Negative Regulator Of Autophagy Research Articles

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

Negative Regulation of Autophagy during Macrophage Infection by Mycobacterium bovis BCG via Protein Kinase C Activation.

Mycobacterium tuberculosis (Mtb) employs various strategies to manipulate the host's cellular machinery, overriding critical molecular mechanisms such as phagosome-lysosome fusion, which are crucial for its destruction. The Protein Kinase C (PKC) signaling pathways play a key role in regulating phagocytosis. Recent research in Interferon-activated macrophages has unveiled that PKC phosphorylates Coronin-1, leading to a shift from phagocytosis to micropinocytosis, ultimately resulting in Mtb destruction. Therefore, this study aims to identify additional PKC targets that may facilitate Mycobacterium bovis (M. bovis) infection in macrophages. Protein extracts were obtained from THP-1 cells, both unstimulated and mycobacterial-stimulated, in the presence or absence of a general PKC inhibitor. We conducted an enrichment of phosphorylated peptides, followed by their identification through mass spectrometry (LC-MS/MS). Our analysis revealed 736 phosphorylated proteins, among which 153 exhibited alterations in their phosphorylation profiles in response to infection in a PKC-dependent manner. Among these 153 proteins, 55 are involved in various cellular processes, including endocytosis, vesicular traffic, autophagy, and programmed cell death. Importantly, our findings suggest that PKC may negatively regulate autophagy by phosphorylating proteins within the mTORC1 pathway (mTOR2/PKC/Raf-1/Tsc2/Raptor/Sequestosome-1) in response to M. bovis BCG infection, thereby promoting macrophage infection.

G-quadruplexes in MTOR and induction of autophagy

G-quadruplex (G4) structures have emerged as singular therapeutic targets for cancer and neurodegeneration. Autophagy, a crucial homeostatic mechanism of the cell, is often dysregulated in neurodegenerative diseases and cancers. We used QGRS mapper to identify 470 G4 sequences in MTOR, a key negative regulator of autophagy. We sought to identify a functional context by leveraging the effect of G4-targeting ligands on MTOR G4 sequences. The effect of Bis-4,3, a G4 selective dimeric carbocyanine dye, was compared with the known G4-stabilizing activity of the porphyrin, TMPyP4 in HeLa and SHSY-5Y cells. Our results show that treatment with G4-selective ligands downregulates MTOR RNA and mTOR protein expression levels. This is the first report describing G4 motifs in MTOR. This study indicates a possible role of G4 stabilizing ligands in induction of autophagy by downregulation of mTOR levels, albeit not precluding MTOR independent pathways.

Activation of interleukin 33-NFκB axis in granulosa cells during atresia and its role in disposal of atretic follicles†.

It has been previously shown that the cytokine interleukin 33 is required for two processes, i.e., autophagic digestion of granulosa cells and recruitment of macrophages into atretic follicles, for full disposal of atretic follicles. Now, this study shows that activation of interleukin 33-suppression of tumorigenicity 2-Nuclear Factor ĸB (NFκB) axis in granulosa in early atretic follicles may regulate those two events. Injection of human chorionic gonadotropin has been shown to induce a transient peak of interleukin 33 expression with synchronized atresia. In this model, interleukin 33-independent expression of suppression of tumorigenicity 2 in granulosa cells was detected in early atretic follicles before macrophage invasion. The activation of NFκB pathway in ovaries was further demonstrated in vivo in Tg mice with luciferase-reporter for NFκB activation; the activation was microscopically localized to granulosa cells in early atretic follicles. Importantly, antibody blockage of interleukin 33 or interleukin 33 Knock-out (KO) (Il33-/-) not only inhibited NFκB activity in ovaries, but it also altered expression of two key genes, i.e., reduction in proinflammatory interleukin6 (IL6) expression, and a surge of potential autophagy-inhibitory mammalian target of rapamycin (mTOR) expression in atretic follicles. By contrast, apoptosis and other genes, such as interleukin1β (IL1β) were not affected. In conclusion, in parallel to apoptosis, atresia signals also trigger activation of the interleukin 33-suppression of tumorigenicity 2-NFκB pathway in granulosa, which leads to (1) down-regulated expression of mTOR that is a negative regulator of autophagy and (2) up-regulated expression of proinflammatory IL6.

Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as cysteine (Cys) in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed phosphatidylinositol-3 kinase (PI3K)–Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor (EGF) receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K–Akt signaling by MVK reversed EGF-induced negative regulation of autophagy, and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K–Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K–Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

MiR-2765 involved in ammonia nitrogen stress via negative regulation of autophagy in shrimp

MicroRNA (miRNA) is a highly conserved non-coding tiny endogenous RNA molecule that regulates various cellular functions by inhibiting mRNA translation or promoting the degradation of proteins. In this study, we identified a specific miRNA (designed as Pva-miR-2765) from Penaeus vannamei, which widely distributed in different tissues of shrimp, with the highest concentration found in the intestine. Through fluorescence in situ hybridization (FISH), we observed that Pva-miR-2765 is primarily located in the cytoplasm. Interestingly, we found that the expression of Pva-miR-2765 significantly decreased in hemocytes, hepatopancreas and gill under ammonia nitrogen stress. Furthermore, when Pva-miR-2765 was silenced, the autophagy level in shrimp significantly increased. Additionally, Pva-miR-2765 was found to promote pathological damage in the hepatopancreas of shrimp. Subsequently, correlation analysis revealed a negative relationship between the expression of Pva-miR-2765 and PvTBC1D7. To confirm this interaction, we conducted a dual luciferase reporter gene assay, which demonstrated that Pva-miR-2765 inhibit the expression of PvTBC1D7 by interacting with its 3’UTR. And the expression level of PvTBC1D7 in shrimp decreased significantly under ammonia nitrogen stress in Pva-miR-2765 overexpressed. Our findings suggest that Pva-miR-2765 can reduce autophagy in P. vannamei by inhibiting the regulation of PvTBC1D7, thereby participating in the oxidative stress of shrimp caused by ammonia nitrogen stress.

Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: relevance to Parkinson’s disease

Dopaminergic degeneration is a central feature of Parkinson’s disease (PD), but glial dysfunction may accelerate or trigger neuronal death. In fact, astrocytes play a key role in the maintenance of the blood–brain barrier and detoxification. 6-hydroxydopamine (6OHDA) is used to induce PD in rodent models due to its specific toxicity to dopaminergic neurons, but its effect on astrocytes has been poorly investigated. Here, we show that 6OHDA dose-dependently impairs autophagy in human U373 cells and primary murine astrocytes in the absence of cell death. LC3II downregulation was observed 6 to 48 h after treatment. Interestingly, 6OHDA enhanced NRH:quinone oxidoreductase 2 (NQO2) expression and activity in U373 cells, even if 6OHDA turned out not to be its substrate. Autophagic flux was restored by inhibition of NQO2 with S29434, which correlated with a partial reduction in oxidative stress in response to 6OHDA in human and murine astrocytes. NQO2 inhibition also increased the neuroprotective capability of U373 cells, since S29434 protected dopaminergic SHSY5Y cells from 6OHDA-induced cell death when cocultured with astrocytes. The toxic effects of 6OHDA on autophagy were attenuated by silencing NQO2 in human cells and primary astrocytes from NQO2−/− mice. Finally, the analysis of Gene Expression Omnibus datasets showed elevated NQO2 gene expression in the blood cells of early-stage PD patients. These data support a toxifying function of NQO2 in dopaminergic degeneration via negative regulation of autophagy and neuroprotection in astrocytes, suggesting a potential pharmacological target in PD.

A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms. Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS. Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy. Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.

