Bay41-4109-induced aberrant polymers of hepatitis b capsid proteins are removed via STUB1-promoted p62-mediated macroautophagy.

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients.

Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins.

Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection.

Ubiquitin ligase MARCH5 localizes to peroxisomes to regulate pexophagy.

Mitochondria and peroxisomes are independent but functionally closely related organelles. A few proteins have been characterized as dual-organelle locating proteins with distinct or similar roles on mitochondria and peroxisomes. MARCH5 is a mitochondria-associated ubiquitin ligase best known for its regulatory role in mitochondria quality control, fission, and fusion. Here, we used a proximity tagging system, PUP-IT, and identified new interacting proteins of MARCH5. Our data uncover that MARCH5 is a dual-organelle locating protein that interacts with several peroxisomal proteins. PEX19 binds the transmembrane region on MARCH5 and targets it to peroxisomes. On peroxisomes, MARCH5 binds and mediates the ubiquitination of PMP70. Furthermore, we find PMP70 ubiquitination and pexophagy induced by mTOR inhibition are blocked in the absence of MARCH5. Our study suggests novel roles of MARCH5 on peroxisomes.

Prolyl oligopeptidase acts as a link between chaperone-mediated autophagy and macroautophagy.

The accumulation of aggregated α-synuclein (α-syn) has been identified as the primary component of Lewy bodies that are the pathological hallmarks of Parkinson's disease (PD). Several preclinical studies have shown α-syn aggregation, and particularly the intermediates formed during the aggregation process to be toxic to cells. Current PD treatments only provide symptomatic relief, and α-syn serves as a promising target to develop a disease-modifying therapy for PD. Our previous studies have revealed that a small-molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, increases α-syn degradation by accelerating macroautophagy (MA) leading to disease-modifying effects in preclinical PD models. However, α-syn is also degraded by chaperone-mediated autophagy (CMA). In the present study, we tested the effects of PREP inhibition or deletion on CMA activation and α-syn degradation. HEK-293 cells were transfected with α-syn and incubated with 1 & 10 µM KYP-2047 for 24 h. Both 1 & 10 µM KYP-2047 increased LAMP-2A levels, induced α-syn degradation and reduced the expression of Hsc70, suggesting that the PREP inhibitor prevented α-syn aggregation by activating the CMA pathway. Similarly, KYP-2047 increased the LAMP-2A immunoreactivity and reduced the Hsc70 levels in mouse primary cortical neurons. When LAMP-2A was silenced by a siRNA, KYP-2047 increased the LC3BII/LC3BI ratio and accelerated the clearance of α-syn. Additionally, KYP-2047 induced CMA effectively also when MA was blocked by bafilomycin A1. Based on our results, we suggest that PREP might function as a core network node in MA-CMA crosstalk, and PREP inhibition can reduce α-syn levels via both main autophagy systems.

Serum supplementation during bovine embryo culture affects their development and proliferation through macroautophagy and endoplasmic reticulum stress regulation.

Serum supplementation during bovine embryo culture has been demonstrated to promote cell proliferation and preimplantation embryo development. However, these desirable outcomes, have been associated with gene expression alterations of pathways involved in macroautophagy, growth, and development at the blastocyst stage, as well as with developmental anomalies such as fetal overgrowth and placental malformations. In order to start dissecting the molecular pathways by which serum supplementation of the culture medium during the preimplantation stage promotes developmental abnormalities, we examined blastocyst morphometry, inner cell mass and trophectoderm cell allocations, macroautophagy, and endoplasmic reticulum stress. On day 5 post-insemination, > 16 cells embryos were selected and cultured in medium containing 10% serum or left as controls. Embryo diameter, inner cell mass and trophectoderm cell number, and macroautophagy were measured on day 8 blastocysts (BL) and expanded blastocysts (XBL). On day 5 and day 8, we assessed transcript level of the ER stress markers HSPA5, ATF4, MTHFD2, and SHMT2 as well as XBP1 splicing (a marker of the unfolded protein response). Serum increased diameter and proliferation of embryos when compared to the no-serum group. In addition, serum increased macroautophagy of BL when compared to controls, while the opposite was true for XBL. None of the genes analyzed was differentially expressed at any stage, except that serum decreased HSPA5 in day 5 > 16 cells stage embryos. XBP1 splicing was decreased in BL when compared to XBL, but only in the serum group. Our data suggest that serum rescues delayed embryos by alleviating endoplasmic reticulum stress and promotes development of advanced embryos by decreasing macroautophagy.

