Search Results (157)

Herpes simplex virus type 1 (HSV-1) has a global prevalence of 64%. Established antiviral drugs, such as acyclovir (ACV), have been successfully used over the past decades. However, due to growing viral resistance against approved antivirals and the lack of effective vaccines, new concepts are essential to target HSV-1 infections. Here, we present data on the inhibitory effect of the procaine-based substance ProcCluster^® (PC) in reducing HSV-1 replication in vitro. Non-toxic PC concentrations significantly decreased HSV-1 replication in infected cells. Immunofluorescence microscopy revealed an accumulation of viral proteins in early and recycling endosomes, resulting in reduced viral release. The combination of PC with ACV resulted in an enhanced antiviral effect. Based on these results, PC alone, as well as in combination with ACV, appears to be a promising substance with antiviral potential against HSV-1 infections. Full article

VE-cadherin (VE-cad) membrane stability and localization regulates adhesion formation and actin cytoskeleton dynamics in angiogenesis and vascular remodeling and requires the heparan sulfate proteoglycan (HSPG), Syndecan-4 (Sdc4). This study characterizes the interactions of the heparin receptor, Transmembrane protein-184A (TMEM184A), and Sdc4 in bovine aortic endothelial cells (BAOECs) and the regenerating Zebrafish (ZF) caudal fin and measures the effect of siRNA TMEM184A KD (siTMEM) and TMEM184A overexpression (TMEM OE) on VE-cad levels and localization in confluent and sub-confluent cultured BAOECs. Additionally, we examined the effect of siTMEM on key Rab GTPase trafficking regulators and migrating BAOECs in scratch wound healing assays. We demonstrated that TMEM184A and Sdc4 colocalize in BAOECs and that Sdc4 OE increases colocalization in an HS chain dependent manner, while both Tmem184a and Sdc4 cooperate synergistically in ZF fin angiogenic and tissue repair. We also showed that siTMEM decreases VE-cad membrane and cytoplasmic levels, while increasing scratch wound migration rates. However, TMEM OE cells show increased vesicle formation and VE-cad trafficking and membrane recovery. These findings characterize TMEM184A-Sdc4 cooperation in angiogenesis and indicate a dual function of TMEM184A in signaling and trafficking in vascular cells that promotes VE-cad recovery and membrane localization. Full article

The Rab family of small guanosine triphosphatases (GTPases) are nucleotide-dependent switches. Mutations in Rabs can result in human diseases. Rab7a and Rab7b transition from early endosomes to lysosomes and are presumed to function similarly. Most studies look at Rab7a, less on Rab7b, with the underlying assumption they function similarly. There have yet to be articles comparing them side by side. Whilst cloning Rab7 homologues, we identified splice isoforms for Rab7b only. These splice isoforms, Rab7b2 and Rab7bx8 lacking different exons, have not been previously characterized but suggest alternative function(s) for Rab7b. Thus, we hypothesize that Rab7 homologues have distinct functions. Here, we compare Rab7a and Rab7b nucleotide mutants locked in GDP-bound (Rab7T22N), GTP-bound (Rab7Q67L), nucleotide-free (Rab7aN125I/Rab7bN124I) states and characterized localization of the Rab7b splice isoforms. HeLa cells were transiently transfected with fluorescently tagged Rab7 reporters. Confocal images were processed with ImageJ and analyzed with SPSS. Rab7a and Rab7b nucleotide mutants were significantly different to one another. Approximately 50% of Rab7b splice isoform-expressing cells had aggregated vesicles, which were phenotypically different from Rab7b vesicles. Rab7a and Rab7b vesicles shared approximately 60% colocalization with each other, while Rab7b vesicles preferentially localized to the Trans Golgi Network. Our results suggest Rab7b is distinct from Rab7a, and Rab7b splice isoforms have different biological functions. Full article

