pFind Studio: a computational solution for mass spectrometry-based proteomics
2016
Journal of Proteomics2016. Xiong, YH et al.
Chinese Acad Agr Sci, State Key Lab Biol Plant Dis & Insect Pests, Inst Plant Protect, Beijing 100193, Peoples R China.
ABSTRACT:Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in the gene transcription regulation. Here, we report high quality proteome-scale data for lysine-acetylation (Mac) sites and Mac proteins in rice (Oryza sativa). A total of 1337 Kac sites in 716 Mac proteins with diverse biological functions and subcellular localizations were identified in rice seedlings. About 42% of the sites were predicted to be localized in the chioroplast. Seven putative acetylation motifs were detected. Phenylalanine, located in both the upstream and downstream of the Kac sites, is the most conserved amino acid surrounding the regions. In addition, protein interaction network analysis revealed that a variety of signaling pathways are modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways are significantly enriched. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Kac functions in growth, development, and abiotic and biotic stress responses in rice and other plants. Biological significance: Rice is one of the most important crops consumption and is a model monocot for research. In this study, we combined a highly sensitive immune-affinity purification method (used pan anti-acetyl-lysine antibody conjugated agarose for immunoaffinity acetylated peptide enrichment) with high-resolution LC-MS/MS. In total, we identified 1337 Kac sites on 716 Mac proteins in rice cells. Bioinformatic analysis of the acetylome revealed that the acetylated proteins are involved in a variety of cellular functions and have diverse subcellular localizations. We also identified seven putative acetylation motifs in the acetylated proteins of rice. In addition, protein interaction network analysis revealed that a variety of signaling pathways were modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways were significantly enriched. To our knowledge, the number of Kac sites we identified was 23-times greater and the number of Kac proteins was 16-times greater than in a previous report. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Mac functions in growth, development and responses to abiotic and biotic stresses in rice or other plants. (C) 2016 Elsevier B.V. All rights reserved.
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Nature2016. Wang, T et al.
Chinese Acad Sci, Inst Genet & Dev Biol, State Key Lab Mol & Dev Biol, Beijing 100101, Peoples R China.
ABSTRACT:Sexual reproduction requires recognition between the male and female gametes. In flowering plants, the immobile sperms are delivered to the ovule-enclosed female gametophyte by guided pollen tube growth. Although the female gametophyte-secreted peptides have been identified to be the chemotactic attractant to the pollen tube(1-3), the male receptor(s) is still unknown. Here we identify a cell-surface receptor heteromer, MDIS1-MIK, on the pollen tube that perceives female attractant LURE1 in Arabidopsis thaliana. MDIS1, MIK1 and MIK2 are plasma-membrane-localized receptor-like kinases with extracellular leucine-rich repeats and an intracellular kinase domain. LURE1 specifically binds the extracellular domains of MDIS1, MIK1 and MIK2, whereas mdis1 and mik1 mik2 mutant pollen tubes respond less sensitively to LURE1. Furthermore, LURE1 triggers dimerization of the receptors and activates the kinase activity of MIK1. Importantly, transformation of AtMDIS1 to the sister species Capsella rubella can partially break down the reproductive isolation barrier. Our findings reveal a new mechanism of the male perception of the female attracting signals.
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Journal of Proteomics2016. Hung, CW et al.
Univ Kiel, Inst Expt Med, Systemat Prote & Bioanalyt, Kiel, Germany.
