Polycomb group (PcG) proteins safeguard cell identity by maintaining target genes in a transcriptionally repressed state through depositing two histone marks, H2A lysine-119 ubiquitination (H2AK119ub1) and H3 lysine-27 trimethylation (H3K27me3). BAP1 and UTX are thought to erase these marks during transcription activation, but their exact roles remain unclear. Here we observe a rapid loss of H2AK119ub1, followed by a reduction in H3K27me3 levels at promoters upon transcription induction, coinciding with a swift dissociation of PRCs. With an acute degradation system, we show that neither BAP1 nor UTX is required for transcription activation or the dissolution of PcG repressive marks, though BAP1 depletion leads to elevated steady-state H2AK119ub1 levels. Furthermore, while UTX regulates enhancer activation independently of its demethylase activity, BAP1 contributes to this process through a dual role: its scaffolding function supports MLL4 occupancy at nascent enhancers, while its catalytic activity restricts H2AK119ub1 accumulation that would otherwise impede enhancer maturation. Together, these findings establish that BAP1 and UTX are dispensable for transcription-driven erasure of Polycomb marks but are required for enhancer establishment, with BAP1 acting through coordinated scaffolding and catalytic mechanisms.

Lignin depolymerization by white-rot fungi generates diverse aromatic compounds derived from hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. Although the metabolic pathways for G- and H-unit-derived aromatics have been studied, the enzymatic step responsible for the oxidative decarboxylation of the S-unit intermediate syringic acid (SA) has remained unknown. Here, we identify PcMNX1, a group A flavoprotein monooxygenase (FPMO) from the white-rot fungus Phanerochaete chrysosporium, as the enzyme catalyzing this missing step. Recombinant PcMNX1 catalyzed the NAD(P)H-dependent oxidative decarboxylation of SA to dimethoxyhydroquinone (DMHQ) and also converted other lignin-derived aromatics, including vanillic acid and 4-hydroxybenzoic acid, with markedly higher catalytic efficiency than the closely related enzyme GsMNX1 from Gelatoporia (Ceriporiopsis) subvermispora. The crystal structure of PcMNX1 was determined at 2.00 Å resolution, revealing a typical group A FPMO fold with FAD bound in the "out" conformation. Structure-guided mutagenesis demonstrated that His247 functions as the catalytic base required for decarboxylative hydroxylation. Comparative structural analysis with bacterial 3-hydroxybenzoate 6-hydroxylase (3HB6H) indicated that subtle substitutions in active-site residues alter substrate positioning and reaction outcomes. Consistent with this hypothesis, introduction of PcMNX1-type residues into 3HB6H conferred decarboxylation activity toward lignin-derived aromatics. Furthermore, enlargement of the PcMNX1 active-site cavity through the L264A substitution markedly enhanced SA conversion. Together, these findings demonstrate that PcMNX1 catalyzes the oxidative decarboxylation of SA and reveal how subtle active-site remodeling diversifies the catalytic repertoire of closely related group A FPMOs involved in lignin-derived aromatic metabolism.

Liver disease in Alpha-1 antitrypsin deficiency (AATD) is caused by the toxic accumulation of mutant Z alpha-1 antitrypsin (Z-AAT) within the endoplasmic reticulum (ER) of hepatocytes. Livers from PiZ transgenic mice expressing the human Z-AAT and AATD patients were both found to have increased p62/SQSTM1, a multifunctional protein involved in protein homeostasis, consistent with previous reports. However, whether p62/SQSTM1 is a marker of Z-AAT globules or plays an active role in Z-AAT proteostasis is unclear. The goal of this study was to elucidate the involvement of p62/SQSTM1 in the formation of Z-AAT globules that are responsible for liver injury in AATD. In the present study, we found that p62/SQSTM1 decorated ubiquitin-positive, Periodic-Acid Shiff-diastase-resistant Z-AAT globules and interacted with Z-AAT at the ER-cytosol interface. Genetic ablation of p62/SQSTM1 in PiZ mice (PiZ;p62-/-) led to marked reduction in hepatic Z-AAT globules and polymers, and decreased serum Z-AAT, highlighting a central role for p62/SQSTM1 in disease pathogenesis. Moreover, hepatocyte-specific somatic deletion of the ubiquitin-association (UBA) domain of p62/SQSTM1 reduced Z-AAT aggregation. Furthermore, KEAP1 was identified as a binding partner of p62/SQSTM1-Z-AAT complex, leading to nuclear translocation and activation of NRF2. Inhibition of KEAP1-p62/SQSTM1 interaction reduced the abundance of p62 and phosphorylated p62, decreased intracellular Z-AAT, and redistributed NRF2 to the cytoplasm. In conclusion, this study identifies p62/SQSTM1 as a regulator of Z-AAT proteostasis and link Z-AAT/p62 accumulation to KEAP1 sequestration and NRF2 pathway activation in liver disease due to Z-AAT.

