Xiong Y, Tomares DT, Guo J
… +8 more, Sato K, Zeng L, Tian Y, Su M, Albis A, Pant A, Pappu RV, Su X
Nat Chem Biol
· 2026 Jul · PMID 41760814
·
Full text
Biomolecular condensates are membraneless bodies that organize biochemical reactions typically within cells. However, the roles of condensates in extracellular space-where conditions differ substantially from intracellul...Biomolecular condensates are membraneless bodies that organize biochemical reactions typically within cells. However, the roles of condensates in extracellular space-where conditions differ substantially from intracellular space-remain poorly understood. Here we report that mast cell extracellular granules (MCEGs), a stable membraneless entity, are condensates assembled through electrostatic interactions between glycosaminoglycans and polyamines. Disrupting polyamine synthesis or trafficking blocks MCEG formation and compromises the storage of proteases and cytokines. Granules reconstituted with heparin and spermine are sufficient to enrich mediators such as carboxypeptidase A3 (CPA3) and tumor necrosis factor (TNF), maintaining an elevated pH and higher concentrations of calcium and zinc compared to the extracellular milieu. This unique environment enhances CPA3 enzymatic activity. Furthermore, the granules increase TNF binding and its bioactivity toward endothelial cells. Together, we reveal MCEGs as functionally active biomolecular condensates with distinct biochemical and immunological properties; MCEGs are formed through sugar-metabolite interactions, expanding the mechanisms of condensate assembly beyond classical protein-protein and protein-RNA interactions.
Cysteine is one of the rarest amino acids yet exerts a profound influence on biology through the exceptional chemistry of its thiol group. Tunable acidity, high nucleophilicity and access to multiple oxidation states pos...Cysteine is one of the rarest amino acids yet exerts a profound influence on biology through the exceptional chemistry of its thiol group. Tunable acidity, high nucleophilicity and access to multiple oxidation states position cysteine as both a dominant cellular redox buffer and a privileged regulatory site. Chemoproteomics has revealed a vast, dynamic cysteine redoxome in which oxidative post-translational modifications act as sensors, switches and buffers across metabolism, signaling and stress responses, respectively. This study advances the following three frameworks: (1) separating intrinsic reactivity from redox sensitivity and regulatory function; (2) using probe chemistry to capture metastable intermediates with site-level precision; and (3) integrating ratiometric measurements with occupancy, exposure and flux to decode redox dynamics. Case studies show how ratiometric chemoproteomics resolves distinct oxoform kinetics, links enzymatic repair to function and exposes the cysteine redoxome as a dynamic regulatory layer and frontier for therapeutic targeting.
The lack of organelle-targeted neurotransmitter probes limits understanding of their intracellular roles. Here we created an organelle-targeted neurotransmitter nanoprobe using specific molecule-trapped DNA nanostructure...The lack of organelle-targeted neurotransmitter probes limits understanding of their intracellular roles. Here we created an organelle-targeted neurotransmitter nanoprobe using specific molecule-trapped DNA nanostructures for multiple recognition effects. In particular, we designed phenylboronic acid derivatives for chemical reaction and hydroxymethyl groups for forming hydrogen bonding with norepinephrine (NE), which were confined into tetrahedral DNA nanostructures with the optimized spatial effects, achieving the specific and rapid NE identification. Moreover, cyanine 3 providing built-in correction was designed for accurate NE quantification and the HaloTag ligand was synthesized for HaloTag protein targeting onto the organelle membrane, which were bonded to tips of the DNA nanostructure. The developed nanotrap demonstrated high selectivity, fast response (~50 ms), good stability and biocompatibility for organelle NE imaging. Using this tool, we discovered that traumatic brain injury triggers NE bursts in the endoplasmic reticulum, inducing endoplasmic reticulum (ER) stress, altering ER-mitochondrial protein regulation, promoting mitophagy and mitochondrial dysfunction and ultimately causing neuronal death.
Reactive sulfur species (RSS) are increasingly recognized as important bioactive agents across phyla. Application of newly developed chemical tools, detection methods and multi-omics techniques has uncovered new and spec...Reactive sulfur species (RSS) are increasingly recognized as important bioactive agents across phyla. Application of newly developed chemical tools, detection methods and multi-omics techniques has uncovered new and specific roles of endogenous RSS and revealed that a number of biological actions previously attributed to reactive oxygen species or reactive nitrogen species can also be mediated by RSS. This Review describes the versatile chemical biology of RSS with focus on persulfide formation on cysteine residues. We examine their pro-oxidant and antioxidant capacities, involvement in redox signaling and metabolic pathways, stress responses, and their role in the pathophysiology of major disease groups, including cardiovascular and neurodegenerative diseases and cancer. We also provide a critical discussion of available detection methods and potential pharmacological and genetic approaches to adjusting persulfide levels. We cover current knowledge and its limitations, along with practical recommendations for advancing persulfide-based therapeutic interventions.
