Membrane Localization Research Articles

Deficiency of KIF15 contributes to oxaliplatin-induced cold hypersensitivity by limiting Annexin A2 and enhancing TRPA1 localization in DRG neuronal membrane

Deficiency of KIF15 contributes to oxaliplatin-induced cold hypersensitivity by limiting Annexin A2 and enhancing TRPA1 localization in DRG neuronal membrane

A Comparative Analysis of Sialic Acid Binding Proteins for Better Understanding of their Role in Host Pathogen Interaction and Human Health: A Bioinformatics Perspective

Sialic acids, especially N-acetylneuraminic acid, serve as pivotal molecular interfaces between host tissues and invading pathogens by modulating attachment, immune evasion, and metabolic processes. In this study, we performed a comprehensive bioinformatics analysis of sialic acid binding proteins (SABPs) to unravel their structural diversity, evolutionary distribution, and functional roles across bacteria and viruses. A curated dataset of 209 SABPs was retrieved from the UniProt database, with inclusion criteria emphasizing annotated sialic acid binding activity and sequence completeness. Notably, 208 proteins in this collection are backed by experimentally determined 3D structures, enabling in-depth examination of their binding sites and catalytic domains. Gene Ontology (GO) analyses revealed that these proteins are predominantly involved in critical biological processes related to host-pathogen interaction, such as virion attachment to host cells, membrane fusion, and carbohydrate metabolism. Molecular function terms were dominated by host cell surface receptor binding, carbohydrate binding, and hydrolase activities targeting glycosyl bonds. Cellular component annotations highlighted membrane localization and extracellular secretions, consistent with roles in mediating initial host contact and pathogen dissemination. Taxonomic assessments showed a strong representation of Orthomyxoviridae, underlining the importance of hemagglutinin and neuraminidase in influenza viruses, while bacterial families such as Clostridiaceae and Staphylococcaceae contributed SABPs involved in sialic acid catabolism and toxin-mediated virulence. Family-level clustering pinpointed hemagglutinins, neuraminidases (glycosyl hydrolases GH33 and GH34), and immunoglobulin superfamily members—further emphasizing the widespread evolutionary adoption of sialic acid recognition. These findings collectively underscore the centrality of SABPs in host-pathogen interactions and offer a valuable resource for future research aimed at leveraging sialic acid biology for therapeutic and diagnostic advancements.

Chloride intracellular channel (CLIC) protein function in S1P-induced Rac1 activation requires membrane localization of the C-terminus, but not thiol-transferase nor ion channel activities.

We have established a novel and evolutionarily-conserved function for chloride intracellular channel proteins (CLICs) in regulating Rho/Rac GTPases downstream of G protein-coupled receptors (GPCRs). Endothelial CLIC1 and CLIC4 are rapidly and transiently re-localized from the cytoplasm to the plasma membrane in response to the GPCR ligand sphingosine-1-phosphate (S1P), and both CLICs are required to activate Rac1 in response to S1P, but how they perform this function remains unknown. Biochemical studies suggest that CLICs act as non-specific ion channels and/or as glutathione-S-transferases, dependent on N-terminal features, in vitro . Here we investigate CLIC functional domains and membrane localization requirements for their function in S1P-mediated Rac1 signaling. Structure-function analyses of CLIC function in endothelial cells demonstrate that CLIC1 and CLIC4-specific functions reside at their C-termini, and that the CLIC4 N-terminus encodes determinants required for S1P-induced re-localization to the plasma membrane but is dispensable for S1P-induced Rac1 activation when the C-terminus is localized to the plasma membrane via a heterologous signal. Our results demonstrate that the postulated ion channel and thiol-transferase activities of CLICs are not required for Rac1 activation and suggests that sequences in the CLIC C-termini are critical for this function. Given the importance of S1P signaling in vascular biology and disease, our work establishes a platform to further our understanding of the membrane-localized proteins required to link GPCR activity to Rho/Rac regulation.

