Characterisation of the site-specific monoPEGylated rhG-CSF analogue pegteograstim.

We describe the characterisation of a novel monoPEGylated recombinant human granulocyte colony-stimulating factor analogue, pegteograstim (Neulapeg), prepared by site-specific 20 kDa maleimide-PEG conjugation. An additional cysteine was inserted between Gly136 and Ala137 of filgrastim (methionyl human granulocyte colony-stimulating factor) for site-specific PEGylation, and Cys18 of filgrastim was replaced with Ser18 to prevent unwanted PEGylation. Pegteograstim was produced by Escherichia coli and purified by cation exchange chromatography, and its structural, physicochemical, biological and immunological properties were investigated. Male Sprague-Dawley rats were administered pegteograstim (100 µg/kg) and the pharmacokinetics and pharmacodynamics compared with those of filgrastim. The results of long-term stability testing of pegteograstim revealed no significant change in its quality attributes at 2-8 °C for 36 months. In addition, pegteograstim was stable under the accelerated conditions (25 ± 2 °C, RH of 60 ± 5%) for 6 months. The site-specific monoPEGylated pegteograstim is a highly pure, stable and novel drug for long-lasting treatment of chemotherapy-induced neutropenia.

Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage-Gated Kv7.2 Potassium Channels.

Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signaling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP2 ), Ca2+ /calmodulin, and phosphorylation. Recent studies found that the PIP2 sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP2 -binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST-fusion proteins exposed to recombinant protein kinases by using LC-MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3ß could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein-protein and protein-lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567.

Transcriptomic and Proteomic Analysis of Arion vulgaris--Proteins for Probably Successful Survival Strategies?

The Spanish slug, Arion vulgaris, is considered one of the hundred most invasive species in Central Europe. The immense and very successful adaptation and spreading of A. vulgaris suggest that it developed highly effective mechanisms to deal with infections and natural predators. Current transcriptomic and proteomic studies on gastropods have been restricted mainly to marine and freshwater gastropods. No transcriptomic or proteomic study on A. vulgaris has been carried out so far, and in the current study, the first transcriptomic database from adult specimen of A. vulgaris is reported. To facilitate and enable proteomics in this non-model organism, a mRNA-derived protein database was constructed for protein identification. A gel-based proteomic approach was used to obtain the first generation of a comprehensive slug mantle proteome. A total of 2128 proteins were unambiguously identified; 48 proteins represent novel proteins with no significant homology in NCBI non-redundant database. Combined transcriptomic and proteomic analysis revealed an extensive repertoire of novel proteins with a role in innate immunity including many associated pattern recognition, effector proteins and cytokine-like proteins. The number and diversity in gene families encoding lectins point to a complex defense system, probably as a result of adaptation to a pathogen-rich environment. These results are providing a fundamental and important resource for subsequent studies on molluscs as well as for putative antimicrobial compounds for drug discovery and biomedical applications.

Amphetamine action at the cocaine- and antidepressant-sensitive serotonin transporter is modulated by αCaMKII.

Serotonergic neurotransmission is terminated by reuptake of extracellular serotonin (5-HT) by the high-affinity serotonin transporter (SERT). Selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine or escitalopram inhibit SERT and are currently the principal treatment for depression and anxiety disorders. In addition, SERT is a major molecular target for psychostimulants such as cocaine and amphetamines. Amphetamine-induced transport reversal at the closely related dopamine transporter (DAT) has been shown previously to be contingent upon modulation by calmodulin kinase IIα (αCaMKII). Here, we show that not only DAT, but also SERT, is regulated by αCaMKII. Inhibition of αCaMKII activity markedly decreased amphetamine-triggered SERT-mediated substrate efflux in both cells coexpressing SERT and αCaMKII and brain tissue preparations. The interaction between SERT and αCaMKII was verified using biochemical assays and FRET analysis and colocalization of the two molecules was confirmed in primary serotonergic neurons in culture. Moreover, we found that genetic deletion of αCaMKII impaired the locomotor response of mice to 3,4-methylenedioxymethamphetamine (also known as "ecstasy") and blunted d-fenfluramine-induced prolactin release, substantiating the importance of αCaMKII modulation for amphetamine action at SERT in vivo as well. SERT-mediated substrate uptake was neither affected by inhibition of nor genetic deficiency in αCaMKII. This finding supports the concept that uptake and efflux at monoamine transporters are asymmetric processes that can be targeted separately. Ultimately, this may provide a molecular mechanism for putative drug developments to treat amphetamine addiction.

