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Synthetic Control of Protein Degradation during Cell Proliferation and Developmental Processes Jonathan Trauth, Johannes Scheffer, Sophia Hasenjäger, and Christof Taxis*
ACS Omega 2019.4:2766-2778. Downloaded from pubs.acs.org by 79.110.19.160 on 02/07/19. For personal use only.
Department of Biology/Genetics, Philipps-University Marburg, Karl-vom-Frisch-Str. 8, 35032 Marburg, Germany ABSTRACT: Synthetic tools for the control of protein function are valuable for biomedical research to characterize cellular functions of essential proteins or if a rapid switch in protein activity is necessary. The ability to tune protein activity precisely opens another level of investigations that is not available with gene deletion mutants. Control of protein stability is a versatile approach to influence the activity of a target protein by its cellular abundance. Diverse strategies have been developed to achieve efficient proteolysis using external inducers or differentiation-coupled signals. The latter is especially important for the inactivation of a protein during a developmental process. Recently, several approaches to achieve this have been engineered. In this article, we present current synthetic tools for regulation of protein stability that allow fine-tuning of protein abundance, their advantages and disadvantages with an emphasis on methods applicable in the context of cell differentiation and development. We give an outlook toward future developments and discuss main applications of these tools.
1. INTRODUCTION 1.1. Strategies To Achieve Temporal Control of Protein Function. The basic strategy to achieve synthetic control of protein activity is to regulate the abundance of a protein, the catalytic activity of an enzyme, the localization of a protein, or the interactions between proteins. Control of protein abundance has the advantage of generality over the other methods with a tradeoff in terms of switching time, which is the delay between adding an inducer and observing the actual change in the cell. The lack in speed in the case of targeted proteolysis is evoked by the necessity to generate or degrade the selected protein, whereas localization, protein− protein interaction, or direct interference with protein activity enables near instant switching times (Figure 1).1 A gene deletion is maybe the most radical solution toward control of protein abundance and is used in cases where the function of the target protein is not essential for cell viability. However, temporal control is not easily achievable in this case and a graded change of protein activity is not possible at all. During a developmental process, a deletion mutant of a protein with a function in the initial phase or at an early developmental stage does not permit investigation of protein functions during later stages. A well-established solution is regulated gene deletion with the Cre-LoxP system during a differentiation process that relies on the expression of the Cre recombinase in selected tissues at a specific time point.2 A modern variation is to use a regulated CRISPR/Cas9 tool to remove the target gene at a specific time point.3,4 Alternatives are synthetic control of gene expression, e.g., by a tetracycline-responsive expression system or regulation of translation, for example by using an aptamer; in both cases the abundance of a protein of interest is controlled.5 These methodologies have the advantage that the target protein itself is not modified and many different © 2019 American Chemical Society
Figure 1. Strategies for synthetic control of protein activity. Diverse approaches have been developed to achieve synthetic control of protein activity. Interference with protein biosynthesis targets steps like transcription, translation, or RNA stability. Protein activity can be directly controlled by inhibitors, regulation of localization, or interference of protein−protein interactions. Abundance of a protein can be regulated via targeted proteolysis. Approaches that directly target protein activity act in a very short time-period; methods relying on biosynthesis or protein degradation require a longer switching time.