MAGED2 Enhances Expression and Function of NCC at the Cell Surface via cAMP Signaling Under Hypoxia

Mutations in MAGED2 cause transient antenatal Bartter syndrome (tBS) characterized by excessive amounts of amniotic fluid due to impaired renal salt transport via NKCC2 and NCC, high perinatal mortality, and pre-term birth. Surprisingly, renal salt handling completely normalizes after birth. Previously, we demonstrated that, under hypoxic conditions, MAGED2 depletion enhances endocytosis of GalphaS (Gαs), reducing adenylate cyclase (AC) activation and cAMP production. This impaired cAMP signaling likely contributes to the dysfunction of salt transporters NKCC2 and NCC, explaining salt wasting and the subsequent recovery with renal oxygenation after birth. In this study, we show that MAGED2 depletion significantly decreases both total cellular and plasma membrane NCC expression and activity. We further demonstrate that MAGED2 depletion disrupts NCC trafficking by reducing exocytosis, increasing endocytosis, and promoting lysosomal degradation via enhanced ubiquitination. Additionally, forskolin (FSK), which increases cAMP production by activating AC, rescues NCC expression and localization in MAGED2-depleted cells. Conversely, MAGED2 overexpression increases NCC expression and membrane localization, although this effect is diminished in Gαs-depleted cells, indicating that Gαs acts downstream of MAGED2. In summary, our findings reveal the essential role of MAGED2 in regulating NCC function and trafficking under hypoxic conditions, providing new insights into the mechanisms behind salt loss in tBS and identifying potential therapeutic targets.

A temperature-inducible protein module for control of mammalian cell fate.

Inducible protein switches are currently limited for use in tissues and organisms because common inducers cannot be controlled with precision in space and time in optically dense settings. Here, we introduce a protein that can be reversibly toggled with a small change in temperature, a stimulus that is both penetrant and dynamic. This protein, called Melt (Membrane localization using temperature) oligomerizes and translocates to the plasma membrane when temperature is lowered. We generated a library of Melt variants with switching temperatures ranging from 30 °C to 40 °C, including two that operate at and above 37 °C. Melt was a highly modular actuator of cell function, permitting thermal control over diverse processes including signaling, proteolysis, nuclear shuttling, cytoskeletal rearrangements and cell death. Finally, Melt permitted thermal control of cell death in a mouse model of human cancer. Melt represents a versatile thermogenetic module for straightforward, non-invasive and spatiotemporally defined control of mammalian cells with broad potential for biotechnology and biomedicine.

Super-Resolution Fluorescence Imaging Reveals the Mechanism of NRP1 Clustering on Non-Small-Cell Lung Cancer Membranes.

Neuropilin 1 (NRP1) is upregulated in various types of malignant tumors, especially non-small-cell lung cancer (NSCLC). However, the precise mechanisms for membrane localization and regulation are not fully understood. Observations from super-resolution microscopy have revealed that NRP1 tends to form nanoscale clusters on the cell membrane, with these clusters varying significantly in size and density across different regions. Further research has shown that stimulation by hepatocyte growth factor (HGF) can reorganize the distribution of NRP1, reducing the number of small clusters while promoting the formation of larger ones. This suggests a propensity for internalization after activation. Additionally, dual-color dSTORM imaging has demonstrated a certain degree of colocalization between NRP1 and c-MET, indicating that c-MET plays an important role in stabilizing NRP1 clusters. This study provides new insights into the mechanism behind NRP1's clustered distribution on cell membranes and paves the way for developing more effective therapeutic strategies targeting NRP1 within tumors.

Melatonin protects aged oocytes from depalmitoylation-mediated quality reduction by promoting PPT1 degradation and antioxidation.

