Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Stinn, Anne; Furkert, Jens; She., Kaufmann; Moura-Alves, Pedro; Kolbe, Michael

doi:10.3390/bios11030060

Cited by 8 publications

(5 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Eventually we acquired soluble and stable protein using a genetic construct that produces FliB fused to the C-terminal of truncated FleB protein (FleBt), a Yersinia enterocolitica flagellin protein lacking D0 domain ( S5A Fig ). The N-terminal his-tagged FleBt tag has been used previously to obtain sufficient amounts of soluble and folded proteins, which are notoriously difficult to produce [ 20 ]. It is also worth to mention that FleB shares a conserved D1 domain with the substrate flagellin of FliB ( S3 Fig ) and therefore might also contribute to stabilize FliB in the fused protein.…”

Section: Resultsmentioning

confidence: 99%

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

et al. 2021

Self Cite

View full text Add to dashboard Cite

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.

show abstract

Section: Resultsmentioning

confidence: 99%

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…Mouse His-ARNT (mARNT 85-465 Δ274–297 C256S Δ351–358) expression was adapted from Huang et al and Schulte et al , Protein expression was induced by addition of 0.5 mM IPTG and incubation overnight. Bacterial pellets were resuspended in 5× volume of lysis buffer (50 mM Na 3 PO 4 , 300 mM NaCl, 15 mM imidazole, 10% (v/v) glycerol, 1 mM TCEP, 0.2% Triton X-100 with 0.5 mM AEBSF, and 5 U/mL benzonase) and lysed using a cell disrupter (TS0.75, Constant System) at 1.35 kbar and one pass.…”

Section: Methodsmentioning

confidence: 99%

The Highly Potent AhR Agonist Picoberin Modulates Hh-Dependent Osteoblast Differentiation

et al. 2022

Self Cite

View full text Add to dashboard Cite

Identification and analysis of small molecule bioactivity in target-agnostic cellular assays and monitoring changes in phenotype followed by identification of the biological target are a powerful approach for the identification of novel bioactive chemical matter in particular when the monitored phenotype is disease-related and physiologically relevant. Profiling methods that enable the unbiased analysis of compound-perturbed states can suggest mechanisms of action or even targets for bioactive small molecules and may yield novel insights into biology. Here we report the enantioselective synthesis of natural-product-inspired 8-oxotetrahydroprotoberberines and the identification of Picoberin, a low picomolar inhibitor of Hedgehog (Hh)-induced osteoblast differentiation. Global transcriptome and proteome profiling revealed the aryl hydrocarbon receptor (AhR) as the molecular target of this compound and identified a cross talk between Hh and AhR signaling during osteoblast differentiation.

show abstract

“…Dysregulation of the AHR pathway is associated with a variety of maladies, including autoimmune diseases and cancer. AHR is thus a promising target for drug development and host-directed therapy [ 113 ].…”

Section: Metabolites and Molecular Mechanisms In The Kynurenine Pathwaymentioning

confidence: 99%

Kynurenine Pathway Metabolites as Biomarkers in Alzheimer’s Disease

Liang

Xie

et al. 2022

Disease Markers

View full text Add to dashboard Cite

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that deteriorates cognitive function. Patients with AD generally exhibit neuroinflammation, elevated beta-amyloid (Aβ), tau phosphorylation (p-tau), and other pathological changes in the brain. The kynurenine pathway (KP) and several of its metabolites, especially quinolinic acid (QA), are considered to be involved in the neuropathogenesis of AD. The important metabolites and key enzymes show significant importance in neuroinflammation and AD. Meanwhile, the discovery of changed levels of KP metabolites in patients with AD suggests that KP metabolites may have a prominent role in the pathogenesis of AD. Further, some KP metabolites exhibit other effects on the brain, such as oxidative stress regulation and neurotoxicity. Both analogs of the neuroprotective and antineuroinflammation metabolites and small molecule enzyme inhibitors preventing the formation of neurotoxic and neuroinflammation compounds may have potential therapeutic significance. This review focused on the KP metabolites through the relationship of neuroinflammation in AD, significant KP metabolites, and associated molecular mechanisms as well as the utility of these metabolites as biomarkers and therapeutic targets for AD. The objective is to provide references to find biomarkers and therapeutic targets for patients with AD.

show abstract

Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Cited by 8 publications

References 31 publications

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction

The Highly Potent AhR Agonist Picoberin Modulates Hh-Dependent Osteoblast Differentiation

Kynurenine Pathway Metabolites as Biomarkers in Alzheimer’s Disease

Contact Info

Product

Resources

About