Nature Chemical Biology

G-protein-coupled receptors (GPCRs) signal through multiple heterotrimeric G proteins, {beta}-arrestins, GPCR kinases (GRKs), and downstream effectors, whose combinatorial interactions shape cellular responses. These events are typically measured with separate assay formats that each capture only part of the network, making comparison across signaling layers difficult. Here, we consolidate a broad set of previously reported GPCR signaling interactions and assay concepts, together with newly designed sensors, into a single NanoBiT split-luciferase framework, allowing multiple layers of signal transduction to be examined side by side in living cells. We show that rational sensor engineering, in particular the positioning of NanoBiT fragments and targeted modification of the tagged proteins, is essential for detecting transient protein-protein interactions. The framework implements assays for G-protein dissociation, {beta}-arrestin recruitment, conformational activation and trafficking, and GRK recruitment, and extends to G-protein-effector and inter-effector interactions across diverse G, G{beta}, G{gamma}, {beta}-arrestin, GRK, adenylyl cyclase, PLC{beta}, and RhoGEF subtypes. It also enables real-time monitoring of the difficult-to-access G12/13-RhoGEF-RhoA pathway. Together, these assays provide a unified NanoBiT readout for systematic, side-by-side dissection of GPCR signaling.

Biomedical research overlooks most genes in favor of a well-studied minority, yet whether analogous blind spots exist in metabolomics remains unknown. We show that reductive amination, forming secondary amines from aldehydes or ketones and amines, generates a previously hidden class of metabolites we term alkamines. Multiplexed synthesis of 8,475 alkamines combined with MS/MS searches across 1.7 billion spectra identified 1,626 candidates across multiple species and organs. Of these, 56 were confirmed in biological samples, including 27 steroid- and 12 drug-derived alkamines matching prescription patterns. Notably, 77% of synthesized alkamines are absent from PubChem. This combinatorial logic likely explains why alkamines have evaded detection and suggests drug metabolism frameworks substantially underestimate drug-derived metabolite diversity. Reductive amination is an overlooked route modifying steroids, bile acids, and xenobiotics.

Lipids exhibit extraordinary molecular diversity, yet tools to selectively manipulate defined lipid classes in living cells are lacking. Here we show that lipid tail structure biases metabolic fate, enabling the design of synthetic lipid analogs with programmable metabolic selectivity. This approach enables selective cellular production of distinct lipid species or subclasses, including types of neutral lipids, phospholipids, sphingolipids, and ether lipids, without genetic or enzymatic perturbation. We further couple metabolic selectivity to chemical functionalization using bifunctional lipids, in which one modification directs metabolic flux and a second enables bioorthogonal tagging. Using this strategy, we achieve selective in situ labeling of different lipid pools in living cells. Together, our work establishes a chemical biology strategy that enables unprecedented precision in modulating, functionalizing, and rewiring the mammalian lipidome.

Bisbenzylisoquinoline alkaloids (bisBIAs) are pharmacologically valuable plant metabolites with complex stereochemical architectures, yet the catalytic principles governing their assembly have remained largely unclear. Here, we elucidate the enzymatic pathway to cyclic bisBIAs and uncover a non-canonical redox-mediated mechanism for post-assembly stereochemical control. We identify cytochrome P450 enzymes that catalyze regioselective oxidative dimerization and macrocyclization of benzylisoquinoline monomers, establishing the macrocyclic scaffold. Subsequent stereochemical specification is achieved by a paired oxidase-reductase module that selectively epimerizes a single stereocenter through a transient imine formation, converting (R,S)-configured intermediates to (S,S)-products. Reconstitution of the pathway in yeast enabled production of both native bisBIAs and non-natural analogs, demonstrating pathway modularity and engineering potential. These results establish the biochemical principle underlying bisBIA biosynthesis and provide a framework for programmable biosynthesis of these complex natural products.

Extracellular proteases are important signaling molecules in coagulation, inflammation, cell migration, and pain. Dysregulation of extracellular protease activity is common in diseases that perturb these critical functions. Engineering cells to sense and respond programmatically to protease activity has applications in biosensing, cell-based screening for protease activity, and therapeutics. Here we report synthetic protease-activated receptors (SynPARs) based on engineered, auto-inhibited G protein-coupled receptors (GPCRs). Relief of autoinhibition by proteolysis enables receptor activation by an exogenous or tethered agonist to generate transgene expression, real-time fluorescence, or endogenous G-protein signaling. We demonstrate SynPAR modularity with diverse secreted proteases, establish a cell-based SynPAR library selection to optimize protease recognition sequences, and control neuronal activity in response to protease activity. Finally, we use SynPARs in the dorsal root ganglion of mice to counteract hyperalgesia produced by trypsin activity, rewiring neurons to produce an analgesic response to a pain-inducing stimulus. Our study establishes SynPAR as a versatile and modular platform for recording, sensing, and responding to pericellular proteolysis. This fills a critical gap in protease-sensing tools and lays the groundwork for protease-activated genetic and cell-based medicines.

