Search Results (21)

Short linear motifs (SLiMs), such as PDZ-binding motifs (PDZbms), are compact interaction modules that mediate transient, specific protein–protein interactions. While PDZbms are well characterized in viral pathogenesis, subverting host protein functions, their role in bacterial systems requires further study. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that mainly infects macrophages. The type VII secretion system (T7SS) of Mtb secretes a subset of effector proteins (Esx) involved in virulence. By using molecular docking and support vector machine-based prediction, we analyzed PDZbm occurrence in T7SS Esx effector proteins and their ability to bind human PDZ domain-containing proteins. We identified PDZbms in most of the Esx proteins studied, with EsxA and EsxG showing the best PDZ-dependent interaction with syntenin-1, a host scaffold protein involved in vesicular trafficking and immune signaling. Additional Esx proteins were predicted to engage other host PDZ proteins. Proteome-wide analysis of Mtb H37Rv revealed that 23.1% of expressed proteins with ≥50 amino acids contained a C-terminal PDZbm. Gene Ontology and Reactome pathway enrichment revealed their involvement in processes related to bacterial and bacterial–host interactions, including redox balance, immunomodulation, and membrane localization, at various stages of infection. Our results support the existence of a PDZbm-mediated interface between Mtb and the human host, extending the PDZbm mimicry hypothesis beyond viruses to bacterial systems as an immune evasion strategy. This work may open multiple research lines focused on experimental validation and the development of a comparative PDZbm catalogue to uncover conserved virulence mechanisms that may guide the design of host-directed therapeutics. Full article

Extracellular vesicles, which carry bioactive cargos such as proteins, RNAs, and lipids, represent promising drug delivery vehicles owing to their biocompatibility, low immunogenicity, and inherent tissue-targeting capabilities. To address the current limitations in controlled cargo loading, we developed an abscisic acid (ABA)-inducible proximity system that directs proteins into exosomes during biogenesis. We engineered exosomal scaffolds by fusing the ABA receptor PYL1 to EV-enriched proteins—including BASP1, CD9, PTGFRN, and a truncated form PTGFRNΔ687—thereby creating docking sites within the exosomal lumen, while the target cargo (e.g., EGFP, firefly luciferase, or Cas9) was tagged with the ABI1 phosphatase domain. We demonstrate that ABA administration in producer cells induces PYL1–ABI1 complex formation, which recruits ABI1-fused cargo for selective encapsulation into EVs. Among the scaffolds tested, BASP1–PYL1 proved the most effective, enabling robust, ABA-dependent enrichment of cargo proteins. Purified EVs maintained canonical morphology, size, and marker expression (CD63, syntenin-1, CD9), confirming preserved biogenesis. Critically, these loaded exosomes efficiently delivered functional cargo to recipient cells, enabling Cas9/sgRNA-mediated genome editing. Together, our findings establish an ABA-triggered molecular switch for controllable EV protein loading, providing a versatile platform for next-generation therapeutic delivery. Full article

The National Institute of Standards and Technology (NIST) is developing analytical methods to characterize extracellular vesicles (EVs) to support the urgent need for standardized EV reference materials (RMs). This study used orthogonal techniques, cryogenic electron microscopy (Cryo-EM), particle tracking analysis (PTA), asymmetrical flow field-flow fractionation (AF⁴), and microfluidic resistive pulse sensing (MRPS), to evaluate particle size distributions (PSDs) and particle number concentrations (PNCs) of human mesenchymal stem cells (MSCs) and LNCaP prostate cancer cell EVs. Proteomic profiles were assessed by mass spectrometry (MS), and microRNA (miRNA) content of LNCaP EVs was evaluated by small RNA-seq at two independent laboratories. A commercial green fluorescent protein exosome served as a control, except in Cryo-EM, proteomic, and miRNA analyses. Cryo-EM, regarded as the gold standard for morphological resolution, served as PSD reference. PSDs from all methods skewed larger than Cryo-EM, with MRPS closest, AF⁴ most divergent, and PTA intermediate with broader distributions. All techniques reported broad PSDs (30 nm to >350 nm) with PNCs decreasing with increasing particle size, except for AF⁴. Quantitative discrepancies in PNCs reached up to two orders of magnitude across methods and cell sources. MS identified global and EV-specific proteins, including syntenin-1 and tetraspanins CD9, CD63, and CD81. RNA-seq revealed notable inter-laboratory variation. These findings highlight the variability across measurement platforms and emphasize the need for reproducible methods to support NIST’s mission of developing reliable EV reference materials. Full article

