Background: Cultured primary mouse hepatocytes undergo drastic phenotypic and metabolic reprogramming, while the temporal rules and regulatory machinery of membrane phospholipid remodeling remain elusive. Methods: Relying on a 0–72 h time-series in vitro culture system, this study integrated multi-omics technologies to dissect the temporal dynamics of phospholipid remodeling in hepatocytes. Through phosphatidylethanolamine N-methyltransferase (PEMT) knockout, exogenous PEMT expression, and methionine deprivation, we examined the association of PEMT status and methionine availability with phospholipid remodeling. Results: In vitro cultivation reduces intracellular total phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) through three coordinated events: suppressed transcription of phospholipid synthetic genes hinders de novo synthesis, elevated lipid hydrolysis consumes cellular phospholipids, and extracellular phospholipids accumulate in the culture medium from 12 to 48 h. These jointly trigger ordered remodeling of PC/PE balance, acyl chain length and fatty acid unsaturation. PEMT knockout was associated with PE retention without worsening hepatocyte dedifferentiation, PEMT exogenous expression raises PC content and PC/PE ratio yet cannot rescue culture-dominated lipid structural shifts. Methionine depletion depleted cellular methionine, S-adenosylmethionine (SAM) and S-adenosyl-L-homocysteine (SAH), producing selected lipid changes that partially overlapped with lipid phenotypes of PEMT knockout. Conclusion: In short, culture duration was the dominant factor associated with the fundamental phospholipid remodeling trajectory, and PEMT status and methionine availability, were associated with selective differences in lipid composition.
CD97 is an adhesion G-protein-coupled receptor encoded by ADGRE5 that integrates extracellular signals (including cell adhesion, ligand binding, and mechanical stimulation) with intracellular signal transduction. Recent structural studies have further elucidated tethered/intramolecular agonist (TIA)/Stachel recognition and engagement of the seven-transmembrane domain (7TMD), activation-associated 7TMD conformational changes, and G-protein coupling, including the structural basis for the preferential coupling of CD97 to G13. Currently, antibody–drug conjugates (ADCs) targeting CD97 are supported by in vitro proof-of-concept evidence, whereas chimeric antigen receptor (CAR) strategies have shown antitumor activity in animal models of glioblastoma (GBM) and acute myeloid leukemia (AML). Existing research indicates that CD97 is involved in maintaining stem-like states, invasion and metastasis, metabolic adaptation, and stress survival in certain tumors, and its function varies depending on tumor type and cellular environment. Because CD97 is also expressed in normal immune cells and various nonhematopoietic tissues, systemic targeted therapy may be limited by on-target/off-tumor toxicity. This article reviews the latest advances in CD97 structure and signal transduction, and explores its tumor-related functions, biomarker value, evidence for ADC and CAR-related therapies, as well as early exploratory directions involving RNA-mediated downregulation and structure-guided interventions.
Neuroinflammation within the tumor microenvironment (TME) of central nervous system (CNS) neoplasms, particularly glioblastoma (GBM), is no longer viewed merely as a reactive phenomenon but rather as a major driver of gliomagenesis and malignant transformation. This process involves a shift from acute immune activation to a chronic, sterile state that reshapes the CNS borders and immune niches to favor tumor evasion. This narrative review provides a comprehensive mechanistically focused analysis of the mechanisms governing the inflammatory stroma in primary and metastatic brain neoplasms. It critically examines the ontogeny and transcriptomic profile of myeloid and glial populations, dismantling the binary M1/M2 polarization model in favor of a continuum of functional states determined by metabolic and oxygenation gradients. It also analyzes intracellular signaling cascades, the subversion of innate immunity sensors such as the cGAS-STING pathway, the epigenetic reprogramming of stromal cells, and the role of extracellular vesicles. The electrochemical integration of tumor cells into neuronal circuits via glutamatergic synapses and connexin 43 gap junction coupling is addressed in detail, defining the mitogenic impact of neuronal activity on the tumor. The inflammatory profiles of IDH-wildtype and IDH-mutant gliomas and of secondary brain metastases are contrasted. Finally, the correlates of functional neuroimaging, liquid biopsies, and resistance mechanisms to conventional therapies are analyzed, including the GIANT and SENIPERA clinical trials, CARv3-TEAM-E bivalent cellular immunotherapy preconditioned with the LDC + R regimen, and the accelerated approval of dordaviprone (Modeyso) in H3 K27M-mutant diffuse midline gliomas.
