Chemistry

A modular degron library for artificial circuits in mammalian cells

Choice and characterization of the degron library

First, we assembled a library of protein modifications that affect the in vivo half-life of proteins to which they’re fused (Fig. 1a). This library comprises a variety of Ub tags, as described beneath, along with a number of Ub-independent degradation tags. The covalent attachment of Ub to mobile proteins has been proven to mediate the proteasomal degradation of each short- and long-lived proteins, in addition to to take part within the removing of irregular and denatured proteins3. Two main pathways goal the proteins for degradation both by way of the N-end rule pathway or the Ub-fusion degradation (UFD) pathway (Supplementary Fig. 1a, b). The N-end rule implies that the N-terminal amino acid of a protein determines its metabolic in vivo half-life8,28,29. Our library comprises a set of 20 such Ub tags (UbR, UbP, UbW, UbH, UbI, UbK, UbQ, UbV, UbL, UbD, UbN, UbG, UbY, UbT, UbS, UbF, UbA, UbC, UbE, UbM) with an intact C-terminal isopeptidase web site (Supplementary Fig. 1a). Moreover, the library comprises one other set of ten Ub tags (3xUbVR, 3xUbVV, 2xUbVR, 2xUbVV, UbAR, UbVV, UbVR, UbAV, 2xUbAR, 2xUbAV) concentrating on proteins to the UFD pathway30 (Supplementary Fig. 1b). In these instances, the Gly76 residue of Ub is mutated and the C-terminal isopeptidase web site is now not acknowledged by deubiquitinating enzymes. Lastly, 4 extra tags have been included within the library: lacS, PEST, 2xPEST, and PESTmod. These degrons affect the half-lives of proteins both by way of the Ub-proteasome pathway, as described for the Escherichia coli Lac repressor-derived spacer30, or by way of a Ub-independent pathway10,31 (PEST, 2xPEST, and PESTmod) (Supplementary Fig. 1c).

Fig. 1Fig. 1

Design and set-up of the protein tag library. a Schematic of the constructs forming the library. The core of the constructs consists of a transcription issue (TF) fused to the photoconvertible protein Dendra2. The protein modifications (tag) affecting the degradation charge of the constructs are fused to TF-Dendra2 both on the N- or on the C- terminus. b To characterize the degron library, every degron was used to tag a tetracycline-dependent transactivator (tTA), which binds to its cognate Tet response component (TRE) situated on the 5′- finish of a minimal promoter positioned in entrance of the human secreted embryonic alkaline phosphatase (SEAP) reporter gene (PTRE-SEAP-pA). c Full library of 36 protein modifications. HEK-293 cells (three × 104) have been transfected with the totally different constructs (PhCMV-tag-tTA-Dendra2-pA, containing the tetracycline-dependent transactivator tTA) and the reporter gene (PTRE-SEAP-pA). SEAP ranges have been profiled after 24 hours (h) within the tradition supernatant. The degrons marked in daring are the six ubiquitin (Ub) fusion constructs bearing the tags 3xUbVR, UbR, UbK, UbD, UbS, and UbM, which have been chosen as offering consultant degradation patterns. The information symbolize the imply ± SD (n  =three unbiased experiments). Supply knowledge for this determine is on the market within the Supply knowledge file

