A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:proto¬chlorophyllide oxidoreductase (POR) A

NADPH: protochlorophyllide oxido reductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Amongthem are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radio labeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of35S-Oep16/Ptc16 is stimulated by GTP.35S-Oep16/Ptc16forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photo oxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.

Estructura de una red esencial de señalización que regula la homeostasis iónica en plantas

Las plantas son organismos sésiles que han desarrollado la capacidad para detectar variaciones sutiles en su ambiente y producir respuestas adaptativas mediante rutas de señalización. Los estímulos causados por el estrés biótico y abiótico son numerosos y dependiendo del tiempo de exposición y su intensidad, pueden reducir la tasa de crecimiento de las plantas y la producción. Los cambios en la concentración del calcio citosólico libre constituyen una de las primeras reacciones intracelulares a las situaciones de estrés abiótico. En esta situación, el calcio actúa como segundo mensajero y las variaciones en su concentración son descodificadas por proteínas de unión a calcio. Las más conocidas son las manos-EF y los dominios C2. Los dominios C2 han sido descritos como dominios de unión a lípidos dependientes de calcio. Estos dominios se consideran proteínas periféricas solubles en agua que se asocian de manera reversible a los lípidos de la membrana mediante una o dos regiones funcionales: el sitio de unión a calcio y el sitio polibásico. A pesar de que se conoce la estructura molecular de algunos dominios C2, se desconocen aspectos relacionados como las reglas que dirigen su forma de interaccionar con los diferentes fosfolípidos y proteínas, la posición que ocupan en la bicapa lipídica y su papel en la transmisión de señales. En esta tesis se ha estudiado una proteína de Arabidopsis thaliana (At3g17980) representativa de una nueva familia de proteínas con dominios C2, que consiste únicamente de un dominio C2. Esta proteína, llamada AtC2.1, ha sido clonada en el vector pETM11, expresada en E. coli y purificada a homogeneidad en dos pasos cromatográficos. Se obtuvieron cristales de AtC2.1 de buena calidad mediante técnicas de difusión de vapor. La proteína fue co-cristalizada con calcio, fosfocolina (POC) y el fosfolípido 1,2-dihexanoil-sn-glicero-3-fosfo-L-serina (PSF). Se recogieron ocho conjuntos de datos de difracción de rayos X empleando radiación sincrotrón. Los cristales difractaron hasta 1.6 Å de resolución. Siete de ellos pertenecían al grupo ortorrómbico P212121, con las dimensiones de la celdilla unidad a = 35.3, b = 88.9, c = 110.6 Å, y un cristal pertenecía al grupo espacial monoclínico C2, con a = 124.84, b = 35.27, c = 92.32 Å y = 121.70º. La estructura se resolvió mediante la técnica MR-SAD utilizando el cinc como dispersor anómalo. La estructura cristalina mostró que la molécula forma un dímero en el que cada protómero se pliega como un dominio C2 típico, con la topología tipo II y presenta una inserción de 43 aminoácidos que la diferencia de los dominios C2 conocidos. El mapa de densidad electrónica mostró dos átomos de calcio por protómero. Se resolvieron las estructuras de AtC2.1 en complejo con POC o PSF. En ambos complejos, el análisis cristalográfico detectó máximos de densidad electrónica en la región correspondiente al sitio polibásico formado por las hebras 2, 3 5 y el lazo 3. Éstos se interpretaron correctamente como dos moléculas de POC y un átomo de cinc, en un complejo, y como la cabeza polar del PSF en el otro. AtC2.1 define un sitio de interacción con lípidos dependiente de cinc. En conclusión, en este trabajo se presenta la estructura tridimensional de AtC2.1, miembro representativo de una familia de proteínas de Arabidopsis thaliana, identificadas como proteínas que interaccionan con los receptores de ABA. Estas proteínas están constituidas únicamente por un dominio C2. El análisis conjunto de los datos biofísicos y cristalográficos muestra que AtC2.1 es un sensor de calcio que une lípidos usando dos sitios funcionales. Estos datos sugieren un mecanismo de inserción en membrana dependiente de calcio que trae consigo la disociación de la estructura dimérica y, por consiguiente, un cambio en las propiedades de superficie de la molécula. Este mecanismo proporciona las bases del reconocimiento y transporte de los receptores de ABA y/o otras moléculas a la membrana celular. Plants are sessile organisms that have developed the capacity to detect slight variations of their environment. They are able to perceive biotic and abiotic stress signals and to transduce them by signaling pathways in order to trigger adaptative responses. Stress factors are numerous and, depending on their exposition time and their concentration, can reduce plant growth rate, limiting the productivity of crop plants. Changes in the cytosolic free calcium concentration are observed as one of the earliest intracellular reactions to abiotic stress signals. Calcium plays a key role as a second messenger, and calcium concentration signatures, called calcium signals, are decodified by calcium binding proteins. The main calcium binding structures are the EF-hand motif and the C2 domains. C2 domain is a calcium dependent lipid-binding domain of approximately 130 amino acids. C2 domain displays two functional regions: the Ca-binding region and the polybasic cluster. Both of them can interact with the membrane phospholipids. Despite the number of C2 domain 3D structures currently available, questions about how they interact with the different target phospholipids, their precise spatial position in the lipid bilayer, interactions with other proteins and their role in transmitting signals downstream, have not yet been explored. In this work we have studied an uncharacterized protein from Arabidopsis thaliana (At3g17980) consisting of only a single C2 domain, as member of a new protein C2-domain family. This protein called AtC2.1 was cloned into the pETM11 vector and expressed in E. coli, allowing the purification to homogeneity in two chromatographic steps. Good quality diffracting crystals were obtained using vapor-diffusion techniques. Crystals were co-crystalized with calcium; phosphocholine (POC) and/or the phospholipid 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (PSF). Eight data set were collected with synchrotron radiation. Crystals diffracted up to 1.6 Å resolution and seven of them belong to the orthorhombic space group P212121, with unit-cell parameters a = 35.3, b = 88.9, c = 110.6 Å. Another crystal was monoclinic, space group C2, with a = 124.84, b = 35.27, c = 92.32 Å and = 121.70º. The structural model was solved by MR-SAD using Zn2+ as anomalous scatterer. The crystal structure shows that the molecule is a dimer. Each monomer was folded as a canonical C2 domain with the topology II with a 43 residues insertion. The electron density map reveals two calcium ions per molecule. Structures of AtC2.1, complexed with POC and PSF, have been solved. Well-defined extra electron densities were found, in both complexes, within the concave surface formed by strands 2, 3, 5 and loop 3 of AtC2.1. These densities were clearly explained by the presence of the two POC molecules, one zinc atom and head groups of PSF, occupying the cavity of the polybasic site. AtC2.1 defines a new metal dependent lipid-binding site into the polybasic site. In conclusion, in this thesis it is presented the molecular structure of AtC2.1, a representative member of a family of Arabidopsis thaliana C2 domain proteins, of unknown function, but identified as a molecular interacting unit of the ABA receptors. The joint analyses of the biophysical and crystallographic data show that AtC2.1 is a calcium sensor that binds lipids in two sites and suggest a model of calcium-dependent membrane insertion mechanism that will involve either dimer dissociation or a strong rearrangement of the dimeric structure. This mechanism may be the basis for the recognition and delivery of ABA receptors or other protein molecules to cell membranes.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa)

