Brain neurons rely predominantly on glucose as an energy source and express glucose transporter 3 (GLUT3) as their principal glucose transporter. Unlike GLUT4, which undergoes insulin-stimulated membrane translocation in peripheral tissues, GLUT3 mediates largely constitutive and relatively insulin-independent glucose uptake in neurons. Although GLUT3 is essential for neuronal functions, its regulatory mechanisms are not fully understood. Microtubule affinity-regulating kinase 4 (MARK4), a member of the AMP-activated protein kinase-related kinase family, has been implicated in Alzheimer's disease and metabolic regulation in peripheral tissues. Here, we show that MARK4 knockdown in primary neurons reduces GLUT3 surface expression and consequently decreases glucose uptake. While MARK4 knockdown did not alter the intracellular distribution of GLUT3 or mitochondria, it reduced mitochondrial abundance. We further found that MARK4 activity is negatively regulated by the insulin/IGF-1-GSK3β signaling axis in primary neurons. Despite these acute metabolic effects in cultured neurons, Mark4-null mice exhibited no overt morphological abnormalities in the brain. Together, these findings identify MARK4 as a regulator of neuronal glucose uptake by modulating GLUT3 surface expression and suggest that MARK4 functions as a modulatory rather than essential component of neuronal metabolic homeostasis.
Supersulfides, a class of catenated sulfur-containing biomolecules, are increasingly recognized as key regulators of redox signaling, mitochondrial function, and inflammatory responses. Recent evidence suggests that lysosomes, central organelles for intracellular degradation and nutrient sensing, are closely linked to supersulfide metabolism through lysosomal acidification, cysteine metabolism, and autophagy. Conversely, supersulfides modulate lysosomal activity and inflammatory responses. This review summarizes recent progress in supersulfide biology and lysosomal regulation and discusses evidence supporting functional interactions between these systems. We propose the lysosome-supersulfide axis as a new concept in cellular homeostasis and metabolic regulation.
Abstract Fucosylated haptoglobin (Fuc-Hp) has been recognized as a cancer-associated glycoform. We previously established a monoclonal antibody, 10-7G mAb, which detects both Fuc-Hp and its precursor prohaptoglobin (proHp). Recent studies have suggested that proHp itself may serve as a cancer-associated biomarker with clinical implications distinct from those of Fuc-Hp. Importantly, proHp is produced during inflammation and cancer progression and may exert biological activities different from mature haptoglobin (Hp). These observations highlight the need for a reagent that can specifically detect proHp. However, to date, no antibody strictly specific to proHp has been available. In this study, we generated a monoclonal antibody, 21-4F mAb, which specifically recognizes human proHp without cross-reactivity to mature Hp. Using 21-4F mAb, we developed a proHp-specific enzyme-linked immunosorbent assay and integrated it with assays for several types of Hp, enabling independent quantification of three Hp-related isoforms. Serum analyses of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer revealed distinct alterations among these isoforms, and their combined use provided the highest diagnostic performance. Taken together, 21-4F mAb offers a novel analytical tool for investigating the pathophysiological significance of proHp and may contribute to the development of refined biomarker panels for cancer diagnosis and disease monitoring.
Synapses are the fundamental computational units of the brain. Although synapses are classically classified according to the neurotransmitter they use, glutamatergic excitatory synapses also exhibit marked heterogeneity in their molecular composition and structure. Understanding this diversity requires comprehensive approaches that go beyond small-scale, candidate-based analyses of synapses. Proteomic analyses of biochemically enriched synaptic fractions have provided unbiased, quantitative measurements of synaptic protein composition, revealing differences across brain regions, cell types, developmental stages, and physiological and pathological states. Proteomics has also revealed the composition and remodeling of synaptic protein complexes and post-translational modifications. In parallel, synaptome mapping, an imaging-based approach for the large-scale in situ analysis of synapses across the brain, has emerged as a form of spatial omics. In this approach, synapses labeled with a small set of synaptic proteins are systematically imaged across the brain. Synaptome mapping reveals whole-brain variation in synapse molecular identity and structural features, including differences in synapse size, shape, and spatial distribution in vivo. Together, these approaches show that excitatory synapses are diversified across anatomical, cellular, developmental, activity-dependent, and disease-related contexts. These complementary perspectives will advance our understanding of the synaptic mechanisms underlying neural computation, behavior, and brain disorders.
Fornicata is a group of eukaryotes adapted to anaerobic and microaerophilic environments. These organisms generate ATP anaerobically through substrate-level phosphorylation. ADP-forming acetyl-CoA synthetase (ACS) is one of the key enzymes of this process. While ACS characteristics in the parasite Giardia intestinalis have been studied, those in free-living species remain unknown. Here, we investigated the ATP-generating activities of recombinant ACS (rACS) of free-living fornicates Aduncisulcus paluster and Kipferlia bialata and compared them with Gasterophilus intestinalis. All rACSs exhibited the highest activity toward the substrate acetyl-CoA and the next toward n-propionyl-CoA. For acetyl-CoA, rApACS exhibited higher affinity (lower KM) and lower catalytic turnover (kcat) than the other two that displayed comparable kinetic profiles. For n-propionyl-CoA, rApACS also exhibited lower KM and kcat than the other two, while rGiACS exhibited far higher kcat than the other two, indicating that rGiACS could utilize n-propionyl-CoA effectively. For the substrate ADP, rKbACS exhibited the highest kcat among the three. These results suggest that ACSs from free-living fornicates are active ATP-generating enzymes, and that during the evolution of fornicates, the ATP-generating activity has been conserved, while the subtle changes in kinetic properties have occurred on their ACSs.
Methylglyoxal (MGO), a highly reactive dicarbonyl compound generated mainly by glycolysis, is a major precursor of advanced glycation end products (AGEs). Although lysine-derived AGEs, such as Nε-(carboxyethyl)lysine (CEL), have been widely used as markers of MGO-mediated protein modification, increasing evidence suggests that MGO preferentially modifies arginine residues. However, the biochemical significance of arginine-derived AGEs remains poorly understood because of limited analytical and immunochemical methods for their detection. In this study, we investigated the formation and quantitative significance of Nω-(carboxyethyl)arginine (CEA), an underexplored MGO-derived arginine adduct. CEA was chemically synthesized and structurally characterized by NMR and mass spectrometry. Analysis of human tissues revealed that CEA accumulated at high levels in cataractous lenses, comparable to MG-H1 and higher than CEL levels. A monoclonal antibody specific for CEA was developed and validated using ELISA, showing no cross-reactivity with related AGEs. CEA was generated in a time-dependent manner in bovine serum albumin incubated with MGO, whereas CEL formation was minimal. CEA levels were higher than those of CEL in MGO-modified proteins, indicating its preferential formation under physiological conditions. These findings indicate that CEA is a reliable marker for evaluating MGO-mediated protein degeneration and dicarbonyl stress in vitro and in vivo.
Non-canonical disulfide bonds are a hallmark of the VHH domain of heavy-chain antibodies, with predominant bonds between CDRs 1 and 3 and between framework FR2 and CDR3. The former interloop disulfide bond is advantageous for stability and antigen-binding affinity, albeit not indispensable. Importantly, mutations in the cysteine of the interloop disulfide bond of CDRs 1 and 3 can sometimes preserve antigen-binding activity. In contrast, the functional role of the interloop disulfide bond between FR2 and CDR3 remains largely understudied. In this study, we investigated the replacement of the disulfide bond between FR2 and CDR3 with cysteine mutations in aliphatic amino acids (alanine, valine, and isoleucine). These results indicate that this interloop disulfide bond contributes to the structural stabilization of VHH but is not required for antigen binding, consistent with the findings for the disulfide bond between CDRs 1 and 3. Furthermore, the disulfide bond between FR2 and CDR3 was not critical for maintaining reversibility following heat-induced unfolding. Given the prevalence of FR2-CDR3 interloop disulfide bonds in VHHs from llamas and alpacas, these findings provide valuable insights into VHH engineering and applications.
