Characterizing the interaction of the mammalian eIF4E-related protein 4EHP with 4E-BP1

Eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) represses translation initiation by binding to eukaryotic initiation factor 4E (eIF4E). 4E-BP1 also binds to the eIF4E homologous protein (4EHP). We show that eIF4E-binding mutants of 4E-BP1 (Y54A and L59A) fail to form heterodimeric complexes with wild-type 4EHP. In addition, the W95A mutant of 4EHP, similar to a homologous mutation in eIF4E, inhibits its binding to wild-type 4E-BP1. Interestingly, 4EHP over-expression instigates a negative feedback loop that inhibits upstream signaling to 4E-BP1 and ribosomal protein S6 kinase 1 (S6K1) whereas the 4E-BP1-binding-deficient mutant of 4EHP(W95A) was unable to trigger this feedback loop. Thus, the interaction of 4EHP with 4E-BP1 is necessary for this observed impaired signaling to 4E-BP1 and S6K1.     

Formation of 4-ethoxy-4'-nitrosodiphenylamine in the reaction of the phenacetin metabolite 4-nitrosophenetol with glutathione

Phenacetin, a constituent of several analgesic and antipyretic formulations has been made responsible for a variety of toxic and carcinogenic actions. 4-Nitrosophenetol, the N-oxydation product of intermediate 4-phenetidine, forms methemoglobin and binds covalently to sulfhydryl groups of proteins and glutathione. In the reaction of 4-nitrosophenetol with glutathione and other thiols an intermediate so-called "semimercaptal" is formed from which N-(thiol-S-yl)-4-phenetidine S-oxide, N-(thiol-S-yl)-4-phenetidine and 4-phenetidine derive. Besides thiol adducts, a yellow compound is formed which was isolated as a pure crystalline product (elemental analysis) and identified by FAB-MS, EI-MS, 13C-, 1H-NMR, and UV-VIS spectroscopy as 4-ethoxy-4'-nitrosodiphenylamine. This nitrosoarene is formed by an unknown mechanism from 4-nitrosophenetol and 4-phenetidine under liberation of ethanol. In human erythrocytes this compound is easily reduced to 4-amino-4'-ethoxydiphenylamine (FAB-MS, EI-MS, 13C-NMR). During the reaction of 4-nitrosophenetol with red cells only traces of 4-ethoxy-4'-nitrosodiphenylamine were formed, whereas up to 10% appeared as the reduction product 4-amino-4'-ethoxydiphenylamine. This latter compound is unstable in red cells and is metabolized further to unidentified products.     

Macrosatellite epigenetics: the two faces of DXZ4 and D4Z4

Almost half of the human genome consists of repetitive DNA. Understanding what role these elements have in setting up chromatin states that underlie gene and chromosome function in complex genomes is paramount. The function of some types of repetitive DNA is obvious by virtue of their location, such as the alphoid arrays that define active centromeres. However, there are many other types of repetitive DNA whose evolutionary origins and current roles in genome biology remain unknown. One type of repetitive DNA that falls into this class is the macrosatellites. The relevance of these sequences to disease is clearly demonstrated by the 4q macrosatellite (D4Z4), whereupon contraction in the size of the array is associated with the onset of facioscapulohumeral muscular dystrophy. Here, I describe recent findings relating to the chromatin organization of D4Z4 and that of the X-linked macrosatellite DXZ4, highlighting the fact that these enigmatic sequences share more than a similar name.     

Epigenetic regulation of the INK4b-ARF-INK4a locus

The INK4b-ARF-INK4a locus encodes for two cyclin-dependent kinase inhibitors, p15^INK4b and p16^INK4a and a regulator of the p53 pathway, ARF. In addition ANRIL, a non-coding RNA, is also transcribed from the locus. ARF, p15^INK4b and p16^INK4a are well-established tumor suppressors which function is frequently disabled in human cancers. Recent studies showed that single nucleotide polymorphisms mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including cardiovascular disease, ischemic stroke, type 2 diabetes, frailty and Alzheimer’s disease. The INK4b-ARF-INK4a locus is regulated by Polycomb repressive complexes (PRCs), and its expression can be invoked by activating signals. Other epigenetic modifiers such as the histone demethylases JMJD3 and JHDM1B, the SWI/SNF chromatin remodeling complex and DNA methyltransferases regulate the locus interplaying with PRCs. In view of the intimate involvement of the INK4b-ARF-INK4a locus on disease, to understand its regulation is the first step for manipulate it to therapeutic benefit.     

A monoclonal antibody against the sulfidopeptide leukotrienes LTC4, LTD4 and LTE4.

