Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy driven predominantly by oncogenic KRAS mutations, which enforce extensive metabolic reprogramming to support tumor progression. Although ketone body catabolism has emerged as a critical metabolic adaptation in PDAC, the signaling mechanisms that link mutant KRAS to the ketolytic machinery remain largely unexplored. Here, we identify a previously unrecognized, context-dependent role of oncogenic KRAS in driving ketone body utilization. We show that KRAS mutation alone is insufficient to fully activate ketolysis; instead, it primes the ketolytic pathway in a manner that requires cooperative input from additional tumor microenvironment signals. Mechanistically, oncogenic KRAS engages a downstream signaling cascade that leads to specific post-translational modifications of key mitochondrial enzymes. These modifications enhance the flux of ketone body catabolism, thereby increasing acetyl-CoA and ATP production and promoting pancreatic cancer cell proliferation and xenograft tumor growth. In a clinical PDAC cohort, activation of this KRAS-dependent ketolytic axis was elevated in KRAS-mutant tumors compared with KRAS wild-type cases, albeit with a trend that requires further validation. Collectively, our findings define a conditional dependency of mutant KRAS on cooperative signals to drive ketone body catabolism, linking oncogenic signaling to mitochondrial ketone metabolism in PDAC. This study expands our understanding of KRAS-driven metabolic reprogramming and highlights the ketolytic pathway as a context-dependent vulnerability for therapeutic intervention in pancreatic cancer.

Fibronectin (FN) and transglutaminase 2 (TG2) engage in a high-affinity interaction. This interaction affects anchoring, migration and survival of cells, and high cellular expression of TG2 is associated with metastasis potential of cancers. Previous work has mapped the interaction to involve the N-terminal part of TG2 and an N-terminally located 45 kDa domain of FN (FN45). In particular, the I8 module of FN45 is reported to be necessary and sufficient for TG2 binding. Here, we present the cryo-EM structure of a complex between human TG2 and FN45 at 2.8 Å resolution. In the structure, the distal region of the TG2 N-terminal domain binds the FN45 I8 module, whereas the proximal N-terminal region and adjacent catalytic core segment bind the FN45 I7 module and, to a lesser extent, the FN45 II2 module. The FN45 I7 and FN45 I8 modules have about equal buried surface areas in their interaction with TG2. A glycine residue at position 127 of TG2 is crucial for interaction with Trp553 of the FN45 I8 module. Mutation of this residue to isoleucine, which is carried by transglutaminase 3 that does not bind FN45, abrogates the binding of FN45 to TG2. This finding suggests that the interaction of the FN45 I7 module with TG2 is contingent on the FN45 I8 module making an interaction with TG2. Further, this finding highlights the region surrounding residue 127 of TG2 as a promising target for structure-based design of anticancer drugs aimed at disrupting the TG2-FN interaction.

The increase in flooding events due to climate change is having devastating effects on food security. Plants can respond to submergence by reducing their energy requirements and switching their metabolism from oxidative phosphorylation to fermentation. This response is promoted by group VII ethylene response factors (ERF-VIIs) whose stability is dependent on oxygen availability; in normoxia, plant cysteine oxidase (PCO) enzymes catalyze oxidation of ERF-VII N-terminal Cys residues to trigger ERF-VII degradation via the Cys/Arg-N-degron pathway. In hypoxia, reduced PCO activity prevents ERF-VII degradation, enabling acclimation to anaerobic conditions. We have proposed previously that reducing PCO activity by enzyme engineering could enhance ERF-VII stabilization and improve submergence tolerance. Here, we describe the rational design and detailed in vitro analysis of a range of PCO variants based on the most active PCO isoform in Arabidopsis thaliana, AtPCO4. We introduced single substitutions of specific amino acids in the active site and analysed their impact on enzyme catalysis, substrate affinity and iron binding capacity. We selected two variants with minimally or severely reduced activity (C173A and Y183F, respectively) and investigated their effect in an Arabidopsis model. Following submergence, survival and recovery were both improved in plants expressing the AtPCO4 variants compared to AtPCO4 wildtype. This proof-of-concept study demonstrates the potential to engineer just one amino acid in PCOs to improve flood resilience in plants and has implications for enhancing submergence tolerance in agronomically important crop species.