applications exist for the most important model systems. However, in all of these cases, protein stability dictates the target-inactivation time. One solution to this problem is targeted proteolysis with a conditional, portable degradation sequence, termed degron. This minimal, transferable element is sufficient for recognition and degradation by a proteolytic apparatus. Thus, usage of a conditional degron results in fastinactivation kinetics of the selected target protein after administration of the appropriate inducer. Received: October 30, 2018 Accepted: January 22, 2019 Published: February 6, 2019 2766
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1.2. Regulated Proteolysis by the Ubiquitin-Proteasome System. An important part of protein degradation in eukaryotes is carried out by the ubiquitin-proteasome system (Figure 2). The 76 amino acid protein ubiquitin (Ub) is used
substrates and break Ub-chains up reverse protein ubiquitylation. The active protease is the proteasome, a multisubunit complex containing a proteolytic core, a ring of AAA-ATPases that unfolds the substrate, and a lid complex, which binds the polyubiquitin chain and harbors a deubiquitylating enzyme for Ub recycling. Substrate degradation is initiated by recognition of the polyubiquitylated substrate by the proteasome. An unfolded stretch of amino acids in the substrate is grabbed by the AAA-ATPases, and the amino acid chain is fed into the interior of the proteolytic core. ATP-hydrolysis fuels unfolding and transfer of the substrate into the proteolytic core by the AAA-ATPases. The active peptidases in the proteolytic core cleave the protein into small peptides, whereas ubiquitin is removed from the substrate by deubiquitylating enzymes. Certain degrons like the C-terminus of the murine ornithine decarboxylase (ODC) are directly recognized by the proteasome itself, presumably by the same subunits that recognize polyubiquitin chains. This leads to ubiquitinindependent proteasomal degradation of the substrate.6,7,10
Figure 2. Ubiquitin-proteasome system. Details are given in the text. Ub: ubiquitin; E1: ubiquitin-activating enzyme; E2: ubiquitinconjugating enzyme; E3: ubiquitin-protein ligase; and DUB: deubiquitylating enzyme. A Degron (degradation sequence) is a portable sequence that contains the minimal element in a protein that is sufficient for recognition and degradation by a proteolytic apparatus. In the case of proteasomal degradation, a specific degron might initiate polyubiquitylation by an E3 or might target the substrate directly to the proteasome.
2. CONDITIONAL CONTROL OF PROTEIN DEGRADATION 2.1. Targeted Degradation Controlled by Temperature Shift. The classical approach to regulate protein stability depends on the heat-inducible degron developed in the lab of Varshavsky.11,12 It is based on the generation of a degron at the N-terminus of a protein using the ubiquitinfusion technique. Co-translational removal of a Ub by a deubiquitylating enzyme leads to exposure of an arginine or another destabilizing amino acid like phenylalanine at the Nterminus of a temperature-sensitive dihydrofolate reductase (DHFR) mutant (Figure 3). At 37 °C, the E3 Ubr1, or homologous proteins in higher eukaryotes,13,14 recognizes the destabilizing amino acid, which leads to ubiquitylation and rapid proteasomal degradation of the DHFR as well as proteins fused to it. At lower temperature (23 °C), the fusion protein is stable. This heat-inducible degron has been used in budding yeast in numerous studies and for high-throughput generation of temperature-sensitive alleles of essential yeast proteins.15−17 Furthermore, the approach has been transferred to fission yeast, in cultured mouse cells as well as in complex multicellular organisms like fruit flies and plants after further degron modifications.18−22 Importantly, adaptations of the restrictive and permissive temperatures have been used to adjust the system to the needs of the used model organism.22−24 Even though temperature is a global signal, developmental specific phenotypes were successfully induced by selecting cell-type specific targets or used as a tool to regulate cell ablation via expression of the temperature degronmodified cytotoxic ribonuclease barnase.22,23,25 Nevertheless,
as a post-translational protein modification that induces, among other regulatory functions, proteasomal degradation. Ubiquitin-dependent proteolysis involves an enzymatic cascade that leads to ubiquitylation of the substrate protein. Ub is activated by ubiquitin-activating enzymes (E1) in an ATPrequiring step. During this step, a thioester bond is formed between the active cysteine of the E1 and the terminal glycine of Ub. Next, the activated Ub is transferred onto an ubiquitinconjugating enzyme (E2); again, Ub is bound to a cysteine with a thioester bond. An ubiquitin-protein ligase (E3) selects the substrate and catalyzes, together with the E2, the transfer of Ub onto the substrate. A minimal, transferable motif in a substrate that is recognized by the E3 and is sufficient to confer degradation is called degron.6−8 In most cases, degrons consist of two parts, a reversibly attached polyubiquitin tag and an unfolded region in the substrate.9 Usually, E3s catalyze the formation of an isopetide bond between the terminal glycine of Ub and the side chain of a substrate-lysine. Further Ub transfer onto a lysine of substratebound Ub leads to formation of ubiquitin chains that constitute the signal for proteasomal proteolysis. Deubiquitylating enzymes (DUBs) that cleave Ub moieties from
