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BMC Veterinary Research May 2022Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune...
BACKGROUND
Sheep are an important livestock species worldwide and an essential large-animal model for animal husbandry and veterinary research. Understanding fundamental immune indicators, especially T-lymphocyte parameters, is necessary for research on sheep diseases and vaccines, to better understand the immune response to bacteria and viruses for reducing the use of antibiotics and improving the welfare of sheep. We randomly selected 36 sheep of similar ages to analyze cell-related immune indicators in peripheral blood mononuclear cells (PBMCs). The proportions of CD4 and CD8 T cells in PBMCs were detected by flow cytometry. We used Concanavalin A (Con A) and Phorbol-12-myristate-13-acetate (PMA)/Ionomycin to stimulate PBMCs, and measured the expression of IFN-γ, IL-4, and IL-17A using enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISpot). Simultaneously, PMA/Ionomycin/brefeldin A (BFA) was added to PBMCs, then the expression of IFN-γ, IL-4, and IL-17A was detected by flow cytometry after 4 h of culturing. In addition, we observed the proliferation of PBMCs stimulated with Con A for 3, 4, and 5 days.
RESULTS
The proportions of CD4 T lymphocytes (18.70 ± 4.21%) and CD8 T lymphocytes (8.70 ± 3.65%) were generally consistent among individuals, with a CD4/CD8 ratio of 2.40 ± 0.79. PBMCs produced high levels of IFN-γ, IL-4, and IL-17A after stimulation with PMA/Ionomycin and Con A. Furthermore, PMA/Ionomycin stimulation of PBMC yielded significantly higher cytokine levels than Con A stimulation. Flow cytometry showed that the level of IFN-γ (51.49 ± 11.54%) in CD8 T lymphocytes was significantly (p < 0.001) higher than that in CD4 T lymphocytes (14.29 ± 3.26%); IL-4 (16.13 ± 6.81%) in CD4 T lymphocytes was significantly (p < 0.001) higher than that in CD8 T lymphocytes (1.84 ± 1.33%), There was no difference in IL-17A between CD4 (2.83 ± 0.98%) and CD8 T lymphocytes (1.34 ± 0.67%). The proliferation of total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes continued to increase between days 3 and 5; however, there were no significant differences in proliferation between the cell types during the stimulation period.
CONCLUSIONS
Evaluating primary sheep immune indicators, especially T lymphocytes, is significant for studying cellular immunity. This study provided valuable data and theoretical support for assessing the immune response of sheep to pathogens and improving sheep welfare.
Topics: Animals; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Flow Cytometry; Interleukin-17; Interleukin-4; Ionomycin; Leukocytes, Mononuclear; Lymphocyte Activation; Sheep; Tetradecanoylphorbol Acetate
PubMed: 35513847
DOI: 10.1186/s12917-022-03268-7 -
Diabetologia Nov 2023Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested...
AIMS/HYPOTHESIS
Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca signalling with defective insulin maturation remain incompletely understood.
METHODS
We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised.
RESULTS
βS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca levels and glucose-stimulated Ca synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking.
CONCLUSIONS/INTERPRETATION
Taken together, these data highlight an important link between ER Ca homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway.
DATA AVAILABILITY
RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).
Topics: Mice; Humans; Animals; Proinsulin; Insulin-Secreting Cells; Diabetes Mellitus, Type 2; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Insulin; Glucose; Islets of Langerhans
PubMed: 37537395
DOI: 10.1007/s00125-023-05979-4 -
Developmental Biology Sep 2014Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain...
Myoblast fusion (a critical process by which muscles grow) occurs in a multi-step fashion that requires actin and membrane remodeling; but important questions remain regarding the spatial/temporal regulation of and interrelationship between these processes. We recently reported that the Rho-GAP, GRAF1, was particularly abundant in muscles undergoing fusion to form multinucleated fibers and that enforced expression of GRAF1 in cultured myoblasts induced robust fusion by a process that required GAP-dependent actin remodeling and BAR domain-dependent membrane sculpting. Herein we developed a novel line of GRAF1-deficient mice to explore a role for this protein in the formation/maturation of myotubes in vivo. Post-natal muscles from GRAF1-depleted mice exhibited a significant and persistent reduction in cross-sectional area, impaired regenerative capacity and a significant decrease in force production indicative of lack of efficient myoblast fusion. A significant fusion defect was recapitulated in isolated myoblasts depleted of GRAF1 or its closely related family member GRAF2. Mechanistically, we show that GRAF1 and 2 facilitate myoblast fusion, at least in part, by promoting vesicle-mediated translocation of fusogenic ferlin proteins to the plasma membrane.
Topics: Animals; Biological Transport; Brefeldin A; Cell Fusion; Cell Membrane; Cells, Cultured; GTPase-Activating Proteins; Golgi Apparatus; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Development; Muscle Fibers, Skeletal; Muscle Proteins; Myoblasts; Protein Synthesis Inhibitors; RNA Interference; RNA, Small Interfering; Vesicular Transport Proteins
PubMed: 25019370
DOI: 10.1016/j.ydbio.2014.06.025 -
PLoS Pathogens Sep 2023The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses...
The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.
Topics: Humans; Enterovirus; Monomeric GTP-Binding Proteins; ADP-Ribosylation Factor 1; HeLa Cells; Poliovirus; Viral Proteins; Enterovirus Infections; Antigens, Viral; Brefeldin A; Guanine Nucleotide Exchange Factors
PubMed: 37721955
DOI: 10.1371/journal.ppat.1011673 -
Bioengineered Feb 2022Cervical cancer is a common gynecological malignancy, and miR-133b is an abnormally expressed cervical cancer gene, which suggests that miR-133b may be involved in the...
Cervical cancer is a common gynecological malignancy, and miR-133b is an abnormally expressed cervical cancer gene, which suggests that miR-133b may be involved in the occurrence and development of cervical cancer. However, the underlying mechanism is still unclear. miR-133b was overexpressed or silenced in the cervical cancer cell line C33A. Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (ARFGEF1) was combined with overexpression of miR-133b in C33A cells. Cell Counting Kit-8, clone formation, and Transwell assays were performed to determine the influence of miR-133b and ARFGEF1 on clone formation, proliferation, migration, and invasion of C33A cells. The interaction between miR-133b and ARFGEF1 was verified using a luciferase reporter assay. Finally, the mRNA and protein expression of miR-133b and ARFGEF1 in the tumor and adjacent normal tissues of cervical cancer patients was detected by real-time quantitative PCR, Western blotting, and immunohistochemistry. The results indicated that miR-133b up-regulation suppressed the proliferation, invasion, migration, and clone formation abilities of C33A cells ( < 0.05). However, silence of miR-133b promoted the proliferation, invasion, and migration of C33A cells ( < 0.05). Clone formation ability of C33A cells was also elevated by miR-133b deficiency ( < 0.05). Moreover, miR-133b interacted with ARFGEF1 and repressed ARFGEF1 expression in C33A cells ( < 0.05). ARFGEF1 overexpression weakened miR-133b overexpression-mediated inhibition of proliferation, invasion, and migration of C33A cells ( < 0.05). miR-133b expression was decreased, and ARFGEF1 was up-regulated in tumor tissues of cervical cancer patients ( < 0.05). All results revealed that miR-133b suppresses cervical cancer progression by inhibiting proliferation, invasion, and migration of cervical cancer cells via targeting ARFGEF1. Thus, our study determined the mechanism of miR-133b in cervical cancer, and confirmed miR-133b/ARFGEF1 may become a potential therapeutic target for cervical cancer.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Guanine Nucleotide Exchange Factors; Humans; MicroRNAs; Neoplasm Invasiveness; Neoplasm Proteins; RNA, Neoplasm; Uterine Cervical Neoplasms
PubMed: 35048795
DOI: 10.1080/21655979.2022.2027063 -
MBio Apr 2024The STimulator of INterferon Genes (STING) constitutes a major DNA-sensing pathway that restricts HSV-1 infection in different models by activating type I interferon and...
The STimulator of INterferon Genes (STING) constitutes a major DNA-sensing pathway that restricts HSV-1 infection in different models by activating type I interferon and pro-inflammatory responses. To counteract STING, HSV-1 has evolved numerous strategies including mechanisms to interfere with its oligomerization, post-translational modifications, and downstream signaling. Previously, we demonstrated that STING is packaged in extracellular vesicles (EVs) produced from HSV-1-infected cells. These EVs activated antiviral responses in uninfected recipient cells and suppressed a subsequent HSV-1 infection in a STING-dependent manner. Here, we provide information on the packaging of STING in EVs and its exocytosis. We found that STING exocytosis did not occur in CD63 knockdown cells supporting that STING follows the CD63 exocytosis pathway. Consistently, we found that STING co-localized with CD63 in cytoplasmic globular structures and exosomal STING and CD63 co-fractionated. Both golgicide A and brefeldin A prevented STING exocytosis during HSV-1 infection suggesting that STING trafficking through the Golgi is required. A STING ligand was insufficient for STING exocytosis, and downstream signaling through TBK1 was not required. However, STING palmitoylation and tethering to the ER by STIM1 were required for STING exocytosis. Finally, we found that HSV-1 replication/late gene expression triggered CD63 exocytosis that was required for STING exocytosis. Surprisingly, HSV-2 strain G did not trigger CD63 or STING exocytosis as opposed to VZV and HCMV. Also, EVs from HSV-1(F)- and HSV-2(G)-infected cells displayed differences in their ability to restrict these viruses. Overall, STING exocytosis is induced by certain viruses and shapes the microenvironment of infection.IMPORTANCEExtracellular vesicles (EVs) are released by all types of cells as they constitute a major mechanism of intercellular communication. The packaging of specific cargo in EVs and the pathway of exocytosis are not fully understood. STING is a sensor of a broad spectrum of pathogens and a key component of innate immunity. STING exocytosis during HSV-1 infection has been an intriguing observation, raising questions of whether this is a virus-induced process, the purpose it serves, and whether it is observed after infection with other viruses. Here, we have provided insights into the pathway of STING exocytosis and determined factors involved. STING exocytosis is a virus-induced process and not a response of the host to the infection. Besides HSV-1, other herpes viruses triggered STING exocytosis, but HSV-2(G) did not. HSV-1 EVs displayed different restriction capabilities compared with HSV-2(G) EVs. Overall, STING exocytosis is triggered by viruses to shape the microenvironment of infection.