To characterize the degron library, every degron was used to tag a tTA, which binds to its cognate Tet response component (TRE) situated on the 5′-end of a minimal promoter positioned in entrance of the human secreted embryonic alkaline phosphatase (SEAP) reporter gene (pMM130, PTRE-SEAP-pA) (Fig. 1b). Furthermore, fusion of the photoactivable fluorescent protein Dendra2 to the tagged transcription issue tTA enabled us to measure the in vivo half-lives of the modified transcription components and thus to find out the affect of the degrons thereon32,33. These constructs have been expressed in human embryonic kidney cells (HEK-293) (Fig. 1c). The degradation sample of the tTA-Dendra2 assemble differed relying upon the fused degron, offering an 18-fold vary of SEAP expression, starting from 5 U/L SEAP (3xUbVR) to 91 U/L SEAP (UbM) after 24 h in tradition. An untagged assemble (pCHX17, PhCMV-tTA-Dendra2-pA) was inserted to benchmark the expression stage of the fusion protein with none degron. The totally different quantities of SEAP reporter within the cell tradition supernatant weren’t resulting from cytotoxic results of the degrons, as their expression in HEK-293 cells didn’t have an effect on the cell viability (Supplementary Fig. 2a). Six consultant Ub-fusion tags (Fig. 1c, Supplementary Fig. 2a) have been chosen for additional examine: 3xUbVR, UbR, UbK, UbD, UbS, and UbM. Using these six degrons fused to tTA and Dendra2 in two different broadly used human cell strains (human mesenchymal stem cells (hMSCs) (Supplementary Fig. 2b) and human cervical carcinoma cells (HeLa) (Supplementary Fig. 2c)), and one rodent cell line (Chinese language hamster ovary (CHO-K1) (Supplementary Fig. 2nd)) confirmed that the degradation patterns of the constructs have been reproducible, indicating a broad applicability of this technique.

To rule out the likelihood that the totally different ranges of SEAP expression have been resulting from variations within the DNA transfection effectivity, the six Ub degrons fused to tTA have been linked to an mCherry fluorescent protein by way of a self-cleaving P2A peptide34 (Supplementary Fig. 3a). Fluorescence microscopy revealed a fair distribution of mCherry fluorescence within the cell inhabitants, whereas the expression of Dendra2 was extremely depending on the character of the fused degradation tag (Supplementary Fig. 3b). Thus, the variations within the Dendra2 fluorescence sample enabled us to visualise the affect of the degrons on the expression of the photoactivable protein Dendra2 (Supplementary Fig. 3b). Furthermore, SEAP expression ranges have been assessed in parallel (Supplementary Fig. 3c) and confirmed the identical conduct because the Dendra2 fluorescence in these cell populations (Supplementary Fig. 3d).

The totally different degradation patterns have been confirmed to be steady (Supplementary Fig. 4a). Moreover, the six degrons fused to tTA and Dendra2 have been cloned downstream of normal promoters to verify their broad applicability in varied organic contexts (Supplementary Fig. 4b). Apart from the human cytomegalovirus immediate-early promoter (PhCMV) proven in Fig. 1c, we examined the simian virus 40 enhancer and early promoter (PSV40) (Supplementary Fig. 4c), the human elongation factor-1 alpha promoter (PhEF1α) (Supplementary Fig. 4d), and the murine phosphoglycerate kinase 1 promoter (PPGK) (Supplementary Fig. 4e). The induction ranges of the totally different promoters have been related, supporting the transcriptional independence of the tags. A management experiment utilizing proteasome inhibitor AdaAhx3L3VS35 was run with HEK-293 cells transfected with the six degrons fused to tTA and Dendra2, to analyze whether or not the inhibition of proteasome exercise results in an accumulation of the Ub-tagged Dendra2 constructs. Addition of AdaAhx3L3VS (50 μM) 10 h previous to fluorescence microscopy (Supplementary Fig. 5a) successfully prevented the degradation of the tagged constructs (Supplementary Fig. 5b). The incubation with AdaAhx3L3VS resulted in an Eight-fold improve of the unstable 3xUbVR-tagged assemble, whereas 1.Eight-fold induction was noticed for the steady UbM-tTA-Dendra2 fusion assemble (Supplementary Fig. 5b).

