Class I phosphoinositide 3-OH kinase (PI3K) signaling is central to animal growth and metabolism, and pathological disruption of this pathway affects cancer and diabetes. However, the specific spatial/temporal dynamics and signaling roles of its minor lipid messenger, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), are not well understood. This owes principally to a lack of tools to study this scarce lipid. Here we developed a high-sensitivity genetically encoded biosensor for PI(3,4)P2, demonstrating high selectivity and specificity of the sensor for the lipid. We show that despite clear evidence for class II PI3K in PI(3,4)P2-driven function, the overwhelming majority of the lipid accumulates through degradation of class I PI3K-produced PIP3 However, we show that PI(3,4)P2 is also subject to hydrolysis by the tumor suppressor lipid phosphatase PTEN. Collectively, our results show that PI(3,4)P2 is potentially an important driver of class I PI3K-driven signaling and provides powerful new tools to begin to resolve the biological functions of this lipid downstream of class I and II PI3K.

Human mitochondrial methionine transfer RNA (hmtRNA(Met)(CAU)) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f(5)C(34)). The role of this modification in (hmtRNA(Met)(CAU)) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

Genetic code expansion with unnatural amino acids (UAAs) has significantly broadened the chemical repertoire of proteins. Applications of this method in mammalian cells include probing of molecular interactions, conditional control of biological processes, and new strategies for therapeutics and vaccines. A number of methods have been developed for transient UAA mutagenesis in mammalian cells, each with unique features and advantages. All have in common a need to deliver genes encoding additional protein biosynthetic machinery (an orthogonal tRNA/tRNA synthetase pair) and a gene for the protein of interest. In this study, we present a comparative evaluation of select plasmid-based genetic code expansion systems and a detailed analysis of suppression efficiency with different UAAs and in different cell lines.

Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components.

Due to their high stability and specificity in living cells, fluorescently labeled nanobodies are perfect probes for visualizing intracellular targets at an endogenous level. However, intrabodies bind unrestrainedly and hence may interfere with the target protein function. Here, we report a strategy to prevent premature binding through the development of photo-conditional intrabodies. Using genetic code expansion, we introduce photocaged amino acids within the nanobody-binding interface, which, after photo-activation, show instantaneous binding of target proteins with high spatiotemporal precision inside living cells. Due to the highly stable binding, light-guided intrabodies offer a versatile platform for downstream imaging and regulation of target proteins.

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

Many proteins harboring low complexity or intrinsically disordered sequences (IDRs) are capable of undergoing liquid-liquid phase separation to form mesoscale condensates that function as biochemical niches with the ability to concentrate or sequester macromolecules and regulate cellular activity. Engineered disordered proteins have been used to generate programmable synthetic membraneless organelles in cells. Phase separation is governed by the strength of interactions among polypeptides with multivalency enhancing phase separation at lower concentrations. Previously, we and others demonstrated enzymatic control of IDR valency from multivalent precursors to dissolve condensed phases. Here, we develop noncovalent strategies to multimerize an individual IDR, the RGG domain of LAF-1, using protein interaction domains to regulate condensate formation in vitro and in living cells. First, we characterize modular dimerization of RGG domains at either terminus using cognate high-affinity coiled-coil pairs to form stable condensates in vitro. Second, we demonstrate temporal control over phase separation of RGG domains fused to FRB and FKBP in the presence of dimerizer. Further, using a photocaged dimerizer, we achieve optically induced condensation both in cell-sized emulsions and within live cells. Collectively, these modular tools allow multiple strategies to promote phase separation of a common core IDR for tunable control of condensate assembly.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and non-traditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with co-administered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously following Boolean logic. OFF-switch adaptors represent a robust new approach for precision targeting of universal CAR T cells with potential for enhanced safety.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Controlled manipulation of proteins and their function is important in almost all biological disciplines. Here, we demonstrate control of protein activity with light. We present two different applications-light-triggered transcription and light-triggered protease cleavage-both based on the same concept of protein mislocation, followed by optochemically triggered translocation to an active cellular compartment. In our approach, we genetically encode a photocaged lysine into the nuclear localization signal (NLS) of the transcription factor SATB1. This blocks nuclear import of the protein until illumination induces caging group removal and release of the protein into the nucleus. In the first application, prepending this NLS to the transcription factor FOXO3 allows us to optochemically switch on its transcription activity. The second application uses the developed light-activated NLS to control nuclear import of TEV protease and subsequent cleavage of nuclear proteins containing TEV cleavage sites. The small size of the light-controlled NLS (only 20 amino acids) minimizes impact of its insertion on protein function and promises a general approach to a wide range of optochemical applications. Since the light-activated NLS is genetically encoded and optically triggered, it will prove useful to address a variety of problems requiring spatial and temporal control of protein function, for example, in stem-cell, developmental, and cancer biology.

