Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

Shin, Yeong Ju; Park, Seung Kyun; Jung, Yoo Jung; Kim, Ye Na; Kim, Ki Sung; Park, Ok Kyu; Kwon, Seung‐Hae; Jeon, Sung Ho; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Hwang, Byung Joon

doi:10.1038/srep14269

Cited by 99 publications

(132 citation statements)

References 20 publications

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“…As a specific and generalizable approach for rapid protein suppression, several genetic strategies for rendering proteins the targets of ligand-induced decay have been established in mammalian cells 169–171 , D. melanogaster 172 , zebrafish 173,174 and C. elegans 175,176 , which have been reviewed elsewhere 177 . One example of this approach, known as deGradFP, involves targeting GFP-tagged proteins with a GFP-specific nanobody fused to a domain that targets the protein for proteasome-mediated degradation.…”

Section: Lof Approaches Across Organismsmentioning

confidence: 99%

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

et al. 2016

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Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

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Section: Lof Approaches Across Organismsmentioning

confidence: 99%

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

et al. 2016

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“…Other scaffolds may also work, provided that they are small, fold efficiently in the reducing cytosolic environment, and exhibit high affinity (low µM to nM) and specificity towards the intended target (Sha, Salzman, Gupta, & Koide, 2017). There have been several reports describing successful fusions between DBPs and truncated E3 ubiquitin ligases (Caussinus et al, 2011; Portnoff et al, 2014; Shin et al, 2015). In our hands, the flexibility and solubility of human CHIP are advantageous properties in the context of uAbs; hence, these protocols will focus on the implementation of CHIP-based uAbs.…”

Section: Strategic Planningmentioning

confidence: 99%

Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion

Baltz

Stephens

DeLisa

2018

CP Chemical Biology

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A number of techniques now exist for decreasing the expression of cellular proteins without the need for genomic modification. One such technique involves engineered protein chimeras that combine the ubiquitination activity of E3 ubiquitin ligases with the binding affinity and substrate specificity of designer binding proteins (DBPs). These chimeras, called "ubiquibodies," are capable of selectively and controllably steering virtually any protein to the ubiquitin proteasome pathway (UPP) for degradation, making ubiquibodies a powerful addition to the protein knockout toolbox. A distinguishing feature of ubiquibodies is their modularity-simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Moreover, by employing DBPs that recognize particular protein states (e.g., active versus inactive conformation, mutant versus wild-type, post-translational modification), it becomes possible to deplete certain protein subpopulations while sparing others. This protocol outlines the steps necessary to design and functionally evaluate ubiquibodies for customizable silencing of cellular proteins. © 2018 by John Wiley & Sons, Inc.

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“…One approach traps target proteins from maturing out of the ER (B€oldicke, 2017). In the deGradFP system, GFP-tagged targets are instead recruited for proteosomal degradation, via E3-ubiquitin ligases fused to the GBP Nb (Caussinus et al, 2012; Shin et al, 2015). …”

Section: Introductionmentioning

confidence: 99%

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos

Schrankel

Gökirmak

Lee

et al. 2019

Echinoderms, Part B

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Single-domain antibodies, also known as nanobodies, are small antigen-binding fragments (~15 kDa) that are derived from heavy chain only antibodies present in camelids (VHH, from camels and llamas), and cartilaginous fishes (VNAR, from sharks). Nanobody V-like domains are useful alternatives to conventional antibodies due to their small size, and high solubility and stability across many applications. In addition, phage display, ribosome display, and mRNA/cDNA display methods can be used for the efficient generation and optimization of binders in vitro. The resulting nanobodies can be genetically encoded, tagged, and expressed in cells for in vivo localization and functional studies of target proteins. Collectively, these properties make nanobodies ideal for use within echinoderm embryos. This chapter describes the optimization and imaging of genetically encoded nanobodies in the sea urchin embryo. Examples of live-cell antigen tagging (LCAT) and the manipulation of green fluorescent protein (GFP) are shown. We discuss the potentially transformative applications of nanobody technology for probing membrane protein trafficking, cytoskeleton re-organization, receptor signaling events, and gene regulation during echinoderm development.

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Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

Cited by 99 publications

References 20 publications

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion

Generation, expression and utilization of single-domain antibodies for in vivo protein localization and manipulation in sea urchin embryos

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