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Zhang, Yujing, Xinyue Yang, Yang Hu, and Xin Huang. "Integrated Bioinformatic Investigation of EXOSCs in Hepatocellular Carcinoma Followed by the Preliminary Validation of EXOSC5 in Cell Proliferation." International Journal of Molecular Sciences 23, no. 20 (October 12, 2022): 12161. http://dx.doi.org/10.3390/ijms232012161.
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The Exosome complex (EXOSC) is a multiprotein complex that was originally discovered as the machinery of RNA degradation. Interestingly, recent studies have reported that EXOSC family members (EXOSCs) are associated with various human diseases, including cancers. It will be interesting to investigate whether EXOSCs are related to the processes of hepatocellular carcinoma (HCC). In this study, multiple public databases and experimental validation were utilized to systemically investigate the role of EXOSCs, especially EXOSC5, in HCC. It is worth considering that the mRNA and protein levels of many EXOSCs were elevated in HCC, although there were some differences in the results from different database analyses. The over-expression of EXOSCs could predict HCC to some extent, as evidenced by the positive correlation between the elevated EXOSCs and alpha fetoprotein (AFP) levels, as well as with a high accuracy, as shown by the receiver operating characteristic curve analysis. Additionally, higher mRNA expressions of specific EXOSCs were significantly related to clinical cancer stage, shorter overall survival and disease-free survival in HCC patients. A moderate mutation rate of EXOSCs was also observed in HCC. Furthermore, a gene functional enrichment analysis indicated that EXOSCs were mainly involved in the metabolism of RNA. Moreover, we revealed that the expression of EXOSCs is remarkably related to immune cell infiltration. Finally, EXOSC5 was upregulated in HCC tissues and cell lines, promoting cell growth and proliferation via activated signal transducer and activator of transcription 3 (STAT3). The bioinformatic analyses, following verification in situ and in vitro, provided a direction for further functions and underlying mechanism of EXOSCs in HCC.
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Müller, Juliane S., David T. Burns, Helen Griffin, Graeme R. Wells, Romance A. Zendah, Benjamin Munro, Claudia Schneider, and Rita Horvath. "RNA exosome mutations in pontocerebellar hypoplasia alter ribosome biogenesis and p53 levels." Life Science Alliance 3, no. 8 (June 11, 2020): e202000678. http://dx.doi.org/10.26508/lsa.202000678.
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The RNA exosome is a ubiquitously expressed complex of nine core proteins (EXOSC1-9) and associated nucleases responsible for RNA processing and degradation. Mutations in EXOSC3, EXOSC8, EXOSC9, and the exosome cofactor RBM7 cause pontocerebellar hypoplasia and motor neuronopathy. We investigated the consequences of exosome mutations on RNA metabolism and cellular survival in zebrafish and human cell models. We observed that levels of mRNAs encoding p53 and ribosome biogenesis factors are increased in zebrafish lines with homozygous mutations of exosc8 or exosc9, respectively. Consistent with higher p53 levels, mutant zebrafish have a reduced head size, smaller brain, and cerebellum caused by an increased number of apoptotic cells during development. Down-regulation of EXOSC8 and EXOSC9 in human cells leads to p53 protein stabilisation and G2/M cell cycle arrest. Increased p53 transcript levels were also observed in muscle samples from patients with EXOSC9 mutations. Our work provides explanation for the pathogenesis of exosome-related disorders and highlights the link between exosome function, ribosome biogenesis, and p53-dependent signalling. We suggest that exosome-related disorders could be classified as ribosomopathies.
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McIver, Skye C., Yoon-A. Kang, Andrew W. DeVilbiss, Chelsea A. O'Driscoll, David T. Yang, Saghi Ghaffari, and Emery H. Bresnick. "The RNA-Degrading Exosome Complex Is an Endogenous Suppressor of Erythroid Maturation." Blood 124, no. 21 (December 6, 2014): 2659. http://dx.doi.org/10.1182/blood.v124.21.2659.2659.
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Abstract Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. We demonstrated that the master regulator of erythropoiesis, GATA-1, induces expression of genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1-mediated transcriptional activation. We conducted studies to assess whether the GATA-1/Foxo3 cooperativity is restricted to the control of autophagy, or if it more broadly impacts the erythroid cell transcriptome. Analysis of the GATA-1/Foxo3-dependent transcriptome in erythroid cells revealed a target gene ensemble extending beyond autophagy, but representing only a small fraction of the complex GATA-1-dependent target gene ensemble. GATA-1/Foxo3 repressed expression of genes encoding two exosome complex components, Exosc5 and Exosc8. The exosome complex functions in one of the major RNA degradation pathways in diverse cell types, mediates splicing and degradation of mRNAs and non-coding RNAs, and functions in epigenetic gene regulation. As the role of the exosome complex in erythropoiesis, and more broadly in hematopoiesis, had not been described previously, we conducted biological and mechanistic studies to determine whether the endogenous exosome complex has important roles in the development and/or function of erythroid cells. Strikingly, downregulating expression of endogenous exosome components, Exosc8, Exosc9 and the catalytic component Dis3 dramatically increased the percentage of primary erythroid precursor cells in the R4 (polychromatic and orthrochromatic orthrochromatic) population from 1% in control cells to 30%, 28% and 16% respectively. We have extended these initial findings to explore key mechanistic and biological questions. Using the exosome complex high-resolution crystal structure as a guide, we are conducting loss-of-function studies to establish whether additional exosome complex components that serve structural roles in the complex (Exosc4 and Exosc7), bind RNA substrates (Exosc1), and degrade RNAs (Dis3L and Exosc10) are also important determinants of erythroid maturation. Initial studies indicate that multiple components suppress maturation, but differ quantitatively in their importance. Studies are underway to test the hypothesis that downregulating Exosc8 or Exosc9 severely disrupts the integrity of the exosome complex, whereas certain other components are less critical for complex integrity and function. We have developed a co-immunoprecipitation assay to measure interactions between endogenous exosome complex components in erythroid cells. This assay is being used to establish the role of the various exosome complex subunits in complex integrity in cultured and primary erythroid cells. Under conditions in which downregulating Exosc8 or Exosc9 induced erythroid maturation, expression of the established regulators of erythropoiesis GATA-1, FOG-1, or KLF1 was unaffected. To test the hypothesis that the exosome complex downregulates a cohort of critical RNAs, including regulatory non-coding RNAs, required for erythroid maturation, we are conducting studies to identify direct exosome complex targets in erythroid cells at distinct stages of maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program. Furthermore, since the exosome complex had not been shown previously to regulate any aspect of hematopoiesis, this work expands the biological repertoire of exosome complex-dependent processes. Disclosures No relevant conflicts of interest to declare.
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Beheshtian, Maryam, Zohreh Fattahi, Mahsa Fadaee, Raheleh Vazehan, Payman Jamali, Elham Parsimehr, Mahboubeh Kamgar, et al. "Identification of disease‐causing variants in the EXOSC gene family underlying autosomal recessive intellectual disability in Iranian families." Clinical Genetics 95, no. 6 (May 14, 2019): 718–25. http://dx.doi.org/10.1111/cge.13549.
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McIver, Skye C., Koichi R. Kasumura, Elsa Davids, Yoon-A. Kang, Peng Liu, David T. Yang, and Emery H. Bresnick. "Orchestrating Developmental Signaling to Balance Erythroblast Proliferation and Differentiation." Blood 128, no. 22 (December 2, 2016): 699. http://dx.doi.org/10.1182/blood.v128.22.699.699.
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Abstract The highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs. How this post-transcriptional RNA- and gene-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in mice, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation versus differentiation of erythroid precursor cells. Using shRNAs to downregulate exosome complex subunits, we developed conditions that disrupt exosome complex integrity and analyzed functional consequences. We discovered that erythroid precursor cells derived from E14.5 murine fetal liver exhibited significantly decreased survival when erythropoietin (Epo) was removed from the culture media. Live cells decreased from 66% in the control to 24% (p = 2 x 10-5) in the Exosc8-knockdown condition. The maturation-associated increase (1.5 fold, p = 0.003) in the Ter119+CD71+ (R3: early/late basophilic erythroblast) population upon Exosc8 knockdown did not occur without exogenous Epo. Thus, the survival and differentiation of erythroid precursors after downregulating Exosc8 is critically dependent on Epo. We tested whether Exosc8 downregulation impaired stem cell factor (SCF)-mediated proliferation/amplification of erythroid precursors. Using a phospho-flow cytometry assay, we quantitated the capacity of SCF or Epo to instigate cell signaling using the shared downstream substrate Akt. SCF induced maximal Akt phosphorylation in immature Ter119-CD71high erythroblasts (5.5 fold, p = 3 x 10-6). As erythroid maturation progressed to Ter119+CD71high, the SCF response was diminished. Exosc8 downregulation abrogated SCF-mediated induction of phospho-Akt. Epo induced maximal Akt phosphorylation in Ter119+CD71high erythroblasts, and Exosc8 downregulation accelerated acquisition of this signaling response. Whereas Epo did not affect Akt phosphorylation in control Ter119-CD71high erythroblasts, Epo increased phospho-Akt 4 fold (p = 0.003) upon Exosc8 downregulation. Thus, downregulating Exosc8 abrogated SCF signaling that supports erythroid precursor proliferation/amplification, while precociously inducing Epo-dependent pro-differentiation signaling. As downregulating Exosc8 abrogated SCF signaling, we tested whether the exosome complex or individual subunits confer Kit expression or promote signaling via a post-receptor mechanism. Downregulating Exosc8 or Exosc9 reduced Kit surface expression in the R2 (proerythroblast and early basophilic) cell population [14.6 (p = 3.6 x 10-4) and 6 fold (p= 1.3 x 10-6) decrease, respectively]. Exosc8 downregulation also reduced Kit mRNA and primary transcript levels at all erythroid maturation stages. By 24 h post-infection with shExosc8-expressing retrovirus, Kit protein decreased 10 fold (p = 0.046) in Ter119- erythroid precursor cells. We tested whether the Exosc8 requirement for Kit primary transcript, mRNA and protein expression involved alterations in the distribution of transcriptionally-competent serine 5-phosphorylated RNA polymerase II (Pol II) at Kit. Using quantitative ChIP analysis with control and Exosc8-knockdown Ter119- cells, Exosc8 downregulation reduced phospho-Ser5 Pol II occupancy within the coding region (+5 kb) and 3' UTR, but not at the promoter. Furthermore, Exosc9 occupied multiple regions of the Kit locus. These results support a model in which the exosome complex confers Kit expression and stem cell factor/Kit signaling via a transcriptional mechanism. Functioning as a gatekeeper of the SCF - Epo developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts. Studies are underway to establish the full repertoire of exosome complex targets in the developing erythroblast, how these targets relate to exosome complex targets in hematopoietic stem and progenitor cells and exosome complex-dependent strategies for translational applications. Disclosures No relevant conflicts of interest to declare.