A monoclonal antibody (1A-LDR1) against sulfidopeptide leukotrienes (LT) is described. The mAb shows a nearly identical detection limit of about 0.04 ng for LTC4, LTD4, LTE4 and NacLTE4 in standard fluid phase RIA. Steric modifications, however, diminish the sensitivity, as determined for the examples 5-epi-LTC4, 6-epi-LTC4, 5,6-epi-LTC4 and 11-trans-LTC4. No crossreactivity could be observed for LTB4. Crossreactions with components of the LT peptide chain such as L-cysteine or glutathione, as well as with arachidonic acid, were not detectable. In assessing the accuracy of the LT-RIA, recovery experiments with supernatants of mouse peritoneal macrophages and incubates of gastric mucosa showed a good correlation of r = 0.993 and 0.990, respectively. Results of an inhibition experiment with mouse peritoneal macrophages, incubated with several concentrations of indomethacin and nordihydroguaiaretic acid (NDGA), support the reliability of RIA and ELISA. The new LT-mAB allows an almost complete detection of peptide leukotrienes in one assay.     

Photochemical reactions of 4-thiouridine disulfide and 4-benzylthiouridine--the involvement of the 4-pyrimidinylthiyl radical.

The reactions of a disulfide and a benzylsulfide derived from 4-thiouridine were studied in aqueous acetonitrile using stationary and laser flash photolysis methods. Irradiation of the compounds results in specific cleavage of the S-S bond in the disulfide and the S-CH(2) bond in the sulfide. Identical pyrimidine-derived intermediates were observed in the transient absorption spectra (lambda(max) = 420 nm, epsilon(max) approximately 2500 M(-1) cm(-1)) recorded for both compounds in laser flash photolysis experiments. The intermediate was identified as the 4-pyrimidinylthiyl radical. Irradiation of the disulfide in the absence of oxygen gives 4-thiouridine while the sulfide under identical conditions produced, additionally, 3-benzyl-4-thiouridine as a stable photoproduct. The formation of the latter photoproduct provides evidence for the existence of the N-centered 4-thioxopyrimidynyl radical formed from the initially produced S-centered (thiyl) radical. The 4-thiouridine is formed from the radicals generated in the primary photochemical step by an H abstraction reaction from the solvent (acetonitrile) or from additives (alcohols) that were purposely added. Interestingly, in contrast to the benzylsulfide, the photoreaction of the disulfide is quenched by molecular oxygen with the concomitant formation of uridine. However it appears that uridine is not produced as a result of the reaction of the radicals with oxygen. A mechanism is proposed for the photochemical transformations of the disulfide and benzylsulfide derived from 4-thiouridine. The proposed mechanism is based on the structures of the identified stable photoproducts, the values of the photoreaction quantum yields determined under differing irradiation conditions, and the flash photolysis results.     

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.     

Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr(591) and Arg(594)) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4 alpha-PDD and different 4 alpha-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4 alpha-phorbol esters and the TM3/4 region of TRPV4.     

Chicken microtubule-associated protein 4 (MAP4): a novel member of the MAP4 family

 Chicken gizzard smooth muscle has often been used as a source of proteins of the contractile and cytoskeletal apparatus. In the present study, we isolated a hitherto unknown doublet of proteins, with apparent molecular weights of 200 kDa, from embryonic chicken gizzard and showed its association with the microtubular cytoskeleton by cosedimentation with microtubules (MTs) and by immunofluorescence staining of cultured cells. Immunoblot analysis also revealed the ubiquitous expression of this protein in all embryonic chicken tissues examined. Molecular cloning techniques allowed its identification as the chicken homologue of the microtubule-associated protein 4 (MAP4), known from mammalian species, and revealed approximately 90% of its amino acid sequence. MAP4 is the major MAP of non-neuronal tissues and cross-species comparisons clearly demonstrated its highly conserved overall structure, consisting of a basic C-terminal MT-binding region and an acidic N-terminal projection domain of unknown function. Despite these conserved features, overall sequence homologies to its mammalian counterparts are rather low and focused to distinct regions of the molecule. Among these are a conserved 18-amino acid motif, which is known to mediate binding to MTs and a part of the MT-binding domain known as the proline-rich region, which is thought to be the regulatory domain of MAP4. The N-terminal 59 amino acids are a conserved and unique feature of the MAP4 sequence and might be an indication that MAP4 performs other functions besides the enhancement of MT assembly. Accepted: 13 March 1996     

Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4

The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.     

