Rekombinante Expression und vergleichende biochemische Charakterisierung der Isoformen C beta1 und C beta2 der katalytischen Untereinheit der cAMP-abhängigen Proteinkinase

Die cAMP-abhängige Proteinkinase (PKA) ist das Schlüsselenzym der cAMP-Signalübertragung. Im Ruhezustand bilden die Isoformen der regulatorischen (R) und katalytischen Untereinheiten (C) Heterotetramere, cAMP setzt daraus die aktiven C-Untereinheiten frei. Unser Wissen über die Funktion der C-Untereinheiten basiert fast ausschließlich auf Daten, die an C alpha erhoben wurden. Über die biochemischen Eigenschaften und die biologische Rolle der C beta-Isoform und ihrer Spleißvarianten ist nur sehr wenig bekannt. Völlig unklar ist bislang, ob und wie die C beta-Untereinheiten zur Weichenstellung in der cAMP-Signalkette beitragen können. Die Spleißvariante C beta2 unterscheidet sich von C beta1 im Exon 1 und ist mit 46 kDa die größte C-Untereinheit in Säugern. In dieser Arbeit konnte mit der Expression in Sf9-Zellen erstmals ein System etabliert werden, das es erlaubt, das C beta2-Protein katalytisch aktiv zu gewinnen und homogen zu reinigen. C beta2 wurde biochemisch mit den C alpha- und C beta1-Untereinheiten vergleichend charakterisiert. Dabei konnte bestätigt werden, dass C beta2 wesentliche Eigenschaften einer katalytischen Untereinheit der PKA aufweist. Allerdings unterscheiden sich die drei Isoformen signifikant in ihren katalytischen Konstanten - ein möglicher Ansatzpunkte für eine isoformabhängige Feinregulation der Signalweitergabe. Interaktionsanalysen mittels Oberflächenplasmonresonanz (SPR) zeigten erstmals, dass C beta1 und C beta2 mit allen Isoformen der R-Untereinheit assoziieren können. Verglichen mit C alpha weisen aber sowohl C beta1 als auch C beta2 verringerte Dissoziationsraten von den Untereinheiten RI alpha und RII alpha auf – dies könnte Konsequenzen für die Regulation der verschiedenen Isoformen in der Zelle haben. Weiterhin scheinen C alpha und C beta unterschiedliche Substrate zu bevorzugen. Während einige Substrate, wie z.B. CREB, unterschiedslos durch alle drei Isoformen phosphoryliert wurden, zeigte sich, dass das basische Myelinprotein und Histon H3 durch C beta1 und C beta2 deutlich geringer phosphoryliert werden als durch C alpha. Darüber hinaus konnte in Histonpräparationen eine inhibitorische Aktivität charakterisiert werden, die nur gegen C beta1 und C beta2, nicht aber gegen C alpha gerichtet ist. Sie weist Eigenschaften eines Proteins auf. Die Entdeckung eines solchen isoformspezifischen Inhibitors könnte der Schlüssel zu einem neuen Verständnis der spezifischen Signalweiterleitung sein. Es bleibt weiteren Arbeiten vorbehalten, zu klären, wie diese Regulation im zellulären Kontext stattfindet und weitere beteiligte Komponenten zu identifizieren. Zusammen mit der isoformspezifischen inhibitorischen Aktivität weisen die beobachtete Substratselektivität und die potentiell frühere Aktivierbarkeit und Abschaltung von C beta-Untereinheiten schon jetzt auf komplexe und vielfältige Möglichkeiten der Zelle hin, mit Hilfe der C beta Isoformen cAMP-Signale selektiv zu verarbeiten

Chemical-free lysis and fractionation of cells by use of surface acoustic waves for sensitive protein assays