Oocyte aging is closely related to a decline in female fertility, accompanied by increased reactive oxygen species levels and changes in protein posttranslational modifications. However, the role of protein palmitoylation in oocyte aging has not been investigated. In the present study, a new association between redox and palmitoylation in aging oocytes was found. We found that the protein level of palmitoyl-protein thioesterase 1 (PPT1), a depalmitoylation enzyme, was increased in maternally aged mice oocytes and follicular fluid of aged (age >35 years) patients with decreased ovarian reserve (DOR). Elevated PPT1 led to decreased S-palmitoylation levels in oocytes, which impaired oocyte maturation and spindle formation. Tubulin was identified as a critical palmitoylated protein regulated by PPT1, whose palmitoylation was also decreased by advanced age, accompanied by abnormalities in membrane localization and microtubule polymerization. Melatonin was found to down-regulate excessive PPT1 and rescue PPT1-induced damage in mouse oocytes, not only by regulating oxidative stress, but also by binding with PPT1 to regulate its lysosomal degradation. In summary, our data demonstrate that PPT1 participates in oocyte aging by regulating tubulin palmitoylation, providing evidence that oxidative stress regulates protein palmitoylation and revealing a novel mechanism of oocyte aging.

The grapevine ABC transporter B family member 15 is a trans-resveratrol transporter out of grapevine cells.

Stilbenes, particularly trans-resveratrol, play a highly relevant defense role in grapevines as phytoalexin is induced in response to stress. Metabolism and transport of stilbenes can be conveniently investigated in grapevine cell culture since large amounts of trans-resveratrol are accumulated in the extracellular medium upon treatment with the elicitor methylated cyclodextrin, either alone or combined with methyl jasmonate. A proteomic approach on grapevine cell membrane fractions was performed to find trans-resveratrol transporter candidates. The candidate VvABCB15 was functionally characterized. Its stable expression in both yeast and Silybum marianum cells' heterologous systems led to increased trans-resveratrol transport in these hosts. Transient expression in Vitis cells showed an enhanced absorbent- or elicitor-assisted accumulation of extracellular trans-resveratrol in VvABCB15-expressing or VvGSTU10/VvABCB15-co-expressing cell suspension cultures. Experiments of transient expression in Vitis cell suspensions using light-switchable stilbene synthase (pHYH::VvSTS3) and VvABCB15 further confirmed the candidate's role as a trans-resveratrol transporter. VvABCB15-YFP fusion proteins in Nicotiana leaf showed localization in the plasma membrane, consistent with a functional role in trans-resveratrol transport. This is the first report to provide evidence for the involvement of an ABC transporter B type, VvABCB15, in trans-resveratrol transport to the extracellular medium of grapevine cells.

Assessment of Self-Activation and Inhibition of Wheat Coiled-Coil Domain Containing NLR Immune Receptor Yr10CG.

Plant immunity is largely governed by nucleotide-binding leucine-rich repeat receptor (NLR). Here, we examine the molecular activation and inhibition mechanisms of the wheat CC-type NLR Yr10CG, a previously proposed candidate for the Yr10 resistance gene. Though recent studies have identified YrNAM as the true Yr10 gene, Yr10CG remains an important NLR in understanding NLR-mediated immunity in wheat. In this study, we found that the overexpression of either the full-length Yr10CG or its CC domain in Nicotiana benthamiana did not trigger cell death, suggesting a robust autoinhibitory mechanism within Yr10CG. However, we observed that mutations in the conserved MHD motif, specifically D502G, activated Yr10CG and induced cell death. Structural modeling indicated that this mutation disrupted key interactions within the MHD motif, promoting local flexibility and activation. We further explored the effector recognition potential of Yr10CG by creating chimeric proteins with Sr50 domains, revealing that both the NB-ARC and LRR domains are necessary for effector recognition, while the CC domain likely functions in downstream immune signaling. Additionally, disrupting membrane localization through an L11E mutation abolished Yr10CG self-activation, suggesting a requirement for membrane association in immune activation. Our findings contribute to the understanding of CC-NLR activation and autoinhibition mechanisms, highlighting the potential of Yr10CG in NLR engineering for crop resistance improvement.

Biotinylated Viscosity Sensitive Cell Membrane Probe for Targeted Imaging and Precise Visualization of Tumor Cells and Tumors.