Methylation of DNA, histones, and RNA is central to the regulation of circadian rhythms, yet the biochemical origin of the methyl groups driving these modifications has received comparatively little attention in circadian biology. This review explores the bidirectional crosstalk between the methyl cycle and the mammalian circadian clock. We describe how S-adenosylmethionine-dependent epigenetic and epitranscriptomic modifications constitute essential layers of circadian gene regulation, and how the clock orchestrates the rhythmic expression of one-carbon metabolism enzymes and oscillations in S-adenosylmethionine availability. The direct interaction between the S-adenosylhomocysteine hydrolase AHCY and the core clock component BMAL1 at circadian gene promoters emerges as a molecular nexus linking methyl group supply to clock-driven transcription. We further discuss how the methyl cycle occupies a privileged position within the circadian entrainment hierarchy, acting as both a target of nutritional zeitgebers in peripheral tissues and a potential source of metabolic feedback to the central pacemaker, and how dietary perturbation of the methyl cycle disrupts circadian rhythms. Finally, we discuss how this crosstalk is implicated in metabolic liver disease, cancer, neurological disorders, and aging. Together, these findings position the circadian clock as a sensitive readout of nutritional methyl metabolic status, with broad implications for chronobiology and nutrigenomics.
Estrogen-related receptor γ (ERRγ) is an orphan nuclear receptor whose molecular functions in transcriptional regulation remain incompletely understood. The androgen receptor (AR) is a ligand-dependent transcription factor that plays a pivotal role in prostate cancer by regulating gene expression through androgen-response elements. Its activity is tightly controlled by receptor conformation and coactivator recruitment. Although ERRγ has been implicated in the suppression of prostate cancer progression, whether it modulates AR signaling remains unclear. In this study, we examined the functional and molecular interplay between ERRγ and AR using a heterologous expression system in HeLa cells. We found that ERRγ suppresses AR-mediated transcriptional activation in a dose-dependent manner on ARE-containing promoters. Mechanistically, ERRγ interacted with ligand-activated AR in the nucleus and disrupted the intramolecular interaction between the N-terminal domain (NTD) and the C-terminal ligand-binding domain (LBD), which is required for full receptor activation. This interference did not affect AR nuclear translocation. Furthermore, ERRγ functionally competed with major transcriptional coactivators, including steroid receptor coactivators-3 (SRC-3) and p300, thereby attenuating coactivator-enhanced AR-dependent transcription. Collectively, these findings demonstrate that ERRγ functions as a novel corepressor of AR by destabilizing receptor conformation and blocking coactivator recruitment, providing new mechanistic insights into the regulation of AR-mediated transcription.
Metabolism is a complex consortium of chemical reactions, consuming and releasing energy. In addition, cellular respiration is a pivotal catabolic process of nutrients using oxygen to generate ATP. Dynamics of chromatin structure are fundamental to genomic events in all cellular processes. Therefore, chromatin consumes robust cellular energy formed in metabolism. To regulate cellular processes, chromatin functions and metabolism need to be tightly coordinated in response to environmental changes including nutrition availability. Indeed, molecular oxygen directly impacts DNA and chromatin modification and influences cell fate. Herein, we review fundamentals of chromatin dynamics, nucleosome modifications, and roles of chromatin dynamics in spatiotemporal regulation of cellular energy homeostasis.
The circadian clock generates ~24-hour rhythms in physiology by coordinating gene expression programs, but temporal mRNA profiles often fail to predict their protein function rhythms. This gap reflects regulatory layers beyond transcription, including rhythmic translation, protein stability, subcellular localization and post-translational modifications that collectively determine the circadian rhythms of protein abundance and activity. Here, we summarize evidence supporting a shift from RNA-level descriptions to protein-level frameworks and readouts of the circadian rhythms. Here, we highlight three topics: (i) protein abundance rhythms as informative but incomplete readouts, (ii) widespread circadian control of nuclear localization and phosphorylation that can occur without changes in total protein levels, and (iii) multi-tissue proteomic comparisons that reveal how circadian rhythms are organized differently across tissues. We then discuss how recent data-independent acquisition-based, high-throughput mass spectrometry accelerates cross-study reuse and hypothesis generation, as illustrated by a mouse circadian proteome atlas and an interactive portal enabled by Orbitrap Astral mass spectrometer. Together, these advances motivate 'functional chronobiology', linking proteome dynamics to mechanism and disease-relevant physiology.