ABSTRACT:Bone morphogenetic protein 1 (BMP-1) is an essential metalloproteinase to trigger extracellular matrix assembly and organogenesis. Previous structural studies on the refolded catalytic domain of BMP-1 produced in E. coli have suggested the existence of a rare vicinal disulfide linkage near the active site. To confirm that this was not an artifact of the refolding procedure, the full-length human BMP-1 produced in mammalian cells was investigated via sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution LC-MS/MS analysis to interrogate the post-translational modifications. Ten disulfide linkages of BMP-1, including the vicinal disulfide linkage C-185-C-186 could be unambiguously identified. Further, around 50% of this vicinal disulfide bond was found to be modified by N-ethylmaleimide (NEM), a cysteine protease inhibitor supplied when the BMP-1-containing medium was collected, suggesting that this bond was highly unstable. In the absence of NEM, BMP-1 has a higher tendency to form aggregates, but after aggregate removal, C-185 and C-186 are almost quantitatively engaged in the vicinal disulfide bond and BMP-1 activity remains unchanged. In addition, three consensus N-glycosylation sites at N-142, N-363, and N-599 could be identified together with a previously unknown O-glycosylation site and an Asn-hydroxylation. Significance: An in-depth characterization of post-translational modifications of the full-length human BMP-1 produced in mammalian cells by MS was performed. A rare vicinal disulfide bond in the catalytic domain could be confirmed for the first time by mass spectrometry along with nine other proposed disulfide linkages of mature BMP-1. This vicinal disulfide bond can transiently open to form covalent adducts with the cysteine protease inhibitor (NEM) supplied in cell medium during protein harvesting. Further, we report a previously unknown O-glycosylation site and Asn-hydroxylation site, indicating a novel feature of BMP-1 in the EGF domain. The study clearly outlines the benefit of in-depth characterization of overexpressed proteins to deduce important protein modifications. (C) 2016 Elsevier B.V. All rights reserved.
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Molecular Cell2016. Cretu, C et al.
Max Planck Inst Biophys Chem, Res Grp Macromol Crystallog, Fassberg 11, D-37077 Gottingen, Germany.
ABSTRACT:SF3b is a heptameric protein complex of the U2 small nuclear ribonucleoprotein (snRNP) that is essential for pre-mRNA splicing. Mutations in the largest SF3b subunit, SF3B1/SF3b155, are linked to cancer and lead to alternative branch site (BS) selection. Here we report the crystal structure of a human SF3b core complex, revealing how the distinctive conformation of SF3b155's HEAT domain is maintained by multiple contacts with SF3b130, SF3b10, and SF3b14b. Protein-protein crosslinking enabled the localization of the BS-binding proteins p14 and U2AF65 within SF3b155's HEAT-repeat superhelix, which together with SF3b14b forms a composite RNA-binding platform. SF3b155 residues, the mutation of which leads to cancer, contribute to the tertiary structure of the HEAT superhelix and its surface properties in the proximity of p14 and U2AF65. The molecular architecture of SF3b reveals the spatial organization of cancer-related SF3b155 mutations and advances our understanding of their effects on SF3b structure and function.
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NATURE STRUCTURAL & MOLECULAR BIOLOGY2016. Kloet, SL et al.
Radboud Univ Nijmegen, Radboud Inst Mol Life Sci, Fac Sci, Dept Mol Biol, Nijmegen, Netherlands.
ABSTRACT:Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independently of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to, and decreased expression of, NPC-enriched Ring1b-target genes. In summary, our integrative analyses uncovered dynamic PcG subcomplexes and their widespread colocalization with active chromatin marks during differentiation.
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Cell2016. Fernandez-Martinez, J et al.
Rockefeller Univ, Lab Cellular & Struct Biol, New York, NY 10065 USA.
ABSTRACT:The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.
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Science2016. Reinhard Rauhut, Patrizia Fabrizio, Olexandr Dybkov et al.
Department of Cellular Biochemistry, Max Planck Institute (MPI) for Biophysical Chemistry, Am Fassberg 11, D-37077 Gttingen, Germany.
ABSTRACT:Theactivatedspliceosome(B-act) is in a catalytically inactive state and is remodeled into a catalytically active machine bytheRNA helicase Prp2, butthemechanism is unclear. Here, we describe a 3D electron cryomicroscopy structureoftheSaccharomycescerevisiaeB-act complex at 5.8-angstrom resolution. Our model reveals that in B-act,thecatalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, andthe5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. Thefirst-step nucleophile-thebranchsite adenosine-is sequestered withintheHsh155 HEAT domain and is held 50 angstroms away fromthe5' ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberatethefirst-step reactants for catalysis.
