Hu L, Lin J, Sun L
… +18 more, Berezuk AM, Tuttle KS, Zhu X, Seo HS, Dhe-Paganon S, Li P, Sun Y, Ni L, Zhang J, Tan D, Wakimoto H, Cahill DP, Bai X, Luo X, Asara JM, Subramaniam S, Shan Y, Wu X
Gain-of-function mutations of isocitrate dehydrogenase 1 (IDH1) lead to oncometabolite (R)-2-hydroxyglutarate production, contributing to the tumorigenesis of multiple human cancers. While fatty acid biosynthesis is crit...Gain-of-function mutations of isocitrate dehydrogenase 1 (IDH1) lead to oncometabolite (R)-2-hydroxyglutarate production, contributing to the tumorigenesis of multiple human cancers. While fatty acid biosynthesis is critical for IDH1-mutant tumor growth, the underlying mechanisms remain unclear. Here, leveraging chemical probes and chemoproteomic profiling, we identified that oncogenic IDH1-R132H is uniquely autopalmitoylated at C269, which is not observed in wild-type IDH1. This modification responds to fatty acids and regulates R132H enzymatic activity by enhancing substrate and cofactor binding, as well as dimerization. Loss of C269 palmitoylation reverses IDH1-R132H-induced metabolic reprogramming and hypermethylation phenotypes and impairs cell transformation. Interestingly, C269 autopalmitoylation occurs within a hydrophobic pocket, targeted by a clinical IDH1-mutant inhibitor (LY3410738). Our study reveals that autopalmitoylation, conferred by the IDH1 mutation, links fatty acid metabolism to the regulation of IDH1 mutant activity and represents a druggable vulnerability in IDH1-mutant cancers.
Huang B, Jin M, Cui G
… +24 more, Wang Z, Wang F, Chen M, Zhu L, Li Y, Yang X, Li R, Wu J, Zhai L, He Y, Yang J, Ding X, Wang Q, Xv Z, Ouyang Y, Li J, Yang Y, Li K, Lou Z, Mer G, Zhang J, Chen Y, Yuan J, Zhong C
The Warburg effect leads to increased lactate production and promotes cancer progression but the underlying mechanisms remain unclear. Here, we found that lactate activates the MAPK pathway through ERK lactylation, which...The Warburg effect leads to increased lactate production and promotes cancer progression but the underlying mechanisms remain unclear. Here, we found that lactate activates the MAPK pathway through ERK lactylation, which promotes cancer progression. We identified GCN5 as the lactyltransferase responsible for ERK lactylation. Activated ERK phosphorylates GCN5, increasing its lactyltransferase activity toward ERK and establishing a positive feedback loop that amplifies lactate-mediated cancer progression. We provide evidence that lactylation of ERK at residue K231 weakens its interaction with MEK, thereby promoting ERK dimerization and activation. We developed a cell-penetrating peptide that specifically inhibits ERK lactylation. This peptide impairs tumor growth in KRAS-mutant cancer models. Taken together, our findings reveal a molecular mechanism by which lactate accelerates cancer progression through the ERK-GCN5 lactylation-phosphorylation cascade and suggest a strategy to disrupt ERK lactylation in RAS-ERK-driven cancers.
Lipid droplets (LDs) dynamically interact with other organelles, such as mitochondria, in surveillance of cellular metabolic homeostasis. The transient nature of LDs, however, poses technical challenges to snapshot molec...Lipid droplets (LDs) dynamically interact with other organelles, such as mitochondria, in surveillance of cellular metabolic homeostasis. The transient nature of LDs, however, poses technical challenges to snapshot molecular information underlying these interactions. Herein, we present a small-molecule-based photocatalytic protein proximity labeling method (named LipoID) to enable in situ labeling, capturing and profiling of the LD-interacting proteome. This method is enabled by a set of LD-targeting probes designed to catalyze protein modifications nearby LDs using nucleophilic substrates. Profiled by liquid chromatography-tandem mass spectrometry, LipoID identifies tethered interorganellar interactions, particularly with mitochondria, in addition to reliable capture of validated LD biomarkers (for example, perilipins (PLINs)). Coupled with comparative proteomics, LipoID discovers mitochondrial voltage-dependent anion channel 3 as a potential regulator of LD-mitochondria proximity through interacting with PLIN3 on LDs. Further metabolomics analysis suggested remodeled lipid metabolism in line with the LD-mitochondria interaction. Together, LipoID enables in situ profiling of the LD interactome and reveals interorganellar regulation.
