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Design of proteolytic-cleavage-responsive CC interaction modules a, Design of the proteolytic-cleavage-responsive CC rearrangement for reconstitution of the functional split protein. After linker cleavage, an autoinhibitory coil is replaced by a displacer segment with higher binding affinity to reconstitute the split effector/reporter. b, ABA- and rapamycin-inducible activation, demonstrated in HEK293T cells measured 30 min after induction with the indicated concentration of ABA or rapamycin. fLuc, firefly luciferase; nRLU, normalized relative light units; n and c prefixes, N and C fragments. c, Design of a proteolytic-cleavage-inactivated module (logical negation). A protease-cleavage site is introduced between the CC-forming segments and split effector/reporter domain. After cleavage of the linker, split luciferase dissociates. d, Decrease in luciferase activity after cotransfection of logical negation functions with plasmids encoding specific proteases. Values in b and d are the means from four cell cultures ±s.d. and are representative of two independent experiments. Transfection plasmid mixtures are listed in Supplementary Table 1.
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Cellular signal transduction is predominantly based on protein interactions and their post-translational modifications, which enable a fast response to input signals. Owing to difficulties in designing new unique protein–protein interactions, designed cellular logic has focused on transcriptional regulation; however, that process has a substantiall...
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... In particular, compared to the more conventional transcriptional control, protein-level controls hold advantages for potential clinical use, such as fast operation, compact single-transcript delivery and context-independent performance 12 . Notably, orthogonal viral proteases have emerged as useful post-translational tools due to their ability to control the activity, degradation and localization of target proteins with high substrate specificity [13][14][15][16] . These orthogonal proteases are highly versatile and can be combined to implement robust sense-and-response behaviors in mammalian cells. ...
Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. Although protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. In this study, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins. We sourced a specific human protease and its FDA-approved inhibitor. We engineered cytokines (IL-2, IL-6 and IL-10) whose activities can be activated and abrogated by proteolytic cleavage. We used species specificity and re-localization strategies to orthogonalize the cytokines and protease from the human context that they would be deployed in. hDIRECT should enable local cytokine activation to support a variety of cell-based therapies, such as muscle regeneration and cancer immunotherapy. Our work offers a proof of concept for the emerging appreciation of humanization in synthetic biology for human health.
... We also prepared GSDMD TEV where the caspase-1/11 cleavage site was mutated into the Tobacco etch virus protease (TEVp) recognition site which enables controlled cleavage of GSDMD and induction of pore formation using TEVp. TEVp is a highly specific protease recognizing a sequence of seven amino acid residues, minimally interferes with the existing cellular chassis, and is nontoxic to mammalian cells 45 , therefore suitable for use in the setup preferring the orthogonality facilitated by the non-endogenous enzyme. ...
... Thus, we developed two systems that trigger GSDMD-based pyroptosis upon the addition of a small molecule utilizing ligand-dependent dimerization (Fig. 3). The first system is based on chemically regulated split TEVp, with complementary split fragments fused to FKBP and FRB dimerization domains, whose heterodimerization is inducible using rapamycin or its analog (rapalog) AP21967 (Fig. 3a) 45 . The pore-forming capacity was assessed using LDH and PI assays upon transfection of constructs into HEK293T cells (Fig. 3b). ...
... We developed a toolbox of engineered GSDMD variants that can induce pyroptosis spontaneously when introduced into tumors or on-demand, controlled by a non-toxic small molecule. These systems independently of endogenous inflammasome components enable pyroptosis induction with different levels of efficiency and regulation for future integration into more complex systems 45,84 . When applied to relatively large tumors, the induction of ICD with GSDMD variants proved to be potent in B16F10 melanoma tumor surveillance with around 20-25% long-term survivors regardless of whether NT GSDMD was introduced (Fig. 2) or GSDMD cleavage was induced by the addition of small molecule (Fig. 3). ...