Lastly, the six chosen tags have been fused to the broadly used Streptococcus pyogenes useless Cas9 protein (dCas9) (Supplementary Fig. 6a, b) and to its synergistic activation MCP-VPR assemble36 (Supplementary Fig. 6c, d), to verify the universality of the degrons for protein expression management. The nuclease dCas9 is a big protein that targets particular areas of DNA within the presence of gRNA, whereas the RNA-binding coat protein (MCP) provided with a VP64-p65-Rta (VPR) tripartite activator targets the gRNA itself, forming a protein advanced with a transcription factor-like motion. The addition of the stabilizing UbM tag to dCas9 resulted in a 65-fold improve of SEAP expression, in contrast with that noticed when the unstable 3xUbVR-dCas9 assemble was used (Supplementary Fig. 6b). Furthermore, the induction of SEAP expression was 130-fold higher for the UbM-tagged MCP-VPR, in contrast with the 3xUbVR-tagged MCP-VPR assemble (Supplementary Fig. 6d).

Tuning dose–response dynamics of the Tet system by degrons

To validate that our methodology permits to fine-tune the response of artificial networks to environmental stimuli, we examined the impact of adjusting protein half-life on the dynamic vary of the dose–response curves within the well-established Tet system to see whether or not the response to an enter sign may very well be fine-tuned. With a hard and fast SEAP reporter (pMM130, PTRE-SEAP-pA), the focus of the Tet by-product doxycycline was different and the dose-dependent inductions of 3xUbVR, UbR, UbK, UbD, UbS, and UbM fused to tTA and Dendra2 have been examined (Fig. 2a). Utilizing the Tet-OFF system (Fig. 2a, b), we obtained a 15-fold change within the dose–response curves of the six tTA-Dendra2 fusion constructs, masking the vary from 12 U/L (3xUbVR) to 184 U/L (UbM). Improve of the repressive doxycycline enter resulted in regular declines (Fig. 2b) to minima ranging between 12 U/L (3xUbVR) and 17 U/L (UbM).

Fig. 2Fig. 2

Tunability of the dose–response curves with six chosen protein tags. a Schematic of the six chosen tags (3xUbVR, UbR, UbK, UbD, UbS, UbM) fused to tTA-Dendra2 and uncovered to growing concentrations of doxycycline (dox). b HEK-293 cells (three × 104) have been transfected with the totally different constructs (PhCMV-tag-tTA-Dendra2-pA) and the reporter gene (PTRE-SEAP-pA). SEAP ranges within the tradition supernatant have been profiled after 24 h. c Schematic of the six chosen tags (3xUbVR, UbR, UbK, UbD, UbS, UbM) fused to rtTA-Dendra2 and uncovered to growing concentrations of dox. d HEK-293 cells (three × 104) have been transfected with the totally different constructs (PhCMV-tag-rtTA-Dendra2-pA) and the reporter gene (PTRE-SEAP-pA). SEAP ranges within the tradition supernatant have been profiled after 24 h. e Schematic of the six chosen tags (3xUbVR, UbR, UbK, UbD, UbS, UbM) fused to the autoactivating PTRE-tag-tTA-Dendra2-pA assemble and uncovered to growing concentrations of dox. f HEK-293 cells (three × 104) have been transfected with the totally different constructs (PTRE-tag-tTA-Dendra2-pA) and the reporter gene (PTRE-SEAP-pA). SEAP ranges within the tradition supernatant have been profiled after 24 h. The information symbolize the imply ± SD (n = three unbiased experiments) measured in triplicate. Supply knowledge for this determine is on the market within the Supply knowledge file

We additional launched a reverse tTA (rtTA) as a substitute of tTA (Fig. 2c, d). Utilizing rtTA, SEAP ranges ranged from 9 U/L (3xUbVR) to 20 U/L (UbM) within the absence of doxycycline. Because the inducer focus was raised, SEAP manufacturing elevated to the vary of 10 U/L (3xUbVR) to 210 U/L (UbM). The six degrons confirmed considerably totally different dose–response curves, confirming that their attribute results are retained within the transactivator-based Tet system.

