Kinase activity of SOBIR1 and BAK1 is required for immune signalling
Burgh, Aranka M. Van Der; Postma, Jelle ; Robatzek, Silke ; Joosten, Matthieu H.A.J. - \ 2019
Molecular Plant Pathology 20 (2019)3. - ISSN 1464-6722 - p. 410 - 422.
BAK1/SERK3 - Cf-4 - immunity - phosphorylation - RLK - RLP - SOBIR1
Leucine-rich repeat-receptor-like proteins (LRR-RLPs) and LRR-receptor-like kinases (LRR-RLKs) trigger immune signalling to promote plant resistance against pathogens. LRR-RLPs lack an intracellular kinase domain, and several of these receptors have been shown to constitutively interact with the LRR-RLK Suppressor of BIR1-1/EVERSHED (SOBIR1/EVR) to form signalling-competent receptor complexes. Ligand perception by LRR-RLPs initiates recruitment of the co-receptor BRI1-Associated Kinase 1/Somatic Embryogenesis Receptor Kinase 3 (BAK1/SERK3) to the LRR-RLP/SOBIR1 complex, thereby activating LRR-RLP-mediated immunity. We employed phosphorylation analysis of in planta-produced proteins, live cell imaging, gene silencing and co-immunoprecipitation to investigate the roles of SOBIR1 and BAK1 in immune signalling. We show that Arabidopsis thaliana (At) SOBIR1, which constitutively activates immune responses when overexpressed in planta, is highly phosphorylated. Moreover, in addition to the kinase activity of SOBIR1 itself, kinase-active BAK1 is essential for AtSOBIR1-induced constitutive immunity and for the phosphorylation of AtSOBIR1. Furthermore, the defence response triggered by the tomato LRR-RLP Cf-4 on perception of Avr4 from the extracellular pathogenic fungus Cladosporium fulvum is dependent on kinase-active BAK1. We argue that, in addition to the trans-autophosphorylation of SOBIR1, it is likely that SOBIR1 and BAK1 transphosphorylate, and thereby activate the receptor complex. The signalling-competent cell surface receptor complex subsequently activates downstream cytoplasmic signalling partners to initiate RLP-mediated immunity.
The genetic background of bovine αs1- and αs2-casein phosphorylation
Fang, Zih-Hua - \ 2017
Wageningen University. Promotor(en): E. Verrier; H. Bovenhuis, co-promotor(en): P. Martin; M.H.P.W. Visker. - Wageningen : Wageningen University - ISBN 9789463438148 - 141
dairy cattle - alpha-s1-casein - alpha-s2-casein - phosphorylation - milk composition - milk proteins - genetic variation - genetic factors - animal genetics - melkvee - alfa-s-1-caseïne - alfa-s-2-caseïne - fosforylering - melksamenstelling - melkeiwitten - genetische variatie - genetische factoren - diergenetica
Phosphorylation of caseins (CN) is a crucial post-translational modification allowing caseins to aggregate as micelles. The formation and stability of casein micelles are important for transporting abundant minerals to the neonate and manufacturing of dairy products. Therefore, it is of great interest to explore variation in degrees of phosphorylation of caseins and study to what extent genetic and other factors contribute to this variation. This thesis aimed to investigate the genetic background of bovine milk protein composition with a focus on phosphorylation of αs1- and αs2-CN. Two studies were conducted to quantify phosphorylation levels of αs1- and αs2-CN: one in French Montbéliarde using liquid chromatography coupled with electrospray ionization mass spectrometry and the other in Dutch Holstein Friesian using capillary zone electrophoresis. In French Montbéliarde, in addition to the known isoforms αs1-CN-8P and-9P and αs2-CN-10P to -13P, three new phosphorylation isoforms were detected, namely αs2-CN-9P, αs2-CN-14P, and αs2-CN-15P. Relative concentrations of the phosphorylation isoforms varied considerably among cows. Phenotypic correlations showed that isoforms phosphorylated at higher degrees (αs1-CN-9P and αs2-CN-12P to -14P) correlated negatively with isoforms phosphorylated at lower degrees (αs1-CN-8P, αs2-CN-10P, and -11P). Furthermore, it was shown that αs1- and αs2-CN phosphorylation profiles changed across parity and lactation, and exploitable genetic variation for the phosphorylation degrees