Cisplatin-based chemotherapy is a standard treatment for non-small cell lung cancer (NSCLC), but drug resistance poses a major clinical challenge. Stress-adaptive mechanisms such as stress granule (SG) formation are increasingly recognized alternative pathways that facilitate cancer cell survival. Here, we identify the RNA-binding protein, family with sequence similarity 120A (FAM120A), as a SG-associated factor that drives cisplatin resistance in NSCLC. FAM120A expression was markedly elevated in cisplatin-resistant NSCLC cell lines and clinical tumor specimens, and was essential for SG formation and cell survival following cisplatin-induced stress. We found that the intrinsically disordered RNA-binding domain of FAM120A is essential for its incorporation into SGs and for its cytoprotective function. Using enhanced Cross-Linking Immunoprecipitation sequencing (eCLIP-seq) data and RNA immunoprecipitation-qPCR (RIP-qPCR), we identified the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) as a key FAM120A interacting partner. MALAT1 levels were reduced upon FAM120A depletion, and overexpression of MALAT1 was sufficient to restore cisplatin resistance in these cells. These findings suggest that MALAT1 is an RNA species that is stabilized by FAM120A and involved in the cellular response to chemotherapy. Targeting this regulatory mechanism may offer new therapeutic strategies to overcome cisplatin resistance in NSCLC.

Phospholipase A (PLA) and acyltransferases coordinate glycerophospholipid remodeling to maintain membrane diversity and function. The phospholipase A and acyltransferase (PLAAT) family combines PLA1/PLA2 with N- and O-acyltransferase activities, generating N-acylethanolamines with diverse bioactivities and enabling acyl-CoA-independent remodeling. PLAAT3 has been identified as a causative gene for human lipodystrophy. In addition to adipocyte dysfunction, PLAAT3-deficient mice develop cataracts due to impaired organelle degradation in lens fiber cells. In non-mammalian vertebrates such as zebrafish, which lack PLAAT3, PLAAT1 is highly expressed in the lens, and its deficiency similarly causes cataract-like abnormalities by blocking organelle clearance. A recent study reported that PLAAT1 promotes cardiolipin production in cultured cells, indicating a role in mitochondrial membrane lipid metabolism; however, its direct involvement in mitochondrial dynamics remains unclear. To address this, Sikder et al. (J. Biochem. 175:101-113, 2023) established a doxycycline-inducible mouse PLAAT1 expression system in HEK293 cells. Catalytically active PLAAT1 rapidly induced mitochondrial fragmentation and peroxisome loss, independently of changes in Drp1, Mfn2, and Opa1 expression. These findings reveal a previously unrecognized role of PLAAT1 in regulating organelle dynamics and maintaining cellular homeostasis.

Microglia, the central nervous system's resident macrophages, are critical for immune defense, protecting neurons during infection. Their role in postnatal brain development, particularly after injury, remains unclear. Nucling, a protein up-regulated during cardiac muscle differentiation, regulates NF-κB, influencing apoptosis and cell proliferation. In this study, we examined the role of Nucling in microglial activation using wild-type (WT) and Nucling-knockout (KO) neonatal mice subjected to poly(I:C), a viral mimic. Poly(I:C) treatment increased Iba1-positive microglia in both genotypes; however, KO mice showed a significantly exaggerated response in both cortical and hippocampal regions. Furthermore, while proinflammatory M1 markers (iNOS, CD86, TNFα, IL-6) were upregulated in both WT and KO mice, the anti-inflammatory M2 marker Arginase 1 (Arg1) was induced in WT but significantly suppressed in KO mice, indicating impaired M2 polarization. These findings suggest that Nucling is essential for maintaining microglial polarization, supporting immunological processes against pathogens and aiding central nervous system development.