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Yin, Shang-Jun, Guo-Ying Qian, Jun-Mo Yang, Jinhyuk Lee, and Yong-Doo Park. "Detection of Melanogenesis and Anti-Apoptosis-Associated Melanoma Factors: Array CGH and PPI Mapping Integrating Study." Protein & Peptide Letters 28, no. 12 (December 2021): 1408–24. http://dx.doi.org/10.2174/0929866528666211105112927.
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Background: We investigated melanogenesis- and anti-apoptosis-related melanoma factors in melanoma cells (TXM1, TXM18, A375P, and A375SM). Objective: To find melanoma associated hub factor, high-throughput screening-based techniques integrating with bioinformatics were investigated. Methods: Array CGH analysis was conducted with a commercial system. Total genomic DNAs prepared individually from each cell line with control DNA were properly labeled with Cy3-dCTP and Cy5-dCTP and hybridizations and subsequently performed data treatment by the log2 green (G; test) to red (R; reference) fluorescence ratios (G/R). Gain or loss of copy number was judged by spots with log2-transformed ratios. PPI mapping analysis of detected candidate genes based on the array CGH results was conducted using the human interactome in the STRING database. Energy minimization and a short Molecular Dynamics (MD) simulation using the implicit solvation model in CHARMM were performed to analyze the interacting residues between YWHAZ and YWHAB. Results: Three genes (BMP-4, BFGF, LEF-1) known to be involved in melanogenesis were found to lose chromosomal copy numbers, and Chr. 6q23.3 was lost in all tested cell lines. Ten hub genes (CTNNB1, PEX13, PEX14, PEX5, IFNG, EXOSC3, EXOSC1, EXOSC8, UBC, and PEX10) were predicted to be functional interaction factors in the network of the 6q23.3 locus. The apoptosis-associated genes E2F1, p50, BCL2L1, and BIRC7 gained, and FGF2 lost chromosomal copy numbers in the tested melanoma cell lines. YWHAB, which gained chromosomal copy numbers, was predicted to be the most important hub protein in melanoma cells. Molecular dynamics simulations for binding YWHAB and YWHAZ were conducted, and the complex was predicted to be energetically and structurally stable through its 3 hydrogen-bond patterns. The number of interacting residues is 27. Conclusion: Our study compares genome-wide screening interactomics predictions for melanoma factors and offers new information for understanding melanogenesis- and anti-apoptosis-associated mechanisms in melanoma. Especially, YWHAB was newly detected as a core factor in melanoma cells.
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Di Giovambattista, Anna Paola, Itxaropena Jácome Querejeta, Purificación Ventura Faci, Gerardo Rodríguez Martínez, and Feliciano Ramos Fuentes. "Familial EXOSC3-related pontocerebellar hypoplasia." Anales de Pediatría (English Edition) 86, no. 5 (May 2017): 284–86. http://dx.doi.org/10.1016/j.anpede.2016.09.004.
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Li, Baoyu, Xuehong Xian, Xinwei Lin, Luo Huang, Ailin Liang, Hongwei Jiang, and Qimei Gong. "Hypoxia Alters the Proteome Profile and Enhances the Angiogenic Potential of Dental Pulp Stem Cell-Derived Exosomes." Biomolecules 12, no. 4 (April 14, 2022): 575. http://dx.doi.org/10.3390/biom12040575.
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Dental pulp stem cells (DPSCs) and their exosomes (Exos) are effective treatments for regenerative medicine. Hypoxia was confirmed to improve the angiogenic potential of stem cells. However, the angiogenic effect and mechanism of hypoxia-preconditioned DPSC-Exos are poorly understood. We isolated exosomes from DPSCs under normoxia (Nor-Exos) and hypoxia (Hypo-Exos) and added them to human umbilical vein endothelial cells (HUVECs). HUVEC proliferation, migration and angiogenic capacity were assessed by CCK-8, transwell, tube formation assays, qRT-PCR and Western blot. iTRAQ-based proteomics and bioinformatic analysis were performed to investigate proteome profile differences between Nor-Exos and Hypo-Exos. Western blot, immunofluorescence and immunohistochemistry were used to detect the expression of lysyl oxidase-like 2 (LOXL2) in vitro and in vivo. Finally, we silenced LOXL2 in HUVECs and rescued tube formation with Hypo-Exos. Hypo-Exos enhanced HUVEC proliferation, migration and tube formation in vitro superior to Nor-Exos. The proteomics analysis identified 79 proteins with significantly different expression in Hypo-Exos, among which LOXL2 was verified as being upregulated in hypoxia-preconditioned DPSCs, Hypo-Exos, and inflamed dental pulp. Hypo-Exos partially rescued the inhibitory influence of LOXL2 silence on HUVEC tube formation. In conclusion, hypoxia enhanced the angiogenic potential of DPSCs-Exos and partially altered their proteome profile. LOXL2 is likely involved in Hypo-Exos mediated angiogenesis.
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Zhang, Yi, Yi Qin, Michael Chopp, Chao Li, Amy Kemper, Xianshuang Liu, Xinli Wang, Li Zhang, and Zheng Gang Zhang. "Ischemic Cerebral Endothelial Cell–Derived Exosomes Promote Axonal Growth." Stroke 51, no. 12 (December 2020): 3701–12. http://dx.doi.org/10.1161/strokeaha.120.031728.
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Background and Purpose: Cerebral endothelial cells (CECs) and axons of neurons interact to maintain vascular and neuronal homeostasis and axonal remodeling in normal and ischemic brain, respectively. However, the role of exosomes in the interaction of CECs and axons in brain under normal conditions and after stroke is unknown. Methods: Exosomes were isolated from CECs of nonischemic rats and is chemic rats (nCEC-exos and isCEC-exos), respectively. A multicompartmental cell culture system was used to separate axons from neuronal cell bodies. Results: Axonal application of nCEC-exos promotes axonal growth of cortical neurons, whereas isCEC-exos further enhance axonal growth than nCEC-exos. Ultrastructural analysis revealed that CEC-exos applied into distal axons were internalized by axons and reached to their parent somata. Bioinformatic analysis revealed that both nCEC-exos and isCEC-exos contain abundant mature miRNAs; however, isCEC-exos exhibit more robust elevation of select miRNAs than nCEC-exos. Mechanistically, axonal application of nCEC-exos and isCEC-exos significantly elevated miRNAs and reduced proteins in distal axons and their parent somata that are involved in inhibiting axonal outgrowth. Blockage of axonal transport suppressed isCEC-exo–altered miRNAs and proteins in somata but not in distal axons. Conclusions: nCEC-exos and isCEC-exos facilitate axonal growth by altering miRNAs and their target protein profiles in recipient neurons.
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McGuffie, Eileen M., Jennifer E. Fraylick, Debra J. Hazen-Martin, Timothy S. Vincent, and Joan C. Olson. "Differential Sensitivity of Human Epithelial Cells to Pseudomonas aeruginosa Exoenzyme S." Infection and Immunity 67, no. 7 (July 1, 1999): 3494–503. http://dx.doi.org/10.1128/iai.67.7.3494-3503.1999.
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ABSTRACT Exoenzyme S (ExoS) is an ADP-ribosyltransferase produced and directly translocated into eukaryotic cells by the opportunistic pathogen Pseudomonas aeruginosa. Model systems that allow bacterial translocation of ExoS have found ExoS to have multiple effects on eukaryotic cell function, affecting DNA synthesis, actin cytoskeletal structure, and cell matrix adherence. To understand mechanisms underlying differences observed in cell sensitivities to ExoS, we examined the effects of bacterially translocated ExoS on multiple human epithelial cell lines. Of the cell lines examined, confluent normal kidney (NK) epithelial cells were most resistant to ExoS, while tumor-derived cell lines were highly sensitive to ExoS. Analysis of the mechanisms of resistance indicated that cell association as well as an intrinsic resistance to morphological alterations were associated with increased resistance to ExoS. Conversely, increased sensitivity to ExoS appeared to be linked to epithelial cell growth, with tumor cells capable of undergoing non-contact-inhibited, anchorage-independent growth all being sensitive to ExoS, and NK cells becoming sensitive to ExoS when subconfluent and growing. Consistent with the possibility that growth-related, actin-based structures are involved in sensitivity to ExoS, scanning electron microscopy revealed cellular extensions from sensitive, growing cells to bacteria, which were not readily evident in resistant cells. In all studies, the severity of effects of ExoS on cell function directly correlated with the degree of Ras modification, indicating that sensitivity to ExoS in some manner related to the efficiency of ExoS translocation and its ADP-ribosylation of Ras. Our results suggest that factors expressed by growing epithelial cells are required for the bacterial contact-dependent translocation of ExoS; as normal epithelial cells differentiate into polarized confluent monolayers, expression of these factors is altered, and cells in turn become more resistant to the effects of ExoS.
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Taniue, Kenzui, Tanzina Tanu, Yuki Shimoura, Shuhei Mitsutomi, Han Han, Rika Kakisaka, Yusuke Ono, et al. "RNA Exosome Component EXOSC4 Amplified in Multiple Cancer Types Is Required for the Cancer Cell Survival." International Journal of Molecular Sciences 23, no. 1 (January 2, 2022): 496. http://dx.doi.org/10.3390/ijms23010496.
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The RNA exosome is a multi-subunit ribonuclease complex that is evolutionally conserved and the major cellular machinery for the surveillance, processing, degradation, and turnover of diverse RNAs essential for cell viability. Here we performed integrated genomic and clinicopathological analyses of 27 RNA exosome components across 32 tumor types using The Cancer Genome Atlas PanCancer Atlas Studies’ datasets. We discovered that the EXOSC4 gene, which encodes a barrel component of the RNA exosome, was amplified across multiple cancer types. We further found that EXOSC4 alteration is associated with a poor prognosis of pancreatic cancer patients. Moreover, we demonstrated that EXOSC4 is required for the survival of pancreatic cancer cells. EXOSC4 also repressed BIK expression and destabilized SESN2 mRNA by promoting its degradation. Furthermore, knockdown of BIK and SESN2 could partially rescue pancreatic cells from the reduction in cell viability caused by EXOSC4 knockdown. Our study provides evidence for EXOSC4-mediated regulation of BIK and SESN2 mRNA in the survival of pancreatic tumor cells.
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Yan, Litao, and Xing Wu. "Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral regeneration activity." Cell Biology and Toxicology 36, no. 2 (December 9, 2019): 165–78. http://dx.doi.org/10.1007/s10565-019-09504-5.