Cancer is a global health challenge that urgently requires more sensitive and effective cancer detection methods. Fluorescence imaging with small molecule fluorescent probes has shown great promise for cancer detection but most of the developed probes lack active tumor cell targeting, which makes them unable to selectively target tumors, thereby reducing the accuracy of in vivo tumor detection. Herein, we report a novel probe Bio-S that combines a viscosity-sensitive and cell membrane targetable fluorescent group with biotin for targeted imaging and precise visualization of tumor cells and tumors. Bio-S exhibits sensitive fluorescence changes for viscosity at ∼660 nm and excellent cell membrane localization and imaging ability (red fluorescence, wash-free, and long-term imaging). Moreover, compared with the nonbiotinylated control probe C6-S, the biotinylated Bio-S can specifically target tumor cell membranes, thereby achieving much higher selectivity and sensitivity in distinguishing tumor cells from normal cells. Mice imaging experiments show that tail vein injection of Bio-S can target tumors and monitor lung cancer metastasis at the in vivo level. Therefore, this work provides an effective new strategy and tool for tumor-targeted detection and precise diagnosis.

Attenuating ABHD17 isoforms augments the S-acylation and function of NOD2 and a subset of Crohn's disease-associated NOD2 variants.

NOD2 is an intracellular innate immune receptor that detects bacterial peptidoglycan fragments. Although nominally soluble, some NOD2 is associated with the plasma membrane and endosomal compartments for microbial surveillance. This membrane targeting is achieved through post-translational S-acylation of NOD2 by the protein acyltransferase ZDHHC5. Membrane attachment is necessary to initiate a signaling cascade in response to cytosolic peptidoglycan fragments. Ultimately, this signaling results in the production of antimicrobial peptides and pro-inflammatory cytokines. In most cases, S-acylation is a reversible post-translational modification with removal of the fatty acyl chain catalyzed by one of several acyl protein thioesterases. Deacylation of NOD2 by such an enzyme will displace it from the plasma membrane and endosomes, thus preventing signaling. To identify the enzymes responsible for NOD2 deacylation, we used engineered cell lines with RNA interference and small-molecule inhibitors. These approaches were combined with confocal microscopy, acyl-resin-assisted capture, immunoblotting, and cytokine multiplex assays. We identified α/β-hydrolase domain-containing protein 17 isoforms (ABHD17A, ABHD17B, and ABHD17C) as the acyl protein thioesterases responsible for NOD2 deacylation. Inhibiting ABHD17 increased the plasma membrane localization of wild-type NOD2 and a subset of poorly acylated Crohn's disease-associated variants. This enhanced NOD2 activity, increasing NF-κB activation and pro-inflammatory cytokine production in epithelial cells. These findings demonstrate that ABHD17 isoforms are negative regulators of NOD2. The results also suggest that targeting ABHD17 isoforms could restore functionality to specific Crohn's disease-associated NOD2 variants, offering a potential therapeutic strategy.

Prenylation-dependent membrane localization of a deubiquitinating enzyme and its role in regulating G protein-mediated signaling in yeast.

Miy1 is a highly conserved de-ubiquitinating enzyme in yeast with MINDY1 as its human homolog. Miy1 is known to act on K48-linked polyubiquitin chain, but its biological function is unknown. Miy1 has a putative prenylation site, suggesting it as a membrane-associated protein that may contribute to the regulation of cell signaling. Here, we demonstrate that Miy1 is localized in the plasma membrane and nuclear periphery. Mutating the putative prenylation site in Miy1 or disrupting the farnesyltransferase activity impairs its localization. Consistent with a role of Miy1 in regulating the ubiquitination status of membrane proteins, the miy1Δ mutants exhibit a higher level of ubiquitinated conjugates at the plasma membrane. To examine a role of Miy1 in regulating cell signaling across plasma membrane, we focused on the pheromone response, as both Ste2, the receptor for mating pheromone, and Gpa1, the cognate Gα protein of Ste2, are well-known to be regulated by ubiquitination. We find that Miy1 interacts with Gpa1, regulates its level of ubiquitination and abundance. Pheromone-induced MAP kinase Fus3 activation is also altered in the MIY1-disrupted mutants. The findings demonstrate that Miy1 is a membrane-associated deubiquitinating enzyme and a regulator of G protein-mediated signaling.

Uvaol attenuates TGF-β1-induced epithelial-mesenchymal transition in human alveolar epithelial cells by modulating expression and membrane localization of β-catenin.

The epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells change into mesenchymal cells with fibroblast-like characteristics. EMT plays a crucial role in the progression of fibrosis. Classical inducers associated with the maintenance of EMT, such as TGF-β1, have become targets of several anti-EMT therapeutic strategies. Natural products from the pentacyclic triterpene class have emerged as promising elements in inhibiting EMT. Uvaol is a pentacyclic triterpene found in olive trees (Olea europaea L.) known for its anti-inflammatory, antioxidant, and antiproliferative properties. Yet, its effect on the TGF-β1-induced EMT in alveolar epithelial cells is unknown. The present study aimed to investigate the impact of uvaol upon TGF-β1-induced EMT in a cultured A549 human alveolar epithelial cell line, a classic in vitro model for studies of EMT. Changes in cell shape were measured using phase-contrast and confocal microscopy, whereas protein expression levels were measured using immunofluorescence, flow cytometry, and Western blotting. We also performed wound scratch experiments to explore its effects on cell migration. Uvaol had no significant cytotoxic effects on A549 cells. By contrast, the changes in the cell morphology consistent with TGF-β1-induced EMT were largely suppressed by treatment with uvaol. In addition, increased contents of mesenchymal markers, namely, vimentin, N-cadherin, and fibronectin in TGF-β1-induced A549 cells, were downregulated by uvaol treatment. Furthermore, the TGF-β1-induced migration of A549 cells was significantly suppressed by uvaol. Mechanistically, uvaol prevented the nuclear translocation of β-catenin and reduced the TGF-β1-induced levels of ZEB1 in A549 cells. These results provide compelling evidence that uvaol inhibits EMT by regulating proteins related to the mesenchymal profile in human alveolar epithelial cells, likely by modulating β-catenin and ZEB1 levels.

Vitamin K-dependent gamma-carboxyglutamic acid protein 1 promotes pancreatic ductal adenocarcinoma progression through stabilizing oncoprotein KRAS and tyrosine kinase receptor EGFR.

Vitamin K-dependent γ-glutamic acid carboxylation (Gla) proteins are calcium-binding and membrane-associated, participating in coagulation, bone turnover, and cancer biology. The molecular function of transmembrane proline-rich Gla proteins (PRRGs) remains unexplored. Analysis of pancreatic ductal adenocarcinoma (PDAC) datasets, including transcription profiles, clinical data, and tissue microarrays, was conducted to evaluate PRRG1 expression and its clinical relevance. PDAC cell lines with overexpressed, knockdown, and mutated PRRG1 were developed to study biological functions and pathways using RNA-seq, co-immunoprecipitation with mass spectrometry, Western blotting, and immunofluorescence. In vivo xenograft and orthotopic models assessed PRRG1's impact on PDAC progression, with and without warfarin treatment. PRRG1 was significantly upregulated in PDAC compared to normal pancreas, correlating with poorer patient survival. PRRG1 knockdown reduced PDAC cell proliferation, anchorage-independent growth in vitro, and tumor growth in vivo. PRRG1 localized at the plasma membrane, interacted with the HECT E3 ligase NEDD4 via the C-terminal PPXY motif, and promoted NEDD4 self-ubiquitination, reducing its protein levels. PRRG1 knockdown elevated NEDD4, destabilizing the oncoprotein KRAS and receptor EGFR, and attenuating downstream signaling and macropinocytosis under nutrient deprivation. The vitamin K-dependent Gla modification of PRRG1 was crucial for its membrane localization and pro-tumorigenic effects, and was inhibited by low-dose warfarin, a clinical vitamin K antagonist. This study identifies PRRG1 as a key regulator of pro-tumorigenic signaling in PDAC, suggesting the potential of repurposing the anticoagulant warfarin as a therapeutic strategy. PRRG1 is identified as the transmembrane Gla protein mediating PDAC malignancy. PRRG1 recruits and induces self-ubiquitination of membrane-anchoring E3 ligase NEDD4. PRRG1 exerts a protective role toward KRAS and EGFR by inhibiting NEDD4. The anticoagulant warfarin can be utilized to inhibit PRRG1 and PDAC advancement.

