Search Results (597)

Background: During infection with the cytomegalovirus (CMV), the membrane system of the infected cell is remodelled into a megastructure called the assembly compartment (AC). These extensive changes may involve the manipulation of the host cell proteome by targeting a pleiotropic function of the cell such as ubiquitination (Ub). In this study, we investigate whether the Ub system is required for the establishment and maintenance of the AC in murine CMV (MCMV)-infected cells Methods: NIH3T3 cells were infected with wild-type and recombinant MCMVs and the Ub system was inhibited with PYR-41. The expression of viral and host cell proteins was analyzed by Western blot. AC formation was monitored by immunofluorescence with confocal imaging and long-term live imaging as the dislocation of the Golgi and expansion of Rab10-positive tubular membranes (Rab10 TMs). A cell line with inducible expression of hemagglutinin (HA)-Ub was constructed to monitor ubiquitination. siRNA was used to deplete host cell factors. Infectious virion production was monitored using the plaque assay. Results: The Ub system is required for the establishment of the infection, progression of the replication cycle, viral gene expression and production of infectious virions. The Ub system also regulates the establishment and maintenance of the AC, including the expansion of Rab10 TMs. Increased ubiquitination of WASHC1, which is recruited to the machinery that drives the growth of Rab10 TMs, is consistent with Ub-dependent rheostatic control of membrane tubulation and the continued expansion of Rab10 TMs. Conclusions: The Ub system is intensively utilized at all stages of the MCMV replication cycle, including the reorganization of the membrane system into the AC. Disruption of rheostatic control of the membrane tubulation by ubiquitination and expansion of Rab10 TREs within the AC may contribute to the development of a sufficient amount of tubular membranes for virion envelopment. Full article

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

Purpose: The adenoid microbiota plays a key role in adenoid hypertrophy (AH). This study explored the molecular epidemiology and antimicrobial resistance of Haemophilus. Influenzae (H. influenzae) strains in pediatric AH patients. Methods: Retrospective analysis of pediatric AH patients undergoing endoscopic adenoidectomy. Adenoid tissue samples were cultured to screen for pathogens. H. influenzae strains were identified by 16S rRNA sequencing and serotyped via q-PCR. Multilocus sequence typing (MLST) and ftsI gene analysis were conducted using PubMLST. β-lactamase genes (bla_TEM-1, bla_ROB-1) were detected by PCR, and antibiotic susceptibility testing (AST) was performed using the Etest method. For imipenem-resistant strains, the acrRAB efflux pump gene cluster and ompP2 porin gene were sequenced and compared with those of the wild-type strain Rd KW20. Results: Over 8 months, 56 non-duplicate H. influenzae strains were isolated from 386 patients. The detection rate was highest in children under 5 years (30.5%) compared to those aged 5–10 years (13.4%) and 10–15 years (8.7%). Of 49 sub-cultured strains, all were non-typeable H. influenzae (NTHi). MLST identified 22 sequence types (STs) and 13 clonal complexes (CCs), with CC11 (26.5%), CC3 (14.3%), and CC107 (14.3%) being predominant. Common STs included ST103 (22.4%), ST57 (10.2%), and ST107 (10.2%). Most strains belonged to the ftsI group III-like+ (57.1%). β-lactamase positivity was 98.0% (48/49), with bla_TEM-1 (95.9%) and bla_ROB-1 (18.4%) detected. AST showed low susceptibility to ampicillin (10.2%), amoxicillin–clavulanate (34.7%), azithromycin (12.2%), and trimethoprim–sulfamethoxazole (14.3%). Among the β-lactamase-positive strains, 44/48 were β-lactamase-positive ampicillin-resistant (BLPAR); none were β-lactamase-negative ampicillin-resistant (BLNAR). Imipenem susceptibility was 91.8% (45/49). No carbapenemases were found in the imipenem-resistant strains, but mutations in acrRAB (88.12–94.94% identity) and ompP2 (77.10–82.94% identity) were observed. Conclusions: BLPAR NTHi strains of CC11 are major epidemic strains in pediatric AH. Imipenem resistance in H. influenzae likely results from porin mutations rather than carbapenemase activity. Enhanced surveillance of H. influenzae’s role in AH and its resistance patterns is warranted. Full article