CDC42 is involved in multiple signaling pathways, including actin organization and polarity. We previously reported progressive sensorineural hearing loss (SNHL) in inner ear hair cell (HC)-specific Cdc42-knockout (Atho1-Cre+/-;Cdc42flox/flox) mice. However, the phenotype was milder than expected, suggesting possible redundancy with other Rho-family GTPases. Thereafter, Takenouchi-Kosaki syndrome (TKS), caused by de novo CDC42 mutations and manifesting as SNHL, was reported, in which the p.Y64C mutation was speculated to be constitutively active. However, the relationship between CDC42 status and hearing phenotypes in TKS remains unclear. Using cell models, mouse models, and patient data, we propose that impaired and/or dysregulated cycling between GDP/inactive and GTP/active forms, through either loss-of-function or constitutive activation, can lead to SNHL. Furthermore, to test redundancy, we generated HC-specific Cdc42;RhoQ double-knockout (Atho1-Cre+/-;Cdc42flox/flox;RhoQflox/flox) mice, which revealed synergistic roles of CDC42 and RHOQ in cochlear HCs. Supporting this synergy, MDCK cells with CDC42 and RHOQ double knockdown showed greater phospho-cofilin, a key regulator of actin turnover, elevation than single knockdowns.

The carboxyl terminus of Hsp70-interacting protein (CHIP, encoded by STUB1) combines co-chaperone and E3 ubiquitin ligase activities to regulate protein quality control. Heterozygous mutations in STUB1 cause spinocerebellar ataxia type 48 (SCA48), a progressive cerebellar ataxia with variable extrapyramidal and cognitive features. To understand the molecular basis of this variability, we systematically analyzed 13 SCA48-associated variants spanning the TPR and U-box domains through recombinant protein biochemistry and cellular models. TPR variants retained intrinsic ligase activity but showed significantly reduced HSP70 binding, impaired substrate ubiquitination, and decreased stability. Conversely, U-box variants abolished ligase function, promoted the formation of high-molecular-weight oligomers, and often increased CHIP levels while only partially impairing co-chaperone activity. Many mutants displayed temperature-sensitive defects and defective stress-induced nuclear translocation. Principal component analysis revealed distinct biochemical clustering specific to each domain. RNA-seq following STUB1 knockdown modeled CHIP insufficiency and showed preserved HSF1-dependent transactivation, but loss of CHIP's capacity to amplify ubiquitination, chaperone function, and stress-related transcriptional programs. Meta-analysis of 87 SCA48 patients linked TPR-like biochemical signatures to upper motor neuron involvement and U-box-like profiles to prominent dysarthria. Overall, these data indicate that SCA48 results from domain-specific disruption of CHIP's dual functions, producing varying degrees of CHIP insufficiency and/or gain-of-toxic effects that together contribute to the phenotypic diversity observed across patients. This work refines the mechanistic framework for SCA48 pathogenesis and highlights strategies for therapeutic modulation of residual CHIP activity.

Antibiotic tolerance and persistence contribute to the emergence of antimicrobial resistance, yet strategies to reverse these phenotypes remain limited. Our previous work revealed that the naturally occurring nucleoside adenosine can reverse antibiotic tolerance in diverse bacterial strains by modulating cellular energetics. Here, we define the mechanism underlying this potentiation, identifying adenosine metabolism as a driver of cytoplasmic alkalinization and proton motive force (PMF) generation. Using RNA sequencing, metabolite assays, and pH-sensitive fluorescent reporters, we show that adenosine is catabolized by purine nucleoside phosphorylase (deoD) to yield ribose-1-phosphate, which enters the pentose phosphate pathway. This metabolic flux stimulates the electron transport chain, leading to proton translocation, increased cytoplasmic pH, and enhanced PMF. Disruption of key metabolic enzymes (deoD, deoB, tktAB) or the inhibition of enolase abolishes both alkalinization and antibiotic sensitization. Using a respiratory-deficient mutant, we demonstrate that aerobic respiration is the primary driver of alkalinization and gentamicin potentiation, although adenosine can partially increase membrane potential independently of oxidative phosphorylation. These findings support a model in which adenosine metabolism promotes aminoglycoside uptake via PMF-driven transport, sensitizing tolerant bacteria to killing. Our work implicates the ribose moiety of adenosine as a key metabolic lever for reversing tolerance. Broader exploration of nucleoside-based adjuvants across bacterial species and antibiotic classes may reveal generalizable strategies to enhance antibiotic efficacy against recalcitrant infections.

