Inhibition of Biosynthesis of Human Endothelin B Receptor by the Cyclodepsipeptide Cotransin

Westendorf, Carolin; Schmidt, Antje; Coin, Irene; Furkert, Jens; Ridelis, Ingrid; Zampatis, Dimitris E.; Rutz, Claudia; Wiesner, Burkhard; Rosenthal, Walter; Beyermann, Michael; Schülein, Ralf

doi:10.1074/jbc.m111.239244

Cited by 16 publications

(17 citation statements)

References 28 publications

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“…Apratoxin A, a cyanobacterial metabolite, has been shown to inhibit cotranslational translocation in vitro (Liu et al, 2009), but the blocked step is unknown. Furthermore a group of closely related cyclic heptadepsipeptide inhibitors including HUN-7293, CAM741 (Besemer et al, 2005) and a simplified version thereof called cotransin (Garrison et al, 2005) have been found to inhibit cotranslational translocation of VCAM1 and other specific substrates (Maifeld et al, 2011;Westendorf et al, 2011). Photoaffinity labeling has identified Sec61a as the target (MacKinnon et al, 2007), which was confirmed by isolation of resistance mutations in SEC61A1 (MacKinnon et al, 2014).…”

Section: Introductionmentioning

confidence: 83%

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

Junne

Wong

Studer

et al. 2015

Journal of Cell Science

116

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A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61a1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co-and posttranslationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.

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Section: Introductionmentioning

confidence: 83%

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

Junne

Wong

Studer

et al. 2015

Journal of Cell Science

116

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“…Moreover, these cells are easy to wash because of their epithelial morphology. For the few sensitive proteins reported, the IC 50 values for cotransin-mediated biosynthesis inhibition were in a range of 0.5–5 μM [ 4 , 6 ]. To identify less-sensitive proteins and to discriminate the latter from completely resistant proteins, we used a cotransin concentration of 30 μM for our study which is a saturating concentration taking the reported IC 50 values into account.…”

Section: Resultsmentioning

confidence: 99%

“…Proteins were reduced using dithiothreitol (DTT) (5 mM, 30 min, 55°C) and alkylated using iodoacetamide (15 mM, 30 min, room temperature) in the dark. Separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as described previously [ 6 ].…”

Section: Methodsmentioning

confidence: 99%

“…The originally described group of cotransin-sensitive proteins is formed by vascular cell adhesion molecule 1 (VCAM1), P-selectin, angiotensinogen, β-lactamase and the G protein-coupled corticotropin-releasing factor receptor type 1, an integral membrane protein [ 4 ]. Recently, another G protein-coupled receptor, namely the endothelin B receptor was shown to be cotransin-sensitive [ 6 ]. Interestingly, no SAS-containing protein was found among this original subset of proteins.…”

Section: Introductionmentioning

confidence: 99%

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Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

et al. 2015

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The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

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“…The tag thus seems to be ideally suited to study protein trafficking between different subcellular compartments. Another pc‐FP, namely the Kaede protein, was already used to study real time GPCR recycling [9] and biosynthesis [10]. Kaede seems not to oligomerize when fused to membrane proteins but is prone to tetramerize in its soluble form [9].…”

Section: Discussionmentioning

confidence: 99%

Use of Kikume green–red fusions to study the influence of pharmacological chaperones on trafficking of G protein‐coupled receptors

Ridelis¹,

Schmidt²,

Teichmann³

et al. 2012

FEBS Letters

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Keywords:Photoconvertible fluorescent proteins G protein-coupled receptor Vasopressin V 2 receptor Pharmacological chaperone Post-translational rescue Co-translational rescue a b s t r a c tIn this study we demonstrate that the photoconvertible monomeric Kikume green-red (mKikGR) protein is suitable to study trafficking of G protein-coupled receptors. Taking mKikGR-tagged mutants of the vasopressin V 2 receptor (V 2 R) as models, we analyzed whether the V 2 R-specific pharmacological chaperone SR121463B influences receptor folding on a co-or post-translational level. Misfolded mKikGR-tagged V 2 Rs were completely photoconverted in the early secretory pathway yielding a red receptor population (already synthesized receptors) and an arising green receptor population (newly synthesized receptors). Trafficking of both receptor populations could be rescued by treatment with SR121463B demonstrating that the substance can act co-and post-translationally.

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Inhibition of Biosynthesis of Human Endothelin B Receptor by the Cyclodepsipeptide Cotransin

Cited by 16 publications

References 28 publications

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

Decatransin, a novel natural product inhibiting protein translocation at the Sec61/SecY translocon

Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

Use of Kikume green–red fusions to study the influence of pharmacological chaperones on trafficking of G protein‐coupled receptors

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