Inflammasomes are defense complexes that utilize cytokines and immunogenic cell death (ICD) to stimulate the immune system against pathogens. Inspired by their dual action, we present cytokine-armed pyroptosis as a strategy for boosting immune response against diverse types of tumors. To induce pyroptosis, we utilize designed tightly regulated gasdermin D variants comprising different pore-forming capabilities and diverse modes of activation, representing a toolbox of ICD inducers. We demonstrate that the electrogenic transfer of ICD effector-encoding plasmids into mouse melanoma tumors when combined with intratumoral expression of cytokines IL-1β, IL-12, or IL-18, enhanced anti-tumor immune responses. Careful selection of immunostimulatory molecules is, however, imperative as a combination of IL-1β and IL-18 antagonized the protective effect of pyroptosis by IFNγ-mediated upregulation of several immunosuppressive pathways. Additionally, we show that the intratumoral introduction of armed pyroptosis provides protection against distant tumors and proves effective across various tumor types without inducing systemic inflammation. Deconstructed inflammasomes thus serve as a powerful, tunable, and tumor-agnostic strategy to enhance antitumor response, even against the most resilient types of tumors.
... For the parallel dimeric modules, we selected two heterodimeric pairs (P3-P4 and P5-P6) designed, characterized, and used in previous studies. [6,7,[20][21][22][23][24] Among the sets of antiparallel homodimeric building blocks in the CC toolbox, we opted for the APH [25] module, which shows high stability. [26] The chosen modules enable two different permutations of the polypeptide sequence, designed to fold into the tetrahedral shape (Scheme 1a, b, c). ...
Versatile DNA and polypeptide‐based structures have been designed based on complementary modules. However, polypeptides can also form higher oligomeric states. We investigated the introduction of tetrameric modules as a substitute for coiled‐coil dimerization units used in previous modular nanostructures. Tetramerizing helical bundles can run in parallel or antiparallel orientation, expanding the number of topological solutions for modular nanostructures. Furthermore, this strategy facilitates the construction of nanostructures from two identical polypeptide chains. Importantly, tetrameric modules substantially stabilized protein nanostructures against air–water interface denaturation, enabling the determination of the first cryo‐electron microscopy three‐dimensional structure of a coiled‐coil‐based nanostructure, confirming the designed agreement of the modules forming a tetrahedral cage.
... Synthetic biological regulations design can also be conducted at the translation level, which has a shorter time scale than the tr anscription le v el (Fink et al. 2019 ). In this section, we pr ovide an ov ervie w of differ ent orthogonal tr anslation r egulations, including ribosomes , ribos witches , and aminoac yl-tRN A synthetase (aaRS)-tRNA pairs based on non-canonical amino acids (ncAAs). ...
... Specifically, using the diversity and pr ogr ammability of vir al pr oteases, a combinable protein system (circuits of hacked orthogonal modular proteases, CHOMP) was de v eloped (Fig. 5 a) for the construction of different synthetic logics . In order to r egulate pr otein circuits mor e quic kl y and accur atel y, Fink et al. obtained split-pr oteasecleavable orthogonal-CC-based (SPOC) logic circuits, which could quic kl y r espond to small molecule inducers within a few minutes (Fink et al. 2019 ). The complete activation of functional enzymes can be ac hie v ed when the other recombinant enzyme has str onger matc hing ability and paired implementation (Fig. 5 b). ...
Microbes compete and cooperate with each other via a variety of chemicals and circuits. Recently, to decipher, simulate or reconstruct microbial communities, many researches have been engaged in engineering microbiomes with bottom-up synthetic biology approaches for diverse applications. However, they have been separately focused on individual perspectives including genetic circuits, communications tools, microbiome engineering, or promising applications. The strategies for coordinating microbial ecosystems based on different regulation circuits have not been systematically summarized, which calls for a more comprehensive framework for the assembly of microbial communities. In this review, we summarize diverse cross-talk and orthogonal regulation modules for de novo bottom-up assembling functional microbial ecosystems, thus promoting further consortia-based applications. Firstly, we review the cross-talk communication-based regulations among various microbial communities from intra-species and inter-species aspects. Then, orthogonal regulations are summarized at metabolites, transcription, translation, and post-translation levels, respectively. Furthermore, to give more details for better design and optimize various microbial ecosystems, we propose a more comprehensive design-build-test-learn (cDBTL) procedure including function specification, chassis selection, interaction design, system build, performance test, modelling analysis, and global optimization. Finally, current challenges and opportunities are discussed for the further development and application of microbial ecosystems.
