Search Results (140)

Insulin-regulated glucose uptake is a central mechanism in maintaining systemic glucose homeostasis, primarily occurring in skeletal muscle and adipose tissue. This process relies on the insulin-stimulated translocation of the glucose transporter, GLUT4, from specialized intracellular compartments, known as GLUT4 storage vesicles (GSVs), to the plasma membrane. Disruption of this pathway is a hallmark of insulin resistance and a key contributor to the pathogenesis of type 2 diabetes. Recent advances have provided critical insights into both the insulin signalling cascades and the complex biogenesis, as well as the trafficking and fusion dynamics of GSVs. This review synthesizes the current understanding of the molecular mechanisms governing GSV mobilization and membrane fusion, highlighting key regulatory nodes that may become dysfunctional in metabolic disease. By elucidating these pathways, we propose new therapeutic avenues targeting GSV trafficking to improve insulin sensitivity and combat type 2 diabetes. Full article

Background: Diabetic foot ulcers (DFUs) are a severe complication of diabetes and are characterized by impaired wound healing and a high amputation risk. Exosomes—which are nanovesicles carrying proteins, RNAs, and lipids—mediate intercellular communication in wound microenvironments, yet their biomarker potential in DFUs remains underexplored. Methods: We analyzed transcriptomic data from GSE134431 (13 DFU vs. 8 controls) as a training set and validated findings in GSE80178 (6 DFU vs. 3 controls). A sum of 7901 differentially expressed genes (DEGs) of DFUs were detected and intersected with 125 literature-curated exosome-related genes (ERGs) to yield 51 candidates. This was followed by GO/KEGG analyses and a PPI network construction. Support vector machine–recursive feature elimination (SVM-RFE) and the Boruta random forest algorithm distilled five biomarkers (DIS3L, EXOSC7, SDC1, STX11, SYT17). Expression trends were confirmed in both datasets. Analyses included nomogram construction, functional and correlation analyses, immune infiltration, GSEA, gene co-expression and regulatory network construction, drug prediction, molecular docking, and RT-qPCR validation in clinical samples. Results: A nomogram combining these markers achieved an acceptable calibration (Hosmer–Lemeshow p = 0.0718, MAE = 0.044). Immune cell infiltration (CIBERSORT) revealed associations between biomarker levels and NK cell and neutrophil subsets. Gene set enrichment analysis (GSEA) implicated IL-17 signaling, proteasome function, and microbial infection pathways. A GeneMANIA network highlighted RNA processing and vesicle trafficking. Transcription factor and miRNA predictions uncovered regulatory circuits, and DGIdb-driven drug repurposing followed by molecular docking identified Indatuximab ravtansine and heparin as high-affinity SDC1 binders. Finally, RT-qPCR validation in clinical DFU tissues (n = 5) recapitulated the bioinformatic expression patterns. Conclusions: We present five exosome-associated genes as novel DFU biomarkers with diagnostic potential and mechanistic links to immune modulation and vesicular transport. These findings lay the groundwork for exosome-based diagnostics and therapeutic targeting in DFU management. Full article

Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies. Full article

Introduction: The interface between T cells and the tumor microenvironment, termed the ‘immunological synapse’, consists of multiple checkpoint protein pairs co-expressed on both sides of the synapse. mir-16, a microRNA from a widely known tumor-suppressor family of miRNAs, was previously shown by us to be downregulated in melanoma. As other miRNAs from this family have been shown to directly target checkpoint proteins, here we investigated whether miR-16 influences the expression patterns of checkpoint proteins in melanoma. Methods: Single-cell gene expression data from the melanoma microenvironment were retrieved from a public database. Melanoma cell lines were established from metastatic lesions and transiently transfected with an hsa-miR-16-5p-mimic RNA or a mir-16-expressing plasmid. The mRNA expression profiles were analyzed using an Affymetrix microarray. Direct targets of miR-16 were identified by luciferase reporter assays. Protein levels were assessed by Western blotting. Results: Bioinformatic analysis revealed that the expression levels of eight checkpoint mRNAs, known to be present on the melanoma side of the immunological synapse, were highly correlated. Four of these mRNAs contained putative binding sites for the miR-15/16 family. miR-16 expression was significantly reduced in melanoma cells, compared to normal melanocytes. Luciferase reporter assays demonstrated that miR-16 directly targets the 3′ untranslated regions (3′UTRs) of CD40, CD80. The mRNAs downregulated following miR-16 overexpression were highly enriched for genes involved in autophagy, vesicle-mediated transport, and the regulation of protein membrane localization. Among these, VTI1B and SMPD1 were confirmed to be direct targets of miR-16. Transient overexpression of miR-16 resulted in a significant reduction in SMPD1 and VTI1B levels in melanoma cell lines. Conclusions: Our findings suggest that miR-16 potentially modulates melanoma tumorigenesis, metastasis and immunogenicity by altering the composition of checkpoint proteins at the immunological synapse and by regulating cellular pathways associated with intracellular trafficking and transmembrane protein presentation. Full article

Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is marked by the pathological accumulation of amyloid-β plaques and tau neurofibrillary tangles, both of which disrupt neuronal communication and function. Emerging evidence highlights the role of extracellular vesicles (EVs) as key mediators of intercellular communication, particularly in the propagation of pathological proteins in AD. Among the regulatory factors influencing EV composition and function, neuraminidase 1 (NEU1), a lysosomal sialidase responsible for desialylating glycoproteins has gained attention for its involvement in EV glycosylation. This review explores the role of NEU1 in modulating EV glycosylation, with particular emphasis on its influence on immune modulation and intracellular trafficking pathways and the subsequent impact on intercellular signaling and neurodegenerative progression. Altered NEU1 activity has been associated with abnormal glycan profiles on EVs, which may facilitate the enhanced spread of amyloid-β and tau proteins across neural networks. By regulating glycosylation, NEU1 influences EV stability, targeting and uptake by recipient cells, primarily through the desialylation of surface glycoproteins and glycolipids, which alters the EV charge, recognition and receptor-mediated interactions. Targeting NEU1 offers a promising therapeutic avenue to restore EV homeostasis and reduces pathological protein dissemination. However, challenges persist in developing selective NEU1 inhibitors and effective delivery methods to the brain. Furthermore, altered EV glycosylation patterns may serve as potential biomarkers for early AD diagnosis and monitoring. Overall, this review highlights the importance of NEU1 in AD pathogenesis and advocates for deeper investigation into its regulatory functions, with the aim of advancing therapeutic strategies and biomarker development for AD and related neurological disabilities. Full article

Background/Objectives: Activation of the Toll-like receptor 7 (TLR-7) plays an important role in the pathogenesis of many autoimmune diseases and viral infections. Although we have previously observed inflammation-mediated dysregulation of placental transporters, the role of TLR-7 has not been examined. Using the TLR-7 agonist, imiquimod (IMQ), we evaluated transporter expression in IMQ-treated pregnant rats and ex vivo in cultured rat placental explants. Methods: We administered 5 mg/kg (IP) of IMQ to pregnant Sprague Dawley rats on gestational day (GD) 14. The expression levels of inflammatory biomarkers and transporters were measured in maternal and fetal tissues by qRT-PCR and immunodetection methods, and effects on the placental proteome were assessed using LC/MS/MS. The involvement of TLR-7 was confirmed in rat placental explants. Results: IMQ administration resulted in Irf7 induction and increased levels of IL-6, Tnf-α, and type-I/II interferon pathways in maternal liver and placenta, which is consistent with TLR-7 activation. Proteomic profiling revealed IMQ-mediated activation of pathways involved in immune response, vesicle trafficking, and oxidative stress. Significantly decreased placental, hepatic, and renal protein expression of P-glycoprotein (PGP) was seen in the IMQ group. Likewise, TLR-7 activation using single-stranded RNA resulted in an induction of inflammatory biomarkers and downregulation of PGP in rat placental explants. Conclusions: We demonstrated that the activation of TLR-7 signaling during pregnancy reduces the expression of PGP in placenta and maternal tissues. Further studies are warranted, as decreased protein expression could result in decreased activity and altered fetal exposure to its substrates. Full article

