Search Results (1,519)

Aggregates of misfolded α-synuclein proteins are key markers of Parkinson’s disease. The protein α-synuclein (aSyn) is an intrinsically disordered protein (IDP) and therefore lacks a single stable 3D structure, instead sampling multiple conformations in solution. It is primarily located in presynaptic terminals and is thought to help regulate synaptic vesicle trafficking and neurotransmitter release. ASyn proteins have three domains: an N-terminal domain, a hydrophobic non-amyloid-β component (NAC) core implicated in aggregation, and a proline-rich C-terminal domain. Asyn proteins with truncated C-terminal domains are known to be prone to aggregation and suggest that understanding domain–domain interactions in aSyn monomers could help elucidate the role of the flanking domains in modulating protein structure. To this end, we used Gaussian accelerated molecular dynamics (GAMD) to simulate wild-type (WT), N-terminal truncated (ΔN), C-terminal truncated (ΔC), and isolated NAC domain (isoNAC) aSyn protein variants. Using clustering and contact analysis, we found that removal of the N-terminal domain led to increased contacts between NAC and C-terminal domains and the formation of inter-domain β-sheets. Removal of either flanking domain also resulted in increased compactness of every domain. We also found that the contacts between flanking domains in the WT protein result in an electrostatic potential (ESP) that may lead to favorable interactions with anionic lipid membranes. Removal of the C-terminal domain disrupts the ESP in a way that could result in over-stabilized protein–membrane interactions. These results suggest that cooperation between the flanking domains may modulate the protein’s structure in a way that helps maintain elongation and creates an ESP that may aid favorable interactions with the membrane. Full article

Background/Objectives: Subunit vaccines composed of purified proteins and adjuvants offer excellent safety, but often generate short-lived immunity due to rapid antigen clearance and limited antigen-presenting cell engagement. Sustained, localized delivery of antigen and adjuvant may improve the magnitude and durability of the immune response without compromising safety. This study evaluated an in-situ polymerizing type I oligomeric collagen (Oligomer) scaffold to localize antigen/adjuvant at the injection site and prolong antigen presentation. Methods: Mice were immunized intramuscularly with ovalbumin (OVA) and CpG oligonucleotide adjuvant delivered alone or co-formulated with Oligomer. Antibody response and inflammation at the injection site were assessed post-booster at early (Day 32) and late (Day 68) time points. Antigen retention and dendritic cell trafficking to draining lymph nodes were evaluated using fluorescently labeled OVA. Results: The Oligomer scaffold retained vaccine antigen at the injection site without eliciting a material-mediated foreign body response. Co-delivery of OVA and CpG within the scaffold enhanced germinal center activity, increased follicular helper T cells and germinal center B cells, and skewed CD4⁺ T cells toward a Th1 phenotype. Humoral responses were greater and more durable, with higher OVA-specific IgG, IgG1, and IgG2a titers and an increased number of bone marrow antibody-secreting cells persisting through Day 68. Antigen-positive dendritic cells, including both resident and migratory subsets, were elevated in draining lymph nodes, indicating enhanced antigen transport. No anti-mouse collagen I antibodies were detected, confirming the maintenance of collagen self-tolerance. Conclusions: The Oligomer delivery platform functioned as a localized, immunotolerant vaccine depot, sustaining antigen availability and immune cell engagement. This spatiotemporal control enhanced germinal center responses and generated a more robust, durable humoral immune response, supporting its potential to improve subunit vaccine efficacy while maintaining an excellent safety profile. Full article