Scheller L, Giordano Attianese GMP, Castellanos-Rueda R
… +16 more, Di Roberto RB, Barden M, Triboulet M, Hafezi M, Ash S, Shui S, Cribioli E, Reichenbach P, Maillard J, Marchand A, Georgeon S, Wolf B, Abken H, Reddy S, Correia BE, Irving M
Chimeric antigen receptor (CAR) T cell therapy is constrained by on-target, off-tumor toxicities and cellular exhaustion because of chronic antigen exposure. CARs incorporating small-molecule controlled on- and off-switc...Chimeric antigen receptor (CAR) T cell therapy is constrained by on-target, off-tumor toxicities and cellular exhaustion because of chronic antigen exposure. CARs incorporating small-molecule controlled on- and off-switches can enhance both safety and therapeutic efficacy but their design is limited by the scarcity of nonimmunogenic protein elements responsive to nonimmunosuppressive, clinically approved drugs with favorable pharmacodynamics. Here we combine rational design and library-based optimization of a protein-protein interaction (PPI) of human origin to develop venetoclax-controlled drug-regulated off-switch PPI (DROP)-CARs. DROP-CARs enable dose-dependent release of the tumor-targeting scFv and consequent reduction in T cell binding to the tumor cell. Additionally, we present proof of concept for a dual DROP-CAR controlled by different small molecules, as well as for logic-gated synthetic receptors enabling STAT3 signaling. We demonstrate in vitro and in vivo function of DROP-CAR T cells and conclude that the approach holds promise for clinical application.
Zhou JX, Shao ZY, Zhang L
… +19 more, Guo JN, Wang M, Xu Q, Wang YQ, Xu Q, Zhou D, Ren SX, Yu YH, Lu ZH, Pang GZ, Cao Y, Liu YL, Zhou B, Ji HB, Chen YH, Wu HP, Xu GL, Zhang L, Du YR
Mechanosensitive PIEZO channels are thought to open via tension-induced flattening of peripheral transmembrane arm domains, yet the structural basis of this activation remains unclear. Here, by leveraging hybrid-resoluti...Mechanosensitive PIEZO channels are thought to open via tension-induced flattening of peripheral transmembrane arm domains, yet the structural basis of this activation remains unclear. Here, by leveraging hybrid-resolution molecular dynamics simulations, we uncover how large-scale PIEZO2 arm movements funnel into subtle gating motions in the central pore under physiological tension. Arm flattening correlates with anticlockwise rotation of the pore relative to the arms and with clockwise twisting of inner pore helices. These clockwork motions open the pore in a two-step fashion, yielding a fully conducting state and a stable subconducting state populated at a low tension, which was detected electrophysiologically. The fully open PIEZO2 pore is walled by both lipids and amino acids and recapitulates minimal pore size, conductance, ion selectivity and outward rectification of chloride currents measured electrophysiologically. These findings provide structural insights into PIEZO2 gating and demonstrate hybrid-resolution molecular dynamics as a powerful approach to study large-scale membrane protein dynamics and guide drug discovery.
Shaum JB, Muñoz I Ordoño M, Steen EA
… +31 more, Wenge DV, Cheong H, Janowski J, Hunkeler M, Bilotta EM, Rutter ZJ, Barta PA, Thornhill AM, Milosevich N, Hargis LM, Bishop TR, Carter TR, da Camara B, Hinterndorfer M, Dada L, He WJ, Offensperger F, Furihata H, Schweber SR, Hatton C, Wen Y, Cravatt BF, Engle KM, Donovan KA, Melillo B, Kitamura S, Ciulli A, Armstrong SA, Fischer ES, Winter GE, Erb MA
Nat Chem Biol
· 2026 Feb · PMID 41699285
·
Full text
Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While the discovery of heterobifunctional CIPs is expedited by rational design strat...Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry. While the discovery of heterobifunctional CIPs is expedited by rational design strategies, molecular glues have relied predominantly on serendipity. We hypothesized that preexisting ligands could be systematically decorated with chemical modifications to discover compounds that recruit proteins to a composite protein-ligand interface. Using sulfur(VI) fluoride exchange-based high-throughput chemistry (HTC) to install 3,163 structurally diverse building blocks onto ENL (eleven-nineteen leukemia) and BRD4 (bromodomain-containing protein 4) ligands, we screened each analog for degrader activity. This revealed dHTC1, an ENL degrader that recruits CRL4 complex through an extended interface of protein-protein contacts and only engages CRBN after pre-forming the ENL:dHTC1 complex. We also identified dHTC3, a molecular glue that selectively dimerizes BRD4 bromodomain 1 to SCF, an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and new effectors for proximity pharmacology.