Chaperon-mediated autophagy can regulate diquat-induced apoptosis by inhibiting α-synuclein accumulation cooperatively with macroautophagy.

α-Synuclein, which is associated with Parkinson's disease, is cleared by the ubiquitin-proteasome system and autophagy lysosome system. Chaperon-mediated autophagy (CMA) and macroautophagy are major subtypes of autophagy and play a critical role in pesticide-induced α-synucleinopathy. In this study, we explored the role of CMA in diquat (DQ)-induced α-synucleinopathy and characterized the relationship between CMA and macroautophagy in the clearance of pathologic α-synuclein for the prevention of DQ neurotoxicity. DQ was cytotoxic to SH-SY5Y cells in a concentration-dependent manner, as shown by decreased cell viability and increased cytotoxicity. DQ treatment was also found to induce autophagy such as CMA and macroautophagy by monitoring the expression of Lamp2A and microtubule-associated protein 1A/1B light chain 3B (LC3-II) respectively. Following DQ treatment, SH-SY5Y cells were found to have induced phosphorylated and detergent-insoluble α-synuclein deposits, and MG132, a proteasome inhibitor, effectively potentiated both CMA and macroautophagy for preventing α-synuclein aggregation. Interestingly, CMA impairment by Lamp2A-knock down decreased the LC3II expression compared to in DQ-treated cells transfected with control siRNA. In Lamp2-knock down cells, pathologic α-synuclein was increased 12 h after DQ treatment, but there was no change observed at 24 h. In DQ-treated cells, macroautophagy by 3-methyladenine and bafilomycin inhibition increased Lamp2A expression, indicating an increase in CMA activity. In addition, CMA modulation affected apoptosis, and inhibiting lysosome activity by NH4Cl increased apoptosis in DQ-treated cells. An increase in autophagy was confirmed to compensate for the decrease in lysosome activity. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, significantly enhanced the macroautophagy response of DQ-exposed cells without alterations in Lamp2A expression. Our results suggest that CMA can regulate DQ-induced α-synucleinopathy cooperatively with macroautophagy, and crosstalk between macroautophagy and CMA plays an important role in DQ-induced cytotoxicity. Taken together, autophagy modulation may be a useful treatment strategy in pesticide-induced neurodegenerative disorders through preventing α-synucleinopathy.

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope.

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

Crosstalk Between NDP52 and LUBAC in Innate Immune Responses, Cell Death, and Xenophagy.

Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia.

Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. One of the most common mutations that results in AML occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3). Previous studies have demonstrated that AML cells expressing FLT3-internal tandem duplication (ITD) are more sensitive to the proteasome inhibitor bortezomib (Bz) than FLT3 wild-type cells, with this cytotoxicity being mediated by autophagy (Atg). Here, we show that proteasome inhibition with Bz results in modest but consistent proteaphagy in MOLM-14 leukemic cells expressing the FLT3-ITD mutation, but not in OCI-AML3 leukemic cells with wild-type FLT3. Chemical inhibition of Atg with bafilomycin A simultaneously blocked proteaphagy and resulted in the accumulation of the p62 Atg receptor in Bz-treated MOLM-14 cells. The use of ubiquitin traps revealed that ubiquitin plays an important role in proteasome-Atg cross-talk. The p62 inhibitor verteporfin blocked proteaphagy and, importantly, resulted in accumulation of high molecular weight forms of p62 and FLT3-ITD in Bz-treated MOLM-14 cells. Both Atg inhibitors enhanced Bz-induced apoptosis in FLT3-ITD-driven leukemic cells, highlighting the therapeutic potential of these treatments.