Optineurin (OPTN) is a multifunctional adaptor protein that regulates diverse cellular processes, including inflammatory signaling, autophagy, vesicular trafficking, and immune responses. This multifaceted role of OPTN is made possible by the presence of a complex structure comprising multiple domains that interact with different proteins to exert various functions important for modulating key signaling processes. Mutations in OPTN are linked with several human pathologies including glaucoma, Paget’s disease of bone, Crohn’s disease, and neurodegenerative diseases such as amyotrophic lateral sclerosis, and dementia. Emerging evidence suggests that OPTN has a complex and context-dependent role in cancer biology as well. It is upregulated in pancreatic ductal adenocarcinoma and hepatocellular carcinoma but downregulated in lung and colorectal cancers, indicating its dual role as a potential oncogene or tumor suppressor depending on the cellular environment. Additionally, OPTN plays a critical role in preventing immune evasion in colorectal cancer by maintaining interferon-gamma receptor 1 (IFNGR1) expression and supporting dendritic cell-mediated T-cell priming, thereby enhancing antitumor immune responses. Despite its significance in oncogenic pathways and immune regulation, the therapeutic potential of targeting OPTN in cancer remains largely unexplored. This review aims to provide a comprehensive understanding of OPTN’s pleiotropic functions, highlighting its role in autophagy, inflammation, immune surveillance, and cancer progression. By elucidating its diverse regulatory mechanisms, we seek to encourage further research into the therapeutic implications of OPTN in cancer treatment and immunotherapy. Full article

Background/Objectives: Extracellular Vesicles (EVs) have shown great promise as diagnostic and therapeutic tools, as well as pharmacological nanocarriers. Various strategies are being explored to develop EVs for monitoring, imaging, loading with pharmacological agents, and surface decoration with tissue-specific ligands. EVs derived from Mesenchymal Stromal Cells (MSC-EVs) are of particular interest both as therapeutics per se and as natural nanocarriers for the targeted delivery of biotherapeutics. Methods: In this study, we investigated the ability of different tags to deliver a reporter protein into canine MSC-EVs with the aim of identifying the most effective endogenous loading mechanism. To this aim, canine MSCs were engineered to express the Green Fluorescent Protein (GFP) fused to CD63, Syntenin-1, TSG101, and the palmitoylation signal of Lck, which were expected to promote GFP incorporation into EVs. Overexpression of tagged GFP in canine MSCs was confirmed by Western blotting and examined by confocal microscopy and transmission electron microscopy to map intracellular localization. Results: All tags were able to deliver GFP into EVs. Syntenin-1 showed relatively high loading efficiency and secretion index but exhibited a diffuse localization pattern in the transfected cells. The palmitoylation signal showed low loading efficiency and localization specificity. TSG101 displayed a morphological pattern consistent with specific localization in endosomal structures, but its low expression level prevented further evaluations. Finally, CD63 showed the highest expression efficiency, as GFP-CD63 levels were approximately 5-fold higher than untagged GFP. Conclusions: In conclusion, CD63 emerged as the most suitable tag for canine MSC-EV engineering. Indeed, even if the secretion index favours Syntenin-1, CD63’s higher abundance in the lysate suggests its substantial post-secretion uptake. Further studies aimed at elucidating CD63’s specific contribution and identifying the domains involved in vesicle trafficking could provide valuable insights into EV bioengineering. Full article

Neuronal membrane capacitance (Cm) has traditionally been viewed as a static biophysical property determined solely by the geometric and dielectric characteristics of the lipid bilayer. Recent discoveries have fundamentally challenged this perspective, revealing that Cm exhibits robust circadian oscillations that profoundly influence neural computation and behavior. These rhythmic fluctuations in membrane capacitance are orchestrated by intrinsic cellular clocks through coordinated regulation of molecular processes including transcriptional control of membrane proteins, lipid metabolism, ion channel trafficking, and glial-mediated extracellular matrix remodeling. The dynamic modulation of Cm directly impacts the membrane time constant (τm = RmCm), thereby altering synaptic integration windows, action potential dynamics, and network synchronization across the 24 h cycle. At the computational level, circadian Cm oscillations enable neurons to shift between temporal summation and coincidence detection modes, optimizing information processing according to behavioral demands throughout the day–night cycle. These biophysical rhythms influence critical aspects of cognition including memory consolidation, attention, working memory, and sensory processing. Disruptions in normal Cm rhythmicity are increasingly implicated in neuropsychiatric and neurodegenerative disorders, including depression, schizophrenia, Alzheimer’s disease, and epilepsy, where altered membrane dynamics compromise neural circuit stability and information transfer. The integration of circadian biophysics with chronomedicine offers promising therapeutic avenues, including chronotherapeutic strategies that target membrane properties, personalized interventions based on individual chronotypes, and environmental modifications that restore healthy biophysical rhythms. This review synthesizes evidence from molecular chronobiology, cellular electrophysiology, and systems neuroscience to establish circadian Cm regulation as a fundamental mechanism linking molecular timekeeping to neural computation and behavior. Full article