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AbstractAnimal and clinical studies have shown that mesenchymal stem cells (MSCs) play an important role in cartilage repair. The therapeutic effect of mesenchymal stem cells based therapies has been increasingly demonstrated to exosome-mediated paracrine secretion. Here, we investigated the cellular processes and mechanism of exosomes produced by conventional 2D culture (2D-Exos) and exosomes produced from 3D culture (3D-Exos) of umbilical MSCs (U-MSCs) in a hollow-fiber bioreactor for the treatment of cartilage repair. We found that the yield of 3D-Exos was 7.5-fold higher than that of 2D-Exos. The in vitro experiments indicated that both 2D-Exos and 3D-Exos can stimulate chondrocyte proliferation, migration, and matrix synthesis, and inhibit apoptosis, with 3D-Exos exerting a stronger effect than 2D-Exos. This effect was partly attributed to the activation of transforming growth factor beta 1 and Smad2/3 signaling. The injection of 2D-Exos and 3D-Exos showed enhanced gross appearance and attenuated cartilage defect; however, 3D-Exos showed a superior therapeutic effect than 2D-Exos. In summary, our study provides novel insights into the chondroprotective effects of exosomes produced from 3D culture of U-MSCs in a hollow-fiber bioreactor. Because of its promising biological function and high yield, 3D-Exos may become a promising therapeutic method for the treatment of cartilage defects.
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McGuffie, Eileen M., Dara W. Frank, Timothy S. Vincent, and Joan C. Olson. "Modification of Ras in Eukaryotic Cells by Pseudomonas aeruginosa Exoenzyme S." Infection and Immunity 66, no. 6 (June 1, 1998): 2607–13. http://dx.doi.org/10.1128/iai.66.6.2607-2613.1998.
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ABSTRACT Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa388 and an isogenic mutant strain, 388ΔexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388ΔexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388ΔexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.
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Ferguson, Michael W., Jill A. Maxwell, Timothy S. Vincent, Jack da Silva, and Joan C. Olson. "Comparison of the exoS Gene and Protein Expression in Soil and Clinical Isolates of Pseudomonas aeruginosa." Infection and Immunity 69, no. 4 (April 1, 2001): 2198–210. http://dx.doi.org/10.1128/iai.69.4.2198-2210.2001.
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ABSTRACT Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis ofPseudomonas aeruginosa. To gain further understanding of how ExoS might contribute to P. aeruginosa survival and virulence, ExoS expression and the structural gene sequence were determined in P. aeruginosa soil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity. ExoS structural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced by P. aeruginosa or residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral to P. aeruginosa survival in the soil and suggest that, in the transition of P. aeruginosa from the soil to certain clinical settings, the loss of ExoS expression is favored.
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Olson, Joan C., Jennifer E. Fraylick, Eileen M. McGuffie, Katherine M. Dolan, Timothy L. Yahr, Dara W. Frank, and Timothy S. Vincent. "Interruption of Multiple Cellular Processes in HT-29 Epithelial Cells by Pseudomonas aeruginosaExoenzyme S." Infection and Immunity 67, no. 6 (June 1, 1999): 2847–54. http://dx.doi.org/10.1128/iai.67.6.2847-2854.1999.
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ABSTRACT Exoenzyme S (ExoS), an ADP-ribosylating enzyme produced by the opportunistic pathogen Pseudomonas aeruginosa, is directly translocated into eukaryotic cells by bacterial contact. Within the cell, ExoS ADP-ribosylates the cell signaling protein Ras and causes inhibition of DNA synthesis and alterations in cytoskeletal structure. To further understand the interrelationship of the different cellular effects of ExoS, functional analyses were performed on HT-29 epithelial cells after exposure to ExoS-producing P. aeruginosa 388 and the non-ExoS-producing strain 388ΔS. Two different mechanisms of morphological alteration were identified: (i) a more-transient and less-severe cell rounding caused by the non-ExoS-producing strain 388ΔS and (ii) a more-severe, long-term cell rounding caused by ExoS-producing strain 388. Long-term effects of ExoS on cell morphology occurred in conjunction with ExoS-mediated inhibition of DNA synthesis and the ADP-ribosylation of Ras. ExoS was also found to cause alterations in HT-29 cell function, leading to the loss of cell adhesion and microvillus effacement. Nonadherent ExoS-treated cells remained viable but had a high proportion of modified Ras. While microvillus effacement was detected in both 388- and 388ΔS-treated cells, effacement was more prevalent and rapid in cells exposed to strain 388. We conclude from these studies that ExoS can have multiple effects on epithelial cell function, with more severe cellular alterations associated with the enzymatic modification of Ras. The finding that ExoS had greater effects on cell growth and adherence than on cell viability suggests that ExoS may contribute to the P. aeruginosa infectious process by rendering cells nonfunctional.
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Slavotinek, Anne, Doriana Misceo, Stephanie Htun, Linda Mathisen, Eirik Frengen, Michelle Foreman, Jennifer E. Hurtig, et al. "Biallelic variants in the RNA exosome gene EXOSC5 are associated with developmental delays, short stature, cerebellar hypoplasia and motor weakness." Human Molecular Genetics 29, no. 13 (June 5, 2020): 2218–39. http://dx.doi.org/10.1093/hmg/ddaa108.
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Abstract The RNA exosome is an essential ribonuclease complex required for processing and/or degradation of both coding and non-coding RNAs. We identified five patients with biallelic variants in EXOSC5, which encodes a structural subunit of the RNA exosome. The clinical features of these patients include failure to thrive, short stature, feeding difficulties, developmental delays that affect motor skills, hypotonia and esotropia. Brain MRI revealed cerebellar hypoplasia and ventriculomegaly. While we ascertained five patients, three patients with distinct variants of EXOSC5 were studied in detail. The first patient had a deletion involving exons 5–6 of EXOSC5 and a missense variant, p.Thr114Ile, that were inherited in trans, the second patient was homozygous for p.Leu206His and the third patient had paternal isodisomy for chromosome 19 and was homozygous for p.Met148Thr. The additional two patients ascertained are siblings who had an early frameshift mutation in EXOSC5 and the p.Thr114Ile missense variant that were inherited in trans. We employed three complementary approaches to explore the requirement for EXOSC5 in brain development and assess consequences of pathogenic EXOSC5 variants. Loss of function for exosc5 in zebrafish results in shortened and curved tails/bodies, reduced eye/head size and edema. We modeled pathogenic EXOSC5 variants in both budding yeast and mammalian cells. Some of these variants cause defects in RNA exosome function as well as altered interactions with other RNA exosome subunits. These findings expand the number of genes encoding RNA exosome subunits linked to human disease while also suggesting that disease mechanism varies depending on the specific pathogenic variant.
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Bridge, Dacie R., Karen H. Martin, Elizabeth R. Moore, Wendy M. Lee, James A. Carroll, Claudia L. Rocha, and Joan C. Olson. "Examining the Role of Actin-Plasma Membrane Association in Pseudomonas aeruginosa Infection and Type III Secretion Translocation in Migratory T24 Epithelial Cells." Infection and Immunity 80, no. 9 (June 11, 2012): 3049–64. http://dx.doi.org/10.1128/iai.00231-12.
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ABSTRACTThe opportunistic pathogenPseudomonas aeruginosatargets wounded epithelial barriers, but the cellular alteration that increases susceptibility toP. aeruginosainfection remains unclear. This study examined how cell migration contributes to the establishment ofP. aeruginosainfections using (i) highly migratory T24 epithelial cells as a cell culture model, (ii) mutations in the type III secretion (T3S) effector ExoS to manipulateP. aeruginosainfection, and (iii) high-resolution immunofluorescent microscopy to monitor ExoS translocation. ExoS includes both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) activities, andP. aeruginosacells expressing wild-type ExoS preferentially bound to the leading edge of T24 cells, where ExoS altered leading-edge architecture and actin anchoring in conjunction with interrupting T3S translocation. Inactivation of ExoS GAP activity allowedP. aeruginosato be internalized and secrete ExoS within T24 cells, but as with wild-type ExoS, translocation was limited in association with disruption of actin anchoring. Inactivation of ExoS ADPRT activity resulted in significantly enhanced T3S translocation byP. aeruginosacells that remained extracellular and in conjunction with maintenance of actin-plasma membrane association. Infection withP. aeruginosaexpressing ExoS lacking both GAP and ADPRT activities resulted in the highest level of T3S translocation, and this occurred in conjunction with the entry and alignment ofP. aeruginosaand ExoS along actin filaments. Collectively, in using ExoS mutants to modulate and visualize T3S translocation, we were able to (i) confirm effector secretion by internalizedP. aeruginosa, (ii) differentiate the mechanisms underlying the effects of ExoS GAP and ADPRT activities onP. aeruginosainternalization and T3S translocation, (iii) confirm that ExoS ADPRT activity targeted a cellular substrate that interrupted T3S translocation, (iv) visualize the ability ofP. aeruginosaand ExoS to align with actin filaments, and (v) demonstrate an association between actin anchoring at the leading edge of T24 cells and the establishment ofP. aeruginosainfection. Our studies also highlight the contribution of ExoS to the opportunistic nature ofP. aeruginosainfection through its ability to exert cytotoxic effects that interrupt T3S translocation andP. aeruginosainternalization, which in turn limit theP. aeruginosainfectious process.
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Zhang, Yichi, Hanjing Zhangdi, Xinsheng Nie, Lijuan Wang, Zhuzhi Wan, Hao Jin, Ronghui Pu, et al. "Exosomes Derived from BMMSCs Mitigate the Hepatic Fibrosis via Anti-Pyroptosis Pathway in a Cirrhosis Model." Cells 11, no. 24 (December 10, 2022): 4004. http://dx.doi.org/10.3390/cells11244004.
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Researchers increasingly report the therapeutic effect of exosomes derived from rat bone marrow mesenchymal stem cells (Exos-rBMMSC) on liver disease, while the optimal dose of Exos-rBMMSC in liver cirrhotic treatment has not been reported. In this study, we aimed to explore the efficacy and dose of Exos-rBMMSC in a hepatic cirrhosis rat model. The therapeutic effects of a low dose, medium dose and high dose of Exos-rBMMSC were assessed by liver function tests and histopathology. After four-weeks of Exos-rBMMSC therapy, pyroptosis-related expression levels in the medium dose and the high dose Exos-rBMMSC groups were significantly decreased compared to those in the liver cirrhosis group (p < 0.05). The hepatic function assay and histopathology results showed significant improvement in the medium dose and the high dose Exos-rBMMSCs groups. The localization of PKH67-labeled Exos-rBMMSC was verified microscopically, and these particles were coexpressed with the PCNA, NLRP3, GSDMD and Caspase-1. Our results demonstrated that Exos-rBMMSC accelerated hepatocyte proliferation and relieved liver fibrosis by restraining hepatocyte pyroptosis. More importantly, we confirmed that the high dose of Exos-rBMMSC may be the optimal dose for liver cirrhosis, which is conducive to the application of Exos-rBMMSC as a promising cell-free strategy.
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Rocha, Claudia L., Elizabeth A. Rucks, Deanne M. Vincent, and Joan C. Olson. "Examination of the Coordinate Effects of Pseudomonas aeruginosa ExoS on Rac1." Infection and Immunity 73, no. 9 (September 2005): 5458–67. http://dx.doi.org/10.1128/iai.73.9.5458-5467.2005.