N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (NAGPA), also known as the uncovering enzyme, catalyzes the final step in mannose-6-phosphate (M6P) signal generation for lysosomal enzyme trafficking. Despite its central role, the enzyme's architecture, catalytic mechanism, and the contribution of its C-terminal region remain incompletely defined. Here, we combine solution biophysics, cryo-electron microscopy, structural modeling, and a quantitative cell-based assay to characterize human NAGPA. We show that NAGPA forms a noncovalent dimer in solution, resolving prior uncertainty regarding disulfide-linked higher-order assemblies. The structure reveals an elongated dimer composed of two catalytic cores and two C-terminal EGF-like stalks, semi-rigid in nature, that likely position the catalytic domains ∼5 nm from the membrane. A structure determined in the presence of the substrate analog GlcNAc-1-phosphate captures GlcNAc and phosphate in the active site, identifying an invariant DGGGS motif that is critical for substrate recognition and enzyme catalysis. Based on these observations, we propose a substrate-assisted SNi-like mechanism for cleavage of the glycosidic C-O bond between GlcNAc and M6P. Functional assays show that the membrane-tethered full-length NAGPA is more active than the isolated catalytic core, and that mutations in the hinge linking the catalytic domain to the C-terminal stalk reduce activity. Together, these findings establish a structural and mechanistic framework for understanding NAGPA function in lysosomal enzyme targeting.

The pathogenesis of endometrial cancer (EC) remains poorly understood. This study aimed to delineate the expression profile of LON peptidase N-terminal domain and ring finger 2 (LONRF2) in EC and to elucidate its mechanistic role in modulating the proliferative, metabolic, and invasive capacities of EC cells. LONRF2 expression was analyzed in EC tissues and cell lines. EC cells overexpressing LONRF2 were evaluated for malignant phenotypes. Aldolase A (ALDOA) was identified as a LONRF2-interacting protein via immunoprecipitation and nano-liquid chromatography-tandem mass spectrometry. Their regulatory relationship was examined by assessing ALDOA stability and ubiquitination. Functional exploration of the LONRF2/ALDOA axis involved combined LONRF2 knockdown, ALDOA knockdown, ALDOA knockout, or treatment with the ALDOA inhibitor itaconate in EC cells to assess malignant phenotypes, along with a xenograft model to evaluate tumor growth. LONRF2 was downregulated in clinical EC tissue and cell lines. LONRF2 overexpression inhibited the growth, glycolysis, migration, and invasion of KLE cells. LONRF2 interacted with ALDOA and induced its ubiquitination and degradation. In line with this mechanism, the enhanced malignant phenotypes caused by LONRF2 silencing were reversed by either knocking down ALDOA or administering itaconate. ALDOA-knockout cells confirmed that itaconate suppresses EC cell growth, glycolysis, migration, and invasion by targeting ALDOA. Consistently, itaconate abolished the tumor-promoting effect of LONRF2 silencing in a xenograft model. This study identifies the LONRF2/ALDOA axis as a key driver of EC progression, primarily by regulating glycolysis. Targeting this axis, particularly LONRF2, presents a promising therapeutic strategy for EC.

Iron-sulfur clusters are essential cofactors assembled in bacteria by the Suf pathway through a series of transient protein-protein interactions that transfer sulfur from L-cysteine to a scaffold complex. While early steps in persulfide transfer are well characterized, the mechanism of sulfur delivery to the SufBC2D scaffold has remained unresolved. Here, we report the first structure of the SufBC2D-SufE complex, capturing the final step in persulfide transfer in the Suf pathway. The structure reveals coordinated conformational changes in both SufB and SufE that expose the otherwise buried C254 acceptor site on SufB and position the SufE C51 loop beneath the SufB-SufD axis. Biochemical analysis demonstrates that substitutions in the globally conserved 220s β-strand of SufB enhance SufE binding affinity and persulfide transfer rates, consistent with stabilization of a locally rearranged, transfer-competent conformation. Together, these results support a model in which conformational gating regulates persulfide transfer, providing a mechanism for controlling access to reactive sulfur intermediates.