Drebrin depletion alters neurotransmitter receptor levels in protein complexes, dendritic spine morphogenesis and memory-related synaptic plasticity in the mouse hippocampus.

Drebrin an actin-bundling key regulator of dendritic spine genesis and morphology, has been recently proposed as a regulator of hippocampal glutamatergic activity which is critical for memory formation and maintenance. Here, we examined the effects of genetic deletion of drebrin on dendritic spine and on the level of complexes containing major brain receptors. To this end, homozygous and heterozygous drebrin knockout mice generated in our laboratory and related wild-type control animals were studied. Level of protein complexes containing dopamine receptor D1/dopamine receptor D2, 5-hydroxytryptamine receptor 1A (5-HT1(A)R), and 5-hydroxytryptamine receptor 7 (5-HT7R) were significantly reduced in hippocampus of drebrin knockout mice whereas no significant changes were detected for GluR1, 2, and 3 and NR1 as examined by native gel-based immunoblotting. Drebrin depletion also altered dendritic spine formation, morphology, and reduced levels of dopamine receptor D1 in dendritic spines as evaluated using immunohistochemistry/confocal microscopy. Electrophysiological studies further showed significant reduction in memory-related hippocampal synaptic plasticity upon drebrin depletion. These findings provide unprecedented experimental support for a role of drebrin in the regulation of memory-related synaptic plasticity and neurotransmitter receptor signaling, offer relevant information regarding the interpretation of previous studies and help in the design of future studies on dendritic spines.

Radiation protection from whole-body gamma irradiation (6.7 Gy): behavioural effects and brain protein-level changes by an aminothiol compound GL2011 in the Wistar rat.

GL2011 is a naturally occurring thiol compound and a series of thiol compounds have been proposed as radioprotectors. Radioprotective efficacy of a triple intraperitoneal dose of GL2011 of 100 mg/kg body weight of Wistar rats, 30 min prior to and 3 and 6 h following irradiation (6.7 Gy) was evaluated. Four groups of animals were used, vehicle-treated non-irradiated (VN), GL2011-treated and irradiated (GI), GL2011-treated and non-irradiated (GN) and vehicle-treated and irradiated (VI) (n = 30 per group). The radioprotective efficacy of GL2011 was determined by measuring 28-day survival and intestinal crypt cell survival. Neuroprotection in terms of behaviour was evaluated using the behavioural observational battery, open field test and elevated plus maze paradigm. An RNA microarray was carried out in order to show differences at the RNA level between VI and VN groups. Brain protein changes were identified using a gel-based proteomics method and major brain receptor complex levels were determined by blue-native gels followed by immunoblotting. 28-Day survival rate in VI was 30 %, in GI survival was 93 %, survival of VN and GN was 100 %. Jejunal crypt cell survival was significantly enhanced in GI. Protein-level changes of peroxiredoxin-5, Mn-superoxide dismutase 2, voltage-dependent anion-selective channel protein 1, septin 5 and dopamine D2 receptor complex levels were paralleling radiation damage and protection. Taken together, the findings demonstrate that GL2011 improves survival rates and jejunal crypt survival, provides partial neuroprotection at the behavioural level and modulates proteins known to be involved in protection against oxidative stress-mediated cell damage.

Characterization of α-l-Iduronidase (Aldurazyme®) and its complexes.

