A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain.

Phosphoprotein phosphatase 2A (PP2A) is one of the four major protein serine/threonine phosphatases found in all eukaryotic cells. We have shown that the 36-kDa catalytic subunit of PP2A is carboxyl methylated in eukaryotic cells, and we have previously identified and purified a novel methyltransferase (MTase) that is responsible for this modification. Here, we describe a novel protein carboxyl methyl-esterase (MEase) from bovine brain that demethylates PP2A. The enzyme has been purified to homogeneity as a monomeric 46-kDa soluble protein. The MEase is highly specific for PP2A. It does not catalyze the demethylation of other protein or peptide methylesters. Moreover, MEase activity is dramatically inhibited by nanomolar concentrations of okadaic acid, a specific inhibitor of PP2A. From these results, we conclude that PP2A methylation is controlled by two specific enzymes, a MTase and a MEase. Since PP2A methylation is highly conserved in eukaryotes ranging from human to yeast, it is likely that this system plays an important role in phosphatase regulation.

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Crystal structure of a human TATA box-binding protein/TATA element complex.

The TATA box-binding protein (TBP) is required by all three eukaryotic RNA polymerases for correct initiation of transcription of ribosomal, messenger, small nuclear, and transfer RNAs. The cocrystal structure of the C-terminal/core region of human TBP complexed with the TATA element of the adenovirus major late promoter has been determined at 1.9 angstroms resolution. Structural and functional analyses of the protein-DNA complex are presented, with a detailed comparison to our 1.9-angstroms resolution structure of Arabidopsis thaliana TBP2 bound to the same TATA box.

P-glycoprotein: a major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and humans.

The P-glycoprotein (Pgp) efflux pump can influence the hepatocellular concentration of xenobiotics that are modulators and substrates of cytochrome P4503A (CYP3A). We tested the hypothesis that Pgp is a determinant of drug-inducible expression of CYP3A. The magnitude of CYP3A induction by rifampicin was compared in the human parental colon carcinoma cell line LS 180/WT (wild type) and in two derivative clones overexpressing the human multidrug resistance gene MDR1 (also designated PGY1) because of either drug selection (LS 180/ADR) or transfection with MDRI cDNA (LS 180/MDR). In both MDR1 cDNA-overexpressing clones, rifampicin induction of CYP3A mRNA and protein was decreased and required greater rifampicin concentrations compared with parental cells. The role of Pgp in regulation of CYP3A expression in vivo was analyzed in mice carrying a targeted disruption of the mdr1a mouse gene. Oral treatment with increasing doses of rifampicin resulted in elevated drug levels in the livers of mdr1a (-/-) mice compared with mdr1a (+/+) mice at all doses. Consistent with the enhanced accumulation of rifampicin in mdr1a (-/-) mice, lower doses of rifampicin were required for induction of CYP3A proteins, and the magnitude of CYP3A induction was greater at all doses of rifampicin in mdr1a (-/-) mice compared with mdr1a (+/+) mice. We conclude that Pgp-mediated transport is a critical element influencing the CYP3A inductive response.

A unique role for the Rb protein in controlling E2F accumulation during cell growth and differentiation.

Examination of the interactions involving transcription factor E2F activity during cell growth and terminal differentiation suggests distinct roles for Rb family members in the regulation of E2F accumulation. The major species of E2F in quiescent cells is a complex containing the E2F4 product in association with the Rb-related p130 protein. As cells enter the cell cycle, this complex disappears, and there is a concomitant accumulation of free E2F activity of which E2F4 is a major component. E2F4 then associates with the Rb-related p107 protein as cells enter S phase. Rb can be found in interactions with each E2F species, including E2F4, during G1, but there appears to be a limited amount of Rb with respect to E2F, likely due to the maintenance of most Rb protein in an inactive state by phosphorylation. A contrasting circumstance can be found during the induction of HL60 cell differentiation. As these cells exit the cell cycle, active Rb protein appears to exceed E2F, as there is a marked accumulation of E2F-Rb interactions, involving all E2F species, including E2F4, which is paralleled by the conversion of Rb from a hyperphosphorylated state to a hypophosphorylated state. These results suggest that the specific ability of Rb protein to interact with each E2F species, dependent on concentration of active Rb relative to accumulation of E2F, may be critical in cell-growth decisions.

Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease.

There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

In vivo selection of basic region-leucine zipper proteins with altered DNA-binding specificities.

A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.

Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.

Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells.

Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density-dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density-dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low-density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp -650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells.

Expression of the C terminus of the amyloid precursor protein alters growth factor responsiveness in stably transfected PC12 cells.