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ABSTRACT Exoenzyme S (ExoS) is a bifunctional toxin directly translocated into eukaryotic cells by the Pseudomonas aeruginosa type III secretory (TTS) process. The amino-terminal GTPase-activating (GAP) activity and the carboxy-terminal ADP-ribosyltransferase (ADPRT) activity of ExoS have been found to target but exert opposite effects on the same low-molecular-weight G protein, Rac1. ExoS ADP-ribosylation of Rac1 is cell line dependent. In HT-29 human epithelial cells, where Rac1 is ADP-ribosylated by TTS-ExoS, Rac1 was activated and relocalized to the membrane fraction. Arg66 and Arg68 within the GTPase-binding region of Rac1 were identified as preferred sites of ExoS ADP-ribosylation. The modification of these residues by ExoS would be predicted to interfere with Rac1 inactivation and explain the increase in active Rac1 caused by ExoS ADPRT activity. Using ExoS-GAP and ADPRT mutants to examine the coordinate effects of the two domains on Rac1 function, limited effects of ExoS-GAP on Rac1 inactivation were evident in HT-29 cells. In J774A.1 macrophages, where Rac1 was not ADP-ribosylated, ExoS caused a decrease in the levels of active Rac1, and this decrease was linked to ExoS-GAP. Using immunofluorescence staining of Rac1 to understand the cellular basis for the targeting of ExoS ADPRT activity to Rac1, an inverse relationship was observed between Rac1 plasma membrane localization and Rac1 ADP-ribosylation. The results obtained from these studies have allowed the development of a model to explain the differential targeting and coordinate effects of ExoS GAP and ADPRT activity on Rac1 within the host cell.
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Liu, Xiang, Jiazichao Tu, Ziqin Zhou, Bingxin Huang, Jianrong Zhou, and Jimei Chen. "TMAO-Activated Hepatocyte-Derived Exosomes Are Widely Distributed in Mice with Different Patterns and Promote Vascular Inflammation." Cardiology Research and Practice 2022 (February 14, 2022): 1–9. http://dx.doi.org/10.1155/2022/5166302.
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Background. Trimethylamine-N-oxide (TMAO) has been shown to be an important player in cardiovascular disease (CVD) by promoting vascular inflammation and endothelial dysfunction. We recently found that exosomes (Exos) released from TMAO-activated hepatocytes (TMAO-Exos) could significantly induce inflammation and endothelial dysfunction. However, understandings of how are the Exos secreted by hepatocytes, where are they distributed in vivo, and what effects will they have on vascular inflammation remain limited. The present study aimed to explore the hub genes involved in the production of TMAO-Exos and their distributions in vivo and effects on inflammation. Methods. The transcriptome profiles of primary rat hepatocytes stimulated with TMAO were obtained from the GSE135856 dataset in the Gene Expression Omnibus repository, and the hub genes associated with Exos were screened and verified by qPCR. Next, Exos derived from TMAO-treated hepatocytes were isolated using differential centrifugation and given intravenously to mice. After 24 h, the distributions of DiI-labelled Exos were visualized with a fluorescence microscope, and the levels of proinflammatory genes in the aorta were detected by qPCR. Results. Phgdh, Mdh2, Echs1, Rap2a, Gpd1l, and Slc3a2 were identified as hub genes that may be involved in the production of TMAO-Exos. And TMAO-Exos were found to be efficiently taken up by cardiomyocytes, hepatocytes, and endothelial cells in the aorta and gastrocnemius muscle. Furthermore, TMAO-Exos, but not control-Exos, could significantly promote the mRNA expressions of Tnf, Icam1, Sele, and Cox-2 in the aorta. Conclusions. We provided clues about how TMAO may stimulate hepatocytes to produce Exos and further offered evidence that Exos secreted by TMAO-treated hepatocytes could be widely distributed in vivo and promote vascular inflammation.
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Wang, Xiwei, Zheng Xiang, Yinping Liu, Chunyu Huang, Yujun Pei, Xia Wang, Hui Zhi, et al. "Exosomes derived from Vδ2-T cells control Epstein-Barr virus–associated tumors and induce T cell antitumor immunity." Science Translational Medicine 12, no. 563 (September 30, 2020): eaaz3426. http://dx.doi.org/10.1126/scitranslmed.aaz3426.
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Treatment of life-threatening Epstein-Barr virus (EBV)–associated tumors remains a great challenge, especially for patients with relapsed or refractory disease. Here, we found that exosomes derived from phosphoantigen-expanded Vδ2-T cells (Vδ2-T-Exos) contained death-inducing ligands (FasL and TRAIL), an activating receptor for natural killer (NK) cells (NKG2D), immunostimulatory ligands (CD80 and CD86), and antigen-presenting molecules (MHC class I and II). Vδ2-T-Exos targeted and efficiently killed EBV-associated tumor cells through FasL and TRAIL pathways and promoted EBV antigen–specific CD4 and CD8 T cell expansion. Administration of Vδ2-T-Exos effectively controlled EBV-associated tumors in Rag2−/−γc−/− and humanized mice. Because expanding Vδ2-T cells and preparing autologous Vδ2-T-Exos from cancer patients ex vivo in large scale is challenging, we explored the antitumor activity of allogeneic Vδ2-T-Exos in humanized mouse cancer models. Here, we found that allogeneic Vδ2-T-Exos had more effective antitumor activity than autologous Vδ2-T-Exos in humanized mice; the allogeneic Vδ2-T-Exos increased the infiltration of T cells into tumor tissues and induced more robust CD4 and CD8 T cell–mediated antitumor immunity. Compared with exosomes derived from NK cells (NK-Exos) with direct cytotoxic antitumor activity or dendritic cells (DC-Exos) that induced T cell antitumor responses, Vδ2-T-Exos directly killed tumor cells and induced T cell–mediated antitumor response, thus resulting in more effective control of EBV-associated tumors. This study provided proof of concept for the strategy of using Vδ2-T-Exos, especially allogeneic Vδ2-T-Exos, to treat EBV-associated tumors.
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Gao, Xiang, Xiao-Tian Zhang, and Song Chen. "Protective effect of human umbilical cord mesenchymal stem cell-derived exosomes on rat retinal neurons in hyperglycemia through the brain-derived neurotrophic factor/TrkB pathway." International Journal of Ophthalmology 14, no. 11 (November 18, 2021): 1683–89. http://dx.doi.org/10.18240/ijo.2021.11.06.
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AIM: To explore whether human umbilical cord mesenchymal stem cell (hUCMSC)-derived exosomes (hUCMSC-Exos) protect rat retinal neurons in high-glucose (HG) conditions by activating the brain-derived neurotrophic factor (BDNF)-TrkB pathway. METHODS: hUCMSC-Exos were collected with differential ultracentrifugation methods and observed by transmission electron microscopy. Enzyme-linked immunosorbent assays (ELISAs) was used to quantify BDNF in hUCMSC-Exos, and Western blot was used to identify surface markers of hUCMSC-Exos. Rat retinal neurons were divided into 4 groups. Furthermore, cell viability, cell apoptosis, and TrkB protein expression were measured in retinal neurons. RESULTS: hUCMSCs and isolated hUCMSC-Exos were successfully cultured. All hUCMSC-Exos showed a diameter of 30 to 150 nm and had a phospholipid bimolecular membrane structure, as observed by transmission electron microscopy. ELISA showed the BDNF concentration of hUCMSCs-Exos was 2483.16±281.75. hUCMSCs-Exos effectively reduced the apoptosis of retinal neuron rate and improved neuron survival rate, meanwhile, the results of immunofluorescence verified the fluorescence intensity of TrKB in neurons increased. And all above effects were reduced by treated hUCMSCs-Exos with BDNF inhibitors. hUCMSC-Exos effectively reduced the apoptosis rate of retinal neurons by activating the BDNF-TrkB pathway in a HG environment. CONCLUSION: In the HG environment, hUCMSC-Exos could carry BDNF into rat retinal neurons, inhibiting neuronal apoptosis by activating the BDNF-TrkB pathway.
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Moll, Tobias, Valerie Odon, Calum Harvey, Mark O. Collins, Andrew Peden, John Franklin, Emily Graves, et al. "Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication." Life Science Alliance 6, no. 1 (October 14, 2022): e202201449. http://dx.doi.org/10.26508/lsa.202201449.
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New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host–virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression ofEXOSC2and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations withinEXOSC2in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.
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Rocha, Claudia L., Jenifer Coburn, Elizabeth A. Rucks, and Joan C. Olson. "Characterization of Pseudomonas aeruginosa Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages." Infection and Immunity 71, no. 9 (September 2003): 5296–305. http://dx.doi.org/10.1128/iai.71.9.5296-5305.2003.
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ABSTRACT Pseudomonas aeruginosa exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using P. aeruginosa strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione S-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.
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Liu, Huan, Ming Shen, De Zhao, Dan Ru, Yourong Duan, Chenhuan Ding, and He Li. "The Effect of Triptolide-Loaded Exosomes on the Proliferation and Apoptosis of Human Ovarian Cancer SKOV3 Cells." BioMed Research International 2019 (May 21, 2019): 1–14. http://dx.doi.org/10.1155/2019/2595801.
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Triptolide has been proven to possess anticancer efficacy; however, its application in the clinical practice was limited by poor water solubility, hepatotoxicity, and nephrotoxicity. In this study, a triptolide-loaded exosomes delivery system (TP-Exos) was constructed and its effects on the proliferation and apoptosis of SKOV3 cells in vitro and in vivo were observed. SKOV3-exosomes (SK-Exos) were collected by ultracentrifugation and ultrafiltration centrifugation. TP-Exos was constructed by sonication and ultrafiltration centrifugation. SK-Exos and TP-Exos were characterized by transmission electron microscopy, western blotting, nanoparticle-tracking analysis, and high-performance liquid chromatography. Cellular uptake of exosomes, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, bromodeoxyuridine (BrdU) cell proliferation assay, and cell apoptosis experiment were used to study the effect of TP-Exos on ovarian cancer in vitro. Tumor-targeting study of exosomes, monitoring the tumor volume of mice, and TdT-mediated dUTP Nick-End labeling (TUNEL) assay were used to evaluate the effect of TP-Exos on ovarian cancer in vivo. The toxicity of TP-Exos in vivo was evaluated by liver and kidney function and histopathology of major organs (heart, liver, spleen, lung, kidney, and ovary). The results revealed that TP-Exos not only have the general characteristics of exosomes but also have high drug encapsulation efficiency. Besides, PKH26 labeled exosomes (PKH26-Exos) could be uptaken by SKOV3 cells, and Dir labeled exosomes (Dir-Exos) could be enriched to the tumor site of tumor bearing mice. Furthermore, the cytotoxic and apoptotic effects on SKOV3 cells of TP-Exos were weaker than those of free TP, and tumor cell proliferation inhibition and tumor growth inhibition were stronger than that of free TP. Moreover, TP-Exos have toxic effect on liver and spleen. In conclusion, the TP-Exos could be a promising strategy for ovarian cancer, but they need to be further optimized to attenuate the damage to liver and spleen.