The murine retrovirus integration site 1 (MRVI1) gene encodes an endoplasmic reticulum (ER)-associated membrane protein involved in calcium signalling, yet its molecular interaction network remains largely undefined. Here, we employed TurboID-based proximity labelling to construct the first comprehensive map of MRVI1-associated proteins in mammalian cells. This analysis identified >700 candidate interactors, including ER-localized factors and components of intracellular trafficking, consistent with the subcellular localization and signalling role of MRVI1. To investigate oncogenic modulation, we examined how co-expression of NPM-ALK-a constitutively active tyrosine kinase implicated in lymphoid malignancies-reshapes the MRVI1 interactome. Quantitative proteomics revealed that while the overall composition of MRVI1-associated proteins was largely preserved, a subset of interactions was selectively enhanced or attenuated by NPM-ALK. The association of MRVI1 with several signalling-related proteins was enhanced by NPM-ALK, including 12 proteins that have all been previously implicated in cancer-related pathways. In contrast, proteins whose interaction with MRVI1 was suppressed were functionally enriched in the Gene Ontology term 'negative regulation of apoptotic process'. Notably, anti-apoptotic regulators such as DDB1, PHB2 and NOTCH2 showed significantly reduced proximity labelling, suggesting that MRVI1 may participate in apoptosis-related networks disrupted during oncogenic transformation. Together, our findings demonstrate that MRVI1 forms a functionally diverse protein network that can be selectively remodelled by oncogenic signalling. This study not only uncovers potential mechanisms by which MRVI1 contributes to transformation but also provides a valuable proteomic resource for future investigation of MRVI1 function and regulation.

Macromolecular crowding is a fundamental property of the intracellular environment that influences protein folding, enzymatic activity, and phase behavior. Disruptions to the homeostasis of macromolecular crowding can drive pathological processes, such as aberrant liquid-liquid phase separation and protein aggregation, which are central features of several neurodegenerative diseases. However, tools for quantifying crowding and aggregation remain limited. Here, we describe moxCRONOS, a Förster resonance energy transfer (FRET)-based biosensor that enables the quantitative measurement of macromolecular crowding and protein condensation. moxCRONOS retains the optical properties of the original CRONOS sensor but offers enhanced stability in oxidative environments, such as within the endoplasmic reticulum or under sodium arsenite treatment, allowing for direct comparison of crowding levels across organelles regardless of redox conditions. Moreover, when fused to dipeptide repeat proteins associated with C9ORF72-linked neurodegeneration, moxCRONOS detects aggregation-prone states-especially in cells expressing glycine-alanine (GA) repeats. Using fluorescence-activated cell sorting, we achieved sensitive and quantitative detection of heterogeneous high-FRET cell populations containing GA aggregates. FRET signal intensity increased upon treatment with a molecular crowding agent or a proteasome inhibitor. These findings establish moxCRONOS as a versatile biosensor for investigating both physiological macromolecular crowding and pathological protein aggregation, with significant potential for disease modeling and therapeutic screening.

Liquid-liquid phase separation (LLPS) is a fundamental organizing principle in biology, driving the formation of membraneless compartments and thereby orchestrating a vast array of biochemical reactions in a spatiotemporal manner. LLPS is mediated by weak, multivalent interactions between biomolecules. While intrinsically disordered regions (IDRs) are widely recognized as major drivers of LLPS, coiled-coils, one of the most ubiquitous protein motifs, are emerging as functionally distinct, versatile contributors. This review systematically explores the multifaceted roles of coiled-coils in LLPS, highlighting their capabilities that contrast with those of IDRs. A key feature distinguishing coiled-coils is their ability to span an exceptionally broad range of interaction affinities, from picomolar to millimolar levels. This vast dynamic range allows them to operate across a continuous functional spectrum-from serving as high-affinity oligomerization platforms to acting as modules that mediate weak, transient interactions-a functional duality not recapitulated by IDRs. Through this inherent tunability, coiled-coils can play a pivotal role in modulating both the propensity for phase separation and the material properties of the resultant condensates.

Recent advances in mass spectrometry-based proteomics have enabled increasingly precise characterization of protein modifications in clinical specimens. Among these, glycosylation is one of the most structurally complex and biologically informative post-translational modifications, reflecting cellular differentiation and disease states. Ohashi et al. (J. Biochem. 2024; 175: 561-572) performed a site-specific N-glycosylation analysis of LAMP1 in breast cancer tissue samples, demonstrating the feasibility of targeted glycoproteomics in patient-derived specimens and revealing tumor-associated glycoform heterogeneity. Their study exemplifies how focusing on a single glycoprotein target can provide detailed insight into disease-specific glycan remodeling within the tumor microenvironment. In this commentary, I discuss the significance of such targeted approaches in the broader context of clinical glycoproteomics and highlight their potential contribution to cancer biomarker discovery and precision medicine. Continued integration of glycoproteomic data with genomic and clinical information is expected to further advance our understanding of tumor biology and therapeutic response.

