Nature chemical biology最新文献

Cell-free systems are powerful synthetic biology technologies that can recapitulate gene expression and sensing without the complications of living cells. Cell-free systems can perform more advanced functions when genetic circuits are incorporated. Here we expand cell-free biosensing by engineering a highly specific isothermal amplification circuit called polymerase strand recycling (PSR), which leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We first construct simple PSR circuits to detect different RNA targets with high specificity. We then interface PSR circuits to amplify signals from allosteric transcription factor-based biosensors for small molecule detection. A double equilibrium model of transcription factor–DNA/ligand binding predicts that PSR can improve biosensor sensitivity, which we confirm experimentally by improving the limits of detection by 10-fold to submicromolar levels for two biosensors. We believe this work expands the capabilities of cell-free circuits and demonstrates PSR’s potential for diverse applications in biotechnology.

Heat shock factor 1 (HSF1) is the critical orchestrator of cell responses to heat shock, and its dysfunction is linked to various diseases. HSF1 undergoes phase separation upon heat shock, and its activity is regulated by post-translational modifications (PTMs). The molecular details underlying HSF1 phase separation, temperature sensing and PTM regulation remain poorly understood. Here, we discovered that HSF1 exhibits temperature-dependent phase separation with a lower critical solution temperature behavior, providing a new conceptual mechanism accounting for HSF1 activation. We revealed the residue-level molecular details of the interactions driving the phase separation of wild-type HSF1 and its distinct PTM patterns at various temperatures. The mapped interfaces were validated experimentally and accounted for the reported HSF1 functions. Importantly, the molecular grammar of temperature-dependent HSF1 phase separation is species specific and physiologically relevant. These findings delineate a chemical code that integrates accurate phase separation with physiological body temperature control in animals.

Cell surface receptor-targeted protein degraders hold promise for drug discovery. However, their application is restricted because of the complexity of creating bifunctional degraders and the reliance on specific lysosome-shuttling receptors or E3 ubiquitin ligases. To address these limitations, we developed an autophagy-based plasma membrane protein degradation platform, which we term AUTABs (autophagy-inducing antibodies). Through covalent conjugation with polyethylenimine (PEI), the engineered antibodies acquire the capacity to degrade target receptors through autophagy. The degradation activities of AUTABs are self-sufficient, without necessitating the participation of lysosome-shuttling receptors or E3 ubiquitin ligases. The broad applicability of this platform was then illustrated by targeting various clinically important receptors. Notably, combining specific primary antibodies with a PEI-tagged secondary nanobody also demonstrated effective degradation of target receptors. Thus, our study outlines a strategy for directing plasma membrane proteins for autophagic degradation, which possesses desirable attributes such as ease of generation, independence from cell type and broad applicability.

Ribozymes that catalyze site-specific RNA modification have recently gained increasing interest for their ability to mimic methyltransferase enzymes and for their application to install molecular tags. Recently, we reported SAMURI as a site-specific alkyltransferase ribozyme using S-adenosylmethionine (SAM) or a stabilized analog to transfer a methyl or propargyl group to N³ of an adenosine. Here, we report the crystal structures of SAMURI in the postcatalytic state. The structures reveal a three-helix junction with the catalytic core folded into four stacked layers, harboring the cofactor and the modified nucleotide. Detailed structure–activity analyses explain the cofactor scope and the structural basis for site selectivity. A structural comparison of SAMURI with SAM riboswitches sheds light on how the synthetic ribozyme overcomes the strategies of natural riboswitches to avoid self-methylation. Our results suggest that SAM and its analogs may serve as substrates for various RNA-catalyzed reactions, for which the corresponding ribozymes remain to be identified.

Engineering cells to sense and respond to environmental cues often focuses on maximizing gene regulation at the single-cell level. Inspired by population-level control mechanisms like the immune response, we demonstrate dynamic control and amplification of gene regulation in bacterial populations using programmable plasmid-mediated gene transfer. By regulating plasmid loss rate, transfer rate and fitness effects via Cas9 endonuclease, F conjugation machinery and antibiotic selection, we modulate the fraction of plasmid-carrying cells, serving as an amplification factor for single-cell-level regulation. This approach expands the dynamic range of gene expression and allows orthogonal control across populations. Our platform offers a versatile strategy for dynamically regulating gene expression in engineered microbial communities.