N-glycosylation of ACTRIIB enhances protein stability leading to rapid cell proliferation and strong resistance to docetaxel in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a malignant tumor predominantly influenced by Epstein-Barr virus infection and genetic factors. The transforming growth factor-beta (TGF-β) superfamily is implicated in various cellular processes, including tumorigenesis. This study aimed to detect the role of one TGF-β superfamily member activin receptor type IIB (ACTRIIB) in NPC. This study analyzed NPC datasets, including GSE12452, GSE102349, and GSE53819. ACTRIIB expression and N-glycosylation levels were assessed by western blot, real-time PCR, immunofluorescence, and immunohistochemistry in NPC cells and tissues. As indicated by the datasets, ACTRIIB was significantly upregulated in NPC tissues, and the up-regulation was associated with poor prognosis. This study confirmed the N-glycosylation of ACTRIIB primarily at the forty-second amino acid, an asparagine. The N-glycosylation of ACTRIIB promoted the localization of ACTRIIB to the cell membrane and prevented the degradation of the protein by lysosomes, through which ACTRIIB activated the downstream Smard1/2 to promote tumor cell proliferation and invasion. Inhibition of N-glycosylation or knockdown of ACTRIIB resulted in reduced cell proliferation and invasion and increased the cell sensitivity to docetaxel. In conclusion, N-glycosylation of ACTRIIB was a critical post-translational modification that enhanced protein stability and induced membrane localization, which facilitates the functions of ACTRIIB in cell proliferation and invasion in NPC. Inhibition of ACTRIIB N-glycosylation could potentially serve as a therapeutic strategy to improve the efficacy of chemotherapy in NPC.

Synergism of Fusobacterium periodonticum and N-nitrosamines promote the formation of EMT subtypes in ESCC by modulating Wnt3a palmitoylation

ABSTRACT N-Nitrosamine disinfection by-products (NAs-DBPs) have been well proven for its role in esophageal carcinogenesis. However, the role of intratumoral microorganisms in esophageal squamous cell carcinoma (ESCC) has not yet been well explored in the context of exposure to NAs-DBPs. Here, the multi-omics integration reveals F. periodonticum (Fp) as “facilitators” is highly enriched in cancer tissues and promotes the epithelial mesenchymal transition (EMT)-like subtype formation of ESCC. We demonstrate that Fp potently drives de novo synthesis of fatty acids, migration, invasion and EMT phenotype through its unique FadAL adhesin. However, N-nitrosomethylbenzylamine upregulates the transcription level of FadAL. Mechanistically, co-immunoprecipitation coupled to mass spectrometry shows that FadAL interacts with FLOT1. Furthermore, FLOT1 activates PI3K-AKT/FASN signaling pathway, leading to triglyceride and palmitic acid (PA) accumulation. Innovatively, the results from the acyl-biotin exchange demonstrate that FadAL-mediated PA accumulation enhances Wnt3A palmitoylation on a conserved cysteine residue, Cys-77, and promotes Wnt3A membrane localization and the translocation of β-catenin into the nucleus, further activating Wnt3A/β-catenin axis and inducing EMT phenotype. We therefore propose a “microbiota-cancer cell subpopulation” interaction model in the highly heterogeneous tumor microenvironment. This study unveils a mechanism by which Fp can drive ESCC and identifies FadAL as a potential diagnostic and therapeutic target for ESCC.

Sigma 1 Receptor and Its Pivotal Role in Neurological Disorders.

Sigma 1 receptor (S1R) is a multifunctional, ligand-activated protein located in the membranes of the endoplasmic reticulum (ER). It mediates a variety of neurological disorders, including epilepsy, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease. The wide neuroprotective effects of S1R agonists are achieved by a variety of pro-survival and antiapoptotic S1R-mediated signaling functions. Nonetheless, relatively little is known about the specific molecular mechanisms underlying S1R activity. Many studies on S1R protein have highlighted the importance of maintaining normal cellular homeostasis through its control of calcium and lipid exchange between the ER and mitochondria, ER-stress response, and many other mechanisms. In this review, we will discuss S1R different cellular localization and explain S1R-associated biological activity, such as its localization in the ER-plasma membrane and Mitochondrion-Associated ER Membrane interfaces. While outlining the cellular mechanisms and important binding partners involved in these processes, we also explained how the dysregulation of these pathways contributes to neurodegenerative disorders.