Alpha-l-Iduronidase(IDUA) was the first enzyme replacement therapy approved for mucopolysaccharidosis type I and the corresponding recombinant protein drug, Aldurazyme®, is commercially available. In the frame of gel-based mass spectrometrical characterization of protein drugs, we intended to identify protein sequence and possible protein modifications. Moreover, we were interested in which aggregation/complex form Aldurazyme® would exist, which complexes were enzymatically active and in which form the naturally occurring enzyme would be present in the brain. Aldurazyme® was run on 2DE gel electrophoresis, spots were excised, in-gel digested with several proteases and identified by nano-LC-ESI-MS/MS on an ion trap. IDUA-activity was determined by a fluorometric principle. Blue-native gel electrophoresis with subsequent immunoblotting was carried out to show the presence of protein complexes. The protein was unambiguously identified by 100% sequence coverage; several amino acid substitutions were detected and protein modifications were novel phosphorylations on S59 and S482, histidine methylation at H572 and provide evidence for already known N-glycosylations. Four Aldurazyme® complexes that all were enzymatically active, were observed while a single complex was observed for the physiologically occurring IDUA in the brain. The findings are relevant for understanding chemistry, physiology, pharmacology and medicine of IDUA, design of further and interpretation of previous work.

Hippocampal levels and activity of the sodium/potassium transporting ATPase subunit α-3 (AT1A3) are paralleling memory training in the multiple T-maze in the C57BL/6J mouse.

Although the sodium/potassium transporting ATPase subunit alpha-3 (AT1A3) has been linked to memory mechanisms in rodents, regulation of this ATPase in terms of activity and complex levels by memory performance in a land maze has not been shown so far. It was therefore the aim of the study to link memory retrieval in the multiple T-Maze (MTM) to AT1A3 protein levels and activity. C57BL/6J mice were trained in the MTM and euthanized 6h following memory retrieval. Hippocampal membrane proteins were prepared by ultracentrifugation and run on blue native gel electrophoresis (BN-PAGE). Enzyme activity was evaluated using an in-gel method. AT1A3 protein was characterized using mass spectrometry (nano-LC-ESI-MS/MS). On BN-PAGE a single band was observed at 240 kDa, which corresponds to the dimeric form of the enzyme. Higher levels of AT1A3 complex were seen in trained mice. Also ATPase activity was higher in trained mice, and was observed both at 110 and at 240 kDa. Mass spectrometry unambiguously identified AT1A3 with 98.91% sequence coverage. A series of novel AT1A3 phosphorylation sites were detected. Taken together, it was shown that increased AT1A3 protein levels for the dimer as well as AT1A3 activity represented by the monomer and the dimer were paralleling memory training in the MTM. This may be relevant for understanding the role of the catalytic hydrolysis of ATP coupled with the exchange of sodium and potassium ions across the plasma membrane that generates the electrochemical gradient of sodium and potassium ions. Herein, we provide evidence for a possible role of AT1A3 in memory mechanisms and support previous findings using different animal models for memory formation.

Hippocampal glutamate transporter 1 (GLT-1) complex levels are paralleling memory training in the Multiple T-maze in C57BL/6J mice.

The glutamate transporter 1 (GLT-1) is essential for glutamate uptake in the brain and associated with various psychiatric and neurological disorders. Pharmacological inhibition of GLT-1 results in memory deficits, but no study linking native GLT-1 complexes was published so far. It was therefore the aim of the study to associate this highly hydrophobic, eight transmembrane spanning domains containing transporter to memory training in the Multiple T-maze (MTM). C57BL/6J mice were used for the spatial memory training experiments, and trained mice were compared to untrained (yoked) animals. Mouse hippocampi were dissected out 6 h after training on day 4, and a total enriched membrane fraction was prepared by ultracentrifugation. Membrane proteins were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) with subsequent Western blotting against GLT-1 on these native gels. Moreover, GLT-1 complexes were identified by mass spectrometry (nano-LC-ESI-MS/MS). Animals learned the MTM task and multiple GLT-1 complexes were detected at apparent molecular weights of 242, 480 and 720 kDa on BN-PAGE Western blotting. GLT-1 complex levels were significantly higher in the trained group as compared to yoked controls, and antibody specificity was verified by immunoblotting on multidimensional gels. Hippocampal GLT-1 was unambiguously identified by mass spectrometry with high sequence coverage, and glycosylation was observed. It is revealed that increased GLT-1 complex levels are paralleling and are linked to spatial memory training. We provide evidence that signal termination, represented by the excitatory amino acid transporter GLT-1 complexes, is involved in spatial memory mechanisms.