A Brief Review of SPINK1 Studies

SPINK1, a trypsin inhibitor secreted by the pancreas, is closely associated with the development of pancreatitis. Additionally, SPINK1 exhibits multiple biological functions, acting as a trypsin inhibitor, a growth factor-like protein, and a negative regulator of autophagy. This paper reviews the role of the SPINK1 gene and its variants in pancreatitis development, its expression as TATI in cancer, and its role in oncogenesis.

CSNK2 suppresses autophagy by activating FLN-NHL-containing TRIM proteins

ABSTRACT Macroautophagy/autophagy is a tightly regulated cellular process integral to homeostasis and innate immunity. As such, dysregulation of autophagy is associated with cancer, neurodegenerative disorders, and infectious diseases. While numerous factors that promote autophagy have been characterized, the key mechanisms that prevent excessive autophagy are less well understood. Here, we identify CSNK2/CK2 (casein kinase 2) as a negative regulator of autophagy. Pharmacological inhibition of CSNK2 activity or siRNA-mediated depletion of CSNK2 increased basal autophagic flux in cell lines and primary human lung cells. Vice versa, ectopic expression of CSNK2 reduced autophagic flux. Mechanistically, CSNK2 interacted with the FLN (filamin)-NHL domain-containing tripartite motif (TRIM) family members TRIM2, TRIM3 and TRIM71. Our data show that recruitment of CSNK2 to the C-terminal NHL domain of TRIM3 lead to its robust phosphorylation at serine 661 by CSNK2. A phosphorylation-defective mutant of TRIM3 was unable to reduce autophagosome numbers indicating that phosphorylation by CSNK2 is required for TRIM-mediated autophagy inhibition. All three TRIMs facilitated inactivation of the ULK1-BECN1 autophagy initiation complex by facilitating ULK1 serine 757 phosphorylation. Inhibition of CSNK2 promoted autophagy upon influenza A virus (IAV) and measles virus (MeV) infection. In line with this, targeting of CSNK2 or depletion of TRIM2, TRIM3 or TRIM71 enhanced autophagy-dependent restriction of IAV, MeV and human immunodeficiency virus 1 (HIV-1). Thus, our results identify the CSNK2-TRIM2, -TRIM3, -TRIM71 axis as a key regulatory pathway that limits autophagy. Targeting this axis may allow for therapeutic induction of autophagy against viral infections and in diseases associated with dysregulated autophagy. Abbreviation: ATG5: autophagy related 5; BafA1: bafilomycin A1; BECN1: beclin 1; CCD: coiled-coil domain; CSNK2/CK2: casein kinase 2; CSNK2A1: casein kinase 2 alpha 1; CSNK2A2: casein kinase 2 alpha 2; CSNK2B: casein kinase 2 beta; FLN: filamin; HeLa GL: HeLa cells stably expressing eGFP-LC3B; HIV-1: human immunodeficiency virus 1; IAV: influenza A virus; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3; MeV: measles virus; MTOR: mechanistic target of rapamycin kinase; RING: really interesting new gene; SQSTM1/p62: sequestosome 1; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1

EXTH-21. FKBP38 REGULATES SELF-RENEWAL AND SURVIVAL OF GBM NEUROSPHERES

Abstract Glioblastoma (GBM) is the most common malignant primary brain tumor. The outcome is dismal, despite the multimodal therapeutic approach that includes surgical resection followed by radiation and chemotherapy. The quest for novel therapeutic targets to treat GBM is underway. FKBP38, a member of the immunophilin family of proteins, is a multidomain protein that plays an important role in the regulation of cellular functions including apoptosis and autophagy. In this study, we tested the role of FKBP38 in GBM tumor biology. Expression of FKBP38 is upregulated in the patient-derived primary GBM neurospheres (GBMNS) compared to normal human astrocytes. Attenuation of FKBP38 expression decreased the viability of GBMNS and increased the caspase 3/7 activity indicating that FKBP38 is required for the survival of GBMNS. Further, the depletion of FKBP38 significantly reduced the number of neurospheres that were formed implying that FKBP38 regulates the self-renewal of GBMNS. Also, the transient knockdown of FKBP38 increased the LC3-II/I ratio suggesting the induction of autophagy with the depletion of FKBP38. Further investigation showed that the negative regulation of autophagy by FKBP38 in GBMNS is mediated through the JNK/C-Jun-PTEN-AKT pathway. In vivo, FKBP38 depletion significantly extended the survival of tumor-bearing mice. Overall, our results suggest targeting FKBP38 imparts an anti-GBM effect by inducing apoptosis and autophagy and thus can be a potential therapeutic target for GBM therapy.

Hepatocyte-specific regulation of autophagy and inflammasome activation via MyD88 during lethal Ehrlichia infection.

Hepatocytes play a crucial role in host response to infection. Ehrlichia is an obligate intracellular bacterium that causes potentially life-threatening human monocytic ehrlichiosis (HME) characterized by an initial liver injury followed by sepsis and multi-organ failure. We previously showed that infection with highly virulent Ehrlichia japonica (E. japonica) induces liver damage and fatal ehrlichiosis in mice via deleterious MyD88-dependent activation of CASP11 and inhibition of autophagy in macrophage. While macrophages are major target cells for Ehrlichia, the role of hepatocytes (HCs) in ehrlichiosis remains unclear. We investigated here the role of MyD88 signaling in HCs during infection with E. japonica using primary cells from wild-type (WT) and MyD88-/- mice, along with pharmacologic inhibitors of MyD88 in a murine HC cell line. Similar to macrophages, MyD88 signaling in infected HCs led to deleterious CASP11 activation, cleavage of Gasdermin D, secretion of high mobility group box 1, IL-6 production, and inflammatory cell death, while controlling bacterial replication. Unlike macrophages, MyD88 signaling in Ehrlichia-infected HCs attenuated CASP1 activation but activated CASP3. Mechanistically, active CASP1/canonical inflammasome pathway negatively regulated the activation of CASP3 in infected MyD88-/- HCs. Further, MyD88 promoted autophagy induction in HCs, which was surprisingly associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), a known negative regulator of autophagy. Pharmacologic blocking mTORC1 activation in E. japonica-infected WT, but not infected MyD88-/- HCs, resulted in significant induction of autophagy, suggesting that MyD88 promotes autophagy during Ehrlichia infection not only in an mTORC1-indpenedent manner, but also abrogates mTORC1-mediated inhibition of autophagy in HCs. In conclusion, this study demonstrates that hepatocyte-specific regulation of autophagy and inflammasome pathway via MyD88 is distinct than MyD88 signaling in macrophages during fatal ehrlichiosis. Understanding hepatocyte-specific signaling is critical for the development of new therapeutics against liver-targeting pathogens such as Ehrlichia.

FKBP38 Regulates Self-Renewal and Survival of GBM Neurospheres.

Glioblastoma is the most common malignant primary brain tumor. The outcome is dismal, despite the multimodal therapeutic approach that includes surgical resection, followed by radiation and chemotherapy. The quest for novel therapeutic targets to treat glioblastoma is underway. FKBP38, a member of the immunophilin family of proteins, is a multidomain protein that plays an important role in the regulation of cellular functions, including apoptosis and autophagy. In this study, we tested the role of FKBP38 in glioblastoma tumor biology. Expression of FKBP38 was upregulated in the patient-derived primary glioblastoma neurospheres (GBMNS), compared to normal human astrocytes. Attenuation of FKBP38 expression decreased the viability of GBMNSs and increased the caspase 3/7 activity, indicating that FKBP38 is required for the survival of GBMNSs. Further, the depletion of FKBP38 significantly reduced the number of neurospheres that were formed, implying that FKBP38 regulates the self-renewal of GBMNSs. Additionally, the transient knockdown of FKBP38 increased the LC3-II/I ratio, suggesting the induction of autophagy with the depletion of FKBP38. Further investigation showed that the negative regulation of autophagy by FKBP38 in GBMNSs is mediated through the JNK/C-Jun-PTEN-AKT pathway. In vivo, FKBP38 depletion significantly extended the survival of tumor-bearing mice. Overall, our results suggest that targeting FKBP38 imparts an anti-glioblastoma effect by inducing apoptosis and autophagy and thus can be a potential therapeutic target for glioblastoma therapy.