of αs1- and αs2-CN (defined as the proportion of higher-degree isoforms in αs1- and αs2-CN, respectively) exist. In Dutch Holstein Friesian, three αs2-CN isoforms, namely αs2-CN-10P to -12P, and the phosphorylation degrees of αs1- and αs2-CN were quantified. High intra-herd heritabilities were estimated for individual αs2-CN phosphorylation isoforms and the phosphorylation degrees of αs1- and αs2-CN (ranging from 0.54 to 0.89). This suggests that genetic factors contribute substantially to observed differences in αs1- and αs2-CN phosphorylation profiles. The highly positive correlation between the phosphorylation degrees of αs1- and αs2-CN (0.94) suggest that phosphorylation of αs1- and αs2-CN is related. Additionally, a total of 10 regions, distributed across Bos taurus autosomes (BTA) 1, 2, 6, 9, 11, 14, 15, 18, 24 and 28, were detected to be associated with individual αs1- and αs2-CN phosphorylation isoforms and their phosphorylation degrees. Regions on BTA1, 6, 11 and 14 were associated with multiple traits studied. Two quantitative trait loci (QTL) regions were detected on BTA1: one affecting αs2-CN production, and the other affecting αs1-CN PD and αs2-CN PD. The QTL region on BTA6 affected only individual αs2-CN isoforms. The QTL region on BTA11 and 14 affected relative concentrations of αs2-CN-10P and αs2-CN-11P, αs1-CN PD and αs2-CN PD. Results suggested that effects of identified genomic regions on αs1-CN PD and αs2-CN PD are probably due to changes in milk synthesis and phosphorus secretion in milk.
Jnk1 in murine hepatic stellate cells is a crucial mediator of liver fibrogenesis
Zhao, G. ; Hatting, M. ; Nevzorova, Y.A. ; Peng, J. ; Hu, W.Y. ; Boekschoten, M.V. ; Müller, M.R. - \ 2014
Gut 63 (2014)7. - ISSN 0017-5749 - p. 1159 - 1172.
signal-transduction pathway - tnf-alpha - pathogenic role - fibrosis - activation - mice - proliferation - induction - injury - phosphorylation
Objective The c-Jun N-terminal kinase-1 (Jnk1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the Jnk1-dependent effect on liver fibrogenesis. Design Jnk1f/f wildtype (WT), Jnk1-/- and Jnk1¿hepa (hepatocyte-specific deletion of Jnk1) mice were subjected to (i) bile duct ligation (BDL) and (ii) CCl4-induced liver fibrosis. Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs), studied their activation in vitro and investigated human diseased liver samples. Results Phosphorylated Jnk was expressed in myofibroblasts, epithelial and inflammatory cells during the progression of fibrogenesis in humans and mice. In mice, liver transaminases, alkaline phosphatase, bilirubin and liver histology revealed reduced injury in Jnk1-/- compared with WT and Jnk1¿hepa mice correlating with lower hepatocyte cell death and proliferation. Consequently, parameters of liver fibrosis such as Sirius red staining and collagen IA1 and a-smooth muscle actin expression were downregulated in Jnk1-/- compared with WT and Jnk1¿hepa livers, 4 weeks after CCl4 or BDL. BMT experiments excluded bone marrow–derived cells from having a major impact on the Jnk1-dependent effect on fibrogenesis, while primary HSCs from Jnk1-/- livers showed reduced transdifferentiation and extracellular matrix production. Moreover, Jnk1 ablation caused a reduced lifespan and poor differentiation of HSCs into matrix-producing myofibroblasts. Conclusions Jnk1 in HSCs, but not in hepatocytes, significantly contribute to liver fibrosis development, identifying Jnk1 in HSCs as a profibrotic kinase and a promising cell-directed target for liver fibrosis.
Nod factor receptors form heteromeric complexes and are essential for intracellular infection in Medicago nodules
Moling, S. ; Pietraszewska-Bogiel, A. ; Postma, M. ; Fedorova, E.E. ; Hink, M.A. ; Limpens, E.H.M. ; Gadella, T.W.J. ; Bisseling, T. - \ 2014
The Plant Cell 26 (2014)10. - ISSN 1040-4651 - p. 4188 - 4199.
rhizobium-leguminosarum - n-2-fixing symbiosomes - root-nodules - kinase - truncatula - arabidopsis - lyk3 - phosphorylation - perception - nodulation
Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules.