Protein L-isoaspartyl O-methyltransferase inhibits amyloid beta fibrillogenesis in vitro.

Fibrillar aggregates of beta-amyloid peptide (Abeta) are major constituents of the senile plaques found in the brains of patients suffering from Alzheimer's disease (AD). Previous studies have shown that spontaneous isomerization or racemization of aspartyl residues in Abeta peptides leads to conformational changes in the secondary structure and increased aggregative ability of the peptides. Protein L-isoaspartyl O-methyltransferase (PIMT, EC 2.1.1.77) is a repairing enzyme converting L-isoaspartyl/D-aspartyl residues in damaged proteins to normal L-aspartyl residues. In this study it was investigated, whether PIMT is able to modulate Abeta fibrillogenesis in vitro by methylation of isoaspartyl residue using purified 5Abeta and PIMT. A Thioflavin-T (Th-T) binding assay conducted after aging Abeta in vitro (37 degrees C, pH 7.4 in PBS) revealed that PIMT inhibited the increase of fluorescence caused by amyloid fibrillogenesis. Western blot analysis revealed that high molecular Abeta aggregates (> 200 kDa) only occurred during Abeta incubation, while they were reduced in response to incubation with PIMT and AdoMet. Additionally, circular dichroism (CD) showed that the beta-sheet structure was increased in Abeta peptides in a time-dependent fashion, while PIMT suppressed the beta-sheet transition after 24 h. Finally, transmission electron microscopy (TEM) revealed that PIMT reduced the size of the Abeta aggregates and induced a different pathway, leading to the formation of amorphous structures. Taken together, these findings indicate that isoaspartyl methylation leads to partial blockade of fibrillogenesis of Abeta by inhibiting the beta transition in the Abeta peptide.

A serotonin receptor 1A containing complex in hippocampus of PWD/PhJ mice is linked to training effects in the Barnes maze.

Although the involvement of the serotonin 1A receptor (5-HT(1A) R) in memory has been shown by several groups there is no information about the 5-HT(1A) R complex but rather the monomeric form. Moreover, there is insufficient information on the characterization of the antigen, the 5-HT(1A) R. PWD/PhJ mice, a wild-caught strain that was inbred at The Jackson Laboratories were used for the experiments. The Barnes maze (BM) paradigm for the evaluation of spatial memory was chosen because of experience with this setting in our laboratory. Mice were sacrificed 6 h following the probe trial on day 12, hippocampi were extirpated, proteins extracted and run on blue native gels with subsequent immunoblotting with a specific antibody against the mouse 5-HT(1A) R. Densitometry of the single band from the immunoblots was carried out. The receptor from the complex was identified by mass spectrometry (nano-LC-ESI-MS/MS). A serotonin receptor 1A complex was identified by immunoblotting at the apparent molecular weight of 480 kDa indicating the presence of a homopolymer as denaturation only revealed a single band at approx. 50 kDa. 5-HT(1A) R levels were significantly higher in the trained group as compared to yoked controls. The hippocampal 5-HT(1A) R of the trained group was unambiguously identified. Taken together, a serotonin receptor 1A homopolymer is associated with memory training effects in the BM using PWD/PhJ mice. This observation shows that a specific complex rather than a receptor subunit as previously shown is involved in memory process and the receptor protein was characterized.