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Publikationen - Molekulare Signalverarbeitung

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Publikation

Antolín-Llovera, M.; Petutsching, E. K.; Ried, M. K.; Lipka, V.; Nürnberger, T.; Robatzek, S.; Parniske, M.; Knowing your friends and foes - plant receptor-like kinases as initiators of symbiosis or defence New Phytol. 204, 791-802, (2014) DOI: 10.1111/nph.13117

The decision between defence and symbiosis signalling in plants involves alternative and modular plasma membrane‐localized receptor complexes. A critical step in their activation is ligand‐induced homo‐ or hetero‐oligomerization of leucine‐rich repeat (LRR)‐ and/or lysin motif (LysM) receptor‐like kinases (RLKs). In defence signalling, receptor complexes form upon binding of pathogen‐associated molecular patterns (PAMPs), including the bacterial flagellin‐derived peptide flg22, or chitin. Similar mechanisms are likely to operate during the perception of microbial symbiont‐derived (lipo)‐chitooligosaccharides. The structurally related chitin‐oligomer ligands chitooctaose and chitotetraose trigger defence and symbiosis signalling, respectively, and their discrimination involves closely related, if not identical, LysM‐RLKs. This illustrates the demand for and the challenges imposed on decision mechanisms that ensure appropriate signal initiation. Appropriate signalling critically depends on abundance and localization of RLKs at the cell surface. This is regulated by internalization, which also provides a mechanism for the removal of activated signalling RLKs. Abundance of the malectin‐like domain (MLD)‐LRR‐RLK Symbiosis Receptor‐like Kinase (SYMRK) is additionally controlled by cleavage of its modular ectodomain, which generates a truncated and rapidly degraded RLK fragment. This review explores LRR‐ and LysM‐mediated signalling, the involvement of MLD‐LRR‐RLKs in symbiosis and defence, and the role of endocytosis in RLK function.
Publikation

Antolín-Llovera, M.; Ried, M. K.; Parniske, M.; Cleavage of the SYMBIOSIS RECEPTOR-LIKE KINASE Ectodomain Promotes Complex Formation with Nod Factor Receptor 5 Curr. Biol. 24, 422-427, (2014) DOI: 10.1016/j.cub.2013.12.053

Plants form root symbioses with fungi and bacteria to improve their nutrient supply. SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK) is required for phosphate-acquiring arbuscular mycorrhiza, as well as for the nitrogen-fixing root nodule symbiosis of legumes [1] and actinorhizal plants [2, 3], but its precise function was completely unclear. Here we show that the extracytoplasmic region of SYMRK, which comprises three leucine-rich repeats (LRRs) and a malectin-like domain (MLD) related to a carbohydrate-binding protein from Xenopus laevis [4], is cleaved to release the MLD in the absence of symbiotic stimulation. A conserved sequence motif—GDPC—that connects the MLD to the LRRs is required for MLD release. We discovered that Nod factor receptor 5 (NFR5) [5, 6, 7, 8] forms a complex with the SYMRK version that remains after MLD release (SYMRK-ΔMLD). SYMRK-ΔMLD outcompeted full-length SYMRK for NFR5 interaction, indicating that the MLD negatively interferes with complex formation. SYMRK-ΔMLD is present at lower amounts than MLD, suggesting rapid degradation after MLD release. A deletion of the entire extracytoplasmic region increased protein abundance, suggesting that the LRR region promotes degradation. Curiously, this deletion led to excessive infection thread formation, highlighting the importance of fine-tuned regulation of SYMRK by its ectodomain.
Publikation

Ried, M. K.; Antolín-Llovera, M.; Parniske, M.; Spontaneous symbiotic reprogramming of plant roots triggered by receptor-like kinases eLife 3, e03891, (2014) DOI: 10.7554/eLife.03891

Symbiosis Receptor-like Kinase (SYMRK) is indispensable for the development of phosphate-acquiring arbuscular mycorrhiza (AM) as well as nitrogen-fixing root nodule symbiosis, but the mechanisms that discriminate between the two distinct symbiotic developmental fates have been enigmatic. In this study, we show that upon ectopic expression, the receptor-like kinase genes Nod Factor Receptor 1 (NFR1), NFR5, and SYMRK initiate spontaneous nodule organogenesis and nodulation-related gene expression in the absence of rhizobia. Furthermore, overexpressed NFR1 or NFR5 associated with endogenous SYMRK in roots of the legume Lotus japonicus. Epistasis tests revealed that the dominant active SYMRK allele initiates signalling independently of either the NFR1 or NFR5 gene and upstream of a set of genes required for the generation or decoding of calcium-spiking in both symbioses. Only SYMRK but not NFR overexpression triggered the expression of AM-related genes, indicating that the receptors play a key role in the decision between AM- or root nodule symbiosis-development.
Publikation

Den Herder, G.; Yoshida, S.; Antolín-Llovera, M.; Ried, M. K.; Parniske, M.; Lotus japonicus E3 Ligase SEVEN IN ABSENTIA4 Destabilizes the Symbiosis Receptor-Like Kinase SYMRK and Negatively Regulates Rhizobial Infection Plant Cell 24, 1691-1707, (2012) DOI: 10.1105/tpc.110.082248