Plant exosomes exhibit high stability and easy absorption, and have emerged as promising bioactive tools due to their potential health benefits and biomedical applications. Saffron tepals contain abundant metabolites with potential therapeutic properties and were used for exosome extraction by ultracentrifugation and gradient purification. The exosomes showed an average particle size of 151.5 ± 79.6 nm and exhibited a spherical morphology. Five well-conserved miRNAs—miR157, miR166, miR168, miR396, and miR398—were identified in the exosomes, which are involved in the coordination of growth and physiological plant responses with endogenous and environmental abiotic and biotic signals, and their potential targets in mammals are upregulated in specific cancer types and associated with inflammation. Proteome analysis revealed an enrichment of proteasome proteins, ribosomal proteins, and proteins involved in the cytoskeleton, transport across the membrane (ABC transporters), and vesicle trafficking (RAB GTPases, TM9SF and Coatomer subunits). Metabolite analyses showed mainly anthocyanins. The exosomes have selective stimulatory activity on macrophages, increasing the expression of surface molecules (CD80 and CD86), and cytokines (IL-1β, IL-6, and TNF-α), but not the levels of IL-10. Overall, these results indicated that saffron flowers are an effective and abundant source of exosomes as new nanomedicines for human health. Full article

Protein phosphorylation is a crucial regulatory mechanism in cellular homeostasis. The human cytomegalovirus (HCMV) incorporates protein phosphatase 1 (PP1) into its tegument, yet the biological relevance and mechanisms of this incorporation remain unclear. Our study offers the first characterization of the PP1 interactome during HCMV infection and its alterations. Using co-immunoprecipitation, mass spectrometry, and quantitative proteomics, we identified 159 high-confidence interacting proteins (HCIPs) in the PP1 interactome, consisting of 126 human and 33 viral proteins. We observed significant temporal changes in the PP1 interactome following HCMV infection, including the altered interactions of PP1 regulatory subunits. Further analysis highlighted the central roles of these PP1 interacting proteins in intracellular trafficking, with particular emphasis on the trafficking protein particle complex and Rab GTPases, which are crucial for the virus’s manipulation of host cellular processes in virion assembly and egress. Additionally, our study on the noncatalytic PP1 inhibitor 1E7-03 revealed a decrease in PP1’s interaction with key HCMV proteins, supporting its potential as an antiviral agent. Our findings suggest that PP1 docking motifs are critical in viral–host interactions and offer new insights for antiviral strategies. Full article

The TRAPP (TRAnsport Protein Particle) protein complex is a multi-subunit complex involved in vesicular transport between intracellular compartments. The TRAPP complex plays an important role in endoplasmic reticulum-to-Golgi and Golgi-to-plasma membrane transport, as well as autophagy. TRAPP complexes comprise a core complex, TRAPPI, and the association of peripheral protein subunits to make two complexes, known as TRAPPII and TRAPPIII, which act as Guanine Nucleotide Exchange Factors (GEFs) of Rab11 and Rab1, respectively. Rab1 and Rab11 are GTPases that mediate cargo selection, packaging, and delivery during pre- and post-Golgi transport in the secretory pathway. Rab1 is also required for the first step of macroautophagy, a cellular recycling pathway. Pathogenic variants in genes encoding protein subunits of the TRAPP complex are associated with a range of rare but severe neurological, skeletal, and muscular disorders, collectively called TRAPPopathies. Disease-causing variants have been identified in multiple subunits of the TRAPP complex; however, little is known about the underlying disease mechanisms. In this review, we will provide an overview of the current knowledge surrounding disease-associated variants of the TRAPP complex subunits, propose new insights into the underlying disease pathology, and suggest future research directions into the underlying disease mechanisms. Full article

RAB11, a pivotal RabGTPase, regulates essential cellular processes such as endocytic recycling, exocytosis, and autophagy. The protein was implicated in various human diseases, including cancer, neurodegenerative disorders, viral infections, and podocytopathies. However, a small-molecular inhibitor is lacking. The complexity and workload associated with potential assays make conducting large-scale screening for RAB11 challenging. We employed a tiered approach for drug discovery, utilizing deep learning-based computational screening to preselect compounds targeting a specific pocket of RAB11 protein with experimental validation by an in vitro platform reflecting RAB11 activity through the exocytosis of GFP. Further validation included the exposure of Drosophila by drug feeding. In silico pre-screening identified 94 candidates, of which 9 were confirmed using our in vitro platform for Rab11 activity. Focusing on compounds with high potency, we assessed autophagy, which independently requires RAB11, and validated three of these compounds. We further analyzed the dose–response relationship, observing a biphasic, potentially hormetic effect. Two candidate compounds specifically caused a shift in Rab11 vesicles to the cell periphery, without significant impact on Rab5 or Rab7. Drosophila larvae exposed to another candidate compound with predicted oral bioavailability exhibited minimal toxicity, subcellular dispersal of endogenous Rab11, and a decrease in RAB11-dependent nephrocyte function, further supporting an inhibitory role. Taken together, the combination of computational screening and experimental validation allowed the identification of small molecules that modify the function of Rab11. This discovery may further open avenues for treating RAB11-associated disorders. Full article