We exploit the mechanical action of surface acoustic waves (SAW) to differentially lyse human cancer cells in a chemical-free manner. The extent to which cells were disrupted is reported for a range of SAW parameters, and we show that the presence of 10 μm polystyrene beads is required to fully rupture cells and their nuclei. We show that SAW is capable of subcellular fractionation through the chemical-free isolation of nuclei from whole cells. The concentration of protein was assessed in lysates with a sensitive microfluidic antibody capture (MAC) chip. An antibody-based sandwich assay in a microfluidic microarray format was used to detect unlabeled human tumor suppressor protein p53 in crude lysates, without any purification step, with single-molecule resolution. The results are digital, enabling sensitive quantification of proteins with a dynamic range >4 orders of magnitude. For the conditions used, the efficiency of SAW-induced mechanical lysis was determined to be 12.9% ± 0.7% of that for conventional detergent-based lysis in yielding detectable protein. A range of possible loss mechanisms that could lead to the drop in protein yield are discussed. Our results show that the methods described here are amenable to an integrated point-of-care device for the assessment of tumor protein expression in fine needle aspirate biopsies

Cardiac hypertrophy is inhibited by a local pool of cAMP regulated by phosphodiesterase 2

Rationale: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodelling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A (PKA) signalling appears to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signalling microdomains. Objective: How individual cAMP microdomains impact on cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results: Using pharmacological and genetic manipulation of PDE activity we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy whereas increasing cAMP levels via PDE2 inhibition is anti-hypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of PKA isoforms we demonstrate that the anti-hypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a PKA type II subset leading to phosphorylation of the nuclear factor of activated T cells (NFAT). Conclusions: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo and its inhibition may have therapeutic applications

Artistler nasıl dinlenirler?:Bir köşede Bedia Ferdi yün örerken, ötede Vasfi ile Hazım..

Taha Toros Arşivi, Dosya No: 152-Vasfi Rıza Zobuİstanbul Kalkınma Ajansı (TR10/14/YEN/0033) İstanbul Development Agency (TR10/14/YEN/0033

Magnetite-doped polydimethylsiloxane (PDMS) for phosphopeptide enrichment

Reversible phosphorylation plays a key role in numerous biological processes. Mass spectrometry-based approaches are commonly used to analyze protein phosphorylation, but such analysis is challenging, largely due to the low phosphorylation stoichiometry. Hence, a number of phosphopeptide enrichment strategies have been developed, including metal oxide affinity chromatography (MOAC). Here, we describe a new material for performing MOAC that employs a magnetite-doped polydimethylsiloxane (PDMS), that is suitable for the creation of microwell array and microfluidic systems to enable low volume, high throughput analysis. Incubation time and sample loading were explored and optimized and demonstrate that the embedded magnetite is able to enrich phosphopeptides. This substrate-based approach is rapid, straightforward and suitable for simultaneously performing multiple, low volume enrichments

An inactivating caspase 11 passenger mutation originating from the 129 murine strain in mice targeted for c-IAP1

A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117–121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1−/− animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context

Phosphodiesterase 2A2 regulates mitochondria clearance through Parkin-dependent mitophagy

Programmed degradation of mitochondria by mitophagy, an essential process to maintain mitochondrial homeostasis, is not completely understood. Here we uncover a regulatory process that controls mitophagy and involves the cAMP-degrading enzyme phosphodiesterase 2A2 (PDE2A2). We find that PDE2A2 is part of a mitochondrial signalosome at the mitochondrial inner membrane where it interacts with the mitochondrial contact site and organizing system (MICOS). As part of this compartmentalised signalling system PDE2A2 regulates PKA-mediated phosphorylation of the MICOS component MIC60, resulting in modulation of Parkin recruitment to the mitochondria and mitophagy. Inhibition of PDE2A2 is sufficient to regulate mitophagy in the absence of other triggers, highlighting the physiological relevance of PDE2A2 in this process. Pharmacological inhibition of PDE2 promotes a ‘fat-burning’ phenotype to retain thermogenic beige adipocytes, indicating that PDE2A2 may serve as a novel target with potential for developing therapies for metabolic disorders

