pFind Studio: a computational solution for mass spectrometry-based proteomics
2018
Cell2018. Mashtalir, N et al.
Dana Farber Canc Inst, Dept Pediat Oncol, Boston, MA 02215 USA.
ABSTRACT:Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human ca; and developmental disorders. To date, the modular organization and path- ways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionailry conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.
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NATURE2018. Kim, SJ et al.
Univ Calif San Francisco, Dept Pharmaceut Chem, Dept Bioengn & Therapeut Sci, San Francisco, CA 94158 USA.
ABSTRACT:Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.
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Nature2018. Zhao, QC et al.
Tsinghua Univ, Sch Pharmaceut Sci Life Sci, Beijing 100084, Peoples R China.
ABSTRACT:The mechanosensitive Piezo channels function as key eukaryotic mechanotransducers. However, their structures and mechanogating mechanisms remain unknown. Here we determine the three-bladed, propeller-like electron cryo-microscopy structure of mouse Piezol and functionally reveal its mechanotransduction components. Despite the lack of sequence repetition, we identify nine repetitive units consisting of four transmembrane helices each-which we term transmembrane helical units (THUs) -which assemble into a highly curved blade-like structure. The last transmembrane helix encloses a hydrophobic pore, followed by three intracellular fenestration sites and side portals that contain pore-property-determining residues. The central region forms a 90 A-long intracellular beam-like structure, which undergoes a lever-like motion to connect THUs to the pore via the interfaces of the C-terminal domain, the anchor-resembling domain and the outer helix. Deleting extracellular loops in the distal THUs or mutating single residues in the beam impairs the mechanical activation of Piezol. Overall, Piezol possesses a unique 38-transmembrane-helix topology and designated mechanotransduction components, which enable a lever-like mechanogating mechanism.
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Nature2018. Vos, SM et al.
Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT:Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Honto sapiens DSIF, PAF and SPT6 was determined at 3.1 A resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminaldomain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
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Nature2018. Vos, SM et al.
Max Planck Inst Biophys Chem, Dept Mol Biol, Gottingen, Germany.
ABSTRACT:Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 angstrom resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.
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Nature2018. Zhou, P et al.
Natl Inst Biol Sci, Beijing, Peoples R China.
ABSTRACT:Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-kappa B signalling(1). Recent studies indicate that the bacterial metabolite D-glycero-beta-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-kappa B signalling in host cytosol(2-4), but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-beta-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-kappa B activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-kappa B. A CRISPR-Cas9 screen showed that activation of NF-kappa B by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.
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Nature2018. Pao, KC et al.
Univ Dundee, MRC Prot Phosphorylat & Ubiquitylat Unit, Dundee, Scotland.
ABSTRACT:Ubiquitination is initiated by transfer of ubiquitin (Ub) from a ubiquitin-activating enzyme (E1) to a ubiquitin-conjugating enzyme (E2), producing a covalently linked intermediate (E2-Ub)(1). Ubiquitin ligases (E3s) of the 'really interesting new gene' (RING) class recruit E2-Ub via their RING domain and then mediate direct transfer of ubiquitin to substrates(2). By contrast, 'homologous to E6-AP carboxy terminus' (HECT) E3 ligases undergo a catalytic cysteine-dependent transthiolation reaction with E2-Ub, forming a covalent E3-Ub intermediate(3,4). Additionally, RING-between-RING (RBR) E3 ligases have a canonical RING domain that is linked to an ancillary domain. This ancillary domain contains a catalytic cysteine that enables a hybrid RING-HECT mechanism(5). Ubiquitination is typically considered a post-translational modification of lysine residues, as there are no known human E3 ligases with non-lysine activity. Here we perform activity-based protein profiling of HECT or RBR-like E3 ligases and identify the neuron-associated E3 ligase MYCBP2 (also known as PHR1) as the apparent single member of a class of RING-linked E3 ligase with esterification activity and intrinsic selectivity for threonine over serine. MYCBP2 contains two essential catalytic cysteine residues that relay ubiquitin to its substrate via thioester intermediates. Crystallographic characterization of this class of E3 ligase, which we designate RING-Cys-relay (RCR), provides insights into its mechanism and threonine selectivity. These findings implicate non-lysine ubiquitination in cellular regulation of higher eukaryotes and suggest that E3 enzymes have an unappreciated mechanistic diversity.
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Cell2018. Haselbach, D et al.
Max Planck Inst Biophys Chem, Dept Struct Dynam, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT:The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human B-act spliceosome at 3.4 angstrom resolution. In the B-act state, the spliceosome is activated but not catalytically primed, so that it is functionally blocked prior to the first catalytic step of splicing. The spliceosomal core is similar to the yeast B-act spliceosome; important differences include the presence of the RNA helicase aquarius and peptidyl prolyl isomerases. To examine the overall dynamic behavior of the purified spliceosome, we developed a principal component analysis-based approach. Calculating the energy landscape revealed eight major conformational states, which we refined to higher resolution. Conformational differences of the highly flexible structural components between these eight states reveal how spliceosomal components contribute to the assembly of the spliceosome, allowing it to generate a dynamic interaction network required for its subsequent catalytic activation.
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nature structural & molecular biology2018. Lucas Kuhlen, Patrizia Abrusci, Steven Johnson et al.
Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
ABSTRACT:Exportofproteins throughtypeIIIsecretionsystems is critical for motility and virulenceofmany major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to formthecoreofanexportgate intheinner membrane, but theirstructure, assembly and location withinthefinal nanomachine remain unclear. Here, we presentthecryoelectron microscopystructureoftheSalmonella Typhimurium FliP-FliQ-FliR complex at 4.2 angstrom. Noneofthesubunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fittingofthestructureinto reconstructionsofintactsecretionsystems, combined with cross-linking, localizetheexportgate as acorecomponentoftheperiplasmic portionofthemachinery. This study thereby identifiestheexportgate as a key elementofthesecretionchannel and implies that it primesthehelical architectureofthecomponents assembling downstream.
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NATURE COMMUNICATIONS2018. Teng, YQ et al.
Univ Pittsburgh, Dept Microbiol & Mol Genet, Sch Med, 450 Technol Dr,523 Bridgeside Point 2, Pittsburgh, PA 15219 USA.
ABSTRACT:Actively transcribed regions of the genome are protected by transcription-coupled DNA repair mechanisms, including transcription-coupled homologous recombination (TC-HR). Here we used reactive oxygen species (ROS) to induce and characterize TC-HR at a transcribed locus in human cells. As canonical HR, TC-HR requires RAD51. However, the localization of RAD51 to damage sites during TC-HR does not require BRCA1 and BRCA2, but relies on RAD52 and Cockayne Syndrome Protein B (CSB). During TC-HR, RAD52 is recruited by CSB through an acidic domain. CSB in turn is recruited by R loops, which are strongly induced by ROS in transcribed regions. Notably, CSB displays a strong affinity for DNA:RNA hybrids in vitro, suggesting that it is a sensor of ROS-induced R loops. Thus, TC-HR is triggered by R loops, initiated by CSB, and carried out by the CSB-RAD52-RAD51 axis, establishing a BRCA1/2-independent alternative HR pathway protecting the transcribed genome.
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