Letters pubs.acs.org/acschemicalbiology
Light-Activated Proteolysis for the Spatiotemporal Control of Proteins Quentin Delacour,†,‡,§ Chenge Li,†,‡,§ Marie-Aude Plamont,†,‡,§ Emmanuelle Billon-Denis,†,‡,§ Isabelle Aujard,†,‡,§ Thomas Le Saux,†,‡,§ Ludovic Jullien,†,‡,§ and Arnaud Gautier*,†,‡,§ Department of Chemistry, École Normale Supérieure−PSL Research University, 24 rue Lhomond, F-75005 Paris, France Sorbonne Universités, UPMC Univ Paris 06, UMR 8640 PASTEUR, F-75005 Paris, France § CNRS, UMR 8640 PASTEUR, F-75005 Paris, France † ‡
S Supporting Information *
ABSTRACT: The regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation system obtained by transposing components of the plant auxindependent degradation pathway in mammalian cells. We developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control.
C
on photocontrolling the interaction between the protein of interest and the ubiquitin−proteasome system. To have a high and general control on the degradation process, we placed under light control the ubiquitination step and, more particularly, the recognition of the substrate by ubiquitin ligase. Our approach is based on the auxin-inducible degradation system, which exploits components of the plant auxindependent degradation pathway to create conditional degradation pathways in nonplant eukaryotic cells.11 Auxin binds to the F-box transport inhibitor response 1 (TIR1) protein and promotes the interaction between the E3 ubiquitin ligase SCF(Skp1/Cul1/F-box protein)-TIR1 and the AUX/IAA transcription repressors via their auxin-inducible degron (AID) sequence.12,13 As the SCF system is well conserved through eukaryotic species, heterologous expression of TIR1 in eukaryotic cells leads to the formation of a functional auxindependent artificial E3 ubiquitin ligase complex SCF-TIR1, which promotes the fast degradation of proteins fused to AID in the presence of auxin. In this Letter, we present the development of photoactivatable auxins that can act as lightactivatable inducers of degradation when combined with the auxin-inducible degradation system (Figure 1a). We show in particular that rapid and local photorelease of auxin can promote ubiquitin-dependent degradation of AID-tagged proteins in mammalian cells in a temporally and spatially controlled manner.
ontrolling the activity of proteins with light offers new opportunities to study biological processes with high spatiotemporal resolution.1,2 The gain in resolution compared to traditional genetic and pharmacological control methods results from the light illumination itself whose intensity can be tuned with submicrometric and submillisecond resolutions. To regulate the activity of a protein of interest with light, various strategies can be envisioned. First, one can rely on lightactivatable modules attached to the protein:3−6 activation of these modules with light induces a change of the protein activity and therefore a biological response. A second strategy consists in manipulating the cellular concentration of the protein by, for instance, the control of its synthesis using lightactivatable transcription factors.7,8 Compared to the first strategy, the photocontrol of transcription is more general as it enables to control a priori any proteins. However, the timing between the photoactivation and the effect depends thus on several pretranslational mechanisms and can therefore exceed hours, which decreases the time resolution of the approach. Conversely, one can also envision regulating protein levels via proteolysis, the reverse mechanism of protein synthesis. Lightinduced removal of a specific protein can inhibit (if the target is an activator) or activate (if the target is an inhibitor) specific biological functions with good spatiotemporal control. Recently, light-activatable degrons were developed by coupling peptide sequences known to promote fast degradation with light-sensitive LOV domains: upon light activation the degradation sequence is exposed, enabling its interaction with the cellular degradation machinery and promoting hence the degradation of the target protein.9,10 In this Letter, we present a general strategy to promote the degradation of a protein of interest with light. We directly relied © XXXX American Chemical Society