Protein ubiquitination critically regulates biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for protein degradation, ubiquitination also modulates enzymatic activity....Protein ubiquitination critically regulates biological processes through both proteolytic and nonproteolytic mechanisms. While classically known for protein degradation, ubiquitination also modulates enzymatic activity. However, current mechanisms of ubiquitination-mediated enzymatic modulation are spatially constrained near enzyme-substrate interfaces. Here, we report a unique ubiquitination-mediated regulatory paradigm that activates the Polycomb repressive deubiquitinase (PR-DUB) complex from a site distal to the enzyme-substrate interface. We found that ASXL1 K351 monoubiquitination promotes nucleosomal H2AK119Ub deubiquitination by stabilizing the PR-DUB catalytic pocket, thereby increasing catalytic velocity (V) without affecting substrate affinity (K). Structurally, ubiquitin at ASXL1 K351 bridges the BAP1 and ASXL1 subunits, functioning as a cross-bracing 'glue' that constrains their conformational dynamics without altering the nucleosome-binding interface. Molecular dynamics and hydrogen-deuterium exchange mass spectrometry revealed that this modification locks PR-DUB in a catalytic state poised for substrate cleavage. This study reveals a unique ubiquitin function of intersubunit fastening through a molecular glue effect and clarifies the mechanism of PR-DUB activation.
Engineering synthetic intrinsically disordered proteins (synIDPs) enables regulation of biomolecular condensation and protein solubility. However, limited understanding of how sequence-dependent interaction cooperativity...Engineering synthetic intrinsically disordered proteins (synIDPs) enables regulation of biomolecular condensation and protein solubility. However, limited understanding of how sequence-dependent interaction cooperativity relates to the fitness impacts of synIDPs on endogenous cellular processes constrains our design capability. Here, to circumvent this design challenge, we present a systematic directed evolution method for the evolution of synIDPs capable of mediating diverse phase behaviors in living cells. The selection methods allow us to evolve a toolbox of synIDPs with distinct phase behaviors and thermoresponsive features in living cells, leading to the evolution of synthetic condensates. The reverse-selection method further allows us to select synIDPs as solubility tags. We demonstrate the applications of the evolved synIDPs in protein circuits to (1) regulate intracellular protein activity and (2) reverse antibiotic resistance. Our systematic evolution and selection strategies provide a versatile platform for developing synIDPs for broad applications in synthetic biology and biotechnology.
Temozolomide is a standard-of-care therapeutic agent for glioblastoma. However, persons who initially respond well often experience a notable reduction in efficacy over time, with the underlying mechanisms remaining uncl...Temozolomide is a standard-of-care therapeutic agent for glioblastoma. However, persons who initially respond well often experience a notable reduction in efficacy over time, with the underlying mechanisms remaining unclear. Here we demonstrate that the reduced response to temozolomide correlates with decreased chromatin accessibility, marked by reduced H3K27ac modification and alterations in chromatin loops. Mechanistically, temozolomide treatment upregulates histone deacetylase 1 (HDAC1) expression. Intriguingly, increased HDAC1 forms condensates independently of its deacetylase function. These condensates arise from multivalent interactions within the intrinsically disordered region and specific interactions with CCCTC-binding factor (CTCF), facilitating resistance to temozolomide by promoting the assembly of DNA repair complexes, even in the absence of direct deacetylase activity of HDAC1. Through phase-separation-based screening, we identified resminostat as an effective disruptor of HDAC1-CTCF condensates, thereby restoring temozolomide sensitivity in patient-derived xenograft models. Our findings introduce deacetylase-independent HDAC1 condensation as a distinct mechanism regulating temozolomide response, providing valuable insights into potential therapeutic strategies.