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Wu, X. R., P. Lan, X. J. Wu, and X. Gao. "P064 Exosomes from mesenchymal stromal cells reduce murine colonic inflammation via a macrophage-dependent mechanism." Journal of Crohn's and Colitis 14, Supplement_1 (January 2020): S166. http://dx.doi.org/10.1093/ecco-jcc/jjz203.193.
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Abstract Background Conventional treatments for inflammatory bowel disease (IBD) have multiple potential side effects. Therefore, alternative treatments are desperately needed. Exosomes from mesenchymal stromal cells (MSC-Exos) show potent immunomodulatory activities and protective effects in several diseases. Methods MSC-Exos were isolated from human bone marrow-derived mesenchymal stromal cells. Experimental colitis was induced by administration of dextran sulphate sodium (DSS) in C57BL/6 mice (male, 6–8 weeks), or 2, 4, 6-trinitrobenzenesulfonic acid (TNBS) in Balb/c mice (male, 7–9 weeks). Macrophage depletion was performed via intraperitoneal injection of clodronate liposomes. Colitic mice were treated intravenously with MSC-Exos or saline, and mortality and diverse disease signs were tracked. Results Systemic administration of MSC-Exos significantly mitigated colitis in mice. MSC-Exos treatment downregulated inflammatory responses, maintained intestinal barrier integrity and polarised M2 macrophages. Infused MSC-Exos mainly acted on colonic macrophages and their beneficial effect was blocked by macrophage depletion. Particularly, MSC-Exos were enriched in proteins involved in regulating multiple biological processes associated with the anti-colitic benefit of MSC-Exos. Conclusion MSC-Exos are critical regulators of immune/inflammatory responses and may be promising candidates for IBD treatment
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Wang, Xiwei, Yanmei Zhang, Xiaofeng Mu, Chloe Ran Tu, Yuet Chung, Sai Wah Tsao, Godfrey Chi-Fung Chan, et al. "Exosomes derived from γδ-T cells synergize with radiotherapy and preserve antitumor activities against nasopharyngeal carcinoma in immunosuppressive microenvironment." Journal for ImmunoTherapy of Cancer 10, no. 2 (February 2022): e003832. http://dx.doi.org/10.1136/jitc-2021-003832.
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BackgroundRadiotherapy is the first-line treatment for patients nasopharyngeal carcinoma (NPC), but its therapeutic efficacy is poor in some patients due to radioresistance. Adoptive T cell-based immunotherapy has also shown promise to control NPC; however, its antitumor efficacy may be attenuated by an immunosuppressive tumor microenvironment. Exosomes derived from γδ-T cells (γδ-T-Exos) have potent antitumor potentials. However, it remains unknown whether γδ-T-Exos have synergistic effect with radiotherapy and preserve their antitumor activities against NPC in an immunosuppressive tumor microenvironment.Methodsγδ-T-Exos were stained with fluorescent membrane dye, and their interactions with NPC were determined both in vitro and in vivo. NPC cell deaths were detected after treatment with γδ-T-Exos and/or irradiation. Moreover, effects of γδ-T-Exos on radioresistant cancer stem-like cells (CSCs) were determined. The therapeutic efficacy of combination therapy using γδ-T-Exos and irradiation on NPC tumor progression was also monitored in vivo. Finally, the tumor-killing and T cell-promoting activities of γδ-T-Exos were determined under the culture in immunosuppressive NPC supernatant.Resultsγδ-T-Exos effectively interacted with NPC tumor cells in vitro and in vivo. γδ-T-Exos not only killed NPC cells in vitro, which was mainly mediated by Fas/Fas ligand (FasL) and death receptor 5 (DR5)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathways, but also controlled NPC tumor growth and prolonged tumor-bearing mice survival in vivo. Furthermore, γδ-T-Exos selectively targeted the radioresistant CD44+/high CSCs and induced profound cell apoptosis. The combination of γδ-T-Exos with radiotherapy overcame the radioresistance of CD44+/high NPC cells and significantly improved its therapeutic efficacy against NPC in vitro and in vivo. In addition, γδ-T-Exos promoted T-cell migration into NPC tumors by upregulating CCR5 on T cells that were chemoattracted by CCR5 ligands in the NPC tumor microenvironment. Although NPC tumor cells secreted abundant tumor growth factor beta to suppress T-cell responses, γδ-T-Exos preserved their direct antitumor activities and overcame the immunosuppressive NPC microenvironment to amplify T-cell antitumor immunity.Conclusionsγδ-T-Exos synergized with radiotherapy to control NPC by overcoming the radioresistance of NPC CSCs. Moreover, γδ-T-Exos preserved their tumor-killing and T cell-promoting activities in the immunosuppressive NPC microenvironment. This study provides a proof of concept for a novel and potent strategy by combining γδ-T-Exos with radiotherapy in the control of NPC.
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HENRIKSSON, Maria L., Charlotta SUNDIN, Anna L. JANSSON, Åke FORSBERG, Ruth H. PALMER, and Bengt HALLBERG. "Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo." Biochemical Journal 367, no. 3 (November 1, 2002): 617–28. http://dx.doi.org/10.1042/bj20020714.
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Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.
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Yang, Hongwei, Meng Cong, Weixiao Huang, Jin Chen, Min Zhang, Xiaosong Gu, Cheng Sun, and Huilin Yang. "The Effect of Human Bone Marrow Mesenchymal Stem Cell-Derived Exosomes on Cartilage Repair in Rabbits." Stem Cells International 2022 (September 8, 2022): 1–12. http://dx.doi.org/10.1155/2022/5760107.
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Mesenchymal stem cells (MSCs) have shown chondroprotective effects in cartilage repair. However, side effects caused by MSC treatment limit their application in clinic. As a cell-free therapy, MSC-derived exosomes (EXOs) have attracted much more attention in recent years. In the present study, we prepared EXOs from human bone marrow mesenchymal stem cells (hBMSCs) and examined their therapeutic potentials in cartilage repair. Our results showed that the prepared extracellular vesicles exhibit classical features of EXOs, such as cup-like shape, around 100 nm diameter, positive protein markers (CD81, TSG101, and Flotillin 1), and ability of internalization. In primary chondrocytes, the treatment of hBMSC-EXOs markedly increases cell viability and proliferation in a dose-dependent manner. Moreover, wound healing assay showed that hBMSC-EXOs accelerate cell migration in primary chondrocytes. JC-1 staining revealed that the mitochondrial membrane potential was enhanced by hBMSC-EXOs, indicating cell apoptosis was decreased in the presence of hBMSC-EXOs. In rabbits with articular cartilage defects, local administration with hBMSC-EXOs facilitates cartilage regeneration as evidenced by gross view and hematoxylin-eosin (H&E) and Saf-O/Fast Green staining. In addition, the International Cartilage Repair Society (ICRS) score was increased by the application of hBMSC-EXOs. Overall, our data indicate that the treatment with hBMSC-EXOs is a suitable cell-free therapy for treating cartilage defects, and these benefits are likely due to improved cell proliferation and migration in chondrocytes.
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HENRIKSSON, Maria L., Roland ROSQVIST, Maxim TELEPNEV, Hans WOLF-WATZ, and Bengt HALLBERG. "Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras." Biochemical Journal 347, no. 1 (March 27, 2000): 217–22. http://dx.doi.org/10.1042/bj3470217.
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We have examined the functional consequences of ADP-ribosyltransferase modification of Ras by the exoenzyme S (ExoS) protein of Pseudomonas aeruginosa. ExoS has been shown previously to ADP-ribosylate a number of proteins, including members of the Ras superfamily, which play an essential role in the processes of cell proliferation, differentiation, motility and cell division. HeLa and NIH3T3 cells were infected with ExoS protein, which was delivered via the type III secretion system of the heterologous host Yersinia pseudotuberculosis. Infection of mammalian cells with ExoS results in a change in the ratio of GTP/GDP bound directly to Ras in vivo. This ADP-ribosylation of Ras in vivo is mediated by the C-terminal domain of ExoS. Further, ExoS ADP-ribosylation of Ras in vivo inhibits activation of Ras and the ability to interact with the Ras binding domain of Raf upon stimulation with epidermal growth factor (EGF). In the present study, we show that ExoS activity does not interfere with EGF receptor phosphorylation itself, nor with the formation of a Grb2-activated Shc complex upon EGF stimulation, consistent with ExoS blockage of this mitogenic signalling pathway at the level of Ras. This is further supported by our observation of a substantial inhibition of extracellular signal-regulated kinase and protein kinase B/Akt kinase activation in response to EGF upon ExoS infection. In conclusion, in the present study, the consequences of ExoS infection on Ras effector pathway in vivo have been defined.
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Angus, Annette A., David J. Evans, Joseph T. Barbieri, and Suzanne M. J. Fleiszig. "The ADP-Ribosylation Domain of Pseudomonas aeruginosa ExoS Is Required for Membrane Bleb Niche Formation and Bacterial Survival within Epithelial Cells." Infection and Immunity 78, no. 11 (August 23, 2010): 4500–4510. http://dx.doi.org/10.1128/iai.00417-10.
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ABSTRACT Pseudomonas aeruginosa can establish a niche within the plasma membrane of epithelial cells (bleb niches) within which bacteria can survive, replicate, and swim at speeds detectable by real-time phase-contrast imaging. This novel virulence strategy is dependent on the bacterial type three secretion system (T3SS), since mutants lacking the T3SS needle or known T3SS effectors localize to perinuclear vacuoles and fail to replicate. Here, we determined which of the three effectors (ExoS, ExoT, or ExoY) were required for bleb niche formation and intracellular replication. PAO1 strains with mutations in exoS, exoT, exoY, or combinations thereof were compared to wild-type and complemented strains. P. aeruginosa exoS mutants, but not exoT or exoY mutants, lost the capacity for bleb niche formation and intracellular replication. Complementation with exoS rescued both phenotypes, either in the background of an exoS mutant or in a mutant lacking all three known effectors. Complementation with activity domain mutants of exoS revealed that the ADP-ribosyltransferase (ADP-r) activity of ExoS, but not the Rho-GAP activity nor the membrane localization domain (MLD) of ExoS, was required to elicit this phenotype. Membrane bleb niches that contained P. aeruginosa also bound annexin V-enhanced green fluorescent protein (EGFP), a marker of early apoptosis. These data show that P. aeruginosa bleb niches and intracellular survival involve ExoS ADP-r activity and implicate a connection between bleb niche formation and the known role(s) of ExoS-mediated apoptosis and/or Rab GTPase inactivation.