Cell-surface degrader platforms typically require target-specific engineering and have therefore been applied to a relatively small set of protein targets. Here we report Z-TAC, a strategy that enables plug-and-play conversion of existing IgG antibodies into cell-surface protein degraders. Across multiple targets from distinct protein families, Z-TAC induced efficient and sustained degradation of both individual receptors and receptor combinations. For a multi-pass membrane receptor lacking selective antagonists, Z-TAC mediated complete receptor degradation and functional inhibition, demonstrating the ability of this platform to overcome the limitations of conventional pharmacological approaches. This study delineates a generalizable and scalable strategy for functional perturbation of the cell-surface proteome.

Commercial penicillin production has relied on microbial fermentation for more than 80 years. Here, we engineered the plant, Nicotiana benthamiana, to produce penicillin G in its leaves by transient expression of up to seven fungal biosynthetic genes. Remarkably, all recombinant proteins localize to the analogous subcellular compartments without engineering signal peptide sequences or post-translational modification sites. Although non-ribosomal peptide synthetases occur widely in fungi and bacteria to produce a plethora of specialized metabolites, their evolutionary distribution does not extend to plants. Our results now open a new metabolic frontier for natural product synthesis, and offer possibilities to address global health concerns through an alternative biotechnology platform for fungal-derived pharmaceutical production.

Inducible control of protein activity with temporal precision is essential for understanding and engineering dynamic cellular behaviors. However, current inducible molecular tools largely rely on overexpression of target proteins, which often disrupts the signaling pathways and cellular functions under investigation. A generalizable method to achieve inducible control of endogenous proteins in mammalian cells remains an unmet need. Here, we present a versatile platform based on engineered streptavidin biomolecular condensates to trap and release endogenously tagged proteins. By tagging endogenous loci with a short streptavidin-binding peptide via CRISPR knock-in, our synthetic streptavidin condensates efficiently partition and functionally inhibit the tagged endogenous proteins. The sequestered cargo protein is rapidly released upon the addition of biotin, restoring protein activity within minutes. We demonstrated the broad applicability of this system by controlling diverse endogenous targets: the anterograde motor KIF5B and retrograde motor DYNC1H1, which regulate intracellular vesicle trafficking, and the Arp2/3 complex subunit ARPC3, which regulates actin dynamics. Furthermore, we developed a dual-inducible system based on rapamycin-dependent condensation of streptavidin, enabling both rapid sequestration and release of endogenous proteins at user-defined time points. Altogether, this engineered streptavidin condensate platform provides a robust, rapid, and scalable approach for manipulating endogenous protein function under physiologically relevant conditions in both basic and translational research.

Engineered bacterial routes for oxidation of non-native alcohols face three challenges: Nicotinamide-dependent enzymes are coupled to cellular redox metabolism, nicotinamide-independent aryl-alcohol oxidases (AAOs) usually express poorly in bacteria, and aldehyde products are rapidly modified by host enzymes. Here, we address these limitations by engineering aldehyde-retaining Escherichia coli for discovery and application of soluble bacterial AAOs. Screening 51 candidates revealed a high-expression sequence cluster containing enzymes that are active on diverse aromatic and furan-based alcohols. Pairing the top-performing AAO with designer pathways in aldehyde-retaining cells enabled modular C-N and C-C bond forming cascades starting from supplied alcohols. By making both the oxidase and its product compatible with the host, this work advances air-driven oxidation of diverse alcohols as a programmable entry point to aldehyde-derived chemistry in engineered bacteria.

Covalent chemistry has transformed small-molecule drug discovery, yet analogous strategies for proteins remain largely inaccessible because covalent warheads cannot be readily integrated into biologics. Conventional genetic code expansion requires engineering a dedicated aminoacyl-tRNA synthetase for each new amino acid, rendering broad warhead screening impractical. Here we introduce AminoX, a platform that bypasses this limitation through direct tRNA acylation, enabling site-specific incorporation of chemically diverse non-standard amino acids (nsAAs), including covalent warhead nsAAs compatible with scalable biologic manufacturing and multifunctional nsAAs. Using a pooled mRNA display workflow, we screened more than 2,000 warhead-position combinations in machine learning-designed de novo miniproteins targeting CTLA-4, enabling parallel interrogation of covalent chemistry, linker geometry, and incorporation site. We confirmed covalent engagement on cells together with enhanced functional blockade. Finally, we demonstrate multifunctional nsAAs that combine covalent warheads with fluorogenic reporters for real-time detection of target engagement, as well as dual nsAA incorporation for macrocyclization and fluorescent imaging of covalent binding on cell surfaces. By uniting synthetic biology, chemical biology, generative protein design, and high-throughput functional selection, AminoX compresses covalent protein engineering timelines by orders of magnitude, accelerating the development of next-generation therapeutics, biosensors, and chemical probes.