Histone acetyltransferases (HATs) modify chromatin to regulate gene expression. Instead of acting alone, HATs function in complexes with other proteins, leading to variations in substrate specificity, genomic localizatio...Histone acetyltransferases (HATs) modify chromatin to regulate gene expression. Instead of acting alone, HATs function in complexes with other proteins, leading to variations in substrate specificity, genomic localization and cellular function. To understand the complex-dependent roles of HATs, we present a chemical approach to specifically dissociate ATAC (Ada-two-A-containing) HAT complex from chromatin without perturbing other complexes. Rather than targeting the shared HAT enzyme, we developed chemical inhibitors for an ATAC-specific subunit, YEATS2. The most effective inhibitor, LS-170, specifically reduced the chromatin occupancy of the ATAC complex, decreased the ATAC-dependent histone acetylation level and downregulated the expression of ATAC-governed genes, leading to significantly suppressed tumor growth in a lung cancer mouse model. This study not only sheds light on the regulatory roles of the ATAC HAT complex in gene transcription but also provides evidence that the chemical inhibition of the ATAC complex can be a promising therapeutic strategy.
Serianni VM, Škerlová J, Dubánková AK
… +8 more, Škríba A, Šváchová H, Vučková T, Filimoněnko A, Fábry M, Řezáčová P, Kouba T, Cahova H
Nat Chem Biol
· 2026 Jun · PMID 41513851
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RNA capped with dinucleoside polyphosphates has been discovered in bacteria and eukaryotes only recently. The likely mechanism of this specific capping involves direct incorporation of dinucleoside polyphosphates by RNA...RNA capped with dinucleoside polyphosphates has been discovered in bacteria and eukaryotes only recently. The likely mechanism of this specific capping involves direct incorporation of dinucleoside polyphosphates by RNA polymerase as noncanonical initiating nucleotides. However, how these compounds bind into the active site of RNA polymerase during transcription initiation is unknown. Here, we explored transcription initiation in vitro, using a series of DNA templates in combination with dinucleoside polyphosphates and model RNA polymerase from Thermus thermophilus. We observed that the transcription start site can vary on the basis of the compatibility of the specific template and dinucleoside polyphosphate. Cryo-electron microscopy structures of transcription initiation complexes with dinucleoside polyphosphates revealed that both nucleobase moieties can pair with the DNA template. The first encoded nucleotide pairs in a canonical Watson-Crick manner, whereas the second nucleobase pairs noncanonically in a reverse Watson-Crick manner. Our work provides a structural explanation of how dinucleoside polyphosphates initiate RNA transcription.
Lipid phosphate phosphatases (LPPs) catalyze the dephosphorylation of a broad range of bioactive lipid phosphates, including lysophosphatidic acid and sphingosine-1-phosphate, playing essential roles in embryonic vasculo...Lipid phosphate phosphatases (LPPs) catalyze the dephosphorylation of a broad range of bioactive lipid phosphates, including lysophosphatidic acid and sphingosine-1-phosphate, playing essential roles in embryonic vasculogenesis, cell differentiation and inflammation. Here we present the cryo-electron microscopic structure of human LPP1 as a tetramer with C4 symmetry. We capture the phosphohistidine intermediate state by using vanadate as a phosphate analog, where vanadate is coordinated by positively charged residues from three conserved motifs (C1, C2 and C3). Structural investigations of LPP1 variants with mutations in two catalytic histidine residues confirm that the histidine in the C2 motif facilitates phosphate bond cleavage. Enzymatic assays validate our structural observations. Additionally, a phosphatidylinositol 4,5-bisphosphate (PIP) molecule was discovered in the LPP1 structure, underscoring a potential regulatory role for PIP in the catalytic activity of LPP1.