While the chaperonin-containing TCP-1 (CCT) complex is essential for proteostasis, the distinct roles of individual subunits in tumor immune regulation remain unclear. Here, we identify CCT7 as a previously unrecognized regulator of immune evasion in lung adenocarcinoma (LUAD). Integrative analyses of TCGA and GEO cohorts revealed that CCT7 is markedly upregulated in LUAD and is associated with poor patient prognosis. Functional studies demonstrated that CCT7 knockdown inhibited tumor cell proliferation and migration and enhanced cisplatin-induced apoptosis, yet paradoxically impaired T-cell activation. Mechanistically, transcriptomic and biochemical analyses revealed that CCT7 depletion activated the DR5–MKK4–JNK–c-Jun signaling cascade, resulting in the transcriptional upregulation of PD-L1. Disruption of DR5 or JNK signaling effectively abrogated PD-L1 induction. In contrast, CCT2 depletion exerted the opposite effect by suppressing the DR5–JNK–c-Jun–PD-L1 signaling axis and enhancing T-cell activation. Collectively, these findings reveal unexpected functional divergence among TRiC/CCT subunits and identify the CCT7–DR5–JNK–c-Jun signaling axis as a previously unrecognized mechanism regulating PD-L1-mediated immune evasion, highlighting the potential therapeutic relevance of this signaling axis in LUAD.
Fabry disease is a rare X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, resulting in deficient α-galactosidase A activity and progressive accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Although lysosomal substrate storage represents the primary molecular defect, accumulating evidence indicates that disease progression is driven by interconnected mechanisms, including chronic inflammation, oxidative stress, endothelial dysfunction, and impaired autophagy, leading to progressive multisystem involvement. The marked clinical heterogeneity of Fabry disease, together with nonspecific early manifestations, frequently delays diagnosis and complicates patient stratification and therapeutic decision-making. While advances in biomarkers, genetic testing, and imaging have improved disease recognition, current diagnostic approaches remain insufficient to fully capture disease complexity. Precision medicine is therefore emerging as a promising strategy through the integration of clinical, molecular, imaging, and multi-omics data. In this context, artificial intelligence (AI) offers novel opportunities for early diagnosis, biomarker discovery, risk stratification, and prediction of therapeutic response. This review provides an integrated overview of the molecular mechanisms, inflammatory pathways, clinical manifestations, and precision diagnostic strategies underlying Fabry disease, highlighting how AI-driven approaches may accelerate the transition toward more accurate, personalized, and predictive disease management.
Leukemia progression is increasingly shaped by reciprocal interactions between leukemic cells and the bone marrow microenvironment, yet the extracellular regulatory networks associated with these interactions remain incompletely understood. Here, we investigated the biological context associated with the antileukemic activity of LCC-10 (NSC765599), a synthetic biphenyl benzamide derivative, using an integrated pharmacogenomic and structure-guided computational framework. Antiproliferative activity was first characterized using the NCI-60 screen and subsequently integrated with pharmacogenomic response similarity analysis, baseline transcriptomic profiling, similarity-based target prediction, systems-level network analysis, molecular docking, coarse-grained molecular dynamics simulations, comparative in silico ADMET evaluation, and zebrafish embryo developmental toxicity assessment. LCC-10 exhibited potent antiproliferative activity across leukemia cell lines, with submicromolar GI50 values in five of six models. Computational analyses converged on a matrix metalloproteinase (MMP)-associated extracellular matrix (ECM) regulatory network, with MMP2 and MMP9 among the recurrently implicated candidates. Structure-guided analyses suggested structural compatibility of LCC-10 with representative MMP catalytic domains but did not establish direct biochemical inhibition or target engagement. Comparative in silico ADMET analyses supported the predicted developability profile of LCC-10, whereas zebrafish embryo assays indicated concentration-dependent developmental tolerability within the tested range. Collectively, these findings associate LCC-10 with an MMP-associated ECM regulatory network in leukemia while defining this relationship as a hypothesis requiring direct experimental validation. This integrated framework provides a rationale for subsequent biochemical, target-engagement, and functional studies to clarify the molecular basis of LCC-10 activity.
