Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation

Yang, Jianhong; Li, Yong; Yan, Wei; Liu, Weimin; Qiu, Qiang; Ye, Haoyu; Chen, Lijuan

doi:10.1074/jbc.ra118.006325

Cited by 39 publications

(54 citation statements)

References 34 publications

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“…However, the tubulin inhibitors identified to date have no effects on tubulin protein levels. Previous experiments by our group showed that small molecules covalently bound to the Cys239 site, including T0070906, T007-1, and T138067, promoting tubulin degradation (Yang et al, 2019). These small molecules were collectively classified as the third type of tubulin inhibitor-tubulin degradation agent.…”

Section: Discussionmentioning

confidence: 96%

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The Natural Compound Withaferin A Covalently Binds to Cys239 ofβ-Tubulin to Promote Tubulin Degradation

Yang

Yan

et al. 2019

Mol Pharmacol

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Withaferin A (WIT) is a natural product possessing a wide range of pharmacologic activities. Previous studies have reported covalent binding of WIT to tubulin and down-of tubulin protein levels although the underlying mechanisms remain to be established. In the current investigation, we showed that WIT induces down-regulation of tubulin in a post-transcriptional manner, suggestive of direct and potent activity in tubulin degradation. The N,N9-ethylene bis(iodoacetamide) assay and competitive binding experiments with four colchicine sitetargeted tubulin inhibitors further revealed that WIT interacts with the colchicine site of tubulin to promote degradation. WIT irreversibly inhibited tubulin polymerization, and mass spectrometry results disclosed binding to cysteine at position 239 (Cys239) and Cys303 sites of b-tubulin. Interestingly, WIT promoted degradation of the b-tubulin isoforms containing Cys239 [b2, b4, and b5(b)] but had no effect on those containing Ser239 (b3 and b6). Moreover, a C239S but not C303S mutation in b-tubulin completely abolished the degradation effect of WIT, suggesting that the Cys239-WIT covalent bond accounts for this activity. Our collective results clearly demonstrate that covalent interactions between WIT and Cys239 of b-tubulin promote tubulin degradation, supporting its potential utility as a therapeutic compound. SIGNIFICANCE STATEMENT Withaferin A, a natural product possessing a wide range of pharmacologic activities, covalently binds to Cys239 of b-tubulin near the colchicine site, and the WIT-Cys239 covalent bond accounts for WIT-induced tubulin degradation, fully clarifying the underlying mechanisms and supporting its potential utility a therapeutic compound.

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Section: Discussionmentioning

confidence: 96%

“…Our data showed that WIT forms covalent bonds with Cys303 and Cys239 of b-tubulin. Moreover, the bond between WIT and Cys239, but not Cys303, was responsible for tubulin degradation, providing another example of a small molecule that acts as a covalent modifier of Cys239 in b-tubulin to promote tubulin degradation (Yang et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

The Natural Compound Withaferin A Covalently Binds to Cys239 ofβ-Tubulin to Promote Tubulin Degradation

Yang

Yan

et al. 2019

Mol Pharmacol

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“…That some of the active compound-cysteine interactions led to protein degradation without requiring a separate E3 ligase-directing ligand (e.g., EV-3-mediated degradation of BIRC2/3) underscores the potential for covalent modification by small molecules to directly affect protein stability in cells (Foulkes et al, 2018;Jones, 2018;Schreiber, 2019;Yang et al, 2019). We do not yet understand how EV-3 promotes BIRC2 and BIRC3 degradation, but this could involve disruption of protein-protein interactions proximal to the compound-modified cysteines in these proteins.…”

Section: Discussionmentioning

confidence: 99%

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Vinogradova

Lazar

Suciu

et al. 2019

Preprint

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Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

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“…We have done three biological repeats. Then the following label-free quantitative proteomic analysis of these samples were carried out following the procedure as described previously [13].…”

Section: Label Free Quantitative Proteomicsmentioning

confidence: 99%

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect

Yang

et al. 2020

Preprint

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Microtubule, composed of αβ-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs mainly target β-tubulin to inhibit microtubule dynamic instability (MDI) while agents binding to α-tubulin are less well characterized and also the molecular mechanism of MDI is far from being articulated. Cevipabulin, an oral microtubule-active antitumor clinical candidate, is widely accepted as a microtubule stabilizing agent (MSA) but binds to the microtubule-destabilization vinblastine site on β-tubulin and this unusual phenomenon has so far failed to be explained. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on α-tubulin (named the seventh site) which located at the region spatially corresponding to the vinblastine site on β-tubulin. Interestingly, cevipabulin exhibits two unique site-dependent functions. Cevipabulin binding to the seventh site promotes tubulin degradation through interaction of the non-exchengeable GTP to reduce tubulin stability. Cevipabulin binding to the vinblastine site enhances longitudinal interactions but inhibits lateral interactions of tubulins, thus inducing tubulin protofilament polymerization (but not microtubule polymerization like MSAs), and then tangling into irregular tubulin aggregates. Importantly, the tubulin-cevipabulin structure is an intermediate between “bent” and “straight” tubulins and the involved bent-to-straight conformation change will be helpful to fully understand the molecular mechanism of tubulin assembly. Our findings confirm cevipabulin is not an MSA and shed light on the development of a new generation of anti-microtubule drugs targeting the novel site on α-tubulin and also provide new insights into MDI.

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Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation

Cited by 39 publications

References 34 publications

The Natural Compound Withaferin A Covalently Binds to Cys239 ofβ-Tubulin to Promote Tubulin Degradation

The Natural Compound Withaferin A Covalently Binds to Cys239 ofβ-Tubulin to Promote Tubulin Degradation

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect

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