Goetzke FW, Bernard SM, Ju CW
… +7 more, Pollock J, DeMeester KE, Gross J, Simon GM, He C, Melillo B, Cravatt BF
Nat Chem Biol
· 2026 May · PMID 41507545
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Adaptors serve as hubs to regulate diverse protein complexes in cells. This multitude of functions can complicate the study of adaptors, as their genetic disruption may simultaneously impair the activities of several com...Adaptors serve as hubs to regulate diverse protein complexes in cells. This multitude of functions can complicate the study of adaptors, as their genetic disruption may simultaneously impair the activities of several compositionally distinct complexes (or adaptor 'complexoforms'). Here we describe the chemical proteomic discovery of bicyclopyrrolidine acrylamide stereoprobes that react with C100 of the methyltransferase (MT) adaptor TRMT112 in human cells. Curiously, the stereoprobes showed negligible reactivity with uncomplexed recombinant TRMT112 and we found that this interaction was restored exclusively in the presence of METTL5 but not other MTs. A cocrystal structure revealed stereoprobe binding to a composite pocket proximal to C100 of TRMT112 that is templated by METTL5 and absent in other TRMT112:MT complexes. Structural rearrangements promoted by stereoprobe binding in turn lead to allosteric agonism of METTL5, thus revealing how covalent ligands targeting a pleiotropic adaptor can confer partner-specific functional effects through reactivity with a single complexoform.
Nat Chem Biol
· 2026 May · PMID 41501183
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Cellular lipid metabolism is subject to strong homeostatic regulation, but the players involved in and mechanisms underlying these pathways remain largely uncharacterized. Here we develop a 'feeding-fishing' approach cou...Cellular lipid metabolism is subject to strong homeostatic regulation, but the players involved in and mechanisms underlying these pathways remain largely uncharacterized. Here we develop a 'feeding-fishing' approach coupling membrane editing using optogenetic lipid-modifying enzymes (feeding) with organelle membrane proteomics through proximity labeling (fishing) to elucidate molecular players and pathways involved in the homeostasis of phosphatidic acid (PA), a multifunctional lipid central to glycerolipid metabolism. This approach identified several PA-metabolizing enzymes and lipid transfer proteins enriched in and depleted from PA-fed membranes. Mechanistic analysis revealed that PA homeostasis in the cytosolic leaflets of the plasma membrane and lysosomes is mediated by both local PA metabolism and the action of lipid transfer proteins that carry out interorganelle lipid transport before subsequent metabolism. More broadly, the interfacing of membrane editing to controllably modify membrane lipid composition with organelle membrane proteomics using proximity labeling represents a strategy for revealing mechanisms governing lipid homeostasis.
Engineering DNA polymerases to efficiently synthesize artificial or noncognate nucleic acids remains an essential challenge in synthetic biology. Here we describe an evolutionary campaign designed to convert a family of...Engineering DNA polymerases to efficiently synthesize artificial or noncognate nucleic acids remains an essential challenge in synthetic biology. Here we describe an evolutionary campaign designed to convert a family of highly selective DNA polymerases into an unnatural homolog with strong RNA synthesis activity. Starting from a homologous recombination library, a short evolutionary path was achieved using a single-cell droplet-based microfluidic selection strategy to produce C28, a newly engineered polymerase that can synthesize RNA with a rate of ~3 nt s and of >99% fidelity. C28 is capable of long-range RNA synthesis, reverse transcription and chimeric DNA-RNA amplification using the PCR. Despite strong discrimination against other genetic systems, C28 readily accepts several 2'F and base-modified RNA analogs. Together, these findings highlight the power of directed evolution as an approach for reprogramming DNA polymerases with activities that could help drive future applications in biotechnology and medicine.