This study utilized a genetically engineered mouse model deficient in the small GTPase Rap1A (knockout/Rap1A-null) to understand the biological role of Rap1A in the heart. We examined differential protein expression in the left ventricle of Rap1A-null versus wild-type control C57BL/6 male mice (~5 months) using proteomics (nanoLC-MS/MS quantitative analysis), and in the whole heart of aged male mice (~16 months) using MAL-DI-TOF/TOF mass spectrometry. Additionally, we used an experimental model of acute cardiovascular stress and assessed the impact on heart tissue histology, gene expression and mortality risk. Rap1A-deficient hearts showed reduced size and reduced heart and left ventricular weights. Significantly reduced gene expression of extracellular matrix collagen type I and collagen type III was present under baseline and cardiovascular stress conditions. Assessment of the proteomic profile identified a crucial role of Rap1A in promoting healthy ventricular myocardium, as its deficiency exhibited increased impact on cytoskeletal, mitochondrial, metabolic and contractile protein expression in young and aged mice. In young Rap1A-deficient mice, overrepresentation analysis revealed markers myosin heavy chain 7 (β-MHC) and alpha-actinin-2 (α-actinin-2) associated with cardiomyopathies, and upon cardiac stress, showed mortality risk compared to controls. Altogether, these findings provide important insights into the role of Rap1A in cardiac structure and remodeling under basal and stress conditions in male mice.
Cancer immunotherapy has transformed the treatment of multiple malignancies; however, primary and acquired resistance remain major clinical challenges. Because effective immune recognition depends on the repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules, increasing attention has focused on the antigen processing and presentation pathway as a therapeutic target to enhance tumor immunogenicity. Among its key regulators, the endoplasmic reticulum (ER) aminopeptidases ERAP1 and ERAP2 shape the MHC-I immunopeptidome by trimming peptide precursors before antigen presentation. Beyond this canonical function, accumulating evidence indicates that ERAP aminopeptidases are multifunctional proteins involved in inflammation, angiogenesis, ER stress responses, cell migration, and tumor-intrinsic signaling. These moonlighting activities suggest that ERAP enzymes influence cancer progression through both immune-dependent and immune-independent mechanisms. Recent advances in medicinal chemistry have enabled the development of selective ERAP1 inhibitors, leading to the first clinical evaluation of this therapeutic strategy and providing early clinical evidence that pharmacological modulation of antigen processing may complement existing immunotherapies. In this review, we summarize the multiple functions of ERAP aminopeptidases in cancer, discuss their role in regulating adaptive and innate immune responses, and highlight emerging therapeutic strategies and future challenges for exploiting ERAP-targeted interventions in precision immuno-oncology.
This manuscript is the corrected version of a previously published paper. Glucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and is well studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase–activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface abundance of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a two-fold decrease in cell-surface GLUT1 abundance, concomitant with a 40% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 and also S565 in its paralogue TBC1D1 as candidate WNK1 phosphorylation sites. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell-surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in GLUT1 trafficking and glucose uptake and may have potential impact for the understanding of metabolic dysregulation, as observed in many cancer cells or insulin-responsive cell types.
