Apoprotein A-1 is a cofactor independent substrate of protein kinase C

Apoprotein A-1 (apo A-1), the predominant protein constituent of high density lipoproteins (HDL), was phosphorylated by protein kinase C (PKC). Optimal phosphorylation of lipid-free apo A-1 occurs in the absence of calcium, phosphatidyl serine (PS), and diolein (DO). However, HDL-bound apo A-1 was not phosphorylated by PKC. Furthermore, addition of either native or reconstituted HDL particles to lipid-free apo A-1 resulted in a concentration-dependent inhibition of phosphorylation. It appears that the phosphorylatable sites on apo A-1 are involved in hydrophobic interaction with the lipids of HDL. Apo A-1 is a novel substrate of PKC because it does not require calcium and lipid cofactors for optimal phosphorylation.     

Molecular characterization of a fourth isoform of the catalytic subunit of protein phosphatase 2A from Arabidopsis thaliana

We have recently reported the existence of multiple isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) in Arabidopsis thaliana and the molecular cloning of cDNAs encoding three of these proteins (PP2A-1, PP2A-2, PP2A-3). The reported cDNA encoding PP2A-3 was truncated at the 5 terminus, lacking a short fragment of the N-terminal coding sequence. We have now isolated a near full-length cDNA encoding the entire PP2A-3 protein (313 residues). The clone includes 188 nucleotides of 5-untranslated region, where a 44 bp long poly(GA) track is found. We also describe the cloning of a cDNA encoding a fourth isoform of PP2A (PP2A-4). The polypeptide contains 313 residues being 98% identical to PP2A-3 and only 80% identical to both PP2A-1 and PP2A-2. The mRNA for PP2A-4 is 1.4 kb in length and, although predominantly expressed in roots, it is also found in other organs. It is concluded that in A. thaliana the isoforms of PP2A can be grouped in two extremely conserved subfamilies.     

Synthesis,glycosylation and rapid secretion of a glycoprotein by Achlya a primitive eucaryote

Achlya ambisexualis, a water mold, secretes several glycoproteins during exponential growth. Among these is a major protein of 39 000 daltons (protein A-39) which is secreted very rapidly. Protein A-39 is detected among the soluble cellular proteins labeled for 5 min. However, after longer labeling times, an additional 95 000 dalton glycoprotein was immunoprecipitated from among the cytoplasmic proteins by antiserum against protein A-39. This antiserum reacted with a single 37 000 dalton protein from the in vitro translation products of poly(A)-containing RNA in a wheat germ cell-free system which is cleaved to a faster moving component in the presence of dog pancreatic membranes. Immunoprecipitated, chain-completion products of polysomes also show a 37 000 dalton peptide which does not bind to lectins, indicating absence of co-translational cleavage and glycosylation.Tunicamycin inhibits the appearance of the 95 000 dalton protein. Several immunoprecipitable proteins, including protein A-39, having sizes identical to the secretory proteins accumulate in the cytoplasm in the presence of this inhibitor. A short pulse with [³H]glucosamine followed by a chase showed that incorporation in protein A-39 increases while that in 95 000 dalton protein is decreasing. These results suggest that the 95 000 dalton glycoprotein may serve as a glycosyl donor to secretory protein A-39.     

Molecular dynamics simulation studies of a protein-RNA complex with a selectively modified binding interface

The RNA recognition motif (RRM) is one of the most common RNA binding domains. We have investigated the contribution of three highly conserved aromatic amino acids to RNA binding by the N-terminal RRM of the U1A protein. Recently, we synthesized a modified base (A-4CPh) in which a phenyl group is tethered to adenine using a linker of 4 methylene groups. The substitution of this base for adenine in the target RNA selectively stabilizes the complex formed with a U1A protein in which one of the conserved aromatic amino acids is replaced with Ala (Phe56Ala). In this article, we report molecular dynamics (MD) simulations that probe the structural consequences of the substitution of A-4CPh for adenine in the wild type and Phe56Ala U1A-RNA complexes and in the free RNA. The simulations suggest that A-4CPh stabilizes the complex formed with Phe56Ala by adopting a folded conformation in which the tethered phenyl group fills the site occupied by the phenyl group of Phe56 in the wild-type complex. In contrast, an extended conformation of A-4CPh is predicted to be most stable in the complex formed with the wild-type protein. The calculations indicate A-4CPh is in an extended conformation in the free RNA. Therefore, preorganizing the structure of the phenyl-tethered base for binding may improve both the affinity and specificity of the RNA containing A-4CPh for the Phe56Ala U1A protein. Taken together, the previous experimental work and the calculations reported here suggest a general design strategy for altering RRM-RNA complex stability.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Selection and characterization of a new class of peptide exosite inhibitors of coagulation factor VIIa

