Translational control of human p53 expression in yeast mediated by 5′-UTR–ORF structural interaction

We have expressed human p53 cDNA in the yeast Saccharomyces cerevisiae and shown that the level of production and the length of the p53 protein depends on the presence of untranslated mRNA regions (UTRs). The expression of the ORF alone leads to a p53 protein of correct size (53 kDa) that accumulates to high levels, concomitantly with the presence of a small amount of a p40 protein (40 kDa). However, when either the entire 5′-UTR and a part of the 3′- or 5′-UTR alone is used, this leads to the production of small amounts of the 40 kDa truncated form only. The p40 protein corresponds to a truncated form of p53 at the C-terminal extremity since it reacts only with a monoclonal antibody recognising the N-terminal epitope. This effect on the amount and length of p53 protein had no correlation at the mRNA level, suggesting that translational control probably occurs through the 5′-UTR. We propose a model of structural interaction between this UTR and a part of the ORF mRNA for the regulation of p53 expression in this heterologous context.

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (ΨSI) catalyzes the conversion of uridine to Ψ at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. ΨSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and ΨSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for ΨS.

Interaction of 5-lipoxygenase with cellular proteins

5-Lipoxygenase (5LO) plays a pivotal role in cellular leukotriene synthesis. To identify proteins interacting with human 5LO, we used a two-hybrid approach to screen a human lung cDNA library. From a total of 1.5 × 107 yeast transformants, nine independent clones representing three different proteins were isolated and found to specifically interact with 5LO. Four 1.7- to 1.8-kb clones represented a 16-kDa protein named coactosin-like protein for its significant homology with coactosin, a protein found to be associated with actin in Dictyostelium discoideum. Coactosin-like protein thus may provide a link between 5LO and the cytoskeleton. Two other yeast clones of 1.5 kb encoded transforming growth factor (TGF) type β receptor-I-associated protein 1 partial cDNA. TGF type β receptor-I-associated protein 1 recently has been reported to associate with the activated form of the TGF β receptor I and may be involved in the TGF β-induced up-regulation of 5LO expression and activity observed in HL-60 and Mono Mac 6 cells. Finally, three identical 2.1-kb clones contained the partial cDNA of a human protein with high homology to a hypothetical helicase K12H4.8 from Caenorhabditis elegans and consequently was named ΔK12H4.8 homologue. Analysis of the predicted amino acid sequence revealed the presence of a RNase III motif and a double-stranded RNA binding domain, indicative of a protein of nuclear origin. The identification of these 5LO-interacting proteins provides additional approaches to studies of the cellular functions of 5LO.

Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor.

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme⋅substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

Enhancer-dependent interaction between 5' and 3' splice sites in trans.

Splice-site selection and alternative splicing of nuclear pre-mRNAs can be controlled by splicing enhancers that act by promoting the activity of upstream splice sites. Here we show that RNA molecules containing a 3' splice site and enhancer sequence are efficiently spliced in trans to RNA molecules containing normally cis-spliced 5' splice sites or to normally trans-spliced spliced leader RNAs from lower eukaryotes. In addition, we show that this reaction is stimulated by (Ser + Arg)-rich splicing factors that are known to promote protein-protein interactions in the cis-splicing reaction. Thus, splicing enhancers facilitate the assembly of protein complexes on RNAs containing a 3' splice site, and this complex is sufficiently stable to functionally interact with 5' splice sites located on separate RNAs. This trans-splicing is mediated by interactions between (Ser + Arg)-rich splicing factors bound to the enhancer and general splicing factors bound to the 5' and 3' splice sites. These same interactions are likely to play a crucial role in alternative splicing and splice-site selection in cis.

Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand–DNA complexes: The interaction between DNA and calichearubicin B

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (φ) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand–DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin γ1I. The unwinding angle for CRB calculated by this method is −5.3 ± 0.5°. Its Ka value is 0.20 × 106 M−1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Translation of cytochrome f is autoregulated through the 5′ untranslated region of petA mRNA in Chlamydomonas chloroplasts

A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing. Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits. This autoregulation is mediated by an interaction, either direct or indirect, between the 5′ untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein. This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented. When expressed from a chimeric mRNA containing the atpA 5′ untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation. Conversely, the level of antibiotic resistance conferred by a chimeric petA-aadA-rbcL gene was shown to depend on the state of assembly of cytochrome b6f complexes and on the accumulation of the C-terminal domain of cytochrome f. We discuss the possible ubiquity of the CES process in organellar protein biogenesis.

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.

Preferential interaction of the his pause RNA hairpin with RNA polymerase β subunit residues 904–950 correlates with strong transcriptional pausing

RNA secondary structures (hairpins) that form as the nascent RNA emerges from RNA polymerase are important components of many signals that regulate transcription, including some pause sites, all ρ-independent terminators, and some antiterminators. At the his leader pause site, a 5-bp-stem, 8-nt-loop pause RNA hairpin forms 11 nt from the RNA 3′ end and stabilizes a transcription complex conformation slow to react with NTP substrate. This stabilization appears to depend at least in part on an interaction with RNA polymerase. We tested for RNA hairpin interaction with the paused polymerase by crosslinking 5-iodoUMP positioned specifically in the hairpin loop. In the paused conformation, strong and unusual crosslinking of the pause hairpin to β904–950 replaced crosslinking to β′ and to other parts of β that occurred in nonpaused complexes prior to hairpin formation. These changes in nascent RNA interactions may inhibit reactive alignment of the RNA 3′ end in the paused complex and be related to events at ρ-independent terminators.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate

The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+ CCR-5+ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

Inhibition of the electrostatic interaction between β-amyloid peptide and membranes prevents β-amyloid-induced toxicity

The accumulation of β-amyloid peptides (Aβ) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated Aβ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent Aβ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of Aβ1-42 and the toxic fragment Aβ25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic β-sheet conformation by Aβ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of Aβ1-40 and Aβ25-35 to negatively charged lipid vesicles and Aβ induced cell toxicity. These results suggest that Aβ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.

Interaction of human apurinic endonuclease and DNA polymerase β in the base excision repair pathway

Mutagenic abasic (AP) sites are generated directly by DNA-damaging agents or by DNA glycosylases acting in base excision repair. AP sites are corrected via incision by AP endonucleases, removal of deoxyribose 5-phosphate, repair synthesis, and ligation. Mammalian DNA polymerase β (Polβ) carries out most base excision repair synthesis and also can excise deoxyribose 5-phosphate after AP endonuclease incision. Yeast two-hybrid analysis now indicates protein–protein contact between Polβ and human AP endonuclease (Ape protein). In vitro, binding of Ape protein to uncleaved AP sites loads Polβ into a ternary complex with Ape and the AP-DNA. After incision by Ape, only Polβ exhibits stable DNA binding. Kinetic experiments indicated that Ape accelerates the excision of 5′-terminal deoxyribose 5-phosphate by Polβ. Thus, the two central players of the base excision repair pathway are coordinated in sequential reactions.