An inhibitor of the microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells.

The microsomal triglyceride (TG) transfer protein (MTP) is a heterodimeric lipid transfer protein that catalyzes the transport of triglyceride, cholesteryl ester, and phosphatidylcholine between membranes. Previous studies showing that the proximal cause of abetalipoproteinemia is an absence of MTP indicate that MTP function is required for the assembly of the apolipoprotein B (apoB) containing plasma lipoproteins, i.e., very low density lipoproteins and chylomicrons. However, the precise role of MTP in lipoprotein assembly is not known. In this study, the role of MTP in lipoprotein assembly is investigated using an inhibitor of MTP-mediated lipid transport, 2-[1-(3, 3-diphenylpropyl)-4-piperidinyl]-2,3-dihydro-1H-isoindol-1-o ne (BMS-200150). The similarity of the IC50 for inhibition of bovine MTP-mediated TG transfer (0.6 microM) to the Kd for binding of BMS-200150 to bovine MTP (1.3 microM) strongly supports that the inhibition of TG transfer is the result of a direct effect of the compound on MTP. BMS-200150 also inhibits the transfer of phosphatidylcholine, however to a lesser extent (30% at a concentration that almost completely inhibits TG and cholesteryl ester transfer). When BMS-200150 is added to cultured HepG2 cells, a human liver-derived cell line that secretes apoB containing lipoproteins, it inhibits apoB secretion in a concentration dependent manner. These results support the hypothesis that transport of lipid, and in particular, the transport of neutral lipid by MTP, plays a critical role in the assembly of apoB containing lipoproteins.

Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.

One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.

The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.

Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.

Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.

New approach for inhibiting Rev function and HIV-1 production using the influenza virus NS1 protein.

The Rev protein of HIV-1, which facilitates the nuclear export of HIV-1 pre-mRNAs, has been a target for antiviral therapy. Here we describe a new strategy for inhibiting Rev function and HIV-1 replication. In contrast to previous approaches, we use a wild-type rather than a mutant Rev protein and covalently link this Rev sequence to the NS1 protein of influenza A virus, a protein that inhibits the nuclear export of mRNAs. The NS1 protein contains an RNA-binding domain mutation (RM), so that the only functional RNA-binding domain in the chimeric protein (NS1RM-Rev) is in the Rev protein sequence. In the presence of the NS1RM-Rev chimeric protein, HIV-1 pre-mRNAs were retained in, rather than exported from, the nucleus. In addition, this chimeric protein effectively inhibited Rev function in trans in transfection experiments and effectively inhibited the production of HIV-1 in tissue culture cells transfected with an infectious molecular clone of HIV-1 DNA. The inhibitory activities of the NS1RM-Rev chimera were at least equivalent to those of the Rev M10 mutant protein, which has been considered to be the prototype trans inhibitor of Rev function and is currently in phase I clinical trials for the treatment of AIDS patients. We discuss (i) the potential for increasing the inhibitory activity of NS1-Rev chimeras against HIV-1 and (ii) the need for additional studies to evaluate these chimeras for the treatment of AIDS.

The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice.

The DNA-binding activity of AP-1 proteins is modulated, in vitro, by a posttranslational mechanism involving reduction oxidation. This mode of regulation has been proposed to control both the transcriptional activity and the oncogenic potential of Fos and Jun. Previous studies revealed that reduction of oxidized Fos and Jun by a cellular protein, Ref-1, stimulates sequence-specific AP-1 DNA-binding activity. Ref-1, a bifunctional protein, is also capable of initiating the repair of apurinic/apyrymidinic sites in damaged DNA. The relationship between the redox and DNA repair activities of Ref-1 is intriguing; both activities have been suggested to play an important role in the cellular response to oxidative stress. To investigate the physiological function of Ref-1, we used a gene targeting strategy to generate mice lacking a functional ref-1 gene. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities. In contrast, homozygous mutant mice, lacking a functional ref-1 gene, die during embryonic development. Detailed analysis indicates that death occurs following blastocyst formation, shortly after the time of implantation. Degeneration of the mutant embryos is clearly evident at embryonic day 5.5. These findings demonstrate that Ref-1 is essential for early embryonic development.

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

The alpha subunit of the karyopherin heterodimer functions in recognition of the protein import substrate and the beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of karyopherin beta's binding sites both for Ran-GTP and for karyopherin alpha. We discovered that karyopherin beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and karyopherin alpha bind to overlapping sites on karyopherin beta. Having a higher affinity to the overlapping site, Ran-GTP displaces karyopherin alpha and binds to karyopherin beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for karyopherin beta to a cluster of basic residues analogous to those previously shown to constitute karyopherin alpha's binding site to karyopherin beta.

A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137). To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion of the nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.

Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain.

The hypothesis that age-associated impairment of cognitive and motor functions is due to oxidative molecular damage was tested in the mouse. In a blind study, senescent mice (aged 22 months) were subjected to a battery of behavioral tests for motor and cognitive functions and subsequently assayed for oxidative molecular damage as assessed by protein carbonyl concentration in different regions of the brain. The degree of age-related impairment in each mouse was determined by comparison to a reference group of young mice (aged 4 months) tested concurrently on the behavioral battery. The age-related loss of ability to perform a spatial swim maze task was found to be positively correlated with oxidative molecular damage in the cerebral cortex, whereas age-related loss of motor coordination was correlated with oxidative molecular damage within the cerebellum. These results support the view that oxidative stress is a causal factor in brain senescence. Furthermore, the findings suggest that age-related declines of cognitive and motor performance progress independently, and involve oxidative molecular damage within different regions of the brain.

Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors.

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

Mapping the protein regions responsible for the functional specificities of the Arabidopsis MADS domain organ-identity proteins.

The Arabidopsis MADS domain proteins AP1, AP3, PI, and AG specify floral organ identity. All of these proteins contain a MADS domain required for DNA binding and dimerization; a region termed L (linker between MADS domain and K domain), which plays an important role in dimerization specificity; the K domain, named for its similarity to the coiled-coil domain of keratin; and a C-terminal region of unknown function. To determine which regions of these proteins are responsible for their abilities to specify different organs, we have made a number of chimeric MADS box genes. The in vivo function of these chimeric genes was investigated by ectopic expression in transgenic Arabidopsis plants. The four proteins fall into two classes on the basis of regions responsible for their functional specificities. The L region and K domain define the functional specificities of AP3 and PI, while the MADS domain and L region define the functional specificities of AP1 and AG.

Didemnin binds to the protein palmitoyl thioesterase responsible for infantile neuronal ceroid lipofuscinosis.

The marine natural product didemnin B, currently in clinical trials as an antitumor agent, has several potent biological activities apparently mediated by distinct mechanisms. Our initial investigation of didemnin B resulted in the discovery of its GTP-dependent binding of the translation elongation factor EF1 alpha. This finding is consistent with the protein synthesis inhibitory activity of didemnin B observed at intermediate concentrations. To begin to dissect the mechanisms involved in the cytostatic and immunosuppressive activities of didemnin B, observed at low concentrations, additional didemnin-binding proteins were sought. Here we report the purification of a 36-kDa glycosylated didemnin-binding protein from bovine brain lysate. Cloning of the human cDNA encoding this protein revealed a strong sequence similarity with palmitoyl protein thioesterase (PPT), an enzyme that removes palmitate from H-Ras and the G alpha s subunits of heterotrimeric GTP-binding proteins in vitro. Mutations in PPT have recently been shown to be responsible for infantile neuronal ceroid lipofuscinosis, which is a severe brain disorder characterized by progressive loss of brain function and early death.