A new series of peptide inhibitors of human Factor VIIa (FVIIa) has been identified and affinity matured from naive and partially randomized peptide phage libraries selected against the immobilized tissue factor x Factor VIIa (TF x FVIIa) complex. These "A-series" peptides contain a single disulfide bond and a 13-residue minimal core required for maximal affinity. They are exemplified by peptide A-183 (EEWEVLCWTWETCER), which binds at a newly identified exosite on the FVIIa protease domain, described in the accompanying report [Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531]. A-183 was obtained from a trypsin digest of A-100-Z, a recombinant protein comprising A-183 and the Z domain of protein A. Surprisingly, A-183 was a very potent inhibitor of TF x FVIIa, inhibiting activation of Factor X (FX) and Factor IX and amidolytic activity of Chromozym t-PA with IC50 values of 1.6 +/- 1.2, 3.5 +/- 0.3, and 8.5 +/- 3.5 nM, respectively. Kinetic analysis revealed that A-183 was a partial (hyperbolic) mixed-type inhibitor of FX activation having a Ki of 200 pM as well as a partial competitive inhibitor of amidolytic activity. The A-series peptides were also specific and potent inhibitors of TF-dependent clotting as measured in a prothrombin time (PT) clotting assay and had no effect on the TF-independent activated partial thromboplastin time. At saturating concentrations of peptide, the maximal extent by which A-183 and A-100-Z inhibited the rate of FX activation was 78 +/- 3 and 89 +/- 6%, respectively. The degree of inhibition of the rate of FX activation correlated with a maximum fold prolongation in the PT assay of 1.8-fold for A- 183 and 3.3-fold for A-100-Z. The A-series peptides represent a new class of peptide exosite inhibitors that are capable of attenuating, rather than completely inhibiting, the activity of TF x FVIIa, potentially leading to anticoagulants with an increased therapeutic window.     

Effects of a new hypoglycemic agent A-4166 on glucose metabolism in rat adipocytes and muscle tissues

The effects of the oral administration of a non-sulfonylurea hypoglycemic agent, the phenylalanine derivative A-4166, on serum insulin and glucose levels and glucose metabolism in isolated rat adipocytes and slices of muscle tissues were studied. An increase in serum insulin and a decrease in glucose levels were observed 30 minutes after A-4166 administration to rats fed basal or high fat diet. No changes in basal glucose transport in isolated fat cells were observed after the administration of A-4166. The effect of in vitro added insulin was, however, stronger in rats fed basal diet and treated with A-4166. An elevation of the membrane glucose transporter GLUT 4 was observed in rats treated with A-4166. An increase of basal lipogenesis, measured by incorporation of radiocarbon labeled glucose into lipids, was noted in adipocytes from rats fed high fat diet. The addition of insulin was followed by stimulation of lipogenesis in rats fed basal diet, however, this hormone had no effect in rats fed high fat diet. The administration of A-4166 did not affect the basal or insulin stimulated lipogenesis. Basal glucose oxidation in the diaphragm was not influenced by high fat diet or by A-4166 treatment. In the soleus muscle, basal glucose oxidation was decreased in rats fed high fat diet, and treatment with A-4166 increased the glucose oxidation up to values observed in the control basal diet fed rats. These results indicate that the administration of A-4166 can affect glucose metabolism in muscle tissue and the sensitivity of adipocytes to insulin.     

In vivo characterization of A-192411: a novel fungicidal lipopeptide (II)

The ability of the novel antifungal cyclic hexalipopetide A-192411 to treat fungal infections in rodents is presented. Efficacy was demonstrated against Candida albicans as both prolonged survival of systemically infected mice and clearance of viable yeasts from kidneys. The efficacy of A-192411, administered intraperitoneally, was equivalent to amphotercin B at a 4-fold lower dose by weight in the systemic candidiasis models in mice, while the efficacy of A-192441 administered intravenously was equivalent to amphotercin B by weight in the Candida pyelonephritis model in rats. A-192411 also slightly prolonged the survival of immunocompromised mice infected systemically with Aspergillus fumigatus.     

Genetic determination of high-density lipoprotein-cholesterol and apolipoprotein A-1 plasma levels in a family study of cardiac catheterization patients.

Plasma levels of two lipoprotein risk factors, high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-1 (apo A-1), have been shown to be negatively associated with the risk of developing coronary artery disease, and several reports have examined familial factors in HDL-C and apo A-1 levels. A number of studies suggest that shared genes influence familial resemblance of these lipoprotein levels far more than do shared environments. Possible mechanisms for the inheritance of these two risk factors (HDL-C and apo A-1 plasma levels) are explored using data from 390 individuals in 69 families ascertained through probands undergoing diagnostic cardiac catheterization. Segregation analysis was used to test a series of specific models of inheritance. Evidence for single-locus control of apo A-1 levels, with Mendelian transmission of a dominant allele leading to elevated apo A-1 levels, was seen in these families, although there was additional correlation among sibs present. This locus accounted for 48.6% and 37.2% of the total variation in apo A-1 levels in males and females, respectively. Similar evidence of segregation at a single locus controlling HDL-C levels was not seen in these families.     

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.     

