A neurosteroid analogue photolabeling reagent labels the colchicine‐binding site on tubulin: A mass spectrometric analysis

Chen, Zi Wei; Chen, Li Hai; Akentieva, Natalia; Lichti, Cheryl F.; Darbandi, Ramin; Hastings, Randy; Covey, Douglas F.; Reichert, David E.; Townsend, R. Reid; Evers, Alex S.

doi:10.1002/elps.201100434

Cited by 17 publications

(22 citation statements)

References 33 publications

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“…It is also possible that 6-AziP undergoes fragmentation during electrospray ionization, which would result in an adduct weight for 6-AziP different from that with simple loss of two nitrogens (Brunner, 1993) and would confound the search protocols. Previous studies with 6-AziP-labeled proteins and synthetic peptides detected peptides containing an intact 6-AziP moiety and failed to detect fragmented 6-AziP adducts (Chen et al, 2012a). Therefore, it is unlikely that we failed to detect photolabeled peptides because of systematic 6-AziP fragmentation during electrospray ionization.…”

Section: Identification Of Neurosteroid Photoincorporationmentioning

confidence: 73%

“…6-azipregnanolone (6-AziP) is a neurosteroid-analog photolabeling reagent that was shown previously to label VDAC and ␤-tubulin covalently (Darbandi-Tonkabon et al, 2003, 2004Chen et al, 2012a). Studies that used tritiated 6-AziP to label rat brain membranes and separated the photolabeled proteins with native gel electrophoresis showed that 6-AziP also labeled native GABA A receptors (data not shown).…”

Section: Introductionmentioning

confidence: 93%

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Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABA_A Receptor

et al. 2012

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Accumulated evidence suggests that neurosteroids modulate GABA A receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3␣,5␤)-6-azipregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His 8 -␤3 homomeric GABA A receptors. 6-AziP inhibited 3 H](3␤,5␤)-6-AziP. Highresolution mass spectrometric analysis of affinity-purified, 6-AziPlabeled His 8 -␤3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole ␤3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.

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Section: Identification Of Neurosteroid Photoincorporationmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 93%

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABA_A Receptor

et al. 2012

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“…However, it is also recognized that none of these molecular targets have satisfied the criteria of being both necessary and sufficient to produce the complete anesthetic state. Other plausible targets have included mitochondria, tubulin, and the synaptic vesicle transport and release machinery (17,(55)(56)(57)(58). For example, SNAP-25 and syntaxin specifically bind propofol (7).…”

Section: Discussionmentioning

confidence: 99%

Common general anesthetic propofol impairs kinesin processivity

Bensel

Guzik-Lendrum

Masucci

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Propofol is the most widely used i.v. general anesthetic to induce and maintain anesthesia. It is now recognized that this small molecule influences ligand-gated channels, including the GABA A receptor and others. Specific propofol binding sites have been mapped using photoaffinity ligands and mutagenesis; however, their precise target interaction profiles fail to provide complete mechanistic underpinnings for the anesthetic state. These results suggest that propofol and other common anesthetics, such as etomidate and ketamine, may target additional protein networks of the CNS to contribute to the desired and undesired anesthesia end points. Some evidence for anesthetic interactions with the cytoskeleton exists, but the molecular motors have received no attention as anesthetic targets. We have recently discovered that propofol inhibits conventional kinesin-1 KIF5B and kinesin-2 KIF3AB and KIF3AC, causing a significant reduction in the distances that these processive kinesins can travel. These microtubule-based motors are highly expressed in the CNS and the major anterograde transporters of cargos, such as mitochondria, synaptic vesicle precursors, neurotransmitter receptors, cell signaling and adhesion molecules, and ciliary intraflagellar transport particles. The single-molecule results presented show that the kinesin processive stepping distance decreases 40-60% with EC 50 values <100 nM propofol without an effect on velocity. The lack of a velocity effect suggests that propofol is not binding at the ATP site or allosteric sites that modulate microtubule-activated ATP turnover. Rather, we propose that a transient propofol allosteric site forms when the motor head binds to the microtubule during stepping.anesthesia | allosteric inhibitor | microtubule | etomidate | ketamine

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“…24,25 This fact has prompted us to examine the series of other biologically active colchicine derivatives, i.e.…”

Section: Maldi Mass Spectramentioning

confidence: 99%

Mass spectrometric study of colchicine and its synthetic derivatives 10-alkylthiocolchicines

Kurek

Nowakowska

Bartkowiak

2015

ACSi

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The mass spectra behaviour of five 10-alkylthiocolchicine derivatives has been studied using electron ionization and MALDI-TOF MS techniques. Fragmentation patterns of colchicines derivatives after EI have been investigated, as well as mass spectra after collision-induced dissociation (CID) MALDI have been used to gain structural information. To the best of our knowledge, this is the first time that the MALDI MS/MS data for colchicine and its alkylthio derivatives have been described. It has been shown also that the data derived from mass spectra can be used for identification/quantitative determination of natural and modified alkaloids of colchicine group. The detailed fragmentation pathways proposed here could be helpful for the characterization of other colchicines of these type. The utility of different ionization techniques for analysis of compounds of this class has been evaluated. Due to the cytotoxic activity towards tumour cell lines, 10-alkylthiocolchicines may be considered as the active ingredients of anticancer agents. If these compounds find use in medical treatment, their distribution in organism and their metabolism will have to be monitored by spectroscopic or spectrometric methods. The characteristic fragment ions may be used by Selected Reaction Monitoring method for determination of colchicine analogues.

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A neurosteroid analogue photolabeling reagent labels the colchicine‐binding site on tubulin: A mass spectrometric analysis

Cited by 17 publications

References 33 publications

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABA_A Receptor

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABA_A Receptor

Common general anesthetic propofol impairs kinesin processivity

Mass spectrometric study of colchicine and its synthetic derivatives 10-alkylthiocolchicines

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A neurosteroid analogue photolabeling reagent labels the colchicine‐binding site on tubulin: A mass spectrometric analysis

Cited by 17 publications

References 33 publications

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABAA Receptor

Common general anesthetic propofol impairs kinesin processivity

Mass spectrometric study of colchicine and its synthetic derivatives 10-alkylthiocolchicines

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Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABA_A Receptor

Neurosteroid Analog Photolabeling of a Site in the Third Transmembrane Domain of the β3 Subunit of the GABA_A Receptor