Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

doi:10.3389/fpls.2018.00130

Cited by 36 publications

(30 citation statements)

References 34 publications

(42 reference statements)

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“…Cid protein (CENH3 in Drosophila) was depleted during spermatogenesis using the deGradFP system (Caussinus et al 2011 ) based on the proteasome-mediated targeted degradation of the protein of interest. This principle may be applied in crops too, as targeted protein degradation by the 26S-proteasome was successfully demonstrated in Nicotiana tabacum (Baudisch et al 2018 ). The identification of genes other than CENH3 which could be used to trigger the formation of haploids is challenging, as a large genetic screen for haploidy inducers in A. thaliana has failed to identify any suitable mutants (Portemer et al 2015 ).…”

Section: Generation Of Haploids Using Targeted Centromere Manipulatiomentioning

confidence: 99%

State-of-the-art and novel developments of in vivo haploid technologies

et al. 2018

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The ability to generate (doubled) haploid plants significantly accelerates the crop breeding process. Haploids have been induced mainly through the generation of plants from cultivated gametophic (haploid) cells and tissues, i.e., in vitro haploid technologies, or through the selective loss of a parental chromosome set upon inter- or intraspecific hybridization. Here, we focus our review on the mechanisms responsible for the in vivo formation of haploids in the context of inter- and intraspecific hybridization. The application of a modified CENH3 for uniparental genome elimination, the IG1 system used for paternal as well as the BBM-like and the patatin-like phospholipase essential for maternal haploidy induction are discussed in detail.

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Section: Generation Of Haploids Using Targeted Centromere Manipulatiomentioning

confidence: 99%

State-of-the-art and novel developments of in vivo haploid technologies

et al. 2018

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“…Together, this work greatly expands the Trim-Away technology toolbox beyond the use of standard antibodies and allows more flexibility when designing protein depletion experiments utilising TRIM21. In addition to Trim-Away there are a growing number of methods available for acutely controlling endogenous protein levels (Baudisch et al, 2018;Caussinus et al, 2012;Clift et al, 2017;Clift et al, 2018;Deng et al, 2020;Fulcher et al, 2016;Gross et al, 2016;Ibrahim et al, 2020;Ju Shin et al, 2015;Lim et al, 2020;Ludwicki et al, 2019;Marschall et al, 2014;Portnoff et al, 2014;Traub, 2019;Yamaguchi et al, 2019). However, our work here on TRIM21 provides the first mechanistic understanding of how an E3 ligase can be re-directed to degrade diverse non-canonical substrates.…”

Section: Discussionmentioning

confidence: 99%

Substrate-induced clustering activates Trim-Away of pathogens and proteins

Zeng

Santos

Mukadam

et al. 2020

Preprint

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SUMMARYTrim-Away is a powerful new technology that exploits off-the-shelf antibodies and the E3 RING ligase and cytosolic antibody receptor TRIM21 to carry out rapid protein depletion. How TRIM21 is catalytically-activated upon substrate engagement during either its normal immune function or when re-purposed for targeted protein degradation is unknown. Here we show that a mechanism of substrate-induced clustering triggers intermolecular dimerization of the RING domain to switch on the ubiquitination activity of TRIM21 and induce an antiviral response or drive Trim-Away. We harness this mechanism to expand the Trim-Away toolbox with highly-active TRIM21-nanobody chimeras that can also be controlled optogenetically. This work provides a mechanism for cellular activation of TRIM RING ligases and has important implications for targeted protein degradation technologies.

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“…Heterologous expression of ZIF-1 in worms or zebrafish can also degrade ZF1-tagged proteins and could be adapted to more systems 15,38 . Fusing an anti-GFP nanobody to endogenous ubiquitin ligase adaptors like ZIF-1 enables spatial control of degradation of GFP-tagged proteins in C. elegans, Drosophila, plants, and zebrafish [38][39][40][41] , which allows existing GFP-tagged reporters to be used for degron protection assays. Heterologous expression of the auxininducible degron (AID) and TIR1 ligase adaptor is also used in various animal models 18,33,42 .…”

Section: Discussionmentioning

confidence: 99%

Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures

Beer

Fazeli

Irmisch

et al. 2018

Preprint

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Visualization of specific organelles in tissues over background fluorescence can be challenging, especially when reporters localize to multiple structures. Instead of trying to identify proteins enriched in specific membrane-wrapped structures, we used a selective degradation approach to remove reporters from the cytoplasm or nucleus of C. elegans embryos and mammalian cells. We demonstrate specific labelling of organelles using degron-tagged reporters, including extracellular vesicles, as well as individual neighbouring membranes.These degron-tagged reporters facilitate long-term tracking of released cell debris and cell corpses, even during uptake and phagolysosomal degradation. We further show that degron protection assays can probe the topology of the nuclear envelope and plasma membrane during cell division, giving insight into protein and organelle dynamics. As endogenous and heterologous degrons are used in bacteria, yeast, plants, and animals, degron approaches can enable the specific labelling and tracking of proteins, vesicles, organelles, cell fragments, and cells in many model systems.

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Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

Cited by 36 publications

References 34 publications

State-of-the-art and novel developments of in vivo haploid technologies

State-of-the-art and novel developments of in vivo haploid technologies

Substrate-induced clustering activates Trim-Away of pathogens and proteins

Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures

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