Molecular model of the outward facing state of the human multidrug resistance protein 4 (MRP4/ABCC4)

ATP-binding cassette (ABC) transporter multidrug resistance protein 4 (MRP4, ABCC4) is involved in multidrug resistance (MDR), which is an increasing challenge to the treatment of cancer and infections. We have constructed a molecular model of ABCC4 based on the outward facing Sav1866 crystal structure using molecular modeling techniques. Amino acids reported by ICMPocketFinder to take part in substrate translocation were among others Glu103 (TMH1), Ser328 (TMH5), Gly359 (TMH6), Arg362 (TMH6), Val726 (TMH7), and Leu987 (TMH12), and their corresponding amino acids in ABCB1 (P-glycoprotein) have been reported to be involved in drug binding according to site-directed mutagenesis studies. The ABCC4 model may be used as a working tool for experimental studies on ABCC4 and design of more specific membrane transport modulating agents (MTMA).     

eIF4E/4E-BP dissociation and 4E-BP degradation in the first mitotic division of the sea urchin embryo

The mRNA's cap-binding protein eukaryotic translation initiation factor (eIF)4E is a major target for the regulation of translation initiation. eIF4E activity is controlled by a family of translation inhibitors, the eIF4E-binding proteins (4E-BPs). We have previously shown that a rapid dissociation of 4E-BP from eIF4E is related with the dramatic rise in protein synthesis that occurs following sea urchin fertilization. Here, we demonstrate that 4E-BP is destroyed shortly following fertilization and that 4E-BP degradation is sensitive to rapamycin, suggesting that proteolysis could be a novel means of regulating 4E-BP function. We also show that eIF4E/4E-BP dissociation following fertilization is sensitive to rapamycin. Furthermore, while rapamycin modestly affects global translation rates, the drug strongly inhibits cyclin B de novo synthesis and, consequently, precludes the completion of the first mitotic cleavage. These results demonstrate that, following sea urchin fertilization, cyclin B translation, and thus the onset of mitosis, are regulated by a rapamycin-sensitive pathway. These processes are effected at least in part through eIF4E/4E-BP complex dissociation and 4E-BP degradation.     

Molecular modeling of ErbB4/HER4 kinase in the context of the HER4 signaling network helps rationalize the effects of clinically identified HER4 somatic mutations on the cell phenotype

In the ErbB/HER family of receptor tyrosine kinases, the deregulation of the EGFR/ErbB1/HER1, HER2/ErbB2, and HER3/ErbB3 kinases is associated with several cancers, while the HER4/ErbB4 kinase has been shown to play an anti-carcinogenic role in certain tumors. We present molecular and network models of HER4/ErbB4 activation and signaling in order to elucidate molecular mechanisms of activation and rationalize the effects of the clinically identified HER4 somatic mutants. Our molecular-scale simulations identify the important role played by the interactions within the juxtamembrane region during the activation process. Our results also support the hypothesis that the HER4 mutants may heterodimerize but not activate, resulting in blockage of the HER4-STAT5 differentiation pathway, in favor of the proliferative PI3K/AKT pathway. Translating our molecular simulation results into a cellular pathway model of wild type versus mutant HER4 signaling, we are able to recapitulate the major features of the PI3K/AKT and JAK/STAT activation downstream of HER4. Our model predicts that the signaling downstream of the wild type HER4 is enriched for the JAK-STAT pathway, whereas downstream of the mutant HER4 is enriched for the PI3K/AKT pathway. HER4 mutations may hence constitute a cellular shift from a program of differentiation to that of proliferation.     

Catabolism of 4-Hydroxyacids and 4-Hydroxynonenal via 4-Hydroxy-4-phosphoacyl-CoAs

4-Hydroxyacids are products of ubiquitously occurring lipid peroxidation (C₉, C₆) or drugs of abuse (C₄, C₅). We investigated the catabolism of these compounds using a combination of metabolomics and mass isotopomer analysis. Livers were perfused with various concentrations of unlabeled and labeled saturated 4-hydroxyacids (C₄ to C₁₁) or 4-hydroxynonenal. All the compounds tested form a new class of acyl-CoA esters, 4-hydroxy-4-phosphoacyl-CoAs, characterized by liquid chromatography-tandem mass spectrometry, accurate mass spectrometry, and ³¹P-NMR. All 4-hydroxyacids with five or more carbons are metabolized by two new pathways. The first and major pathway, which involves 4-hydroxy-4-phosphoacyl-CoAs, leads in six steps to the isomerization of 4-hydroxyacyl-CoA to 3-hydroxyacyl-CoAs. The latter are intermediates of physiological β-oxidation. The second and minor pathway involves a sequence of β-oxidation, α-oxidation, and β-oxidation steps. In mice deficient in succinic semialdehyde dehydrogenase, high plasma concentrations of 4-hydroxybutyrate result in high concentrations of 4-hydroxy-4-phospho-butyryl-CoA in brain and liver. The high concentration of 4-hydroxy-4-phospho-butyryl-CoA may be related to the cerebral dysfunction of subjects ingesting 4-hydroxybutyrate and to the mental retardation of patients with 4-hydroxybutyric aciduria. Our data illustrate the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.