Artificial metalloenzymes (ArMs) integrated within whole cells have emerged as promising catalysts; however, their sensitivity to metal centers remains a systematic challenge, resulting in diminished activity and turnover. Here we address this issue by inducing in cellulo liquid–liquid phase separation through a self-labeling fusion protein, HaloTag–SNAPTag. This strategy creates membraneless, isolated liquid condensates within Escherichia coli as protective compartments for the assembly of ArMs using the same fusion protein. The approach allows for high ArM loading and stabilization by localizing the ArMs within the phase-separated regions. Consequently, the performance of ArM-based whole-cell catalysts is improved, with a demonstrated turnover per cell of up to 7.1 × 10⁹ for the olefin metathesis reaction. Furthermore, we apply this to an engineered E. coli system in live mice, where host bacterial cells confine the metal catalytic species, and in a mouse colorectal cancer model, where ArM-containing whole-cell catalysts mediate concurrent reactions to activate prodrugs.

Intricate coupling between metabolism and protein post-translational modifications (PTMs) has emerged as a fundamental aspect of cellular regulation. Recent studies demonstrate that protein modifications can originate from diverse metabolites, and that their regulation is closely tied to the cellular metabolic state. Here we explore recently uncovered PTMs, including the concept of ‘modification of a modification’, as well as associated feedback and feedforward regulatory mechanisms, in which modified proteins impact not only related metabolic pathways but also other signaling cascades affecting physiology and diseases. The recently uncovered role of nucleus-localized metabolic enzymes for histone modifications additionally highlights the importance of cell-compartment-specific metabolic states. We further comment on the utility of untargeted metabolomics and proteomics for previously unrecognized PTMs and associated metabolic patterns. Together, these advances have uncovered a dynamic interplay between metabolism and PTMs, offering new perspectives for understanding metabolic regulation and developing targeted therapeutic strategies.

Opioid receptors, a subfamily of G protein-coupled receptors (GPCRs), are key therapeutic targets. In the canonical GPCR activation model, agonist binding is required for receptor–G protein complex formation, while antagonists prevent G protein coupling. However, many GPCRs exhibit basal activity, allowing G protein association without an agonist. The pharmacological impact of agonist-free receptor–G protein complexes is poorly understood. Here we present biochemical evidence that certain κ-opioid receptor (KOR) inverse agonists can act via KOR–G_i protein complexes. To investigate this phenomenon, we determined cryo-EM structures of KOR–G_i protein complexes with three inverse agonists: JDTic, norBNI and GB18, corresponding to structures of inverse agonist-bound GPCR–G protein complexes. Remarkably, the orthosteric binding pocket resembles the G protein-free ‘inactive’ receptor conformation, while the receptor remains coupled to the G protein. In summary, our work challenges the canonical model of receptor antagonism and offers crucial insights into GPCR pharmacology.

O-Fucosylation plays crucial roles in various essential biological events. Alongside the well-established O-fucosylation of epidermal growth factor-like repeats by protein O-fucosyltransferase 1 (POFUT1) and thrombospondin type 1 repeats by POFUT2, we recently identified a type of O-fucosylation on the elastin microfibril interface (EMI) domain of Multimerin-1 (MMRN1). Here, using AlphaFold2 screens, co-immunoprecipitation, enzymatic assays combined with mass spectrometric analysis and CRISPR–Cas9 knockouts, we demonstrate that FUT10 and FUT11, originally annotated in UniProt as α1,3-fucosyltransferases, are actually POFUTs responsible for modifying EMI domains; thus, we renamed them as POFUT3 and POFUT4, respectively. Like POFUT1/2, POFUT3/4 function in the endoplasmic reticulum, require folded domain structures for modification and participate in a non-canonical endoplasmic reticulum quality control pathway for EMI domain-containing protein secretion. This finding expands the O-fucosylation repertoire and provides an entry point for further exploration in this emerging field of O-fucosylation.