XCT 790 is a pharmacological aggrephagy inducer in a yeast model of proteotoxicity.

Aggrephagy is a selective autophagic degradation intracellular mechanism that clears toxic misfolded protein aggregates such as α-synuclein. Here, we identify and demonstrate that the small molecule, XCT 790 alleviates α-synuclein-mediated adverse effects in a yeast model of proteotoxicity. XCT 790 induced general autophagy and also enhanced starvation-induced autophagy. Mechanistically, we showed that XCT 790 clears toxic α-synuclein aggregates in an autophagy-dependent manner. Interestingly, XCT 790 did not demonstrate a synergistic effect on autophagy induction in the presence of another autophagy inducer such as 6-Bio.

lncRNA GAS5 inhibits malignant progression by regulating macroautophagy and forms a negative feedback regulatory loop with the miR­34a/mTOR/SIRT1 pathway in colorectal cancer.

Long non­coding RNA growth arrest specific 5 (GAS5) exerts inhibitory effects through the modulation of several target microRNAs (miRs) in cancer. However, its potential roles and underlying relationship during colorectal cancer (CRC) progression are unclear. Therefore, we explored the role of the negative feedback loop formed by the GAS5/miR­34a axis and mammalian target of rapamycin/sirtuin 1 (mTOR/SIRT1) pathway on macroautophagy and apoptosis in CRC. Expression of GAS5, miR­34a, SIRT1 and mTOR in CRC patients and cell lines was detected by quantitative reverse transcription polymerase chain reaction. Online bioinformatic analysis was used to predict the downstream miRs of GAS5. Luciferase assay and western blotting were performed to demonstrate miR­34a as a downstream target gene of GAS5 in CRC cells. The effects of the GAS5/miR­34a axis on apoptosis, macroautophagy, and the mTOR/SIRT1 pathway were assessed by flow cytometry, transmission electron microscopy and western blotting, respectively. Our results suggested that GAS5 was downregulated and acted as a molecular sponge of miR­34a during CRC progression. miR­34a participated in regulating GAS5­suppressed CRC cell macroautophagy and induced apoptosis through the mTOR/SIRT1 pathway. GAS5­mediated macroautophagy was maintained in an equilibrium state that might have a protective effect on CRC cell apoptosis. The mTOR signaling pathway suppressed GAS5 expression and formed a negative regulation feedback loop with miR­34a in CRC cells. Our results suggested that the GAS5/miR­34a/SIRT1/mTOR negative regulatory feedback loop mediated CRC cell macroautophagy, and maintained the cells in an autonomous equilibrium state, but not excessive activation state, which functions as a strong antiapoptotic phenotype during human CRC progression.

A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers.

Macroautophagy/autophagy plays a critical role in the pathogenesis of various human diseases including neurodegenerative disorders such as Parkinson disease (PD) and Huntington disease (HD). Chemical autophagy inducers are expected to serve as disease-modifying agents by eliminating cytotoxic/damaged proteins. Although many autophagy inducers have been identified, their precise molecular mechanisms are not fully understood because of the complicated crosstalk among signaling pathways. To address this issue, we performed several chemical genomic analyses enabling us to comprehend the dominancy among the autophagy-associated pathways followed by an aggresome-clearance assay. In a first step, more than 400 target-established small molecules were assessed for their ability to activate autophagic flux in neuronal PC12D cells, and we identified 39 compounds as autophagy inducers. We then profiled the autophagy inducers by testing their effect on the induction of autophagy by 200 well-established signal transduction modulators. Our principal component analysis (PCA) and clustering analysis using a dataset of "autophagy profiles" revealed that two Food and Drug Administration (FDA)-approved drugs, memantine and clemastine, activate endoplasmic reticulum (ER) stress responses, which could lead to autophagy induction. We also confirmed that SMK-17, a recently identified autophagy inducer, induced autophagy via the PRKC/PKC-TFEB pathway, as had been predicted from PCA. Finally, we showed that almost all of the autophagy inducers tested in this present work significantly enhanced the clearance of the protein aggregates observed in cellular models of PD and HD. These results, with the combined approach, suggested that autophagy-activating small molecules may improve proteinopathies by eliminating nonfunctional protein aggregates.Abbreviations: ADK: adenosine kinase; AMPK: AMP-activated protein kinase; ATF4: activating transcription factor 4; BECN1: beclin-1; DDIT3/CHOP: DNA damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2 subunit alpha; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FDA: Food and Drug Administration; GSH: glutathione; HD: Huntington disease; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; JAK: Janus kinase, MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MAP2K/MEK: mitogen-activated protein kinase kinase; MAP3K8/Tpl2: mitogen-activated protein kinase kinase kinase 8; MAPK: mitogen-activated protein kinase; MPP+: 1-methyl-4-phenylpyridinium; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; NAC: N-acetylcysteine; NGF: nerve growth factor 2; NMDA: N-methyl-D-aspartate; PCA: principal component analysis; PD: Parkinson disease; PDA: pancreatic ductal adenocarcinoma; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PMA: phorbol 12-myristate 13-acetate; PRKC/PKC: protein kinase C; ROCK: Rho-associated coiled-coil protein kinase; RR: ribonucleotide reductase; SIGMAR1: sigma non-opioid intracellular receptor 1; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: Transcription factor EB; TGFB/TGF-ß: Transforming growth factor beta; ULK1: unc-51 like autophagy activating kinase 1; XBP1: X-box binding protein 1.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

