Nanoelectrospray based synthesis of large, transportable membranes with integrated membrane proteins.

Membrane proteins tend to be difficult to study since they need to be integrated into a lipid bilayer membrane to function properly. This study presents a method to synthesize a macroscopically large and freely transportable membrane with integrated membrane proteins which is useful for studying membrane proteins and protein complexes in isolation. The method could serve as a blueprint for the production of larger quantities of functionalised membranes for integration into technical devices similar to the MinION DNA sequencer.

Lipid Discovery by Combinatorial Screening and Untargeted LC-MS/MS.

We present a method for the systematic identification of picogram quantities of new lipids in total extracts of tissues and fluids. It relies on the modularity of lipid structures and applies all-ions fragmentation LC-MS/MS and Arcadiate software to recognize individual modules originating from the same lipid precursor of known or assumed structure. In this way it alleviates the need to recognize and fragment very low abundant precursors of novel molecules in complex lipid extracts.

Structural basis for targeting the chromatin repressor Sfmbt to Polycomb response elements.

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt.

DMSO enhances electrospray response, boosting sensitivity of proteomic experiments.

We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware or software and at no additional cost.

Principles of electrospray ionization.

Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized.

Principles of electrospray ionization.

Electrospray ionization is today the most widely used ionization technique in chemical and bio-chemical analysis. Interfaced with a mass spectrometer it allows to investigate the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized.

p38 MAPK controls prothrombin expression by regulated RNA 3' end processing.

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation.

Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB.

Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes.

Quantitative proteomics in biological research.

Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article reviews the different methods used to extract quantitative information from mass spectra.

RBM5/Luca-15/H37 regulates Fas alternative splice site pairing after exon definition.

RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death.

Translation initiation by the c-myc mRNA internal ribosome entry sequence and the poly(A) tail.

Eukaryotic mRNAs possess a poly(A) tail that enhances translation via the (7)mGpppN cap structure or internal ribosome entry sequences (IRESs). Here we address the question of how cellular IRESs recruit the ribosome and how recruitment is augmented by the poly(A) tail. We show that the poly(A) tail enhances 48S complex assembly by the c-myc IRES.

Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate.

Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts.

A compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export from the nucleus.

PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A.

Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes.

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro.

Combination of peptide OFFGEL fractionation and label-free quantitation facilitated proteomics profiling of extraocular muscle.

Several label-free quantitation strategies have been introduced that obliterate the need for expensive isotopically labeled molecules. However label-free approaches have considerably higher demands in respect of repeatability of sample preparation and fractionation than multiplexing isotope labeling-based strategies. OFFGEL fractionation promises the necessary separation efficiency and repeatability.

Splicing factors stimulate polyadenylation via USEs at non-canonical 3' end formation signals.

The prothrombin (F2) 3' end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3' UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function.

Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25.

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation-induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin-binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L-plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT).

Geminin cleavage during apoptosis by caspase-3 alters its binding ability to the SWI/SNF subunit Brahma.

Geminin has been proposed to coordinate cell cycle and differentiation events through balanced interactions with the cell cycle regulator Cdt1 and with homeobox transcription factors and chromatin remodeling activities implicated in cell fate decisions. Here we show that Geminin is cleaved in primary cells and cancer cell lines induced to undergo apoptosis by a variety of stimuli. Geminin targeting is mediated by caspase-3 both in vivo and in vitro.

Quantitative proteomics profiling of sarcomere associated proteins in limb and extraocular muscle allotypes.

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only allow the analysis of a small fraction of proteins at a time.

The sec14 homology module of neurofibromin binds cellular glycerophospholipids: mass spectrometry and structure of a lipid complex.

Neurofibromin is the protein product of the tumor suppressor gene NF1, alterations of which are responsible for the pathogenesis of the common disorder Neurofibromatosis type I (NF1). The only well-characterized function of neurofibromin is its RasGAP activity, contained in the central GAP related domain (GRD). By solving the crystal structure of a 31 kDa fragment at the C-terminal end of the GRD we have recently identified a novel bipartite lipid-binding module composed of a Sec14 homologous and a previously undetected pleckstrin homology (PH)-like domain.

Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A.

Legionella pneumophila, the causal agent of Legionnaires' disease, is an intracellular parasite and invades and proliferates within different eukaryotic cells, including human alveolar macrophages. After several 100-fold multiplication within host cells, the pathogens are released for new invasion by induction of apoptosis or necrosis. Here we report that L.

IRAG mediates NO/cGMP-dependent inhibition of platelet aggregation and thrombus formation.

Defective regulation of platelet activation/aggregation is a predominant cause for arterial thrombosis, the major complication of atherosclerosis triggering myocardial infarction and stroke. A central regulatory pathway conveying inhibition of platelet activation/aggregation is nitric oxide (NO)/cyclic GMP (cGMP) signaling by cGMP-dependent protein kinase I (cGKI). However, the regulatory cascade downstream of cGKI mediating platelet inhibition is still unclear.

HURP is part of a Ran-dependent complex involved in spindle formation.

Background: GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described.

Results: Here we identify a complex that is required for the conversion of aster-like to spindle-like structures.

A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities.

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pho is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, we purified Pho protein complexes from Drosophila embryos and found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt.

NuSAP, a mitotic RanGTP target that stabilizes and cross-links microtubules.

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes.