Multimerization and phase separation represent two paradigms for organizing receptor tyrosine kinases (RTKs). However, their functional distinctions from the perspective of biomolecular organization remain unclear. Here, we present CORdensate, a light-controllable condensation system combining two synergistic photoactuators: oligomeric Cry2 and heterodimeric LOVpep/ePDZ. Engineering single-chain photoswitches, we achieve four biomolecular organization patterns ranging from monomerization to phase separation. CORdensate exhibits constant assembly and disassembly kinetics. Applying CORdensate to mimic pathogenic RTK granules establishes the role of phase separation in activating ALK and RET. Moreover, assembling ALK and RET through varying organization patterns, we highlight the superior organizational ability of phase separation over multimerization. Additionally, CORdensate-based RTK granules suggest that phase separation broadly and robustly activates RTKs. This study introduces a optogenetic tool for investigating biomolecular condensation.

Opioid receptors are expressed in virtually all neural loci contributing to the experience of pain. Due to this widespread expression, the contribution of specific cell types to the analgesic properties and deleterious side effects of opioids remains incompletely understood. Linking the activity of specific receptors in defined cells to behavioral or physiological processes remains a major challenge of translational pharmacology. In this study, we describe the development of drugs acutely restricted by membrane tethering (DART) antagonists that contain an antagonist naloxone moiety linked to a Halo-tag reactive group. The optimized Naloxo-DART displayed robust blockade of a MOR agonist only when cells co-expressed a Halo-tagged membrane tether. We use the Naloxo-DART delivered in vivo to selectively block MORs in locus coeruleus neurons in brain slide preparations. The Naloxo-DART provides a powerful approach for elucidating the physiological roles of MORs expressed in specific neuronal populations with acute spatiotemporal control.

Ubiquitin (Ub) is a protein post-translational modifier that controls proteostasis through mechanisms spanning transcription, translation, and protein degradation. Ub conjugation occurs through a cascade of three enzyme classes (E1, E2, and E3s) involving >1,000 proteins that regulate the ubiquitination of cellular proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized. Here, we develop highly selective and potent pan-UBE2D/UBCH5 inhibitors by targeting the RING- and backside-binding sites with engineered linked-domain proteins. In HeLa cells, these inhibitors phenocopy the knockdown of UBE2D by enhancing chemosensitivity to cisplatin. Whole-cell proteomics reveals that ∼20% of the identified proteins are more abundant, and most do not have altered mRNA levels, suggesting that their protein turnover is regulated by UBE2D. Enrichment analysis of the altered mRNAs indicates that the linked-domain proteins trigger the unfolded protein response. These precision tools will enable new studies probing UBE2D's cellular roles and help to deconvolute complex Ub regulatory networks.

WD40 repeat-containing protein 5 (WDR5) is a core component of the SET1/mixed lineage leukemia (MLL) complex that regulates gene expression via H3K4 methylation and plays a key role in maintaining oncogenic gene expression programs, particularly in MLL1-rearranged leukemias. In this study, we leveraged a microprotein, endogenous microprotein binder of WDR5 (EMBOW), to develop peptide-based inhibitors that specifically targeted WDR5. Through comprehensive biophysical analyses and high-resolution structural studies, we revealed that EMBOW mainly bound to the WDR5 interaction (WIN) site of WDR5. Structure-guided optimization led to the development of EMBOW-derived peptides, notably Ac7, which exhibited high affinity for WDR5 (K = 9.17 ± 4.01 nM). These peptides effectively inhibited H3K4 methylation, suppressed oncogenic gene expression, and impeded leukemia cell proliferation in vitro. Importantly, in xenograft mouse models, Ac7 demonstrated significant anti-tumor activity with low toxicity. This work offers a promising strategy for targeting epigenetic regulators with peptide-based therapeutics, providing a foundation for innovative treatments in leukemia.

Stress granules (SGs) are stress-induced membraneless organelles whose dynamics are tightly regulated by protein interactions and modifications. However, whether SUMOylation directly targets SG core proteins G3BP1/2 and which ligase is involved remains unclear, partly due to their transient and membraneless nature. To investigate this SUMOylation and its ligase, we applied our low-concentration formaldehyde crosslinking (lcFAX) method to stabilize SGs and enhance analysis. Using lcFAX-MS, we identified TRIM28 as a previously undefined SG-associated protein and showed that it SUMOylates G3BP1 at K287 and G3BP2 at K281, establishing a critical mechanism regulating SG dynamics that ultimately impacts cellular ROS and apoptosis. In addition, lcFAX-seq provides insights into SG RNA composition. Altogether, our study uncovers an essential role for TRIM28-mediated SUMOylation in modulating SG dynamics. TRIM28 may act as a versatile regulator, and with the aid of lcFAX, this mechanism could be further explored across diverse membraneless organelles and regulatory pathways.

Mutant isocitrate dehydrogenases (IDH1/IDH2) catalyze the conversion of α-ketoglutarate (αKG) to D-2-hydroxyglutarate (D2HG), a hallmark of many lower-grade gliomas. Elevated D2HG levels promote tumorigenesis through epigenetic reprogramming and immunosuppressive mechanisms, although paradoxically, D2HG can also inhibit tumor growth. To explore D2HG's biological functions, we developed genetically encoded D2HG biosensors (DHsers) based on the prokaryotic transcriptional regulator DhdR. Structural analysis of DhdR, including its apo form, D2HG-bound complex, and DNA-bound complex, revealed that D2HG binding induces DhdR conformational changes that regulate DNA interaction. Leveraging these insights, we engineered biosensors (DHsers) that detect a wide range of concentrations of D2HG (0.3-30 mM) with high sensitivity. We also established a standardized protocol for quantifying subcellular D2HG levels in living cells. Notably, STING activation promotes D2HG production, suggesting a role of D2HG in immune modulation. Our findings reveal D2HG-induced transcriptional regulation in prokaryotes, offering a platform for studying the role of D2HG in cellular metabolism and tumorigenesis.

Dysregulation of cysteine-dependent processes is implicated in many diseases, including cancer. Despite the importance of cysteine in crucial cellular functions, including protein synthesis, redox balance, and glutathione production, a lack of efficient assays to measure cellular cysteine has limited efforts to identify agents that affect physiological cysteine levels. We employed circular permutation to engineer a fluorescent sensor that changes conformation upon cysteine binding. Biochemical experiments showed that this sensor is selective for cysteine, operating in the 10 μM-10 mM range. To demonstrate the sensor's applicability, we performed high-throughput screens for compounds that reduce cellular cysteine. Liquid chromatography of cell extracts validated the effect of two hit compounds, and mechanistic investigations showed that one was dependent on the anticancer target, xCT. Future application of this sensor in cell biology and drug discovery will advance understanding of cysteine metabolism and drive the development of therapeutics that restore cysteine homeostasis.

Emerging evidence suggests that autophagy is activated during exercise, mediating the benefits of exercise. However, the molecular mechanisms underlying the regulation of skeletal muscle autophagy during exercise are incompletely understood. Here, we show lactate severs as a positive regulator of autophagy in myocytes and its levels increase rapidly in response to a single bout of exercise. Mice with low lactate levels due to the lack of myocyte lactate dehydrogenase A exhibit significant abnormalities in skeletal muscle, including impaired autophagy. Our mechanistic study demonstrates that lactate enhances autophagy by inactivating mTOR complex 1 (mTORC1) through promoting mTOR lactylation at lysine 921 (K921) in myocytes. Accordingly, mutation of mTOR at K921 site causes sustained mTORC1 activation, leading to defects in skeletal muscle autophagy. Thus, our work uncovers a previously undescribed physiological action of lactate in the regulation of mTORC1-controlled skeletal muscle autophagy during acute exercise, which involves a lactylation-based post-translational modification mechanism.

Phenotypic screens carried out with functional genomics or small molecules have led to novel biological insights, revealed previously unknown targets for drug discovery programs, and provided starting points for the development of first-in-class therapies. Despite being valuable research tools, genetic and compound screening also have significant limitations. This perspective aims to shed a light on those limitations and provide mitigation strategies when available, with a goal of helping phenotypic screening practitioners gain an understanding of how and when to best utilize either approach.

Food as medicine shows promise for disease intervention or treatment. Here, we found phytate, an active ingredient of plant-based diets, exhibits properties in mitigating radiotherapy-related complications. Oral gavage of phytate restored hematogenic organ atrophy, elevated peripheral blood neutrophils and white blood cells, reduced inflammation, and improved gastrointestinal (GI) integrity in irradiated mice. Phytate intake modulated the gut microbiota, facilitating the colonization of symbiotic Parasutterella in GI tract, thus combating intestinal radiation toxicity. In vitro assays and untargeted metabolomics identified 3-phenyllactic acid (PLA) and N-acetyl-L-leucine (NL) as functional metabolites produced by Parasutterella. In vitro, ex vivo, and in vivo models showed that PLA induces M2-like polarization in macrophages, while NL reduced oxidative stress, both counteracting radiation toxicity and working synergistically. Our findings offer mechanistic insights into phytate for alleviating radiation-associated complications and suggest that Parasutterella and its metabolites might be employed as promising probiotics or postbiotics for cancer patients undergoing radiotherapy.

In an interview with Dr. Mishtu Dey, the editor-in-chief of Cell Chemical Biology, the authors of the research article entitled "TRIM28-mediated SUMOylation of G3BP1/2 regulates stress granule dynamics" share insights about their work and reflect on their scientific field and their journeys as researchers.

In this issue of Cell Chemical Biology, Zhang et al. report the identification of a high-affinity EMBOW-derived inhibitor of WDR5, Ac7, which demonstrates in-cell target engagement and in vivo antileukemic efficacy. The microprotein-inspired inhibitor potently blocks the WDR5-MLL1 interaction, suppressing H3K4 methylation and transcription of target genes in mixed lineage leukemia (MLL)-rearranged leukemia.

Aberrant Wnt signaling activation occurs in various cancers but has limited druggable targets. In this issue of Cell Chemical Biology, He et al. established a double death trap Wnt reporter system. Combined with genome-wide CRISPR screening, this approach identified STT3A as an essential Wnt signaling regulator with therapeutic potential.

Abnormalities in the Wnt pathway are major drivers of cancer. RNF43 loss-of-function mutations are frequently detected in aggressive cancers lacking targeted therapies, underscoring the need to uncover key regulators and targets of this pathway. Using a double death trap (DDT) Wnt reporter and genome-wide CRISPR screen, we identified STT3A as an essential regulator of Wnt signaling. Genetic and pharmacological inhibition of STT3A suppressed aberrant Wnt activity caused by RNF43/ZNRF3 loss. Importantly, suppression of STT3A blocked the growth of RNF43-deficient cancer cell lines, patient-derived organoids, and spontaneous tumors. Mechanistically, STT3A regulates Wnt/β-catenin signaling via LRP6, but not LRP5. Glycosylation of LRP6 by STT3A is required for Wnt ligand binding. Notably, STT3A depletion displayed milder effects on bone homeostasis, as supported by phenotypes in STT3A-deficient patients. Together, this study established STT3A as a critical Wnt regulator through LRP6 glycosylation and a therapeutic target for RNF43-deficient cancers.

Cannabis-derived compounds, particularly the non-psychoactive cannabidiol (CBD), hold significant potential for pain management. However, CBD's hydrophobicity limits systemic brain delivery, constraining both its therapeutic efficacy and mechanistic investigation. Here, we present an inclusion-complex-enhanced nano-micelle formulation (CBD-IN) that significantly improves systemic absorption and elevates brain CBD levels. A single dose of CBD-IN fully suppresses allodynia and hyperalgesia in a mouse model of neuropathic pain, without impairing normal sensorimotor or cognitive functions. This rapid and robust analgesic effect enables in-depth investigation of underlying neural mechanisms. Activity-dependent genetic mapping reveals that CBD-IN selectively reduces allodynia-associated neuronal activation across the somatosensory system. Complementary calcium imaging in spinal nociceptive and somatosensory corticospinal neurons further demonstrates pathophysiological state-dependent neural suppression by CBD. These results demonstrate that nano-formulated CBD delivers rapid and effective analgesia by selectively suppressing pathological hyperactivity throughout the somatosensory system, offering a promising therapeutic strategy for neuropathic pain and other disorders involving circuit-level disinhibition.