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

Microglia-mediated chronic neuroinflammation is a common pathological feature of Parkinson’s disease (PD). Strong evidence suggests that activated microglia can lesion neurons by releasing exosomes. However, the mechanisms of exosome release from activated microglia remain unclear. We recently revealed a key role of complement receptor 3 (CR3) in regulating microglial activation in the process of progressive neurodegeneration. This study aimed to investigate whether CR3 can regulate exosome release from activated microglia, as well as the underlying mechanisms. We found that LPS, an inducer of microglial M1 activation, induced exosome release from activated microglia. Inhibition of exosome synthesis suppressed LPS-induced microglial activation, gene expression of proinflammatory factors, and related neurotoxicity. Silencing or knocking out CR3 attenuated LPS-induced exosome release in microglia. NADPH oxidase (NOX2) was further identified as a downstream signal of CR3, mediating microglial exosome release and related neurotoxicity. CR3 silencing blocked LPS-induced NOX2 activation and superoxide production through inhibition of p47^phox phosphorylation and membrane translocation. Moreover, NOX2 activation elicited by PMA or supplementation of H₂O₂ recovered exosome release from CR3-silenced microglia. Subsequently, we demonstrated that the CR3-NOX2 axis regulates syntenin-1 to control microglial exosome release. Finally, we observed that the expression of CR3 was increased in the brain of LPS-treated mice, and genetic ablation of CR3 significantly reduced LPS-induced NOX2 activation, microglial M1 polarization, and exosome production in mice. Overall, our findings revealed a critical role of the CR3-NOX2 axis in controlling microglial exosome release and related neurotoxicity through syntenin-1, providing a novel target for the development of a therapeutic strategy for neuroinflammation-mediated neurodegeneration. Full article

MDA-9/Syntenin, a key scaffolding protein and a molecular hub involved in a diverse range of cell signaling responses, has proved to be a challenging target for the design and discovery of small molecule probes. In this paper, we report on the design and synthesis of small molecule ligands of this key protein. Genetic algorithm-based computational design and the five–eight step synthesis of three molecules led to ligands with affinities in the range of 1–3 µM, a 20–60-fold improvement over literature reports. The design and synthesis strategies, coupled with the structure-dependent gain or loss in affinity, afford the deduction of principles that should guide the design of advanced probes of MDA-9/Syntenin. Full article

Background/Objectives: A key role of extracellular vesicles (EVs) is mediating both cell–cell and cell–stroma communication in pathological/physiological conditions. EVs from resistant tumor cells can transport different molecules like P-glycoprotein (P-gp), acting as a shuttle between donor and recipient cells, resulting in a phenotypic change. The aim of our work was to isolate, characterize, and inhibit the release of EVs in two multidrug resistance (MDR) cancer models: MCF-7R (breast cancer cell line) and HL-60R (acute myeloid leukemia cell line). Methods: The existence of P-gp in EVs from MDR cells was confirmed by Western blotting assays. The characterization of EVs was carried out by evaluating the size using NTA and the presence of specific markers such as CD63, Hsp70 and Syntenin. The ability of HL-60R and MCF-7R to perform horizontal transfer of P-gp via EVs to sensitive cells was assessed using three different methods. The acquisition of resistance and its inhibition in recipient cells was confirmed by MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Results: Our data showed that cell lines (MDR) release P-gp-loaded EVs, unlike sensitive cells. The acquisition of resistance determined by the incorporation of P-gp into the membrane of sensitive cells was confirmed by the reduced cytotoxic activity of doxorubicin. Natural compounds such as curcumin, lupeol, and heptacosane can block vesicular transfer and restore the sensitivity of HL-60 and MCF-7 cells. Conclusions: Our study demonstrates that natural inhibitors able to reverse this mechanism may represent a new therapeutic strategy to limit the propagation of the resistant phenotype. Full article

