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Biomedical subjects

N Noy

Publications and source records attributed to N Noy.

At least 19 recordsLinked to original sources
PubMed2001

Critical roles of PPAR beta/delta in keratinocyte response to inflammation.

The immediate response to skin injury is the release of inflammatory signals. It is shown here, by use of cultures of primary keratinocytes from wild-type and PPAR beta/delta(-/-) mice, that such signals including TNF-alpha and IFN-gamma, induce keratinocyte differentiation. This cytokine-dependent cell differentiation pathway requires up-regulation of the PPAR beta/delta gene via the stress-associated kinase cascade, which targets an AP-1 site in the PPAR beta/delta promoter. In addition, the pro-inflammatory cytokines also initiate the production of endogenous PPAR beta/delta ligands, which are essential for PPAR beta/delta activation and action. Activated PPAR beta/delta regulates the expression of genes associated with apoptosis resulting in an increased resistance of cultured keratinocytes to cell death. This effect is also observed in vivo during wound healing after an injury, as shown in dorsal skin of PPAR beta/delta(+/+) and PPAR beta/delta(+/-) mice.

Animals↗
PubMed2001

Effects of acyclo-retinoic acid and lycopene on activation of the retinoic acid receptor and proliferation of mammary cancer cells.

The biochemical mechanisms underlying the inhibitory effects of lycopene, the main tomato carotenoid, on the growth of cancer cells are largely unknown. It has been hypothesized that lycopene derivatives may act as ligands for a nuclear receptor in analogy to retinoic acid, the hormone derived from beta-carotene. The inhibition of human mammary cancer (MCF-7) cell growth and the transactivation of the retinoic acid receptor (RAR) reporter gene by synthetic acyclo-retinoic acid, the open chain analog of retinoic acid, was compared to the effects of lycopene and retinoic acid in the same systems. Acyclo-retinoic acid activated the DR-5 retinoic acid response element with a approximately 100-fold lower potency than retinoic acid. This effect was independent of cotransfection with the RARalpha receptor. Lycopene exhibited only very modest activity in this system. In contrast to the results from the transactivation studies, acyclo-retinoic acid, retinoic acid, and lycopene inhibited cell growth with a similar potency. Preincubation with each of the three compounds slowed down cell cycle progression from G1 to S phase. In summary, acyclo-retinoic acid inhibited cancer cell growth and interacted with RAR. However, it exhibited low affinity for RAR and a correspondingly low efficacy in activating this receptor, indicating that RAR does not mediate the growth inhibitory effect of the compound. In addition, the concentrations of acyclo-retinoic acid and of lycopene required for inducing inhibition of cell growth were similar, suggesting that acyclo-retinoic acid is unlikely to be the active metabolite of lycopene.

Antineoplastic Agents↗
PubMed2001

Localization of the RAR interaction domain of cellular retinoic acid binding protein-II.

The pleiotropic effects of retinoic acid (RA) in mammalian cells are mediated by two classes of proteins: the retinoic acid receptors (RAR), and cellular retinoic acid binding proteins (CRABP-I and CRABP-II). The high conservation across species and the differential expression patterns of the two CRABPs suggest that they serve distinct biological functions. We previously showed that CRABP-II, but not CRABP-I, delivers RA to RAR through direct protein-protein interactions between the binding protein and the receptor. "Channeling" of RA between CRABP-II and RAR markedly facilitates the formation of the holo-receptor and, as a consequence, enhances the transcriptional activity of RAR in cells. Here, we localize the region of CRABP-II that mediates the interactions of this protein with RAR. Comparison between the electrostatic surface potential of CRABP-I and II revealed the presence of a sole region displaying a dramatic potential change between the two isoforms. Examination of the underlying model revealed that the change stemmed from CRABP-I/CRABP-II substitution of three spatially aligned residues E75Q, K81P, and E102 K, located on a protrusion above the entrance to the ligand binding pocket of the protein. Substituting the corresponding CRABP-II residues onto CRABP-I conferred upon this protein the ability to channel RA to RAR and to enhance the transcriptional activity of RAR in cells. Conversely, converting these amino acid residues in CRABP-II to the homologous CRABP-I residues resulted in loss of the ability of CRABP-II to interact with RAR and to augment the receptor's activity. The data demonstrate that the surface region of CRABP-II containing residues Gln75, Pro81, and Lys102 is necessary and sufficient for mediating the interactions of this protein with RAR, facilitating the formation of the holo-receptor, and enhancing the transcriptional activity of RA.