Subsequent, we examined self-activating constructs wherein the PhCMV promoter was changed with a TRE promoter (PTRE-tag-Dendra2-pA) (Fig. 2e). Utilizing the SEAP reporter (pMM130, PTRE-SEAP-pA) in parallel, the autoactivated dose–response curves may very well be assessed in the identical method as used for the unique PhCMV promoter constructs. As within the Tet-OFF system, the curves began from clearly distinct maximal charges starting from 5 U/L (3xUbVR) to 202 U/L (UbM) and fell to three U/L (3xUbVR) to 7 U/L (UbM), reflecting the totally different degradation patterns of the proteins (Fig. 2f). The totally different induction behaviors weren’t a results of totally different expression strengths (e.g., resulting from totally different promoters), however have been moderately brought on by the altered dose–response curves. Collectively, our outcomes help the concept our modular degron library can modulate the protein expression profile and due to this fact the conduct of advanced biologic programs.

Measuring the half-lives of the degron-tagged tTA

Data of protein half-lives is a vital prerequisite for the artificial building and practical evaluation of dynamic organic programs37. Right here, to be able to assess the half-lives of the tagged tTA transactivators, we fused them to Dendra2 (Fig. 3a), a green-to-red photoconvertible protein used for the photolabeling and monitoring of proteins38. The photoconversion circumstances for HEK-293 cells have been optimized, and to exclude the affect of cytotoxicity of the photoconverting laser mild, cell viability was assessed after 24 h (Supplementary Fig. 7a).

Fig. threeFig. 3

Willpower of the protein half-lives of the six chosen constructs. a Earlier than photoconversion, the chromophore of Dendra2 exists in an equilibrium between impartial (nonfluorescent) and anionic (inexperienced fluorescent) states. Excitation of the impartial chromophore with mild at 405 nm ends in photoconversion to a purple fluorescent state. b Fluorescence microscopy photos of cells transfected with PhCMV-3xUbVR-tTA-Dendra2-pA and PhCMV-UbM-tTA-Dendra2-pA earlier than and after photoconversion with 405 nm mild. The picture intensities have been all stretched by the identical issue, whereas sustaining the identical depth ratio for higher visibility. White scale bars correspond to 50 µm. c HEK-293 cells (three × 104) have been transfected with the six chosen tags (3xUbVR, UbR, UbK, UbD, UbS, UbM) and the half-lives of the constructs have been decided by time-lapse microscopy. Photoconversion was performed after 24 h with blue mild and fluorescence photos have been recorded each 10 min. The information symbolize the imply (n = three unbiased experiments) measured in triplicate. Supply knowledge for this determine is on the market within the Supply knowledge file

Half-lives have been decided by transfecting the six chosen constructs (3xUbVR, UbR, UbK, UbD, UbS, and UbM) into HEK-293 cells and photoconverting the proteins 24 h after transfection. Fluorescence microscopic photos pre- and post-irradiation at 405 nm of HEK-293 cells transfected with the 3xUbVR- and the UbM-tagged constructs are proven in Fig. 3b. Time-lapse fluorescence microscopy was used to comply with protein degradation within the cells by measuring the fluorescence depth lower of photoconverted Dendra2 (Fig. 3c). Imply fluorescence depth was measured each 10 min for a minimum of 16 h, to acquire excessive time decision. The half-lives of the degron-tagged tTA transactivators have been within the order 2.Eight h < 5.four h < 6.four h < 6.5 h < 11.7 h < 34.1 h for the UbR, 3x UbVR, UbK, UbD, UbS, and UbM constructs, respectively (Fig. 3c and Supplementary Fig. 4b).

