Alkyl cinnamates as regulator for the C1 domain of protein kinase C isoforms

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Natural product curcumin is known to interact with PKC isoforms through the C1 domain and modulate PKC activity. The reported results demonstrate that the symmetric curcumin molecule might act as two separate units during its recognition of C1 domains. To understand the importance of the two halves of curcumin in PKC binding and to develop effective PKC regulators, we synthesized a series of alkyl cinnamates (1-8), characterized absorption and fluorescence properties and measured binding affinities with the C1b subdomains of PKC isoforms. The binding parameters of the monomeric compounds and liposomes containing compounds confirmed their interaction with the C1b subdomains of PKCδ and PKCθ. The molecular docking analysis with PKCδ and PKCθ C1b subdomains revealed that the alkyl cinnamates form hydrogen bond with the backbone of the protein at the same binding site as that of diacylglycerol and phorbol esters. The results show that the alkyl cinnamates bind to the activator binding site of PKCs and both methoxy and hydroxyl groups play important roles in the binding process.     

Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKCθ. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers. The calculated EC(50) values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.     

Karavilagenin C derivatives as antimalarials

Karavilagenin C (1), a cucurbitane-type triterpenoid, previously isolated from the aerial parts of Momordica balsamina, was acylated with different alkanoyl, aroyl and cinnamoyl chlorides/anydrides, yielding ten new mono or diesters, karavoates F (7) and H-P (8-16). Furthermore, the new compound cucurbalsaminol C (17) was isolated from the same plant. Their structures were assigned by spectroscopic methods, including 2D NMR experiments. Compounds 1 and 17 and the acyl derivatives 8-16 along with other five esters (2-6, karavoates A-E), previously prepared from 1, were evaluated for their in vitro antimalarial activity against the chloroquine-sensitive (3D7) and the chloroquine-resistant (Dd2) strains of Plasmodium falciparum. Compound 1 exhibited a moderate activity and 17 was inactive. However, a remarkable antiplasmodial activity was observed for most of karavilagenin C alkanoyl and monoaroyl/cynamoyl derivatives. Karavoates B, D, E, I, and M were the most active, displaying IC₅₀ values similar to those found for chloroquine, particularly against the resistant strain (IC₅₀ <0.6 ??M). Structure-activity relationships (SAR) are discussed. Moreover, the preliminary toxicity toward human cells of compounds 1-17 was also evaluated in breast cancer cell line (MCF-7). Most of the esters showed no toxicity, displaying, in general, much higher selectivity index values than those obtained for the parent compound.     

Immunochemical identification of the ADP-ribosyltransferase in botulinum C1 neurotoxin as C3 exoenzyme-like molecule

Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22,000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1,000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.     

Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx

Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C.     

Protein C defects as the basis of a thrombophilic state

Protein C defects often cause a pronounced tendency to thrombosis. This paper reports on observations of five families suffering from distinct thrombophilia due to a protein C defect.     

Diacylglycerols as activators of protein kinase C

Diacylglycerols are generated in the membrane as the result of extracellular signals and are able to stimulate the activity of protein kinase C, acting as membrane second messengers. Diacylglycerols are recognized by protein kinases C through the C1 domain and established models propose that they will stabilize the translocation of the protein to the membrane. However, diacylglycerols also act by modulating the physical properties of the membrane, thus favouring the translocation of the enzyme. This is done through alteration of the membrane surface curvature, dehydration of the surface and the separation of phospholipid surface groups. Good correlations have been observed between the physical state of the membrane and protein kinase C activity.     

S-Sulfocysteine as a Source of the Sulfur Atom of Cephalosporin C

S. lignicolum acid proteinases A-1, A-2, and B (S-PI-insensitive) were examined for the structure of the active sites by the method of Nakatani using PAD as a probe. We attempted to screen for substances that release zinc(II)-PAD from the complex of zinc(II)-PAD-S-PI-insensitive acid proteinases. Angiotensin I released zinc(II)-PAD from the complex as well as from the complex of S-PI-sensitive acid proteinases. Angiotensin I is a good substrate for these enzymes regardless of S-PI-sensitivity. These results suggest that the two carboxyl groups capable of binding to zinc(II)-PAD are at the active site regions of S. lignicolum enzymes and that they are involved in their catalytic actions.     