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Lykken, Guinevere L., GuoZhou Chen, Evan D. Brutinel, Lingling Chen, and Timothy L. Yahr. "Characterization of ExsC and ExsD Self-Association and Heterocomplex Formation." Journal of Bacteriology 188, no. 19 (October 1, 2006): 6832–40. http://dx.doi.org/10.1128/jb.00884-06.
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ABSTRACT Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is induced by calcium depletion and is positively regulated by the ExsA transcriptional activator and negatively regulated by the ExsD antiactivator. Under conditions permissive for expression of the T3SS, the negative regulatory activity of ExsD is antagonized by a direct binding interaction with ExsC. In the present study, the ExsC-ExsD binding interaction was characterized. Individually, both ExsC and ExsD form self-associated complexes, as judged by bacterial monohybrid and gel filtration experiments. A mixture of purified ExsC and ExsD readily formed a complex that elutes from gel filtration medium as a single included peak. The calculated molecular weight of the ExsC-ExsD complex is consistent with a complex containing multiple copies of ExsC and ExsD. Isothermic titration calorimetry experiments found formation of the ExsC-ExsD complex to be thermodynamically favorable, with a Kd of ∼18 nM and a likely binding ratio of 1:1. To identify amino acid residues important for the regulatory activities of ExsC and ExsD, self-association, and complex formation, charged-cluster mutagenesis was performed. Two of the resulting ExsD charged-cluster mutants (DM2 and DM3) demonstrated a hyperrepressive phenotype for expression of the T3SS. By two-hybrid and copurification assays, the DM3 mutant was found to be impaired in its interaction with ExsC. This finding demonstrates that the binding of ExsC to ExsD is required for transcriptional induction of the T3SS under calcium-limiting growth conditions.
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Cheng, Hai-Ping, and Graham C. Walker. "Succinoglycan Production by Rhizobium meliloti Is Regulated through the ExoS-ChvI Two-Component Regulatory System." Journal of Bacteriology 180, no. 1 (January 1, 1998): 20–26. http://dx.doi.org/10.1128/jb.180.1.20-26.1998.
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ABSTRACT The Rhizobium meliloti exoS gene is involved in regulating the production of succinoglycan, which plays a crucial role in the establishment of the symbiosis between R. melilotiRm1021 and its host plant, alfalfa. TheexoS96::Tn5 mutation causes the upregulation of the succinoglycan biosynthetic genes, thereby resulting in the overproduction of succinoglycan. Through cloning and sequencing, we found that the exoS gene is a close homolog of theAgrobacterium tumefaciens chvG gene, which has been proposed to encode the sensor protein of the ChvG-ChvI two-component regulatory system, a member of the EnvZ-OmpR family. Further analyses revealed the existence of a newly discovered A. tumefaciens chvI homolog located just upstream of the R. meliloti exoS gene. R. meliloti ChvI may serve as the response regulator of ExoS in a two-component regulatory system. By using ExoS-specific antibodies, it was found that the ExoS protein cofractionated with membrane proteins, suggesting that it is located in the cytoplasmic membrane. By using the same antibodies, it was shown that the exoS96::Tn5 allele encodes an N-terminal truncated derivative of ExoS. The cytoplasmic histidine kinase domain of ExoS was expressed in Escherichia coli and purified, as was the R. meliloti ChvI protein. The ChvI protein autophosphorylated in the presence of acetylphosphate, and the ExoS cytoplasmic domain fragment autophosphorylated at a histidine residue in the presence of ATP. The ChvI protein was phosphorylated in the presence of ATP only when the histidine kinase domain of ExoS was also present. We propose a model for regulation of succinoglycan production by R. meliloti through the ExoS-ChvI two-component regulatory system.
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Soliman, Hagar M., Ghada A. Ghonaim, Shaza M. Gharib, Hitesh Chopra, Aya K. Farag, Mohamed H. Hassanin, Abdalrazeq Nagah, et al. "Exosomes in Alzheimer’s Disease: From Being Pathological Players to Potential Diagnostics and Therapeutics." International Journal of Molecular Sciences 22, no. 19 (October 6, 2021): 10794. http://dx.doi.org/10.3390/ijms221910794.
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Exosomes (EXOs) were given attention as an extracellular vesicle (EV) with a pivotal pathophysiological role in the development of certain neurodegenerative disorders (NDD), such as Parkinson’s and Alzheimer’s disease (AD). EXOs have shown the potential to carry pathological and therapeutic cargo; thus, researchers have harnessed EXOs in drug delivery applications. EXOs have shown low immunogenicity as natural drug delivery vehicles, thus ensuring efficient drug delivery without causing significant adverse reactions. Recently, EXOs provided potential drug delivery opportunities in AD and promising future clinical applications with the diagnosis of NDD and were studied for their usefulness in disease detection and prediction prior to the emergence of symptoms. In the future, the microfluidics technique will play an essential role in isolating and detecting EXOs to diagnose AD before the development of advanced symptoms. This review is not reiterative literature but will discuss why EXOs have strong potential in treating AD and how they can be used as a tool to predict and diagnose this disorder.
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Zhao, Bin, Xiaodong Li, Xiaomin Shi, Xueqin Shi, Wei Zhang, Gaofeng Wu, Xujie Wang, Linlin Su, and Dahai Hu. "Exosomal MicroRNAs Derived from Human Amniotic Epithelial Cells Accelerate Wound Healing by Promoting the Proliferation and Migration of Fibroblasts." Stem Cells International 2018 (July 25, 2018): 1–10. http://dx.doi.org/10.1155/2018/5420463.
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Previous work in our laboratory demonstrated that exosomes derived from human amniotic epithelial cells (hAECs) accelerated wound healing by promoting the proliferation and migration of fibroblasts. It is reported that exosomes, which are carriers of the microRNAs (miRNAs) and proteins, play an important role in the regulation of cell-to-cell communication. However, it is still unclear precisely which molecule or which group of molecules carried within hAEC-derived exosomes (hAEC-Exos) mediated wound healing. Here, we explored purified hAEC-Exos together with either proteinase K (PROse) or RNase A on the effect of fibroblasts and cutaneous wound healing. Our experiments demonstrated that hAEC-Exos were positive for exosomal markers CD9, CD63, and CD81. Also, we found that hAEC-Exos could be internalized by fibroblasts and then stimulated cell migration and proliferation. However, the promotive effect of hAEC-Exos was abolished by pretreating hAEC-Exos with RNase A, not PROse. Importantly, in vivo wound healing assay showed that local injection of hAEC-Exos or PROse pretreated hAEC-Exos at skin wounds significantly accelerated wound healing. Our findings revealed an important role of exosomal miRNAs in wound healing.
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Hu, Hui, Xiaowei Hu, Lin Li, Yan Fang, Yan Yang, Jingjing Gu, Jiadong Xu, and Lisheng Chu. "Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Promote Angiogenesis in Ischemic Stroke Mice via Upregulation of MiR-21-5p." Biomolecules 12, no. 7 (June 24, 2022): 883. http://dx.doi.org/10.3390/biom12070883.
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Exosomes derived from bone mesenchymal stem cells (BMSC-Exos) are one of the main factors responsible for the therapeutic effects of BMSCs. The study aimed to investigate whether BMSC-Exos could promote angiogenesis in ischemic stroke mice via miR-21-5p. In ischemic stroke mice, the therapeutic effects of BMSC-Exos were evaluated by neurological functions and infarct volume. Microvessel density was detected by BrdU/vWF immunofluorescence staining. In in vitro experiments, the proangiogenic effects of BMSC-Exos were assessed via proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). The miR-21-5p inhibitor was transfected into BMSCs using Lipofectamine 2000. miR-21-5p expression was detected by qRT-PCR. The expression levels of VEGF, VEGFR2, Ang-1, and Tie-2 were determined by Western blot. BMSC-Exos significantly improved neurological functions and reduced infarct volume, upregulated microvessel density, and miR-21-5p expression after cerebral ischemia. In vitro assays revealed that BMSC-Exos enhanced HUVECs functions including proliferation, migration, and tube formation. BMSC-Exos increased the expression levels of VEGF, VEGFR2, Ang-1, and Tie-2. However, the proangiogenic effects of BMSC-Exos on HUVECs were reversed by the miR-21-5p inhibitor. These results suggest that BMSC-Exos could promote angiogenesis via miR-21-5p upregulation, making them an attractive treatment strategy for stroke recovery.
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Zhou, Shiyu, Yu Lan, Yuqun Li, Zhenxing Li, Jinding Pu, and Liping Wei. "Hypoxic Tumor-Derived Exosomes Induce M2 Macrophage Polarization via PKM2/AMPK to Promote Lung Cancer Progression." Cell Transplantation 31 (January 2022): 096368972211069. http://dx.doi.org/10.1177/09636897221106998.
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Hypoxia is a major regulator of tumor aggressiveness and metastasis in cancer progression. Exosomes (exos) play an important role in the communication between lung cancer and hypoxic microenvironment. However, the underlying mechanisms are largely undefined. Exos were isolated from A549 cells under hypoxia conditions. Transmission electron microscopy and nanoparticle tracking analysis were carried out to characterize exos. CCK-8 assay, flow cytometry, Western blot, wound healing, and transwell assays were performed to assess the proliferation, apoptosis, migration, and invasion of A549 cells, respectively. The M2 polarization of macrophages was evaluated by RT-qPCR and Western blot analysis. In vivo nude mice model was established to determine the regulatory effect of hypoxia/exos on the progression of lung cancer. Hypoxic A549 cell-derived exos (hypoxia/exos) promoted the proliferation and migration, and inhibited the apoptosis in A549 cells. The expression of PKM2 was significantly upregulated in hypoxia/exos. Hypoxic exosomal PKM2 induced M2 polarization of macrophages by activating AMPK pathway. Co-culture with hypoxia/exos-treated macrophages enhanced the migration, invasion, and epithelial-mesenchymal transition (EMT) in A549 cells. Moreover, treatment with hypoxia/exos facilitated the tumor growth and lung metastasis of A549 cells. Our findings reveal that hypoxic exosomal PKM2 induces M2 macrophage polarization via AMPK pathway, and thus exerts a simulative effect on the growth and metastasis of lung carcinoma.
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Rui, Ke, Ziwei Shen, Na Peng, Futao Zhao, Yuan Tang, Shiyi Liu, Xinyi Xu, et al. "Olfactory ecto-mesenchymal stem cell-derived exosomes ameliorate murine Sjögren’s syndrome via suppressing Tfh cell response." Rheumatology and Immunology Research 3, no. 4 (December 1, 2022): 198–207. http://dx.doi.org/10.2478/rir-2022-0035.