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Science2016. Dmitry E. Agafonov, Berthold Kastner, Olexandr Dybkov et al.
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Gttingen, Germany.
ABSTRACT:TheU4/U6.U5triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structureofthe1.8-megadaltonhumantri-snRNPat a resolutionof7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structuresoftri-snRNPcomponents intotheEM density map and validated them by protein cross-linking. Our model reveals howthespatial organizationofBrr2 RNA helicase prevents prematureU4/U6RNA unwinding in isolatedhumantri-snRNPs and howtheubiquitin C-terminal hydrolase-like protein Sad1 likely tethersthehelicase Brr2 to its preactivation position. Comparisonofour model with cryo-EM three-dimensional structuresoftheSaccharomyces cerevisiaetri-snRNPand Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and thatthescaffolding protein Prp8 is also rearranged to accommodatethespliceosome's catalytic RNA network.
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NATURE2016. Wu, S et al.
Tsinghua Univ, Sch Life Sci, Beijing Adv Innovat Ctr Struct Biol, Minist Educ,Key Lab Prot Sci, Beijing 100084, Peoples R China.
ABSTRACT:Ribosome biogenesis is a highly complex process in eukaryotes, involving temporally and spatially regulated ribosomal protein (r-protein) binding and ribosomal RNA remodelling events in the nucleolus, nucleoplasm and cytoplasm(1,2). Hundreds of assembly factors, organized into sequential functional groups(3,4), facilitate and guide the maturation process into productive assembly branches in and across different cellular compartments. However, the precise mechanisms by which these assembly factors function are largely unknown. Here we use cryo-electron microscopy to characterize the structures of yeast nucleoplasmic pre-60S particles affinity-purified using the epitope-tagged assembly factor Nog2. Our data pinpoint the locations and determine the structures of over 20 assembly factors, which are enriched in two areas: an arc region extending from the central protuberance to the polypeptide tunnel exit, and the domain including the internal transcribed spacer 2 (ITS2) that separates 5.8S and 25S ribosomal RNAs. In particular, two regulatory GTPases, Nog2 and Nog1, act as hub proteins to interact with multiple, distant assembly factors and functional ribosomal RNA elements, manifesting their critical roles in structural remodelling checkpoints and nuclear export. Moreover, our snapshots of compositionally and structurally different pre-60S intermediates provide essential mechanistic details for three major remodelling events before nuclear export: rotation of the 5S ribonucleoprotein, construction of the active centre and ITS2 removal. The rich structural information in our structures provides a framework to dissect molecular roles of diverse assembly factors in eukaryotic ribosome assembly.
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SCIENCE2016. Wan, RX et al.
Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Tsinghua Peking Joint Ctr Life Sci, Sch Life Sci,Minist Educ,Key Lab Prot Sci, Beijing 100084, Peoples R China.
ABSTRACT:Splicing of precursor messenger RNA is accomplished by a dynamic megacomplex known as the spliceosome. Assembly of a functional spliceosome requires a preassembled U4/U6.U5 tri-snRNP complex, which comprises the U5 small nuclear ribonucleoprotein (snRNP), the U4 and U6 small nuclear RNA (snRNA) duplex, and a number of protein factors. Here we report the three-dimensional structure of a Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at an overall resolution of 3.8 angstroms by single-particle electron cryomicroscopy. The local resolution for the core regions of the tri-snRNP reaches 3.0 to 3.5 angstroms, allowing construction of a refined atomic model. Our structure contains U5 snRNA, the extensively base-paired U4/U6 snRNA, and 30 proteins including Prp8 and Snu114, which amount to 8495 amino acids and 263 nucleotides with a combined molecular mass of similar to 1 megadalton. The catalytic nucleotide U80 from U6 snRNA exists in an inactive conformation, stabilized by its base-pairing interactions with U4 snRNA and protected by Prp3. Pre-messenger RNA is bound in the tri-snRNP through base-pairing interactions with U6 snRNA and loop I of U5 snRNA. This structure, together with that of the spliceosome, reveals the molecular choreography of the snRNAs in the activation process of the spliceosomal ribozyme.
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