Dagmar Selke

Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C

The standardized extract from Ginkgo biloba (EGb 761) is used for the treatment of dementia. Because of allergenic and genotoxic effects, ginkgolic acids are restricted in EGb 761 to 5 ppm. The question arises whether ginkgolic acids also have neurotoxic effects. In the present study, ginkgolic acids caused death of cultured chick embryonic neurons in a concentration-dependent manner, in the presence and in the absence of serum. Ginkgolic acids-induced death showed features of apoptosis as we observed chromatin condensation, shrinkage of the nucleus and reduction of the damage by the protein synthesis inhibitor cycloheximide, demonstrating an active type of cell death. However, DNA fragmentation detected by the terminal-transferase-mediated ddUTP-digoxigenin nick-end labeling (TUNEL) assay and caspase-3 activation, which are also considered as hallmarks of apoptosis, were not seen after treatment with 150 microM ginkgolic acids in serum-free medium, a dose which increased the percentage of neurons with chromatin condensation and shrunken nuclei to 88% compared with 25% in serum-deprived, vehicle-treated controls. This suggests that ginkgolic acid-induced death showed signs of apoptosis as well as of necrosis. Ginkgolic acids specifically increased the activity of protein phosphatase type-2C, whereas other protein phosphatases such as protein phosphatases 1A, 2A and 2B, tyrosine phosphatase, and unspecific acid- and alkaline phosphatases were inhibited or remained unchanged, suggesting protein phosphatase 2C to play a role in the neurotoxic effect mediated by ginkgolic acids.

Protein phosphatase type 2C active at physiological Mg2+: stimulation by unsaturated fatty acids

Type 2C serine/threonine protein phosphatases (PP2C) so far require unphysiologically large amounts of Mg2+ ions for activity. Activators and inhibitors are not available, targeting subunits unknown. Studying the regulation of PP2C isozymes in bovine retinae, we found that the activity of PP2C increased specifically by the addition of mono‐ and polyunsaturated fatty acids. Activation was most pronounced at low Mg2+ levels (10‐fold stimulation of PP2Cα by 0.5 mM arachidonic acid at 0.7 mM Mg2+). Sensitivity of PP2Cβ was 30–50% less, revealing for the first time enzymatic differences among the PP2C isozymes. Combining unsaturated fatty acids with physiological Mg2+ concentrations resulted in PP2C activity that by far exceeded the dephosphorylation rates obtained otherwise. This suggests that PP2C activity has been severely underestimated in the past. In the presence of fatty acids, Ca2+ ions became inhibitory in the micromolar range. We conclude that unsaturated fatty acids may play a role in the regulation of PP2C activity.

ATP-citrate lyase as a substrate of protein histidine phosphatase in vertebrates.

The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes.

Protein Histidine Phosphatase: A Novel Enzyme with Potency for Neuronal Signaling

The importance of reversible phosphorylation for neuronal signaling and cell survival is well recognized. Knowledge in vertebrates, however, is so far limited to O-phosphates from serine, threonine, and tyrosine. The authors describe an enzyme acting on N-phosphates. It is the first protein histidine phosphatase identified in vertebrates. This histidine phosphatase is ubiquitously expressed in mammalian tissues including brain. Characterization and sequencing showed a yet unknown protein with no similarity to other phosphatases. In Caenorhabditis elegans, the homolog of this histidine phosphatase was exclusively expressed in neurons, suggesting a distinct role of reversible histidine phosphorylation in neuronal functions.

PROTEIN PHOSPHATASE TYPE-2C ISOZYMES PRESENT IN VERTEBRATE RETINAE : PURIFICATION, CHARACTERIZATION, AND LOCALIZATION IN PHOTORECEPTORS

Posttranslational modification of proteins by kinases and phosphatases plays an important role in the regulation of cellular signaling in general and neurochemistry in particular. This also applies to vertebrate photoreceptors where phosphorylation of rhodopsin causes uncoupling from the signal transduction cascade. Functional activity of rhodopsin is restored after substitution of the bleached photopigment 11‐cis‐retinal and by dephosphorylation of the opsin moiety. Phosphatases type‐1 and type‐2A have been identified in vertebrate retinae. Recently, we have shown by molecular cloning that two isozymes of protein phosphatase type‐2C (PP2C, PPM) do exist in retinal tissue. In this report, we have purified PP2Cα and PP2Cβ from bovine retinae. Thirty to 40% of PP2C was recovered in the cytosolic fraction. Biochemical properties of native and heterologously expressed recombinant enzymes were similar. Enzymatic activity is strictly dependent on the presence of Mg2+. Addition of Ca2+ ions inhibits Mg2+‐sustained activity. Antiserum raised against a C‐terminal peptide of PP2Cβ specifically labeled the outer segments of rod photoreceptor cells. PP2C protein levels were significantly reduced in RCS rats, which develop age‐dependent photoreceptor degeneration comparable to the hereditary disease retinitis pigmentosa. Although the retinal substrate(s) remain to be identified, the results suggest that PP2C modulates cellular components of the phototransduction machinery. J. Neurosci. Res. 51:328–338, 1998.