Scope: Today, about 2–8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Methods and results: Sera from 42 Spanish lettuce-allergic patients were obtained from pa-tients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Conclusion: Two new major lettuce allergens—a thaumatin-like protein and an aspartyl protease—have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

Molecular physiology of nickel and cobalt homeostasis in Rhizobium leguminosarum.

Transition metals such as Fe, Cu, Mn, Ni, or Co are essential nutrients, as they are constitutive elements of a significant fraction of cell proteins. Such metals are present in the active site of many enzymes, and also participate as structural elements in different proteins. From a chemical point of view, metals have a defined order of affinity for binding, designated as the Irving-Williams series (Irving and Williams, 1948) Mg2+ menor que Mn2+ menor que Fe2+ menor que Co2+ menor que Ni2+ menor que Cu2+mayor queZn2+ Since cells contain a high number of different proteins harbouring different metal ions, a simplistic model in which proteins are synthesized and metals imported into a ?cytoplasmic soup? cannot explain the final product that we find in the cell. Instead we need to envisage a complex model in which specific ligands are present in definite amounts to leave the right amounts of available metals and protein binding sites, so specific pairs can bind appropriately. A critical control on the amount of ligands and metal present is exerted through specific metal-responsive regulators able to induce the synthesis of the right amount of ligands (essentially metal binding proteins), import and efflux proteins. These systems are adapted to establish the metal-protein equilibria compatible with the formation of the right metalloprotein complexes. Understanding this complex network of interactions is central to the understanding of metal metabolism for the synthesis of metalloenzymes, a key topic in the Rhizobium-legume symbiosis. In the case of the Rhizobium leguminosarum bv viciae (Rlv) UPM791 -Pisum sativum symbiotic system, the concentration of nickel in the plant nutrient solution is a limiting factor for hydrogenase expression, and provision of high amounts of this element to the plant nutrient solution is required to ensure optimal levels of enzyme synthesis (Brito et al., 1994).

