Regulation of ferroptotic cancer cell death by GPX4.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

Super-multiplex vibrational imaging.

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Peptide-Dependent Growth in Yeast via Fine-Tuned Peptide/GPCR-Activated Essential Gene Expression.

Building multicellular microbial consortia that communicate with each other and perform programmed functionalities is the next milestone for synthetic biology. Achieving cell-cell communication within these communities requires programming of the transduction of an extracellular signal into a customized intracellular response. G-protein-coupled receptors (GPCRs) are attractive candidates for engineering signal transduction as they can sense extracellular events with high sensitivity and specificity and transduce them into complex intracellular programs. We recently developed a scalable cell-cell communication language based on fungal mating GPCRs and their secreted peptide ligands. This language allows the assembly of engineered yeast strains into multicellular communication networks and allows them to be made interdependent by peptide signaling. In peptide signaling, one cell secretes a peptide that supports the growth of another cell at nanomolar concentrations, a scalable approach for engineering interdependence. Here we address the challenge of correlating the doubling time of Saccharomyces cerevisiae cells with an increasing external peptide concentration by linking GPCR activation to the expression of an essential gene. The required fine-tuning of downstream signaling is achieved via the transcriptional titration of a set of orthogonal GPCR-activated transcription factors, a series of corresponding promoters with different output dynamics, and the use of chemically recoded peptide ligands with varying activation potentials. As such, our work establishes three control points that allow the tuning of the basal and maximal activation of the GPCR response, fold change activation, and response sensitivity. The presented results enable the implementation of peptide-dependent and peptide-tunable growth but could also facilitate the design and calibration of more complex GPCR-controlled synthetic functionality in the future.

Genetic Code Expansion: A Brief History and Perspective.

Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.

Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP.

Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent noncanonical amino acid 3-((6-acetylnaphthalen-2-yl)amino)-2-aminopropanoic acid (ANAP) into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells, and they exhibited pathophysiological function. A spectral imaging potassium depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells.

Interrogation of Eukaryotic Stop Codon Readthrough Signals by in Vitro RNA Selection.

RNA signals located downstream of stop codons in eukaryotic mRNAs can stimulate high levels of translational readthrough by the ribosome, thereby giving rise to functionally distinct C-terminally extended protein products. Although many readthrough events have been previously discovered in Nature, a broader description of the stimulatory RNA signals would help to identify new reprogramming events in eukaryotic genes and provide insights into the molecular mechanisms of readthrough. Here, we explore the RNA reprogramming landscape by performing in vitro translation selections to enrich RNA readthrough signals de novo from a starting randomized library comprising >1013 unique sequence variants. Selection products were characterized using high-throughput sequencing, from which we identified primary sequence and secondary structure readthrough features. The activities of readthrough signals, including three novel sequence motifs, were confirmed in cellular reporter assays. Then, we used machine learning and our HTS data to predict readthrough activity from human 3'-untranslated region sequences. This led to the discovery of >1.5% readthrough in four human genes (CDKN2B, LEPROTL1, PVRL3, and SFTA2). Together, our results provide valuable insights into RNA-mediated translation reprogramming, offer tools for readthrough discovery in eukaryotic genes, and present new opportunities to explore the biological consequences of stop codon readthrough in humans.

Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches.

Protein synthesis in eukaryotes is regulated by diverse reprogramming mechanisms that expand the coding capacity of individual genes. Here, we exploit one such mechanism, termed -1 programmed ribosomal frameshifting (-1 PRF), to engineer ligand-responsive RNA switches that regulate protein expression. First, efficient -1 PRF stimulatory RNA elements were discovered by in vitro selection; then, ligand-responsive switches were constructed by coupling -1 PRF stimulatory elements to RNA aptamers using rational design and directed evolution in Saccharomyces cerevisiae. We demonstrate that -1 PRF switches tightly control the relative stoichiometry of two distinct protein outputs from a single mRNA, exhibiting consistent ligand response across whole populations of cells. Furthermore, -1 PRF switches were applied to build single-mRNA logic gates and an apoptosis module in yeast. Together, these results showcase the potential for harnessing translation-reprogramming mechanisms for synthetic biology, and they establish -1 PRF switches as powerful RNA tools for controlling protein synthesis in eukaryotes.

d-Amino Acid-Mediated Translation Arrest Is Modulated by the Identity of the Incoming Aminoacyl-tRNA.

A complete understanding of the determinants that restrict d-amino acid incorporation by the ribosome, which is of interest to both basic biologists and the protein engineering community, remains elusive. Previously, we demonstrated that d-amino acids are successfully incorporated into the C-terminus of the nascent polypeptide chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA (peptidyl-d-aa-tRNA) donor substrate, however, partition into subpopulations that either undergo translation arrest through inactivation of the ribosomal peptidyl-transferase center (PTC) or remain translationally competent. The proportion of each subpopulation is determined by the identity of the d-amino acid side chain. Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA) acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery demonstrates that it is the pairing of the peptidyl-d-aa-tRNA donor and the aa-tRNA acceptor that determines the activity of the PTC. Moreover, we provide evidence that both the amino acid and tRNA components of the aa-tRNA acceptor contribute synergistically to the extent of arrest. The results of this work deepen our understanding of the mechanism of d-amino acid-mediated translation arrest and how cells avoid this precarious obstacle, reveal similarities to other translation arrest mechanisms involving the PTC, and provide a new route for improving the yields of engineered proteins containing d-amino acids.