Lactoferrin is a multifunctional protein mainly involved in the immune defense of organisms against various pathogens. It has been reported that intestinal inflammation was reduced by lactoferrin administration. However, the precise mechanism underlying lactoferrin's involvement in intestinal inflammation is not yet fully understood. In this study, we purified a mouse intestinal lactoferrin-binding protein with a molecular mass of ~ 400 kDa that was expressed in the small intestine and colon. Sequence analysis revealed that the intestinal lactoferrin-binding protein represented an ortholog of rat immunoglobulin G fragment crystallizable gamma-binding protein (IgGFcγBP). N-linked glycans of lactoferrin were not necessary for binding to IgGFcγBP. After reduction, IgGFcγBP was separated into 120, 70, 65, 60, and 55 kDa proteins, but these did not bind to lactoferrin. The expression of IgGFcγBP was lost in a mouse model of dextran sodium sulfate induced colitis and restored during the convalescence period of colitis, suggesting a role in mucosal protection and immune regulation. Furthermore, we discuss potential links between IgGFcγBP and mucin-associated microbiota which may contribute to lactoferrin's immunomodulatory effects. These findings provide new insights into the interaction between lactoferrin, mucosal immunity, and gut microbiota.

Mitochondria contain their own DNA (mtDNA), which is essential for respiratory function. Multiple copies of mtDNA are assembled into dot-like structures called nucleoids. Nucleoids move dynamically within mitochondria, and their size and distribution are influenced by mitochondrial membrane fission and fusion. However, the molecular mechanisms and their pathophysiological significance, particularly in vivo, remain largely unknown. Here, we identify a novel role for ubiquinone, as well as natural quinones lacking electron-carrying capacity, in the organization of nucleoids and respiratory complexes, independent of their conventional roles. These quinones facilitate the association and packaging of mtDNA on the cardiolipin-enriched mitochondrial inner membrane. This quinone-dependent maintenance of nucleoids protects against mitochondrial dysfunction and heart failure induced by the anticancer drug doxorubicin. Our RNAi screen identifies a set of genes involved in mitochondrial diseases that exhibit nucleoid deformation, suggesting a novel therapeutic approach targeting mitochondrial nucleoids for various pathological conditions associated with mitochondrial dysfunction.

Pancreatic β cells maintain glucose homeostasis through insulin production, and their loss underlies both type 1 and type 2 diabetes. Among the signaling systems that govern β-cell biology, insulin and insulin-like growth factor (IGF) receptor pathways have long attracted attention as intrinsic modulators of β-cell growth, survival, and secretory competence. However, the physiological and pathological relevance of these receptors in β cells remains uncertain, reflecting model-specific discrepancies and the complex interplay between local autocrine and systemic endocrine effects. Recent analyses have expanded this view, revealing the coexistence of insulin receptor-dependent and insulin receptor-independent regulatory modules that govern β-cell adaptation to metabolic stress. Furthermore, molecular regulators, including inceptor and IGF2R, reshape our understanding of insulin/IGF receptor signaling as a flexible, adaptive network. Together, these insights suggest that precise modulation of receptor networks may hold the key to unlocking endogenous β-cell regenerative capacity.

Type 2 diabetes, which is closely linked to obesity, results from complex genetic and environmental interactions. Despite high heritability estimates, genome-wide association studies have not fully explained the heritability, suggesting the involvement of epigenetic mechanisms. This review highlights two distinct genome-independent pathways for intergenerational transmission of diabetic risk: (1) epigenetic transmission via gametes, whereby parental exposures induce heritable epigenetic changes in germ cells, and (2) developmental programming, in which prenatal or early postnatal environments shape the metabolism of offspring. Both processes are increasingly understood to involve epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNAs. These epigenetic modifications have been suggested to contribute to intergenerational disease transmission in both animal and human studies. Understanding these mechanisms is essential for developing preventive strategies targeting the intergenerational risk of metabolic diseases.