Background: Differences in gene expression between inflammatory-like (iEOS-like) and resident-like (rEOS-like) eosinophil subtypes, and in eosinophil-derived serum mediators, may reflect eosinophil functional activity and their potential role in the pathogenesis of allergic asthma (AA). Methods: Twenty-three patients with non-severe AA and thirteen healthy subjects (HS) were examined. AA patients underwent a bronchial allergen challenge (BAC) with Dermatophagoides pteronyssinus and were re-evaluated 24 h later. Blood eosinophils were isolated by gradient centrifugation and magnetic separation, followed by subtyping based on CD62L expression. Gene expression was assessed by TaqMan-based quantitative PCR. Serum eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), Galectin-10 (Gal10), and NADPH oxidase 2 (NOX2) were measured using ELISA. Results: Blood eosinophil subtypes from AA patients showed significantly higher expression of CLC, ECP, EPX, EDN, MBP, ALOX5, NOX2, and TGF-β1 compared to those from HS (p < 0.05), with no significant changes in LTA4H and LTC4S. iEOS-like cells in AA patients exhibited higher CLC and EPX expression compared to rEOS-like cells, (p < 0.05). Following BAC, CLC, MBP, and TGF-β1 expression increased in both eosinophil subtypes, while EPX and NOX2 increased only in iEOS-like cells (all p < 0.05). Serum ECP, EDN, and Gal10 concentrations were elevated in AA compared to HS and further increased after BAC (all p < 0.05); serum NOX2 remained unchanged. Conclusions: BAC induces a late-phase eosinophilic response in AA characterized by a partially eosinophil subtype-specific increase in gene expression and in circulating eosinophil-derived mediators. An increase in Gal10, observed at the CLC transcript level in eosinophils and in serum—but not in ECP or EDN—suggests the existence of mediator-specific mechanisms that regulate gene expression and extracellular release.
Despite major advances in lipid-lowering therapies, a significant unmet need remains, particularly for patients with homozygous familial hypercholesterolemia (HoFH), severe heterozygous familial hypercholesterolemia (HeFH), and those who fail to achieve guideline-recommended LDL-C targets. Nucleic acid-based therapeutics have emerged as a transformative approach for treating hypercholesterolemia. Antisense oligonucleotides and small interfering RNAs (siRNAs) have demonstrated durable hepatic gene silencing and have led to approved therapies, while gene replacement and in vivo genome-editing strategies offer the potential for long-lasting, and possibly one-time, interventions. In parallel, microRNAs (miRNAs) have attracted increasing interest because of their ability to coordinately regulate multiple genes involved in lipoprotein metabolism, cholesterol transport, and lipid homeostasis. Human genetic studies further support the importance of miRNA-mediated regulation, exemplified by a rare ~2.5 kb deletion in the distal LDLR 3′UTR (“del2.5”) that disrupts miRNA-binding sites and is associated with lifelong low LDL-C levels. This review summarizes recent advances, mechanisms of action, clinical progress, and remaining challenges across antisense oligonucleotides, siRNAs, gene therapy, genome editing, and emerging miRNA-based therapeutics for hypercholesterolemia. As an example of the latter approach, the liver-directed miR-30c analog C2 has demonstrated preclinical activity by coordinately reducing hepatic lipoprotein secretion and lipogenesis while enhancing cholesterol elimination, resulting in reduced LDL-C and atherosclerosis. However, it must be noted that these findings remain preclinical, and further optimization of delivery, pharmacokinetics, safety, and long-term efficacy will be required before clinical evaluation. Continued advances in RNA chemistry, targeted delivery, and genome engineering are expected to further expand the therapeutic landscape for dyslipidemia and cardiovascular disease.
Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have emerged as an important class of medications for managing type 2 diabetes and obesity, with cardiovascular outcome trials demonstrating reductions in major adverse cardiovascular events (MACEs) for several agents. The SELECT trial, which enrolled 17,604 participants with established cardiovascular disease and overweight or obesity but without diabetes, showed a 20% reduction in MACEs with semaglutide compared with placebo. Importantly, mediation analyses indicated that weight loss accounted for approximately one-third of this cardiovascular benefit, suggesting that additional mechanisms contribute substantially to the observed risk reduction. The temporal dissociation between weight loss and early MACEs reduction supports the hypothesis that GLP-1RAs exert direct vascular protective effects independent of their metabolic actions. This review synthesizes current evidence on the thromboinflammatory mechanisms through which GLP-1RAs may exert their cardiovascular benefits, with particular emphasis on the neutrophil–NET axis, platelet function, and plaque stabilization. Overall, thromboinflammation represents a biologically reasonable candidate mechanism, but its contribution to the cardiovascular benefit of GLP-1RAs treatment is an open question. Still, residual treatment effects cannot be attributed specifically to thromboinflammation, because metabolic, renal, hemodynamic, and vascular pathways, among others, may also contribute.
SLAMF1 encodes CD150, an immunoregulatory receptor involved in lymphocyte activation, T–B-cell interactions, and humoral immune responses. The SLAMF1 promoter polymorphism rs2295613(G>A) was previously associated with systemic lupus erythematosus (SLE) susceptibility in a Chinese case–control cohort. Here, we investigated the regulatory activity of rs2295613 in the transformed B-cell lines Raji and MP1 and in primary human CD19+ B cells. The rs2295613(A)-containing reporter showed higher promoter activity than the rs2295613(G)-containing reporter in all three cellular systems. Bioinformatic analysis predicted that the G to A substitution strengthens a pre-existing MYC-compatible motif. Substitutions disrupting the motif-containing region attenuated the rs2295613(A)-associated increase in reporter activity and reduced enrichment of the promoter fragment in anti-c-MYC DNA pull-down assays. Partial siRNA-mediated reduction in MYC mRNA also decreased the activity of the rs2295613(A)-containing reporter in Raji cells. Together, these findings identify rs2295613 as a functional SLAMF1 promoter variant in B-cell reporter systems and support a contribution of c-MYC-associated regulation to the enhanced activity of the rs2295613(A)-containing promoter.
Hispanics of Mexican American descent in South Texas show a very high prevalence of MASLD, with some studies reporting rates as high as 50% in adults. However, assessment of genetic risk factors underlying this prevalence is complicated by a high co-occurrence of other metabolic disorders and variable endogenous and exogenous environmental risk factors. To map the transcriptomic architecture of MASLD hepatic steatosis risk, we conducted an epidemiological-scale investigation using human induced pluripotent stem cell (iPSC)-derived hepatocyte cultures from 193 participants in our longitudinal South Texas Family Study (STFS). iPSC-based models offer greater power to map genetic risk factors by experimentally controlling for confounding organismal and environmental factors. We combined transcriptome-wide gene expression analysis with high-content cellular measurements of neutral lipids to define a core hepatic steatosis MASLD phenotype at baseline (vehicle-treated) and following a lipid challenge. The additive genetic heritability of hepatic steatosis measures was 0.44 (p-value = 0.03) at baseline and 0.42 (p-value = 0.03) at post-lipid challenge. Multivariable linear regression comparing each gene’s expression against hepatic steatosis measures identified 1070 genes at baseline and 1229 genes post-lipid challenge, whose expression showed a transcriptome-wide statistically significant association (standardized |β| ≥ 0.24; Bonferroni-corrected p-value ≤ 0.001) with baseline and post-lipid challenge hepatic steatosis measures, respectively. Functional annotation and pathway enrichment analyses of these genes implicated a broad range of hepatocellular functions, mapping an overall transcriptomic architecture of MASLD-associated steatosis risk in Mexican Americans. The genes whose expression was positively correlated with hepatic steatosis measures suggest a direct role of variation in fatty acid (FA) and cholesterol uptake, de novo lipogenesis (DNL), and carbohydrate shunts in hepatic steatosis risk, as well as a cellular stress-associated and high-turnover metabolic state marked by elevated FA-oxidation and ketogenesis. In contrast, the genes whose expression was inversely correlated with hepatic steatosis measures suggest a significant role of the cellular cytoskeleton, hepatocyte epithelial integrity, and endosomal and autophagic clearance machinery in steatosis risk.
