Rabies virus phosphoprotein P5 binding to BECN1 regulates self-replication by BECN1-mediated autophagy signaling pathway.
ABSTRACT: BACKGROUND:Rabies virus (RABV) is reported to encode five phosphoproteins (P), which are involved in viral genomic replication, axonal transport, oxidative stress, interferon antagonism, and autophagy induction. However, the functions of the different P proteins are poorly understood. METHODS:Immunofluorescence staining and western blot were performed to detect the autophagy activity, the form of ring-like structure, and the colocalization of BECN1 and P. Co-immunoprecipitation was performed to detect the interaction between P and BECN1. QRT-PCR and TCID50 assay were performed to detect the replication level of RABV. Small interfering RNA was used to detect the autophagy signaling pathway. RESULTS:We found that P5 attaches to N-terminal residues 1-139 of BECN1 (beclin1) on the BECN1 ring-like structure through amino acid residues 173-222 of P5. Subsequently, we found that P5-induced autophagosomes did not fuse with lysosomes. Becn1 silencing did not recover P5 overexpression-induced promotion of RABV replication. Mechanistically, RABV protein P?N82 (P5) induced incomplete autophagy via the BECN1-mediated signaling pathway. CONCLUSIONS:Our data indicate that P5 binding to the BECN1 ring benefits RABV replication by inducing BECN1 signaling pathway-dependent incomplete autophagy, which provides a potential target for antiviral drugs against RABV. Video abstract.
Project description:Autophagy is an essential component of host immunity and used by viruses for survival. However, the autophagy signaling pathways involved in virus replication are poorly documented. Here, we observed that rabies virus (RABV) infection triggered intracellular autophagosome accumulation and results in incomplete autophagy by inhibiting autophagy flux. Subsequently, we found that RABV infection induced the reduction of CASP2/caspase 2 and the activation of AMP-activated protein kinase (AMPK)-AKT-MTOR (mechanistic target of rapamycin) and AMPK-MAPK (mitogen-activated protein kinase) pathways. Further investigation revealed that BECN1/Beclin 1 binding to viral phosphoprotein (P) induced an incomplete autophagy via activating the pathways CASP2-AMPK-AKT-MTOR and CASP2-AMPK-MAPK by decreasing CASP2. Taken together, our data first reveals a crosstalk of BECN1 and CASP2-dependent autophagy pathways by RABV infection.
Project description:Originally identified as an E3 ligase regulating toll-like receptor (TLR) signaling, ring finger protein 216 (RNF216) also plays an essential role in autophagy, which is fundamental to cellular homeostasis. Autophagy dysfunction leads to an array of pathological events, including tumor formation. In this study, we found that RNF216 was upregulated in human colorectal cancer (CRC) tissues and cell lines, and was associated with progression of CRC. RNF216 promoted CRC cell proliferation and migration in vitro and in vivo, largely by enhancing proteasomal degradation of BECN1, a key autophagy regulator and tumor suppressor. RNF216 restricted CRC cell autophagy through BECN1 inhibition under nutritional starvation conditions. RNF216 knockdown increased the autophagy, limiting CRC cell proliferation and migration. Moreover, BECN1 knockdown or autophagy inhibition restored proliferation and migration of RNF216-knockdown CRC cells. Collectively, our results suggested that RNF216 promoted CRC cell proliferation and migration by negatively regulating BECN1-dependent autophagy. This makes RNF216 as a potential biomarker and novel therapeutic target for inhibiting CRC development and progression.