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

The phosphorylation of Rab10 (pT73-Rab10) by LRRK2 promotes the establishment of epithelial cell polarity by controlling the trafficking to the primary cilia membrane of cilia-resident proteins and signaling proteins. Previous studies have identified a site in the globular tail domain of MYO5A that specifically binds to only the phosphorylated form of Rab10. In this work, we have demonstrated that pT73-Rab10 does not associate with the globular tail of MYO5B. We have mapped the putative binding site to a required three amino acids (MEN, 1473–1475) in the MYO5A globular tail domain that are not found in the MYO5B globular tail. Substitution of the MEN amino acid sequence found in MYO5A into the paralogous position in the MYO5B globular tail conferred the ability to associate with pT73-Rab10. The results demonstrate that the interactors with MYO5A and MYO5B are not completely overlapping and that the interaction of pT73-Rab10 is specific to the MYO5A globular tail domain. Full article

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and effective countermeasures remain limited. The EBOV-encoded major matrix protein VP40 is essential for viral assembly, budding, and particle release, making it a promising target for antiviral drug development. However, no approved drugs currently target the viral particle formation process. In this study, we established a simple and highly sensitive screening system to evaluate VP40-mediated virus-like particle (VLP) formation under biosafety level −2 conditions. The system uses the HiBiT luminescence-based reporter fused to VP40, allowing for the detection of VP40 release. Our results demonstrate that the HiBiT sequence fused at the N-terminus [HiBiT-VP40 (N)] retains VP40′s ability to form VLPs, supporting its use as a functional reporter. Furthermore, we validated the system by assessing the role of Rab11-dependent trafficking in VP40-mediated budding and by evaluating the effect of nocodazole, a microtubule depolymerizer, on VLP release. This novel screening system provides a convenient and reliable platform for screening potential inhibitors targeting the late stages of EBOV infection, including viral particle formation and release. Additionally, its potential adaptability to other filoviruses suggests wide applicability in the discovery and development of additional novel therapeutic agents. Full article

This manuscript assesses the critical role of exosomal PD-L1 (ExoPD-L1) in immune suppression, tumor progression, and resistance to therapy. ExoPD-L1 has been identified as a key mediator of tumor immune evasion, contributing to systemic immunosuppression beyond the tumor microenvironment (TME) due to its capacity to travel to distant anatomical sites. In this context, the review aims to elaborate on the mechanisms by which exosomal PD-L1 interacts with T cell receptors and modulates both the tumor microenvironment and immune responses, impacting patient outcomes. We further explore emerging therapeutic strategies that target ExoPD-L1 to enhance the effectiveness of immunotherapy. Blocking ExoPD-L1 offers a novel approach to counteracting immune escape in cancer. Promising strategies include inhibiting exosome biogenesis with GW4869 or Rab inhibitors, neutralizing ExoPD-L1 with targeted antibodies, and silencing PD-L1 expression through RNA interference (RNAi) or CRISPR-based methods. While each approach presents certain limitations, their integration into combination therapies holds significant potential to improve the efficacy of immune checkpoint inhibitors. Future research should focus on optimizing these strategies for clinical application, with particular attention to improving delivery specificity and minimizing off-target effects. Full article

Bone is a preferred site for disseminated tumor cells, yet the molecular mechanisms that prepare the skeletal microenvironment for metastatic colonization are only beginning to be understood. At the heart of this process are extracellular vesicles (EVs), nano-sized, lipid-encapsulated particles secreted by cancer cells and stromal components. This review consolidates current findings that position EVs as key architects of the bone-metastatic niche. We detail the biogenesis of EVs and their organotropic distribution, focusing on how integrin patterns and bone-specific ligands guide vesicle homing to mineralized tissues. We then outline the sequential establishment of the pre-metastatic niche, driven by EV-mediated processes including fibronectin deposition, stromal cell reprogramming, angiogenesis, neurogenesis, metabolic reconfiguration, and immune modulation, specifically, the expansion of myeloid-derived suppressor cells and impaired lymphocyte function. Within the bone microenvironment, tumor-derived EVs carrying microRNAs and proteins shift the balance toward osteoclastogenesis, inhibit osteoblast differentiation, and disrupt osteocyte signaling. These alterations promote osteolytic destruction or aberrant bone formation depending on tumor type. We also highlight cutting-edge imaging modalities and single-EV omics technologies that resolve EV heterogeneity and identify potential biomarkers detectable in plasma and urine. Finally, we explore therapeutic approaches targeting EVs, such as inhibition of nSMase2 or Rab27A, extracorporeal EV clearance, and delivery of engineered, bone-targeted vesicles, while addressing translational challenges and regulatory considerations. This review offers a roadmap for leveraging EV biology in predicting, preventing, and treating skeletal metastases by integrating advances across basic biology, bioengineering, and translational science. Full article