Figure 3. Heat-inducible degron. Exposure of an N-degron coupled with a temperature-sensitive dihydrofolate reductase (DHFRts) renders a modified substrate temperature sensitive with regard to protein stability. Abbreviations: R: arginine; F: Phenylalanine; K: lysine. 2767
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Figure 4. (A) Stability regulation of destabilizing domain (DD) and ligand-induced degron (LID) by shield-1. The small-molecule shield-1 stabilizes DD, whereas it destabilizes LID. (B) The auxin-induced degron (AID). Usage of AID requires the expression of TIR1, which binds to the conserved SCF (Skp1, Cullin and F-box) complex. The degron sequence (auxin-induced degron, AID) is bound by TIR1 in the presence of auxin or derivatives thereof. (C) The split ubiquitin for the rescue of function (SURF) system. The rapamycin-induced heterodimerization of FKBP and mutated FRB are used to reconstitute the N-terminal ubiquitin fragment (UbN) with the C-terminal fragment (UbC). The reconstituted Ub is removed by a deubiquitylating enzyme (DUB), resulting in the unmodified substrate. In the absence of rapamycin, the substrate is constitutively degraded due to the destabilizing FRB variant. (D) The small-molecule-assisted shutoff (SMASh) concept. The viral peptidase NS3 is fused to the substrate and modified with a degron. Self-removal of NS3 results in an unmodified substrate; inhibition of the peptidase activity by an inhibitor triggers degradation of the fusion protein.
control by auxin.37 The method requires the modification of the target protein with the auxin-inducible degron (AID) derived from an AUX/IAA protein and the heterologous expression of the adapter-protein TIR1 in the selected cell type (Figure 4). The method is used in nonplant cells, since TIR1 and AID as well as auxin are endogenous plant components. Upon administering auxin to the cells, it recruits the TIR1− SCF complex to the target via the AID tag, which triggers ubiquitylation and rapid degradation. It has been successfully used in human cell culture, T. gondii, P. falciparum, Plasmodium berghei, Drosophila melanogaster, and Caenorhabditis elegans.38−44 Similarly, a jasmonate-induced degradation system was established for mammalian cell cultures.45 Limiting factors in some cases may be that two genetic modifications are necessary for the implementation of the AID system, auxin is not completely stable in vivo, and auxin or auxin metabolic byproducts may be toxic.46 Nevertheless, it has been widely used in yeast and other eukaryotes, which argues for the robustness of the method and general applicability. The usage of the AID system during a developmental process targeting a specific cell line has been tested. As expected, expression of TIR1 during a developmental process led to cell-type specific degradation of a target protein in Drosophila embryos in response to auxin.47 Further developments of the original AID system have been implemented in diverse model organisms. Increased degrada-
temperature changes might clash with the optimal conditions for growth of cells or proceeding of a developmental process. 2.2. Protein Stability Regulated by Small Molecules. Many methods for conditional protein degradation utilize small molecules as inducers for conditional degrons. One method has been developed in the lab of Thomas Wandless.26 The rapamycin-analog shield-1 binds the so-called degron domain (DD), preventing its degradation by the proteasome (Figure 4A). Shield-1-dependent regulation of a target protein is achieved by modification of a target with DD at the Nterminus or the C-terminus of a protein.26 Further development of the technique resulted in the ligand-induced degradation (LID), in which shield-1 exposes a degron, which results in proteolysis of the target protein (Figure 4A).27 DD-based approaches and applications with modified degron domains have been undertaken in mammalian cell culture, transgenic mice, the plant Arabidopsis thaliana, as well as the human parasites Toxoplasma gondii and Plasmodium falciparum.28−36 Another small-molecule approach was inspired by the effect of auxin in plant cells. Auxin treatment results in recruitment of certain transcription factors to an SCF (Skp1, Cullin, and Fbox) complex, which contains the adapter-protein TIR1. The SCF polyubiquitylates these transcription factors, which results in proteasomal degradation. This regulatory mechanism was reconstituted in budding yeast to achieve protein-stability 2768
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Figure 5. (A) Proteolysis-targeting chimeric molecule (PROTAC) approach. A chimeric molecule with affinity to the substrate and an E3 targets the substrate for polyubiquitylation and degradation. (B) The substrate is modified with Halotag7 (HaloPROTAC) or an FKBP12 variant (dTag). A HaloPROTAC molecule and a dTag molecule is used to target the substrate to the E3 VHL and cereblon (CRBN), respectively. Polyubiquitylation by a Cullin-RING ligase E3 (CRL) induces proteasomal degradation in both cases.