... To promote the reassembly of the fragments in linear TXTL, we used a pair of antiparallel heterodimeric coiled-coils, P3/ AP4. 25 We fused P3 to the C-terminus of LgBit and AP4 to the N-terminus of smBit to respect the structural orientations of both fragments in the native nanoluc protein. To test this system in linear TXTL, LgBit and smBit were introduced as linear DNA templates. ...
... For example, PM recruitment of a ubiquitin ligase or other post-translational modification enzymes could facilitate targeted protein degradation, phosphorylation, or glycosylation of PM pools of POIs [55][56][57][58][59][60] . Alternatively, PM recruitment of TEV protease during mitosis could be used to enable release of a POI (for example, a transcription factor) tethered to the PM via a linker containing a TEV substrate sequence to affect downstream signalling and/or transcription, potentially integrating with existing systems that leverage protease cleavage for various applications [61][62][63][64][65] . ...
Tools for acute manipulation of protein localization enable elucidation of spatiotemporally defined functions, but their reliance on exogenous triggers can interfere with cell physiology. This limitation is particularly apparent for studying mitosis, whose highly choreographed events are sensitive to perturbations. Here we exploit the serendipitous discovery of a phosphorylation-controlled, cell cycle-dependent localization change of the adaptor protein PLEKHA5 to develop a system for mitosis-specific protein recruitment to the plasma membrane that requires no exogenous stimulus. Mitosis-enabled anchor-away/recruiter system comprises an engineered, 15 kDa module derived from PLEKHA5 capable of recruiting functional protein cargoes to the plasma membrane during mitosis, either through direct fusion or via GFP–GFP nanobody interaction. Applications of the mitosis-enabled anchor-away/recruiter system include both knock sideways to rapidly extract proteins from their native localizations during mitosis and conditional recruitment of lipid-metabolizing enzymes for mitosis-selective editing of plasma membrane lipid content, without the need for exogenous triggers or perturbative synchronization methods.
... One example of such a switch would be a dual-module ZF protein-based switch wherein both modules binding to next to each other on a DNA target sequence leads to the reconstitution of an orthogonal proteaselvi, [58],lvii. If mutations in the DNA are directly targeted, an enzymatic cascade may be required for sufficiently rapid amplification of the mutation "signal" [59]. Such a cascade might increase vector off-target activity, however. ...
Recently concluded, large-scale cancer genomics studies involving multiregion sequencing of primary tumors and paired metastases appear to indicate that many or most cancer patients have one or more “clonal" mutations in their tumors. Clonal mutations are those that are present in all of a patient’s cancer cells. Clonally mutated proteins can potentially be targeted by inhibitors or E3 ligase small molecule glues, but developing new small molecule drugs for each patient is not feasible currently. Achilles Therapeutics is currently the only company specifically targeting clonal mutations. However, they are doing so with tumor-derived T cells. To address the potential limitations of immunotherapy, I have devised another approach for exploiting clonal mutations, which I call “Oncolytic Vector Efficient Replication Contingent on Omnipresent Mutation Engagement” (OVERCOME). The ideal version of OVERCOME would likely employ a bioengineered facultative intracellular bacterium. The bacterium would initially be attenuated, but (transiently) reverse its attenuation upon clonal mutation detection.
... Splittable systems have found widespread applications in diverse fields, including immunotherapy, gene editing, prodrug activation, biosensing, and synthetic biology. [1][2][3][4] In the realm of cancer immunotherapy, splitting strategies have been employed to develop chimeric antigen receptor (CAR) T-cell therapies with improved tumor specificity and reduced offtumor toxicity. 5,6 Furthermore, the engineering of bispecific antibodies has benefited from split designs, enabling enhanced target recognition and recruitment of cytotoxic effector cells. ...