While lipidation is a widely observed strategy to promote membrane permeation, whether the factors governing lipid-driven accumulation are shared across the divergent membranes of mammalian and Gram-negative cells remains unresolved. Here, we apply the Chloroalkane Azide-based Membrane Penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. Through targeted endogenous and exogenous modifications, we further resolve how charge, scaffold composition, and individual envelope barriers shape these patterns. Together, these results establish that lipidation is a context-dependent permeation principle that fundamentally diverges between mammalian and diderm envelopes. By showing that hydrophobic modifications routinely hinder Gram-negative cytosolic entry, this work explains the scarcity of lipidated Gram-negative antimicrobials, exposes the limits of lipophilicity-driven optimization, and redefines the physicochemical boundaries for penetrating the diderm envelope.

In vitro transcription (IVT) systems regulated by allosteric transcription factors (aTFs) are central to emerging cell-free biosensing and synthetic biology platforms, yet their performance is often limited by suboptimal protein-DNA interactions and the need for well-characterized regulatory elements. Here, we report an in vitro evolution strategy to engineer DNA operator sequences that enables tunable aTF-DNA interactions without requiring prior detailed knowledge of the native operator or regulatory mechanism. Using a SELEX-based approach with integrated positive and counter-selection steps, we evolved non-natural operators for the sulfane sulfur-responsive transcriptional repressor SqrR. The selected sequences preserve ligand-responsive allostery, with some sequences exhibiting enhanced binding affinity and reducing transcriptional leakage. Notably, we identify operator with binding behaviors consistent with cooperative recruitment of multiple SqrR dimers, suggesting that sequence architecture can modulate aTF-DNA interactions beyond affinity alone. Incorporation of these operators into IVT circuits improves transcriptional control and dynamic range, enabling the development of ROSALIND-based sensors for sulfane sulfur species, achieving sensitive and selective detection in a fully cell-free format. More broadly, this work establishes operator evolution as a programmable strategy to optimize transcription factor-DNA interactions and expand the design space of transcription-based biosensors, including for systems lacking well-characterized genetic components.

The ribosome's DNA-encoded production of defined polymer sequences is naturally limited to 22 amino acids. Although the translation machinery has the latent capacity to polymerize backbone-modified substrates, including {beta}-amino acids, this potential is constrained by the intrinsic -selectivity of native aminoacyl-tRNA synthetases. Here, we address this limitation by "reverse engineering" the Escherichia coli protein ligase EpmA. Naturally activating (R)-{beta}-lysine, EpmA evolved to discard its tRNA-binding domain in favor of protein recognition. By grafting the anticodon-binding domain of the canonical lysyl-tRNA synthetase, LysRS, onto EpmA, we created the chimeric enzyme chEpmA. To our knowledge, this represents the first successful reprogramming of a protein ligase into a functional aminoacyl-tRNA synthetase. We demonstrate that chEpmA serves as a versatile dual-specificity platform: it efficiently charges tRNAs with the non-canonical backbone (R)-{beta}-lysine, and a single substitution unlocks the scaffold for -substrates, thereby enabling a broad spectrum of post-translational modifications previously inaccessible to genetic code expansion. This repertoire ranges from acylated lysines such as N{varepsilon}-succinyl-(S)- lysine (Ksucc) and bulky modifications such as biocytin to advanced glycation end products (AGEs) including N{varepsilon}-carboxymethyl-(S)- lysine (CML). Our work establishes a structural blueprint for mobilizing non-canonical substrates, paving the way for the biosynthesis of protease-resistant peptidomimetics and next-generation therapeutics.

Molecular glues stabilize weak interactions to impart novel functionalities onto complexes. While plant hormones or drugs are known molecular glues, it is still unknown whether this modality provides endogenous regulation in human cells. Here, we show that purine nucleotides are molecular glues that tether the rate-limiting enzyme of purine biosynthesis, phosphoribosyl-pyrophosphate-amidotransferase (PPAT), to its inhibitor NUDT5. This mechanism allows cells to sense purine levels and establish essential feedback control of their synthesis. Thiopurine chemotherapeutics, in clinical use since the 1950s, act as molecular glues of the same complex, but adopt unique orientations for enhanced function. Distinct from the recognition of many therapeutic glues, metabolic glue pockets can adjust their conformation to significant compound alterations and thereby enable increasing glue potency without sacrificing specificity. Our findings therefore identify endogenous metabolic glues as a mode of nutrient sensing that can be exploited to obtain compounds that rewire metabolic pathways for therapeutic benefit.