tion has been observed with alternative AUX/IAA proteins, minimization of the AID tag, fusion of TIR1 to the SCF subunit Skp1 together with a nuclear targeting sequence, usage of alternative auxin receptors, replacement of the native target protein by a temperature-sensitive variant, and shutdown of target gene expression at the time of auxin addition.48−54 Current developments focus on quantitative characterization and efficient genome integration of the AID system.55−57 In an interesting application, the AID system has been used to regulate apoptosis in cell culture, which is somewhat similar to the usage of temperature degron-modified barnase as cell ablation tool in plants.23,58 Another class of conditional degrons was derived from ubiquitin-independent protein degradation. They originate from investigations of yeast or murine ornithine decarboxylase (ODC) degradation by the proteasome. Murine ODC carries a degron at the C-terminus that is activated upon antizyme binding to ODC; induction of protein degradation has been achieved in mammalian cell culture by fusing ODC to a target and manipulating antizyme levels.59,60 Similarly, yeast ODC carries a degron at the N-terminus that is activated by the expression of yeast antizyme upon addition of a polyamine, e.g., putrescine, spermidine, or spermine.61−65 However, usage of these conditional degrons requires the fusion of ODC to the target; ODC has a size of about 50 kDa, forms a dimer, and influences polyamine levels if the active enzyme is used. A reverse setup is used by the split ubiquitin for the rescue of function (SURF) system (Figure 4C). Here, the target protein is modified at the N-terminus with a mutated variant of the rapamycin binding protein FRB, which acts as degron constantly destabilizing the substrate, and a C-terminal fragment of ubiquitin (UbC). Additionally, a fusion protein consisting of a maltose-binding protein, an N-terminal fragment of ubiquitin (UbN), and the protein FKBP is expressed in cells. The small molecule rapamycin is used to heterodimerize FKBP and mutated FRB, which reconstitutes the two ubiquitin fragments and removal of reconstituted ubiquitin and the FRB degron from the target protein by deubiquitylating enzymes. Thus, the target protein is stabilized in the presence of rapamycin and destabilized in its absence.66
A variation of this system called TShld uses a mutated FKBP, acting as degron, fused with UbC to the target and additional expression of FRB-UbN. Again, addition of rapamycin leads to reconstitution of Ub, followed by removal of the degron from the target and resulting in rescue of the target from proteolysis.67 A similar strategy was pursued with the small-moleculeassisted shutoff (SMASh) concept, in which a degron containing the protease NS3 is constantly removed by autoproteolysis (Figure 4D). Inhibition of the NS3 protease induces proteasomal degradation due to the stable presentation of the degron sequence at the C-terminus of the target protein.68 The advantage of SURF, TShld, and SMASh is that the target protein stays unmodified at the permissive condition. However, the methods might be better suited to induce upregulation of a protein than downregulation, since the constant removal of the degron leaves the protein unmodified and the natural turnover of the target determines the time required for depletion. Thus, the accumulation of the target should be rather quick due to the constant biosynthesis and degradation of the target protein, whereas downregulation might be slow in the case of a stable target protein. 2.3. Proteolysis-Targeting Chimeric Molecules (PROTACs). The first proteolysis-targeting chimeric molecules (PROTACs) were developed almost 20 years ago. They are similar to the AID system, yet no modification of the target protein is necessary to induce degradation (Figure 5A). The PROTAC tethers the target molecule to an E3, which induces polyubiquitylation and proteasomal degradation of the target. Originally, the proteolysis-targeting chimeric molecule 1 (Protac-1) consisted of the IκBα peptide (IPP) GGGGGGDRHDS*GLDS*M (*phosphorylated serine) tethered to ovalicin, which binds the methionine aminopeptidase2. Thus, the chimera targeted the methionine aminopeptidase2 to the SCF complex. However, cellular uptake of Protac-1 seemed unlikely due to the presence of IPP.69 Subsequently, in vivo PROTACs were developed based on membranepermeable peptides or using heterofunctional small-molecules that bind to an E3 and a specific cellular target. These PROTACs consist of three parts: an E3-binding part, a linker, 2769
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Figure 6. (A) Nanobody-induced proteolysis. An SCF-bound nanobody that is directed against GFP targets GFP-modified proteins for degradation. (B) A specific antibody targets the substrate for polyubiquitylation by the E3 TRIM21 and subsequent proteasomal degradation.