The migratory and invasive capabilities of melanoma cells contribute to metastasis. Therefore, targeting the genes driving these processes can support melanoma therapy. Rab27A and Rab27B contribute to tumor formation progression in many types of cancer through various mechanisms, including the secretion of small extracellular vesicles (sEVs). We explored the role of these GTPases in melanoma cell functioning in three RAB27A knockout (KO) cell lines (A375, DMBC12, and SkMel28) and a double RAB27A/B KO A375 cell line. The loss of RAB27A impaired the migration and invasion of DMBC12 and SkMel28 cells; however, the behavior of highly aggressive A375 cells was unaffected. The RAB27A/B double knockout moderately decreased the migratory capacity of A375 cells without disturbing their invasiveness. Additionally, the silencing of RAB27A did not affect the number and mean size of the sEVs, despite some alterations in the protein content of the vesicles. Both Rab27 isoforms can, at least partially, act independently. The potential role of Rab27A in the functioning of melanoma cells depends on the individual character of the cell line, but not on its basal expression, and seems to be unrelated to the secretion of sEVs. Full article

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune cell-mediated joint inflammation and subsequent osteoclast-dependent bone destruction. Collagen antibody-induced arthritis (CAIA) is a useful mouse model for examining the inflammatory mechanisms in human RA. Previously, we identified the novel gene Rab44, which is a member of the large Rab GTPase family and is highly expressed in immune-related cells and osteoclasts. Methods: In this study, we induced CAIA in Rab44-knockout (KO) mice to investigate the effects of Rab44 on inflammation, cell filtration, and bone destruction. Results: Compared with wild-type (WT) mice, Rab44-KO mice showed reduced inflammation in arthritis under CAIA-inducing conditions. Rab44-KO CAIA mice exhibited reduced cell filtration in the radiocarpal joints. Consistent with these findings, Rab44-KO CAIA mice showed decreased mRNA levels of arthritis-related marker genes including genes for inflammation, cartilage turnover, bone formation, and bone absorption markers. Rab44-KO CAIA mice exhibited predominant infiltration of M2-type macrophages at inflammatory sites and reduced bone loss compared to WT CAIA mice. Conclusions: These results indicate that Rab44 deficiency reduces the progression of inflammation in CAIA in mice. Full article

Osteoclasts are bone-resorbing multinucleated giant cells formed by the fusion of monocyte/macrophage lineages. Various small GTPases are involved in the multinucleation and differentiation of osteoclasts. However, the roles of small GTPases regulatory molecules in osteoclast differentiation remain unclear. In the present study, we examined the role of Dennd2c, a putative guanine nucleotide exchange factor for Rab GTPases, in osteoclast differentiation. Knockdown of Dennd2c promoted osteoclast differentiation, resorption, and expression of osteoclast markers. Morphologically, Dennd2c knockdown induced the formation of larger osteoclasts with several protrusions. In contrast, overexpression of Dennd2c inhibited the multinucleation and differentiation of osteoclasts, bone resorption, and the expression of osteoclast markers. Dennd2c-overexpressing macrophages exhibited spindle-shaped mononuclear cells and long thin protrusions. Treatment of Dennd2c-overexpressing cells with the Cdc42 inhibitor ML-141 or the Rac1 inhibitor 6-thio-GTP prevented protrusion formation. Moreover, treatment of Dennd2c-overexpressing cells with the actin polymerization inhibitor latrunculin B restored multinucleated and TRAP-positive osteoclast formation. These results indicate that Dennd2c negatively regulates osteoclast differentiation and multinucleation by modulating protrusion formation in macrophages. Full article