Charcot–Marie–Tooth disease type 1A (CMT1A) is a hereditary condition caused by the duplication of the PMP22 gene. Overexpression of peripheral myelin protein 22 in Schwann cells leads to myelin sheath defects and axonal loss. We have produced a cell model to facilitate studies of the molecular mechanisms involved in PMP22 accumulation and clearance. Our model is a stably transfected, clonal, immortalised human Schwann cell line with overexpressed levels of PMP22 fusion protein. A control-transfected cell line (vector lacking PMP22) was also produced. PMP22-transfected cells had reduced levels of mitosis, with the PMP22 fusion protein concentrated in punctate aggregates in the cytoplasm and expressed at the plasma membranes, which were often irregular and spindly. In contrast, control cells (control-transfected and parent cell lines) generally had smooth and regular plasma membrane morphology. Culturing in the presence of NRG1 and forskolin lead to upregulation of markers of myelination potential in the control cells. These markers were more variable in the cells stably transfected with PMP22, including decreased levels of transcripts of SOX10, JUN, S100B and NGFR, but increased levels of MPZ and EGR2 compared to controls. Using proximity-dependent biotin identification (BioID2), several hundred proteins were identified in the proximity of the overexpressed PMP22, of which 291 significant proteins were only detected in the proximity of PMP22 and not in that of control pull-downs. Among the most significantly enriched PMP22-interacting proteins were integrins alpha-2 (ITGA2) and alpha-7 (ITGA7), which play a role in myelination via their interactions with the extracellular matrix. The presence of ITGA2 in just the PMP22-transfected fraction was confirmed by western blot. Some of the proteins were associated with several enriched molecular pathways, including molecular transport and protein trafficking, and may represent potential therapeutic targets for CMT1A by promoting the degradation and enhanced trafficking of PMP22. Full article

Background/Objectives: Obesity and hyperglycemia predispose patients to respiratory infections. Although the lung is a major organ to utilize glucose, pulmonary glucose homeostasis in type 2 diabetic (T2Dx) subjects remains poorly characterized. We hypothesized that pulmonary glucose transport would be altered during T2Dx, which would be rescued with long-term metformin treatment. Methods: T2Dx was induced by feeding mice a high-fat diet for 16 weeks, with metformin treatment administered during the final 8 weeks. Results: Glucose transporter (GLUT) protein expression and trafficking was quantified by Western blotting and the biotinylated photolabeling assay, respectively. T2Dx mice exhibited obesity, and increased glucose levels in blood and bronchoalveolar lavage (BAL) fluid. T2Dx also significantly decreased protein expression of GLUTs from Class I (i.e., GLUT-2 and -4) and class III (i.e., GLUT-10 and -12) isoforms in lung. Metformin treatment restored the protein expression of GLUT-2, -4, and -10, but not GLUT-12. Pulmonary cell surface expression of GLUT-4 and -8 was also significantly reduced in T2Dx mice and rescued by metformin. Conclusions: These findings suggest that alterations in pulmonary GLUT expression and trafficking during diabetes could contribute to the elevated airway glucose levels and severity of respiratory infections. Metformin treatment restored pulmonary glucose transport during T2Dx. Full article

