Search Results (156)

Orthoebolavirus causes severe Ebola virus disease (EVD) and deadly outbreaks in humans. This infection occurs through macropinocytosis and trafficking to late endosomes or lysosomes that utilize the receptor Niemann–Pick C1 (NPC1) to enter the cell cytoplasm. We designed peptide inhibitors based on the NPC1 receptor to target the NPC1 binding site to block viral entry. The results indicated that the ligand-based peptide inhibitors showed potent inhibition activities in vitro studies against pseudotyped or replication-competent Orthoebolavirus. Therefore, we further evaluated one of them in a mouse model challenged with mice-adapted Ebola viruses, which showed some protection efficacy compared with the control group. This study suggests that ligand-based peptides are encouraging inhibitors in the development of inhibitors against Ebola virus infection. Full article

Annexin A6 (AnxA6) is a predominantly intracellular calcium-dependent membrane-binding multifunctional protein that is also detected extracellularly and in small extracellular vesicles (exosomes). We previously demonstrated that lapatinib resistance in triple-negative breast cancer (TNBC) cells is associated with AnxA6 upregulation and accumulation of cholesterol in late endosomes. Here, we investigated the fate of AnxA6 and cholesterol in lapatinib-resistant (LAP-R) cells and whether extracellular AnxA6 influences TNBC cell survival. We demonstrate that reduced expression of AnxA6 in LAP-R cells decreased the secretion of MCP-1/CCL2, CCL8/IL-8, DKK1, TSP-1, and OPN by antibody arrays. The secretion of exosomes was also markedly reduced in AnxA6-depleted LAP-R cells, while AnxA6 upregulation stimulated the release of MCP-1 and exosomes. Compared to the respective controls, exosome-associated AnxA6, Rab7, and cholesterol levels were increased in exosomes isolated from AnxA6-expressing LAP-R cells. Mechanistically, we demonstrated by co-immunoprecipitation, GST pulldown, and proximity ligation assays that AnxA6 interacts with SNAP23, a component of the membrane fusion machinery. Finally, blocking extracellular AnxA6 with neutralizing antibodies reduced the viability of AnxA6-low TNBC cells but had little effect on AnxA6-high cells. These findings suggest that extracellular AnxA6 is critical for the survival of highly proliferative AnxA6-low basal-like breast cancer cells and that AnxA6 influences TNBC progression by facilitating the secretion of pro-inflammatory cytokines and cholesterol-enriched exosomes. Full article

Bluetongue virus (BTV), the prototype of the genus Orbivirus, infects livestock, causing high morbidity and mortality and impacting global trade. BTV is a non-enveloped, double-capsid virus, composed of seven structural proteins and a genome of 10 double-stranded RNA segments. This manuscript highlights our group’s recent findings on the molecular and structural mechanisms underlying BTV entry and assembly during replication. Viral entry is a stepwise, pH-dependent process. The outermost protein, VP2, attaches to sialic acids and senses the acidic pH of early endosomes, triggering their dissociation. Subsequently, the second outer capsid protein, VP5, undergoes major changes in late endosomes, forming a membrane-penetrating pore that releases the transcriptionally active inner core into the host cytoplasm. Core assembly also proceeds stepwise and requires the accurate packaging of 10 positive-sense RNA segments. These segments form an RNA–RNA interaction network independent of viral proteins, beginning with the smaller segments and guiding the complete genome assortment. The small capsid protein, VP6, interacts with VP3 to facilitate RNA encapsidation. While infectious cores assemble in vitro without non-structural proteins, NS2 is essential for the in vivo formation of viral inclusion bodies via liquid–liquid phase separation, concentrating viral components and promoting genome assembly. These comprehensive characterizations of BTV provide a foundation for future control strategies against related reoviruses. Full article