Understanding how evolutionarily related receptors preserve recognition principles for conserved peptide post-translational modifications (PTMs) while acquiring new selectivity remains central to neuropeptide-G protein-coupled receptor (GPCR) biology. The Aplysia cholecystokinin (CCK) system provides an informative model, as its peptides combine two representative PTM-related features: an amidated aromatic C-terminus (RFamide/DFamide), and a dual-tyrosine sulfation pattern exceeding the single-site architecture typical of vertebrates. Notably, its two receptor subtypes, apCCKR1 and apCCKR2, exhibit distinct sensitivities to ligands carrying different numbers of sulfated tyrosines, yet the molecular basis remains unclear. Here, we first used multiple docking approaches and found that CCK ligands could insert into receptor binding pockets in either N-terminal-in or C-terminal-in orientations. We then applied pose-specific mutagenesis to resolve this ambiguity and established a common C-terminal-in binding mode for apCCK peptides, similar to mammalian CCK/gastrin binding modes observed by cryo-EM. Within this framework, recognition of the C-terminal aromatic motif is hierarchically organized through three receptor positions, comprising a conserved anchor, a CCK-biased layer and a flexible tuning site. Moreover, we uncovered a mechanism for sulfation-number-dependent subtype selectivity: apCCKR1 accommodates an additional sulfate with minimal structural rearrangement, whereas apCCKR2 loses a favorable electrostatic network, disfavoring the disulfated ligand. Comparative analyses across representative lineages identified a conserved sulfate-recognition module alongside a lineage-restricted modifier, enabling inferences regarding the ancestral state of sulfation recognition and how it was differentially remodeled across protostomes and deuterostomes. Together, these findings establish a framework for understanding how homologous receptors may decode peptide chemical features through conserved structural logic and adaptive diversification.

Sphingolipids are fundamental components of the plasma membrane that regulate immune receptor organization and host responses to infection. Here, we identify sphingomyelin synthase 2 (SMS2) as a critical mediator of antifungal host defense against Cryptococcus neoformans. SMS2 deficiency reduces sphingomyelin and increases ceramide levels, altering membrane lipid composition and impairing receptor localization. This is associated with decreased plasma membrane toll-like receptor 2 (TLR2), attenuated signaling, and reduced IL-17A and IFNγ production by γδ T cells. Macrophages lacking SMS2 exhibit impaired phagocytosis, resulting in inefficient clearance of the attenuated C. neoformans Δsgl1 strain. Consequently, SMS2-deficient mice fail to develop effective vaccine-induced protection and show increased susceptibility to secondary infection with wild-type C. neoformans. Mechanistically, bSMase-driven alterations in SM/ceramide balance are associated with disruption of membrane organization and reduced TLR2 and CTxB staining, suggesting that SMS2-dependent sphingomyelin regulation represents a key mechanism linking membrane lipid composition to innate immune signaling, phagocytosis, and antifungal host defense against C. neoformans.

Hepatocytes display notable ploidy diversity, varying both in the number of genomic copies and in the number of nuclei. In adult mice, more than 50% of hepatocytes are tetraploid (4n), which can exist as either mononucleated (1x4n) or binucleated (2x2n) cells. Despite this distinction, these two cell types have traditionally been grouped and studied as a single population. One likely reason for this is that conventional ploidy-sorting methods classify hepatocytes based solely on total DNA content, without distinguishing between mononucleated and binucleated states. Consequently, it remains unclear whether 1x4n and 2x2n hepatocytes are functionally equivalent. In this study, we developed a novel FACS strategy to distinguish and isolate 1x4n and 2x2n hepatocytes. Our approach leverages Hoechst-area to assess total ploidy and Hoechst-height to differentiate mononucleated and binucleated hepatocytes. Transcriptome analysis comparing these two populations revealed enrichment of various metabolism-related gene signatures in 1x4n hepatocytes. Importantly, these metabolic gene expression programs were independent of liver zonation, a well-known driver of metabolic heterogeneity in hepatocytes. Our findings uncover a previously underappreciated layer of diversity in the liver and may provide a new framework for studying the physiological and pathological roles of nuclear configuration in polyploid cells in general.