Received: January 30, 2015 Accepted: May 4, 2015
A
DOI: 10.1021/acschembio.5b00069 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 1. Light-activated ubiquitin-dependent proteolysis. (a) An auxin (in red) protected with a light-removable group (blue circle) acts as photoactivatable inducer of degradation. Upon light illumination, auxin is released, enabling the interaction between the protein of interest fused to the auxin-inducible degron (AID) and an artificial ubiquitin ligase that contains the F-box protein TIR1 as recognition module. The resulting polyubiquitinated protein is targeted to the proteasome, which promotes its degradation. (b) PA-IAA photolysis reaction. (c) Photolysis of PA-IAA. Solutions of PA-IAA (400 μL at 21 μM) were illuminated at 350 nm (integral photon rate 10 nmol·s−1, path length of the light beam 1 cm). Plots show the concentrations of PA-IAA (circle) and IAA (square) in function of time. Least squares fit gave the uncaging quantum yield ϕ. Temperature, 20 °C; solvent, acetonitrile/20 mM Tris buffer (pH 7.5) 1:1. (d) HEK293 cells coexpressing EGFP-AID-NLS and OsTIR1D170E-NLS were incubated with (+) or without (−) 50 μM PA-IAA for 20 min in darkness, then illuminated (+) or not (−) at 365 nm with a photon flux of 100 μmol·s−1·m−2 for 8 min, and finally incubated in the dark for 1 h (lanes 2, 4, and 5). Cells were lysed, and lysates were analyzed by immunoblotting (IB) with the indicated antibodies. Control experiments in which cells coexpressing EGFP-AID-NLS and OsTIR1D170E-NLS were untreated (lane 1) or treated for 1 h with 500 μM IAA (lane 3) are shown. (e) Western blot quantification. Plots represent the EGFP-AID-NLS level normalized by the OsTIR1D170E-NLS level. Data represent the mean ± SD of three independent experiments. The statistical significance was evaluated by a one-way analysis of variance (***p value < 0.001, ****p value < 0.0001).
a photon flux I0 = 100 μmol·s−1·m−2 (respectively, 100 mmol· s−1·m−2). However, PA-IAA2 was not quantitatively photolyzed: only 30% IAA was released upon light illumination at 390 nm (Supplementary Figure 4). This partial release results very likely from the competitive formation of an unreactive side-product through a rearrangement reaction. As such sidereaction could prevent controlling reliably the photorelease of IAA in cells, we performed the rest of our study only with PAIAA, which underwent quantitative photolysis. Before evaluating the ability of PA-IAA to promote protein depletion upon light illumination, we first optimized the auxininducible degradation system to obtain faster protein degradation. Supplementary Text 1 and Supplementary Figures 5 and 6 present the optimization. In brief, we showed that the degradation is optimal when OsTIR1 is properly colocalized with the AID-tagged protein. Moreover, by using OsTIR1D170E variant, we were able to reduce the half-life of AID-tagged protein to 15 min, ensuring 90% of protein depletion within 45 min. With this optimized degradation system, we next tested our ability to promote protein depletion using light. Human embryonic kidney (HEK) 293 cells coexpressing EGFP fused to AID and to a nuclear localization signal (EGFP-AID-NLS) and OsTIR1D170E fused to a nuclear localization signal (OsTIR1D170E-NLS) were exposed to 50 μM PA-IAA for 20 min in darkness, then illuminated 8 min at 365 nm with a hand lamp delivering a photon flux of 100 μmol·s−1·m−2 and finally left in the dark for 1 h at 37 °C. A control experiment without illumination at 365 nm was performed for evaluating the stability of PA-IAA in darkness: in that case, no significant depletion of AID-EGFP-NLS was detected by immunoblotting