Cancer therapy-related acute kidney injury has become an increasingly common challenge as modern treatments prolong survival and increase exposure to potentially nephrotoxic therapies. Decisions regarding therapeutic rechallenge have relied on normalizing serum creatinine and recovering estimated glomerular filtration rate, despite growing evidence that biochemical recovery does not necessarily indicate restoration of kidney integrity or resilience. In this review, we propose biological kidney recovery as a conceptual framework that integrates mechanisms of kidney injury and repair (adaptive and maladaptive) with emerging biomarkers and therapeutic rechallenge. We first summarize the distinct mechanisms of kidney injury induced by platinum-based chemotherapy, immune checkpoint inhibitors, and vascular endothelial growth factor pathway inhibitors, highlighting how these differences influence subsequent repair. We then discuss the cellular and metabolic processes underlying adaptive repair, the transition to maladaptive remodeling, and current approaches for assessing biological recovery through pathology, biomarkers, and multi-omics technologies. Finally, we present a practical framework for individualized therapeutic rechallenge based on an integrated assessment of kidney-, tumor-, and patient-related factors and outline future directions for precision onco-nephrology. By shifting the focus from filtration alone to biological recovery, this framework enables more informed therapeutic rechallenge aimed at preserving both oncologic efficacy and long-term kidney health.
Background: Death-associated protein kinase 1 (DAPK1) is a key regulator of apoptosis and immune responses; however, its prognostic significance in oral cancer remains insufficiently characterized. This study investigated the prognostic relevance of DAPK1 in oral squamous cell carcinoma (OSCC) and examined its associations with immune infiltration and apoptosis-related signaling pathways. Methods: A retrospective translational study design was employed, integrating TCGA-based expression and methylation analyses of 528 head and neck squamous cell carcinoma (HNSCC) tumors, UALCAN epigenetic profiling, GeneMANIA protein–protein interaction mapping, TIMER 2.0 immune correlation analyses in 422 HPV-negative HNSCC patients, and multiplex immunofluorescence validation using a tissue microarray cohort of 82 patients with histologically confirmed OSCC, of whom 75 were eligible for the final analysis at Kaohsiung Veterans General Hospital, Taiwan. Results: In vitro validation using Western blot analysis in FaDu cells showed that epidermal growth factor receptor (EGFR) inhibition with gefitinib induced upregulation of DAPK1 protein expression at 10 μM and increased total caspase-3 expression. Higher DAPK1 signal in whole-field quantification was associated with increased CD4+ and CD8+ T-cell infiltration and enrichment of apoptosis-related pathways. Patients with high DAPK1 expression demonstrated a consistent protective trend for overall survival in a pre-specified fully adjusted primary model (adjusted HR = 0.51, 95% CI: 0.21–1.21, p = 0.126), and exhibited significantly improved survival in a secondary parsimonious model (adjusted HR = 0.41, 95% CI: 0.18–0.91, p = 0.029). Multiplex immunofluorescence further confirmed stronger DAPK1 and caspase-3 staining, along with denser lymphocytic infiltration within the tumor microenvironment. Conclusions: Collectively, these findings suggest that DAPK1 is associated with apoptosis-related signaling, increased immune-cell infiltration, and favorable clinical outcomes in OSCC, although its independent prognostic value requires validation in larger cohorts.
KRAS G12C mutation is a clinically relevant driver in non-small cell lung cancer (NSCLC), yet the signaling networks that modulate malignant behavior in this context remain incompletely defined. In this study, we examined the functional role of FOXD3 and its relationship with NF-κB signaling in KRAS G12C-mutant NSCLC models. Stable FOXD3 overexpression was established in SW1573 and LU65 cells. FOXD3 reduced cell viability, migration, and invasion while increasing caspase 3/7 activity in both cell lines. Transcriptomic profiling in LU65 cells followed by Hallmark enrichment analysis identified TNFα signaling via NF-κB as a prominently altered pathway associated with FOXD3 overexpression. Consistently, NF-κB dual-luciferase assays showed reduced basal NF-κB transcriptional activity in FOXD3-overexpressing cells. TNFα stimulation partially reversed the inhibitory effects of FOXD3 on proliferation, migration, and invasion and attenuated FOXD3-induced apoptosis. In addition, stable FOXD3 overexpression suppressed xenograft growth in vivo. Collectively, these findings support a functional association between FOXD3 overexpression and reduced NF-κB-related transcriptional activity in KRAS G12C-mutant NSCLC models, although the present data do not establish direct causal mediation by NF-κB.