Cytokine interleukin-33 (IL-33) signaling in primary sensory neurons plays a crucial role in pain. However, the underlying molecular mechanisms remain poorly understood. Therefore, we investigated whether IL-33 signaling affects ion channels in nociceptive dorsal root ganglion (DRG) neurons. Herein, we reported that the application of IL-33 enhanced the electrophysiological activity of acid-sensing ion channels (ASICs). IL-33 dose-dependently increased acid-evoked ASIC currents in mouse DRG neurons. IL-33 enhanced the maximum responses of ASICs, whereas the sensitivity to acidic stimuli remained unaffected. This IL-33-induced enhancement of ASIC currents was dependent on suppression of the tumorigenicity 2 (ST2) receptors. The enhancing effect of IL-33 on ASIC currents was prevented by the p38 mitogen-activated protein kinase inhibitor SB202190, but not by the ERK inhibitor U0126 or the JNK inhibitor SP600125, indicating the effect was p38-dependent. Moreover, IL-33 potentiated the action potentials triggered by acidic stimuli. Finally, ASIC3-deficient mice displayed attenuated mechanical hyperalgesia induced by intraplantar or intramuscular injection of IL-33. Our findings revealed that IL-33 enhanced ASIC function via ST2 and the intracellular p38 signaling pathway, which might provide a promising therapeutic approach for pain treatment by targeting IL-33/ST2 signaling.

Emerging evidence indicates that metabolic reprogramming plays a pivotal role in glioblastoma (GBM) progression. Here, we report that the expression of fumarate hydratase (FH) is significantly reduced in GBM patient samples. FH overexpression suppresses the viability, migration, and invasion of GBM cells, whereas FH knockdown or pharmacological inhibition produces the opposite effects. Consistent with FH downregulation, its substrate fumarate accumulates in GBM cells and clinical tumor tissues. As a result, excess fumarate induces succination of TRIM17 at Cys370, thereby reducing TRIM17 protein stability and impairing its E3 ubiquitin ligase activity. Subsequent functional studies identify TRIM17 as a tumor suppressor in GBM. In vivo xenograft models further confirm that FH restrains GBM progression by regulating TRIM17 succination. Collectively, these findings establish FH as a negative regulator of GBM and reveal a fumarate-TRIM17 signaling axis that promotes GBM malignancy through post-translational modification of TRIM17.

MAP2 has been widely used as a marker of neuronal dendrites because of its extensive restriction in the somatodendritic region. Despite that, how the precise localization of such a soluble protein is established and maintained against diffusion has been elusive and long remained a mystery in neuroscience. In this study, using GFP-tagged MAP2 expressed in cultured hippocampal neurons, we discovered a crucial protein region responsible for the localization of MAP2, the serine/proline-rich (S/P) region. Our pulse-chase live-cell imaging revealed the slow but steady migration of MAP2 toward distal dendrites, which was not observed in the presence of a Dynein inhibitor. By integrating these experimental parameters into a mathematical model, we demonstrated that an intermittent active transport mechanism by Dynein is sufficient to explain the observed MAP2 dynamics. In addition, our experiments using a Dynein inhibitor as well as Dynein knockdown, showed that impaired Dynein function leads to mislocalization of MAP2 in the axon. Furthermore, we verified that cytoplasmic Dynein binds to MAP2 through the S/P region in heterologous cells. Finally, we show that the failure of this Dynein-mediated transport mechanism results in the ectopic stabilization of axonal microtubules, thereby disrupting axonal identity. Thus, we propose that the cytoplasmic Dynein recruits and transports free MAP2 toward distal dendrites, thereby maintaining the precise dendritic localization of MAP2. Our findings shed light on the previously unknown mechanism behind MAP2 localization and provide a new direction for soluble protein trafficking research in the field of cell biology of neurons.

