Advances in targeted degradation of endogenous proteins

Röth, Sascha; Fulcher, Luke J.; Sapkota, Gopal P.

doi:10.1007/s00018-019-03112-6

Cited by 83 publications

(74 citation statements)

References 166 publications

(252 reference statements)

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“…Increasingly, targeted protein degradation is being used as an investigative tool in cell biology, with hopes to translate into a therapeutic option for a variety of diseases (28). The efficacy of inducible protein degradation is influenced by a number of factors including protein synthesis rate, binding affinity of protein of interest to E3 ligase complexes, efficiency of ubiquitylation and presence of deubiquitinases (29).…”

Section: Discussionmentioning

confidence: 99%

IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

Sapkota

Dunbar

Macartney

2020

Preprint

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Immunomodulatory imide drugs (IMiDs) bind CRBN, a substrate receptor of the Cul4A E3 ligase complex, enabling neo-substrate recruitment and degradation via the ubiquitin-proteasome system. Here, we report FAM83F as such a neo-substrate. We recently showed that the eight FAM83 proteins (A-H) interact with members of the serine/threonine protein kinase CK1 family, to regulate their subcellular distribution and distinct biological roles. CK1α is a well-established IMiD neo-substrate and we demonstrate here that IMiD-induced FAM83F degradation requires its association with CK1α. Despite all FAM83 proteins interacting with CK1α, no other FAM83 protein is degraded by IMiDs. FAM83F is localised to the plasma membrane, and consistent with this, IMiD treatment results in depletion of both FAM83F and CK1α levels from the plasma membrane. We have recently identified FAM83F as a mediator of the canonical Wnt signalling pathway. The IMiD-induced degradation of FAM83F attenuated Wnt signalling in colorectal cancer cells and removed CK1α from the plasma membrane, mirroring the phenotypes observed with genetic ablation of FAM83F. Intriguingly, in many cancer cell lines, IMiD-induced degradation of CK1α is only modest and incomplete. In line with this observation, the expression of FAM83G, which also binds to CK1α, appears to attenuate the IMiD-induced degradation of CK1α, suggesting a protective role for FAM83G on CK1α. Our findings reveal that the efficiency of target protein degradation by IMiDs, and perhaps other degraders such as PROTACs, relies on the nature of the inherent multiprotein complex in which the target protein exists. Our findings unearth opportunities for developing degraders to target specific protein complexes.

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Section: Discussionmentioning

confidence: 99%

IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

Sapkota

Dunbar

Macartney

2020

Preprint

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“…Targeting at the protein level offers the advantages of targeting protein conformations, post-translational modifications, splice variants, different functional epitopes, targeting individual cellular compartments and interference independently of protein half-life (see details in Table 1). In contrast to knocking out a gene, interference at the protein level furthermore allows tuning the amounts of a target protein instead of allowing only the presence or complete absence of the protein [5,6]. The rapid effects achievable by protein delivery can be used to interfere in a stage-specific way and when time is a relevant factor.…”

Section: Why Target Proteins Directly?mentioning

confidence: 99%

“…The rapid effects achievable by protein delivery can be used to interfere in a stage-specific way and when time is a relevant factor. This is also relevant if the time required to obtain a single clone with a gene knockout is long enough to allow genetic compensation for the modification introduced [6,7]. Delivering proteins directly circumvents stable DNA integration into the genome.…”

Section: Why Target Proteins Directly?mentioning

confidence: 99%

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

et al. 2020

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To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy. Proteins can be targeted directly and very specifically by antibodies, but using antibodies inside cells has so far been considered to be challenging. However, it is possible to deliver antibodies or other proteins into the cytosol using standard laboratory equipment. Physical methods such as electroporation have been demonstrated to be efficient and validated thoroughly over time. The expression of intracellular antibodies (intrabodies) inside cells is another way to interfere with intracellular targets at the protein level. Methodological strategies to target the inside of cells with antibodies, including delivered antibodies and expressed antibodies, as well as applications in the research areas of neurobiology, viral infections and oncology, are reviewed here. Antibodies have already been used to interfere with a wide range of intracellular targets. Disease-related targets included proteins associated with neurodegenerative diseases such as Parkinson's disease (α-synuclein), Alzheimer's disease (amyloid-β) or Huntington's disease (mutant huntingtin [mHtt]). The applications of intrabodies in the context of viral infections include targeting proteins associated with HIV (e.g. HIV1-TAT, Rev, Vif, gp41, gp120, gp160) and different oncoviruses such as human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV) and Epstein-Barr virus, and they have been used to interfere with various targets related to different processes in cancer, including oncogenic pathways, proliferation, cell cycle, apoptosis, metastasis, angiogenesis or neo-antigens (e.g. p53, human epidermal growth factor receptor-2 [HER2], signal transducer and activator of transcription 3 [STAT3], RAS-related RHO-GTPase B (RHOB), cortactin, vascular endothelial growth factor receptor 2 [VEGFR2], Ras, Bcr-Abl). Interfering at the protein level allows questions to be addressed that may remain unanswered using alternative methods. This review addresses why direct targeting of proteins allows unique insights, what is currently feasible in vitro, and how this relates to potential therapeutic applications.Therapeutic antibodies are valuable drugs, which mostly act outside of cells.Reaching the numerous drug targets that reside inside cells by antibodies is possible in vitro and allows unique insights compared with other methods.Applying antibodies inside cells for therapeutic purposes has been explored in animal models and promises specific therapeutic benefits in neurobiology, virology and oncology.

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“…Given the recent advances in CRISPR/Cas9-genome editing technology (Dickinson and 273 Goldstein 2016;Dokshin et al 2018), the auxin-inducible degron (AID) with or without a 274 fluorescent reporter (e.g., GFP or its derivatives) can be inserted into a genomic locus of 275 interest (Röth et al 2019). Though this technology can be applied with ease, there are 276 certain limitations that exist with the use of the natural auxin indole-3-acetic acid (IAA), 277 including its limited solubility in water.…”

Section: Naa Is a Synthetic Alternative To The Natural Auxin Iaa 272mentioning

confidence: 99%

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

Martinez

Kinney

Medwig-Kinney

et al. 2019

Preprint

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As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system, allows for spatial and temporal control of protein degradation, functioning through the activity of a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID method, utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 minutes of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 interacts with C. elegans SKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the Ftz-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

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Advances in targeted degradation of endogenous proteins

Cited by 83 publications

References 166 publications

IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

Rapid degradation ofC. elegansproteins at single-cell resolution with a synthetic auxin

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