Project description:Recently, autophagy has emerged as a critical process in the control of T-cell homeostasis. Given the pivotal role of NF-kappaB in the signaling events of T cells, we have analyzed and unveiled a conserved NF-kappaB binding site in the promoter of the murine and human BECN1 autophagic gene (Atg6). Accordingly, we demonstrate that the NF-kappaB family member p65/RelA upregulates BECN1 mRNA and protein levels in different cellular systems. Moreover, p65-mediated upregulation of BECN1 is coupled to increased autophagy. The newly identified kappaB site in the BECN1 promoter specifically interacts with p65 both in vitro and in living Jurkat cells upon phorbol myristate acetate (PMA)-ionomycin stimulation, where p65 induction is coupled to BECN1 upregulation and autophagy induction. Finally, anti-CD3- and PMA-ionomycin-mediated activation of T-cell receptor signaling in peripheral T cells from lymph nodes of healthy mice results in an upregulation of BECN1 expression that can be blocked by the NF-kappaB inhibitor BAY 11-7082. Altogether, these data suggest that autophagy could represent a novel route modulated by p65 to regulate cell survival and control T-cell homeostasis.
Project description:Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.
Project description:Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs) for defense against numerous viral infections, including classical swine fever virus (CSFV). However, the mechanisms underlying the effect of ISGs on CSFV infection are rarely reported. In this study, we demonstrate that IFN-? treatment induces upregulation of ISG15 and thus attenuates CSFV replication. To determine whether ISG15 is critical for controlling CSFV replication, we established porcine alveolar macrophages (PAMs) with stable overexpression or knockdown of ISG15. Overexpression of Flag-ISG15 significantly prevented CSFV replication, whereas loss of ISG15 led to abnormal proliferation of CSFV. Furthermore, upregulated ISG15 promoted beclin-1 (BECN1) ISGylation and dysfunction and subsequently inhibited autophagy, which is indispensable for CSFV replication. In addition, HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), which functions to catalyze conjugation of ISG15 protein, was confirmed to interact with BECN1. Collectively, these results indicate that IFN-? restricts CSFV replication through ISG15-mediated BECN1 ISGylation and autophagy inhibition, providing insight into the mechanism of CSFV replication control by type I IFN. This mechanism may not be the only antiviral mechanism of ISG15; nonetheless, this study may contribute to the development of CSFV treatment and prevention strategies.
Project description:BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1(-/-) cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.
Project description:The lysosomal degradation pathway, autophagy, is essential for the maintenance of cellular homeostasis. Recently, autophagy has been demonstrated to be required in the process of adipocyte conversion. However, its role in mature adipocytes under physiological and pathological conditions remains unclear. Here, we report a major function of BECN1 in the regulation of basal autophagy in mature adipocytes. We also show that berberine, a natural plant alkaloid, inhibits basal autophagy in adipocytes and adipose tissue of mice fed a high-fat diet via downregulation of BECN1 expression. We further demonstrate that berberine has a pronounced effect on the stability of Becn 1 mRNA through the Mir30 family. These findings explore the potential of BECN1 as a key molecule and a drug target for regulating autophagy in mature adipocytes.
Project description:Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.
Project description:GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A1 treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.
Project description:Multiple factors have been shown to promote the progression of diabetic cardiomyopathy. A link has previously been found between Mir30 and autophagy in cancer cells and in the heart, but the role of Mir30 in diabetic heart has not been studied. Using in vitro and in vivo approaches, we found that the depletion of Mir30c and induction of BECN1 enhanced autophagy in diabetic (db/db) hearts and in cardiomyocytes treated with the fatty acid palmitate. We verified that Mir30c repressed BECN1 expression by direct binding to the BECN1 3' UTRs. Mir30c overexpression inhibited the induction of BECN1 and subsequent autophagy in diabetic hearts and improved cardiac function and structure in diabetic mice. However, these effects were abrogated by BECN1 overexpression. Similarly, Mir30c knockdown resulted in increased BECN1 levels and autophagic flux, aggravating cardiac abnormalities. We also show that SP1, an important transcriptional factor in energy metabolism regulation, is a key upstream activator of Mir30c that binds the promoter region of Mir30c. Our findings indicate that downregulation of Mir30c and subsequent activation of BECN1 promotes autophagy, contributing to the pathogenesis of diabetic cardiomyopathy. This observation suggests a theoretical ground for developing microRNA-based therapeutics against diabetic cardiomyopathy by inhibiting autophagy.