HIF-1α transcriptional activity is enhanced through SUMOylation mediated by CBX4. Despite the recognized importance of the CBX4-HIF-1α axis, the molecular mechanisms governing its regulation remain largely unclear. In this study, phenotypic screening of a 101,254-compound library followed by structural optimization led to the identification of XZA-1, a small molecule capable of disrupting CBX4-mediated HIF-1α transcriptional activation. Mechanistic investigations revealed that XZA-1 activates HADH, a key enzyme in fatty acid β-oxidation, resulting in increased intracellular levels of acetoacetyl-CoA. This metabolite promotes acetoacetylation of CBX4 at lysine 106, thereby reducing its SUMO E3 ligase activity. In a CBX4-overexpressing xenograft model, XZA-1 demonstrated antitumor effects by enhancing CBX4 K106 acetoacetylation. Additionally, elevated levels of CBX4 K106 acetoacetylation were observed in clinical HCC tissues from patients with better overall survival. These findings suggest that acetoacetyl-CoA functions as a potential antitumor metabolite and establish a novel pharmacological approach for modulating HIF-1α transcriptional activity in cancer.

The improper folding and aggregation of tau are linked to several neurodegenerative diseases affecting millions worldwide. However, the pathogenesis of tauopathies remains poorly understood, resulting in limited effective treatments. Here, we employ an integrated chemoproteomic phenotypic strategy to identify druggable targets and corresponding chemical probes for the treatment of tauopathies. We identified and optimized an indole-amine compound that potently and extensively clears tau aggregates in two human tauopathy models. Mechanistic and chemoproteomic studies implicate protein disulfide isomerase 1 (P4HB) as the primary target, forming covalent adducts upon metabolic activation. Knockdown of P4HB reduced tau aggregates in three tauopathy models, including an ex vivo murine neuron preclinical model. Functional characterization revealed the compound induces mild endoplasmic reticulum (ER)-stress responses as assessed by RNA sequencing and whole proteomic profiling. Our findings highlight P4HB as a potential therapeutic target for treatment of tauopathies.

Plasmodium falciparum evades the antimalarial activity of proline-competitive prolyl-tRNA synthetase (PfProRS) inhibitors, such as halofuginone (HFG), by a resistance mechanism termed the adaptive proline response (APR). The APR is characterized by a marked elevation of intracellular proline following drug exposure. Contrary to initial expectations, the APR is not mediated by alterations in canonical proline metabolic pathways involving arginase (P. falciparum arginase [PfARG]) and ornithine aminotransferase (P. falciparum ornithine aminotransferase [PfOAT]). Instead, we identified loss-of-function mutations in the apicomplexan amino acid transporter 2 (P. falciparum apicomplexan amino acid transporter 2 [PfApiAT2]) as the primary genetic driver of this resistance phenotype. Importantly, reversion of these mutations to wild type effectively suppresses the APR, establishing PfApiAT2 as the molecular determinant of this resistance mechanism. The elucidation of the APR significantly advances our understanding of antimalarial drug resistance. By delineating the role of PfApiAT2 in this process, we establish critical insights for the development of strategies to circumvent PfProRS inhibitor resistance for future antimalarial therapies.

The receptor for advanced glycation end products (RAGE) drives inflammation in several chronic diseases. In this issue of Cell Chemical Biology, Theophall et al. built a structural model of the actin polymerase-inducing RAGE-Diaphanous 1 complex and identified a small molecule that disrupts this interaction, enhancing wound healing and reducing inflammation in vivo.

In this issue of Cell Chemical Biology, Bopp et al. discover that malaria parasite resistance to halofuginone is mediated by mutations in PfApiAT2, an amino acid transporter, rather than halofuginone's target prolyl-tRNA synthetase. This rapid and distinctive resistance mechanism highlights amino acid transport as a promising avenue for drug discovery.

Organellophagy receptors have been reported previously, but the underlying mechanisms of their function remain unclear. In a recent issue of Nature Cell Biology, Rudinskiy et al. demonstrated that the intrinsically disordered regions (IDRs) of the receptors function as interchangeable modular codes, driving organelle fragmentation. This provides insights for the exploration of future models of organellophagy receptor function.