Kinetics of PKCε activating and inhibiting llama single chain antibodies and their effect on PKCε translocation in HeLa cells

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing V(max) and/or decreasing K(m) values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.     

Fungi isolated from olive ecosystems and screening of their potential biotechnological use

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for β-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for β-glucosidase, CMCase and lipase); Rhizopus oryzae (for β-glucosidase and lipase); Rhizomucor variabilis (for β-glucosidase, CMCase and polygalacturonase); Mucor fragilis (β-glucosidase, CMCase and lipase); Galactomyces geotrichum (for β-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, β-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and β-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.     

Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Molecular linkage mapping in rye (Secale cereale L.)

A rye linkage map containing clones from rye, wheat, barley, oat and rice genomic and cDNA libraries, known-function genes and microsatellite markers, was created using an F₂ population consisting of 110 F₂-derived F₃ families. Both co-dominant and dominant markers were added to the map. Of all probes screened, 30.8% were polymorphic, and of those polymorphic 79.3% were mapped. The current map contains 184 markers present in all seven linkage groups covering only 727.3 cM. This places a marker about every 3.96 cM on average throughout the map; however, large gaps are still present. The map contains 60 markers that have been integrated from previous rye maps. Surprisingly, no markers were placed between the centromere and C1–1RS in the short arm of 1R. The short arm of chromosome 4 also lacked an adequate number of polymorphic markers. The population showed a remarkable degree of segregation distortion (72.8%). In addition, the genetic distance observed in rye was found to be very different among the maps created by different mapping populations. Received: 10 January 2000 / Accepted: 26 May 2000     

Differential inhibition of fungal oxidosqualene cyclase by 6E and 6Z isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene

Inhibitory properties of 6E (compound 1) and 6Z (compound 2) isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene against oxidosqualene-lanosterol cyclase were assayed on microsomes and whole cells of Saccharomyces cerevisiae and Candida albicans. Only the 6E isomer (compound 1), bearing a correct substrate-like configuration, strongly inhibited the enzyme both in microsomes and cell cultures. The difference between compounds 1 and 2 (which had an unfavorable geometry) was especially evident when measuring [¹⁴C]acetate incorporation into non-saponifiable lipids extracted from treated cells. While isomer Z was totally ineffective at up to 30 μM, in cells treated with 5 μM isomer E, labelled oxidosqualene, the level of which was negligible in the control, rose to over 60% of the non-saponifiable lipids.     

Two polyketide synthases are necessary for 4‐hydroxy‐5‐methylcoumarin biosynthesis in Gerbera hybrida

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4‐hydroxy‐5‐methylcoumarin (HMC). Unlike most coumarins, 5‐methylcoumarins have been suggested to be derived through the acetate‐malonate pathway. All of these polyketide‐derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2‐pyrone synthases (G2PS1–3). The enzymes are chalcone synthase‐like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4‐hydroxy‐6‐methyl‐2‐pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7‐dihydroxy‐5‐methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site‐directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.     

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.     

The TACE zymogen

The tumor necrosis factor-α-converting enzyme (TACE) is a member of the disintegrin family of metallopro-teinases (ADAMs) that plays a central role in the regulated shedding of a host of cell surface proteins. TACE is biosynthesized as a precursor protein with latent proteolytic activity (zymogen). TACE’s zymogen inhibition is mediated by its Pro domain, a 197-amino acid region that serves this function as well as aiding in the secretion of this enzyme through the secretory pathway. We have discovered that a conserved “cysteine switch” consensus motif within TACE’s Pro domain is, contrary to expectations, not required for maintenance of the inactive precursor state or for the secretion of this metalloproteinase in its functional form. The only role for this motif seems to be in decreasing TACE’s susceptibility to proteolytic degradation during its biogenesis and maturation within the secretory pathway. Interestingly, the Pro domain of TACE seems to carry both its inhibitory and secretory functions through the same mechanism: it seems to prevent the Catalytic domain from accessing its native, functional state, resembling the function of true molecular chaperones. Recent evidence suggests that TACE may also be switched out of the active conformation even by small, drug-like molecules such as the synthetic compound SB-3CT. These findings point at the possibility of developing, in the near future, a new generation of anti-inflammatory, noncompetitive TACE inhibitors that would exert negative allosteric modulation over the activity of this key enzyme, mediating several inflammatory diseases and certain cancers.