Overexpression of PLIN5 in skeletal muscle promotes oxidative gene expression and intramyocellular lipid content without compromising insulin sensitivity
Bosma, M. ; Sparks, L.M. ; Hooiveld, G.J.E.J. ; Jorgensen, J.A. ; Houten, S.M. ; Schrauwen, P. ; Kersten, A.H. ; Hesselink, M.K.C. - \ 2013
Biochimica et Biophysica Acta. Molecular and Cell Biology of Lipids 1831 (2013)4. - ISSN 1388-1981 - p. 844 - 852.
fatty-acid oxidation - perilipin 5 - weight-loss - ppar-alpha - in-vivo - resistance - protein - mitochondria - carnitine - phosphorylation
Aims/hypothesis: While lipid deposition in the skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like the skeletal muscle. Here we investigated the effects of PLIN5 overexpression - in comparison with the effects of PLIN2 - on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity. Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet Eight days after electroporation, insulin-mediated glucose uptake in the skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays. Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function. Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PUNS in the skeletal muscle promoted expression of a cluster of genes under control of PPAR alpha and PGC1 alpha involved in FA catabolism and mitochondrial oxidation. (C) 2013 Elsevier B.V. All rights reserved.
Diacylglycerol kinase counteracts protein kinase C-mediated inactivation of the EGF receptor
Baal, J. van; Widt, J. de; Divecha, N. ; Blitterswijk, W.J. van - \ 2012
International Journal of Biochemistry and Cell Biology 44 (2012)11. - ISSN 1357-2725 - p. 1791 - 1799.
growth-factor receptor - signal-transduction - coupled receptors - phosphorylation - cells - src - transactivation - activation - mechanisms - expression
Epidermal growth factor receptor (EGFR) activation is negatively regulated by protein kinase C (PKC)signaling. Stimulation of A431 cells with EGF, bradykinin or UTP increased EGFR phosphorylation at Thr654 in a PKC-dependent manner. Inhibition of PKC signaling enhanced EGFR activation, as assessed by increased phosphorylation of Tyr845 and Tyr1068 residues of the EGFR. Diacylglycerol is a physiological activator of PKC that can be removed by diacylglycerol kinase (DGK) activity. We found, in A431 and HEK293 cells, that the DGK isozyme translocated from the cytosol to the plasma membrane, where it co-localized with the EGFR and subsequently moved into EGFR-containing intracellular vesicles. This translocation was dependent on both activation of EGFR and PKC signaling. Furthermore, DGK physically interacted with the EGFR and became tyrosine-phosphorylated upon EGFR stimulation. Overexpression of DGK attenuated the bradykinin-stimulated, PKC-mediated EGFR phosphorylation at Thr654, and enhanced the phosphorylation at Tyr845 and Tyr1068. SiRNA-induced DGK downregulation enhanced this PKC-mediated Thr654 phosphorylation. Our data indicate that DGK translocation and activity is regulated by the concerted activity of EGFR and PKC and that DGK attenuates PKC-mediated Thr654 phosphorylation that is linked to desensitisation of EGFR signaling
The Arabidopsis thaliana SERK1 kinase domain spontaneously refolds to an active state in vitro
Toorn, M. aan den; Huijbers, M.M.E. ; Vries, S.C. de; Mierlo, C.P.M. van - \ 2012
PLoS ONE 7 (2012)12. - ISSN 1932-6203
embryogenesis receptor kinase-1 - intrinsically disordered proteins - smooth-muscle myosin - to-order transition - molecular recognition - signal-transduction - structural basis - activation loop - gras proteins - phosphorylation
Auto-phosphorylating kinase activity of plant leucine-rich-repeat receptor-like kinases (LRR-RLK's) needs to be under tight negative control to avoid unscheduled activation. One way to achieve this would be to keep these kinase domains as intrinsically disordered protein (IDP) during synthesis and transport to its final location. Subsequent folding, which may depend on chaperone activity or presence of interaction partners, is then required for full activation of the kinase domain. Bacterially produced SERK1 kinase domain was previously shown to be an active Ser/Thr kinase. SERK1 is predicted to contain a disordered region in kinase domains X and XI. Here, we show that loss of structure of the SERK1 kinase domain during unfolding is intimately linked to loss of activity. Phosphorylation of the SERK1 kinase domain neither changes its structure nor its stability. Unfolded SERK1 kinase has no autophosphorylation activity and upon removal of denaturant about one half of the protein population spontaneously refolds to an active protein in vitro. Thus, neither chaperones nor interaction partners are required during folding of this protein to its catalytically active state.
