Time-resolved protein activation by proximal decaging in living systems

Wang, Jie; Yuan, Lingyun; Liu, Yanjun; Zheng, Siqi; Wang, Xin; Zhao, Jingyi; Yang, Fan; Zhang, Gong; Wang, Chu; Chen, Peng R.

doi:10.1038/s41586-019-1188-1

Cited by 163 publications

(165 citation statements)

References 73 publications

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“…In order to abrogate any potential for substrate sequestration by a caged phosphatase, we next explored the possibility of optically controlling the phosphatase:substrate interface. While optical control of protein active sites through site-specific caging group installation has been used to study a wide range of enzymatic functions (e.g., kinase 3–6 , polymerase 30 , nuclease 31 , recombinase 32 , helicase 33 , and others) 34 , optical control of protein-protein interactions has remained underexplored. In this context of transitioning from optical control of enzymatic function to optical control of protein-protein interactions, caged amino acid mutagenesis represents an advantage over other optogenetic approaches, since the position of the caging group is simply defined by the position of the TAG codon and thus can be readily moved to other sites on the protein, e.g., from the active site to the protein surface.…”

Section: Resultsmentioning

confidence: 99%

“…Optical control of the kinase interaction motif will be useful for placing other phosphatase-MAPK interactions under optical control since electrostatic interactions are most often the main determinants of these high affinity interactions. More broadly, light-regulation of catalytic sites through introduction of caged amino acids has been demonstrated numerous times; however, the use of caged amino acids to control protein-protein interactions remains underdeveloped 6,42–44 and extension of this approach to other scaffolding proteins will further facilitate the optical dissection of cell-signaling networks.…”

Section: Resultsmentioning

confidence: 99%

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Optical control of protein phosphatase function

Courtney

Deiters

2019

Nat Commun

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Protein phosphatases are involved in embryonic development, metabolic homeostasis, stress response, cell cycle transitions, and many other essential biological mechanisms. Unlike kinases, protein phosphatases remain understudied and less characterized. Traditional genetic and biochemical methods have contributed significantly to our understanding; however, these methodologies lack precise and acute spatiotemporal control. Here, we report the development of a light-activated protein phosphatase, the dual specificity phosphatase 6 (DUSP6 or MKP3). Through genetic code expansion, MKP3 is placed under optical control via two different approaches: (i) incorporation of a caged cysteine into the active site for controlling catalytic activity and (ii) incorporation of a caged lysine into the kinase interaction motif for controlling the protein-protein interaction between the phosphatase and its substrate. Both strategies are expected to be applicable to the engineering of a wide range of light-activated phosphatases. Applying the optogenetically controlled MKP3 in conjunction with live cell reporters, we discover that ERK nuclear translocation is regulated in a graded manner in response to increasing MKP3 activity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Optical control of protein phosphatase function

Courtney

Deiters

2019

Nat Commun

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“…[13] Compared with the diverse bioorthogonal ligations,t he researches on the bioorthogonal cleavages are limited. Them ost representative examples include light-induced bond cleavage, [14] transition-metal-catalyzed deallylation or depropargylation, [15] the specific IED-DA-induced click-and-release reaction, [16] hydrazinolysis, [17] reduction of diazo [18] and disulfide compounds, [19] and acidinduced hydrolysis of silyl esters. [20] Unfortunately,t he conditions for some of bioorthogonal cleavages are harsh, which may affect the activity of biomolecules to some extent.…”

Section: Introductionmentioning

confidence: 99%

Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho‐Dithiophenols

Liu

Yang

et al. 2020

Angewandte Chemie

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A bioorthogonal ligation and cleavage method via reactions of chloroquinoxalines (CQ) and ortho‐dithiophenols (DT) is presented. Double nucleophilic substitutions of ortho‐dithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo[5,6][1,4]dithiino[2,3‐b]quinoxaline with strong built‐in fluorescence together with release of the other functional molecules. Three cleavable linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates. The CQ‐DT bioorthogonal reactions can be applied for the bioorthogonal ligations, bioorthogonal cleavages, and trans‐tagging of proteins, and show advantages of readily accessible unnatural orthogonal groups, appealing reaction kinetics (k2≈1.3 m−1 s−1), excellent biocompatibility of orthogonal groups, and high stability of conjugates. This complements previous bioorthogonal reactions and is a new route for protein‐fishing applications and in‐gel fluorescence analysis.

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“…However, UAA mutagenesis requires the use of nonendogenous protein translation machinery and can be inefficient and slow to produce the biosensor. 23,24 In the past decade, there have been significant advances in using "self-labeling" enzyme fragments, including SNAP-tag and Halo-tag, to attach dyes to proteins in living cells. These altered enzyme fragments covalently incorporate substrates bearing dyes.…”

Section: Introductionmentioning

confidence: 99%

Using SNAP-tag for facile construction of dye-based biosensors in living cells

Pinkin

Liu

Pimenta

et al. 2020

Preprint

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Fluorescent biosensors based on environment-sensitive dyes have important advantages over alternative methodologies such as FRET, including the potential for enhanced brightness, elimination of bleaching artifacts, and more possibilities for multiplexing. However, such biosensors have been difficult to use because they required proteins to be covalently labeled and reintroduced into cells. Recent development of self-labeling enzymes that covalently react with membrane-permeable dyes (e.g. SNAP-tag) provide an opportunity to easily generate dye-based biosensors within cells. Here, we generate a new biosensor for Cdc42 activation by positioning SNAP-tag between Cdc42 and a peptide that binds selectively to active Cdc42. We generate a membrane-permeable Nile Red derivative that exhibits 50-fold fluorescence enhancement upon covalent labeling of the biosensor, then optimize the biosensor so the dye undergoes a 20 nm emission shift upon Cdc42 activation, enabling ratiometric imaging with a single dye. The biosensor, named SNAPsense Cdc42, is validated by examining its response to known regulatory proteins and studying Cdc42 activation during protrusion in living cells. Variants using other dyes are also presented.

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Time-resolved protein activation by proximal decaging in living systems

Cited by 163 publications

References 73 publications

Optical control of protein phosphatase function

Optical control of protein phosphatase function

Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho‐Dithiophenols

Using SNAP-tag for facile construction of dye-based biosensors in living cells

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