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1.
The specificity of protein-protein interactions is encoded in those parts of the sequence that compose the binding interface. Therefore, understanding how changes in protein sequence influence interaction specificity, and possibly the phenotype, requires knowing the location of binding sites in those sequences. However, large-scale detection of protein interfaces remains a challenge. Here, we present a sequence- and interactome-based approach to mine interaction motifs from the recently published Arabidopsis thaliana interactome. The resultant proteome-wide predictions are available via www.ab.wur.nl/sliderbio and set the stage for further investigations of protein-protein binding sites. To assess our method, we first show that, by using a priori information calculated from protein sequences, such as evolutionary conservation and residue surface accessibility, we improve the performance of interface prediction compared to using only interactome data. Next, we present evidence for the functional importance of the predicted sites, which are under stronger selective pressure than the rest of protein sequence. We also observe a tendency for compensatory mutations in the binding sites of interacting proteins. Subsequently, we interrogated the interactome data to formulate testable hypotheses for the molecular mechanisms underlying effects of protein sequence mutations. Examples include proteins relevant for various developmental processes. Finally, we observed, by analysing pairs of paralogs, a correlation between functional divergence and sequence divergence in interaction sites. This analysis suggests that large-scale prediction of binding sites can cast light on evolutionary processes that shape protein-protein interaction networks. 相似文献
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Active DNA demethylation plays crucial roles in the regulation of gene expression in both plants and animals. In Arabidopsis thaliana, active DNA demethylation is initiated by the ROS1 subfamily of 5-methylcytosine-specific DNA glycosylases via a base excision repair mechanism. Recently, IDM1 and IDM2 were shown to be required for the recruitment of ROS1 to some of its target loci. However, the mechanism(s) by which IDM1 is targeted to specific genomic loci remains to be determined. Affinity purification of IDM1- and IDM2- associating proteins demonstrated that IDM1 and IDM2 copurify together with two novel components, methyl-CpG-binding domain protein 7 (MBD7) and IDM2-like protein 1 (IDL1). IDL1 encodes an α-crystallin domain protein that shows high sequence similarity with IDM2. MBD7 interacts with IDM2 and IDL1 in vitro and in vivo and they form a protein complex associating with IDM1 in vivo. MBD7 directly binds to the target loci and is required for the H3K18 and H3K23 acetylation in planta. MBD7 dysfunction causes DNA hypermethylation and silencing of reporter genes and a subset of endogenous genes. Our results suggest that a histone acetyltransferase complex functions in active DNA demethylation and in suppression of gene silencing at some loci in Arabidopsis. 相似文献
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Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte–derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function. 相似文献
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Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates. 相似文献
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Frank Baumann Jens Pahnke Ivan Radovanovic Thomas Rülicke Juliane Bremer Markus Tolnay Adriano Aguzzi 《PloS one》2009,4(9)
The prion consists essentially of PrPSc, a misfolded and aggregated conformer of the cellular protein PrPC. Whereas PrPC deficient mice are clinically healthy, expression of PrPC variants lacking its central domain (PrPΔCD), or of the PrP-related protein Dpl, induces lethal neurodegenerative syndromes which are repressed by full-length PrP. Here we tested the structural basis of these syndromes by grafting the amino terminus of PrPC (residues 1–134), or its central domain (residues 90–134), onto Dpl. Further, we constructed a soluble variant of the neurotoxic PrPΔCD mutant that lacks its glycosyl phosphatidyl inositol (GPI) membrane anchor. Each of these modifications abrogated the pathogenicity of Dpl and PrPΔCD in transgenic mice. The PrP-Dpl chimeric molecules, but not anchorless PrPΔCD, ameliorated the disease of mice expressing truncated PrP variants. We conclude that the amino proximal domain of PrP exerts a neurotrophic effect even when grafted onto a distantly related protein, and that GPI-linked membrane anchoring is necessary for both beneficial and deleterious effects of PrP and its variants. 相似文献
11.