Modeling and analysis of flux distributions in the two branches of the phosphotransferase system in Pseudomonas putida
Kremling, A. ; Plufger-Grau, K. ; Silva-Rocha, R. ; Puchalka, J. ; Martins Dos Santos, V.A.P. ; Lorenzo, V. de - \ 2012
BMC Systems Biology 6 (2012). - ISSN 1752-0509 - 25 p.
escherichia-coli - catabolite repression - pu promoter - metabolism - iia(ntr) - protein - pathways - phosphorylation - bacteria - nitrogen
BACKGROUND: Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterialphosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents asignaling unit that acts as a sensory element and measures the activity of the central metabolism.Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr),which communicate with each other by phosphate exchange. Recent experimental results showed a cross talkbetween the two branches. However, the functional role of the crosstalk remains open. RESULTS: A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one ofthe PTS proteins, for different environmental conditions and different strain variants. Additionally, data fromflux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based onthe calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructosecould be determined. CONCLUSION: Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major partof the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a newrole of the second branch of the PTS and will serve as a basis for further experimental studies
Chikungunya Virus nsP3 Blocks Stress Granule Assembly by Recruitment of G3BP into Cytoplasmic Foci
Fros, J.J. ; Domeradzka, N.E. ; Baggen, J. ; Geertsema, C. ; Flipse, J. ; Vlak, J.M. ; Pijlman, G.P. - \ 2012
Journal of Virology 86 (2012)19. - ISSN 0022-538X - p. 10873 - 10879.
sindbis virus - infected-cells - phosphorylation - translation - complexes - region
Chikungunya virus nonstructural protein nsP3 has an essential but unknown role in alphavirus replication and interacts with Ras-GAP SH3 domain-binding protein (G3BP). Here we describe the first known function of nsP3, to inhibit stress granule assembly by recruiting G3BP into cytoplasmic foci. A conserved SH3 domain-binding motif in nsP3 is essential for both nsP3-G3BP interactions and viral RNA replication. This study reveals a novel role for nsP3 as a regulator of the cellular stress response
Unbiased Selective Isolation of Protein N-Terminal Peptides from Complex Proteome Samples Using Phospho Tagging PTAG) and TiO2-based Depletion
Mommen, G.P.M. ; Waterbeemd, B. van de; Meiring, H.D. ; Kersten, G. ; Heck, A.J.R. ; Jong, A.P.J.M. de - \ 2012
Molecular and Cellular Proteomics 11 (2012)9. - ISSN 1535-9476 - p. 832 - 842.
fractional diagonal chromatography - positional proteomics - in-vivo - proteolytic events - cleavage products - identification - phosphorylation - phosphoproteomics - fragmentation - acetylation
A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO2) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with formaldehyde at the protein level, after which the proteins are digested and the newly formed internal peptides modified with the PTAG reagent glyceraldhyde-3-phosphate in nearly perfect yields (> 99%). The resulting phosphopeptides are depleted through binding onto TiO2, keeping exclusively a set of N-acetylated and/or N-dimethylated terminal peptides for analysis by LC-MS/MS. Analysis of peptides derivatized with differentially labeled isotopic analogous of the PTAG reagent revealed a high depletion efficiency (> 95%). The method enabled identification of 753 unique N-terminal peptides (428 proteins) in N. meningitidis and 928 unique N-terminal peptides (572 proteins) in S cerevisiae. These included verified neo-N-termini from subcellular relocalized membrane and mitochondrial proteins. The presented PTAG approach is therefore a novel versatile and robust method for mass spectrometry-based N-proteome analysis and identification of protease-generated cleavage products
Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation
Jensen, K.D.C. ; Wang, Y. ; Tait Wonjo, E.D. ; Shastri, A.J. ; Hu, K. ; Cornel, L. ; Boedec, E. ; Ong, Y.C. ; Chien, Y.H. ; Hunter, C.A. ; Boothroyd, J.C. ; Saeij, J.P.J. - \ 2011
Cell Host & Microbe 9 (2011)6. - ISSN 1931-3128 - p. 472 - 483.