A Yeast Three Hybrid Assay for Metabolic Engineering of Tetracycline Derivatives.

Metabolic engineering stands to transform the discovery and production of a wide range of chemicals, but metabolic engineering currently demands considerable resource investments that restrict commercial application. To facilitate the applicability of metabolic engineering, general high-throughput and readily implemented technologies are needed to assay vast libraries of strains producing desirable chemicals. Toward this end, we describe here the development of a yeast three hybrid (Y3H) assay as a general, high-throughput, versatile and readily implemented approach for the detection of target molecule biosynthesis. Our system detects target molecule biosynthesis through a change in reporter gene transcription that results from the binding of the target molecule to a modular protein receptor. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). Various tetracycline derivatives can be detected using our assay, whose versatility enables its use both as a screen and a selection to match the needs and instrumentation of a wide range of end users. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and nonproducer strains of the natural product tetracycline TAN-1612. The Y3H assay is superior to state-of-the-art HPLC-MS methods in throughput and limit of detection of tetracycline derivatives. Finally, our establishment of the Y3H assay for detecting the biosynthesis of a tetracycline supports the generality of the Y3H assay for detecting the biosynthesis of many other target molecules.

The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center.

The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.

Fluorescence Polarization Assay for Small Molecule Screening of FK506 Biosynthesized in 96-Well Microtiter Plates.

The fluorescence polarization (FP) assay has been widely used to study enzyme kinetics, antibody-antigen interactions, and other biological interactions. We propose that the FP assay can be adapted as a high-throughput and potentially widely applicable screen for small molecules. This is useful in metabolic engineering, which is a promising approach to synthesizing compounds of pharmaceutical, agricultural, and industrial importance using bioengineered strains. There, the development of high-yield strains is often a costly and time-consuming process. This problem can be addressed by generating and testing large mutant strain libraries. However, a current key bottleneck is the lack of high-throughput screens to detect the small molecule products. The FP assay is quantitative, sensitive, fast, and cheap. As a proof of principle, we established the FP assay to screen for FK506 (tacrolimus) produced by Streptomyces tsukubaensis, which was cultivated in 96-well plates. An ultraviolet mutagenized library of 160 colonies was screened to identify strains showing higher FK506 productivities. The FP assay has the potential to be generalized to detect a wide range of other small molecules.

Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy.

The dimeric focal adhesion protein talin contains up to 22 cryptic vinculin binding sites that are exposed by unfolding. Using a novel method to monitor the in situ dynamics of the talin dimer stretch, we find that in contrast to several prevalent talin dimer models the integrin-binding talin N-termini are separated by 162 ± 44 nm on average whereas as expected the C-terminal dimerization domains colocalize and are mobile. Using vinculin tagged by DHFR-TMP Atto655 label, we found that optimal vinculin and vinculin head binding occurred when talin was stretched to 180 nm, while the controls did not bind to talin. Surprisingly, multiple vinculins bound within a single second in narrowly localized regions of the talin rod during stretching. We suggest that talin stretches as an antiparallel dimer and that activates vinculin binding in a cooperative manner, consistent with the stabilization of folded talin by other binding proteins.

The covalent trimethoprim chemical tag facilitates single molecule imaging with organic fluorophores.

Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways.

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Live-cell super-resolution imaging with trimethoprim conjugates.

The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at approximately 20 nm resolution.

A common diaryl ether intermediate for the gram-scale synthesis of oxazine and xanthene fluorophores.

Common ground: copper-catalyzed coupling reactions can be used for the high-yielding preparation of widely used oxazine and xanthene fluorophores from a common diaryl ether intermediate on a gram-scale. This general approach may facilitate the future development of novel fluorophores and probes with unique properties.

Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

We previously demonstrated that we could hijack the fungal pheromone signaling pathway to provide a living yeast biosensor where peptide biomarkers were recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, we demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherwise, would likely be difficult to detect using binding-based assays. We characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases-Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, and Schizosaccharomyces japonicus. Alanine scanning was carried out for the most selective of these-S. cerevisiae and C. albicans-with and without the protease. Two peptide variants were discovered, which showed diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides were then distinguished by utilizing the biosensor strains with and without the protease, which selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor with a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. With further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a wide variety of peptide sequences beyond the alanine scanning carried out here. In the future, we envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications.

Structural analysis of the Asn152Gly mutant of P99 cephalosporinase.

P99 cephalosporinase is a class C ß-lactamase that is responsible in part for the widespread bacterial resistance to ß-lactam antibiotics. Mutations of the conserved active-site residue Asn152 of the enzyme have been shown to alter ß-lactam substrate specificity in vivo. Mutation of Asn152 to a glycine is notable in that it exhibits in vivo substrate-selectivity switching. In order to better understand the structural basis for this observed switch, the X-ray crystal structure of the apo Asn152Gly mutant of P99 was determined to 1.95 Å resolution. Unexpectedly, the artificial C-terminal His(6) tag of a symmetrically-related molecule was observed bound in the active site. The His(6) tag makes several interactions with key active-site residues, as well as with several sulfate ions. Additionally, the overall C-terminus occupies the space left vacant upon the mutation of Asn152 to glycine.

Second-generation covalent TMP-tag for live cell imaging.

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.