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Abstract Objectives To investigate the effect of olfactory ecto-mesenchymal stem cell-derived exosomes (OE-MSC-Exos) on T follicular helper (Tfh) cell response and their implication in treating experimental Sjögrens syndrome (ESS). Methods C57BL/6 mice were immunized with salivary glands (SG) proteins to induce ESS mouse model. OE-MSC-Exos were added to the Tfh cell polarization condition, and the proportion of Tfh cells was detected by FCM. The PD-L1 of OE-MSCs was silenced with small interfering RNA to extract siPD-L1-OE-MSC-Exos. Results We found that transfer of OE-MSC-Exos markedly attenuated disease progression and reduced Tfh cell response in mice with ESS. In culture, OE-MSC-Exos potently inhibited the differentiation of Tfh cells from naïve T cells. Moreover, OE-MSC-Exos expressed high level of the ligand for the programmed cell death protein 1 (PD-L1), knocking down PD-L1 expression in OE-MSC-Exos significantly decreased their capacity to suppress Tfh cell differentiation in vitro. Consistently, transfer of OE-MSC-Exos with PD-L1 knockdown exhibited profoundly diminished therapeutic effect in ESS mice, accompanied with sustained Tfh cell response and high levels of autoantibody production. Conclusion Our results suggest that OE-MSC-Exos may exert their therapeutic effect in ameliorating ESS progression via suppressing Tfh cell response in a PD-L1-dependent manner.
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DiStefano, Tyler J., Keti Vaso, Christopher J. Panebianco, George Danias, Henry N. Chionuma, Kuriakose Kunnath, Stylianos Z. Karoulias, et al. "Hydrogel-Embedded Poly(Lactic-co-Glycolic Acid) Microspheres for the Delivery of hMSC-Derived Exosomes to Promote Bioactive Annulus Fibrosus Repair." CARTILAGE 13, no. 3 (July 2022): 194760352211139. http://dx.doi.org/10.1177/19476035221113959.
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Objective Intervertebral disk degeneration is a prevalent postoperative complication after discectomy, underscoring the need to develop preventative and bioactive treatment strategies that decelerate degeneration and seal annulus fibrosus (AF) defects. Human mesenchymal stem cell–derived exosomes (MSC-Exos) hold promise for cell-free bioactive repair; however, their ability to promote AF repair is poorly understood. The objective of this study was to evaluate the ability of MSC-Exos to promote endogenous AF repair processes and integrate MSC-Exos within a biomaterial delivery system. Design We characterize biophysical and biochemical properties of normoxic (Nx) and hypoxic (Hx) preconditioned MSC-Exos from young, healthy donors and examine their effects on AF cell proliferation, migration, and gene expression. We then integrate a poly(lactic- co-glycolic acid) microsphere (PLGA µSphere) delivery platform within an interpenetrating network hydrogel to facilitate sustained MSC-Exo delivery. Results Hx MSC-Exos led to a more robust response in AF cell proliferation and migration than Nx MSC-Exos and was selected for a downstream protection experiment. Hx MSC-Exos maintained a healthy AF cell phenotype under a TNFα challenge in vitro and attenuated catabolic responses. In all functional assays, AF cell responses were more sensitive to Hx MSC-Exos than Nx MSC-Exos. PLGA µSpheres released MSC-Exos over a clinically relevant timescale without affecting hydrogel modulus or pH upon initial embedment and µSphere degradation. Conclusions This MSC-Exo treatment strategy may offer benefits of stem cell therapy without the need for exogenous stem cell transplantation by stimulating cell proliferation, promoting cell migration, and protecting cells from the degenerative proinflammatory microenvironment.
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Chen, Jianbin, Can Zhang, Shouye Li, Zheming Li, Xiaojing Lai, and Qingqing Xia. "Exosomes Derived from Nerve Stem Cells Loaded with FTY720 Promote the Recovery after Spinal Cord Injury in Rats by PTEN/AKT Signal Pathway." Journal of Immunology Research 2021 (July 14, 2021): 1–13. http://dx.doi.org/10.1155/2021/8100298.
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Background. Spinal cord injury (SCI) remains a challenge owing to limited therapies. The exosome of neural stem cells (NSCs-Exos) and FTY720 transplantation could improve SCI effectively. However, the effect and mechanism of NSCs-Exos combined with FTY720 (FTY720-NSCs-Exos) transplantation in the treatment of SCI are not fully understood. Methods. Sprague Dawley rats (8-week-old) were used to establish the SCI model, followed by the treatment of NSCs-Exos, FTY720, and FTY720-NSCs-Exos. The effect of FTY720, NSCs-Exos, and FTY720-NSCs-Exos combination treatment on hindlimb function, pathological changes, apoptosis activity, and the expression of spinal edema-related proteins and apoptosis-related proteins in SCI models were investigated by BBB scoring, HE staining, TUNEL staining and immunohistochemistry, and Western blotting. Meanwhile, the effect of these treatments on spinal cord microvascular endothelial cells (SCMECs) was detected under hypoxic circumstance. Results. Our results found that FTY720-NSCs-Exos could alleviate pathological alterations and ameliorate the hindlimb function and oxygen insufficiency in model mice after SCI. In addition, exosomes could ameliorate the morphology of neurons, reduce inflammatory infiltration and edema, decrease the expression of Bax and AQP-4, upregulate the expression of claudin-5 and Bcl-2, and inhibit cell apoptosis. At the same time, in vitro experiments showed that FTY720-NSCs-Exos could protect the barrier of SCMECs under hypoxic circumstance, and the mechanism is related to PTEN/AKT pathway. Conclusion. FTY720-NSCs-Exos therapy displayed a positive therapeutic effect on SCI by regulating PTEN/AKT pathway and offered a new therapy for SCI.
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Stirling, Fiona R., and Tom J. Evans. "Effects of the type III secreted pseudomonal toxin ExoS in the yeast Saccharomyces cerevisiae." Microbiology 152, no. 8 (August 1, 2006): 2273–85. http://dx.doi.org/10.1099/mic.0.28831-0.
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Pseudomonas aeruginosa secretes a number of toxins by a type III system, and these are important in virulence. One of them, ExoS, is a bifunctional toxin, with a GTPase-activating protein domain, as well as ADP ribosyltransferase (ADPRT) activity. These two domains have numerous potential cellular targets, but the overall mechanism of ExoS action remains unclear. The effects of ExoS in a simple eukaryotic system, the yeast Saccharomyces cerevisiae, using a tetracycline-regulated expression system were studied. This system allowed controlled expression of ExoS in yeast, which was not possible using a galactose-induced system. ExoS was found to be an extremely potent inhibitor of yeast growth, and to be largely dependent on the activity of its ADPRT domain. ExoS produced a dramatic alteration in actin distribution, with the appearance of large aggregates of cortical actin, and thickened disorganized cables, entirely dependent on the ADPRT domain. This phenotype is suggestive of actin stabilization, which was verified by showing that the cortical aggregates of actin induced by ExoS were resistant to treatment with latrunculin A, an agent that prevents actin polymerization. ExoS increased the numbers of mating projections produced following growth arrest with mating pheromone, and prevented subsequent DNA replication, an effect that is again dependent on the ADPRT domain. Following pheromone removal, ExoS produced altered development of the mating projections, which became elongated with a swollen bud-like tip. These results suggest alternative pathways for ExoS action in eukaryotic cells that may result from activation of small GTPases, and this yeast expression system is well suited to explore these pathways.
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Jia, Jinghua, Yanping Wang, Lei Zhou, and Shouguang Jin. "Expression of Pseudomonas aeruginosa Toxin ExoS Effectively Induces Apoptosis in Host Cells." Infection and Immunity 74, no. 12 (September 11, 2006): 6557–70. http://dx.doi.org/10.1128/iai.00591-06.
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ABSTRACT Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Invasive strains of P. aeruginosa are known to induce apoptosis at a high frequency in HeLa cells and in many other cell lines, a process that is dependent on the ADP-ribosylation (ADPRT) activity of a type III secreted protein ExoS. In our previous report, it was proposed that P. aeruginosa secreting ExoS, upon infection, shuts down host cell survival signal pathways by inhibiting ERK1/2 and p38 activation, and it activates proapoptotic pathways through activation of JNK1/2, leading ultimately to cytochrome c release and activation of caspases. In this study, we demonstrate that the expression of ExoS in HeLa cells by eukaryotic expression vector effectively caused apoptosis in an ADPRT activity-dependent manner, indicating that ExoS alone is sufficient to trigger apoptotic death of host cells independent of any other bacterial factors. By expressing an EGFP-ExoS fusion protein, we were able to directly correlate the death of HeLa cells with the presence of intracellular ExoS and further proved the dependence of this process on both JNK activation and mitochondrial proapoptotic event. The cellular pathway responsible for the ExoS-induced cytotoxicity appears to be well conserved, since the expression of the ADPRT-competent ExoS also induced rapid cell death in the Drosophila melanogaster S2 cell lines. The presented study not only highlights the ability of ExoS ADPRT to modulate host cell signaling, eventually leading to apoptosis, but also establishes ExoS as a valuable tool, in principle, for the elucidation of apoptosis mechanisms.
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Wang, Shan-zheng, Jun Jia, and Chang-hong Chen. "lncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived from Adipose-Derived Stem Cells for the Treatment of Osteoporosis." Stem Cells International 2021 (October 19, 2021): 1–17. http://dx.doi.org/10.1155/2021/7690006.
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Background. Osteoporosis is a worldwide medical and socioeconomic burden characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study was aimed at investigating the role of ADSCs-Exos and a novel long noncoding RNA KCNQ1OT1 played in osteoporosis as well as the underlying mechanism. Methods. Primary osteoblasts were treated with different doses of tumor necrosis factor-α (TNF-α) (0, 1, 2.5, 5, and 10 ng/ml) and then cocultured with ADSCs-Exos or exosome-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3, and Bax was determined by Western blot. Cell viability and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results. TNF-α dose-dependently increased miR-141-5p expression, inhibited viability, and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or cocultured with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of KCNQ1OT1 by luciferase reporter assay. Conclusions. ADSCs-Exos can attenuate cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos have a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos can be promising agents in osteoporosis treatment.
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Yasmin, Lubna, Jeffrey L. Veesenmeyer, Maureen H. Diaz, Matthew S. Francis, Christian Ottmann, Ruth H. Palmer, Alan R. Hauser, and Bengt Hallberg. "Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins." Biochemical Journal 427, no. 2 (March 29, 2010): 217–24. http://dx.doi.org/10.1042/bj20100043.
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14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395–408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the ‘roof’ of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.
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Li, Yanqiao, Guangxing Wang, Qian Wang, Yun Zhang, Lei Cui, and Xin Huang. "Exosomes Secreted from Adipose-Derived Stem Cells Are a Potential Treatment Agent for Immune-Mediated Alopecia." Journal of Immunology Research 2022 (February 3, 2022): 1–14. http://dx.doi.org/10.1155/2022/7471246.