and a target-recruiting moiety.70 The development of PROTACs with high target-specific activity needs considerable effort. Thus, PROTACs are mostly generated for medical applications to induce degradation of disease-causing proteins, e.g., in antitumor therapy. A higher promiscuity might be achieved by further development of in-cell click-formed PROTACs, which provides the opportunity to increase the number of targets.71 This approach circumvents low cell permeability of large PROTACs by in situ generation by intracellular formation of the bifunctional molecule by bioorthogonal click combination of two smaller precursors. Recent variations of the PROTACs methodology use the HaloTag derived from the bacterial dehalogenase and HaloPROTACs with affinity to this tag to recruit the E3complex and induce target protein degradation. In another implementation, proteins are modified with a FKBP12 variant and dTag molecules induce proteasomal degradation through binding to the E3 cereblon and the substrate (Figure 5B).72−74 The dTag system demonstrated Cas9-targeted degronintegration at a specific target gene, followed by conditional regulation of target protein abundance.74 Thus, approaches like high-throughput generation of conditional mutants that have been achieved in yeast15 are feasible in higher eukaryotes in the future as well. 2.4. Antibody-Based Protein Degradation. A dual-use degradation system was developed that targets GFP-fusion proteins with a single-chain antibody modified with a SCFbinding domain, resulting in recruitment of the E3 to the substrate and target protein degradation upon expression of the antibody fusion (Figure 6A).75 This technique takes advantage of the wide availability of GFP-modified proteins as well as the multiple capabilities of GFP as reporter of target protein abundance. The method has been used, sometimes with slight variations, in mammalian cells, plants, zebrafish, and C. elegans.75−78 Recently, a combination of the AID system with the GFP single-chain antibody technique was developed and applied in cell culture and zebrafish embryos to regulate abundance of GFP-tagged proteins by auxin.79 The AID tag was fused to the GFP-directed nanobody vhhGFP4. Expression of the fusion enables to degrade GFP-tagged proteins by addition of auxin to the cells. However, a limiting factor for broad application might be that three manipulations are necessary: expression of TIR1, AID-vhhGFP4, and GFPmodification of the target.
Moreover, the Trim-away system utilizing the E3 TRIM21 that binds the Fc domain of antibodies has been established in mammalian cells. Here, an antibody induces selective substrate ubiquitylation through the antibody-binding E3 TRIM21 and subsequent proteolysis by the proteasome (Figure 6B). However, TRIM21 needs to be expressed in some cell lines and target-gene specific antibodies have to be microinjected into cells,80 which might limit the application of this method to a certain extent, especially during later stages of a developmental process. 2.5. Regulation of Protein Stability by Light. Light as inducer has highly desirable characteristics in terms of quality, quantity, temporal application, and subcellular resolution. Optogenetic tools that rely on DNA-encoded, light-responsive actuators comprise tools for regulation of protein activity in single cells to applications in living animals.81−83 The main challenge for application of these tools during a developmental process or whole animals is the site-specific application of light. A completely different challenge is the usage of optogenetic constructs in plants, as they respond to different light conditions and require light for growth and development, which hampers the usage of optogenetic tools in plants. The optogenetic toolbox to control protein stability contains one type of construct in different flavors: a light receptor domain of the light oxygen voltage (LOV) family modified with a degradation sequence at the C-terminal domain (Figure 7). The LOV2 domains of plant phototropins used for these
Figure 7. Photosensitive degron (psd) module. Degron exposure through structural rearrangements of a photoreceptor induces protein degradation.
