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
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.
Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M. The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases. EMBO J 37, 255-268, (2018) DOI: 10.15252/embj.201797159
The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Bochnia, M.; Ziegler, J.; Sander, J.; Uhlig, A.; Schaefer, S.; Vollstedt, S.; Glatter, M.; Abel, S.; Recknagel, S.; Schusser, G. F.; Wensch-Dorendorf, M.; Zeyner, A. Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture PLoS ONE 10, e0136785, (2015) DOI: 10.1371/journal.pone.0136785
Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.
Schwager, K.M.; Calderón Villalobos, L.I.; Dohmann, E.M.; Willige, B.C.; Knierer, S.; Nill, C.; Schwechheimer, C. Characterization of the VIER F-BOX PROTEINE genes from Arabidopsis reveals their importance for plant growth and development Plant Cell 19(4), 1163-1178, (2007) DOI: 10.1105/tpc.105.040675
E3 ubiquitin ligases (E3s) target proteins for degradation by the 26S proteasome. In SKP1/CDC53/F-box proteintype E3s, substrate specificity is conferred by the interchangeable F-box protein subunit. The vast majority of the 694 F-box proteins encoded by the Arabidopsis thaliana genome remain to be understood. We characterize the VIER F-BOX PROTEINE (VFB; German for FOUR F-BOX PROTEINS) genes from Arabidopsis that belong to subfamily C of the Arabidopsis F-box protein superfamily. This subfamily also includes the F-box proteins TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) proteins and EIN3 BINDING F-BOX proteins, which regulate auxin and ethylene responses, respectively. We show that loss of VFB function causes delayed plant growth and reduced lateral root formation. We find that the expression of a number of auxin-responsive genes and the activity of DR5:ß-glucuronidase, a reporter for auxin reponse, are reduced in the vfb mutants. This finding correlates with an increase in the abundance of an AUXIN/INDOLE-3-ACETIC ACID repressor. However, we also find that auxin responses are not affected in the vfb mutants and that a representative VFB family member, VFB2, cannot functionally complement the tir1-1 mutant. We therefore exclude the possibility that VFBs are functional orthologs of TIR1/AFB proteins.
Calderón Villalobos, L.I.; Nill, C.; Marrocco, K.; Kretsch, T.; Schwechheimer, C. The evolutionarily conserved Arabidopsis thaliana
F-box protein AtFBP7 is required for efficient translation during
temperature stress Gene 392(1-2), 106-116, (2007) DOI: 10.1016/j.gene.2006.11.016
In eukaryotes, E3 ubiquitin ligases (E3s) mediate the ubiquitylation of
proteins that are destined for degradation by the ubiquitin-proteasome
system. In SKP1/CDC53/F-box protein (SCF)-type E3 complexes, the
interchangeable F-box protein confers specificity to the E3 ligase
through direct physical interactions with the degradation substrate. The
vast majority of the approximately 700 F-box proteins from the plant
model organism Arabidopsis thaliana remain to be characterized. Here, we
investigate the previously uncharacterized and evolutionarily conserved
Arabidopsis F-box protein 7 (AtFBP7), which is encoded by a unique gene
in Arabidopsis (At1g21760). Several apparent fbp7 loss-of-function
alleles do not have an obvious phenotype. AtFBP7 is ubiquitously
expressed and its expression is induced after cold and heat stress. When
following up on a reported co-purification of the eukaryotic elongation
factor-2 (eEF-2) with YLR097c, the apparent budding yeast orthologue of
AtFBP7, we discovered a general defect in protein biosynthesis after
cold and heat stress in fbp7 mutants. Thus, our findings suggest that
AtFBP7 is required for protein synthesis during temperature stress.
Calderón Villalobos, L.I.; Kuhnle, C.; Li, H.; Rosso, M.; Weisshaar, B.; Schwechheimer, C. LucTrap vectors are tools to generate luciferase fusions for the quantification of transcript and protein abundance in vivo Plant Physiol 141(1), 3-14, (2006) DOI: 10.1104/pp.106.078097
Proper plant growth and development strongly rely on the plant's ability to respond dynamically to signals and cues from the intra- and extracellular environment. Whereas many of these responses require specific changes at the level of gene expression, in recent years it has become increasingly clear that many plant responses are at least in part also controlled at the level of protein turnover. It is a challenge for signal transduction research to understand how distinct incoming signals are integrated to generate specific changes at the transcript or protein level. The activity of luciferase (LUC) reporters can be detected in nondestructive qualitative and quantitative assays in vivo. Therefore,z LUC reporters are particularly well suited for the detection of changes at the transcript and protein level. To the best of our knowledge, the number of plant transformation vectors for LUC fusions is very limited. In this article, we describe the LucTrap plant transformation vectors that allow generation of targeted and random transcriptional and translational fusions with the modified firefly LUC reporter LUC+. We demonstrate that LucTrap-based fusions can be used to monitor rapid changes in gene expression and protein abundance in vivo.
Calderón Villalobos, L.I.; Kuhnle, C.; Dohmann, E.M.; Li, H.; Bevan, M.; Schwechheimer, C. The evolutionarily conserved TOUGH protein is required for proper development of Arabidopsis thaliana Plant Cell 17,9, 24873-2485, (2005)
In this study, we characterize the evolutionarily conserved TOUGH (TGH) protein as a novel regulator required for Arabidopsis thaliana development. We initially identified TGH as a yeast two-hybrid system interactor of the transcription initiation factor TATA-box binding protein 2. TGH has apparent orthologs in all eukaryotic model organisms with the exception of the budding yeast Saccharomyces cerevisiae. TGH contains domains with strong similarity to G-patch and SWAP domains, protein domains that are characteristic of RNA binding and processing proteins. Furthermore, TGH colocalizes with the splicing regulator SRp34 to subnuclear particles. We therefore propose that TGH plays a role in RNA binding or processing. Arabidopsis tgh mutants display developmental defects, including reduced plant height, polycotyly, and reduced vascularization. We found TGH expression to be increased in the amp1-1 mutant, which is similar to tgh mutants with respect to polycotyly and defects in vascular development. Interestingly, we observed a strong genetic interaction between TGH and AMP1 in that tgh-1 amp1-1 double mutants are extremely dwarfed and severely affected in plant development in general and vascular development in particular when compared with the single mutants.
Schwechheimer, C.; Calderón Villalobos, L.I. Cullin-containing E3 ubiquitin ligases in plant development Curr. Opin. Plant Biol. 7(6), 677-686, (2002)
In eukaryotes, the ubiquitinproteasome system participates in the control of signal transduction events by selectively eliminating regulatory proteins. E3 ubiquitin ligases specifically bind degradation substrates and mediate their poly-ubiquitylation, a prerequisite for their degradation by the 26S proteasome. On the basis of the analysis of the Arabidopsis genome sequence, it is predicted that there are more than 1000 E3 ubiquitin ligases in plants. Several types of E3 ubiquitin ligases have already been characterized in eukaryotes. Recently, some of these E3 enzymes have been implicated in specific plant signaling pathways.