While current influenza vaccines often lack broad protection against antigenically drifted strains, some modified hemagglutinin (HA) protein antigens have shown promise in eliciting broadly neutralizing antibodies against conserved epitopes. During infection, the mildly acidic environment of the late endosome triggers irreversible HA conformational changes resulting in a post-fusion structure with altered antigenicity. While enhancing the stability of other structural class I viral fusion protein antigens has been instrumental in improving the effectiveness of COVID-19 and RSV vaccines, the role of HA stability in influenza vaccine immunogenicity is relatively unclear. Here, we used the nucleoside-modified mRNA-LNP platform to test engineered HA antigens with specific acid-stabilizing mutations (E47K, K58I, R106K, and K153E) in the HA stalk. All mutations increased HA acid stability, but E47K and R106K did not increase immunogenicity. K153E and K58I, but not E47K and R106K, enhanced the cell-surface expression of the HA protein in vitro. In mice, K153E- and K58I-containing mRNA-LNP vaccines elicited increased neutralizing antibody titers against homologous virus. K153E conferred greater protection than wild-type vaccine against lethal heterologous A/PR/8/34 challenge at low doses (0.5–1.0 µg), despite the absence of neutralizing antibodies against the challenge strain. K153E also elicited greater expansion of antigen-specific antibody-secreting cells (ASCs) in the bone marrow, as well as cross-reactive T follicular helper (Tfh) cells in the spleen. For the vaccines studied, increased HA expression was a stronger correlate of mRNA-LNP enhancement than increased HA stability. Full article

Toxin A and B from Clostridioides difficile are the main pathogenicity factors for clinical symptoms of C. difficile infections. Receptor-mediated endocytosis and endosomal escape are required for targeting substrate proteins of the Rho-GTPase family. We previously reported that Toxin B (TcdB) affects endo-lysosomal transport and autophagic flux of target cells. These effects are independent from pathogenic Rho inhibition. Here, we aimed at further characterization of this event by immunofluorescent characterization of the vesicular structures that are affected. We found large aggregates of damaged endolysosomal structures positive for EEA1, LAMP1, CHMP4B and TcdB, as well as an increase in perinuclear concentration of non-mature autophagosomes (amphisomes) positive for SQSTM, Rab7, and LC3B. We investigated whether Rab7, a regulator of late endosome transport, is causative for decreased lysosome function. Although TcdB induced an increase in active Rab7, as tested by an RILP pull-down assay, inhibition of Rab7 did not prevent TcdB-induced decrease in cathepsin D as a surrogate for lysosome dysfunction. It also indicates that the observed increase in Rab7 positive amphisomes is secondary to lysosomal dysfunction. By applying an autoproteolytic deficient mutant of TcdB we proved that the release of the glucosyltransferase domain is mandatory for triggering all of these effects. This suggests that after membrane perforation the toxin remnants leave an open leak in endolysosomes affecting ion homeostasis. Investigation of all large clostridial glucosyltransferases and other toxins revealed lysosomal dysfunction as a general effect of many but not of all toxins that integrate into the endosome membrane. Full article

Background: Intracellular miRNA transfer is an intriguing and lesser-described mode of intracellular communication. Epithelial ovarian carcinoma, of which the high-grade serous histotype represents the most common and deadliest form, is characterized by a microenvironment consisting of tumor and stromal cells, ascitic fluid, and extracellular matrix, presenting a rich milieu of factors that can affect neighboring cells. Methods: We examined the mode of miR transfer in serous ovarian carcinoma cell lines cultured on different extracellular matrix supports both in two-dimensional and three-dimensional formats coupled with traditional, live-cell time-lapse, multiphoton fluorescence imaging modalities, and fluorescence-activated cell sorting approaches. Results: Our data demonstrate that miR can transfer between cells both in culture and in vivo. Moreover, transferred miRNA results in target-specific gene expression changes in recipient cells. Our data indicate that miR transfer occurs via extracellular vesicles, which shuttle from and within the donor and recipient cells via endocytic pathways recruiting sorting, early, late, and recycling endosomes. Conclusions: Our study highlights the phenomenon of miR transfer as a mode of communication between serous ovarian cancer cells, which can affect both treatment and diagnostics of this disease. Full article