Autophagy and apoptosis are two essential mechanisms regulating cell fate. Although distinct, their signaling pathways are closely interconnected through various crosstalk mechanisms. Lipid rafts are described to act as both physical and functional platforms during the early stages of autophagic and apoptotic processes. Only recently has a role for lipid raft-associated molecules in regulating EV biogenesis and release begun to emerge. In particular, lipids of EV membranes are essential components in conferring stability to these vesicles in different extracellular environments and/or to facilitate binding or uptake into recipient cells. In this review we highlight these aspects, focusing on the role of lipid molecules during apoptosis and secretory autophagy pathways. We describe the molecular machinery that connects autophagy and apoptosis with vesicular trafficking and lipid metabolism during the release of EVs, and how their alterations contribute to the development of various diseases, including autoimmune disorders and cancer. Overall, these findings emphasize the complexity of autophagy/apoptosis crosstalk and its key role in cellular dynamics, supporting the role of lipid rafts as new therapeutic targets. Full article

Hydrocephalus is a chronic neurological condition caused by abnormal cerebrospinal fluid (CSF) accumulation, significantly impacting patients’ quality of life. Its causes remain poorly understood, making neurosurgery the primary treatment. Research suggests that hydrocephalus may result from impaired macromolecular clearance, leading to increased osmotic load in the ventricles. Macromolecules are cleared via processes such as transcytosis, involving caveolae- and clathrin-dependent pathways, soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins, and vesicular trafficking. Abnormalities in transcytosis components, such as mutations in alpha-SNAP (α-soluble NSF attachment protein) and SNARE complexes, disrupt membrane organization and vesicle fusion, potentially contributing to hydrocephalus. Other factors, including alpha-synuclein and Rab proteins, may also play roles in vesicle dynamics. Insights from animal models, such as hyh (hydrocephalus with hop gait) mice, highlight the pathological consequences of these disruptions. Understanding transcytosis abnormalities in hydrocephalus could lead to novel therapeutic strategies aimed at enhancing macromolecular clearance, reducing ventricular fluid buildup, and improving patient outcomes. Full article

Endocytosis is a specialized transport mechanism in which the cell membrane folds inward to enclose large molecules, fluids, or particles, forming vesicles that are transported within the cell. It plays a crucial role in nutrient uptake, immune responses, and cellular communication. However, many pathogens exploit the endocytic pathway to invade and survive within host cells, allowing them to evade the immune system and establish infection. Endocytosis can be classified as clathrin-mediated (CME) or clathrin-independent (CIE), based on the mechanism of vesicle formation. Unlike CME, which involves the formation of clathrin-coated vesicles that bud from the plasma membrane, CIE does not rely on clathrin-coated vesicles. Instead, other mechanisms facilitate membrane invagination and vesicle formation. CIE encompasses a variety of pathways, including caveolin-mediated, Arf6-dependent, and flotillin-dependent pathways. In this review, we discuss key features of CIE pathways, including cargo selection, vesicle formation, routes taken by internalized cargo, and the regulatory mechanisms governing CIE. Many viruses and bacteria hijack host cell CIE mechanisms to facilitate intracellular trafficking and persistence. We also revisit the exploitation of CIE by bacterial and viral pathogens, highlighting recent discoveries in entry mechanisms, intracellular fate, and host-pathogen interactions. Understanding how pathogens manipulate CIE in host cells can inform the development of novel antimicrobial and immunomodulatory interventions, offering new avenues for disease prevention and treatment. Full article

Human papillomavirus (HPV) 16 is transported in a retrograde fashion from the cell surface to the Golgi apparatus. Prior to mitosis, the virus loses association with the Golgi and, following nuclear envelope breakdown, is found associated with the condensed mitotic chromatin. The intervening steps have not been well defined. It was previously demonstrated that the virus is transported to the mitotic chromosomes in vesicles. Here, we describe the role of the endoplasmic reticulum (ER) in the post-Golgi trafficking and the importance of the ER-generated coat protein complex II (COPII) anterograde trafficking pathway in HPV infection. HPV pseudovirus (PsV) colocalized with COPII components and silencing of this pathway inhibited HPV infection. Additionally, the inner COPII coat protein, Sec24b, could be biochemically isolated in association with HPV capsid proteins. This study provides insight into the mechanism of post-Golgi HPV trafficking. Full article