Domains of a Transit Sequence Required for in Vivo Import in
Arabidopsis Chloroplasts 总被引:1,自引:0,他引:1
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Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence. 相似文献
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Stefanie J. Mueller Daniel Lang Sebastian N.W. Hoernstein Erika G.E. Lang Christian Schuessele Anton Schmidt Melanie Fluck Desirée Leisibach Christina Niegl Andreas D. Zimmer Andreas Schlosser Ralf Reski 《Plant physiology》2014,164(4):2081-2095
Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as
organelles, namely mitochondria and plastids in plants. During evolution, organellar
proteomes are modified by gene gain and loss, by gene subfunctionalization and
neofunctionalization, and by changes in protein targeting. To date, proteomics data
for plastids and mitochondria are available for only a few plant model species, and
evolutionary analyses of high-throughput data are scarce. We combined quantitative
proteomics, cross-species comparative analysis of metabolic pathways, and
localizations by fluorescent proteins in the model plant Physcomitrella
patens in order to assess evolutionary changes in mitochondrial and
plastid proteomes. This study implements data-mining methodology to classify and
reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study
the effects of postendosymbiotic evolution on organellar pathway partitioning. Our
results indicate that, although plant morphologies changed substantially during plant
evolution, metabolic integration of organelles is largely conserved, with exceptions
in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization
are common in the studied nucleus-encoded gene families of organelle-targeted
proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions
revealed novel proteins at organelle interfaces such as plastid stromules
(stroma-filled tubules) and highlight microcompartments as well as intercellular and
intracellular heterogeneity of mitochondria and plastids. Thus, we establish a
comprehensive data set for mitochondrial and plastid proteomes in moss, present a
novel multilevel approach to organelle biology in plants, and place our findings into
an evolutionary context.Endosymbiosis has enabled and shaped eukaryotic evolution. The engulfment of an ancestral
α-proteobacterium by a presumably archaebacterial host cell stands at the origin of
mitochondrial and eukaryotic evolution over 1.5 billion years ago (Dyall et al., 2004). In plants, the subsequent uptake of a
photosynthetic bacterium between 1.5 and 1.2 billion years ago led to the formation of
chloroplasts (Dyall et al., 2004). Plants thereby
evolved by the integration of three distinct genetic compartments. After the establishment
of endosymbiosis, genes were transferred to a great extent, mainly from mitochondria and
plastids to the nucleus (Bock and Timmis, 2008),
necessitating an orchestrated flux of information in the form of proteins and metabolites
between the compartments of eukaryotic cells to ensure homeostasis, growth, and
development. This communication between organelles is facilitated by physical interactions
(Kornmann et al., 2009), control of protein
import (Ling et al., 2012), and retrograde
signaling (Nargund et al., 2012). During radiation
and diversification, especially of land plants, nuclear genomes substantially changed due
to endosymbiotic and horizontal (Yue et al., 2012)
gene transfer, genome duplication, and gene gain and loss (Duarte et al., 2006; Lang et al.,
2010; Martin, 2010), obtruding the
question of whether these phenomena are linked to alterations in metabolic pathway
partitioning between organelles. Retained paralogs can either introduce a new function
(neofunctionalization) or reconstitute existing functions (subfunctionalization; Duarte et al., 2006), for example by distinct
spatiotemporal expression profiles or distinct subcellular localizations, resulting in the
modulation or introduction of metabolic functions in the respective cellular compartments.