degradation tools bind Flavin mononucleotide (FMN) as cofactor. Blue light induces excitation of FMN, which results in formation of a chemical bond between FMN and a cysteine of the LOV2 domain. This triggers a structural rearrangement, which induces the unfolding of an α-helix at the C-terminus of the LOV2 domain. This structural rearrangement has been 2770
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Figure 8. (A) Tobacco etch virus (TEV) protease-induced protein instability (TIPI) system. Site-directed cleavage by the TEV protease (cleavage site ENLYFQ-F, the hyphen denotes the cleaved peptide bond) deprotects two degrons, which induces degradation of both parts of the processed protein. (B) Dual regulation of protein biosynthesis and protein stability compared to methods targeting biosynthesis or stability of a protein. Regulation of protein abundance by transcriptional or translational control (biosynthesis repression) as well as methods to create a conditional 2771
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Figure 8. continued knockout lead to target protein depletion. The depletion kinetics depends in these cases on the stability of the target protein. Protein destabilization induces a decrease in abundance until a new equilibrium between protein biosynthesis and protein degradation is reached. Usage of a conditional degron combined with regulation of protein biosynthesis removes the target protein in a short time span. (C) Substrate expression regulated by the tetracycline-regulatable promoter system coupled to the ubiquitin-fusion technique to expose an N-degron. The relatively weaker N-degrons leucine or isoleucine have been used as destabilizing amino acid (DAA). The N-degron degradation pathway (Figure 3) is used for the degradation of the substrate protein. (D) Protein destabilization with the phospho-degron. Activation of the mitogen-activated protein kinase Fus3 by extracellular α-factor leads to phosphorylation of the degron, which in turn leads to ubiquitylation by SCFCdc4 and proteasomal degradation of the substrate. (E) External inducer versus internal signal to downregulate a protein at a specific time point during a differentiation process. (I) Many differentiation processes are proceeding at an individual pace within a group of developing cells. Such behavior can not only be encountered in sporulating yeast cells but also during development in higher eukaryotes, e.g., muscle formation. (II) External signals that induce protein degradation target all cells, regardless of the specific cell cycle stage of the individual cell. (III) A cell cycle or developmental stage-specific signal induces degradation of the target protein only in cells at the selected stage. The target remains stable in cells that are in another stage.
maybe the cleanest way to characterize protein functions in a developing organism. Most methods that depend on external signals like heat, small chemicals, or light are not easily applicable in such a setting. Either one component of the degradation pathway is expressed solely in the cell line under investigation or the signal is delivered cell-type specific. The AID system combined with CRISPR/Cas9-based genome editing has been used at different stages of Drosophila development for conditional depletion of endogenous proteins.47 Another method utilizes the tobacco etch virus (TEV) protease. This TEV protease-induced protein instability (TIPI) system has been designed to induce degradation of proteins during a developmental process.92,93 Here, site-directed proteolysis is used to deprotect an N-degron and an ODCderived C-degron. This results in recognition of the N-degron by Ubr1 followed by polyubiquitylation, whereas the C-degron is recognized directly by the proteasome. Thus, degradation of the whole protein by the proteasome is induced (Figure 8A). The usage of two degrons provides flexibility; the degron can be placed at the N-terminus, the C-terminus, or in the middle of the protein. As the TEV protease has a rather low processivity, affinity domains derived from human spliceosome components were implemented in the degron tag and in the protease, which ensures high affinity toward the substrate.92 Although the original implementation used a rather large GFPTDegF tag at the N-terminus, it has been shown that the GFP can be efficiently replaced by a short spacer sequence (MSITSLYKKAGS).94 Cell-type specific expression of the TEV protease has been successfully used in yeast to investigate essential proteins for a role during meiosis and spore formation.95,96 To optimize downregulation of the protein of interest, expression of the target gene has been placed under