Small-molecule displacement of a cryptic degron causes conditional protein degradation

Bonger, Kimberly M.; Chen, Ling-chun; Liu, Corey W.; Wandless, Thomas J.

doi:10.1038/nchembio.598

Cited by 181 publications

(171 citation statements)

References 29 publications

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“…A potential limitation of the Degron-KI approach is that it only works efficiently when applied as an N-terminal fusion (10, and data not shown) and thus requires that proteins can tolerate N-terminal tagging. While Cterminal degron fusion systems have been described (36,37), we found that protein depletion was much less effective compared with the DD-Shld system (data not shown). In addition, for some genes the expression level of DD-tagged protein was reduced compared with the untagged protein, possibly due to slight alteration in protein stability (incomplete stabilization through Shld) or translation efficiency (due to the P2A ribosome skipping site).…”

Section: Discussionmentioning

confidence: 60%

A Chemical Genetics Approach for the Functional Assessment of Novel Cancer Genes

Zhou¹,

Derti²,

Ruddy³

et al. 2015

Cancer Research

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Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches.The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Ka inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes.

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Section: Discussionmentioning

confidence: 60%

A Chemical Genetics Approach for the Functional Assessment of Novel Cancer Genes

Zhou¹,

Derti²,

Ruddy³

et al. 2015

Cancer Research

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“…FK506 induced ID1 at low doses, and a high plateau of activation was rapidly achieved. We then compared the effect of FK506 and rapamycin to another calcineurin inhibitor, cyclosporine, that does not interact with FKBP12 but binds the immunophilin, cyclophilin A, as well as shield-1 (27). Shield-1 binds FKBP12 but does not have any immunosuppressive properties ( Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Spiekerkoetter

Tian²,

Cai³

et al. 2013

J. Clin. Invest.

376

370

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Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH. IntroductionIdiopathic pulmonary arterial hypertension (IPAH) is a rare disorder thought to develop following a genetic and/or environmental insult that triggers endothelial cell (EC) apoptosis, loss of distal vessels, and occlusive vascular remodeling (1). These pathological changes increase resistance to pulmonary flow and cause progressive right heart failure. Current therapies mainly include drugs with vasodilatory properties that improve cardiopulmonary function (2). However, the obliterative vascular pathology usually continues to progress (3), leaving heart-lung transplantation as the only option for many patients. Therefore, new approaches are needed that focus on activating cellular mechanisms to reverse vascular remodeling. One strategy could be to improve function of the bone morphogenetic protein receptor-2 (BMPR2) signaling pathway. Germline mutations causing loss of BMPR2 function are found in >80% of familial and approximately 20% of sporadic cases of IPAH (4, 5). Acquired somatic chromosomal abnormalities in the BMPR2 signaling pathway have also been described (6). The low penetrance of pulmonary arterial hypertension (PAH) found in nonaffected family members with a BMPR2 mutation has been attributed to a higher level of

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“…For example, using a procedure named deGradFP, proteins fused to green fluorescent protein (GFP) can be depleted by expressing a fusion protein consisting of an anti-GFP antibody coupled to an F-box domain; the fusion protein recruits GFP-tagged protein to SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complexes, marking it for ubiquitylation and proteasome-mediated degradation (Caussinus et al, 2012). deGradFP has been shown to degrade target proteins over a period of 2-3 hours (Caussinus et al, 2012), comparable with the kinetics of most other protein degradation strategies (Bonger et al, 2011;Taxis et al, 2009;Zhou et al, 2000). However, many developmental events occur on a much faster timescale.…”

Section: Introductionmentioning

confidence: 98%

“…Strategies include introducing temperature-sensitive protease cleavage sites, or using heterologous adaptor proteins, drugs or light to induce the ubiquitin-mediated degradation of a tagged target protein (Banaszynski et al, 2006;Bonger et al, 2011;Nishimura et al, 2009;Raina and Crews, 2010;Renicke et al, 2013). For example, using a procedure named deGradFP, proteins fused to green fluorescent protein (GFP) can be depleted by expressing a fusion protein consisting of an anti-GFP antibody coupled to an F-box domain; the fusion protein recruits GFP-tagged protein to SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complexes, marking it for ubiquitylation and proteasome-mediated degradation (Caussinus et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Repurposing an endogenous degradation system for rapid and targeted depletion ofC. elegansproteins

et al. 2014

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The capability to conditionally inactivate gene function is essential for understanding the molecular basis of development. In gene and mRNA targeting approaches, protein products can perdure, complicating genetic analysis. Current methods for selective protein degradation require drug treatment or take hours for protein removal, limiting their utility in studying rapid developmental processes in vivo. Here, we repurpose an endogenous protein degradation system to rapidly remove targeted C. elegans proteins. We show that upon expression of the E3 ubiquitin ligase substrate-recognition subunit ZIF-1, proteins tagged with the ZF1 zinc-finger domain can be quickly degraded in all somatic cell types examined with temporal and spatial control. We demonstrate that genes can be engineered to become conditional lossof-function alleles by introducing sequences encoding the ZF1 tag into endogenous loci. Finally, we use ZF1 tagging to establish the site of cdc-42 gene function during a cell invasion event. ZF1 tagging provides a powerful new tool for the analysis of dynamic developmental events.

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Small-molecule displacement of a cryptic degron causes conditional protein degradation

Cited by 181 publications

References 29 publications

A Chemical Genetics Approach for the Functional Assessment of Novel Cancer Genes

A Chemical Genetics Approach for the Functional Assessment of Novel Cancer Genes

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

Repurposing an endogenous degradation system for rapid and targeted depletion ofC. elegansproteins

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