Lastly, to ascertain an in depth understanding of how the degrons influence the expression ranges, we constructed a mathematical mannequin that comes with all vital molecular processes of the system. The mathematical mannequin predicted that the dependency of the SEAP concentrations, 24 h after induction, on the (degron dependent) half-lives of the proteins ought to roughly comply with a Hill curve with a Hill coefficient of two (Supplementary Notes). Once we plotted the experimentally measured SEAP concentrations as a perform of the half-lives (Supplementary Fig. Eight), we certainly discovered a detailed match of our experimental knowledge to this mathematical prediction, which demonstrates that our two measures of protein stability lead to constant experimental insights on the dynamic influence of the totally different degrons.

Engineering a tunable pulse-generator circuit

As a way to assure most versatility of our methodology for the management of protein expression within the context of advanced artificial networks, we built-in our degron library with an artificial pulse generator. Pulse mills are adaptive artificial modules that generate transient mobile responses (or pulses) to sustained exterior or inside triggers (e.g., small molecules), which may activate sure mobile key parts for an outlined interval (pulse size), which can vary from milliseconds39 to a number of hours40. Examples of naturally developed adaptive networks that understand pulse mills embody chemotaxis41,42 and stress response43 in micro organism, and a number of other processes concerned in embryonal growth44, cell cycle regulation45, and responses to extracellular inputs46,47,48,49 in mammalian cells. Additionally within the context of artificial biology, the design of many synthetic networks requires outlined pulsatile dynamics, e.g., within the set-up of oscillators50, circadian clocks51, toggle switches52 or sensor–effector gadgets53. Subsequently, the creation of exactly outlined pulse-generating circuits is indispensable for engineering artificial and dynamic gene networks.

The design of our pulse generator (Fig. 4a) contains (i) a trigger-sensing module (rtTA, sensing doxycycline), (ii) a time-delay component (TtgR, phloretin-dependent transactivator), (iii) a negative-feedback loop (the RNA-binding protein L7Ae), and a dynamic quick fluorescent timer (Quick-FT) reporter (3xUbVR-tagged Quick-FT)54. Upon the addition of doxycycline, rtTA binds to its cognate operator web site and allows expression of the reporter gene Quick-FT (Fig. 4a) and of TtgR in a time-delayed vogue. TtgR itself binds to its operator web site and drives the expression of 3xUbVR, UbR, UbK, UbD, UbS, or UbM-tagged L7Ae, which in flip binds to the C/Dbox of the reporter gene mRNA, thereby repressing its translation to a fluorescent protein.

Fig. fourFig. 4

Pulse generator. a Schematic of the pulse-generator parts. Upon addition of the set off (doxycycline (dox)), the reverse tetracycline-dependent transactivator trigger-sensing module (rtTA, sensing doxycycline) drives the expression of the quick fluorescent timer (Quick-FT) reporter protein and the time-delay TtgR-VP16 component. TtgR-VP16 binds in flip to its cognate operator sequence to drive the expression of the negative-feedback component L7Ae (tagged with the six chosen degrons 3xUbVR, UbR, UbK, UbD, UbS, UbM). To watch the induction of the L7Ae repressor component, a fluorescent protein (Citrine) is hooked up by way of a self-cleaving P2A peptide. L7Ae binds to the C/Dbox of a 3xUbVR-tagged Quick-FT mRNA and represses its translation to a fluorescent protein. The in vivo half-life of L7Ae is altered by the degrons and influences the repression energy of L7Ae. b Time-lapse bright-field and fluorescence microscopy photos of HEK-293 cells transfected with the reporter gene and the totally different negative-feedback parts fused to the 3xUbVR tag. The pictures have been recorded each 20 min for 40 h and present the identical cells on the identical time factors imaged with totally different wavelengths. The picture intensities have been all stretched by the identical issue whereas sustaining the identical depth ratio for higher visibility. White scale bars correspond to 10 µm. c Time-lapse bright-field and fluorescence microscopy photos of HEK-293 cells transfected with the reporter gene and the totally different negative-feedback parts fused to the UbM tag. The pictures have been recorded each 20 min for 40 h and present the identical cells on the identical time factors imaged with totally different wavelengths. The picture intensities have been all stretched by the identical issue whereas sustaining the identical depth ratio for higher visibility. White scale bars correspond to 10 µm. d Imply pulse durations of the totally different pulse mills. Information symbolize the imply ± SD (n = 5) measured in duplicate. **P < zero.005, ****P < zero.0001, Scholar’s t-test. Supply knowledge for this determine is on the market within the Supply knowledge file

