the sequence. A flanking Arg residue provided more efficient catalysis than a flanking Lys or Gln residue. Arginines in flanking positions in the helix, in positions four residues before or after the Lys in the sequence, are not as important in catalysis as the Arg of the Arg-Lys pair. The effect of pKa on the catalytic efficiency of the Lys residue in the decarboxylation reaction is well known. The identification of the role of the flanking Arg residue in catalysing decarboxylation, its optimal position, and the importance of conformational stability reported here sets the stage for developing a number of catalytic systems that depend on the formation of imine intermediates, but that lead to different reaction products.

Amino Acid Sequence↗
PubMed2002

Control of lysine reactivity in four-helix bundle proteins by site-selective pKa depression: expanding the versatility of proteins by postsynthetic functionalization.

Five 42-residue polypeptides have been designed to fold into hairpin helix-loop-helix motifs that dimerize to form four-helix bundles, and to serve as protein scaffolds for the elucidation at the molecular level of the principles that control and fine-tune lysine and ornithine reactivities in a protein context. Site-selective control of Lys and Orn reactivity provides a mechanism for addressing directly individual residues and is a prerequisite for the site-selective functionalization of folded proteins. Several lysine and one ornithine residues were introduced on the surface and in the hydrophobic core of the folded motif. The reactivity of each residue was determined by measuring the degree of acylation of the trypsin cleaved fragments by HPLC and mass spectrometry. The most reactive residues were Orn34 and Lys19, both of which were located in d positions in the heptad repeat, and therefore in hydrophobic environments. Upon reaction of the helix-loop-helix dimer KA-I with one equivalent of mono-p-nitrophenyl fumarate, Orn34 was acylated approximately three times more efficiently than Lys19, whereas Lys10 (b position), Lys15 (g position), and Lys33 (c position) remained unmodified. In the sequence KA-I-A(15) Lys15 was replaced by an alanine residue and the selectivity of Orn34 over Lys19 increased to approximately a factor of six, probably because Lys15 had the capacity to reduce the pK(a) value of Lys19 and 85 % of site-selectively monoacylated product was obtained. The pH dependence of the acylation reaction was determined and showed that the pK(a) of the reactive residues were 9.3, more than a pK(a) unit below the magnitude of the corresponding residue in a solvent exposed position. Introducing Lys and Orn residues into a or d positions of the heptad repeat therefore serves as a mechanism of depressing their pK(a) to increase their reactivity site selectively. Extensive NMR and CD spectroscopic analyses showed that the sequences fold according to prediction.

Acylation↗
PubMed2000

The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development.

E2A, HEB and E2-2 genes encode a group of basic helix-loop-helix (bHLH) transcription factors that are structurally and functionally similar. Deletion of the genes encoding either of these proteins leads to early lethality and a block in B lymphocyte development. Evidence for a function in T lymphocyte development has, however, only been reported for E2A and HEB. To further elucidate the role of E2-2 at developmental stages that have proven difficult to study due to the early lethality phenotype of mice defective in E2-2, we generated and analyzed mice conditionally mutated in the E2-2 gene. These mice are mosaic with respect to E2-2 expression, consisting of cells with either one functional and one null mutated E2-2 allele or two null mutated alleles. Using this experimental model, we find that cells with a homozygous null mutated E2-2 gene are under-represented in B lymphocyte as well as T lymphocyte cell lineages as compared to other hematopoietic or non-hematopoietic cell lineages. Our data suggests that E2-2 deficiency leads to a partial block in both B and T lymphocyte development. The block in T cell development appears to occur at an early stage in differentiation, since skewing in the mosaicism is observed already in CD4+8+ double-positive thymocytes.

Alleles↗
PubMed2002

The pT alpha promoter and enhancer are direct targets for transactivation by E box-binding proteins.

The pre-T alpha (pT alpha) gene encodes a protein that forms the pre-TCR together with the newly expressed TCR beta chain during a defined stage of early T cell development. The restricted expression of this gene has been suggested to depend on a promoter and an enhancer element containing E boxes, which are potential binding sites for basic helix-loop-helix (bHLH) proteins. We here report that increased expression of E47 and E12 mRNA in CD4(-)CD8(-)TCR(-) thymocytes correlates with the initiation of pT alpha transcription. Furthermore, electrophoretic mobility shift assays suggested that E47 binds directly to both the pT alpha promoter and enhancer in vitro. Ectopic expression of E47 in non-lymphoid HeLa cells resulted in a dose- and template-dependent activation of these control elements. In addition to this, ectopic expression of E47 in combination with the ets-protein Pu.1 and/or the Pu.1-interacting protein(Pip-1), resulted in a synergistic effect on the activity of the pT alpha control elements. Thus, we suggest that E47, Pu.1 and Pip-1 are directly involved in the regulation of pT alpha expression in early T cell development.