The Lotus japonicus SYMBIOSIS RECEPTOR-LIKE KINASE (SYMRK) is required for symbiotic signal transduction upon stimulation of root cells by microbial signaling molecules. Here, we identified members of the SEVEN IN ABSENTIA (SINA) E3 ubiquitin-ligase family as SYMRK interactors and confirmed their predicted ubiquitin-ligase activity. In Nicotiana benthamiana leaves, SYMRK–yellow fluorescent protein was localized at the plasma membrane, and interaction with SINAs, as determined by bimolecular fluorescence complementation, was observed in small punctae at the cytosolic interface of the plasma membrane. Moreover, fluorescence-tagged SINA4 partially colocalized with SYMRK and caused SYMRK relocalization as well as disappearance of SYMRK from the plasma membrane. Neither the localization nor the abundance of Nod-factor receptor1 was altered by the presence of SINA4. SINA4 was transcriptionally upregulated during root symbiosis, and rhizobia inoculated roots ectopically expressing SINA4 showed reduced SYMRK protein levels. In accordance with a negative regulatory role in symbiosis, infection thread development was impaired upon ectopic expression of SINA4. Our results implicate SINA4 E3 ubiquitin ligase in the turnover of SYMRK and provide a conceptual mechanism for its symbiosis-appropriate spatio-temporal containment.
Publikation

Antolín-Llovera, M.; Ried, M. K.; Binder, A.; Parniske, M.; Receptor Kinase Signaling Pathways in Plant-Microbe Interactions Annu. Rev. Phytopathol. 50, 451-473, (2012) DOI: 10.1146/annurev-phyto-081211-173002

Plant receptor-like kinases (RLKs) function in diverse signaling pathways, including the responses to microbial signals in symbiosis and defense. This versatility is achieved with a common overall structure: an extracytoplasmic domain (ectodomain) and an intracellular protein kinase domain involved in downstream signal transduction. Various surfaces of the leucine-rich repeat (LRR) ectodomain superstructure are utilized for interaction with the cognate ligand in both plant and animal receptors. RLKs with lysin-motif (LysM) ectodomains confer recognitional specificity toward N-acetylglucosamine-containing signaling molecules, such as chitin, peptidoglycan (PGN), and rhizobial nodulation factor (NF), that induce immune or symbiotic responses. Signaling downstream of RLKs does not follow a single pattern; instead, the detailed analysis of brassinosteroid (BR) signaling, innate immunity, and symbiosis revealed at least three largely nonoverlapping pathways. In this review, we focus on RLKs involved in plant-microbe interactions and contrast the signaling pathways leading to symbiosis and defense.
Publikation

Schilling, S.; Manhart, S.; Hoffmann, T.; Ludwig, H.-H.; Wasternack, C.; Demuth, H.-U.; Substrate Specificity of Glutaminyl Cyclases from Plants and Animals Biol. Chem. 384, 1583-1592, (2003) DOI: 10.1515/BC.2003.175

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-?homoglutaminyl residue in the peptide H-?homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo( N?-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates gastrin, neurotensin and [GlN1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.
Publikation

Schilling, S.; Niestroj, A. J.; Rahfeld, J.-U.; Hoffmann, T.; Wermann, M.; Zunkel, K.; Wasternack, C.; Demuth, H.-U.; Identification of Human Glutaminyl Cyclase as a Metalloenzyme J. Biol. Chem. 278, 49773-49779, (2003) DOI: 10.1074/jbc.M309077200

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 ± 1 and 295 ± 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 ± 0.02, 6.93 ± 0.03, and 5.60 ± 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.
Publikation

Schilling, S.; Hoffmann, T.; Rosche, F.; Manhart, S.; Wasternack, C.; Demuth, H.-U.; Heterologous Expression and Characterization of Human Glutaminyl Cyclase: Evidence for a Disulfide Bond with Importance for Catalytic Activity Biochemistry 41, 10849-10857, (2002) DOI: 10.1021/bi0260381

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high α-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.
Publikation

Schilling, S.; Hoffmann, T.; Wermann, M.; Heiser, U.; Wasternack, C.; Demuth, H.-U.; Continuous Spectrometric Assays for Glutaminyl Cyclase Activity Anal. Biochem. 303, 49-56, (2002) DOI: 10.1006/abio.2001.5560

The enzymatic conversion of one chromogenic substrate, -glutamine-p-nitroanilide, and two fluorogenic substrates, -glutaminyl-2-naphthylamide and -glutaminyl-4-methylcoumarinylamide, into their respective pyroglutamic acid derivatives by glutaminyl cyclase (QC) was estimated by introducing a new coupled assay using pyroglutamyl aminopeptidase as the auxiliary enzyme. For the purified papaya QC, the kinetic parameters were found to be in the range of those previously reported for other glutaminyl peptides, such as Gln-Gln, Gln-Ala, or Gln-tert-butyl ester. The assay can be performed in the presence of ammonia up to a concentration of 50 mM. Increasing ionic strength, e.g., potassium chloride up to 300 mM, resulted in an increase in enzymatic activity of about 20%. This is the first report of a fast, continuous, and reliable determination of QC activity, even in the presence of ammonium ions, during the course of protein purification and enzymatic analysis.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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