Cell-cell communications involve signal transmission from sending cells to receiving cells expressing specific receptors. Extracellular vesicles (EVs) mediate this process by transporting diverse biomolecules. G-protein-...Cell-cell communications involve signal transmission from sending cells to receiving cells expressing specific receptors. Extracellular vesicles (EVs) mediate this process by transporting diverse biomolecules. G-protein-coupled receptors (GPCRs) are canonical membrane receptors that integrate various extracellular signals into intracellular responses. However, whether and how GPCRs engage in EV-mediated communications remain elusive. Here, we report that adhesion GPCRs (aGPCRs) induce the formation of migrasomes and retractosomes, two newly identified EV subtypes, through their extracellular adhesion-like domains and G-protein signaling. Remarkably, activated receptors undergo ectocytosis into these EVs and are subsequently internalized by receiving cells, eliciting de novo G-protein activation. We further demonstrate that cancer-cell-derived migrasomes transfer aGPCRs such as GPR56 to endothelial cells in vitro and in vivo, thereby enhancing angiogenic potential. Together, our findings uncover that aGPCRs promote migrasome formation and provide a novel mechanism of cell-cell communications through EV-mediated intercellular spread of active GPCRs.
Kroell AS, Hoffmann KH, Motzkus NA
… +9 more, Lemmen N, Happ N, Wolf B, von Bachmann AL, Southern N, Vogd F, Aschenbrenner S, Niopek D, Mathony J
Nat Chem Biol
· 2026 May · PMID 41680487
·
Full text
Thermogenetics enables noninvasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here,...Thermogenetics enables noninvasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli, we generated potent, thermoswitchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41 °C). Extending this strategy to mammalian systems, we engineered CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Lastly, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermosensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.
Subcellular stress profoundly influences protein synthesis. However, both the nature of spatiotemporally restricted chemical cues and local protein responders to these cues remain elusive. Unlocking these mechanisms requ...Subcellular stress profoundly influences protein synthesis. However, both the nature of spatiotemporally restricted chemical cues and local protein responders to these cues remain elusive. Unlocking these mechanisms requires the ability to functionally map in living systems locale-specific stress responder proteins and interrogate how chemical modification of each responder impacts proteome synthesis. We resolved this problem by integrating precision localized electrophile generation and genetic code expansion tools. Upon examination of four distinct subcellular locales, only nuclear-targeted electrophile stress stalled translation. We discovered that NCBP1-a nuclear-resident protein with multifaceted roles in eukaryotic mRNA biogenesis-propagated this nuclear stress signal through a single cysteine (C436) from among its 19 conserved cysteines. This NCBP1(C436)-specific modification elicited alternative splicing of more than 250 genes. Mechanistically, global protein synthesis stall was choreographed by impaired association between electrophile-modified NCBP1(C436) and SF3A1, an essential component of spliceosome, triggering the production of alternatively spliced S6 kinase, whose expression was sufficient to dominantly inhibit protein translation.
Becker AP, Biletch E, Kennelly JP
… +14 more, Hong SG, Julio AR, Villanueva M, Nagari RT, Turner DW, Burton NR, Fukuta T, Cui L, Xiao X, Vellani Z, Nguyen A, Mack JJ, Tontonoz P, Backus KM
The physical properties of cellular membranes are influenced by protein and lipid interactions. In situ proximity labeling interactomic methods are well suited to characterize these dynamic and often fleeting interaction...The physical properties of cellular membranes are influenced by protein and lipid interactions. In situ proximity labeling interactomic methods are well suited to characterize these dynamic and often fleeting interactions. Yet, available methods require distinct chemistries for proteins and lipids. Here we establish a singlet oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids using cell-penetrant photosensitizer reagents. Cholesterol-directed POCA captured known and unprecedented cholesterol-binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by physiologically relevant lipoprotein uptake. Protein-directed POCA accurately mapped intracellular membrane complexes, defined sterol-dependent changes to the interactome of the cholesterol transport protein Aster-B and revealed singlet oxygen-mediated domain-specific Aster crosslinking. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, fulfilling unmet needs in intracellular singlet oxygen-based proximity labeling proteomics.