A novel combination therapy with Cabozantinib and Honokiol effectively inhibits c-Met-Nrf2-induced renal tumor growth through increased oxidative stress

Receptor tyrosine kinase (RTK), c-Met, is overexpressed and hyper active in renal cell carcinoma (RCC). Most of the therapeutic agents mediate cancer cell death through increased oxidative stress. Induction of c-Met in renal cancer cells promotes the activation of redox-sensitive transcription factor Nrf2 and cytoprotective heme oxygenase-1 (HO-1), which can mediate therapeutic resistance against oxidative stress. c-Met/RTK inhibitor, Cabozantinib, has been approved for the treatment of advanced RCC. However, acquired drug resistance is a major hurdle in the clinical use of cabozantinib. Honokiol, a naturally occurring phenolic compound, has a great potential to downregulate c-Met-induced pathways. In this study, we found that a novel combination treatment with cabozantinib + Honokiol inhibits the growth of renal cancer cells in a synergistic manner through increased production of reactive oxygen species (ROS); and it significantly facilitates apoptosis-and autophagy-mediated cancer cell death. Activation of c-Met can induce Rubicon (a negative regulator of autophagy) and p62 (an autophagy adaptor protein), which can stabilize Nrf2. By utilizing OncoDB online database, we found a positive correlation among c-Met, Rubicon, p62 and Nrf2 in renal cancer. Interestingly, the combination treatment significantly downregulated Rubicon, p62 and Nrf2 in RCC cells. In a tumor xenograft model, this combination treatment markedly inhibited renal tumor growth in vivo; and it is associated with decreased expression of Rubicon, p62, HO-1 and vessel density in the tumor tissues. Together, cabozantinib + Honokiol combination can significantly inhibit c-Met-induced and Nrf2-mediated anti-oxidant pathway in renal cancer cells to promote increased oxidative stress and tumor cell death.

A Novel Tri-Hydroxy-Methylated Chalcone Isolated from Chromolaena tacotana with Anti-Cancer Potential Targeting Pro-Survival Proteins.

Chromolaena tacotana (Klatt) R. M. King and H. Rob (Ch. tacotana) contains bioactive flavonoids that may have antioxidant and/or anti-cancer properties. This study investigated the potential anti-cancer properties of a newly identified chalcone isolated from the inflorescences of the plant Chromolaena tacotana (Klatt) R. M. King and H. Rob (Ch. tacotana). The chalcone structure was determined using HPLC/MS (QTOF), UV, and NMR spectroscopy. The compound cytotoxicity and selectivity were evaluated on prostate, cervical, and breast cancer cell lines using the MTT assay. Apoptosis and autophagy induction were assessed through flow cytometry by detecting annexin V/7-AAD, active Casp3/7, and LC3B proteins. These results were supported by Western blot analysis. Mitochondrial effects on membrane potential, as well as levels of pro- and anti-apoptotic proteins were analyzed using flow cytometry, fluorescent microscopy, and Western blot analysis specifically on a triple-negative breast cancer (TNBC) cell line. Furthermore, molecular docking (MD) and molecular dynamics (MD) simulations were performed to evaluate the interaction between the compounds and pro-survival proteins. The compound identified as 2',3,4-trihydroxy-4',6'-dimethoxy chalcone inhibited the cancer cell line proliferation and induced apoptosis and autophagy. MDA-MB-231, a TNBC cell line, exhibited the highest sensitivity to the compound with good selectivity. This activity was associated with the regulation of mitochondrial membrane potential, activation of the pro-apoptotic proteins, and reduction of anti-apoptotic proteins, thereby triggering the intrinsic apoptotic pathway. The chalcone consistently interacted with anti-apoptotic proteins, particularly the Bcl-2 protein, throughout the simulation period. However, there was a noticeable conformational shift observed with the negative autophagy regulator mTOR protein. Future studies should focus on the molecular mechanisms underlying the anti-cancer potential of the new chalcone and other flavonoids from Ch. tacotana, particularly against predominant cancer cell types.