The interaction between SARS-CoV PDZ-binding motifs (PBMs) and cellular PDZs is responsible for virus virulence. The PBM sequence present in the 3a and envelope (E) proteins of SARS-CoV can potentially bind to over 400 cellular proteins containing PDZ domains. The role of SARS-CoV 3a and E proteins was studied. SARS-CoVs, in which 3a-PBM and E-PMB have been deleted (3a-PBM-/E-PBM-), reduced their titer around one logarithmic unit but still were viable. In addition, the absence of the E-PBM and the replacement of 3a-PBM with that of E did not allow the rescue of SARS-CoV. E protein PBM was necessary for virulence, activating p38-MAPK through the interaction with Syntenin-1 PDZ domain. However, the presence or absence of the homologous motif in the 3a protein, which does not bind to Syntenin-1, did not affect virus pathogenicity. Mutagenesis analysis and in silico modeling were performed to study the extension of the PBM of the SARS-CoV E protein. Alanine and glycine scanning was performed revealing a pair of amino acids necessary for optimum virus replication. The binding of E protein with the PDZ2 domain of the Syntenin-1 homodimer induced conformational changes in both PDZ domains 1 and 2 of the dimer. Full article

Despite the understanding of the coronavirus disease-19 (COVID-19), the role of salivary extracellular vesicles (sEVs) in COVID-19 remains unclear. Exploring the proteomic cargo of sEVs could prove valuable for diagnostic and prognostic purposes in assessing COVID-19. The proteomic cargo of sEVs from COVID-19(+) subjects and their healthy close contacts (HCC) was explored. sEVs were isolated by ultracentrifugation from unstimulated saliva samples, and subsequently characterized through nanoparticle tracking, transmission electron microscopy, and Western blot analyses. The proteomic cargo of sEVs was processed by LC-MS/MS. sEVs were morphologically compatible with EVs, with the presence of Syntenin-1 and CD81 EV markers. The sEV pellet showed 1417 proteins: 1288 in COVID-19(+) cases and 1382 in HCC. In total, 124 proteins were differentially expressed in sEVs from COVID-19(+) subjects. “Coronavirus-disease response”, “complement and coagulation cascades”, and “PMN extracellular trap formation” were the most enriched KEGG pathways in COVID-19(+) cases. The most represented biological processes were “Hemoglobin and haptoglobin binding” and “oxygen carrier activity”, and the best-denoted molecular functions were “regulated exocytosis and secretion” and “leucocyte and PMN mediated immunity”. sEV proteomic cargo in COVID-19(+) suggests activity related to immune response processes, oxygen transport, and antioxidant mechanisms. In contrast, in HCC, sEV signature profiles are mainly associated with epithelial homeostasis. Full article

Proteins containing PDZ (post-synaptic density, PSD-95/disc large, Dlg/zonula occludens, ZO-1) domains assemble signaling complexes that orchestrate cell responses. Viral pathogens target host PDZ proteins by coding proteins containing a PDZ-binding motif (PBM). The presence of a PBM in the SARS-CoV-2 E protein contributes to the virus’s pathogenicity. SARS-CoV-2 infects epithelia, but also cells from the innate immune response, including monocytes and alveolar macrophages. This process is critical for alterations of the immune response that are related to the deaths caused by SARS-CoV-2. Identification of E-protein targets in immune cells might offer clues to understanding how SARS-CoV-2 alters the immune response. We analyzed the interactome of the SARS-CoV-2 E protein in human monocytes. The E protein was expressed fused to a GFP tag at the amino terminal in THP-1 monocytes, and associated proteins were identified using a proteomic approach. The E-protein interactome provided 372 partners; only 8 of these harbored PDZ domains, including the cell polarity protein ZO-2, the chemoattractant IL-16, and syntenin. We addressed the expression and localization of the identified PDZ proteins along the differentiation of primary and THP-1 monocytes towards macrophages and dendritic cells. Our data highlight the importance of identifying the functions of PDZ proteins in the maintenance of immune fitness and the viral alteration of inflammatory response. Full article

Syntenin acts as an adaptor and scaffold protein through its two PSD-95, Dlg, and ZO-1 (PDZ) domains, participating in multiple signaling pathways and modulating cellular physiology. It has been identified as an oncogene, promoting cancer development, metastasis, and angiogenesis in various carcinomas. Syntenin-1 is also associated with the production and release of exosomes, small extracellular vesicles that play a significant role in intercellular communication by containing bioactive molecules such as proteins, lipids, and nucleic acids. The trafficking of exosomes involves a complex interplay of various regulatory proteins, including syntenin-1, which interacts with its binding partners, syndecan and activated leukocyte cell adhesion molecule (ALIX). Exosomal transfer of microRNAs, a key cargo, can regulate the expression of various cancer-related genes, including syntenin-1. Targeting the mechanism involving the regulation of exosomes by syntenin-1 and microRNAs may provide a novel treatment strategy for cancer. This review highlights the current understanding of syntenin-1’s role in regulating exosome trafficking and its associated cellular signaling pathways. Full article