Oocyte maturation critically relies on metabolic cooperation from the surrounding granulosa cells (GCs), and the energy demands on GCs change dynamically throughout follicular development. Although estrogen is a principal endocrine signal, its precise role and mechanism in regulating chicken follicular GC energy metabolism remain largely unknown. In our study, high-throughput transcriptomic analysis of GCs from hierarchical follicles following 17β-estradiol (E2) treatment identified 2731 differentially expressed transcripts, including 15 genes mapped to the glycolysis/gluconeogenesis pathway. Subsequent functional assays confirmed that the pro-glycolysis effect of E2, including increased glucose uptake, pyruvate production, lactate production, and intracellular ATP level occurred exclusively in hierarchical GCs but not prehierarchical GCs. Consistent with these results, quantitative real-time PCR and western blotting further showed significant upregulation of key enzymes related to glycolysis in hierarchical GCs. Pharmacological activation/inhibition of estrogen receptors combined with Seahorse glycolytic flux analysis showed that ERα is the key receptor mediating E2-induced glycolysis. In addition, glycolytic inhibition by 2-deoxy-D-glucose showed that glycolysis is the primary, though not exclusive, source of E2-promoted ATP production. Dual-luciferase reporter system combined with estrogen response element (ERE) mutation confirmed that E2 can activate the liver-type phosphofructokinase (PFKL) transcription by targeting the ERE (-128/-110) within its promoter. Overexpression and knockdown experiments further confirmed PFKL as a functionally required downstream effector. In summary, these findings establish the E2-ERα-PFKL axis as a mechanism enhancing glycolysis in chicken hierarchical follicular GCs, providing insight into endocrine control of ovarian energy metabolism during avian follicular development.

Adhesion GPCRs (aGPCRs) are activated by an intramolecular agonist known as the Stachel sequence. In the intact receptor, this sequence interacts with both the GAIN domain and the seven transmembrane domain (7TMD). In contrast, synthetic Stachel-derived peptides used in experimental settings only need to bind to the 7TMD to activate the receptor. We therefore hypothesized that natural Stachel sequences have evolved under dual structural constraints, interacting with both domains, and that removing the GAIN domain constraint could allow the rational design and optimization of more effective ligands. Using a curated dataset of 13,611 vertebrate aGPCR sequences, we performed large-scale conservation and entropy analyses that revealed strong evolutionary constraint at specific Stachel core positions, particularly X[0.47], X[0.48], and X[0.50]. Structure-guided in silico design with ProteinMPNN, applied separately to GAIN- and 7TMD-bound conformations, generated thousands of Stachel variants and demonstrated that single-domain optimization explores sequence space not sampled in natural orthologs. Guided by these analyses, we designed two 11-mer peptides targeting ADGRG2. Both peptides were readily soluble and exhibited substantially increased potency in cAMP assays compared with wild-type-derived Stachel peptides. Structural modeling suggests that backbone constraint at position X[0.45], polar interactions at X[0.48], and hydrophobic anchoring at X[0.50] contribute critically to enhanced 7TMD engagement. Interestingly, analogous design for ADGRF5 did not yield active peptides. Together, our findings support the concept that separating GAIN- and 7TMD-imposed constraints can enable receptor-selective optimization of Stachel-derived ligands. This strategy provides a framework for exploring previously inaccessible sequence space, while highlighting receptor-specific trade-offs between potency and specificity.

Ependymomas are glial tumors that exhibit high resistance to chemotherapy. ZFTA-fusion-positive supratentorial ependymoma (ST-EPN-ZFTA) represents a subclass with a poor prognosis. ST-EPN-ZFTA expresses fusion proteins, which consist of zinc finger translocation-associated (ZFTA) combined with a transcriptional regulator, predominantly RELA. Unlike wild-type RELA, which translocates to the nucleus in a stimulus-dependent manner, ZFTA-RELA constitutively localizes to the nucleus and induces oncogenic gene expression. To identify inhibitors of this aberrant gene expression, 9,600 structurally diverse compounds were screened using a ZFTA-RELA-responsive luciferase reporter system. The screen identified a colchicine derivative, and subsequent evaluation revealed colchicine, a microtubule polymerization inhibitor, as the most potent inhibitor (IC50 of 90 nM). Colchicine attenuated ZFTA-RELA-upregulated endogenous genes, including L1CAM. Colchicine and another microtubule polymerization inhibitor, vinblastine, partially inhibited ZFTA-RELA nuclear localization, similar to the dynein motor protein inhibitors, ciliobrevin D and dynarrestin. This inhibition was mediated through the RELA region of ZFTA-RELA, indicating that RELA retains its microtubule-dependent nuclear import machinery, even in a fusion scenario. Among other ZFTA-fusion proteins, colchicine reduced nuclear localization of ZFTA-NCOA2 and ZFTA-MKL2, but not ZFTA-MAML2 and ZFTA-MAML3. In these fusion proteins, we identified three candidate nuclear localization signals (NLS) exhibiting colchicine-sensitive nuclear localization activity, two within the ZFTA regions absent in ZFTA-RELA and one within MKL2. The results indicate that the microtubule dependence of nuclear localization varies among different ZFTA-fusion partners, which may be attributable to unique NLS sequences in each fusion protein. Our results suggest that microtubule-dependent nuclear transport represents a therapeutic target in ZFTA-fusion-positive tumors.

