Tunable light and drug induced depletion of target proteins

Deng, Wen; Bates, Jack A.; Wei, Hai; Bartoschek, Michael D.; Conradt, Barbara; Leonhardt, Heinrich

doi:10.1038/s41467-019-14160-8

Cited by 36 publications

(29 citation statements)

References 62 publications

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“…Another possibility is the addition of ‘degrons' to the small tag binders ( Natsume and Kanemaki, 2017 ), to achieve specific and temporally controlled degradation of the tagged POIs. This approach has been successfully applied using the anti-FP nanobodies ( Aguilar et al, 2019a ; Beghein and Gettemans, 2017 ; Deng et al, 2020 ; Ingram et al, 2018 ; Prole and Taylor, 2019 ). Here, we expand these findings by demonstrating that a single short HA tag can be used to inactivate POIs using deGradHA, a tool designed to channel HA-tagged POIs to ubiquitin/proteasome dependent degradation.…”

Section: Discussionmentioning

confidence: 99%

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

et al. 2021

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Cellular development and function rely on highly dynamic molecular interactions among proteins distributed in all cell compartments. Analysis of these interactions has been one of the main topics in cellular and developmental research and has been mostly achieved by the manipulation of proteins of interest (POIs) at the genetic level. Although genetic strategies significantly contributed to our current understanding, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would profit from more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, along with advancement in synthetic biology, have contributed to the creation of a new toolbox for direct protein manipulations. Here, we selected a number of short tag epitopes for which protein binders from different scaffolds have been generated and showed that single copies of these tags allowed efficient POIs binding and manipulation in living cells. Using Drosophila, we also find that single short tags can be utilized for POI manipulation in vivo.

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Section: Discussionmentioning

confidence: 99%

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

et al. 2021

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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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“…Another functionalization that can be applied to the small tag binders is the addition of “degrons” (Natsume and Kanemaki, 2017) to achieve specific and temporally-controlled degradation of the tagged POIs. This approach has been successfully applied using the anti-FP nanobodies (Aguilar et al, 2019a; Beghein and Gettemans, 2017; Deng et al, 2020; Ingram et al, 2018; Prole and Taylor, 2019). Here we expand these findings by demonstrating that a single short HA tag can be efficient applied to inactivate POIs using deGradHA, a tool adapted from the deGradFP system and designed to channel HA-tagged POIs to ubiquitin/proteasome dependent degradation.…”

Section: Discussionmentioning

confidence: 99%

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

Viganò

Ell

Kustermann

et al. 2020

Preprint

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Cellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years.Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time-and spacecontrolled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

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Tunable light and drug induced depletion of target proteins

Cited by 36 publications

References 62 publications

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

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

Protein manipulation using single copies of short peptide tags in cultured cells and inDrosophila melanogaster

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