alternative activation - gene-expression - dendritic cells - interferon-gamma - gondii infection - virulence - population - arginase - phosphorylation - resistance
European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-¿B, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection
Synthesis of non-natural carbohydrates from glycerol and aldehydes in a one-pot four-enzyme cascade reaction
Babich, L. ; Hartog, L. ; Falcicchio, P. ; Oost, J. van der - \ 2011
Green Chemistry 13 (2011)10. - ISSN 1463-9262 - p. 2895 - 2900.
bacterial acid-phosphatases - dihydroxyacetone phosphate - organic-synthesis - aldol reactions - chemistry - enzyme - phosphorylation - inhibitors - catalysis - fagomine
A simple procedure has been developed for the synthesis of enantio- and diastereomerically pure carbohydrate analogues from glycerol and a variety of aldehydes in one pot using a four-enzyme cascade reaction. As a proof of concept of the usefulness of this enzymatic catalytic cascade the naturally occurring azasugar D-fagomine was synthesized. This work highlights the potential value of using enzymes in cascade reactions to selectively form complex products that by previous traditional organic chemistry could only be obtained via repeated isolation and purification of intermediates
Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I
Kudo, N.R. ; Anger, M. ; Peters, A. ; Stemmann, O. ; Theussl, H.C. ; Helmhart, W. ; Kudo, H. ; Heyting, C. ; Nasmyth, K. - \ 2009
Journal of Cell Science 122 (2009)15. - ISSN 0021-9533 - p. 2686 - 2698.
sister-chromatid separation - polo-like kinase - synaptonemal complexes - chromosome segregation - xenopus-oocytes - axial elements - fission yeast - anaphase - recombination - phosphorylation
Proteolytic activity of separase is required for chiasma resolution during meiosis I in mouse oocytes. Rec8, the meiosis-specific alpha-kleisin subunit of cohesin, is a key target of separase in yeast. Is the equivalent protein also a target in mammals? We show here that separase cleaves mouse Rec8 at three positions in vitro but only when the latter is hyper-phosphorylated. Expression of a Rec8 variant (Rec8-N) that cannot be cleaved in vitro at these sites causes sterility in male mice. Their seminiferous tubules lack a normal complement of 2 C secondary spermatocytes and 1 C spermatids and contain instead a high proportion of cells with enlarged nuclei. Chromosome spreads reveal that Rec8-N expression has no effect in primary spermatocytes but produces secondary spermatocytes and spermatids with a 4 C DNA content, suggesting that the first and possibly also the second meiotic division is abolished. Expression of Rec8-N in oocytes causes chromosome segregation to be asynchronous and delays its completion by 2-3 hours during anaphase I, probably due to inefficient proteolysis of Rec8-N by separase. Despite this effect, chromosome segregation must be quite accurate as Rec8-N does not greatly reduce female fertility. Our data is consistent with the notion that Rec8 cleavage is important and probably crucial for the resolution of chiasmata in males and females
Physical model of cellular symmetry breaking
Gucht, J. van der; Sykes, C. - \ 2009
Cold Spring Harbor Perspectives in Biology 1 (2009)1. - ISSN 1943-0264 - 11 p.
cortical tension - surface-receptors - actin-filaments - animal-cells - myosin - phosphorylation - movement - dynamics - flow - neuritogenesis
Cells can polarize in response to external signals, such as chemical gradients, cell–cell contacts, and electromagnetic fields. However, cells can also polarize in the absence of an external cue. For example, a motile cell, which initially has a more or less round shape, can lose its symmetry spontaneously even in a homogeneous environment and start moving in random directions. One of the principal determinants of cell polarity is the cortical actin network that underlies the plasma membrane. Tension in this network generated by myosin motors can be relaxed by rupture of the shell, leading to polarization. In this article, we discuss how simplified model systems can help us to understand the physics that underlie the mechanics of symmetry breaking
On the polyelectrolyte brush model of neurofilaments
Zhulina, E.B. ; Leermakers, F.A.M. - \ 2009
Soft Matter 5 (2009)15. - ISSN 1744-683X - p. 2836 - 2840.