The Transport Protein Particle (TRAPP) complex is a highly conserved multi-subunit tethering complex that plays a critical role in membrane trafficking. Mutations in TRAPP complex subunits have been implicated in a growing spectrum of rare genetic disorders, yet the molecular mechanisms underlying variant pathogenicity often remain unclear. Here, we developed a humanized yeast platform to enable systematic functional characterization of TRAPP complex variants of uncertain significance. Using a stepwise gene replacement strategy in Saccharomyces cerevisiae, we constructed a strain in which five yeast TRAPP core subunits were replaced with their human orthologues. The integration of human subunits was validated through quantitative RT-PCR and Western blotting. Growth assays revealed that partial humanization of the core complex recapitulates key functional aspects of TRAPP assembly and enables the functional investigation of variants of uncertain significance in vivo. Structural modeling and clash analysis provided insights into the impact of specific mutations on complex stability and subunit interactions. TRAPPC3 has not yet been definitively associated with human disease. Introduction of TRAPPC3 variants of uncertain clinical significance into the humanized strain resulted in pronounced growth defects and predicted structural clashes. This work demonstrates the power of humanized yeast as a model for elucidating potential genotype–phenotype relationships in TRAPPopathy disorders and provides a versatile platform to support variant interpretation, mechanistic studies, and potential therapeutic screening.
Efficient and reproducible generation of functional stem cell-derived β cells (sBC) remains a major challenge for basic research and cell replacement therapy, partly due to incomplete understanding of endocrine induction during differentiation and challenges with clinical scale-up. Here, we dissect the individual contributions of commonly used endocrine differentiation factors on pancreatic progenitor maintenance, endocrine commitment, and hormone subset generation in a scalable 3D differentiation system. We demonstrated that starting pluripotent stem cell cluster size is a critical determinant for downstream sBC generation. We also verify that a commonly employed combination of endocrine induction molecules efficiently drives endocrine lineage commitment but yields limited β-cell generation. Detailed analysis of the effects of individual endocrine induction molecules revealed distinct effects: EGF or KGF preserved NKX6.1+ progenitors without induction of endocrine differentiation; Notch or BMP inhibition robustly induced endocrine marker expression but concurrently reduced NKX6.1 expression, resulting in predominant generation of glucagon-expressing cells; retinoic acid, thyroid hormone (T3), or TGFβ inhibition maintained high NKX6.1 levels while also promoting efficient insulin+ endocrine differentiation. These findings indicate NKX6.1 protein maintenance as a key determinant of human β-cell generation and show that endocrine differentiation factors exert divergent effects on lineage progression.
Mitochondria are essential organelles for cellular energy production and the regulation of diverse biological processes, including apoptosis, redox homeostasis, and intracellular signaling. Although mitochondrial reactive oxygen species (mtROS) act as critical mediators of these functions, the molecular mechanisms underlying mtROS regulation remain poorly understood. This review summarizes current insights into the role of heat shock protein 47 (HSP47) in mitochondrial oxidative stress and mtROS-mediated cellular responses. In addition to its classical function as an endoplasmic reticulum (ER) chaperone, HSP47 translocates to the mitochondria under oxidative stress conditions. This mitochondrial localization promotes mtROS production, thereby triggering apoptotic pathways and redox-sensitive signal transduction. Furthermore, we examine the mechanistic insights linking HSP47 to mitochondrial function and oxidative stress, highlighting their implications for cellular homeostasis and disease pathogenesis. Overall, these findings establish HSP47 as a novel regulator of mtROS generation, suggesting that the HSP47–mtROS axis represents a promising therapeutic target for oxidative stress-related disorders and a potential role for exploring virus-induced cellular responses in future research.