... 60,61 N-TEV(1-118)/C-TEV (119-242) pairs of TEV proteases were used first for proteinprotein interactions and were later used for the design of fast proteolysis-based signaling and logic circuits in mammalian cells. 4,60 Considering the crucial role of proteases in various biological processes, programmable protease-based systems have been developed for therapeutic protein secretion. 62 ...
Splittable systems have emerged as a powerful approach for the precise spatiotemporal control of biological processes. This concept relies on splitting a functional molecule into inactive fragments, which can be reassembled under specific conditions or stimuli to regain activity. Several binding pairs and orthogonal split fragments are introduced by fusing with other modalities to develop more complex and robust designs. One of the pillars of these splittable systems is modularity, which involves decoupling targeting, activation, and effector functions. Challenges, such as off-target effects and overactivation, can be addressed through precise control. This review provides an overview of the design principles, strategies, and applications of splittable systems across diverse fields including immunotherapy, gene editing, prodrug activation, biosensing, and synthetic biology.
... Notably, when combined with orthogonal split proteases, they enable the formation of quickly responsive and highly modular systems. Two systems are employed this mode of action: split-proteasecleavable orthogonal-CC-based (SPOC) ( Figure 5B) 103 logic and circuits of hacked orthogonal modular proteases (CHOMP) ( Figure 5C). 104 Both SPOC logic and CHOMP use CC dimerization to reconstitute inactive split proteases for active enzymes. ...
... 87 It was also shown that CCs can modulate the safety and efficacy of CAR receptors. 91,92 Furthermore, CCs can be used in allosteric protein regulation (e.g., INSRTR), 101 molecular circuit design, 103,104 and the controlled release of therapeutic proteins from cells. 105,108 They also serve as engineered activators of calcium channels, 110 expanding their utility in modulating cellular functions. ...
Synthetic biology aims to engineer complex biological systems using modular elements, with coiled-coil (CC) dimer-forming modules are emerging as highly useful building blocks in the regulation of protein assemblies and biological processes. Those small modules facilitate highly specific and orthogonal protein-protein interactions, offering versatility for the regulation of diverse biological functions. Additionally, their design rules enable precise control and tunability over these interactions, which are crucial for specific applications. Recent advancements showcase their potential for use in innovative therapeutic interventions and biomedical applications. In this review, we discuss the potential of CCs, exploring their diverse applications in mammalian cells, such as synthetic biological circuit design, transcriptional and allosteric regulation, cellular assemblies, chimeric antigen receptor (CAR) T cell regulation, and genome editing and their role in advancing the understanding and regulation of cellular processes.
... 3 While most efforts have focused on transcriptional regulation because of the relative ease to rewire gene expression to recognize different input signals, [4][5][6] protein-level circuits, in which post-translational modi cations can speci cally modify protein activity, localization, and/or stability, can often achieve much faster signaling processing within minutes. 7,8 Protein degradation is an effective native mechanism used in modulating intracellular information, and plays an essential role in maintaining cellular homeostasis. 9 Repurposing native protein degradation in a synthetic context is gaining attention as a new strategy to manipulate cellular behavior rapidly for a wide range of applications including disease detection and therapy. ...
BioPROTACs are heterobifunctional proteins designed for targeted protein degradation (TPD). They are useful not only for probing protein functions but also offer a therapeutic avenue for modulating disease-related proteins. To extend the use of TPD beyond just protein attenuation, we introduce a synthetic framework for logic-gated, switchable TPD to achieve temporal control of protein content. By exploiting both the cleavage and ligation functionalities of Sortase A (SrtA), we present a new strategy utilizing SrtA as the control input to direct bioPROTAC activity for switchable TPD. Furthermore, by layering the SrtA input with protease gating, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. This new L ogic-gated A dPROM deploying S rtA-mediated E lement R ecombination ( LASER ) platform allows us to expand the possible protein degradation outcomes in mammalian cells using Boolean logic operationsdepending on the input combinations. The flexibility to temporally modulate the level of multiple native intracellular proteins can potentially lead to applications from therapy to diagnostics and biotechnology.