Peroxisomicine A1, a potential antineoplastic agent, causes micropexophagy in addition to macropexophagy.

Peroxisomicine A1 (PA1) is a potential antineoplastic agent with high and selective toxicity toward peroxisomes of tumor cells. Pexophagy is a selective autophagy process that degrades damaged peroxisomes; this process has been studied mainly in methylotrophic yeasts. There are two main modes of pexophagy in yeast: macropexophagy and micropexophagy. Previous studies showed that peroxisomes damaged by a prolonged exposition to PA1 are eliminated by macropexophagy. In this work, Candida boidinii was grown in methanol-containing media, and PA1 was added to the cultures at 2 µg/mL after they reached the mid-exponential growth phase. Samples were taken at 5, 10, 15, 20, and 25 min after the addition of PA1 and processed for ultrastructural analysis. Typical morphological characteristics of micropexophagy were observed: the direct engulfment of peroxisomes by the vacuolar membrane and the presence of the micropexophagic membrane apparatus (MIPA), which mediates the fusion between the opposing tips of the vacuole to complete sequestration of peroxisomes from the cytosol. In conclusion, here we report that, in addition to macropexophagy, peroxisomes damaged by PA1 can be eliminated by micropexophagy. This information is useful to deepen the knowledge of the mechanism of action of PA1 and of that of pexophagy per se.

Icariin Ameliorates Amyloid Pathologies by Maintaining Homeostasis of Autophagic Systems in Aß1-42-Injected Rats.

Macroautophagy, a sole pathway for dysfunctional organelles or aggregated proteins turnover, has been implicated in the early development of Alzheimer's disease (AD). Previous studies have found that reversal of autophagy dysfunction in APP transgenic mice ameliorates amyloid pathologies. Icariin (ICA), the main component from traditional Chinese herb Epimedium brevicornu Maxim., can reduce accumulations of amyloid-ß (Aß) peptide in vivo and in vitro, but the mechanism remains unclear. Here, we explored the effects of ICA on autophagy-lysosomal pathway in intracerebroventricular (icv) injection of human Aß1-42 peptide rats. We demonstrated that feeding the rats with ICA (30 mg/kg, 60 mg/kg and 90 mg/kg rat, per os) for 4 weeks rescued the Aß1-42-induced spatial memory impairments, reduced endogenous rat Aß42 tested by ELISA and decreased Aß accumulation using 6E10 antibody. Furthermore, Aß1-42 induced strong autophagy response, however ICA decreased the levels of microtubule-associated protein 1 light chain 3 (LC3) II/LC3I, Beclin1, Cathepsin D (Cat D) and brain lysosomal Cathepsin D activity. We also observed that ICA enhanced the phosphorylation of protein kinase B (PKB/AKT) and p70 ribosomal protein S6 kinase (p70S6K). In addition, ICA arrested Aß1-42-induced cells loss, mitochondrias damage, nuclear membranes unclear and abundant nucleas chromatin agglutinates in hippocampus, lessened the expression of Cleaved-caspase-3, brain oxidative stress, astroglial activation. These findings suggest that ICA can ameliorate amyloid pathologies with improving autophagy-lysosome function and Chinese materia medica may be potential for AD treatment.