consistent-field theory - terminal tail domain - intermediate-filaments - ionic-strength - nf-m - phosphorylation - conformation - networks - subunit - disease
We use scaling arguments and numerical self-consistent-field theory to highlight the structure of a biological polyelectrolyte (PE) brush formed by projection domains of a triplet neurofilament (NF) protein. The protein chains are coarse-grained on the amino acid level and grouped by their polarity and chargability. In the numerical model the full primary sequence is taken, whereas in the scaling approach sequence-averaged properties are accounted for. We elaborate on the electrostatic regulation of the brush structure and discuss its role in the formation of the NF network inside axons
Unequally redundant RCD1 and SRO1 mediate stress and developmental responses and interact with transcription factors
Jaspers, P. ; Blomster, T. ; Brosché, M. ; Salojärvi, J. ; Ahlfors, R. ; Vainonen, J.P. ; Reddy, R.A. ; Immink, G.H. ; Angenent, G.C. ; Turck, F. ; Overmyer, K. ; Kangasjärvi, J. - \ 2009
The Plant Journal 60 (2009)2. - ISSN 0960-7412 - p. 268 - 279.
arabidopsis-thaliana - gene-expression - adp-ribosylation - salt tolerance - plant defense - wwe domain - cell-death - protein - degradation - phosphorylation
RADICAL-INDUCED CELL DEATH1 (RCD1) is an important regulator of stress and hormonal and developmental responses in Arabidopsis thaliana. Together with its closest homolog, SIMILAR TO RCD-ONE1 (SRO1), it is the only Arabidopsis protein containing the WWE domain, which is known to mediate protein–protein interactions in other organisms. Additionally, these two proteins contain the core catalytic region of poly-ADP-ribose transferases and a conserved C-terminal domain. Tissue and subcellular localization data indicate that RCD1 and SRO1 have partially overlapping functions in plant development. In contrast mutant data indicate that rcd1 has defects in plant development, whereas sro1 displays normal development. However, the rcd1 sro1 double mutant has severe growth defects, indicating that RCD1 and SRO1 exemplify an important genetic principle – unequal genetic redundancy. A large pair-wise interaction test against the REGIA transcription factor collection revealed that RCD1 interacts with a large number of transcription factors belonging to several protein families, such as AP2/ERF, NAC and basic helix–loop–helix (bHLH), and that SRO1 interacts with a smaller subset of these. Full genome array analysis indicated that in many cases targets of these transcription factors have altered expression in the rcd1 but not the sro1 mutant. Taken together RCD1 and SRO1 are required for proper plant development
Phosphorylation and proteome dynamics in pathogen-resistant tomato plants
Stulemeijer, I.J.E. - \ 2008
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten. - [S.l. : S.n. - ISBN 9789085048855 - 208
solanum lycopersicum - passalora fulva - plantenziektekunde - signaaltransductie - fosforylering - eiwitsynthese - kinasen - verdedigingsmechanismen - plant-microbe interacties - gastheer-pathogeen interacties - eiwitexpressieanalyse - resistentieveredeling - solanum lycopersicum - passalora fulva - plant pathology - signal transduction - phosphorylation - protein synthesis - kinases - defence mechanisms - plant-microbe interactions - host pathogen interactions - proteomics - resistance breeding
Microbial plant pathogens impose a continuous threat on global food production. Similar to disease resistance in mammals, an innate immune system allows plants to recognise pathogens and swiftly activate defence. For the work described in this thesis, the interaction between tomato and the extracellular fungal pathogen Cladosporium fulvum serves as a model system to study host resistance and susceptibility in plant-pathogen interactions. Resistance to C. fulvum in tomato plants follows the gene-for-gene hypothesis, which requires the presence of a Cf resistance gene in tomato and presence of the cognate avirulence gene (Avr) in C. fulvum. Upon perception of the Avr by a tomato plant, a typical hypersensitive response (HR) is induced that renders the plant resistant to C. fulvum. In the years preceding this thesis work, most research was focussed on understanding which Avrs are produced by C. fulvum and how these Avrs are actually perceived by resistant plants (Chapter 1). The goal of the work described in this thesis is to reveal downstream signalling cascades triggered upon Avr perception. Therefore, the HR was studied by using a model system in which the Cf-4 protein of tomato and the Avr4 protein from C. fulvum were simultaneously expressed in tomato seedlings. Since the Cf-4/Avr-induced responses are inhibited at 33°C and high humidity, these Cf-4/Avr4 seedlings initiate a synchronized and reproducible HR after incubation at 33°C and a subsequent shift to 20°C, which allows studying downstream responses.