A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation of autophagy in human gastric cells

BackgroundAutophagy plays an important role in carcinogenesis and tumor progression in many cancers, including gastric cancer. Cytotoxin-associated gene A (CagA) is a well-known virulent factor in Helicobacter pylori (H. pylori) infection that plays a critical role in gastric inflammation and gastric cancer development. However, its role in autophagy during these processes remains unclear. Therefore, we aimed to clarify the role of CagA in autophagy in CagA-related inflammation.MethodsWe evaluated the autophagic index of AGS cells infected with wild-type cagA-positive H. pylori (Hp-WT) and cagA-knockout H. pylori (Hp-ΔcagA) and rat gastric mucosal (RGM1) cells transfected with CagA genes. To identify the mechanisms underlying the down regulation of autophagy in AGS cells infected with H. pylori, we evaluated protein and mRNA expression levels of autophagy core proteins using western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). To determine whether autophagy induced the expression of the pro-inflammatory mediator, cyclooxygenase-2 (COX-2), we evaluated COX-2 expression in AGS cells treated with an autophagy inducer and inhibitor and infected with H. pylori. In addition, we evaluated whether COX-2 protein expression in AGS cells influenced beclin-1 (BECN1) expression with si-RNA transfection when infected with H. pylori.ResultsAutophagic flux assay using chloroquine showed that autophagy in AGS cells was significantly suppressed after H. pylori infection. The autophagic index of AGS cells infected with Hp-WT was decreased significantly when compared with that in AGS cells infected with Hp-ΔcagA. The autophagic index of RGM1 cells transfected with CagA was lower, suggesting that CagA inhibits autophagy. In addition, BECN1 expression levels in AGS cells infected with Hp-WT were reduced compared to those in AGS cells infected with Hp-ΔcagA. Furthermore, COX-2 expression in AGS cells infected with H. pylori was controlled in an autophagy-dependent manner. When AGS cells were transfected with small interfering RNA specific for BECN1 and infected with Hp-WT and Hp-ΔcagA, COX-2 was upregulated significantly in cells infected with Hp-ΔcagA.ConclusionsIn conclusion, the H. pylori CagA protein negatively regulated autophagy by downregulating BECN1. CagA-induced autophagy inhibition may be a causative factor in promoting pro-inflammatory mediator production in human gastric epithelial cells.

Opposing roles of RUBCN isoforms in autophagy and memory B cell generation.

RUBCN (also known as Rubicon) was originally identified as a negative regulator of autophagy, a process by which cells degrade and recycle damaged components or organelles and that requires the activity of the class III PI3K VPS34 and the mTORC1 protein complex. Here, we characterized the role of a shorter isoform, RUBCN100, as an autophagy-promoting factor in B cells. RUBCN100 was translated from alternative translation initiation sites and lacked the RUN domain of the longer, previously characterized RUBCN130 isoform. Specific deficiency of RUBCN130 in B cells enhanced autophagy, which promoted memory B cell generation. In contrast to RUBCN130, which is localized in late endosomes and lysosomes and suppresses the enzymatic activity of VPS34, an effect thought to mediated by its RUN domain, RUBCN100 was preferentially located in early endosomes and enhanced VPS34 activity, presumably because of the absence of the RUN domain. Furthermore, RUBCN100, but not RUBCN130, enhanced autophagy and suppressed mTORC1 activation. Our findings reveal that the opposing roles of two RUBCN isoforms are critical for autophagy regulation and memory B cell differentiation.

RUNDC1 inhibits autolysosome formation and survival of zebrafish via clasping ATG14-STX17-SNAP29 complex.