Identification of JAK/STAT signalling components by genome-wide RNA interference

Signalling pathways mediating the transduction of information between cells are essential for development, cellular differentiation and homeostasis. Their dysregulation is also frequently associated with human malignancies. The Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway represents one such signalling cascade whose evolutionarily conserved roles include cell proliferation and haematopoiesis. Here we describe a systematic genome-wide survey for genes required for JAK/STAT pathway activity. Analysis of 20,026 RNA interference (RNAi)-induced phenotypes in cultured Drosophila melanogaster haemocyte-like cells identified interacting genes encoding 4 known and 86 previously uncharacterized proteins. Subsequently, cell-based epistasis experiments were used to classify these proteins on the basis of their interaction with known components of the signalling cascade. In addition to multiple human disease gene homologues, we have found the tyrosine phosphatase Ptp61F and the Drosophila homologue of BRWD3, a bromo-domain-containing protein disrupted in leukaemia. Moreover, in vivo analysis demonstrates that disrupted dBRWD3 and overexpressed Ptp61F function as suppressors of leukaemia-like blood cell tumours. This screen represents a comprehensive identification of novel loci required for JAK/STAT signalling and provides molecular insights into an important pathway relevant for human cancer. Human homologues of identified pathway modifiers may constitute targets for therapeutic interventions.publishe

Screen-printed ultrasonic 2-D matrix array transducers for microparticle manipulation

This paper reports the development of a two-dimensional thick film lead zirconate titanate (PZT) ultrasonic transducer array, operating at frequency approximately 7.5 MHz, to demonstrate the potential of this fabrication technique for microparticle manipulation. All layers of the array are screen-printed then sintered on an alumina substrate without any subsequent patterning processes. The thickness of the thick film PZT is 139 ± 2 μm, the element pitch of the array is 2.3 mm, and the dimension of each individual PZT element is 2 × 2 mm2 with top electrode 1.7 × 1.7 mm2. The measured relative dielectric constant of the PZT is 2250 ± 100 and the dielectric loss is 0.09 ± 0.005 at 10 kHz. Finite element analysis was used to predict the behaviour of the array and to optimise its configuration. Electrical impedance spectroscopy and laser vibrometry were used to characterise the array experimentally. The measured surface motion of a single element is on the order of tens of nanometres with a 10 Vpeak continuous sinusoidal excitation. Particle manipulation experiments have been demonstrated with the array by manipulating Ø10 μm polystyrene microspheres in degassed water. The simplified array fabrication process and the bulk production capability of screen-printing suggest potential for the commercialisation of multilayer planar resonant devices for ultrasonic particle manipulation

Enhanced Fusion Pore Expansion Mediated by the Trans-Acting Endodomain of the Reovirus FAST Proteins

The reovirus fusion-associated small transmembrane (FAST) proteins are virus-encoded membrane fusion proteins that function as dedicated cell–cell fusogens. The topology of these small, single-pass membrane proteins orients the majority of the protein on the distal side of the membrane (i.e., inside the cell). We now show that ectopic expression of the endodomains of the p10, p14, and p15 FAST proteins enhances syncytiogenesis induced by the full-length FAST proteins, both homotypically and heterotypically. Results further indicate that the 68-residue cytoplasmic endodomain of the p14 FAST protein (1) is endogenously generated from full-length p14 protein expressed in virus-infected or transfected cells; (2) enhances syncytiogenesis subsequent to stable pore formation; (3) increases the syncytiogenic activity of heterologous fusion proteins, including the differentiation-dependent fusion of murine myoblasts; (4) exerts its enhancing activity from the cytosol, independent of direct interactions with either the fusogen or the membranes being fused; and (5) contains several regions with protein–protein interaction motifs that influence enhancing activity. We propose that the unique evolution of the FAST proteins as virus-encoded cellular fusogens has allowed them to generate a trans-acting, soluble endodomain peptide to harness a cellular pathway or process involved in the poorly understood process that facilitates the transition from microfusion pores to macrofusion and syncytiogenesis