Our exact characterization of the six degrons described above (3xUbVR, UbR, UbK, UbD, UbS, and UbM) enabled us the design a set of artificial pulse mills with variable pulse lengths. The heart beat-generator parts have been cloned onto two plasmids (pCHX301, PTRE-TtgR-VP16-pA:PhCMV-rtTA-pA and pCHX300, PTtgR1-3xUbVR-L7Ae-P2A-Citrine-pA:PTRE-C/Dbox-3xUbVR-Quick-FT-pA, or pCHX308, PTtgR1-UbM-L7Ae-P2A-Citrine-pA:PTRE-C/Dbox-3xUbVR-Quick-FT-pA, pCHX309, PTtgR1-UbS-L7Ae-P2A-Citrine-pA:PTRE-C/Dbox-3xUbVR-Quick-FT-pA, pCHX310, PTtgR1-UbK-L7Ae-P2A-Citrine-pA:PTRE-C/Dbox-3xUbVR-Quick-FT-pA, pCHX311, PTtgR1-UbR-L7Ae-P2A-Citrine-pA:PTRE-C/Dbox-3xUbVR-Quick-FT-pA, pCHX312, PTtgR1-UbD-L7Ae-P2A-Citrine-pA:PTRE-C/Dbox-3xUbVR-Quick-FT-pA) to ensure the co-localization of all the weather needed for the technology of a fluorescent pulse inside the identical cell. The heart beat-generator parts have been transfected into HEK-293 cells and the ensuing pulse patterns have been noticed by mild microscopy. Citrine was hooked up to L7Ae by way of a self-cleaving peptide P2A, to observe the induction of the repressing L7Ae component (Fig. 4b, Supplementary Fig. 9a, b, Supplementary Motion pictures 1 and a couple of). By making use of the half-life-modulating tags to the negative-feedback component PTtgR1-tag-L7Ae-P2A-Citrine-pA of the heartbeat generator, we might set up a set of expression pulses with imply durations of 10 h (3xUbVR), 9.6 h (UbR), Eight.2 h (UbK), 7.three h (UbD), 7.9 h (UbS), and 6.three h (UbM) (Fig. 4d). Modulation of the protein stability of L7Ae and therefore the negative-feedback component of the heartbeat generator might present shorter pulse lengths for extra steady protein parts such because the UbM-tagged L7Ae protein or longer pulse lengths for the unstable 3xUbVR-tagged L7Ae component.

As a management, the set off module rtTA alone did not generate pulsatile dynamic conduct, confirming the suitability of each the set off module and the reporter Quick-FT (Supplementary Fig. 10a, b, Supplementary Film three). To indicate that each component is important for the proper functioning of the pulsing circuit, we eliminated particular person parts of the heartbeat generator (corresponding to PTRE-TtgR-VP16-pA) (Supplementary Fig. 11a, b, Supplementary Film four) and demonstrated that the negative-feedback module (PTtgR1-tag-L7Ae-pA) alone doesn’t repress the interpretation of the Quick-FT reporter mRNA to a fluorescent protein, whereas the constitutive expression of the negative-feedback module (pCHX255, PhCMV-3xUbVR-L7Ae-pA) absolutely represses the expression of the Quick-FT reporter protein (Supplementary Fig. 12a, b). Supplementary Fig. 13a, b exhibits that the time-delay component TtgR is important for the maturation and accumulation of the Quick-FT reporter protein inside the cells. With out this component, L7Ae represses the interpretation of the reporter protein and the cells fail to provide detectable quantities of Quick-FT.


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