Results. The indole-3-acetic acid (IAA), the natural auxin, binds to the binding pocket of TIR1 via both its side-chain carboxyl group and its indole ring.14 To obtain temporally inactive IAA poised for activation by light, the side-chain carboxyl group was previously caged as a 2-nitrobenzyl ester bearing an additional bulky dimethoxyphenyl group at the benzylic position to hinder the ester function and prevent hydrolysis by plant esterases.15 As the 2-nitrobenzyl is only efficiently cleaved at 280−300 nm, a wavelength range toxic for mammalian cells, we sought for caging groups cleavable at higher and safer wavelengths. We developed bulky versions of the 4,5-dimethoxy-2-nitrobenzyl (DMNB) and the [(7(diethylamino)-coumarin-4-yl]-methyl (DEACM), known to be efficiently cleaved upon illumination within the 350−400 nm range,16,17 by incorporating an extra 2,5-dimethoxyphenyl group on the connecting carbon atom (Supplementary Figure 1). These bulky DMNB and DEACM groups enabled the synthesis of two photoactivatable IAA, dubbed, respectively, PA-IAA and PA-IAA2 (Supplementary Figure 1), displaying the expected absorption properties (Supplementary Figure 2). The stability of PA-IAA and PA-IAA2 in aqueous media in darkness was verified by high-performance liquid chromatography (HPLC) titration (Supplementary Figure 3). We characterized the uncaging of PA-IAA and PA-IAA2 by following the extent of IAA release upon light illumination by HPLC. PA-IAA was quantitatively photocleaved at 350 nm with a resulting uncaging quantum yield ϕ = 0.012 (Figure 1b,c), in accordance with previous reports on similar light-removable protecting groups.16 With an uncaging cross-section εϕ = 6 m2· mol−1, photorelease would occur with half-time (t1/2 = ln 2/ (2.3εϕI0)) of 500 s (respectively, 0.5 s) upon illumination with B
DOI: 10.1021/acschembio.5b00069 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 2. Light-activated protein degradation. (a,b) HEK293 cells coexpressing EGFP-AID-NLS and OsTIR1D170E-NLS were incubated with 50 μM PA-IAA for 20 min. From t = 0, cells were illuminated for 1 s at 365 nm (photon flux at the specimen plan ∼100 mmol·s−1·m−2) every 5 min for 1 h (+ PA-IAA + light). Also shown are control experiments where (i) cells were additionally pretreated for 1 h with 50 μM MG132 (+ PA-IAA + light + MG132), (ii) PA-IAA was omitted (− PA-IAA +light), and (iii) 365 nm illumination was omitted (+ PA-IAA − light) (a) Confocal micrographs of representative cells at various times. (b) Normalized nuclear fluorescence intensity in function of time (mean ± SD, n = 10−30 cells). (c,d) HEK293 cells coexpressing CyclinB1-AID-YFP and OsTIR1 were incubated with 20 μM PA-IAA ± 50 μM MG132. From t = 0, cells were illuminated for 1 s at 365 nm (photon flux at the specimen plan ∼100 mmol·s−1·m−2) every 5 min for 1 h. (c) Confocal micrographs of representative cells at various times. (d) Normalized cytoplasmic fluorescence intensity in function of time (mean ± SD, n = 10−15 cells). Scale bars = 10 μm.
Figure 3. Light-activated protein degradation in single cells. HEK293 cells coexpressing EGFP-AID-NLS and OsTIR1D170E-NLS were incubated with 50 μM PA-IAA for 20 min. From t = 0, single cells were illuminated repeatedly every 10 min with a 405 nm laser on a surface of 200 μm2. Eventually after 1 h, cells were illuminated globally at 365 nm with 1 s pulses every 5 min to induce full degradation. (a) Confocal micrographs of cells before illumination (t = 0 min), after local illumination at 405 nm (t = 60 min) and after global illumination at 365 nm (t = 90 min). The red square shows the region of interest (ROI) illuminated at 405 nm. A full time-lapse is shown in Figure S8. Scale bars = 10 μm. (b) Plots showing the normalized nuclear fluorescence of cells 1−4 in function of time. The arrows indicate the beginning of the local illumination and the beginning of the global illumination. (c) Plots showing the percentage of fluorescence loss in individual cells after the local illumination (t = 60 min) in function of their distance to the illuminated spot (red circles; n = 66 cells from 12 experiments). Data from control experiments where cells were illuminated and imaged in the same conditions but in absence of PA-IAA are shown (blue squares; n = 26 cells from six experiments).