Amino Acid Sequence↗
PubMed2001

Interphotoreceptor retinoid-binding protein contains three retinoid binding sites.

Interphotoreceptor retinoid binding protein (IRBP), the major soluble protein component of the interphotoreceptor matrix, is believed to participate in the visual cycle by transporting retinoids between retinal pigment epithelium and photoreceptor cells in the eye. IRBP can associate with several chemical and isomeric forms of retinoids but displays the highest affinity towards the retinoids that are important in the visual cycle, 11-cis-retinal and all-trans-retinol. It was previously reported that IRBP can associate with 2 mol of all-trans-retinol or 2 mol of 11-cis-retinal per mol of protein. One of the retinoid binding sites, termed 'site 1', was found to display a broad ligand selectivity and to bind either all-trans-retinol or 11-cis-retinal with similar affinities. Here, the retinoid-binding properties of IRBP were further examined. The data demonstrate that IRBP contains three distinct retinoid binding sites. The promiscuous 'site 1', and two additional sites with a stricter selectivity. One of the latter sites appears to be selective towards all-trans-retinol, while the other is specific for 11-cis-retinal.

Binding Sites↗
PubMed2000

The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors.

Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH(2)-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms alpha, delta, zeta, and mu, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKC alpha by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.

3T3 Cells↗
PubMed2000

Retinoid-binding proteins: mediators of retinoid action.

Active vitamin A metabolites, known as retinoids, are essential for multiple physiological processes, ranging from vision to embryonic development. These small hydrophobic compounds associate in vivo with soluble proteins that are present in a variety of cells and in particular extracellular compartments, and which bind different types of retinoids with high selectivity and affinity. Traditionally, retinoid-binding proteins were viewed as transport proteins that act by solubilizing and protecting their labile ligands in aqueous spaces. It is becoming increasingly clear, however, that, in addition to this general role, retinoid-binding proteins have diverse and specific functions in regulating the disposition, metabolism and activities of retinoids. Some retinoid-binding proteins appear to act by sequestering their ligands, thereby generating concentration gradients that allow cells to take up retinoids from extracellular pools and metabolic steps to proceed in energetically unfavourable directions. Other retinoid-binding proteins regulate the metabolic fates of their ligands by protecting them from some enzymes while allowing metabolism by others. In these cases, delivery of a bound retinoid from the binding protein to the 'correct' enzyme is likely to be mediated by direct and specific interactions between the two proteins. One retinoid-binding protein was reported to enhance the ability of its ligand to regulate gene transcription by directly delivering this retinoid to the transcription factor that is activated by it. 'Channelling' of retinoids between their corresponding binding protein and a particular protein target thus seems to be a general theme through which some retinoid-binding proteins exert their effects.

Animals↗
PubMed2000

On the role of the carboxyl-terminal helix of RXR in the interactions of the receptor with ligand.

The retinoid X receptor (RXR), a ligand-inducible transcription factor that is activated by 9-cis-retinoic acid, is a member of the superfamily of nuclear hormone receptors. The ligand-induced transcriptional activity of nuclear receptors is coordinated by their C-terminal region termed the ligand-binding domain. Structural analyses of several nuclear receptors showed that the most dramatic ligand-induced conformational change in these proteins involves a positional shift in the receptors' C-terminal helix, termed helix 12. Consequently, in the liganded state, helix 12 is folded over the entrance to the ligand-binding pocket where it serves as a lid, and it has been proposed that this region functions to stabilize ligand binding by at least some nuclear receptors. Here, to examine the possible role of helix 12 in contributing to the association of RXR with its ligand, the equilibrium and kinetic parameters of the interactions of 9-cis-retinoic acid with RXR and with a deletion mutant lacking helix 12 were measured. Deletion of the region did not significantly alter the ligand-binding affinity of RXR at equilibrium. However, both the rate of dissociation and the rate of association of the RXR-9-cis-retinoic acid complex were significantly slower in the absence of helix 12. Taken together, these observations suggest that helix 12 of RXR facilitates both the entry and the exit of the ligand from the binding pocket without affecting the equilibrium ligand-binding affinity. The results thus point at a previously unsuspected function for this region.

Animals↗
PubMed1999

Distinct roles for cellular retinoic acid-binding proteins I and II in regulating signaling by retinoic acid.