Adaptor Protein-1 (AP-1) is a heterotetrameric essential for intracellular vesicular trafficking and polarized localization of somato-dendritic proteins in neurons. Variants in the AP1G1 gene, encoding the gamma-1 subunit of adaptor-related protein complex 1 (AP1γ1), have recently been associated with Usmani–Riazuddin syndrome (USRISD, MIM#619467), a very rare human genetic disorder characterized by intellectual disability (ID), speech and neurodevelopmental delays. Here we report a novel variant (c.196G>A; p.Gly66Arg) identified by exome sequencing analysis in a young girl showing overlapping clinical features with USRIS, such as motor and speech delay, intellectual disability and abnormal aggressive behavior. In silico analysis of the missense de novo variant suggested an alteration in AP1G1 protein folding. Patient’s fibroblasts have been studied with immunofluorescence techniques to analyze the intracellular distribution of AP-1. Zebrafish are widely regarded as an excellent vertebrate model for studying human disease pathogenesis, given their transparent embryonic development, ease of breeding, high genetic similarity to humans, and straightforward genetic manipulation. Leveraging these advantages, we investigated the phenotype, locomotor behavior, and CNS development in zebrafish embryos following the microinjection of human wild-type and mutated AP1G1 mRNAs at the one-cell stage. Knockout (KO) of the AP1G1 gene in zebrafish led to death at the gastrula stage. Lethality in the KO AP1G1 fish model was significantly rescued by injection of the human wild-type AP1G1 mRNA, but not by transcripts encoded by the Gly66Arg missense allele. The phenotype was also not rescued when ap1g1−/− zebrafish embryos were co-injected with both human wild-type and mutated mRNAs, supporting the dominant-negative effect of the new variant. In this study, we defined the effects of a new AP1G1 variant in cellular and animal models of Usmani–Riazzudin syndrome for future therapeutic approaches. Full article

Tunneling nanotubes (TNTs) are dynamic, actin-based intercellular structures that facilitate the transfer of organelles, including mitochondria, between cells. Unlike other protrusive structures such as filopodia and cytonemes, TNTs exhibit structural heterogeneity and functional versatility, enabling both short- and long-range cargo transport. This review explores the mechanisms underlying mitochondrial transfer via TNTs, with a particular focus on cytoskeletal dynamics and the role of key regulatory proteins such as Miro1, GFAP, MICAL2PV, CD38, Connexin 43, M-Sec, thymosin β4, and Talin 2. Miro1 emerges as a central mediator of mitochondrial trafficking, linking organelle motility to cellular stress responses and tissue repair. We delve into the translational implications of TNTs-mediated mitochondrial exchange in regenerative medicine and oncology, highlighting its potential to restore bioenergetics, mitigate oxidative stress, and reprogram cellular states. Despite growing interest, critical gaps remain in understanding the molecular determinants of TNT formation, the quality and fate of transferred mitochondria, and the optimal sources for mitochondrial isolation. Addressing these questions will be essential for harnessing TNTs and mitochondrial transplantation as therapeutic tools. Full article

The OPTN gene, which encodes the adaptor protein optineurin, is genetically linked to amyotrophic lateral sclerosis and frontotemporal dementia, diseases characterized by chronic microglial activation. Optineurin regulates inflammatory signaling, autophagy, and trafficking, but its role in microglia remains incompletely understood. Here, we used bulk RNA sequencing to profile CRISPR-Cas9-mediated optineurin knockout (KO) and wild-type BV2 microglia under basal conditions and upon LPS stimulation. At baseline, optineurin KO altered ~7% of the transcriptome, with a predominant downregulation of type I interferon and antiviral pathways, suggesting its role in maintaining basal immune readiness. LPS stimulation reprogrammed ~35% of genes in wild-type microglia, inducing immune effectors and suppressing cell cycle regulators, whereas in optineurin-deficient cells, the response was blunted with only ~16% of genes changing relative to the KO baseline. Furthermore, LPS-treated optineurin KO microglia notably diverged from LPS-treated wild-type cells, with ~26% differentially expressed genes (DEGs). This included impaired induction of inflammatory programs and persistence of cell cycle-associated transcripts. Most DEGs in LPS-treated KO cells were unique to this condition, highlighting optineurin-dependent pathways specific to inflammatory challenge. Overall, our study provides a systems-level framework for investigating optineurin in microglia and neurodegeneration, establishing it as a key regulator of the microglial transcriptome, with its loss reshaping innate immune and cell cycle programs. Full article