protein level. As PA-IAA does not absorb beyond 425 nm (Supplementary Figure 3), EGFP could be excited at 488 nm without activating IAA release (Figure 2a,b). For photolyzing PA-IAA at 365 nm, we illuminated the cell dish on a surface of 0.1 mm2 using a 63× objective (NA 1.4) and a microscope metal-halide lamp equipped with a DAPI filter as light source. The photon flux at the specimen plane was ∼100 mmol·s−1· m−2, which enables, according to the uncaging cross-section of PA-IAA, complete photorelease of IAA within few seconds (vide supra). To maintain high IAA intracellular level and obtain efficient degradation, a protocol based on multiple short illumination pulses spaced apart by darkness periods was preferred rather than one single initial pulse. By flashing cells at
(Figure 1d,e). Conversely, cells illuminated at 365 nm showed significant protein depletion, at levels comparable to that observed when cells were treated with IAA (Figure 1d,e), demonstrating that it is possible to specifically photocontrol the cellular stability of proteins fused to AID by photoreleasing IAA. Supplementary Figure 7 shows additional experiments where different concentrations of PA-IAA and durations of illumination were successfully tested. Next, we characterized the kinetics of proteolysis upon photorelease of IAA. We quantified by time-lapse confocal microscopy the timing and extent of depletion of EGFP-AIDNLS coexpressed with OsTIR1D170E-NLS upon photoactivation of PA-IAA. We relied on EGFP fluorescence as reporter of C
DOI: 10.1021/acschembio.5b00069 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
the protein; this process is reversible: as soon as the blue light is off, PSD and B-LID rapidly switch back to their stable dark state. In this Letter, we present an alternative approach to control protein stability with light. We developed a caged auxin (PAIAA) that acts as a photoactivatable inducer of degradation when combined with the auxin-inducible degradation system, which has been shown previously to enable the conditional degradation of various proteins in several eukaryotic systems.11,19 Upon photolysis of PA-IAA with short violet light pulses, IAA is locally released, inducing the interaction between AID-tagged proteins and the artificial SCF-OsTIR1 ubiquitin ligase, which consequently promotes ubiquitination and degradation by the proteasome. We showed that this strategy enabled efficient and fast protein degradation in mammalian cells, with a half-time of ∼20 min comparable to that reported for the B-LID system in similar cellular systems.10 Note that, compared to the B-LID system and the PSD, which may be both limited by the requirement of long-term (∼1 h) blue illumination to keep the LOV domain in a lit active state and ensure complete degradation, the use of a small caged compound like PA-IAA has the advantage of enabling to activate proteolysis with very short (∼1 s) violet light pulses. Additionally, we showed that local photolysis of PA-IAA by short and localized light flashes enabled to control protein degradation at the single cell level, opening new opportunities to manipulate proteasome-regulated cell processes with spatial control in 3D cell cultures, in tissues or in multicellular organisms. Note that the ability of PSD and B-LID to promote degradation with single cell resolution, even though theoretically possible, still remains to be demonstrated: so far, spatially controlled degradation has only been shown macroscopically on yeast lawns using the PSD domain and macroscopic light patterns.9 To conclude, we propose in this study a new method to promote protein degradation with temporal and spatial control relying on the photoactivation of a small caged inducer of degradation. Our study suggests that controlling protein degradation with light might be an efficient strategy for controlling protein activity at the single cell level with temporal control.
365 nm for 1 s every 5 min, we observed significant depletion within 1 h (Figure 2a,b). We determined a protein half-life of 20 ± 3 min, in accordance with the degradation kinetics observed when inducing degradation using free IAA. In control cells pretreated with the proteasome inhibitor MG132, no degradation was observed upon photoactivation of PA-IAA (Figure 2a,b), showing that the observed EGFP loss upon IAA photorelease was indeed due to proteasome degradation and not to photobleaching or inactivation by reactive oxygen species. This confirmed additional control experiments, where no EGFP signal loss was observed in cells untreated with PAIAA but exposed to the same illumination and imaging conditions (Figure 2a,b). This set of experiments showed that we could induce rapid proteasome-dependent protein depletion of AID-tagged protein upon photorelease of IAA. To verify that our approach was general and could enable the local depletion of proteins both nuclear or cytoplasmic, we applied the same protocol as described above to phototrigger the degradation of Cyclin B1, the regulatory subunit of the cyclin-dependent kinase Cdk1 known to be cytosolic in interphase (or in senescent cells).18 We coexpressed in HEK293 cells Cyclin B1 fused to AID and the yellow fluorescent protein (YFP) (CyclinB1-AID-YFP)19 together with cytoplasmic OsTIR1 for optimal degradation. Photorelease of IAA led to a proteasome-dependent degradation of Cyclin B1-AID-YFP (Figure 2c,d). The kinetics of CyclinB1AID-YFP loss was characterized by a half-time of 18 ± 4 min, in good agreement with our kinetic study and previous report.19 Finally, we demonstrated that we could induce specific protein degradation at the single cell level using local photorelease of IAA. We used a 405 nm laser diode as light source to photolyze PA-IAA locally. PA-IAA still absorbs enough at this wavelength to be efficiently uncaged (see Supplementary Figure 3). We incubated cells coexpressing EGFP-AID-NLS and OsTIR1D170E-NLS with 50 μM PA-IAA. Very local IAA bursts were generated by flashing a region of interest (ROI) of 200 μm2 repeatedly every 10 min with the 405 nm laser (Figure 3a−c and Supplementary Figure 9). In these conditions, we observed specific protein degradation only in cells in contact with the uncaging ROI (