Autophagy is a conserved degradation process delivering intracellular components to lysosomes or vacuoles. Yeast studies have been pivotal in identifying autophagy-related (ATG) genes and defining the core machinery essential for autophagosome formation. A recent comprehensive analysis that systematically examined all atg mutants in S. cerevisiae under autophagy-inducing conditions revealed that mutants lacking Atg13, Atg8-conjugation, or Atg12-conjugation components retain partial activity in certain autophagy-related pathways, indicating that these core factors are not strictly essential for autophagy in yeast. In this commentary, we summarize how recent findings reshape our understanding of the flexibility in the essentiality of core autophagy factors and discuss the emerging importance of protein interaction-driven feedback in autophagy regulation.

Giant viruses encode unusual glycosylation machinery distinct from their amoebal hosts, raising fundamental questions about how their glycans are synthesized and diversified. Here we present a comparative glycomic analysis of mimivirus, tokyovirus, and hokutovirus, together with their common host Acanthamoeba castellanii. The main objective of this study was to determine whether giant viruses rely on host-derived N-glycosylation, or alternatively employ virus-encoded pathways to generate lineage-specific O-glycans, and to assess how these processes differ across virus families. N-glycan profiling revealed that all three viruses lack canonical eukaryotic core structures, in contrast to amoebal high-mannose N-glycans carrying pentose and phosphate residues. This finding demonstrates that giant viruses do not exploit the host secretory pathway for N-glycosylation, but instead depend on alternative mechanisms. O-glycan analyses showed lineage-specific patterns: family Marseilleviridae members tokyovirus and hokutovirus, displayed highly similar profiles, with minor virus-specific differences, whereas mimivirus exhibited structurally distinct glycans. Genomic inspection revealed that tokyovirus encodes only five glycosyltransferase-like genes, while A. castellanii harbors candidate enzymes for unusual monosaccharides. These findings clarify the distinct contributions of host and viral pathways and highlight evolutionary diversification of glycosylation among giant viruses.

Rab GTPases are molecular switches that control intracellular vesicular transport by cycling between GDP- and GTP-bound states. Insects encode an insect-specific subset, RabX; Bombyx mori RabX6 (BmRabX6) has been implicated in testis development and neuropeptide secretion, but its structure and mechanism were unknown. Here we report the 3.1 Å crystal structure of BmRabX6 in complex with GDP and Mg2+ (PDB: 9VLB), the first structure of an insect-specific Rab GTPase. BmRabX6 adopts the canonical small GTPase fold with conserved P-loop and Switch I/II, and displays a GDP-binding mode similar to vertebrate Rabs. Two features distinguish BmRabX6. First, the catalytic glutamine required for GTP hydrolysis in typical Rabs is naturally replaced by methionine (Met69) and oriented away from the nucleotide, consistent with obligate GAP-assisted hydrolysis. Second, one residue of the hydrophobic effector-binding triad is histidine (His47), suggesting a potential shift toward a hydrophilic interface-mediated interaction distinct from canonical Rab-effector recognition. AlphaFold3-based complex modeling further identified BmH9J2P5 as a prioritized GTPase-activating protein (GAP) candidate interacting with BmRabX6. These adaptations suggest that BmRabX6 preserves core nucleotide cycling while employing divergent regulatory chemistry tuned to insect physiology. Our structure provides a framework for testing GAP dependence and effector specificity of RabX6 in reproductive and neuronal tissues and illustrates how strategic amino-acid substitutions diversify Rab function.

Cannabinoid-1 receptor (CB1R) is one of the promising targets for treating various diseases, various antagonists, agonists, and reverse agonists targeting CB1R have been synthesized and investigated for clinical use. In this work, we used molecular docking and molecular dynamics (MD) simulations to explore the interaction between CB1R and six antagonists: BNS807, BNS808, and BNS809 derived from template 1 and BNS815, BNS816, BNS825 derived from template 2. Six initial conformations were selected for the subsequent MD simulations using molecular docking and cluster analysis. The binding free energy analysis shows that in the three systems of BNS807-CB1R, BNS808-CB1R, and BNS809-CB1R, the increase of binding affinity is attributed to the nonpolar contributions of residues Val196, Ala120, Phe200, Phe268, Phe380 and Phe381, and large-volume aromatic substituents are favorable for binding, BNS809 with small substituent CH3 could form the hydrogen bond with Gln115. In the three systems based on template 2, Ile105, Ile116, and Phe177 increase the binding affinity of the antagonists to CB1R. Furthermore, the seven-membered and pyrazole ring of BNS816 formed vdW interactions with Phe170, stabilizing the conformation of BNS816-CB1R. These results reveal the interaction patterns of six peripheral antagonists with CB1R, providing theoretical guidance for the design of drug molecules targeting CB1R.