Signal sequences (SS) target newly synthesized proteins to the endoplasmic reticulum and, eventually, the plasma membrane. Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a transmembrane protein with an unusually long SS that interacts with vascular endothelial growth factor receptor (VEGFR)-2 and promotes VEGF-induced endothelial cell signaling, proliferation and migration, as well as angiogenesis. How DCBLD2 interacts with VEGFR-2 to modulate VEGF signaling remains unclear. We generated various constructs containing different combinations of DCBLD2 domains, including its SS, and conducted co-immunoprecipitation and signaling studies in HEK 293T and endothelial cells to identify the smallest DCBLD2 unit that interacts with VEGFR-2. Other proteins containing this unit were identified, and their interactions with VEGFR-2 were evaluated. The DCBLD2 SS interacted with VEGFR-2 and promoted VEGF signaling. The smallest unit in the DCBLD2 SS that interacted with VEGFR-2 was the L5VL5 sequence. Even after the central valine was removed, the L10 sequence mimicked the DCBLD2 SS effect on VEGF-signaling, while shorter or longer multi-leucine sequences were less effective. We identified several other human proteins containing such uncleaved multi-leucine SS, including CD45-associated protein (CD45-AP) and BRICHOS domain containing 5 (BRICD5). Both these proteins could interact with VEGFR-2, and CD45-AP could modulate VEGF signaling in endothelial cells. In conclusion, a multi-leucine motif in SS can directly engage in protein-protein interactions and modulate VEGF signaling. These findings refine the traditional view of SS as merely targeting elements, emphasizing their role in cellular signaling and opening new avenues for research and therapy.

Diabetes is characterized by impaired glucose homeostasis resulting from dysfunction and loss of insulin-producing islet β cells, with oxidative stress emerging as a contributor to β-cell injury. The polyamines (putrescine, spermidine, spermine) regulate cellular stress responses, and their metabolism can generate reactive oxygen species (ROS) and reactive aldehydes via catabolic enzymes such as spermine oxidase (Smox) and polyamine oxidase (Paox). Transcriptomic analyses of mouse and human islets exposed to proinflammatory cytokines revealed induction of the gene encoding Smox but no change in the gene encoding Paox, suggesting a role for Smox in amplifying inflammation-associated stress in islets. In developing zebrafish, the smox gene is expressed in the pancreas and enriched within endocrine cells. Using a β-cell-specific ROS-inducible zebrafish model, smox gene knockdown reduced intra-islet ROS and macrophage recruitment. Metabolomics of zebrafish embryos showed that Smox deficiency did not affect spermine or spermidine levels but led to accumulation of acetylated polyamines, consistent with the rerouting of polyamine flux through alternative acetylation pathways. RNA sequencing of zebrafish embryos demonstrated that Smox deficiency induces distinct transcriptional programs, with enrichment of cell cycle and RNA processing pathways at baseline and reprogramming of stress, translational, and immune-associated pathways during injury. Mechanistically, Smox is the primary polyamine pathway generating ROS in zebrafish, as reducing flux through the Paox-dependent pathway did not reduce ROS production. These findings support a model in which Smox amplifies β-cell stress through peroxide- and aldehyde-mediated toxicity and identify polyamine catabolism as a modifiable pathway in the setting of β-cell injury.

Gelsolin, a Ca2+-dependent actin filament (F-actin) severing protein, plays essential roles in apoptosis, cell motility, and the actin-scavenger system of the bloodstream. It comprises six related domains (G1 to G6) connected by variable linkers (L1 to L5), each contributing distinctly to severing. Individually, neither G1 nor G2G3 severs F-actin, whereas the combined fragment G1-G3 displays potent, Ca2+-independent severing activity, largely mediated by G1, which relies on G2G3 for recruitment to F-actin. The C-terminal fragment G4-G6 neither binds nor severs F-actin but, once recruited via G2G3, contributes to the severing efficiency of full-length gelsolin. However, it remains unclear whether the other domains rely on G2G3 solely for recruitment or also for conformational changes in F-actin that facilitate binding and severing. Here, through a series of cryo-EM structures, we show that G2G3 binds F-actin in a stepwise manner, inducing conformational changes, including a kink in the filament and rotation of a subunit at the binding site, that prime F-actin for binding by the other gelsolin domains and severing. These changes propagate beyond the G2G3 binding site, revealing how F-actin buffers the effects of kinking. Biochemically, substituting G1 with G4 within G1-G3 yields a construct, G4-G3, with substantial severing activity, albeit Ca2+-dependent and lower than that of G1-G3. This result indicates that G4 can substitute for G1 when primed by G2G3, while sequence adaptations make G1 uniquely effective at severing. Collectively, these findings define a dual role of G2G3: recruiting other gelsolin domains and priming F-actin for severing.