Plasmodesmata (PD) are dynamic nanochannels interconnecting plant cells and coordinating development, nutrient distribution, and systemic defense. Their permeability is tightly regulated by callose turnover, PD-localized proteins, lipid microdomains, and endoplasmic reticulum (ER)–plasma membrane (PM) tethers, which together form regulatory nodes that gate symplastic exchange. Increasing evidence demonstrates that effectors from diverse kingdoms—fungi, oomycetes, bacteria, viruses, viroids, phytoplasmas, nematodes, insects, parasitic plants, and symbiotic microbes—converge on these same nodes to modulate PD gating. Pathogens typically suppress callose deposition or destabilize PD regulators to keep channels open, whereas mutualists fine-tune PD conductivity to balance resource exchange with host immunity. This review synthesizes current knowledge of effector strategies that remodel PD architecture or exploit PD for intercellular movement, highlighting novel cross-kingdom commonalities–callose manipulation, reprogramming of PD proteins, lipid rewiring, and co-option of ER-PM tethers. We outline unresolved questions on effector–PD target specificity and dynamics, and identify prospects in imaging, proteomics, and synthetic control of PD. Understanding how effectors reprogram PD connectivity can enable engineering of crops that block pathogenic trafficking while safeguarding beneficial symbioses. Full article

Background: Mesoporous silica nanoparticles (MS NPs) have attracted significant interest for their role in the advancement of drug delivery systems. However, further investigation is needed to unravel the mechanisms behind the shift in organ tropism that occurs with changes in composition. Methods: To shed light on the correlation between their composition and organ-targeting capabilities, a range of MS NPs was synthesized and subsequently administered intravenously to mice. Results: Our results indicate that MS NPs with a pristine -Si-O-Si- framework, or those incorporating -C-C- or –S-S-S-S- bonds, predominantly accumulated in the liver. The shift to lung tropism was observed exclusively in MS NPs that were enriched with –SH groups. Proteomic analysis identified histidine-rich glycoprotein (HRG) as the most prevalent protein associated with liver-preferred MS NPs in serum, while lung-preferred MS NPs, such as thioether-bridged deformable hollow mesoporous organosilica nanoparticles (HSMONs), showed the highest affinity for albumin. Furthermore, the lung-selective HSMONs, endowed with inherent deformability and glutathione-responsive biodegradability, were utilized as systemic nanocarriers for the delivery of gambogic acid (GA). Conclusions: Leveraging albumin absorbing-triggered tumor cell targeting and trafficking, HSMONs conjugated with GA effectively elicited potent antitumor effects in pulmonary tissue. Full article