Moreover, proteins can localize to several subcellular compartments, a phenomenon called
dual or multiple targeting (Yogev and Pines, 2011;
Xu et al., 2013). Consequently, many eukaroytic
metabolic pathways, as well as the plastid and mitochondrial proteomes, are constituted of
a mosaic of proteins of diverse evolutionary origins (Szklarczyk and Huynen, 2010), and evolution has shaped variable organellar
functionalities across taxa. To date, the evolution and variability of postendosymbiotic
metabolic partitioning is largely not characterized on a high-throughput level. So far,
large-scale mitochondrial proteome data sets are only available for the green alga
Chlamydomonas reinhardtii (Atteia et
al., 2009), rice (Oryza sativa; Huang et al., 2009), and the model flowering plant Arabidopsis
(Arabidopsis thaliana; Millar et al.,
2001; Heazlewood et al., 2004), whereas
plastid proteomics in plants is on an advanced level and covers more species (Polyakov et al., 2010; van Wijk and Baginsky, 2011).While higher plants diversified relatively recently but massively, simple moss plants can
be traced back 330 million years (Hubers and Kerp,
2012), identifying them as prime candidates for an evolutionary view of
organellar proteomes and organelle biology at a genome-wide scale. In contrast to
specialized flowering plants, mosses are generalists with few tissues, high metabolic
variability, and ancestral features such as high abiotic stress tolerance (Frank et al., 2007) and few plastid types (Cove, 2005).By integrating quantitative proteomics, multivariate analysis, metabolic pathway maps,
phylogenomics, and localization with fluorescent proteins, we reliably characterize
subcellular proteomes and gene family diversification. Key characteristics of
postendosymbiotic organellar proteome evolution are identified by cross-species comparative
analysis. In support of our high-throughput analyses, we conduct single-protein analyses
and identify proteins that mark microcompartments within organelles and localize to dynamic
contact sites between organelles. These proteins may facilitate the exchange of proteins
and metabolites, while others influence the dynamics of individual chloroplasts and
mitochondria. This study characterizes the mitochondrial and plastid proteomes of moss and
reveals the heterogeneity of organelles within a single cell. 相似文献
13.
We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport. 相似文献
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Yaniv Brandvain Tanja Slotte Khaled M. Hazzouri Stephen I. Wright Graham Coop 《PLoS genetics》2013,9(9)
The shift from outcrossing to self-fertilization is among the most common evolutionary transitions in flowering plants. Until recently, however, a genome-wide view of this transition has been obscured by both a dearth of appropriate data and the lack of appropriate population genomic methods to interpret such data. Here, we present a novel population genomic analysis detailing the origin of the selfing species, Capsella rubella, which recently split from its outcrossing sister, Capsella grandiflora. Due to the recency of the split, much of the variation within C. rubella is also found within C. grandiflora. We can therefore identify genomic regions where two C. rubella individuals have inherited the same or different segments of ancestral diversity (i.e. founding haplotypes) present in C. rubella''s founder(s). Based on this analysis, we show that C. rubella was founded by multiple individuals drawn from a diverse ancestral population closely related to extant C. grandiflora, that drift and selection have rapidly homogenized most of this ancestral variation since C. rubella''s founding, and that little novel variation has accumulated within this time. Despite the extensive loss of ancestral variation, the approximately 25% of the genome for which two C. rubella individuals have inherited different founding haplotypes makes up roughly 90% of the genetic variation between them. To extend these findings, we develop a coalescent model that utilizes the inferred frequency of founding haplotypes and variation within founding haplotypes to estimate that C. rubella was founded by a potentially large number of individuals between 50 and 100 kya, and has subsequently experienced a twenty-fold reduction in its effective population size. As population genomic data from an increasing number of outcrossing/selfing pairs are generated, analyses like the one developed here will facilitate a fine-scaled view of the evolutionary and demographic impact of the transition to self-fertilization. 相似文献
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Cecilia Tamborindeguy Michael S. Bereman Stacy DeBlasio David Igwe Dawn M. Smith Frank White Michael J. MacCoss Stewart M. Gray Michelle Cilia 《PloS one》2013,8(8)
Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are transmitted by aphid vectors. The identification of aphid genes and proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Two cyclophilin B proteins, S28 and S29, were identified previously in populations of
Schizaphisgraminum
that differed in their ability to transmit the RPV strain of Cereal yellow dwarf virus (CYDV-RPV). The presence of S29 was correlated with F2 genotypes that were efficient virus transmitters. The present study revealed the two proteins were isoforms, and a single amino acid change distinguished S28 and S29. The distribution of the two alleles was determined in 12 F2 genotypes segregating for CYDV-RPV transmission capacity and in 11 genetically independent, field-collected
S
. graminum
biotypes. Transmission efficiency for CYDV-RPV was determined in all genotypes and biotypes. The S29 isoform was present in all genotypes or biotypes that efficiently transmit CYDV-RPV and more specifically in genotypes that efficiently transport virus across the hindgut. We confirmed a direct interaction between CYDV-RPV and both S28 and S29 using purified virus and bacterially expressed, his-tagged S28 and S29 proteins. Importantly, S29 failed to interact with a closely related virus that is transported across the aphid midgut. We tested for in vivo interactions using an aphid-virus co-immunoprecipitation strategy coupled with a bottom-up LC-MS/MS analysis using a Q Exactive mass spectrometer. This analysis enabled us to identify a third cyclophilin protein, cyclophilin A, interacting directly or in complex with purified CYDV-RPV. Taken together, these data provide evidence that both cyclophilin A and B interact with CYDV-RPV, and these interactions may be important but not sufficient to mediate virus transport from the hindgut lumen into the hemocoel. 相似文献
18.
Bacterial Cellulose-Binding Domain Modulates in Vitro
Elongation of Different Plant Cells 总被引:4,自引:0,他引:4
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Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL−1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL−1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control. 相似文献
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Protein disulfide isomerases comprise a large family of enzymes responsible for catalyzing the proper oxidation and folding of newly synthesized proteins in the endoplasmic reticulum (ER). Protein disulfide isomerase-related (PDIR) protein (also known as PDIA5) is a specialized member that participates in the folding of α1-antitrypsin and N-linked glycoproteins. Here, the crystal structure of the non-catalytic domain of PDIR was determined to 1.5 Å resolution. The structure adopts a thioredoxin-like fold stabilized by a structural disulfide bridge with a positively charged binding surface for interactions with the ER chaperones, calreticulin and ERp72. Crystal contacts between molecules potentially mimic the interactions of PDIR with misfolded substrate proteins. The results suggest that the non-catalytic domain of PDIR plays a key role in the recognition of protein partners and substrates. 相似文献
20.
Klaas J. van Wijk Giulia Friso Dirk Walther Waltraud X. Schulze 《The Plant cell》2014,26(6):2367-2389
Protein (de)phosphorylation plays an important role in plants. To provide a robust
foundation for subcellular phosphorylation signaling network analysis and
kinase-substrate relationships, we performed a meta-analysis of 27 published and
unpublished in-house mass spectrometry–based phospho-proteome data sets for
Arabidopsis thaliana covering a range of processes,
(non)photosynthetic tissue types, and cell cultures. This resulted in an assembly of
60,366 phospho-peptides matching to 8141 nonredundant proteins. Filtering the data
for quality and consistency generated a set of medium and a set of high confidence
phospho-proteins and their assigned phospho-sites. The relation between single and
multiphosphorylated peptides is discussed. The distribution of p-proteins across
cellular functions and subcellular compartments was determined and showed
overrepresentation of protein kinases. Extensive differences in frequency of pY were
found between individual studies due to proteomics and mass spectrometry workflows.
Interestingly, pY was underrepresented in peroxisomes but overrepresented in
mitochondria. Using motif-finding algorithms motif-x and MMFPh at
high stringency, we identified compartmentalization of phosphorylation motifs likely
reflecting localized kinase activity. The filtering of the data assembly improved
signal/noise ratio for such motifs. Identified motifs were linked to kinases through
(bioinformatic) enrichment analysis. This study also provides insight into the
challenges/pitfalls of using large-scale phospho-proteomic data sets to
nonexperts. 相似文献