Analysis of mRNA coding for blood-stage antigens of a rodent malarial parasite, Plasmodium yoelii: mRNA coding for a possible protective antigen purify as poly A-

We have analyzed mRNA coding for blood-stage antigens of Plasmodium yoelii by using cellfree translation of poly A+ and poly A- RNA in conjunction with immunoprecipitations. Most of the antigens recognized by mouse hyperimmune serum to P. yoelii were coded by poly A+ mRNA ranging in size from 15S to 28S. However, certain P. yoelii antigens, notably those with m.w. greater than 150 kilodaltons (kd), were coded by mRNA that purified as being poly A-. Antigens recognized by a protective monoclonal antibody (McAb) were coded by such operationally poly A- RNA. Three polypeptides apparently coded by different poly A- RNA were immunoprecipitated by this McAb. With the use of another McAb, a poly A+ mRNA of about 19S was identified as coding for a polypeptide of 46 kd synthesized in cellfree translation reactions. The same McAb recognized a 34 kd polypeptide in metabolically labeled polypeptides of P. yoelii. This antigen appeared to be processed in vivo but not in vitro. The observation that some mRNA of P. yoelii purify as being poly A- has significant implications for the construction of cDNA libraries that employ poly A+ mRNA of malarial parasites: if it applies to other species of plasmodia, some potentially important operationally poly A- mRNA may not be represented in such libraries.     

Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody.

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A-56.36 and CB had overlapping binding sites on CA. By screening random phage-displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin-dissociating effect of A-56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage-displayed peptides corresponding to fragments of the two identified regions on CA reacted with A-56.36 and with CB. Our data support the hypothesis that A-56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.     

Biochemical and genetic analysis of a maltopentaose-producing amylase from an alkaliphilic gram-positive bacterium.

Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-180 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases. It appears, therefore, that A-180 has arisen by gene fusion events.     

In vitro preparation and characterization of a 700 nm absorbing chlorophyll-water adduct according to the proposed primary molecular unit in photosynthesis.

1. This study characterized chlorophyll a-H2O adducts in vitro in order to establish their generic relationship to the recently proposed [15, 18-20, 31] primary molecular adduct in photosynthesis. The effects of water titration and temperature on the absorption, fluorescence, excitation, and redox properties of the various in vitro chlorophyll a aggregate species are investigated. 2. From fluorescence measurements, we conclude that the driest chlorophyll a sample contains an equimolar amount of water. This conclusion is consistent with earlier experimental work [2, 3, 14, 17, 31], and clarifies the origin of the controversial [15] Katz model [14] of chlorophyll a-H2O interactions. 3. With increasing water concentration or as the temperature is lowered below room temperature, the A-663 monohydrate chlorophyll a-H2O (species absorbing at 663 nm) is favored at the expense of the A-678 anhydrous aggregate according to the equilibrium 2H2O+chlorophyll a2in equilibrium2 chlorophyll a-H2O. Under excess water conditions, A-663 is converted to A-743 (chlorophyll a-2H2O)n. 4. On slow sample cooling to T less than or approximately 200 degrees K, we observe the growth of A-700 at the expense of A-663. There is a direct correspondence between the increasing (decreasing) absorption by A-700 (A-633) and increasing (decreasing) fluorescence at 720 nm (664 nm). 5. It is concluded that A-700 is most probably the dimer participating in the equilibrium 2 chlorophyll a-H2O in equilibrium (chlorophyll a-H2O)2. The A-700 band consists of two exciton components (separated by approximately 280 cm1) that are interpretable in terms of the dimeric origin of A-700. 6. The deconvoluted A-700 absorption spectrum and the excitation spectrum of the 720 nm fluorescence are compared with the light-minus-dark spectra of P-700. 7. It is found that A-700 is reversibly bleached by I2 (E0 equals 0.54 V). The significance of this observation is discussed in terms of the redox properties of monomeric chlorophyll a and P-700.     

Structural insight into a quinolone-topoisomerase II-DNA complex. Further evidence for a 2:2 quinobenzoxazine-mg2+ self-assembly model formed in the presence of topoisomerase ii.

Quinobenzoxazine A-62176, developed from the antibacterial fluoroquinolones, is active in vitro and in vivo against murine and human tumors. It has been previously claimed that A-62176 is a catalytic inhibitor of mammalian topoisomerase II that does not stabilize the cleaved complex. However, at low drug concentrations and pH 6-7, we have found that A-62176 can enhance the formation of the cleaved complex at certain sites. Using a photocleavage assay, mismatched sequences, and competition experiments between psorospermin and A-62176, we pinpointed the drug binding site on the DNA base pairs between positions +1 and +2 relative to the cleaved phosphodiester bonds. A 2:2 quinobenzoxazine-Mg2+ self-assembly model was previously proposed, in which one drug molecule intercalates into the DNA helix and the second drug molecule is externally bound, held to the first molecule and DNA by two Mg2+ bridges. The results of competition experiments between psorospermin and A-62176, as well as between psorospermin and A-62176 and norfloxacin, are consistent with this model and provide the first evidence that this 2:2 quinobenzoxazine-Mg2+ complex is assembled in the presence of topoisomerase II. These results also have parallel implications for the mode of binding of the quinolone antibiotics to the bacterial gyrase-DNA complex.