ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Rab1b-GBF1-ARF1 secretory pathway axis is required for Birnavirus replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the G olgi c omplex (GC). In this work, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. Analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector G olgi-specific B FA resistance f actor 1 (GBF1), which activates the small GTPase A DP- r ibosylation f actor 1 (ARF1), is required for IBDV replication since inhibiting its activity by treatment with b re f eldin A (BFA) or G olgi c ide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative-mutant T31N over-expression hampered the IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnaviruses-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, being the lack of a transcriptionally active core the main differential feature. This structural trait, among others that resemble the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and have argued the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses. Here, we present original data showing the IBDV-induced GC reorganization and the crosstalk between IBDV and the Rab1b-GBF1-ARF1 mediated intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnaviruses-host cells and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

FBXW2 Inhibits Prostate Cancer Progression and Metastasis Via Promoting EGFR Ubiquitylation and Degradation

Background: The process of ubiquitylation, carried out by a three-step enzymatic cascade, maintains the balance of intracellular proteins and the stability of their functions. FBXW2 is a novel F-box protein, and acts as a tumor suppressor gene in lung cancer by directly degrading substrates or regulating the other E3 ligases. However, whether or how FBXW2 regulates prostate cancer (PCa) tumorigenesis and progression is still unclear.Methods: The expression levels of FBXW2 in PCa tissues and matched non-tumor tissues were detected by immunoblotting and qPCR. Cell counting kit-8, wound-healing assay, invasion assay, apoptosis and cell cycle assays, and animal experiments were performed to identify the function of FBXW2 in PCa cells. Half-life analysis, immunoprecipitation, and the in vivo ubiquitylation assay were performed to determine how FBXW2 regulates EGFR and its downstream signaling pathway.Results: FBXW2 expression was lowly expressed in highly-progressive PCa tissues and PCa cell lines. Augmented FBXW2 expression suppressed PCa cell progression and metastasis by repressing EGFR downstream proteins, while FBXW2 depletion had opposite effects. Overexpression of FBXW2 inhibited tumor growth, and even osteolytic bone destruction. Furthermore, FBXW2 and EGFR protein levels were inversely correlated in PCa cell lines and patient tumor tissues. EGFR protein level and its half-life was extended by FBXW2 depletion. While the basal level of EGFR was decreased, and its half-life was shortened upon overexpression of FBXW2-WT but not its dominant negative mutant. Intriguingly, FBXW2 promoted EGFR ubiquitylation and degradation via its binding motif (TSNNST) on EGFR, thereby elevated FBXW2 abrogated the PCa cell growth and metastasis caused by EGF stimulation.Conclusions: Collectively, our data shows that FBXW2 inhibits PCa cell progression and metastasis by promoting EGFR ubiquitylation and degradation, and would be a novel tumor suppressor for PCa.

HDL promotes adiponectin gene expression via the CAMKK/CAMKIV pathway

Adiponectin (APN) is an adipokine that protects against diabetes and atherosclerosis. High-density lipoprotein (HDL) mediates reverse cholesterol transport, which also protects against atherosclerosis. In this process, the human homolog of the B class type I scavenger receptor (SR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. The level of circulating adiponectin is positively correlated with the serum level of HDL-cholesterol. In this study, we investigated whether HDL stimulates the gene expression of adiponectin through the Ca²+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) cascade. Adiponectin expression was examined using real-time PCR and western blot analysis in 3T3-L1 cells incubated with HDL. CaMKIV activity was assessed by detection of activation loop phosphorylation (at Thr196 residue), and the effect of the constitutively active form, CaMKIVc, on adiponectin promoter activity was investigated. Our results showed that HDL stimulated APN gene expression via hSR-BI/CLA-1. Furthermore, we explored the signaling pathways by which HDL stimulated APN expression in 3T3-L1 cells. The stimulation of APN gene expression by HDL appears to be mediated by CaMKK, as STO-609, a specific inhibitor of CaMKK2, prevents this effect. We revealed that CaMKIVc increased APN gene transcriptional activity, and the CaMKIV dominant negative mutant blocked the effect of HDL on APN promoter activity. Finally, knockdown of hSR-BI/CLA-1 also cancelled the effect of HDL on APN gene expression. These results suggest that HDL has important role to improve the function of adipocytes by activating hSR-BI/CLA-1 and CaMKK/CaMKIV pathway is conceivable as one of the signaling pathways of this activation mechanism.