Our understanding of the molecular mechanisms undergirding artemisinin (ART) resistance in Plasmodium falciparum is currently based on two organizing principles: reduced hemoglobin trafficking into the digestive food vacuole, resulting in lower levels of activated ART, and increased tolerance to ART-induced oxidative stress in the infected erythrocyte. We had previously proposed an extracellular vesicle (EV) export model of ART resistance in P. falciparum. This model predicts that EV abundance will be altered by ART exposure and that the peptide cargo of EVs from the ART-exposed condition will be enriched with aggregation-prone peptides. We tested the predictions of the EV export hypothesis in this study using in vitro culture assays of an ART-resistant transgenic line engineered on a 3D7 background (R561H) and a 3D7 knock-out line (PfVps60KO) with deficient EV production phenotype. EV enrichment was obtained from in vitro parasite culture supernatants via a series of ultracentrifugation and filtration steps, followed by size exclusion chromatography. A quality check on EVs was performed using dynamic light scattering. Liquid chromatography with tandem mass spectrometry was used to determine the proteome cargo from extracted EVs, and parasite peptides were queried for aggregation-prone tendency using open-access software. We report that dihydroartemisinin (DHA) exposure was positively correlated with EV abundance (coefficient estimate = 1038.58, confidence interval of 194.86–1882.30, and p-value = 0.018) and suggests that EV biogenesis is part of the parasite’s response to DHA/ART. Furthermore, our findings suggest the expression of a non-constitutive DHA-induced alternate EV biogenesis pathway as the PfVps60KO was observed to produce the highest number of EVs under DHA exposure. Finally, we show that EVs from both ART-susceptible and resistant parasites under DHA exposure carry a cargo of Chorein N-terminal domain-containing protein (PF3D7_1021700) with a high aggregation-prone index (prion-like domain [PrLD] score = 26.5) out of nine identified parasite peptides. The former of these findings is in concordance with the EV export hypothesis, which posits that the removal of DHA/ART-induced aggregated and/or misfolded peptides is critical to the parasite’s survival under DHA/ART exposure. This observation further implicates EVs in the development of the ART-resistant phenotype. However, the finding of one aggregation-prone peptide out of the nine parasite proteins in the EV cargo does not sufficiently support the EV export hypothesis. Future replicates of this study and further interrogations of the EV export hypothesis are needed. Full article

Originally identified in proteomic-based studies of the Golgi, Golgi phosphoprotein 3 (GOLPH3) is a highly conserved protein from yeast to humans. GOLPH3 localizes to the Golgi through the interaction with phosphatidylinositol-4-phosphate and is required for Golgi architecture and vesicular trafficking. Many studies revealed that the overexpression of GOLPH3 is associated with tumor metastasis and a poor prognosis in several cancer types, including breast cancer, glioblastoma multiforme, and colon cancer. The purpose of this review article is to provide the current progress of our understanding of GOLPH3 molecular and cellular functions, which may potentially reveal therapeutic avenues to inhibit its activity. Specifically, recent papers have demonstrated that GOLPH3 protein functions as a cargo adaptor for COP I-coated intra Golgi vesicles and impinges on Golgi glycosylation pathways. In turn, GOLPH3-dependent defects have been associated with malignant phenotypes in cancer cells. Additionally, the oncogenic activity of GOLPH3 has been linked with enhanced signaling downstream of mechanistic target of rapamycin (mTOR) in several cancer types. Consistent with these data, GOLPH3 controls organ growth in Drosophila by associating with mTOR signaling proteins. Finally, compelling evidence demonstrates that GOLPH3 is essential for cytokinesis, a process required for the maintenance of genomic stability. Full article

