TPT1 (tumor protein,translationally-controlled 1) negatively regulates autophagy through the BECN1 interactome and an MTORC1-mediated pathway

TPT1/TCTP (tumor protein, translationally-controlled 1) is highly expressed in tumor cells, known to participate in various cellular activities including protein synthesis, growth and cell survival. In addition, TPT1 was identified as a direct target of the tumor suppressor TP53/p53 although little is known about the mechanism underlying the anti-survival function of TPT1. Here, we describe a role of TPT1 in the regulation of the MTORC1 pathway through modulating the molecular machinery of macroautophagy/autophagy. TPT1 inhibition induced cellular autophagy via the MTORC1 and AMPK pathways, which are inhibited and activated, respectively, during treatment with the MTOR inhibitor rapamycin. We also found that the depletion of TPT1 potentiated rapamycin-induced autophagy by synergizing with MTORC1 inhibition. We further demonstrated that TPT1 knockdown altered the BECN1 interactome, a representative MTOR-independent pathway, to stimulate autophagosome formation, via downregulating BCL2 expression through activating MAPK8/JNK1, and thereby enhancing BECN1-phosphatidylinositol 3-kinase (PtdIns3K)-UVRAG complex formation. Furthermore, reduced TPT1 promoted autophagic flux by modulating not only early steps of autophagy but also autophagosome maturation. Consistent with in vitro findings, in vivo organ analysis using Tpt1 heterozygote knockout mice showed that autophagy is enhanced because of haploinsufficient TPT1 expression. Overall, our study demonstrated the novel role of TPT1 as a negative regulator of autophagy that may have potential use in manipulating various diseases associated with autophagic dysfunction.     

A fine-tuning mechanism underlying self-control for autophagy: deSUMOylation of BECN1 by SENP3

ABSTRACT

The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3^± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3^+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction.     

The E3 ubiquitin ligase NEDD4 enhances killing of membrane-perturbing intracellular bacteria by promoting autophagy

The E3 ubiquitin ligase NEDD4 has been intensively studied in processes involved in viral infections, such as virus budding. However, little is known about its functions in bacterial infections. Our investigations into the role of NEDD4 in intracellular bacterial infections demonstrate that Mycobacterium tuberculosis and Listeria monocytogenes, but not Mycobacterium bovis BCG, replicate more efficiently in NEDD4 knockdown macrophages. In parallel, NEDD4 knockdown or knockout impaired basal macroautophagy/autophagy, as well as infection-induced autophagy. Conversely, NEDD4 expression promoted autophagy in an E3 catalytic activity-dependent manner, thereby restricting intracellular Listeria replication. Mechanistic studies uncovered that endogenous NEDD4 interacted with BECN1/Beclin 1 and this interaction increased during Listeria infection. Deficiency of NEDD4 resulted in elevated K48-linkage ubiquitination of endogenous BECN1. Further, NEDD4 mediated K6- and K27- linkage ubiquitination of BECN1, leading to elevated stability of BECN1 and increased autophagy. Thus, NEDD4 participates in killing of intracellular bacterial pathogens via autophagy by sustaining the stability of BECN1.     

miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3′ UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.     

ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction

ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.     

Recombinant protein rVP1 upregulates BECN1-independent autophagy,MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration

The monocyte/macrophage is critical for regulating immune and antitumor responses. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus induces apoptosis and inhibits migration/metastasis of cancer cells. Here, we explored the effects of rVP1 on macrophages. Our results showed that rVP1 increased LC3-related autophagosome formation via WIPI1 and WIPI2 in a BECN1-independent manner. rVP1 treatment increased macrophage migration that was attenuated by knockdown of ATG5, ATG7, WIPI1 or WIPI2 and was abolished when both WIPI1 and WIPI2 were depleted. Treatment of macrophages with rVP1 increased matrix metalloproteinase-9 (MMP9) activity and phosphorylated mitogen-activated protein kinase 1/3 (MAPK1/3), two major mediators of cell migration. Knockdown of WIPI1, WIPI2, ATG5 and ATG7 but not BECN1 attenuated the rVP1-mediated increase in MAPK1/3 phosphorylation and MMP9 activity. These results indicated that rVP1 upregulated autophagy, MAPK1/3 phosphorylation and MMP9 activity to promote macrophage migration, which was dependent on WIPI1, WIPI2, ATG5 and ATG7 but not BECN1.     

Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways

In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.     

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

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.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

Roles for the Mitogen-activated Protein Kinase (MAPK) Phosphatase,DUSP1, in Feedback Control of Inflammatory Gene Expression and Repression by Dexamethasone

Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

Caloric restriction and resveratrol promote longevity through the Sirtuin-1-dependent induction of autophagy

Caloric restriction and autophagy-inducing pharmacological agents can prolong lifespan in model organisms including mice, flies, and nematodes. In this study, we show that transgenic expression of Sirtuin-1 induces autophagy in human cells in vitro and in Caenorhabditis elegans in vivo. The knockdown or knockout of Sirtuin-1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in C. elegans. Conversely, Sirtuin-1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in C. elegans. The knockdown or pharmacological inhibition of Sirtuin-1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. Along similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy-proficient nematodes, whereas these beneficial effects on longevity were abolished by the knockdown of the essential autophagic modulator Beclin-1. We conclude that autophagy is universally required for the lifespan-prolonging effects of caloric restriction and pharmacological Sirtuin-1 activators.     

Novel monofunctional platinum (II) complex Mono-Pt induces apoptosis-independent autophagic cell death in human ovarian carcinoma cells,distinct from cisplatin

Failure to engage apoptosis appears to be a leading mechanism of resistance to traditional platinum drugs in patients with ovarian cancer. Therefore, an alternative strategy to induce cell death is needed for the chemotherapy of this apoptosis-resistant cancer. Here we report that autophagic cell death, distinct from cisplatin-induced apoptosis, is triggered by a novel monofunctional platinum (II) complex named Mono-Pt in human ovarian carcinoma cells. Mono-Pt-induced cell death has the following features: cytoplasmic vacuolation, caspase-independent, no nuclear fragmentation or chromatin condensation, and no apoptotic bodies. These characteristics integrally indicated that Mono-Pt, rather than cisplatin, initiated a nonapoptotic cell death in Caov-3 ovarian carcinoma cells. Furthermore, incubation of the cells with Mono-Pt but not with cisplatin produced an increasing punctate distribution of microtubule-associated protein 1 light chain 3 (LC3), and an increasing ratio of LC3-II to LC3-I. Mono-Pt also caused the formation of autophagic vacuoles as revealed by monodansylcadaverine staining and transmission electron microscopy. In addition, Mono-Pt-induced cell death was significantly inhibited by the knockdown of either BECN1 or ATG7 gene expression, or by autophagy inhibitors 3-methyladenine, chloroquine and bafilomycin A₁. Moreover, the effect of Mono-Pt involved the AKT1-MTOR-RPS6KB1 pathway and MAPK1 (ERK2)/MAPK3 (ERK1) signaling, since the MTOR inhibitor rapamycin increased, while the MAPK1/3 inhibitor U0126 decreased Mono-Pt-induced autophagic cell death. Taken together, our results suggest that Mono-Pt exerts anticancer effect via autophagic cell death in apoptosis-resistant ovarian cancer. These findings lead to increased options for anticancer platinum drugs to induce cell death in cancer.     

BECN1/Beclin 1 is recruited into lipid rafts by prion to activate autophagy in response to amyloid β 42

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid β_1–42 (Aβ₄₂)-induced autophagy activation through its interaction with BECN1. Treatment with Aβ₄₂ enhanced autophagy flux in neuronal cells. Aβ₄₂-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aβ₄₂. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aβ₄₂, defining a novel role of PRNP in the regulation of autophagy.