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

The STAR protein QKI-6 is a translational repressor

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3′ untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.

Evidence for a protective role of metallothionein-1 in focal cerebral ischemia

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (P < 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (P = 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N

SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.

Molding a peptide into an RNA site by in vivo peptide evolution

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial α-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an “RNA world.”

Probing the assembly of transcription initiation complexes through changes in σN protease sensitivity

The alternative bacterial σN RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of σN, the assembly of end-labeled Klebsiella pneumoniae σN into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free σN were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that σN can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.

A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2

The prolamin box (P-box) is a highly conserved 7-bp sequence element (5′-TGTAAAG-3′) found in the promoters of many cereal seed storage protein genes. Nuclear factors from maize endosperm specifically interact with the P-box present in maize prolamin genes (zeins). The presence of the P-box in all zein gene promoters suggests that interactions between endosperm DNA binding proteins and the P-box may play an important role in the coordinate activation of zein gene expression during endosperm development. We have cloned an endosperm-specific maize cDNA, named prolamin-box binding factor (PBF), that encodes a member of the recently described Dof class of plant Cys2-Cys2 zinc-finger DNA binding proteins. When tested in gel shift assays, PBF exhibits the same sequence-specific binding to the P-box as factors present in maize endosperm nuclei. Additionally, PBF interacts in vitro with the basic leucine zipper protein Opaque2, a known transcriptional activator of zein gene expression whose target site lies 20 bp downstream of the P-box in the 22-kDa zein gene promoter. The isolation of the PBF gene provides an essential tool to further investigate the functional role of the highly conserved P-box in regulating cereal storage protein gene expression.

An essential role for p300/CBP in the cellular response to hypoxia

p300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. They participate in numerous biological processes, including cell cycle arrest, differentiation, and transcription activation. p300 and/or CBP (p300/CBP) also coactivate CREB. How they participate in these processes is not yet known. In a search for specific p300 binding proteins, we have cloned the intact cDNA for HIF-1α. This transcription factor mediates hypoxic induction of genes encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular endothelial growth factor. Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1α and p300/CBP. Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP. Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by E1A. Hence, p300/CBP–HIF complexes participate in the induction of hypoxia-responsive genes, including one (vascular endothelial growth factor) that plays a major role in tumor angiogenesis. Paradoxically, these data, to our knowledge for the first time, suggest that p300/CBP are active in both transformation suppression and tumor development.

Insulin selectively increases SREBP-1c mRNA in the livers of rats with streptozotocin-induced diabetes

Sterol regulatory element binding proteins (SREBPs) enhance transcription of genes encoding enzymes of cholesterol and fatty acid biosynthesis and uptake. In the current experiments, we observed a decline in the mRNA encoding one SREBP isoform, SREBP-1c, in the livers of rats that were rendered diabetic by treatment with streptozotocin. There was no change in the mRNA encoding SREBP-1a, which is derived from the same gene as SREBP-1c but uses a different promoter. The ratio of SREBP-1c:1a transcripts fell 25-fold from 5:1 in control rats to 0.2:1 in the diabetic animals. The SREBP-1c mRNA rose nearly to normal, and the 1c:1a ratio increased 17-fold when the diabetic rats were treated for 6 h with insulin. These treatments produced no change in the mRNA for SREBP-2, which is encoded by a separate gene. The SREBP-1c mRNA also fell selectively in freshly isolated rat hepatocytes and rose when the cells were treated with insulin. Considered together with recent data on hepatocytes [Foretz, M., Pacot, C., Dugal, I., et al. (1999) Mol. Cell. Biol. 19, 3760–3768], the current in vivo studies suggest that insulin may stimulate lipid synthesis in the liver by selectively inducing transcription of the SREBP-1c gene.