control of a promoter that is inactive during sporulation, which resulted in the sporulationinduced protein depletion (sid).96 Such dual control of gene expression and protein stability results in faster target protein depletion and lower abundance of the target after establishment of the new equilibrium state (Figure 8B). A similar optimization strategy has also been used in connection with ubiquitin-fusion derived N-degrons and a tetracycline-regulatable promoter (Figure 8C) as well as with the AID system.53,97,98 Expression of the TEV protease in higher eukaryotes has been used to study protein function or the necessity for site-directed cleavage showing that in principle, the TIPI system is not restricted to yeast cells. Indeed, successful transfer for in vivo studies in plants with cell-type specific expression of the TEV protease has been achieved.99
used to uncage different degrons derived from murine or human ornithine decarboxylase, as is the case for the photosensitive degron (psd) module or the degron activated by shield-1 in the LID approach.84−86 The ornithine decarboxylase-derived degrons consist of 37 unstructured amino acids with a cysteine-alanine motif located 19 amino acids away from the C-terminus. These degrons are directly recognized by the proteasome; protein degradation is carried out independent of ubiquitin or ubiquitylation with the ODCderived degrons, e.g., the psd module, and ubiquitin-dependent in the case of the LID-degron.7,27 The photosensitive degron (psd) module was established in yeast, and a similar construct was used in mammalian cell culture.84,85 The B-LID domain has been used in mammalian cell culture and zebrafish as a proof of concept study.86 Characterization of protein function based on conditional degrons has been achieved in yeast and C. elegans with the psd module, showing the potential of lightinduced protein degradation in a single-cell organism and an animal.87,88 The investigations in yeast revealed reduced abundance of the target protein modified with the psd module in the permissive state when compared with the endogenous protein.88 Moreover, an optogenetic AID system has been engineered by creating a photoactivatable auxin molecule.89 A short pulse of ultraviolet light is used to uncage photoactivatable auxin, which enables spatiotemporal control of protein degradation. Light delivery in a developing embryo or a living animal is not trivial if cell-type specific inactivation of a target protein is required. However, first steps toward developmental optogenetics have been taken.90 The main advantages of the photosensitive degron and its relatives is the spatial control over illumination that can be achieved as well as the tunability of the signal that allows modulation of target protein abundance with high precision. Furthermore, inactivation of a target is possible at a specific time point or for a specified duration. These advantages will certainly promote optogenetic degradation tools in the future. Current optogenetic tools for protein degradation use blue-light-responsive photoreceptors. For applications in embryos or animals, it will be interesting to create degradation tools based on a phytochrome photoreceptor that are responsive to red or far red light. Red-light photons have lower energy, are less phototoxic to cells, and can penetrate deeper into tissues compared with photons of blue or UV light.91,83 2.6. Developmental Process/Cell Cycle Stage Generated Induction of Protein Instability. Cell lineagespecific targeting of substrates in the background of undisturbed abundance in the rest of the embryo/animal is 2772
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rapamycin
HCV protease inhibitor HyT13, HyT36, HaloPROTACs, dTAGs NSlmb-vhhGFP expression target protein-specific antibody blue-light
SURF, TShld
SMASh
2773
by cell-specific TEV protease expression
no
TEV protease expression
yeast α-factor addition
phospho-degron
difficult (cell-type specific illumination)
by cell-specific NSlmb-vhhGFP expression difficult (cell-type specific antibody injection)
no
no
TIPI system
psd and B-LID
Trim-away
deGradFP
HaloTag, dTAG
no by cell-specific TIR1 expression
shield-1 auxin
no
no
shield-1
destabilizing domain LID AID
no
cell-type specific induction
temperature increase
inducer
ts-degron and lt-degron
method
phospho-degron (6 kDa)
cODC1-TDegF (14 kDa)
LOV2-degron (20 kDa)
none
GFP (25 kDa)
HaloTag (34 kDa), FKBP12F36V (12 kDa)
3xFRB-degron-UbC (58 kDa), FKBP*-UbC (15 kDa) SMASh tag (34 kDa)
FKBP12-degron (13 kDa) AID/IAA7 (25 kDa), mAID (7 kDa)
15 min
15−45 min
45 min
half-life of target protein
about 60 min 45 min
DDFKBP (12 kDa)
degradation kinetics (t1/2 min)