Background: Previous proteomic studies provided valuable information about cataracts, but unclarified issues, such as sex and ethnicity-associated differences, remain. This study aimed to provide additional data on cataract-related proteins regarding age, sex, and cataract type. Methods: Twenty-six female and seven male Turkish cataract patients were screened for visual acuity and dysfunctional lens index. A nano-LC-MS/MS system and Progenesis QI software v3.0 were used for protein identification and quantification. The remaining data were evaluated with SPSS Version 29.0 software. Results: Proteins that showed age-associated changes were mainly involved in cytoskeletal organization. A glyoxalase enzyme, caveolin 1, and HS90B were lower, and RAB8B and ATP6V1B1 were higher in lenses in women. Proteins with lower levels in cataractous lenses than in transparent lenses included filensin and phakinin, concurrent with previous publications, and LCTL, GDI, HSPB1, and EIF4A2, not reported before. Corticonuclear cataracts constituted the only group showing depletions in putatively protective proteins, while the cortical type was the least influenced. ANXA1 and DNHD1 positively, and TCPD, SEC14L2, and PRPS1 proteins negatively correlated with visual acuity. Conclusions: This study revealed cataract-related proteins concurrent with earlier studies and new ones hitherto unreported. Despite the low number of patients investigated, the results merit further research, as these new proteins are highly likely to be involved in cataractogenesis. Full article

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

Advancing age in men significantly contributes to declining sperm fertility. Information on age-related proteomic changes in spermatozoa is limited. This study involved normal fertile Arab men in three age groups: young adult (21–30 years; n = 6), late adult (31–40 years; n = 7), and advanced age (40–51 years; n = 5). Gradient-purified spermatozoa were analyzed using LC-MS/MS and proteomic data were processed using Progenesis QI (QIfp) v3.0 and UniProt/SwissProt. Significantly enriched annotations and clustering of proteins in the proteomic datasets were identified (2-fold change; p < 0.05). A total of 588 proteins were identified, with 93% shared across the three groups. Unique proteins were MYLK4 for the young adult group, PRSS57 for the late adult group, and HMGB4, KRT4, LPGAT1, OXCT2, and MGRN1 for the advanced age group. Furthermore, 261 (44%) proteins were differentially expressed (p < 0.05) across the three groups. Functional enrichment analysis suggested an aging-related significant increase in pathways associated with neurodegenerative diseases and protein folding, alongside decreases in glycolysis/gluconeogenesis, flagellated sperm motility, acetylation, phosphoprotein modifications, oxidation processes, and Ubl conjugation. Cluster analysis highlighted significantly upregulated proteins in young adults (e.g., H2BC1, LAP3, SQLE, LTF, PDIA4, DYNLT2) and late adults (e.g., ATP5F1B, ODF2, TUBA3C, ENO1, SPO11, TEX45, TEKT3), whereas most proteins in the advanced age group exhibited downregulation (e.g., SPESP1, RAB10, SEPTIN4, RAB15, PTPN7, USP5, ANXA1, PRDX1). In conclusion, this study revealed aging-associated proteomic changes in spermatozoa that impact critical processes, including spermatogenesis, motility, metabolism, and fertilization, potentially contributing to fertility decline. These changes provide a molecular framework for developing therapies to preserve sperm proteostasis and enhance fertility in older men. Full article