Camptothecin derivatives are first-line anticancer drugs used worldwide for the treatment of diverse malignant tumors. However, the biosynthetic pathway of camptothecin has remained elusive for five decades. Here, we fully map its entire biosynthetic route. We discovered five key missing enzymes (OpCAR, OpSDR11, OpCS, OpGH1, and OpSTR) via the combination of MALDI mass spectrometry imaging, single-cell RNA sequencing and co-expression analysis. Meanwhile, we demonstrated a free flavin mononucleotide triggered the non-enzymatic 6-5-6 to 6-6-5 fused-ring skeleton rearrangement, filling the last gap in camptothecin biosynthesis. Finally, we validated this identified pathway and achieved the de novo biosynthesis of camptothecin in Saccharomyces cerevisiae. These discoveries uncover the long-standing mystery underlying camptothecin and pave the way for manufacturing camptothecin and its derivatives through synthetic biology approaches.

The lysosome is a major catabolic organelle responsible for the breakdown of both intra- and extracellular substrates1,2. Lysosomal membrane damage mediated by pathologic amyloid fibrils is an area of recent focus3-6. The dipeptide ester LLOMe is typically employed to model lysosomal membrane damage7-11; however its mechanism of membranolysis was previously incompletely understood. Here, in vitro and cell-based analyses, and cryo-electron microscopy and tomography studies reveal LLOMe-derived oligopeptides generated by the lysosomal protease Cathepsin C assemble into cross-{beta}-sheet amyloid fibrils within the lysosome. Additionally, we report lysosome membrane damage triggers the broadly nonspecific dipeptidyl ligase activity of Cathepsin C, facilitating the tagging of proximal proteins within the damaged lysosome lumen with a click chemistry handle: to our knowledge, the first reported localized proximity labeling approach exploiting a fully endogenous, non-engineered enzyme. While Cathepsin C ligase activity has been demonstrated in vitro12,13, our observations of dipeptidyl ligation onto proximal proteins in cells suggests an unexplored role of Cathepsin C in lysosomal biology and broadly exemplifies how other endogenous enzymes might be similarly exploited for proximity labeling. Altogether our results unveil two mechanisms by which dipeptide esters perturb lysosomal homeostasis and provide a roadmap for their utilization toward targeted studies of the lysosome.

Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole-core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the {Omega}perX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 g mL1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

Retro-1 and Retro-2 are structurally distinct small molecules that protect cells from diverse toxins and viruses by disrupting retrograde trafficking, yet their mechanism of action has remained elusive. We show that both compounds target Get3, the ATPase chaperone of the guided entry of tail-anchored proteins (GET) pathway, which mediates biogenesis of tail-anchored SNARE proteins required for retrograde transport to the ER membrane. Cryo-electron microscopy reveals that Retro compounds bind a cryptic pocket in Get3, allosterically stabilizing Get3 in a stalled complex with upstream pathway components. Our work uncovers the GET pathway as an unsuspected vulnerability in pathogen entry, provides clear routes toward compound optimization, and establishes stabilization of dynamic protein complexes as a therapeutic strategy.

Targeted protein degradation (TPD) by PROteolysis TArgeting Chimeras (PROTACs) has emerged as a powerful chemical biology and therapeutic modality, yet many degraders exhibit incomplete target clearance and characteristic rebound kinetics despite continuous exposure. The mechanistic basis for this behavior remains poorly understood. Here we uncover protein age as a previously unrecognized determinant of PROTAC efficacy. Using CG{square}SLENP, a chemical genetics strategy that selectively labels newly synthesized and pre {square}existing proteins within the same living cell, we directly resolve PROTAC{square}induced degradation of distinct intracellular protein populations. Applying this approach to the bromodomain protein BRD4, we show that two mechanistically and structurally distinct PROTACs, dBET6 and MZ{square}1, preferentially degrade pre {square}existing BRD4, while newly synthesized BRD4 is degraded substantially more slowly and incompletely. This age{square}dependent degradation bias is observed in live{square}cell imaging, across compound concentrations and time scales, and for both reporter and endogenous BRD4. These findings reveal that PROTAC{square}mediated degradation is governed not only by target engagement and ternary complex formation, but also by the dynamic balance between protein synthesis and degradation. By identifying temporal proteostasis as a critical parameter in TPD, this work provides a mechanistic framework for incomplete degradation and rebound kinetics and establishes protein maturation state as an important consideration for degrader design and evaluation.