Ludig DL, Zhai X, Rittner A
… +7 more, Gusenda C, Heinz M, Berlage S, Gao N, Jervis AJ, Zhou YJ, Grininger M
Nat Chem Biol
· 2026 Jul · PMID 41501181
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Metazoan fatty acid (FA) synthases (mFASs) facilitate the de novo synthesis of C16- and C18-FAs through iterative extensions within the FA cycle and hydrolytic release. Here we re-engineer mFAS to fine-tune the interplay...Metazoan fatty acid (FA) synthases (mFASs) facilitate the de novo synthesis of C16- and C18-FAs through iterative extensions within the FA cycle and hydrolytic release. Here we re-engineer mFAS to fine-tune the interplay between FA extension and FA hydrolytic release for the targeted production of short- and medium-chain fatty acids. Single amino acid exchanges in the ketosynthase domain can redirect FA product profiles from predominantly C8 (G113W) to C8/C10 (G113F) and C12/C14 (G113M). Integration of a thioreductase domain enables the production of medium-chain fatty aldehydes and alcohols. We apply our approach for controlling chain length in FA biosynthesis to the microbial production of C10- and C12-FAs, translate it into a yeast cell factory and achieve C10/C12-FAs titers of 674 mg l and 67% purity of total free FAs. Our work demonstrates a modular platform for programmable FA synthesis and paves the way toward sustainable bioproduction of valuable oleochemicals.
Capener JL, Schwalm MP, Vasta JD
… +8 more, Michaud A, Teske KA, Marsiglia WM, Huber KVM, Dar AC, Knapp S, Axtman AD, Robers MB
Nat Chem Biol
· 2026 May · PMID 41495225
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Assessing drug-target engagement in living cells is essential for verifying the activity of pharmacological agents. Traditional binding assays often overlook key factors such as permeability, intracellular distribution a...Assessing drug-target engagement in living cells is essential for verifying the activity of pharmacological agents. Traditional binding assays often overlook key factors such as permeability, intracellular distribution and complex formation that influence target occupancy in cells. Bioluminescence resonance energy transfer (BRET)-based probes enable direct, quantitative assessment of small-molecule binding to proteins in live, intact cells. BRET provides sensitive detection of target engagement across a wide range of target classes, including less-tractable proteins in membrane compartments. Compared to other existing methods, BRET offers quantification of drug occupancy at steady state and open system regimens. Recent innovations in this platform have expanded its utility beyond occupancy confirmation to include applications in polypharmacology and mechanism-of-action studies. Here, we provide an updated perspective on BRET target engagement assays as versatile tools for chemical biology and early-stage drug discovery.
The activation of H2B K120 monoubiquitylation (H2BK120ub) by H2B S112 GlcNAcylation (H2BS112GlcNAc) has an important role in regulating transcriptional activation, yet its mechanism remains unclear. Here we chemically sy...The activation of H2B K120 monoubiquitylation (H2BK120ub) by H2B S112 GlcNAcylation (H2BS112GlcNAc) has an important role in regulating transcriptional activation, yet its mechanism remains unclear. Here we chemically synthesized H2BS112GlcNAc-modified nucleosomes and quantitatively evaluated how H2BS112GlcNAc stimulates ubiquitylation by RNF20/RNF40-RAD6A E3-E2 enzymes. Cryo-electron microscopy determination of a chemically trapped RNF20/RNF40-RAD6A-Ub-H2BS112GlcNAc nucleosome complex revealed that the H2BS112GlcNAc moiety interacts with the E2 enzyme RAD6A but not the E3 ligase RNF20/RNF40. Mutagenesis and kinetics analyses demonstrated that H2BS112GlcNAc allosterically stimulates ubiquitin transfer from the RAD6A~Ub thioester to H2B K120 by enhancing the nucleophilicity of H2B K120. Structure‒activity relationship analysis further identified the essential roles of the C2 N-acetyl group and the β-configuration of C1 on the H2BS112GlcNAc moiety. These findings provide the structural evidence of histone posttranslational modification crosstalk involving O-GlcNAcylation and reveal how O-GlcNAcylation can allosterically stimulate enzyme activity through substrate modification.