Autophagy serves as a pro-survival mechanism for a cell or a whole organism to cope with nutrient stress. Our understanding of the molecular regulation of this fusion event remains incomplete. Here, we identified RUNDC1 as a novel ATG14-interacting protein, which is highly conserved across vertebrates, including zebrafish and humans. By gain and loss of function studies, we demonstrate that RUNDC1 negatively modulates autophagy by blocking fusion between autophagosomes and lysosomes via inhibiting the assembly of the STX17-SNAP29-VAMP8 complex both in human cells and the zebrafish model. Moreover, RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation. This also prevents VAMP8 from binding to STX17-SNAP29. We further identified that phosphorylation of RUNDC1 Ser379 is crucial to inhibit the assembly of the STX17-SNAP29-VAMP8 complex via promoting ATG14 homo-oligomerization. In line with our findings, RunDC1 is crucial for zebrafish in their response to nutrient-deficient conditions. Taken together, our findings demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes by clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding.

The yeast transcription factor Stb5 acts as a negative regulator of autophagy by modulating cellular metabolism

ABSTRACT Macroautophagy/autophagy is a highly conserved pathway of cellular degradation and recycling that maintains cell health during homeostatic conditions and facilitates survival during stress. Aberrant cellular autophagy contributes to the pathogenesis of human diseases such as cancer, neurodegeneration, and cardiovascular, metabolic and lysosomal storage disorders. Despite decades of research, there remain unanswered questions as to how autophagy modulates cellular metabolism, and, conversely, how cellular metabolism affects autophagy activity. Here, we have identified the yeast metabolic transcription factor Stb5 as a negative regulator of autophagy. Chromosomal deletion of STB5 in the yeast Saccharomyces cerevisiae enhances autophagy. Loss of Stb5 results in the upregulation of select autophagy-related (ATG) transcripts under nutrient-replete conditions; however, the Stb5-mediated impact on autophagy occurs primarily through its effect on genes involved in NADPH production and the pentose phosphate pathway. This work provides insight into the intersection of Stb5 as a transcription factor that regulates both cellular metabolic responses and autophagy activity. Abbreviations: bp, base pairs; ChIP, chromatin immunoprecipitation; G6PD, glucose-6-phosphate dehydrogenase; GFP, green fluorescent protein; IDR, intrinsically disordered region; NAD, nicotinamide adenine dinucleotide; NADP+, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); ORF, open reading frame; PA, protein A; PCR, polymerase chain reaction; PE, phosphatidylethanolamine; PPP, pentose phosphate pathway; prApe1, precursor aminopeptidase I; ROS, reactive oxygen species; RT-qPCR, real-time quantitative PCR; SD, standard deviation; TF, transcription factor; TOR, target of rapamycin; WT, wild-type.

Regulation of plant autophagy by YWHA/14-3-3 proteins

ATG1/ATG13, a core kinase complex regulator in the macroautophagy/autophagy machinery during autophagosome formation, is modulated in a sophisticated manner by posttranslational ubiquitination to determine proper autophagy levels in eukaryotic cells. However, the mechanisms that regulate the stability and activity of this complex remain elusive. We recently identified two negative regulators of autophagy, 14-3-3λ and 14-3-3k, that help redundantly regulate autophagy by directly associating with SINAT and ATG13s. The specific interaction between the molecular adaptors 14-3-3λ and 14-3-3κ and phosphorylated ATG13a is crucial for SINAT-mediated ubiquitination and degradation of ATG13a, and for maintenance of the ATG1-ATG13 complex. Consistent with the function of 14-3-3s in autophagy, the 14-3-3λ 14-3-3κ double mutant exhibits enhanced tolerance to nutrient deprivation with constitutive induction of autophagy. These findings demonstrate that 14-3-3λ and 14-3-3k coordinate with SINATs to regulate both the homeostasis of ATG13 phosphorylation and the induction of autophagy in plants.