To prevent pathogen proliferation in the resistant plant, defence signalling cascades need to be activated extremely fast upon pathogen recognition. Therefore, many downstream signalling cascades depend on post-translational modifications (PTMs) that allow a rapid, reversible, controlled and highly specific transduction of perceived signals. An overview of the various types of PTMs and their role in the resistance response of plants to pathogens is provided in Chapter 2. In addition, examples are provided of successful pathogens that manipulate PTMs.
Protein phosphorylation seems to play an important role in the Cf-4/Avr4-triggered HR, since Avr4 perception leads to the specific activation of at least three mitogen-activated protein kinases, LeMPK1, -2 and -3, which requires phosphorylation by an upstream kinase (Chapter 3). Each of these three kinases seems to have a different role in downstream defence signalling, since the kinases were shown to have different phosphorylation specificities and therefore most likely have different downstream target substrates. Furthermore, these kinases appear to play a different role with regard to HR and full resistance to C. fulvum in tomato (Chapter 3).
Since protein phosphorylation was shown to play an important role in Cf-4/Avr4-induced defence signalling, the phosphoproteome of Cf-4/Avr4 and control seedlings after HR initiation was studied using a new approach (Chapter 4). This approach led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Quantification revealed 13 phosphoproteins with an altered abundance in the Cf-4/Avr4 seedlings as compared to the control, which implies HR-induced differential phosphorylation of these proteins. Phosphorylation-mediated regulation of the activity of these proteins pointed to a swift decrease in photosynthetic activity upon HR-initiation, which was confirmed by experiments in which the actual efficiency of the photosynthesis in the Cf-4/Avr4 seedlings was determined upon induction of the HR. Furthermore, a shift from aerobic to anaerobic respiration, which possibly results from oxygen depletion caused by a massive oxidative burst consuming large amounts of oxygen, seems to take place upon initiation of the HR. Finally, differential phosphorylation of the four cytoplasmic isoforms of the Hsp90 chaperone protein was observed, suggesting that they play distinct roles during defence signalling (Chapter 4).
In addition to the HR, other associated defence responses are initiated upon recognition of C. fulvum. One of these responses is the secretion of defence-related proteins into the apoplast, which is the environment where C. fulvum operates. Therefore, the dynamics of the apoplastic proteome of resistant, Cf-4-expressing plants and susceptible tomato plants lacking Cf-4, were studied after inoculation with a strain of C. fulvum that secretes Avr4 (Chapter 5). Analysis of the apoplastic proteome revealed a slow accumulation of defence proteins in the apoplast of susceptible plants, which is most likely the result of perception of general elicitors of C. fulvum by tomato. In resistant plants, the same set of proteins accumulates in the apoplast, but this occurs much faster and to higher levels. The accelerated response is caused by the Cf-4/Avr4-initiated HR that also leads to cell death. The HR, in combination with the accelerated protein secretion, renders the plants resistant to C. fulvum. In addition, in susceptible plants C. fulvum seems to specifically downregulate genes encoding cell wall proteins of which the accumulation possibly hampers nutrient and water uptake and thereby proliferation of the pathogen in the tomato apoplast. Possibly, an effector of C. fulvum targets a receptor for general elicitors, thereby suppressing transcription of these genes (Chapter 5).