Oleamide, a Sleep-Inducing Supplement, Upregulates Doublecortin in Hippocampal Progenitor Cells via PPARα

Background: Doublecortin (DCX), a microtubule associated protein, has emerged as a central biomarker of hippocampal neurogenesis. However, molecular mechanisms by which DCX is regulated are poorly understood. Objective: Since sleep is involved with the acquisition of memory and oleamide or 9-Octadecenamide (OCT) is a sleep-inducing supplement in human, we examined whether OCT could upregulate DCX in hippocampal progenitor cells (HPCs). Methods: We employed real-time PCR, western blot, immunostaining, chromatin immunoprecipitation, lentiviral transduction in HPCs, and the calcium influx assay. Results: OCT directly upregulated the transcription of Dcx in HPCs via activation of peroxisome proliferator-activated receptor α (PPARα), a lipid-lowering transcription factor. We observed that, HPCs of Ppara-null mice displayed significant impairment in DCX expression and neuronal differentiation as compared to that of wild-type mice. Interestingly, treatment with OCT stimulated the differentiation process of HPCs in wild-type, but not Ppara-null mice. Reconstruction of PPARα in mouse Ppara-null HPCs restored the expression of DCX, which was further stimulated with OCT treatment. In contrast, a dominant-negative mutant of PPARα significantly attenuated the stimulatory effect of OCT on DCX expression and suppressed neuronal differentiation of human neural progenitor cells. Furthermore, RNA microarray, STRING, chromatin immunoprecipitation, site-directed mutagenesis, and promoter reporter assay have identified DCX as a new target of PPARα. Conclusion: These results indicate that OCT, a sleep supplement, directly controls the expression of DCX and suggest that OCT may be repurposed for stimulating the hippocampal neurogenesis.

The histone deacetylase activity of HDAC1/2 is required to safeguard zygotic genome activation in mice and cattle

ABSTRACTThe genome is transcriptionally inert at fertilization and must be activated through a remarkable developmental process called zygotic genome activation (ZGA). The gene expression pattern formed over the course of ZGA is required for establishing totipotency in early embryos and subsequent development. Substantial epigenetic reprogramming contributes significantly to the pronounced change in gene expression during ZGA, however the mechanism has yet to be resolved. Here, we find histone deacetylase 1 and 2 (HDAC1/2) are critical histone modifiers that regulate ZGA through the histone deacetylase activity. Notably, we show that H3K27ac level declines dramatically during ZGA with a dynamic change in its genome-wide distribution. In mouse embryos, ectopic expression of HDAC1/2 dominant negative mutant leads to a failure of H3K27ac removal and a developmental arrest at 2-cell stage. RNA-seq results reveal a remarkable transcriptomic change with 6565 differentially expressed genes identified. Further analysis shows 64% of down-regulated genes are ZGA genes and 49% of up-regulated genes are developmental genes. Low input ChIP-seq analysis exhibits an increase and decrease of H3K27ac enrichment at the promoter region of up- and down-regulated genes, respectively. Moreover, HDAC1 mutants prohibited removal of broad H3K4me3 domain via impeding the expression of Kdm5s during ZGA. Importantly, the developmental block can be greatly overcome through injection of Kdm5b mRNA and expression of the majority of dysregulated genes partially corrected. Similar functional significance of HDAC1/2 in ZGA is conserved in bovine embryos. Together, we propose that HDAC1/2 is indispensable for mouse and bovine ZGA via creating correct transcriptional repressive and active states.