Dysfunction of the membrane transporter P5B-ATPase 13A2 (ATP13A2) has been linked to neurodegenerative disorders, while its overexpression has been associated with colorectal cancer. ATP13A2 localizes to lysosomes and late endosomes, where it exports polyamines such as spermine into the cytosol. We previously showed that ATP13A2 expression alters lipid homeostasis and reduces the levels of bis(monoacylglycero)phosphate (BMP), an anionic phospholipid essential for lipid digestion in acidic compartments, suggesting that ATP13A2-mediated spermine export may affect lysosomal lipid catabolism. α/β-hydrolase domain-containing 6 (ABHD6), the enzyme responsible for BMP catabolism, was detected by immunofluorescence and immunoblot analysis in SH-SY5Y cells overexpressing human ATP13A2 and treated with spermine. The activities of the lipid-degrading hydrolases acid ceramidase (ACase) and glucocerebrosidase (GCase) were measured using specific fluorogenic substrates. ATP13A2-expressing cells showed higher ABHD6 expression, and spermine treatment promoted its translocation to the cytoplasm. Spermine induced a transient increase in ACase activity, followed by a stronger inhibition in ATP13A2-expressing cells. Moreover, GCase activity was elevated in these cells but also showed greater spermine-induced inhibition. Altogether, these results suggest that ATP13A2-mediated spermine export modulates the lipid digestion capacity of the endolysosomal system and support a functional interplay between polyamine and lipid metabolism in these organelles. Full article

ADP-ribosylation factor (Arf) proteins are small GTPases that regulate intracellular membrane trafficking and actin remodeling through tightly controlled cycles of GTP binding and hydrolysis. Arf1, a central coordinator of Golgi and endosomal transport, and Arf6, which regulates plasma membranes and endosomal dynamics, have both been implicated in late stages of the HIV-1 life cycle. However, the mechanisms by which these GTPases support viral assembly and release remain incompletely defined. Here, we provide direct evidence that both Arf1 and Arf6 are required for efficient trafficking of the HIV-1 Gag polyprotein, assembly, and virion production. Perturbation of Arf1 function using either GTP-locked (Q71L) or GDP-locked (T31N) mutants significantly reduced virus release, impaired Gag association with membrane compartments, and prevented its accumulation at the plasma membrane. Manipulation of Arf1 cycling through the GTPase-activating protein AGAP1 further demonstrated that dynamic transitions between GTP- and GDP-bound states are essential for productive Gag trafficking. Similarly, expression of a constitutively active Arf6 mutant (Q67L) misrouted Gag to intracellular membranes and markedly suppressed virion release. Importantly, disruption of Arf1 or Arf6 activity did not affect the expression, surface levels, or intracellular distribution of the host restriction factor BST-2. Together, these findings identify Arf1- and Arf6-mediated trafficking pathways as critical host determinants of HIV-1 assembly and release and establish that their functions operate independently of BST-2 antagonism. Full article

Lipid nanoparticles (LNPs) have received significant attention as effective RNA carriers in RNA-based therapeutics and vaccines. Particularly, ionizable lipids (ILs) of LNPs play a crucial role in endosomal escape and lipid-mediated RNA delivery owing to their pH-dependent molecular characteristics. Therefore, it is essential to enhance the endosomal escape efficiency of ILs, which is primary bottleneck in the successful cytoplasmic delivery of RNA. However, the molecular-level understanding of the roles and dynamics of ILs during the endosomal escape stage remains unclear. To elucidate this, we utilized coarse-grained (CG) molecular dynamics (MD) simulations. In this simulation, we designed lipid nanodroplets (LNDs) containing D-Lin-MC3-DMA (MC3) and ALC-0315, which have proven effective as LNPs in RNA-based therapeutics and vaccines, respectively, while accounting for the pH environments of early and late endosomes. Also, we formulated lipid bilayers reflecting the composition of early and late endosomal membranes to investigate the fusion process between LNDs and endosomal membranes. Our findings reveal that, irrespective of endosomal membrane composition and LNP types, ILs are the first lipids to enter the endosomal membrane during the fusion, and the flip-flop process of ILs from the inner leaflet to the outer leaflet of the endosomal membrane is a critical step for LNP endosomal escape. More specifically, we observed that protonated ILs predominantly participate in the flip-flop process, while many deprotonated ILs remain clustered and disordered within the intermediate layer of the endosomal membrane. Furthermore, we found that the extent of IL flip-flop varies with the cholesterol content of the endosomal membrane. Additionally, under identical pH conditions, MC3-containing LNDs exhibited a more active IL flip-flop process toward the outer leaflet than ALC-0315-containing LNDs. This observation supports experimental findings that MC3-containing LNPs manifest higher endosomal escape efficiency than ALC-0315-containing LNPs in mRNA delivery studies. The mechanistic insights into the endosomal escape mechanism demonstrated by our simulations could aid in the development of effective ILs. Full article