The pleiotropic effects of retinoic acid (RA) in mammalian cells are mediated by two classes of proteins: the retinoic acid receptors (RAR) and cellular retinoic acid-binding proteins (CRABP-I and CRABP-II). Here we show that expression of CRABP-II, but not CRABP-I, markedly enhanced RAR-mediated transcriptional activation of a reporter gene in COS-7 cells. The equilibrium dissociation constants of complexes of CRABP-I or CRABP-II with RA were found to differ by 2-fold. It is thus unlikely that the distinct effects of the two proteins on transactivation stem from differential ligand-binding affinities. The mechanisms by which RA transfers from the CRABPs to RAR were thus investigated directly. The rate constant for movement of RA from CRABP-II, but not from CRABP-I, to RAR strongly depended on the concentration of the acceptor. The data suggest that transfer of RA from CRABP-I to RAR involves dissociation of the ligand from the binding protein, followed by association with the receptor. In contrast, movement of RA from CRABP-II to the receptor is facilitated by a mechanism that involves direct interactions between CRABP-II and RAR. These findings reveal a striking functional difference between CRABP-I and CRABP-II, and point at a novel mechanism by which the transcriptional activity of RA can be regulated by CRABP-II.

Animals↗
PubMed1999

Ligand selectivity of the peroxisome proliferator-activated receptor alpha.

Peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) are nuclear hormone receptors that play critical roles in regulating lipid metabolism. It is well established that PPARs are the targets for the hypolipidemic synthetic compounds known as peroxisome proliferators, and it has been proposed that various long-chain fatty acids and metabolites of arachidonic acid serve as the physiological ligands that activate these receptors in vivo. However, a persistent problem is that reported values of the equilibrium dissociation constants (Kds) of complexes of PPARs with these ligands are in the micromolar range, at least an order of magnitude higher than the physiological concentrations of the ligands. Thus, the identity of the endogenous ligands for PPAR remains unclear. Here we report on a fluorescence-based method for investigating the interactions of PPAR with ligands. It is shown that the synthetic fluorescent long-chain fatty acid trans-parinaric acid binds to PPARalpha with high affinity and can be used as a probe to monitor protein-ligand interactions by the receptor. Measurements of Kds characterizing the interactions of PPARalpha with various ligands revealed that PPARalpha interacts with unsaturated C:18 fatty acids, with arachidonic acid, and with the leukotriene LTB4 with affinities in the nanomolar range. These data demonstrate the utility of the optical method in examining the ligand-selectivity of PPARs, and resolve a long-standing uncertainty in understanding how the activities of these receptors are regulated in vivo.

Animals↗
PubMed1998

Auto-silencing by the retinoid X receptor.

Gene transcription is often regulated by small ligands, enabling cells to respond to external and metabolic stimuli. Of particular interest are the mechanisms by which hydrophobic hormones modulate the transcriptional activities of proteins of the nuclear receptor superfamily. It was previously shown that, in the absence of ligand, the retinoid X receptor (RXRalpha) forms tetramers with a high affinity and a pronounced positive co-operativity such that tetramers become the receptor's predominant species tat concentrations as low as 60-70 nM. It was shown further that while RXR tetramers are remarkably stable in the absence of ligand, ligand-binding induces their rapid dissociation into smaller species, dimers and monomers. Here, the functional consequences of the self-association properties of RXR were studied by examining two point mutants of RXR that displayed aberrant oligomerization behaviors. One mutant, mRXRalpha-R321A, was found to form tetramers with a wild-type affinity, but these tetramers failed to dissociate upon ligand-binding. This mutant was found to be impaired in its ability to associate with the nuclear receptor co-activator p/CIP and to activate transcription in response to the RXR ligand 9-cis-retinoic acid. The other mutant, mRXRalpha-F318A, self-associated into dimers with a wild-type affinity, but was unable to form tetramers. This mutant displayed substantial transcriptional activity even in the absence of ligand. We previously proposed, based on in vitro studies that RXR acts as an auto-silencer by sequestering itself into tetramers, and that an important role for the ligand in activating this receptor is to release active species, dimers and monomers, from the transcriptionally inactive tetrameric pool. The observations reported here provide in-cell evidence in support of this model and indicate that ligand induced dissociation of tetramers is the first step in signalling by RXR.

