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Publikationen - Molekulare Signalverarbeitung

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Publikation

Picchianti, L.; Sanchez de Medina Hernandez, V.; Zhan, N.; Irwin, N. A.; Groh, R.; Stephani, M.; Hornegger, H.; Beveridge, R.; Sawa‐Makarska, J.; Lendl, T.; Grujic, N.; Naumann, C.; Martens, S.; Richards, T. A.; Clausen, T.; Ramundo, S.; Karagöz, G. E.; Dagdas, Y.; Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy EMBO J. 42, e112053, (2023) DOI: 10.15252/embj.2022112053

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation.
Publikation

Naumann, C.; Heisters, M.; Brandt, W.; Janitza, P.; Alfs, C.; Tang, N.; Toto Nienguesso, A.; Ziegler, J.; Imre, R.; Mechtler, K.; Dagdas, Y.; Hoehenwarter, W.; Sawers, G.; Quint, M.; Abel, S.; Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development Curr. Biol. 32, 2189-2205, (2022) DOI: 10.1016/j.cub.2022.04.005

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi sta-tus are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼2 μmM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.
Publikation

Ziegler, J.; Bochnia, M.; Zeyner, A.; Aminosäurennachweis in geringsten ProbenmengenBestimmung von Hypoglycin A Wiley Analytical Science (2021)

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Publikation

Bochnia, M.; Ziegler, J.; Glatter, M.; Zeyner, A.; Hypoglycin A in cow’s milk—A pilot study Toxins 13, 381, (2021) DOI: 10.3390/toxins13060381

Hypoglycin A (HGA) originating from soapberry fruits (litchi, and ackee) seeds or seedlings from the sycamore maple (SM) tree (related to Sapindaceae) may cause Jamaican vomiting sickness in humans and atypical myopathy in horses and ruminants. A possible transfer into dairy cow’s milk cannot be ruled out since the literature has revealed HGA in the milk of mares and in the offal of captured deer following HGA intoxication. From a study, carried out for another purpose, bulk raw milk samples from four randomly selected dairy farms were available. The cows were pastured in the daytime. A sycamore maple tree was found on the pasture of farm No. 1 only. Bulk milk from the individual tank or milk filling station was sampled in parallels and analyzed for HGA by LC-ESI-MS/MS. Measurable concentrations of HGA occurred only in milk from farm No. 1 and amounted to 120 and 489 nmol/L. Despite low and very variable HGA concentrations, the results indicate that the ingested toxin, once eaten, is transferred into the milk. However, it is unknown how much HGA the individual cow ingested during grazing and what amount was transferred into the bulk milk samples. As a prerequisite for a possible future safety assessment, carry-over studies are needed. Furthermore, the toxins’ stability during milk processing should also be investigated as well.
Preprints

Stephani, M.; Picchianti, L.; Gajic, A.; Beveridge, R.; Skarwan, E.; Sanchez de Medina Hernandez, V.; Mohseni, A.; Clavel, M.; Zeng, Y.; Naumann, C.; Matuszkiewicz, M.; Turco, E.; Loefke, C.; Li, B.; Durnberger, G.; Schutzbier, M.; Chen, H. T.; Abdrakhmanov, A.; Savova, A.; Chia, K.-S.; Djamei, A.; Schaffner, I.; Abel, S.; Jiang, L.; Mechtler, K.; Ikeda, F.; Martens, S.; Clausen, T.; Dagdas, Y.; A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress bioRxiv (2020) DOI: 10.1101/2020.03.18.995316

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control at the ER.
Publikation

Stephani, M.; Picchianti, L.; Gajic, A.; Beveridge, R.; Skarwan, E.; Sanchez de Medina Hernandez, V.; Mohseni, A.; Clavel, M.; Zeng, Y.; Naumann, C.; Matuszkiewicz, M.; Turco, E.; Loefke, C.; Li, B.; Durnberger, G.; Schutzbier, M.; Chen, H. T.; Abdrakhmanov, A.; Savova, A.; Chia, K.-S.; Djamei, A.; Schaffner, I.; Abel, S.; Jiang, L.; Mechtler, K.; Ikeda, F.; Martens, S.; Clausen, T.; Dagdas, Y.; A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress eLife 9, e58396, (2020) DOI: 10.7554/elife.58396

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.
Publikation

Bochnia, M.; Sander, J.; Ziegler, J.; Terhardt, M.; Sander, S.; Janzen, N.; Cavalleri, J.-M. V.; Zuraw, A.; Wensch-Dorendorf, M.; Zeyner, A.; Detection of MCPG metabolites in horses with atypical myopathy PLOS ONE 14, e0211698, (2019) DOI: 10.1371/journal.pone.0211698

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.
Publikation

Bochnia, M.; Scheidemann, W.; Ziegler, J.; Sander, J.; Vollstedt, S.; Glatter, M.; Janzen, N.; Terhardt, M.; Zeyner, A.; Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners Equine Vet. Educ. 30, 24-28, (2018) DOI: 10.1111/eve.12596

Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]‐conjugates) increased in body fluids of the AM horse (MCPA‐carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA‐carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA‐conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA‐conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA‐conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease.
Publikation

Gantner, J.; Ordon, J.; Ilse, T.; Kretschmer, C.; Gruetzner, R.; Löfke, C.; Dagdas, Y.; Bürstenbinder, K.; Marillonnet, S.; Stuttmann, J.; Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system PLOS ONE 13, e0197185, (2018) DOI: 10.1371/journal.pone.0197185

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.
Publikation

Trenner, J.; Poeschl, Y.; Grau, J.; Gogol-Döring, A.; Quint, M.; Delker, C.; Auxin-induced expression divergence between Arabidopsis species may originate within the TIR1/AFB–AUX/IAA–ARF module J. Exp. Bot. 68, 539-552, (2017) DOI: 10.1093/jxb/erw457

Auxin is an essential regulator of plant growth and development, and auxin signaling components are conserved among land plants. Yet, a remarkable degree of natural variation in physiological and transcriptional auxin responses has been described among Arabidopsis thaliana accessions. As intraspecies comparisons offer only limited genetic variation, we here inspect the variation of auxin responses between A. thaliana and A. lyrata. This approach allowed the identification of conserved auxin response genes including novel genes with potential relevance for auxin biology. Furthermore, promoter divergences were analyzed for putative sources of variation. De novo motif discovery identified novel and variants of known elements with potential relevance for auxin responses, emphasizing the complex, and yet elusive, code of element combinations accounting for the diversity in transcriptional auxin responses. Furthermore, network analysis revealed correlations of interspecies differences in the expression of AUX/IAA gene clusters and classic auxin-related genes. We conclude that variation in general transcriptional and physiological auxin responses may originate substantially from functional or transcriptional variations in the TIR1/AFB, AUX/IAA, and ARF signaling network. In that respect, AUX/IAA gene expression divergence potentially reflects differences in the manner in which different species transduce identical auxin signals into gene expression responses.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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