Animals↗
PubMed1995

Expression of the helix-loop-helix gene Id3 during murine embryonic development.

The family of dominant-negative helix-loop-helix (dnHLH) transcriptional modulators consists of four mammalian genes known to date: Id1, Id2, Id3 (Id3 was formerly designated HLH462 or HEIR1 or HLH1R21), and Id4. We have performed a detailed study of Id3 expression in mouse embryos in order to gain insight into the possible developmental control functions of this gene and to compare its expression to that of other mammalian dnHLH genes. Employing RNA in situ hybridization on sectioned mouse embryos of gestational stages E9.5-17.5 and neonatal head, we generally detected a high abundance of transcripts at early stages that gradually declined at most sites toward E15.5. Expression of Id3 in embryos was detected in brain, spinal cord, olfactory system, branchial arches, limbs, sclerotome, endocardiac cushions, the outer lining of the gut, lung, retina, the collecting system of the kidney, and in tooth anlagen. Although the abundance of mRNA decreased toward later stages in most tissues, it remained high in teeth and kidney. This expression pattern suggests that Id3 functions both in undifferentiated tissues and in organs which are in the process of differentiation. When compared to the expression of other dnHLH genes, it becomes obvious that the pattern of Id3 mainly coincides with that of Id1. This may reflect a partial redundancy in gene function. Furthermore, previous experiments suggested a mutually exclusive expression of the proto-oncogene N-myc an Id3. The results of the present study confirm these data.

Animals↗
PubMed1995

Expression of M-twist during postimplantation development of the mouse.

The murine homologue of the Drosophila twist gene has been shown to be essential for head mesenchyme formation and to act as an inhibitor of muscle differentiation. This paper presents a detailed analysis of M-twist expression patterns from day 7 post coitum (p.c.) to day 18 p.c., indicating a more general function of the M-twist gene. At day 7 p.c., M-twist is expressed in the mesoderm outside the primitive streak. Later M-twist message is predominantly found in the somites, the head mesenchyme, the branchial arches, the limbs, and in the mesenchyme underneath the epidermis. Beginning at day 8 p.c., M-twist is mainly expressed in undifferentiated cells committed to muscle and cartilage development: this expression is consistent with a suggested role of M-twist in inhibiting overt muscle and cartilage differentiation. However, during organogenesis, M-twist is expressed in several areas of mesenchyme-epithelia interactions, suggesting additional tissue specific functions.

Animals↗
PubMed2003

The role of class I HLH genes in neural development--have they been overlooked?

Helix-loop-helix (HLH) genes encode for transcription factors affecting a whole variety of developmental programs, including neurogenesis. At least seven functional classes (denoted I to VII) of HLH genes exist, (1) with subclass members exhibiting homo- and heterodimerisation for proper DNA binding and transcriptional regulation of downstream target genes. In the developing nervous system, members of class II, V and VI have been most extensively studied concerning their roles in neural programming. In contrast, the function of class I proteins (such as E12 and E47) is poorly defined and the orthodox view relegates them to general dimerisation duties that are necessary for the activity of the other classes. However, closer scrutiny of the spatiotemporal expression patterns of class I factors, combined with recent biochemical evidence, would suggest that class I proteins possess specific functions during early neural differentiation. This essay supports this possibility, in addition to putting forward the hypothesis that, outside their general dimerisation activity, class I genes have independent roles in regulating neurogenesis.

Animals↗
PubMed1991

The helix-loop-helix domain: a common motif for bristles, muscles and sex.

Three apparently unrelated developmental processes--mammalian myogenesis, the choice of neural fate and sex determination in Drosophila--are controlled by a common mechanism. Most of the genes governing these processes encode transcriptional factors that contain the helix-loop-helix (HLH) motif. This domain mediates the formation of homo- or heterodimers that specifically bind to DNA through a conserved basic region adjacent to the HLH motif. Dimers differ in their affinity for DNA and in their ability to activate transcription from HLH binding-site containing promoters. In addition, the activity of HLH proteins is inhibited by dimerization with another class of HLH proteins that lack a basic domain entirely or have an altered one. These structural properties provide a molecular mechanism to explain the synergistic and antagonistic functional relations among the HLH encoding genes that control several developmental pathways.