Extracellular vesicles (EVs) produced by Fusarium oxysporum f. sp. cubense (Foc) play vital roles in plant–pathogen interactions; however, the isolation of purified Foc TR4-EVs and their pathogenicity and proteomic profiles are not well studied. This study aims to isolate and characterize purified Foc TR4-EVs and compare their pathogenic effects and protein profiles with crude TR4-EVs. Foc TR4-EVs were isolated using ultracentrifugation and purified by iodixanol gradient centrifugation. After characterization and evaluation of the pathogenicity effects on banana leaves, LC-MS/MS was performed to conduct the proteomics assay. Results indicated that Fraction 2 EVs exhibited clearer spherical structures (TEM), excessive abundance (1.70 × 10⁹ particles/mL), greater intensity (400 a.u), mean size (154.5 nm), moderate protein content (333.16 ng/µL), and protein profile (25–77 kDa), which were superior to Fractions 1, 3, and crude EVs. Crude EVs displayed significant background interference with EV structures (TEM), highest abundance (2.11 × 10⁹ particles/mL), lower intensity (7.0 a.u), higher protein content (528.33 ng/µL), and higher molecular weight proteins (55–70 kDa) compared to gradient EVs. A non-significant biocontrol effect of Foc-EVs on the growth of TR4 spores was observed. Pathogenicity assays revealed that crude EVs caused the largest (2.805 cm²), while Fraction 2 (1.386 cm²) and Fraction 3 (1.255 cm²) resulted in moderate lesions on banana leaves. Proteomic analysis identified 807 unique proteins in Fraction 2, enriched in pathways related to EV trafficking and signaling. In comparison, crude EVs contained 179 unique non-EV proteins related to metabolism and secondary metabolites, indicating that non-EV proteins of crude EVs also influence the pathogenicity observed in banana leaves. This study emphasizes the importance of EV purification, with Fraction 2 being a critical focus for future research on Foc EV pathogenicity. Full article

Little brown bats (Myotis lucifugus) cluster in hibernacula sites over winter, in which they use metabolic rate depression (MRD) to facilitate entrance and exit of hibernation. This study used small RNA sequencing and bioinformatic analyses to identify differentially regulated microRNAs (miRNAs) and to predict their downstream effects on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms in the skeletal muscle of torpid M. lucifugus as compared to euthermic controls. We observed a subset of ten miRNAs whose expression changed during hibernation, with predicted functional roles linked to cell cycle processes, downregulation of protein degradation via ubiquitin-mediated proteolysis, downregulation of signaling pathways, including MAPK, p53, mTOR, and TGFβ, and downregulation of cytoskeletal and vesicle trafficking terms. Taken together, our results indicate miRNA regulation corresponding to both widely utilized MRD survival strategies, as well as more hibernation- and tissue-specific roles in M. lucifugus, including skeletal muscle atrophy resistance via myostatin inhibition and insulin signaling suppression. Full article

Extracellular vesicles (EVs) have attracted great attention as promising intracellular drug delivery carriers. While the endocytic pathways of small EVs (sEVs, <200 nm) have been reported, there is limited understanding of large EVs (lEVs, >200 nm), despite their potential applications for drug delivery. Additionally, the low yield of EVs during isolation remains a major challenge in their application. Herein, we aimed to compare the endocytic pathways of sEVs and lEVs using MIA PaCa-2 pancreatic cancer cell-derived EVs as models and to explore the efficiency of their production. The cellular uptake of EVs by MIA PaCa-2 cells was assessed and the pathways were investigated with the aid of endocytic inhibitors. The yield and protein content of sEVs and lEVs from the Integra CELLine culture system and the conventional flasks were compared. Our findings revealed that both sEVs and lEVs produced by the Integra CELLine system entered their parental cells via multiple routes, including caveolin-mediated endocytosis, clathrin-mediated endocytosis, and actin-dependent phagocytosis or macropinocytosis. Notably, caveolin- and clathrin-mediated endocytosis were more prominent in the uptake of sEVs, while actin-dependent phagocytosis and macropinocytosis were significant for both sEVs and lEVs. Compared with conventional flasks, the Integra CELLine system demonstrated a 9-fold increase in sEVs yield and a 6.5-fold increase in lEVs yield, along with 3- to 4-fold higher protein content per 10¹⁰ EVs. Given that different endocytic pathways led to distinct intracellular trafficking routes, this study highlights the unique potentials of sEVs and lEVs for intracellular cargo delivery. The Integra CELLine proves to be a highly productive and cost-effective system for generating EVs with favourable properties for drug delivery. Full article