Regnase-1, encoded by the ZC3H12A gene, is a well-known RNase that suppresses inflammation by degrading the mRNAs of inflammatory cytokines. However, its role in cancer pathogenesis, especially in non-small cell lung cancer (NSCLC), remains poorly understood. Through an analysis of public databases, we found that NSCLC patients with higher ZC3H12A expression levels had a worse prognosis than those with lower levels. To explore the function of Regnase-1 in NSCLC, we knocked out the ZC3H12A gene in NSCLC cell lines and compared their transcriptomes with those of parental cells. This analysis identified the SOX2 pathway as a common pathway suppressed by Regnase-1 deficiency. Consistent with the SOX2 contribution to the cancer stemness, Regnase-1 inhibition impaired oncosphere growth and tumor formation of cell lines derived from adenocarcinoma, squamous cell carcinoma and large cell carcinoma. It was also effective for NRF2-activated NSCLC cells, which are highly resistant to most of the therapeutics. Notably, post-tumorigenic suppression of Regnase-1 significantly inhibited tumor growth, suggesting that Regnase-1 could be a promising therapeutic target for post-tumorigenic treatment of NSCLC. Given recent studies describing that Regnase-1 inhibition enhances anti-cancer immunity, we propose that targeting Regnase-1 could be an ideal strategy for controlling intractable cancers by both suppressing cancer cells and activating anti-cancer immunity.

Tetrahydrobiopterin (BH4) is an essential cofactor for biosynthesis of monoamines and nitric oxide. An excess of BH4 in infiltrated macrophages was reported to cause pain, while a certain level of BH4 is essential for cell survival and proliferation. GTP cyclohydrolase I (GCH) is a rate-limiting enzyme for the de novo synthesis of BH4. Our previous study showed that GCH expression was elevated by an enhancer region containing the C/EBP and Ets binding motifs in macrophage-like RAW264.7 cells when stimulated with lipopolysaccharide (LPS). In this study, we showed that poly(I:C) and R848, Toll-like receptors ligands for RNA viruses, increased GCH expression and BH4 levels in RAW264.7 cells as well as bacterial LPS. We examined the intracellular signaling pathway for the induction of the Gch gene, and found that inhibitors for the NF-кB pathway suppressed the GCH expression by these stimuli. We for the first time identified the region required for LPS-induced GCH expression to be the 5'-untranslted region of exon 1 consisting of 149 bp using a reporter experiment. We also demonstrated that the expression of GCH with LPS was strongly suppressed by an inhibitor of NF-кB in mouse intraperitoneal macrophages in vivo. (192 words).

This study evaluated calcein, a calcium-binding fluorescent dye, as a tool for visualizing elastic structures in connective tissue. Tissue samples from chicken and cow subcutaneous fascia and porcine auricular tissue were stained with calcein and examined by fluorescence microscopy. To assess specificity, the staining pattern was compared with established markers for elastic fibers and calcium deposits. In subcutaneous fascia, calcein labeled fibrous structures that overlapped with fibrillin-1-rich elastic fibers, and the signal disappeared after calcium removal or enzymatic degradation. In porcine auricular tissue, calcein strongly highlighted the outer layers of blood vessels, where elastic fibers are abundant. In contrast, the elastic cartilage region showed no calcein staining despite the presence of elastic fibers confirmed by Modified Verhoeff's staining; it was also negative for calcium deposition and fibrillin-1 immunolabeling. These results indicate that calcein selectively binds to calcium-associated elastic fibers and enables visualization of their structure in fascia. This method is rapid, reversible, and simple, though not applicable to all elastic tissues, making it a useful approach for studying elastic fiber organization in calcium-rich environments.