Bafilomycin-A1 and ML9 Exert Different Lysosomal Actions to Induce Cell Death.

OBJECTIVE: Bafilomycin-A1 and ML9 are lysosomotropic agents, irrespective of cell types. However, the mechanisms of lysosome targeting either bafilomycin-A1 or ML9 are unclear. METHODS: The present research has been carried out by different molecular and biochemical analyses like western blot, confocal imaging and FACS studies, as well as molecular docking. RESULTS: Our data shows that pre-incubation of neonatal cardiomyocytes with ML9 for 4h induced cell death, whereas a longer period of time (24h) with bafilomycin-A1 was required to induce an equivalent effect. Neither changes in ROS nor ATP production is associated with such death mechanisms. Flow cytometry, LC3-II expression levels, and LC3-GFP puncta formation revealed a similar lysosomotropic effect for both compounds. We used a molecular docking approach, that predicts a stronger inhibitory activity against V-ATPase-C1 and C2 domains for bafilomycin-A1 in comparison to ML9. CONCLUSION: Bafilomycin-A1 and ML9 are lysosomotropic agents, involved in cell death events. But such death events are not associated with ATP and ROS production. Furthermore, both the drugs target lysosomes through different mechanisms. For the latter, cell death is likely due to lysosomal membrane permeabilization and release of lysosomal proteases into the cytosol.

A facile forward-genetic screen for Arabidopsis autophagy mutants reveals twenty-one loss-of-function mutations disrupting six ATG genes.

Macroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2 mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790 allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation. Abbreviations: ATG: autophagy-related; ATI: ATG8-interacting protein; Col-0: Columbia-0; DSK2: dominant suppressor of KAR2; EMS: ethyl methanesulfonate; GFP: green fluorescent protein; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; ICL: isocitrate lyase; MLS: malate synthase; NBR1: Next to BRCA1 gene 1; PEX: peroxin; PMDH: peroxisomal malate dehydrogenase; PTS: peroxisomal targeting signal; thiolase: 3-ketoacyl-CoA thiolase; UBA: ubiquitin-associated; WT: wild type.

Biogenesis of Autophagosome in Trichomonas vaginalis during Macroautophagy Induced by Rapamycin-treatment and Iron or Glucose Starvation Conditions.

Autophagy is an adaptive response for cell survival in which cytoplasmic components and organelles are degraded in bulk under normal and stress conditions. Trichomonas vaginalis is a parasite highly adaptable to stress conditions such as iron (IR) and glucose restriction (GR). Autophagy can be traced by detecting a key autophagy protein (Atg8) anchored to the autophagosome membrane by a lipid moiety. Our goal was to perform a morphological and cellular study of autophagy in T. vaginalis under GR, IR, and Rapamycin (Rapa) treatment using TvAtg8 as a putative autophagy marker. We cloned tvatg8a and tvatg8b and expressed and purified rTvAtg8a and rTvAtg8b to produce specific polyclonal antibodies. Autophagy vesicles were detected by indirect immunofluorescence assays and confirmed by ultrastructural analysis. The biogenesis of autophagosomes was detected, showing intact cytosolic cargo. TvAtg8 was detected as puncta signal with the anti-rTvAtg8b antibody that recognized soluble and lipid-associated TvAtg8b by Western blot assays in lysates from stress-inducing conditions. The TvAtg8b signal co-localized with the CytoID and lysotracker labeling (autolysosomes) that accumulated after E-64d treatment in GR parasites. Our data suggest that autophagy induced by starvation in T. vaginalis results in the formation of autophagosomes for which TvAtg8b could be a putative autophagy marker.