Metformin Inhibits BMP9-Induced Proliferation and Differentiation of Human Lung Fibroblasts via AMPK Signaling

Adenosine monophosphosphate-activated protein kinase (AMPK) and its activator metformin were found to be involved in the regulation of fibroblast activation and pulmonary fibrosis. However, the regulatory mechanism has been undetermined. Recently, AMPK has been reported to exert its effect through inhibiting bone morphogenetic protein (BMP) pathway. In this study, HFL-1 cells were treated with metformin or specific AMPKα1 mutants, including constitutively activated mutant (AMPK-CA) and dominant negative mutant (AMPK-DN), combined with BMP9, and then the absorbance of these cells was measured by cell counting kit (CCK)-8 assay. The colony number of HFL-1 cells stimulated by metformin with or without BMP9 was examined by colony formation assay. The protein expressions of differentiated markers (α-smooth muscle actin, collagen I and collagen III) and the key molecules of BMP9 signaling, including activin receptor-like kinase (ALK) 1 and phosphorylated small mother against decapentaplegic (p-Smad)1/5, were also evaluated by western blot. Data revealed that BMP9 induced the proliferation and differentiation of HFL-1 cells which was suppressed by metformin or AMPK-CA. Meanwhile, the effect of metformin on BMP9-induced activation was counteracted by AMPK-DN. In addition, we found that the expressions of ALK1 and p-Smad1/5 induced by BMP9 were attenuated by metformin and AMPK-CA, whereas the inhibitory responses of metformin to the increased ALK1 and p-Smad1/5 were reduced by AMPK-DN. Accordingly, these results suggested that metformin mitigated BMP9-induced proliferation and differentiation of HFL-1 cells, which was achieved partly through the activation of AMPK and inhibition of ALK1/Smad1/5 signaling.

Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of human and murine LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

HGG-43. INTERROGATING THE ROLE OF PEA3 ONCOGENIC TRANSCRIPTION FACTORS IN PEDIATRIC HIGH-GRADE K27M GLIOMAS

Pediatric high-grade gliomas (HGGs) are an aggressive form of pediatric brain tumors which pose a grim five-year survival with little advancement in therapeutic efficacy, often requiring a multimodal therapeutic combination of chemotherapy, resection, and radiation. We have previously shown that proper function of ETS transcription factors is necessary for gliomagenesis in Ras/MAPK-driven pediatric gliomas. It is our hypothesis that ETS transcription factors are necessary for tumor initiation in HGGs by promoting the necessary glial cell fates in glioma. Further, we hypothesize that functional inhibition of ETS proteins following tumor formation will improve survival and outcome in HGG. Functional inhibition of ETS proteins using a competitive dominant-negative mutant was shown to completely rescue neural stem cell depletion, tumor formation and tumor-free survival in two rodent models of HGGs. Mechanistically, we show evidence that Pea3 factors may induce glial-cell fate by promoting Olig2 expression and activation of glial transcriptional programs. Indeed, transcriptomic analysis of ETS-perturbed HGG tumors revealed that Sox9 and Olig2 transcription factor networks were dependent on proper ETS function. Further, we show evidence that Etv5 can directly interact with promoter regions of glial fate master regulators in human primary glioma cell lines. To empirically determine the effect of Pea3 proteins on tumorigenesis, we have created a novel methodology for inducible gain- and loss-of-function genetic interrogation of these factors in vivo. Our survival results and combined single-cell RNA-sequencing of individual groups show that inhibition of the Pea3 family leads to a marked increase in survival in K27M glioma by regulating key features of glioblasts. All in all, our group provides evidence that the ETS family of transcription factors is necessary for glial specification of tumor cells and induce pro-glial transcriptional programs by activating OPC- and astrocyte-specific genes in K27M-driven tumors.

A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.