Wu R, Li N, Wen Z
… +13 more, Wang Y, Li S, Zhou H, Huang Y, Xie X, Zhao X, Wang X, Tao S, Chai G, Wang TA, Li Z, Chen PR, Jing M
Nat Chem Biol
· 2026 Jul · PMID 41495223
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G-protein-coupled receptors (GPCRs) are transmembrane proteins that transduce extracellular stimuli into intracellular events. While central to physiology and drug discovery, approximately 100 GPCRs remain orphan, limiti...G-protein-coupled receptors (GPCRs) are transmembrane proteins that transduce extracellular stimuli into intracellular events. While central to physiology and drug discovery, approximately 100 GPCRs remain orphan, limiting insights into their biology. We establish a generalizable photo-cross-linking-assisted GPCR deorphanization platform that leverages site-specifically incorporated photo-cross-linkers for interface-selective ligand capture from native biological samples. We systematically demonstrate the sensitivity, specificity and broad applicability of our system using multiple GPCR-ligand pairs and further deorphanize GPR50 with the neuropeptide Little-LEN (L-LEN) as its endogenous ligand. L-LEN selectively binds GPR50 and modulates cellular activities through downstream Gα signaling in tissue. In behaving mice, L-LEN functionally coordinates with GPR50 to regulate energy expenditure and thermogenesis, mechanistically through brain-adipose cross-talk, whereas their deficiency increased the likelihood of torpor following challenges. In summary, we develop an efficient platform for GPCR deorphanization from native samples, and the deorphanization of GPR50 provides insights into its function and drug discovery.
Ruan Z, Lee J, Li Y
… +3 more, Orozco IJ, Du J, Lü W
Nat Chem Biol
· 2026 Mar · PMID 41491833
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TRPM5 is a Ca-activated monovalent cation channel essential for taste perception, insulin secretion and gastrointestinal chemosensation. Canonical TRPM5 activation requires Ca binding at two distinct sites: an agonist si...TRPM5 is a Ca-activated monovalent cation channel essential for taste perception, insulin secretion and gastrointestinal chemosensation. Canonical TRPM5 activation requires Ca binding at two distinct sites: an agonist site within the lower vestibule of the S1-S4 pocket in the transmembrane domain (Ca) and a modulatory site in the intracellular domain (Ca) that tunes voltage dependence and agonist sensitivity. Here we characterize CBTA as a noncalcium agonist that binds to the upper vestibule of the S1-S4 pocket, directly above Ca. CBTA alone mimics the dual role of Ca and Ca, merging agonist activation with voltage modulation. CBTA also renders TRPM5 supersensitive to Ca, synergistically hyperactivating the channel even at near-resting Ca levels. We further demonstrate that the inhibitor triphenylphosphine oxide binds the same site but stabilizes a nonconductive state. These opposing effects reveal the upper S1-S4 pocket as a multifunctional regulatory hub integrating activation, inhibition and modulation in TRPM5.
Chen H, Peng H, Ellis T
… +1 more, Ledesma-Amaro R
Nat Chem Biol
· 2026 Jul · PMID 41491832
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In multicellular systems, engineering-controlled cell-cell adhesion and metabolic interdependence are vital for developing complex functionalities. This study introduces a yeast synthetic toolbox for modular cell-cell ad...In multicellular systems, engineering-controlled cell-cell adhesion and metabolic interdependence are vital for developing complex functionalities. This study introduces a yeast synthetic toolbox for modular cell-cell adhesion and cocultures, aiming to overcome the limitations of existing approaches that lack genetic specificity and control. First, a model yeast strain 007Δ is created with seven main flocculation and agglutination genes removed, providing a clean background for synthetic adhesion systems. Then, three distinct adhesion pair systems-Strategy 1, Strategy 2.1 and Strategy 2.2-are established involving yeast flocculation and agglutination proteins and yeast surface display systems. In addition, a quantitative assessment is conducted on the adhesive specificity and strength, alongside the capability of synthetic adhesion to generate patterns. Finally, we successfully demonstrate enhanced bioproduction of the high-value food antioxidant, resveratrol, utilizing synthetic cocultures coupled with cell adhesion systems. We anticipate that this toolkit will emerge as a valuable resource for diverse applications in synthetic biology and biomanufacturing.