Protein S-palmitoylation is a reversible lipid modification that regulates various aspects of protein function, including membrane association, subcellular localization, trafficking, stability, and activity. The depalmitoylase ABHD17A removes palmitate from multiple substrates, but its cellular positioning and the role of its own palmitoylation in regulating its function remain unclear. This study identifies a palmitoylation code within the conserved N-terminal cysteine cluster of ABHD17A, which governs its intracellular distribution and plasma membrane (PM) targeting. N-terminal palmitoylation is essential for PM localization. Through the use of code-restricted mutants, we found that modifications in the middle region (C14, C15) are critical for PM targeting and catalytic activity, while modifications at the front (C10, C11) and rear (C18) influence endosomal routing and delivery to the PM. Alanine scanning revealed that adjacent hydrophobic residues, particularly L9 and F13, are crucial for initial engagement with endomembranes. Sequence analysis and mutagenesis identified two tyrosine-based YXXØ motifs within the alpha/beta hydrolase fold; disruption of the proximal motif (L115A) decreased surface abundance and redirected ABHD17A to autophagosomes, indicating a need for YXXØ-dependent endosomal sorting, likely at the trans-Golgi network. Biochemical assays demonstrated a continuum of acylation states influenced by the palmitoylation code. This requirement for the middle region was conserved in ABHD17B and ABHD17C. Overall, our findings suggest a stepwise mechanism for ABHD17A delivery to the PM, enabling its depalmitoylase activity on membrane-bound substrates. Full article

Perivascular adipose tissue (PVAT) is a unique fat depot that is distributed around blood vessels, contiguous with the vascular adventitia. Due to this proximity, it serves as a local source of adipokines and vasoregulatory factors. Similar to other adipose depots, PVAT is responsive to changes in metabolic state and, at least in mice, can transition to a thermogenic adipocyte phenotype depending on metabolic health. Cardiovascular disease risk is highly correlated with metabolic health and increases substantially in individuals with obesity or metabolic syndrome. Cardiovascular diseases, including atherosclerosis/coronary artery disease, aortic aneurysm, hypertension, arterial stiffening, and heart failure, have been associated with PVAT dysregulation. Understanding the cardiovascular protective effects of healthy PVAT can provide ways to modify disease progression to re-establish functional homeostasis. This review focuses on experimental studies that specifically define a signaling axis between PVAT and the cardiovascular system that provide cardioprotection. Our focus is primarily on the secreted contents of extracellular vesicles that initiate this adipose signaling axis and regulation of extracellular vesicle release by the trafficking molecule, RAB27a. We review the current literature on human and mouse studies and major categories of PVAT-derived signaling components including microRNAs, lipids, and proteins that contribute to cardiovascular homeostasis. Full article

The stimulator of interferon genes (STING) serves as a pivotal signaling hub in innate immunity, orchestrating type I interferon (IFN-I) and pro-inflammatory responses upon detection of cytosolic DNA. While the canonical cyclic GMP-AMP synthase (cGAS)-STING axis has been extensively studied in host defense and sterile inflammation, increasing evidence indicates that STING can also be activated through a variety of both pattern recognition receptors (PRRs)-dependent and PRRs-independent mechanisms. In this review, we comprehensively summarize the molecular pathways through which PRRs—including cGAS, interferon gamma inducible protein 16 (IFI16), DEAD-box helicase 41 (DDX41), and DNA-dependent protein kinase (DNA-PK)—engage and regulate STING activation. Beyond PRRs-triggered pathways, we explore emerging evidence of PRRs-independent STING activation, driven by genetic mutations, endoplasmic reticulum (ER) stress, dysregulated intracellular trafficking, and impaired protein degradation. These mechanisms contribute to the pathogenesis of a broad spectrum of inflammatory and autoimmune disorders affecting multiple organ systems, including the digestive, cardiovascular, renal, pulmonary, and nervous systems. We also highlight the current landscape of pharmacological inhibitors targeting cGAS and STING, categorized according to their mechanisms of action and therapeutic potential. The redundancy and complexity of components within the STING signaling network present challenges in effectively suppressing inflammatory overactivation by targeting a single molecule. Nevertheless, the central role of STING offers multiple opportunities for therapeutic intervention, whether by modulating upstream or downstream signaling elements. This review not only provides a systematic framework for understanding the intricacies of STING signaling, but offers insights into the development of next-generation therapeutics aimed at selectively modulating STING activity in disease contexts. Full article