The yeast Hrq1 protein is an important model for studying the human RECQL4 helicase and RECQL4-associated diseases. In this study, we simultaneously isolated and characterized two stable oligomeric states of Hrq1 expressed in insect cells: a monomeric form (Hrq1mon) and a higher-order oligomeric fraction (Hrq1-oligo). Intriguingly, ATP not only promotes the dissociation of the oligomeric fraction into monomeric or smaller Hrq1-containing species but also markedly stimulates the DNA-unwinding activities of both forms. Furthermore, Hrq1-mediated unwinding is strictly dependent on a forked DNA substrate, and its activity increases with increasing lengths of either the 5'- or 3'-single-stranded DNA tail. Based on the limited unwinding processivity of Hrq1, we propose a "relay-style" mechanism in which an initial Hrq1 monomer partially unwinds the duplex and subsequently dissociates, allowing additional Hrq1 monomers to bind the newly exposed 3' single-stranded region and continue the unwinding reaction. However, this process can be inhibited by ScRPA through competition for the exposed single-stranded DNA. These findings provide new insights into the oligomeric-state-dependent regulation and unwinding mechanism of Hrq1 and may contribute to a broader understanding of RecQ family helicases.

How fungal pathogens coordinate genotoxic stress responses with membrane homeostasis to sustain cellular signaling remains poorly understood. In this study, we characterize Mrv6, a MARVEL domain-containing membrane protein that is prominently induced by genotoxic stress. Intriguingly, rather than functioning as a canonical DNA repair factor, Mrv6 acts as a critical biophysical adjuster governing membrane lipid homeostasis. Deletion of MRV6 triggers a global transcriptional activation of the ergosterol biosynthetic pathway, resulting in aberrant cellular ergosterol accumulation. We demonstrate that this lipid imbalance fundamentally alters plasma membrane homeostasis, resulting in a less permeable architecture. Paradoxically, while this structural alteration inadvertently shields the fungus from exogenous genotoxins, conferring unexpected resistance to methyl methanesulfonate, it simultaneously hinders the intracellular transduction of environmental morphogenetic signals under hypoxic conditions. Consequently, the major hyphal repressor NRG1 remains aberrantly expressed, leading to an attenuated signaling response that significantly impairs virulence. Crucially, targeted genetic suppression of either the lipid biosynthetic node (ERG1) or the downstream signaling node (NRG1) effectively mitigates this biophysical constraint, rescuing both morphogenetic switching and pathogenicity. Collectively, our findings define a novel biochemical paradigm wherein C. albicans utilizes a stress-responsive membrane protein to fine-tune ergosterol remodeling and membrane permeability, structurally coupling stress adaptation to signal transduction.

The gene fusion KIAA1549::BRAF is a common oncogenic driver in pediatric pilocytic astrocytoma. The chimeric fusion protein that comprises most of the type 1 transmembrane KIAA1549 protein fused in its C-terminal cytosolic domain to the truncated BRAF oncogene without its N-terminal autoinhibitory domains. KIAA1549 has a large N-terminal ectodomain that undergoes extensive O-mannosylation directed by the POMT1/2 protein O-mannosyltransferase complex localized in the ER. Here, we investigated how posttranslational modifications contribute to KIAA1549 protein maturation in the secretory pathway. Using a panel of KIAA1549 expression constructs and glycoengineered HEK293 cells, we provide evidence that O-mannosylation of the KIAA1549 ectodomain is required for ER exit, acquisition of complex N-glycans, and subsequent furin-like cleavage in the Golgi. In the absence of POMT1/2 O-mannosylation KIAA1549 is retained in ER and fails to undergo proprotein processing. Our findings confirm and extend our previous studies with the KIAA1549::BRAF fusion protein, where oncogenic transformation was dependent on POMT1/2 O-mannosylation and affected on furin processing. Thus, these intrinsic posttranslational modifications of the KIAA1549 ectodomain are critical determinants of the cellular trafficking that lead to activation of the BRAF oncogene, potentially involving a cascade of cleavage events in the Golgi that ultimately release a truncated but fully active BRAF kinase domain into the cytosol.