Background/Objectives: Bio-produced gold nanoparticles (AuNPs) are effective carriers of short RNAs into specialized mammalian cells. Their potential application is still limited by scarce knowledge on their uptake and intracellular fate. Gold nanoparticles that are not biologically produced (NB-AuNPs) enter specialized cells primarily via clathrin-dependent endocytosis. Unlike the NB-AuNPs, the bio AuNPs possess natural surface coatings that significantly alter the AuNPs properties. Our research aimed to reveal the cellular uptake of the AuNPs with respect to delivering a functional RNA cargo. Methods: The AuNPs were conjugated with short inhibitory RNA specific to miR 135b. Mammary cancer cells 4T1 were pretreated with inhibitors of caveolin- and clathrin-mediated endocytosis and macropinocytosis. AuNPs’ uptake, fate, and miR 135b knock-down were assessed with TEM and qPCR. Results: The AuNPs-antimiR 135b conjugates entered 4T1 cells via all the tested pathways and could be seen inside the cells in early and late endosomes as well as cytoplasm. In contrast to the clathrin-dependent pathway, the caveolae-mediated endocytosis and the macropinocytosis of the AuNPs resulted in the effective targeting and reduction of the miR 135b. Conclusions: The bio-produced AuNPs can effectively enter mammalian cells simultaneously by different endocytic pathways but the delivery of functional cargo is not achieved via the clathrin-dependent endocytosis. Full article

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies due to late diagnosis, poor drug penetration, and intrinsic chemoresistance. Targeted delivery strategies are urgently needed to enhance therapeutic precision while minimizing systemic toxicity. Here, we developed an AS1411 aptamer-functionalized liposomal platform encapsulating siRNA against metastasis-associated protein 2 (MTA2), a chromatin remodeling factor that suppresses the tumor suppressor PTEN and activates PI3K/AKT signaling. The AS1411 aptamer, which binds nucleolin overexpressed on PDAC cells, was conjugated to cationic liposomes via copper-free click chemistry. The resulting AS1411-Lipm[siRNA] exhibited high siRNA encapsulation efficiency, selective uptake by nucleolin-positive PDAC cells, and enhanced endosomal escape. Treatment of MIA PaCa-2 cells with AS1411-Lipm[siRNA] significantly reduced MTA2 expression by ~60%, substantially restored PTEN, and inhibited AKT phosphorylation by ~50%, leading to decreased cell viability, impaired migration by ~75%, and increased apoptosis by ~35%, while sparing nucleolin-negative cells. These findings highlight AS1411-Lipm[siRNA] as a promising platform for selective siRNA delivery and potent molecular inhibition in PDAC therapy. Full article

In eukaryotic cells, vesicle-mediated transport interconnects the endomembrane system. These vesicles are formed by coat proteins via deformation of donor membranes. Here, we constructed a set of fluorescent protein-based markers for major coat protein complexes in the yeast model system, and examined their subcellular localization patterns. Our markers covered COPII, COPI, AP-1, AP-2, AP-3, and retromer complexes. Our live cell imaging demonstrates that COPII puncta were primarily associated with the endoplasmic reticulum (ER), and occasionally with early Golgi. COPI was present on both early Golgi and late Golgi/early endosomes. AP-1 puncta were present on late Golgi/early endosomes. AP-2 was present on plasma membrane (PM)-associated puncta, and around the bud neck. AP-3 puncta were present on late Golgi/early endosomes and on the surface of vacuoles. Retromer was present on the surface of vacuoles, late endosomes, and other perivacuolar puncta. Notably, more than half of AP-1 puncta and AP-3 puncta were not associated with the donor compartments where they are thought to be generated, implying that these were coated transport vesicles. This work provides a convenient tool set for the investigation of vesicular transport in yeast and live cell imaging evidence for the presence of certain coated transport vesicles. Full article