Plant uptake of bicarbonate as measured with the11C isotope

Summary ¹¹C which is cyclotron produced by¹⁴N(P, )¹¹C(half-life 20.1M) was use as a tracer of bicarbonate to determine its movements from a nutrient solution through roots to stems and leaves of bush bean plants (Phaseolus vulgaris L. var. Improved Tendergreen). The short time involved and the high solution pH minimized the need for use of the Hederson Hasselbach equation for activity correction. Quantities of¹¹C did move into roots, stems and leaves with a sharp decreasing gradient (root/stem=14.5, stems/leaves=11.7) More¹¹C moved into plants with KHCO₃ than with NaHCO₃. The (NH₄)₂SO₄ enhanced¹¹C uptake and KNO₃ than with competition indicated possibility of some uptake of HCO ₃ ^– . In an experiment withGalenia pubescens (Eckl. and Zeyh.) Druce, the¹¹C was more readily moved to stems and leaves than in bush bean indicating substantial uptake of HCO ₃ ^– .     

Expanding Role of the Jumonji C Domain as an RNA Hydroxylase

JmjC (Jumonji C) domain-containing proteins are known to be an extensive family of Fe(II)/2-oxoglutarate-dependent oxygenases involved in epigenetic regulation of gene expression by catalyzing oxidative demethylation of methylated histones. We report here that a human JmjC protein named Tyw5p (TYW5) unexpectedly acts in the biosynthesis of a hypermodified nucleoside, hydroxywybutosine, in tRNA^Phe by catalyzing hydroxylation. The finding provides an insight into the expanding role of JmjC protein as an RNA hydroxylase.     

Synthetic peptides as antagonists of the anaphylatoxin C3a.

Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.     

Identification of a new enterotoxin as enterotoxin C

Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481-1485. 1965.-Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain.     

Characterization of the human glomerular C3 receptor as the C3b/C4b complement type one (CR1) receptor

The functional and immunochemical characteristics of the human glomerular C3 receptor were investigated by adherence of sheep erythrocytes (Es) coated with defined C3 fragments and by using polyclonal and/or monoclonal antibodies directed against epitopes expressed on complement receptors CR1, CR2, and CR3. C3b-bearing Es (EsC3b) strongly adhered to glomeruli in frozen kidney sections in a reaction that was selectively inhibited by F(ab')2 anti-CR1 antibodies. There was no adherence of EsC3dg, EsC3d, and EsC3bi in the presence or absence of Ca++ and Mg++ under physiologic buffer conditions. The weak glomerular binding of EsC3bi, which was observed in half-isotonic buffer was selectively suppressed by anti-CR1 antibodies. By indirect immunofluorescence, anti-CR1 antibodies stained all podocytes in glomeruli, whereas no staining of kidney sections was seen with OKM1 and anti-Mol antibodies directed against the alpha-chain of CR3 and with anti-CR2 antibodies anti-B2 and BL13. Solubilization of membrane glycoproteins from freshly isolated glomeruli from three human kidneys, in the presence of 0.1% Nonidet P-40, yielded a material that bound to lentil lectin Sepharose and could accelerate the decay of preformed cell-bound amplification C3 convertase sites in a reaction that was inhibited by anti-CR1 antibodies. The material containing CR1 activity was labeled with 125I, immunoprecipitated with anti-CR1, and analyzed by SDS-PAGE and autoradiography. Anti-CR1 immunoprecipitated a form of CR1 of Mr 205,000 in solubilized glomeruli from three donors, and an additional form of Mr 160,000 in glomeruli from two of the donors. Immunoprecipitation of CR1 from surface-labeled erythrocytes from these individuals demonstrated them to be homozygous for the 205,000 Mr form of the receptor. Whether the 160,000 band represents in vitro or in vivo proteolytic cleavage of CR1, or cell specific-modulation of gene expression of glomerular CR1, remains unclear. Thus, CR1 is the only type of C3 receptor expressed in the human kidney. Glomerular CR1 shares the functional antigenic and biochemical properties of the C3b/C4b CR1 receptor of peripheral blood cells.