The unique secondary messenger 2′3′-cGAMP, produced by cGAS in response to cytosolic dsDNA, plays a critical role in activating innate immunity by binding to and activating STING via cell-intrinsic, autocrine, or paracrine mechanisms. Recently, we identified Rab18 as a novel, STING-independent binder of 2′3′-cGAMP. Binding of 2′3′-cGAMP to Rab18 promotes Rab18 activation and induces cell migration. However, the downstream mechanisms by which 2′3′-cGAMP-induced Rab18 activation regulates cell migration remain largely unclear. Herein, using phospho-profiling analysis, we identify MAPK signaling as a key downstream effector of the 2′3′-cGAMP/Rab18 axis that promotes the expression of FosB2 and drives cell migration. Furthermore, we identify MMP3 as a major transcriptional target of FosB2, through which the 2′3′-cGAMP/Rab18/MAPK/FosB2 signaling pathway positively regulates cell migration. Together, our findings provide new mechanistic insights into how 2′3′-cGAMP signaling controls cell migration and suggest the potential of MAPK inhibitors to block 2′3′-cGAMP-induced migratory responses. Full article

Disorders of vesicular trafficking and genetic defects in autophagy play a critical role in the development of metabolic and neurometabolic diseases. These processes govern intracellular transport and lysosomal degradation, thereby maintaining cellular homeostasis. In this article, we present two siblings with a novel homozygous variant in VPS51 (Vacuolar protein sorting 51) gene (c.1511C>T; p.Thr504Met), exhibiting developmental delay, a thin corpus callosum, severe intellectual disability, epilepsy, microcephaly, hearing loss, and dysphagia. This study aimed to investigate the effects of the novel VPS51 gene variation at the RNA and protein level in fibroblasts derived from patients. A comparative proteomic analysis, which has not been previously elucidated, was performed to identify uncharacterized proteins associated with vesicular trafficking. Furthermore, the impact of disrupted pathways on mitochondria–lysosome contact sites was assessed, offering a thorough pathophysiological evaluation of GARP/EARP (Golgi Associated Retrograde Protein / Endosome Associated Retrograde Protein) complex dysfunction. An analysis of mRNA expression indicated decreased levels of the VPS51 gene, alongside modifications in the expression of autophagy-related genes (LC3B, p62, RAB7A, TBC1D15). Western blotting demonstrated a reduction in VPS51 and autophagy-related protein levels. Proteomic profiling revealed 585 differentially expressed proteins, indicating disruptions in vesicular trafficking, lysosomal function, and mitochondrial metabolism. Proteins involved in mitochondrial β-oxidation and oxidative phosphorylation exhibited downregulation, whereas pathways related to glycolysis and lipid synthesis showed upregulation. Live-cell confocal microscopy revealed a notable increase in mitochondria–lysosome contact sites in patient fibroblasts, suggesting that VPS51 protein dysfunction contributes to impaired organelle communication. The findings indicate that the novel VPS51 gene variation influences intracellular transport, autophagy, and metabolic pathways, offering new insights into its involvement in neurometabolic disorders. Full article

Saliva was used as non-invasive alternative to blood for diagnosing pathophysiological conditions. This study aimed to assess changes in protein profile in people with obesity after bariatric surgery and to assess the impact of exercise on these changes. The saliva proteome was determined from two-dimensional gels of twenty adults (ten people with normal weight and ten people with obesity). The effects of bariatric surgery and exercise were assessed. A decrease in body weight, body mass index, and waist-to-height ratio was observed after bariatric surgery. Low levels of carbonic anhydrase VI (CA-VI), short palate, lung, and nasal epithelium clone 2 (SPLUNC2), and haptoglobin were observed. One month after bariatric surgery, spots of haptoglobin and SPLUNC2 increased, although one CA-VI spot decreased. Zn-alpha-2 glycoprotein, immunoglobulin chains, and actin-related protein-3, which are high in people with obesity, decreased 1 month after bariatric surgery. Five months after bariatric surgery, the most significant change was the amylase decrease. The exercise-induced changes in salivary proteins increased SPLUNC, CA-VI, type S cystatins, actin cytoplasmic 1, and zinc alpha-2 glycoprotein levels and decrease Ig kappa chain C region and Rab GDP dissociation inhibitor beta. It can be concluded that the salivary proteins change between people with normal weight vs. patients with obesity, as well as after bariatric surgery and exercise programmes. Salivary proteins may be useful biomarkers in non-invasive samples for monitoring and assessing the impact of interventions on people with obesity. 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