Combinations of direct-acting antivirals (DAAs) against the hepatitis C virus (HCV) have the potential to revolutionize the HCV therapeutic regime. An integral component of DAA combination therapies is HCV NS5A inhibitors. It has previously been proposed that NS5A DAAs inhibit two functions of NS5A: RNA replication and virion assembly. In this study, we characterize the impact of a prototype NS5A DAA, daclatasvir (DCV), on HCV replication compartment formation. DCV impaired HCV replicase localization and NS5A motility. In order to characterize the mechanism behind altered HCV replicase localization, we examined the impact of DCV on the interaction of NS5A with its essential cellular cofactor, phosphatidylinositol-4-kinase III α (PI4KA). We observed that DCV does not inhibit PI4KA directly, nor does it impair early events of the NS5A-PI4KA interaction that can occur when NS5A is expressed alone. NS5A functions that are unaffected by DCV include PI4KA binding, as determined by co-immunoprecipitation, and a basal accumulation of the PI4KA product, PI4P. However, DCV impairs late steps in PI4KA activation that requires NS5A expressed in the context of the HCV polyprotein. These NS5A functions include hyper-stimulation of PI4P levels and appropriate replication compartment formation. The data are most consistent with a model wherein DCV inhibits conformational changes in the NS5A protein or protein complex formations that occur in the context of HCV polyprotein expression and stimulate PI4P hyper-accumulation and replication compartment formation.

Despite their low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival in host organisms. We demonstrate that PtdIns(3,4)P2 is a major product generated in host cells by the effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4,5)P3 and PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signalling to gain entry and even provoke oncogenic transformation.

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. We report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron-size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to 90% of targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

mTORC1 promotes protein translation, learning and memory, and neuroadaptations that underlie alcohol use and abuse. We report that activation of mTORC1 in the nucleus accumbens (NAc) of mice consuming alcohol promotes the translation of microRNA (miR) machinery components and the upregulation of microRNAs (miRs) expression including miR34a-5p. In parallel, we detected a paradoxical mTORC1-dependent repression of translation of transcripts including Aldolase A, an essential glycolytic enzyme. We found that miR34a-5p in the NAc targets Aldolase A for translation repression and promotes alcohol intake. Our data further suggest that glycolysis is inhibited in the NAc manifesting in an mTORC1-dependent attenuation of L-lactate, the end product of glycolysis. Finally, we show that systemic administration of L-lactate attenuates mouse excessive alcohol intake. Our data suggest that alcohol promotes paradoxical actions of mTORC1 on translation and glycolysis which in turn drive excessive alcohol use.

Controlling protein activity with chemogenetics and optogenetics has proven to be powerful for testing hypotheses regarding protein function in rapid biological processes. Controlling proteins by splitting them and then rescuing their activity through inducible reassembly offers great potential to control diverse protein activities. Building split proteins has been difficult due to spontaneous assembly, difficulty in identifying appropriate split sites, and inefficient induction of effective reassembly. Here we present an automated approach to design effective split proteins regulated by a ligand or by light (SPELL). We develop a scoring function together with an engineered domain to enable reassembly of protein halves with high efficiency and with reduced spontaneous assembly. We demonstrate SPELL by applying it to proteins of various shapes and sizes in living cells. The SPELL server (spell.dokhlab.org) offers an automated prediction of split sites.

The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNA(Met)(CAU), to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl- (A-) sites of the ribosome. The hmtRNA(Met)(CAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f(5)C(34)), and a cytidine substituting for the invariant uridine at position 33 of the canonical U-turn in tRNAs. The structure of the tRNA anticodon stem and loop domain (hmtASL(Met)(CAU)), determined by NMR restrained molecular modeling, revealed how the f(5)C(34) modification facilitates the decoding of AUA at the P- and the A-sites. The f(5)C(34) defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASL(Met)(CAU). The hmtASL(Met)(CAU) exhibited a C-turn conformation that has some characteristics of the U-turn motif. Codon binding studies with both Escherichia coli and bovine mitochondrial ribosomes revealed that the f(5)C(34) facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL binding kinetics. Mitochondrial translation by many organisms, including humans, sometimes initiates with the universal isoleucine codons AUU and AUC. The f(5)C(34) enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f(5)C(34), expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.

The incorporation of unnatural amino acids into proteins through genetic code expansion has been successfully adapted to African claw-toed frog embryos. Six unique unnatural amino acids are incorporated site-specifically into proteins and demonstrate robust and reliable protein expression. Of these amino acids, several are caged analogues that can be used to establish conditional control over enzymatic activity. Using light or small molecule triggers, we exhibit activation and tunability of protein functions in live embryos. This approach was then applied to optical control over the activity of a RASopathy mutant of NRAS, taking advantage of generating explant cultures from Xenopus. Taken together, genetic code expansion is a robust approach in the Xenopus model to incorporate novel chemical functionalities into proteins of interest to study their function and role in a complex biological setting.

Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

In cell and molecular biology, double-stranded circular DNA constructs, known as plasmids, are extensively used to express a gene of interest. These gene expression systems rely on a specific promoter region to drive the transcription of genes either constitutively (i.e., in a continually "ON" state) or conditionally (i.e., in response to a specific transcription initiator). However, controlling plasmid-based expression with high spatial and temporal resolution in cellular environments and in multicellular organisms remains challenging. To overcome this limitation, we have site-specifically installed nucleobase-caging groups within a plasmid promoter region to enable optochemical control of transcription and, thus, gene expression, via photolysis of the caging groups. Through the light-responsive modification of plasmid-based gene expression systems, we have demonstrated optochemical activation of an exogenous fluorescent reporter gene in both tissue culture and a live animal model, as well as light-induced overexpression of an endogenous signaling protein.