The amyloid precursor protein (APP) is a molecule centrally involved in Alzheimer disease pathology, but whose normal function is still poorly understood. To investigate the consequences of increased intracellular production of various regions of APP on cellular physiology, we stably transfected PC12 cells with the C-terminal 100 amino acids of the human APP. In eight transfected clones that express the APP(C100) protein, exposure to nerve growth factor (NGF) did not promote differentiation. Transfectants continued to divide and failed to elaborate extensive neurites, whereas control PC12 cells, mock-transfected PC12 cells, and a nonexpressing transfected cell line did develop neurites and stopped dividing after NGF stimulation. Unlike NGF treatment, treatment with basic fibroblast growth factor profoundly accelerated neurite outgrowth in transfected cells. Also, a dramatic increase in a tyrosine phosphatase activity was noted. Expression and accumulation of APP C100 protein in PC12 cells results in an abnormal response to growth factor stimulation.

In vivo protein-DNA interactions at human DNA replication origin.

Protein-DNA interactions were studied in vivo at the region containing a human DNA replication origin, located at the 3' end of the lamin B2 gene and partially overlapping the promoter of another gene, located downstream. DNase I treatment of nuclei isolated from both exponentially growing and nonproliferating HL-60 cells showed that this region has an altered, highly accessible, chromatin structure. High-resolution analysis of protein-DNA interactions in a 600-bp area encompassing the origin was carried out by the in vivo footprinting technique based on the ligation-mediated polymerase chain reaction. In growing HL-60 cells, footprints at sequences homologous to binding sites for known transcription factors (members of the basic-helix-loop-helix family, nuclear respiratory factor 1, transcription factor Sp1, and upstream binding factor) were detected in the region corresponding to the promoter of the downstream gene. Upon conversion of cells to a nonproliferative state, a reduction in the intensity of these footprints was observed that paralleled the diminished transcriptional activity of the genomic area. In addition to these protections, in close correspondence to the replication initiation site, a prominent footprint was detected that extended over 70 nucleotides on one strand only. This footprint was absent from nonproliferating HL-60 cells, indicating that this specific protein-DNA interaction might be involved in the process of origin activation.

Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult, and polycystic kidney.

Polycystic kidney disease 1 (PKD1) is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease. We have studied PKD1 mRNA, with an RNase protection assay, and found widespread expression in adult tissue, with high levels in brain and moderate signal in kidney. Expression of the PKD1 protein, polycystin, was assessed in kidney using monoclonal antibodies to a recombinant protein containing the C terminus of the molecule. In fetal and adult kidney, staining is restricted to epithelial cells. Expression in the developing nephron is most prominent in mature tubules, with lesser staining in Bowman's capsule and the proximal ureteric bud. In the nephrogenic zone, detectable signal was observed in comma- and S-shaped bodies as well as the distal branches of the ureteric bud. By contrast, uninduced mesenchyme and glomerular tufts showed no staining. In later fetal (>20 weeks) and adult kidney, strong staining persists in cortical tubules with moderate staining detected in the loops of Henle and collecting ducts. These results suggest that polycystin's major role is in the maintenance of renal epithelial differentiation and organization from early fetal life. Interestingly, polycystin expression, monitored at the mRNA level and by immunohistochemistry, appears higher in cystic epithelia, indicating that the disease does not result from complete loss of the protein.

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression.

The retinal protein Nrl belongs to a distinct subfamily of basic motif-leucine zipper DNA-binding proteins and has been shown to bind extended AP-1-like sequence elements as a homo- or heterodimer. Here, we demonstrate that Nrl can positively regulate the expression of the photoreceptor cell-specific gene rhodopsin. Electrophoretic mobility-shift analysis reveals that a protein(s) in nuclear extracts from bovine retina and the Y79 human retinoblastoma cell line binds to a conserved Nrl response element (NRE) in the upstream promoter region of the rhodopsin gene. Nrl or an antigenically similar protein is shown to be part of the bound protein complex by supershift experiments using Nrl-specific antiserum. Cotransfection studies using an Nrl-expression plasmid and a luciferase reporter gene demonstrate that interaction of the Nrl protein with the -61 to -84 region of the rhodopsin promoter (which includes the NRE) stimulates expression of the reporter gene in CV-1 monkey kidney cells. This Nrl-mediated transactivation is specifically inhibited by coexpression of a naturally occurring truncated form of Nrl (dominant negative effect). Involvement of Nrl in photoreceptor gene regulation and its continued high levels of expression in the adult retina suggest that Nrl plays a significant role in controlling retinal function.