Most data described in this thesis have been obtained from Cf-4/Avr4 seedlings in which the HR can be inhibited by incubating the plants at 33°C. The present data suggest that this temperature-sensitivity occurs at the site of signal perception. Possibly, cytoplasmic Hsp90 stabilizes R protein complexes localized at the plasma membrane. Upon high temperature stress, an increased demand for Hsp90 occurs in the cells to stabilize unfolding proteins that play a role in basal cellular processes, which could lead to the release and subsequent degradation of R protein complexes, rendering defence signalling temperature-sensitive (Chapter 6). The temperature-sensitivity of the Cf-4/Avr4-initiated HR provides a very clean and reproducible tool to study the HR, in the absence of the fungus that produces the Avr. Furthermore, the data described in this thesis provide evidence that the Cf-4/Avr4 seedlings recover from the temperature stress before the specific Cf-4/Avr4-triggered HR is initiated. The possibility to separate the events directly associated with the HR from the full resistance response of the plant to the invading fungus, provides new insight into the complexity of plant defence responses and their specific suppression upon successful colonization by C. fulvum (Chapter 6). Comparison of the defence response to other processes that occur in the cell underlines that resistance and HR execution cannot be seen as an independent and separate process in resistant plants that have recognized a pathogen. On the contrary, signalling cascades seem to depend on similar components and on cascades that possibly converge, eventually leading to a similar response (Chapter 6). Finally, an up to date model for the Cf-4/Avr4-triggered HR and resistance is proposed, based on data that have been published before and the results obtained with the research described in this thesis (Chapter 6).
Two approaches to the study of the origin of life
Hengeveld, R. - \ 2007
Acta Biotheoretica 55 (2007)2. - ISSN 0001-5342 - p. 97 - 131.
atmospheric oxygen - evolution - enzymes - redox - phosphorylation - photosynthesis - dehydrogenase - chloroplasts - metabolism - hypothesis
This paper compares two approaches that attempt to explain the origin of life, or biogenesis. The more established approach is one based on chemical principles, whereas a new, yet not widely known approach begins from a physical perspective. According to the first approach, life would have begun with - often organic - compounds. After having developed to a certain level of complexity and mutual dependence within a non-compartmentalised organic soup, they would have assembled into a functioning cell. In contrast, the second, physical type of approach has life developing within tiny compartments from the beginning. It emphasises the importance of redox reactions between inorganic elements and compounds found on two sides of a compartmental boundary. Without this boundary, ¿life¿ would not have begun, nor have been maintained; this boundary - and the complex cell membrane that evolved from it - forms the essence of life.
In vivo hexamerisation and characterisation of the Arabidopsis thaliana AAA ATPase complex using FRET-FLIM and FCS.
Aker, J.C.M. ; Hesselink, R. ; Engel, R. ; Borst, J.W. ; Visser, A.J.W.G. ; Vries, S.C. de - \ 2007
Plant Physiology 145 (2007)2. - ISSN 0032-0889 - p. 339 - 350.
conformational-changes - living cells - protein - p97 - p97/vcp - receptor - membrane - cycle - phosphorylation - degradation
The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes
A self-consistent field analysis of the neurofilament brush with amino-acid resolution
Zhulina, E.B. ; Leermakers, F.A.M. - \ 2007
Biophysical Journal 93 (2007). - ISSN 0006-3495 - p. 1421 - 1430.
nf-h - protein - axons - phosphorylation - organization - disruption - micelles - subunit - domains - surface
Using the numerical model of Scheutjens and Fleer we investigated, on a self-consistent field level, the equilibrium structure of the neurofilament brush formed by the projection domains of NF-H, NF-M and NF-L proteins. Although the actual AA sequences in the projection domains are coarse grained, the different (realistic) solubilities of AA residues and the specific distribution of its intrinsic charges inside the arms of the NF proteins, are taken explicitly into account. We collect strong evidence that the electrostatic interactions are a dominant force that controls the NF brush structure. There exists a remarkable spatial separation of the H, M and L tails. In a dephosphorylated NF we found confined and flower-like conformations for the H and M projection domains, respectively. We demonstrate that the ionization of KSP repeats in NF proteins triggers a conformational transition in the H tail that leads to the expulsion of its terminal (KEP) domain to the periphery of the NF brush. We argue that the phosphorylation of the NF proteins in axons can both increase the interfilament distance and stabilizes cross-bridges between neurofilaments.