Mitochondria are essential for cellular metabolism and homeostasis, and their quality and quantity must therefore be tightly controlled. Mitophagy, a selective form of autophagy targeting mitochondria, contributes to this control by eliminating damaged or superfluous mitochondria. Among the known mitophagy pathways, BNIP3/NIX-dependent mitophagy has emerged as a key mechanism, particularly under hypoxic and metabolic stress. Recent studies have provided important insights into how BNIP3 and NIX are transcriptionally induced, post-translationally regulated and functionally coupled to the core autophagy machinery. These studies have also clarified their roles in isolation membrane tethering, membrane elongation and mitophagosome formation. Beyond its molecular basis, accumulating evidence indicates that BNIP3/NIX-dependent mitophagy contributes to mitochondrial homeostasis, redox balance and cellular stress adaptation. This review summarizes recent progress in understanding the molecular mechanisms and physiological significance of BNIP3/NIX-dependent mitophagy.
Fibroblast growth factor receptor 1 (FGFR1) is a receptor-type tyrosine kinase involved in various human cancers, making it an attractive therapeutic target. To elucidate the complex binding dynamics between FGFR1 and its highly selective inhibitor, dovitinib, we conducted a detailed investigation using a combination of surface plasmon resonance (SPR), 19F-NMR spectroscopy, and molecular dynamics (MD) simulations. Kinetic analysis using SPR suggested that the interaction between FGFR1 and dovitinib is better described by a two-state binding model, implying the potential formation of an initial transient intermediate prior to the formation of a stable complex. We further characterized this interaction using 19F-NMR by leveraging the intrinsic fluorine atoms of dovitinib. When dovitinib was present in excess relative to FGFR1, the 19F-NMR signal exhibited broadening compared to that in the free state, indicating altered dovitinib environments and additional FGFR1-associated states. Furthermore, MD simulations introducing an additional dovitinib molecule to the FGFR1-dovitinib complex indicated the potential for weak and transient interactions in the preferred interaction regions on the FGFR1 surface. These findings highlight the multifaceted nature of kinase-inhibitor interactions and provide valuable biophysical insights that may contribute to future drug discovery efforts targeting receptor tyrosine kinases.
In higher plants, ferredoxin (Fd) is present as distinct isoproteins of photosynthetic type (LFd) and non-photosynthetic type (RFd), which exhibit differential function despite their similarity in the 3D structures. We previously showed that pH-dependency of electron transfer activity with ferredoxin-NADP+ reductase (FNR) was opposite between LFd and RFd, which was explained by the opposite pH-dependent profile of Km for the two Fds, and that the differences of C-terminal residues and 78th residue between the two Fds were partly responsible for the different pH dependency. In this study, we further investigated the determinants responsible for the different pH dependency between the LFd and RFd. Site-directed mutants of Fd, substituted at the residues on the interface with FNR, were prepared. Kinetic analyses using the single site-directed mutants and their combined, multiple mutants showed that combination of the substitutions of Fd residues at 61, 63, 78th and C-terminal region located at the interface with FNR conferred opposite pH-dependency, which indicated that these residues are the enough determinant for their opposite pH dependency of LFd and RFd in the electron transfer reaction with FNR.
RNA-targeted small molecule drug discovery is widely recognized as an important modality. We have demonstrated that NMR spectroscopy is useful for screening small molecules that bind to specific RNA structures. In the previous work, we analysed the changes in the imino proton signals of 47 RNAs with different structures; it was clearly indicated upon addition of three well-known small molecules, risdiplam, NCD, and CPFX and found several combinations of small molecules and RNA structures that showed specific changes. In this study, the structural characteristics of the model RNAs were further analysed by measuring the Tm values. Then, to demonstrate the importance of the NMR-based method, we applied the method to two additional small molecules. In addition to the imino proton signals, the H5-H6 signals were also used to detect the interaction between small molecules and single-stranded regions of RNAs. As a result, two additional examples of specific RNA-small molecule interactions were identified.