Binding Sites↗
PubMed1998

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Amino Acid Sequence↗
PubMed1998

Heterodimer formation by retinoid X receptor: regulation by ligands and by the receptor's self-association properties.

The retinoid X receptor (RXR), a nuclear receptor that is activated by 9-cis-retinoic acid (9cRA), can regulate transcription as a homodimer or as a heterodimer with numerous other receptors. It was previously shown that, in the absence of ligand, RXR self-associates into homotetramers which are transcriptionally silent, and that ligand-binding induces dissociation of RXR tetramers into active species, dimers and monomers. Here, the implications of tetramer formation by RXR for the ability of the receptor to heterodimerize with the retinoic acid and the vitamin D receptors (RAR and VDR) were studied. In addition, the effects of cognate ligands for RXR and for RAR and VDR on formation of the respective heterodimers were examined. The data indicate that RXR subunits that are sequestered in tetramers were not available for interactions with RAR or VDR and, consequently, that in the absence of a RXR ligand, only a small fraction of this receptor became involved in heterodimers. RXR-selective ligands led to tetramer dissociation, but also inhibited the formation of heterodimers, directing a significant fraction of RXR into homodimers. Ligand binding by either heterodimerization partner significantly stabilized the respective heterodimer. Thus, maximal heterodimerization was observed in the presence of both 9cRA, acting to release active RXR species from tetramers, and the partner's cognate ligand, acting to overcome the inhibitory effect of 9cRA on heterodimer formation. These observations suggest that, by modulating protein-protein interactions within homo- and hetero-oligomers of RXR, cognate ligands control the relative distribution of potential RXR-containing complexes, thereby determining the transcriptional pathways that may be invoked under particular conditions in vivo.

Animals↗
PubMed1998

Ligand- and DNA-induced dissociation of RXR tetramers.

Unliganded bacterially expressed RXR alpha lacking the N-terminal region AB (apo-RXR alpha delta AB) was found in solution as an apparent mixture of 165 kDa tetramers and 42 kDa monomers which could be quantitatively separated by gel filtration and non-denaturing gel electrophoresis. Under identical conditions both liganded (holo-) and apo-RAR alpha delta AB were present as single monomeric species. apo-RXR alpha delta AB tetramers, as well as dimers of the apo-RXR ligand binding domain (apo-LBD), dissociated readily into monomers when exposed to their cognate ligand 9-cis retinoic acid (9c-RA). The apo-RXR alpha delta AB tetramer bound only transiently to a cognate DR1 response element, and was converted into DR1-apo-RXR alpha delta AB homodimer complexes indistinguishable from those generated by cooperative DNA binding of apo-RXR alpha delta AB monomers. In the absence of DNA, the addition of 9c-RA greatly accelerated the formation of heterodimers with the apo-RAR alpha delta AB heterodimerization partner. No RXR alpha delta AB or RAR alpha delta AB homodimers could be observed in solution, but upon mixing of the two receptor monomers stable heterodimers could be isolated which bound to DR5 response elements in a highly cooperative manner. In these heterodimers, RXR alpha delta AB interacted with its cognate ligand as efficiently as in RXR alpha delta AB homodimers. The presence of ligand did not alter the stability of RXR alpha delta AB homodimer or RXR alpha delta AB-RAR alpha delta AB heterodimer complexes on DR1 and DR5 response elements, respectively. These in vitro data support a model in which RXR tetramers could serve as an inactive pool with the dual function of: (i) rapidly supplying large amounts of RXR heterodimerization partners upon 9c-RA generation; and (ii) allowing RXR homodimer formation on "accessible" cognate response elements in the absence of 9c-RA. These events may represent a ligand-dependent regulatory mechanism controlling the availability of the promiscuous RXR dimerization partner that is engaged in multiple nuclear receptor signalling pathways.

Amino Acid Substitution↗
PubMed1997

Binding of retinol in both retinoid-binding sites of interphotoreceptor retinoid-binding protein (IRBP) is stabilized mainly by hydrophobic interactions.