Amino Acid Sequence↗
PubMed1997

The SCL/TAL1 gene: roles in normal and malignant haematopoiesis.

SCL (TAL1/TCL5) is a member of the helix-loop-helix family of transcription factors. Originally identified because of its involvement in a tumour-specific chromosomal translocation, overexpression of the SCL gene is the most common molecular abnormality found in human T cell leukaemia. Transgenic models have now formally demonstrated that overexpression of SCL within the T cell lineage is capable of causing malignant transformation. Gene targeting experiments have revealed that the SCL gene is crucial for the development of primitive haematopoiesis in the mouse and is also required for the generation of all adult haematopoietic lineages. Biochemical studies have indicated some of the proteins which interact with SCL and this has refined the hypotheses concerning the mechanisms by which SCL plays a role in leukaemogenesis and haematopoietic development.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Synthesis and conformational properties of protein fragments based on the Id family of DNA-binding and cell-differentiation inhibitors.

Id proteins are dominant negative regulators of the helix-loop-helix (HLH) transcription factors and are important during development, especially by preventing cell differentiation while inducing cell proliferation. In contrast, they are poorly expressed in healthy adults but are found in several tumor types. The Id HLH motif is responsible for the inhibitory activity, whereas not much is known about the role of the N- and C-termini. In the presented work, synthetic peptides reproducing the HLH, the N-terminal region, and the C-terminal region of the Id proteins were characterized by CD. The four HLH sequences built highly stable helical conformations, whereas the N- and C-termini were unstructured, with the exception of an alanine-rich fragment preceding the Id4 HLH motif. Deletion of the loop connecting the two helices led to helix destabilization for all four Id HLH peptides. In addition, modifications of the amino acid composition within the hydrophobic face of the helices of the Id1 HLH peptide induced conformational changes, mostly associated with loss of helix content. Moreover, a fragment containing the helix-2 and the C-terminus of the Id1 protein did not show any helical character. Therefore, both the helix propensity and stability of the HLH domain were shown to be strongly dependent on favorable interhelical contacts. In contrast, it is suggested that the regions beyond this domain could rather play a destabilizing role, for example, by increasing the flexibility of the folded protein.

Amino Acid Motifs↗
PubMed2006

Fourier transform infrared spectroscopic study on the Ca2+ -bound coordination structures of synthetic peptide analogues of the calcium-binding site III of troponin C.

The coordination structures of Ca(2+) ion bound to synthetic peptide analogues of the calcium-binding site III of rabbit skeletal muscle troponin C (TnC) were investigated by Fourier transform infrared (FTIR) spectroscopy. The region of the COO(-) antisymmetric stretching vibration provides information about the coordination modes of a COO(-) group to a metal ion. The 34-residue peptide corresponding to the EF hand motif (helix-loop-helix) showed a band at 1552 cm(-1) in the Ca(2+)-loaded state, indicating that the side-chain COO(-) group of Glu at the 12th position serves as a ligand for Ca(2+) in the bidentate coordination mode. On the other hand, the 13-residue peptide (Ac-DRDADGYIDAEEL-NH(2)) containing the Ca(2+)-binding site III (DRDADGYIDAEE) did not show such spectral patterns in the Ca(2+)-loaded state, meaning that shorter synthetic peptide corresponding to the site III has less or no affinity for Ca(2+). It was found that the 17-residue peptide (Ac-DRDADGYIDAEELAEIF-NH(2)) is the minimum peptide necessary for the interaction of side-chain COO(-)of Glu at the 12th position with Ca(2+) in the bidentate coordination mode. We discuss the relationship between the amino acid length of synthetic peptide analogues and the formation of Ca(2+)-bound coordination structure.

Amino Acid Sequence↗
PubMed2003

Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif-implications for catalyst design.

In our search for a catalyst for the transamination reaction of aspartic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reaction that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18500 deg x cm(2) dmol(-1). Upon addition of 6 mm l-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19900 deg x cm(2) dmol(-1). The corresponding mean residue ellipticities of T-16 were -21200 deg x cm(2) dmol(-1) and -24000 deg x cm(2) dmol(-1). These results show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The (1)H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the (31)P NMR resonance linewidth. The catalytic efficiencies of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show for the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.

Amino Acid Sequence↗