Topics: Humans; Exocytosis; Herpes Simplex; Herpesvirus 1, Human; Immunity, Innate; Membrane Proteins
PubMed: 38470056
DOI: 10.1128/mbio.00373-24 -
Cells Apr 2022Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized...
Neurons are highly polarized cells requiring precise regulation of trafficking and targeting of membrane proteins to generate and maintain different and specialized compartments, such as axons and dendrites. Disruption of the Golgi apparatus (GA) secretory pathway in developing neurons alters axon/dendritic formation. Therefore, detailed knowledge of the mechanisms underlying vesicles exiting from the GA is crucial for understanding neuronal polarity. In this study, we analyzed the role of Brefeldin A-Ribosylated Substrate (CtBP1-S/BARS), a member of the C-terminal-binding protein family, in the regulation of neuronal morphological polarization and the exit of membrane proteins from the Trans Golgi Network. Here, we show that BARS is expressed during neuronal development in vitro and that RNAi suppression of BARS inhibits axonal and dendritic elongation in hippocampal neuronal cultures as well as largely perturbed neuronal migration and multipolar-to-bipolar transition during cortical development in situ. In addition, using plasma membrane (PM) proteins fused to GFP and engineered with reversible aggregation domains, we observed that expression of fission dominant-negative BARS delays the exit of dendritic and axonal membrane protein-containing carriers from the GA. Taken together, these data provide the first set of evidence suggesting a role for BARS in neuronal development by regulating post-Golgi membrane trafficking.
Topics: Axons; Golgi Apparatus; Membrane Proteins; Neurons; trans-Golgi Network
PubMed: 35455998
DOI: 10.3390/cells11081320 -
International Journal of Molecular... Sep 2021Spinal muscular atrophy (SMA) is caused by homozygous () gene deletion, leaving a duplicate gene, , as the sole source of SMN protein. However, a defect in SMN2...
Spinal muscular atrophy (SMA) is caused by homozygous () gene deletion, leaving a duplicate gene, , as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-β and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.
Topics: Calcium Signaling; Cell Line; Endoplasmic Reticulum; Exons; Fibroblasts; Gene Expression; Golgi Apparatus; HEK293 Cells; Humans; Motor Neurons; Muscular Atrophy, Spinal; Protein Transport; RNA Splicing; RNA, Messenger; SMN Complex Proteins
PubMed: 34638572
DOI: 10.3390/ijms221910234 -
Acta Pharmaceutica Sinica. B May 2023Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as...
Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available.
PubMed: 37250169
DOI: 10.1016/j.apsb.2023.03.015 -
Journal of Cell Science Jul 2017Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether...
Tether complexes play important roles in endocytic and exocytic trafficking of lipids and proteins. In yeast, the multisubunit transport protein particle (TRAPP) tether regulates endoplasmic reticulum (ER)-to-Golgi and intra-Golgi transport and is also implicated in autophagy. In addition, the TRAPP complex acts as a guanine nucleotide exchange factor (GEF) for Ypt1, which is homologous to human Rab1a and Rab1b. Here, we show that human TRAPPC13 and other TRAPP subunits are critically involved in the survival response to several Golgi-disrupting agents. Loss of TRAPPC13 partially preserves the secretory pathway and viability in response to brefeldin A, in a manner that is dependent on ARF1 and the large GEF GBF1, and concomitant with reduced caspase activation and ER stress marker induction. TRAPPC13 depletion reduces Rab1a and Rab1b activity, impairs autophagy and leads to increased infectivity to the pathogenic bacterium in response to brefeldin A. Thus, our results lend support for the existence of a mammalian TRAPPIII complex containing TRAPPC13, which is important for autophagic flux under certain stress conditions.
Topics: A549 Cells; ADP-Ribosylation Factor 1; Anti-Bacterial Agents; Antigens, Neoplasm; Autophagy; Brefeldin A; Dysentery, Bacillary; Gene Knockdown Techniques; Golgi Apparatus; Guanine Nucleotide Exchange Factors; HEK293 Cells; HT29 Cells; HeLa Cells; Humans; Shigella flexneri; Vesicular Transport Proteins
PubMed: 28536105
DOI: 10.1242/jcs.199521