Relationship between protein phosphatase type-2C activity and induction of apoptosis in cultured neuronal cells

The cellular composition and concentration of fatty acids are crucial for proliferation and survival. We recently showed stimulation of protein phosphatase type-2C (PP2C) by unsaturated fatty acids. Here, we describe that treatment of cultured chick neurons with 100 microM oleic acid for 24h increased the percentage of damaged neurons to 61+/-9% compared with 25+/-4% in controls. Oleic acid-induced cell death showed features of apoptosis such as chromatin condensation, shrinkage of the nucleus, DNA fragmentation and caspase-3 activation. Extensive studies with a variety of fatty acids revealed a striking correlation between activation of PP2C and induction of apoptosis. Lipophilicity, oxidizability, and an acidic group were required for both effects. In addition, activation of PP2C and induction of apoptosis could discriminate between cis- and trans-conformation of the fatty acids. The results are in favor of PP2C playing an important, yet unidentified role in apoptosis.

Serine / Threonine Protein Phosphatases Type 1, 2A and 2C in Vertebrate Retinae

A number of retinal proteins are phosphorylated by a variety of kinases, resulting in well-known regulatory effects. The identity and role of corresponding phosphatases is less clear. We simultaneously measured the activity of serine/ threonine protein phosphatases type 1, 2A and 2C in bovine retinae. The enzymes were classified according to substrate specificity, divalent cation requirement and the effect of phosphatase subtype-specific inhibitors. The total- and specific activity of phosphatase type 2A was prevalent. Type 2C was 10-fold less abundant. 80% of type 1 and 50% of type 2A and type 2C, respectively, were soluble. An economic purification scheme was developed. We demonstrated the presence of phosphatase isozymes 2Cα and 2Cβ in bovine rod outer segments by enzymatic analysis as well as immunological techniques. The results suggest a yet unknown role of phosphatase type 2C in phototransduction. On the other hand, the immense amount of protein phosphatases found to be soluble – therefore not associated with rod outer segment membranes – points towards participation of these enzymes in the process of visual transduction not considered thus far.

Synthesis of novel N-aroyl- and N-arylsulfonylisothiazole-2-imines by cyclization of thiocyanatovinylaldehyde hydrazones

Abstract The synthesis of N-aroyl- and N-arylsulfonylisothiazole-2-imines 7c - f as well as the corresponding acceptor-substituted 2-aminoisothiazolium sa

Activity of PP2Cβ is Increased by Divalent Cations and Lipophilic Compounds Depending on the Substrate

Publisher Summary The chapter discusses activity of protein phosphatase type-2Cβ (PP2Cβ) that increases by divalent cations and lipophilic compounds depending on the substrate. The α- and β-isozymes of serine/threonine protein phosphatase type-2C (PP2C) belong to the phosphatases of the first generation, described side by side with PP1, PP2A and PP2B. The requirement of Mg 2+ - or Mn 2+ -ions for PP2C activity still holds true. No other mono- or divalent cation was found capable of supporting PP2C activity. It was discovered that PP2C activity is highly active at physiological Mg 2+ concentrations provided certain unsaturated lipophilic compounds were present. The concentration of the lipids necessary, so far, exceeds the concentration of free fatty acids under physiological conditions. However, those assays were performed using casein as a substrate, and casein itself is known to bind free fatty acids, thus reducing the effective concentration to a much lower value. The chapter discusses determination of PP2C-activity including [ 32 P] Casein as a substrate, BAD phosphorylated at Ser-155 as a substrate, and phosphopeptides as substrates. Differences in the dependence of divalent cations for activity and in the effect of lipophilic oxidizable compounds are also discussed.

Oxidation of Acceptor‐substituted Isothiazolium‐2‐imines to Stable Cyclic Sulfin‐ and Sulfonamides with 3‐Hydroperoxy Function

The oxidation of isothiazolium 2-imines 3,5 and their salts 4 to stable 3-hydroperoxy-2,3,4,5,6,7-hexahydro-1,2-benzisothiazole 1-oxides 7 and 1,1-dioxides 8 and 9 as a new class of cyclic sultims and sultams is described. The formation of 3-hydroxysultams 10 and isothiazol-3(2H)one 1,1-dioxides 11 is presented.