In bacterial homologous recombination, the single-stranded DNA (ssDNA)-binding protein (SSB) coats exposed ssDNA to protect it but simultaneously inhibits RecA filament nucleation. RecO, a recombination mediator, overcomes this inhibition by interacting with SSB and displacing it from ssDNA. This SSB-RecO interaction has long been considered to rely primarily on the conserved C-terminal acidic tip (C-tip) of SSB. Here, using domain-truncated SSB variants from Thermus thermophilus HB8, we show that RecO directly binds both the SSB oligonucleotide/oligosaccharide-binding fold (OB-fold) and the C-tip. These two contacts make distinct contributions to RecO's mediator functions. Arg127 in RecO contributes to recognition of the SSB C-tip and to double-stranded DNA (dsDNA) binding, the latter of which is suppressed by the SSB C-tip. In RecO-mediated ssDNA annealing, the SSB C-tip primarily modulates SSB dynamics on ssDNA rather than directly activating RecO. Moreover, stable assembly of the SSB-RecO-RecR ternary complex requires the SSB C-tip. Our findings support a mechanism in which the SSB C-tip modulates RecO function and governs SSB behaviour on ssDNA, whereas the OB-fold provides additional contacts whose functional contributions remain to be elucidated.
Protein phosphorylation, a key post-translational modification, is mediated by various protein kinases. The bacterial PrkA/YeaG is recognized as an atypical protein kinase, but its activity remains elusive. This study investigated the structural and functional characteristics of a PrkA/YeaG homologue, TpkB, from Thermus thermophilus HB8. Using cryo-electron microscopy, the structures of TpkB in apo and AMPPNP-bound forms were determined, revealing a hexameric ring architecture characteristic of AAA + superfamily proteins. The TpkB protomer is comprised of an N-terminal domain, an ATPase domain and an LID domain. The ATPase domain contains conserved sequence motifs associated with ATPase activity, whereas the other domains present a novel fold. TpkB exhibits structural similarity to MoxR family proteins, which possess chaperone-like functions in conjunction with von Willebrand factor A (vWA) domain proteins. Structural and gene neighbourhood analyses suggested a functional link between PrkA/YeaG proteins and vWA domain proteins. Biochemical analyses demonstrated that TpkB exhibited ATPase activity and chaperone-like activity, but lacked detectable protein kinase activity. These findings establish TpkB as a novel member of the AAA+ superfamily with potential chaperone functions, providing new insights into the PrkA/YeaG family.
Neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease, are becoming increasingly prevalent in super-ageing societies. However, the molecular mechanisms by which prion-like proteins undergo aberrant phase transition, aggregation and propagation during neurodegeneration remain incompletely understood. Although these proteins are capable of undergoing liquid-liquid phase separation (LLPS), followed by a sol-gel transition in vitro, the factors that govern their pathological phase transition in vivo remain largely elusive. Notably, many prion-like proteins also possess RNA-binding properties, and accumulating evidence indicates that RNA plays pivotal roles in regulating both LLPS and the subsequent transition to more solid-like states. In this review, we summarize recent advances in the RNA structural biology of neurodegeneration, with particular emphasis on RNA G-quadruplexes (rG4s) as pathological determinants and discuss emerging mechanisms by which RNA structures promote the phase transition and proteinopathy of prion-like proteins.
Pyrogen testing is essential for ensuring pharmaceutical product safety; however, current methods have limitations. The Bacterial Endotoxins Test cannot detect non-endotoxin pyrogens, whereas the Rabbit pyrogen test was phased out for animal welfare reasons. Monocyte activation tests (MAT), using human whole blood samples or peripheral blood mononuclear cells, are effective but suffer from donor variability, biosafety concerns and instability. We aimed to develop a sensitive, rapid and simple alternative pyrogen test by devising a luciferase-based MAT using the Mono-Mac-6 (MM6) cell line. MM6 cells showed higher cytokine responses to endotoxin than THP-1 cells and exhibited comparable or greater sensitivity than human peripheral blood. Introducing an NF-κB-driven NanoLuc® reporter enabled endotoxin detection within 3 h; however, rapid degradation of intracellular luciferase was noted. To overcome this degradation issue, we generated MM6 cells expressing secreted NanoLuc (secNluc), allowing for stable detection between 3 and 12 h after stimulation. The MM6/NFκB-secNluc cells helped detect various pyrogens, including Pam3CSK4, peptidoglycan, polyinosinic-polycytidylic acid sodium salt, lipopolysaccharide, flagellin, macrophage-activating lipopeptide-2 and resiquimod. Adding recombinant human lipopolysaccharide-binding protein enabled establishing a serum-free MAT system, eliminating serum-derived endotoxin contamination. This reporter-based MAT provides a robust platform for rapid endotoxin and NEP detection, offering an improved alternative to conventional MATs.