Mammalian odorant receptors (ORs), a large family of G protein-coupled receptors (GPCRs), rely on conserved intramolecular disulfide bonds for proper folding and their function. However, some ORs lack the typical cysteine in extracellular loop 2 (ECL2), raising questions regarding alternative structural-stabilization mechanisms. Here, we identified and characterized an unconventional disulfide bond between a unique N-terminal cysteine and ECL2 cysteine in OR51E2, the first mammalian OR with an experimentally resolved structure. Using mutagenesis and functional assays in heterologous cells, we demonstrated that this N-terminal cysteine is essential for receptor surface expression and ligand responsiveness. Furthermore, AlphaFold2 structural predictions correlated with experimental data, indicating that alternative disulfide bonds contribute to receptor stability. These findings reveal a flexible disulfide bonding strategy in ORs, suggesting that alternative extracellular cysteine linkages can compensate for missing conserved bonds, thereby supporting receptor folding and functional diversification within the GPCR family.

LIM domain kinase 1 (LIMK1) has been identified as a promising therapeutic target for a variety of conditions, such as chronic pain, open-angle glaucoma, various cancers, schizophrenia, and Fragile X syndrome. However, identifying inhibitors that selectively inhibit LIMK1 over LIM domain kinase 2 (LIMK2) has proven to be challenging. A viable strategy to overcome this difficulty is the development of covalent inhibitors, which can offer both potency and selectivity for LIMK1 due to a reactive cysteine, C349, near the active site absent in its paralog LIMK2. Here we identify an irreversible covalent inhibitor of LIMK1 (cLIMK1i), which is highly selective for LIMK1 over both LIMK2 and a panel of over 100 kinases. A crystal structure of LIMK1 soaked with cLIMK1i reveals it is a type I inhibitor occupying the ATP-binding site with its acrylamide moiety oriented toward the P-loop where C349 resides. Computational modeling supports that the P-loop of LIMK1 can adopt a conformation compatible with covalent bond formation. Biochemical and biophysical characterization of the interaction of cLIMK1i with LIMK1 demonstrates that the covalent bond with LIMK1-C349 is essential for its potent inhibition. These results support covalent inhibition of LIMK1 as a viable strategy for selectively inhibiting LIMK1 over LIMK2 and other kinases.

Water (H2O), the principal component of living organisms including humans, dissociates into H[+] and OH[-] in aqueous environments, and the resulting H[+] concentration determines both cellular pH and the proton motive force (PMF) across cellular membranes. These physicochemical parameters are fundamental regulators of a wide range of biological processes. Optogenetics enables the manipulation of biological and cellular functions using light, typically through the ectopic expression of microbial rhodopsins as photoreceptive proteins in target cells or organs. This review provides a comprehensive overview of optogenetic studies employing H[+] pump rhodopsins in diverse biological systems and highlights their growing relevance to neuroscience, cell biology, bioengineering, and therapeutic research. Notably, optical control of H[+] concentration allows the precise modulation of neural and non-neural activities in animals and bacteria, highlighting the broad applicability and significant potential of H[+] pump rhodopsins for optogenetics. Based on previous and current studies, we discuss how further expansion of the molecular toolkit of H[+] pump rhodopsins could enable increasingly fine-tuned manipulation of intracellular pH dynamics and PMF for probing cellular physiology and designing next-generation therapeutic strategies, and consider emerging directions that extend beyond classical optogenetics toward the optical control of bioenergetic and chemical processes.