Syntenin-1 is a 298 amino acid protein codified by the melanoma differentiation-associated gene-9 (MDA-9). Structurally, it is composed of four domains: N-terminal, PDZ1, PDZ2, and C-terminal. The PDZ domains of syntenin-1 are involved in the stability and interaction with other molecules such as proteins, glycoproteins, and lipids. Domains are also associated with several biological functions such as the activation of signaling pathways related to cell-to-cell adhesion, signaling translation, and the traffic of intracellular lipids, among others. The overexpression of syntenin-1 has been reported in glioblastoma, colorectal, melanoma, lung, prostate, and breast cancer, which promotes tumorigenesis by regulating cell migration, invasion, proliferation, angiogenesis, apoptosis, and immune response evasion, and metastasis. The overexpression of syntenin-1 in samples has been associated with worst prognostic and recurrence, whereas the use of inhibitors such as shRNA, siRNA, and PDZli showed a diminution of the tumor size and reduction in metastasis and invasion. Syntenin-1 has been suggested as a potential biomarker and therapeutic target in cancer for developing more effective diagnostic/prognostic tests or passive/active immunotherapies. Full article

Proteomics analysis of circulating exosomes derived from cancer cells represents a promising approach to the elucidation of cell–cell communication and the discovery of putative biomarker candidates for cancer diagnosis and treatment. Nonetheless, the proteome of exosomes derived from cell lines with different metastatic capabilities still warrants further investigation. Here, we present a comprehensive quantitative proteomics investigation of exosomes isolated from immortalized mammary epithelial cells and matched tumor lines with different metastatic potentials in an attempt to discover exosome markers specific to breast cancer (BC) metastasis. A total of 2135 unique proteins were quantified with a high confidence level from 20 isolated exosome samples, including 94 of the TOP 100 exosome markers archived by ExoCarta. Moreover, 348 altered proteins were observed, among which several metastasis-specific markers, including cathepsin W (CATW), magnesium transporter MRS2 (MRS2), syntenin-2 (SDCB2), reticulon-4 (RTN), and UV excision repair protein RAD23 homolog (RAD23B), were also identified. Notably, the abundance of these metastasis-specific markers corresponds well with the overall survival of BC patients in clinical settings. Together, these data provide a valuable dataset for BC exosome proteomics investigation and prominently facilitate the elucidation of the molecular mechanisms underlying primary tumor development and progression. Full article

Viruses are dependent on host factors in order to efficiently establish an infection and replicate. Targeting the interactions of such host factors provides an attractive strategy to develop novel antivirals. Syntenin is a protein known to regulate the architecture of cellular membranes by its involvement in protein trafficking and has previously been shown to be important for human papilloma virus (HPV) infection. Here, we show that a highly potent and metabolically stable peptide inhibitor that binds to the PDZ1 domain of syntenin inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection by blocking the endosomal entry of the virus. Furthermore, we found that the inhibitor also hampered chikungunya infection and strongly reduced flavivirus infection, which is completely dependent on receptor-mediated endocytosis for their entry. In conclusion, we have identified a novel broad spectrum antiviral inhibitor that efficiently targets a broad range of RNA viruses. Full article

To characterize the mechanisms that govern chemoresistance, we performed a comparative proteomic study analyzing head and neck squamous cell carcinoma (HNSCC) cells: CCL-138 (parental), CCL-138-R (cisplatin-resistant), and cancer stem cells (CSCs). Syntenin-1 (SDCBP) was upregulated in CCL-138-R cells and CSCs over parental cells. SDCBP depletion sensitized biopsy-derived and established HNSCC cell lines to cisplatin (CDDP) and reduced CSC markers, Src activation being the main SDCBP downstream target. In mice, SDCBP-depleted cells formed tumors with decreased mitosis, Ki-67 positivity, and metastasis over controls. Moreover, the fusocellular pattern of CCL-138-R cell-derived tumors reverted to a more epithelial morphology upon SDCBP silencing. Importantly, SDCBP expression was associated with Src activation, poor differentiated tumor grade, advanced tumor stage, and shorter survival rates in a series of 382 HNSCC patients. Our results reveal that SDCBP might be a promising therapeutic target for effectively eliminating CSCs and CDDP resistance. Full article