Background/Objectives: After many years of research and the successful development of therapeutic products by a few industrial actors, the COVID-19 vaccines brought messenger RNAs, as well as other nucleic acid modalities, such as antisense oligonucleotides, small interfering RNA, and aptamers, into the spotlight, eliciting renewed interest from both academia and industry. However, owing to their structure, relative “fragility”, and the (usually) intracellular site of action, the delivery of these therapeutics has frequently proven to be a key limitation, especially when considering endosomal escape, which still needs to be overcome. Methods: By compiling delivery-related data on approved and late clinical-phase ribonucleic acid therapeutics, this review aims to assess the delivery strategies that have proven to be successful or are emerging, as well as areas where more research is needed. Results: In very specific cases, some strategies appeared to be quite effective, such as the N-acetylgalactosamine moiety in the case of liver delivery. Surprisingly, it also appears that for some modalities, efforts in molecular design have led to more “drug-like” properties, enablingthe administration of naked nucleic acids, without any form of encapsulation. This appears to be especially true when local administration, i.e., by injection, is possible, as this provides de facto targeting and a high local concentration, which can compensate for the small proportion of nucleic acids that reach the cytoplasm. Conclusions: Nucleic acid-based therapeutics have come a long way in terms of their physicochemical properties. However, due to their inherent limitations, targeting appears to be crucial for their efficacy, even more so than for traditional pharmaceutical modalities. Full article

Chronic pain affects a significant portion of the population, with fewer than 30% achieving adequate relief from existing treatments. This study describes the humanization methodology and characterization of an effective non-opioid single-chain fragment variable (scFv) biologic that reverses pain-related behaviors, in this case by targeting P2X4. After nerve injury, ATP release activates/upregulates P2X4 receptors (P2X4R) sequestered in late endosomes, triggering a cascade of chronic pain-related events. Nine humanized scFv (hscFv) variants targeting a specific extracellular 13-amino-acid peptide fragment of human P2X4R were generated via CDR grafting. ELISA analysis revealed nanomolar binding affinities, with most humanized molecules exhibiting comparable or superior affinity compared to the original murine antibody. Octet measurements confirmed that the lead, HC3-LC3, exhibited nanomolar binding kinetics (KD = 2.5 × 10⁻⁹ M). In vivo functional validation with P2X4R hscFv reversed nerve injury-induced chronic pain-related behaviors with a single dose (0.4 mg/kg, intraperitoneal) within two weeks. The return to naïve baseline remained durably reduced > 100 days. In independent confirmation, the spared nerve injury (SNI) model was similarly reduced. This constitutes an original method whereby durable reversals of chronic nerve injury pain, anxiety and depression measures are accomplished. Full article

The open reading frame 3a (ORF3a) is a protein important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (160YNSV163, 211YYQL213, and 233YNKI236), and a previous study has indicated that mutation of the 160YNSV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. Here, we have systematically removed all three tyrosine-based motifs and assessed the importance of each motif or combination of motifs in trafficking to the cell surface. Our results indicate that the 160YNSV163 motif alone was insufficient for ORF3a cell-surface trafficking, while the 211YYQL213 motif was the most important. Additionally, an ORF3a with all three YxxΦ motifs disrupted (ORF3a-[ΔYxxΦ]) was not transported to the cell surface, and LysoIP studies indicate that ORF3a but not ORF3a-[ΔYxxΦ] was present in late endosome/lysosome fractions. A growth-curve analysis of different SARS-CoV-2 viruses expressing the different mutant ORF3a proteins revealed no significant differences in virus replication. Finally, the inoculation of K18hACE-2 mice indicated that the SARS-CoV-2 lacking the three YxxΦ motifs was less pathogenic than the unmodified SARS-CoV-2. These results indicate that the tyrosine motifs of ORF3a contribute to cell-surface expression and SARS-CoV-2 pathogenesis Full article