Francisella is a highly infectious gram-negative bacterium that causes tularemia in humans and animals. It can survive and multiply in a variety of cells, including macrophages, dendritic cells, amoebae, and arthropod-derived cells. However, the intracellular life cycle of a bacterium varies depending on the cell type. Shortly after the infection of mammalian cells, the bacterium escapes the phagosome into the cytosol, where it replicates. In contrast, in the amoebae Acanthamoeba castellanii and Hartmannella vermiformis, the bacterium replicates within the membrane-bound vacuole. In recent years, the amoeba Dictyostelium discoideum has emerged as a powerful model to study the intracellular cycle and virulence of many pathogenic bacteria. In this study, we used D. discoideum as a model for the infection and isolation of Francisella novicida-containing vacuoles (FCVs) formed after bacteria invade the amoeba. Our results showed that F. novicida localized in a vacuole after invading D. discoideum. Here, we developed a method to isolate FCV and determined its composition by proteomic analyses. Proteomic analyses revealed 689 proteins, including 13 small GTPases of the Rab family. This is the first evidence of F. novicida-containing vacuoles within amoeba, and this approach will contribute to our understanding of host–pathogen interactions and the process of pathogen vacuole formation, as vacuoles containing bacteria represent direct contact between pathogens and their hosts. Furthermore, this method can be translocated on other amoeba models. Full article

Glaesserella parasuis cytolethal distending toxin (GpCDT) can induce cell cycle arrest and apoptosis. Our laboratory’s previous work demonstrated that GTPase 4b (Rab4b) is a key host protein implicated in GpCDT-induced cytotoxicity. This study investigated the probable involvement of Rab4b in the process. Our study used CRISPR/Cas9 technology to create a Rab4b-knockout cell line. The results showed greater resistance to GpCDT-induced cell cytotoxicity. In contrast, forced Rab4b overexpression increased GpCDT-induced cytotoxicity. Further immunoprecipitation study reveals that GpCDT may bind with Rab4b. In PK-15 cells, GpCDT is transported to the early endosomes and late endosomes, while after knocking out Rab4b, GpCDT cannot be transported to the early endosome via vesicles. Rab4b appears essential for GpCDT-induced cytotoxicity in PK-15 cells. Full article

The transmembrane protein β-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer’s disease (AD). The β-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic β-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD. Full article

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than 692 million cases worldwide and nearly 7 million deaths (August 2023). Severe COVID-19 is characterised in part by vascular thrombosis and a cytokine storm due to increased plasma concentrations of pro-thrombotic proteins such as von Willebrand factor and cytokines secreted from endothelial and T-cells. EFCAB4B is a gene that encodes for two proteins (CRACR2A and Rab46) that play important roles in endothelial and T-cell secretion. In this study, using patient data recorded in the UK Biobank, we demonstrate the importance of variants in the EFCAB4B genetic sequence with COVID-19 fatality. Using logistic regression analysis, we determined that three single-nucleotide polymorphisms (SNPs) in the gene cause missense variations in CRACR2A and Rab46, which are associated with COVID-19 fatality (rs9788233: p = 0.004, odds ratio = 1.511; rs17836273: p = 0.012, odds ratio = 1.433; rs36030417: p = 0.013, odds ratio = 1.393). All three SNPs cause changes in amino acid residues that are highly conserved across species, indicating their importance in protein structure and function. Two SNPs, rs17836273 (A98T) and rs36030417 (H212Q), cause amino acid substitutions in important functional domains: the EF-hand and coiled-coil domain, respectively. Molecular modelling shows minimal impact by the substitution of threonine at position 98 on the structure of the EF-hand. Since Rab46 is a GTPase that regulates both endothelial cell secretion and T-cell signalling, these missense variants may play a role in the molecular mechanisms underlying the thrombotic and inflammatory characteristics observed in patients with severe COVID-19 outcomes. Full article

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1–400 aa), Linker (246–446 aa) and Adh (444–687 aa) to evaluate their role in virulence. The TrophozBro1_1–400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker_226–446 also increased the virulence properties, but with lower effect than the TrophozBro1_1–400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh_444–687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes. Full article