Optimizing encephalomyocarditis virus VP1 protein assembly on pseudorabies virus envelope via US9 protein anchoring

ABSTRACT Live herpesvirus-vectored vaccines are critical in veterinary medicine, but they can sometimes offer insufficient protection due to suboptimal antigen expression or localization. Encephalomyocarditis virus (EMCV) is a significant zoonotic threat, with VP1 protein as a key immunogen on its capsid. To enhance immunogenicity, we explored the use of recombinant pseudorabies virus (rPRV) as a vaccine vector against EMCV. In silico analysis indicated that fusing VP1 with US9 enhances the formation of a type II transmembrane heterodimer. We constructed six rPRV groups expressing different VP1 variants and found that VP1 fused with US9’s C-terminal (US9-VP1) enhances VP1’s membrane localization and its incorporation into the PRV envelope, unlike wild-type VP1. Immunogold electron microscopy illustrated that rPRV with deleted US8 and US9, supplemented with US8 regulatory sequence (rΔ89-U9VP1), improved VP1 incorporation into the viral envelope. Post-immunization, only rΔ89-U9VP1 provided 100% protection against EMCV in mice and induced high levels of virus-neutralizing antibodies in piglets. Additionally, rPRV expressing VP1 stimulated robust T-cell responses, as demonstrated by flow cytometry and ELISpot assays. This study introduces rPRV as a potential EMCV vaccine, demonstrating that the selection of the US9 C-terminal domain and US8 regulatory sequence significantly enhances the presentation of heterologous antigens, improving vaccine efficacy.

Two Novel Mouse Models of Duchenne Muscular Dystrophy with Similar Dmd Exon 51 Frameshift Mutations and Varied Phenotype Severity

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on alternative splicing and mutant exon skipping. Testing of such therapy requires animal models that carry mutations homologous to those found in human patients. Here, we report the generation of two genetically modified mouse lines, named “insT” and “insG”, with distinct mutations at the same position in exon 51 that lead to a frameshift, presumably causing protein truncation. Hemizygous males of both lines exhibit classical signs of muscular dystrophy in all muscle tissues except for the cardiac tissue. However, pathological changes are more pronounced in one of the lines. Membrane localization of the protein is reduced to the point of absence in one of the lines. Moreover, an increase in full-length isoform mRNA was detected in diaphragms of insG line mice. Although further work is needed to qualify these mutations as sole origins of dissimilarity, both genetically modified mouse lines are suitable models of DMD and can be used to test gene therapy based on alternative splicing.

ST8SIA6 Sialylates CD24 to Enhance Its Membrane Localization in BRCA.

CD24, a highly sialylated glycosyl-phosphatidyl-inositol (GPI) cell surface protein that interacts with sialic acid-binding immunoglobulin-like lectins (Siglecs), serves as an innate immune checkpoint and plays a crucial role in inflammatory diseases and tumor progression. Recently, cytoplasmic CD24 has been observed in samples from patients with cancer. However, whether sialylation governs the subcellular localization of CD24 in cancer remains unclear, and the impact of CD24 expression and localization on the clinical prognosis of cancer remains controversial. Here, we performed a systematic pan-cancer analysis of the gene expression levels and clinical correlation of CD24. Our analysis revealed that CD24 was highly expressed in breast tumor tissues and tumor cells, significantly shortening patient survival time. However, this correlation was not evident in other types of cancer. Additionally, a correlation analysis of CD24 levels with sialyltransferases (STs) revealed that ST8SIA6 is the key ST affecting CD24 sialylation. Further investigation demonstrated that ST8SIA6 directly modified CD24, promoting its localization to the cell membrane. Taken together, these findings elucidate, for the first time, the mechanisms by which ST8SIA6 regulates CD24 subcellular localization, providing new insights into the biological functions and applications of CD24.