A new format of electrodes for the electrochemical reduction of cytochromes P450

New approach to the electrochemical reduction of cytochromes P450 (P450s, CYPs) at electrodes chemically modified with appropriate substrates for P450s ("reverse" electrodes) was proposed. The method is based on the analysis of cyclic voltammograms, square-wave voltammograms and amperograms with subsequent determination of electrochemical characteristics such as catalytic current and redox potential. The sensitivity of proposed method is 0.2-1 nmol P450/electrode. The changes of maximal current and of redox potentials in square-wave voltammograms as well as the changes of catalytic current in amperometric experiments proved to be informative and reliable. Planar regime of screen-printed electrodes (strip-type sensors) enabled to utilise 20-60 microl of electrolyte volume. The enzyme-substrate pairs P450 2B4/benzphetamine and P450scc/cholesterol were investigated. Electrochemical parameters of electrodes with unspecific P450 substrates differed considerably from electrodes with appropriate substrates.     

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.     

Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP

Protein misfolding is linked to different neurodegenerative disorders like Alzheimer's disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K-resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115-156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.     

Application of NMR in structural proteomics: screening for proteins amenable to structural analysis

In the time of structural proteomics when protein structures are targeted on a genome-wide scale, the detection of "well-behaved" proteins that would yield good quality NMR spectra or X-ray images is the key to high-throughput structure determination. Already, simple one-dimensional proton NMR spectra provide enough information for assessing the folding properties of proteins. Heteronuclear two-dimensional spectra are routinely used for screenings that reveal structural, as well as binding, properties of proteins. NMR can thus provide important information for optimizing conditions for protein constructs that are amenable to structural studies.     

Fibromyalgia syndrome and chronotype: late chronotypes are more affected

Sleep has strong links to the symptomology of fibromyalgia syndrome (FMS), a diffuse musculoskeletal pain disorder. Information about the involvement of the circadian clock is, however, sparse. In this study, 1548 individuals with FMS completed an online survey containing questions on demographics, stimulant consumption, sleep quality, well-being and subjective pain, chronotype (assessed by the Munich ChronoType Questionnaire, MCTQ), and FMS impact. Chronotype (expressed as the mid-sleep-point on free days, corrected for sleep deficit on workdays, MSF(sc)) significantly correlated with stress-ratings, so-called "memory failures in everyday life," fatigue, FMS impact, and depression but not with anxiety. When chronotypes were categorized into 3 groups (early, intermediate, late), significant group differences were found for sum scores of perceived stress, memory failures in everyday life, fatigue, FMS impact, and depression but not anxiety, with late chronotypes being more affected than early chronotypes. Sleepiness ratings were highest in early chronotypes. Challenges of sleep quality and subjective pain were significantly increased in both early and late chronotypes. The results show that according to their reports, late chronotypes are more affected by fibromyalgia.     

The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger.     

A pressure cooking-based DNA extraction from archival formalin-fixed, paraffin-embedded tissue

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies.     

The design, synthesis, and biological evaluation of potent receptor tyrosine kinase inhibitors

Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models.