Interphotoreceptor retinoid-binding protein (IRBP) is an ocular protein which is believed to participate in the visual cycle by mediating transport of retinoids between pigment epithelium and photoreceptor cells. The molecular mechanism underlying the ability of IRBP to target particular retinoids to the specific cells that are their sites of action and metabolism is not completely clear, and little information is available regarding the structure of the protein's multiple ligand-binding sites. IRBP possesses two retinoid-binding sites, and it was reported that binding of the visual chromophore, 11-cis-retinal, in one of these sites, but not in the other, is tightly regulated by another IRBP ligand, docosahexaenoic acid (Chen, Y., Houghton, L. A., Brenna, J. T., and Noy, N. (1996) J. Biol. Chem. 271, 20507). The two sites are thus functionally distinct. Here, the thermodynamic parameters governing the interactions of retinol with the IRBP retinoid-binding sites were measured. The data demonstrate that the interactions of retinol with both sites are stabilized mainly by hydrophobic interactions, and that the hydroxyl head group of retinol is not involved in formation of protein-ligand complexes. Nevertheless, the data indicate that the two sites are structurally distinct, and that binding of retinol in them occurs by remarkably different modes of interactions.

Animals↗
PubMed1997

The tetramerization region of the retinoid X receptor is important for transcriptional activation by the receptor.

The retinoid X receptor (RXR), a member of the superfamily of hormone nuclear receptors, is a ligand-inducible transcription factor that is activated by the vitamin A derivative 9-cis-retinoic acid. We previously showed that RXR self-associates into tetramers with a high affinity and that ligand binding induces rapid dissociation of receptor tetramers to smaller species. Here, the RXR region that is responsible for mediating tetramer formation is identified. It is shown that this interface, which we term the "tetramerization domain," critically contains two consecutive phenylalanine residues located at the C-terminal region of the receptor. Mutation of these residues is sufficient to disrupt RXR tetramers without affecting the overall fold of the protein or interfering with ligand binding, dimer formation, or DNA binding by the receptor. Nevertheless, the tetramer-impaired mutant was found to be transcriptionally defective. The newly characterized tetramerization domain and the previously identified main dimerization interface of RXR act autonomously to affect separate intersubunit interactions that, overall, lead to formation of tetramers. Protein-protein interactions mediated by the tetramerization domain, but not those that involve the dimerization interface, are disrupted following ligand binding by RXR. Overall, these data attest to the specificity of the interaction and implicate the tetramerization interface in playing a direct role in regulating transcriptional activation by RXR.

Amino Acid Sequence↗
PubMed1997

The DNA binding pattern of the retinoid X receptor is regulated by ligand-dependent modulation of its oligomeric state.

The retinoid X receptor (RXR) regulates target gene transcription via its association with cognate DNA response elements either as a homodimer or as a heterodimer with a number of other nuclear receptors. We previously demonstrated that, in solution, RXR forms tetramers with a high affinity and that ligand binding leads to dissociation of receptor tetramers to smaller species. Here it is shown that RXR tetramers form stable complexes with direct repeats (DR-1 or DR-5) or palindromic (TREpal) response elements. Binding of RXR tetramers to cognate DNA occurs with a significantly higher affinity as compared with dimers. Ligand binding by DNA-bound RXR tetramers results in their dissociation to DNA-bound dimers, a process that is completely reversed upon removal of the ligand. Formation of stable tetramer-DNA complexes requires binding of two oligonucleotides/tetramer. It is proposed that ligand-dependent modulation of the oligomeric state of RXR is a regulatory feature of this nuclear receptor.

Animals↗
PubMed1996

Docosahexaenoic acid modulates the interactions of the interphotoreceptor retinoid-binding protein with 11-cis-retinal.

Rapid transport of retinoids across the interphotoreceptor matrix is a critical part of the visual cycle, since it serves to replenish bleached rhodopsin with its chromophore 11-cis-retinal. The transport of retinoids in the interphotoreceptor matrix is believed to be mediated by the interphotoreceptor retinoid-binding protein (IRBP), a protein that, in addition to possessing two retinoid-binding sites, associates in vivo with long chain fatty acids. Here, the interrelationships between binding of the two types of ligands to IRBP were studied. The composition of fatty acids associated with IRBP in bovine retina was determined, and it was found that polyunsaturated fatty acids constitute a significant fraction of those. It was further found that docosahexaenoic acid, but not palmitic acid, induced a rapid and specific release of 11-cis-retinal from one of the protein's retinoid-binding sites. Based on these results and on the additional observation that a steep concentration gradient of docosahexaenoic acid exists between photoreceptor and pigment epithelium cells, a model for the mechanism by which IRBP may target 11-cis-retinal to photoreceptor cells is proposed.

Allosteric Regulation↗