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Background. Alopecia has become an exceedingly prevalent dermatological disorder. Etiologically, infection (bacterial and fungal infection), inflammation, and immune dysregulation are the main causes of immune-mediated hair loss. Treating hair loss has remained challenging as the available therapies are limited. Exosomes from adipose-derived stem cells (ADSC-Exos) have been used for treating neurodegenerative diseases and autoimmune diseases and in wound-healing treatments. However, the function and mechanism of ADSC-Exos in alopecia treatment remain unclear. This study is aimed at investigating the effects of ADSC-Exos on hair growth in vitro and in vivo for potentially treating immune-mediated alopecia and further exploring the underlying mechanism. Methods. Cell proliferation, migration, and apoptosis of dermal papilla cells (DPCs) that were treated with ADSC-Exos were detected using the cell counting kit-8 (CCK-8) assay, scratch wound-healing assay, and flow cytometry assay, respectively. A C57BL/6 hair-depilated mouse model was established in vivo; then, ADSC-Exos were subcutaneously injected alone or in combined with minoxidil. The effects of ADSC-Exos on hair growth, pathological changes, and the related mechanism were investigated by HE staining, quantitative real‐time PCR (qRT-PCR), western blotting, and RNA sequencing (RNA-seq). Results. ADSC-Exos significantly promoted DPC proliferation and migration while also reducing apoptosis. In addition, compared with the control group, ADSC-Exos-treated mice had better hair growth, more hair follicles (HFs) and thicker dermis. RNA-seq revealed that the miR-22 and TNF-α signaling pathways were markedly downregulated in DPCs after ADSC-Exos treatment. In addition, according to qRT-PCR and western blotting results, the Wnt/β-catenin signaling pathway was activated in the skin of ADSC-Exos-treated mice. Conclusion. ADSC-Exos therapy positively affected the promotion of hair regrowth by regulating miR-22, the Wnt/β-catenin signaling pathway, and the TNF-α signaling pathway, implying that ADSC-Exos could be a promising cell-free therapeutic strategy for immune-mediated alopecia.
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Rui, Shunli, Yi Yuan, Chenzhen Du, Peiyang Song, Yan Chen, Hongyan Wang, Yahan Fan, David G. Armstrong, Wuquan Deng, and Ling Li. "Comparison and Investigation of Exosomes Derived from Platelet-Rich Plasma Activated by Different Agonists." Cell Transplantation 30 (January 1, 2021): 096368972110178. http://dx.doi.org/10.1177/09636897211017833.
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PRP-Exos are nanoscale cup-shaped vesicles that carry a variety of proteins, mRNAs, microRNAs, and other bioactive substances. PRP-Exos can be formed through several induction pathways, which determine their molecular profiles and facilitate their tailormade participation in intercellular communication. Currently, little is known on how the PRP-Exos activation method influences the quality and quantity of PRP-Exos. The present study aims to observe and analyze the number, profile, and growth factors of PRP-Exos through TEM, Nanoflow, and WB after PRP activation and compare the difference in function of PRP-Exos on HUVECs, with different stimuli (calcium gluconate, thrombin, or both). We found that PRP activated with both thrombin and calcium gluconate harvested the highest concentration of exosomes [(7.16 ± 0.46) × 1010 particles/ml], compared to thrombin group [(4.87 ± 0.15) × 1010 particles/ml], calcium gluconate group [(5.85 ± 0.43) × 1010 particles/ml], or saline group [(7.52 ± 0.19) × 109 particles/ml], respectively ( P < 0.05) via Nanoflow analysis. The WB analysis showed that cytokines (VEGF, PDGFBB, bFGF, TGF-β) are differentially encapsulated in PRP-Exos, depending on the PRP stimulus, in which the mixture-PRP-Exos yielded the highest concentration of cytokines. In the function assay of PRP-Exos on HUVECs, the mixture-PRP-Exos promoted HUVECs proliferation, increased HUVECs migration, promoted the formation of vessel-like by HUVECs via the AKT ERK signal pathway more dramatically, compared with other groups. In summary, our studies showed that PRP activated by the mixture of calcium gluconate and thrombin harvested the best quality of exosomes which had the top biological functions. This study provides a protocol for selecting appropriate PRP activators to obtain high-quality exosomes for future applications.
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Wang, Jing, Junwen Wang, Xinyan Li, and Kai Shu. "Cell-Derived Exosomes as Therapeutic Strategies and Exosome-Derived microRNAs as Biomarkers for Traumatic Brain Injury." Journal of Clinical Medicine 11, no. 11 (June 5, 2022): 3223. http://dx.doi.org/10.3390/jcm11113223.
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Traumatic brain injury (TBI) is a complex, life-threatening condition that causes mortality and disability worldwide. No effective treatment has been clinically verified to date. Achieving effective drug delivery across the blood–brain barrier (BBB) presents a major challenge to therapeutic drug development for TBI. Furthermore, the field of TBI biomarkers is rapidly developing to cope with the many aspects of TBI pathology and enhance clinical management of TBI. Exosomes (Exos) are endogenous extracellular vesicles (EVs) containing various biological materials, including lipids, proteins, microRNAs, and other nucleic acids. Compelling evidence exists that Exos, such as stem cell-derived Exos and even neuron or glial cell-derived Exos, are promising TBI treatment strategies because they pass through the BBB and have the potential to deliver molecules to target lesions. Meanwhile, Exos have decreased safety risks from intravenous injection or orthotopic transplantation of viable cells, such as microvascular occlusion or imbalanced growth of transplanted cells. These unique characteristics also create Exos contents, especially Exos-derived microRNAs, as appealing biomarkers in TBI. In this review, we explore the potential impact of cell-derived Exos and exosome-derived microRNAs on the diagnosis, therapy, and prognosis prediction of TBI. The associated challenges and opportunities are also discussed.
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HENRIKSSON, Maria Lena, Ulrika TROLLÉR, and Bengt HALLBERG. "14-3-3 proteins are required for the inhibition of Ras by exoenzyme S." Biochemical Journal 349, no. 3 (July 25, 2000): 697–701. http://dx.doi.org/10.1042/bj3490697.
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14-3-3 proteins play a regulatory role and participate in both signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine ligands, such as Raf-1 kinase and Bad, by recognizing the phosphorylated consensus motif, Arg-Ser-Xaa-pSer-Xaa-Pro (where ‘Xaa’ represents ‘any residue’, and ‘pSer’ is ‘phosphoserine’) . However, 14-3-3 proteins must bind unphosphorylated ligands, such as glycoprotein Ibα and Pseudomonas aeruginosa exoenzyme S (ExoS), since it has been suggested that specific residues of 14-3-3 proteins are required for activation of ExoS. Furthermore, an unphosphorylated peptide derived from a phage display library inhibited the binding of both ExoS and Raf-1 to 14-3-3, and bound within the same conserved amphipathic groove on the surface of 14-3-3 as the Raf-derived phosphopeptide (pS-Raf-259). In the present study we identify the interaction site on ExoS for 14-3-3, and show that ExoS and 14-3-3 do indeed interact in vivo. In addition, we show that this interaction is critical for the ADP-ribosylation of Ras by ExoS, both in vitro and in vivo. Loss of the 14-3-3 binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras, and displays reduced killing activity.
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Teleb, Rasha S., Amal Abdul-Hafez, Amira Othman, Ahmed El-Abd Ahmed, Abdelrahman A. Elsaid, Hattan Arif, Ahmed A. Zarea, et al. "Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness." Cells 12, no. 2 (January 7, 2023): 250. http://dx.doi.org/10.3390/cells12020250.
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Background: Mesenchymal stem cells (MSCs) have been used for ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) to maintain their primitive characters and long-term reconstitution abilities during transplantation. Therapeutic effects of MSCs mainly rely on paracrine mechanisms, including secretion of exosomes (Exos). The objective of this study was to examine the effect of cord blood plasma (CBP)-derived Exos (CBP Exos) and Placental MSCs-derived Exos (MSCs Exos) on the expansion of UCB HSCs to increase their numbers and keep their primitive characteristics. Methods: CD34+ cells were isolated from UCB, cultured for 10 days, and the expanded HSCs were sub-cultured in semisolid methylcellulose media for primitive colony forming units (CFUs) assay. MSCs were cultured from placental chorionic plates. Results: CBP Exos and MSCs Exos compared with the control group significantly increased the number of total nucleated cells (TNCs), invitro expansion of CD34+ cells, primitive subpopulations of CD34+38+ and CD34+38−Lin− cells (p < 0.001). The expanded cells showed a significantly higher number of total CFUs in the Exos groups (p < 0.01). Conclusion: CBP- and placental-derived exosomes are associated with significant ex vivo expansion of UCB HSCs, while maintaining their primitive characters and may eliminate the need for transplantation of an additional unit of UCB.
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Taniue, Kenzui, Yusuke Mizukami, and Nobuyoshi Akimitsu. "Abstract B059: RNA exosome component EXOSC4 amplified in multiple cancer types is required for the pancreatic cancer cell survival via the regulation of BIK and SESN2 mRNA." Cancer Research 82, no. 22_Supplement (November 15, 2022): B059. http://dx.doi.org/10.1158/1538-7445.panca22-b059.
Full textAbstract:
Abstract RNA decay plays a crucial role in the post-transcriptional regulation of gene expression. The RNA exosome is a multi-subunit ribonuclease complex that is evolutionally conserved and the major cellular machinery for the surveillance, processing, degradation, and turnover of diverse RNAs essential for cell viability. The RNA exosome is composed of a catalytically inactive barrel structure of nine core subunits (known as EXO9) that achieves its catalytic activity via the interaction with the exoribonuclease exosome component 10 (EXOSC10), the exo/endo-ribonuclease DIS3, and the two DIS3-like proteins (the exoribonucleases DIS3L and DIS3L2). Moreover, exosome activity is regulated by associated co-factors and RNA-binding adaptor proteins that recruit the exosome to its many targets. Here we performed integrated genomic and clinicopathological analyses of 27 RNA exosome components across 32 tumor types using The Cancer Genome Atlas PanCancer Atlas Studies’ datasets. We discovered that the EXOSC4 gene, which encodes a barrel component of the RNA exosome, was amplified across multiple cancer types. We further found that EXOSC4 alteration is associated with a poor prognosis in pancreatic cancer patients. Moreover, we demonstrated that EXOSC4 is required for the survival of pancreatic cancer cells. EXOSC4 also repressed BIK expression and destabilized SESN2 mRNA by promoting its degradation. Furthermore, knockdown of BIK and SESN2 could partially rescue pancreatic cells from the reduction in cell viability caused by EXOSC4 knockdown. Our study provides evidence for EXOSC4-mediated regulation of BIK and SESN2 mRNA in the survival of pancreatic tumor cells. Citation Format: Kenzui Taniue, Yusuke Mizukami, Nobuyoshi Akimitsu. RNA exosome component EXOSC4 amplified in multiple cancer types is required for the pancreatic cancer cell survival via the regulation of BIK and SESN2 mRNA [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B059.