bims-auttor 2022-08-21 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2022–08–21
thirty-two papers selected by
Viktor Korolchuk, Newcastle University

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.
The role of LC3B in autophagy as an RNA-binding protein.
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.
PINK1-dependent and Parkin-independent mitophagy is involved in reprogramming of glycometabolism in pancreatic cancer cells.
Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.
TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.
Targeting Mitophagy to Promote Apoptosis is a Potential Therapeutic Strategy for Cancer.
Receptor-mediated mitophagy: An emerging therapeutic target in acute kidney injury.
Covalent JNK inhibitor, JNK-IN-8, suppresses tumor growth in triple-negative breast cancer by activating TFEB and TFE3 mediated lysosome biogenesis and autophagy.
Presenilin-1 F105C mutation leads to tau accumulation in human neurons via the Akt/mTORC1 signaling pathway.
PET- and ICT-Based Ratiometric Probe: An Unusual Phenomenon of Morpholine-Conjugated Fluorophore for Mitochondrial pH Mapping during Mitophagy.
Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.
TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.
Pan-cancer gene expression analysis: Identification of deregulated autophagy genes and drugs to target them.
Plant UVRAG interacts with ATG14 to regulate autophagosome maturation and geminivirus infection.
Oleate restores altered autophagic flux to rescue palmitate lipotoxicity in hypothalamic neurons.
Human milk oligosaccharide 2'-fucosyllactose promotes melanin degradation via the autophagic AMPK-ULK1 signaling axis.
Up-regulation of Osh6 boosts an anti-aging membrane trafficking pathway toward vacuoles.
Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.
Histone Acetyltransferase CfGcn5-Mediated Autophagy Governs the Pathogenicity of Colletotrichum fructicola.
Regulation of nuclear DNA damage response by mitochondrial morphofunctional pathway.
The DHAV-1 protein VP1 interacts with PI3KC3 to induce autophagy through the PI3KC3 complex.
Biphasic regulation of osteoblast development via the ERK MAPK-mTOR pathway.
Growth factor receptor signaling induces mitophagy through epitranscriptomic regulation.
AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network.
Nondegradable ubiquitinated ATG9A organizes Golgi integrity and dynamics upon stresses.
METTL14 Regulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells via Inducing Autophagy Through m6A/IGF2BPs/Beclin-1 Signal Axis.
MTP18 inhibition triggers mitochondrial hyperfusion to induce apoptosis through ROS-mediated lysosomal membrane permeabilization-dependent pathway in oral cancer.
Matrine induces autophagy in human neuroblastoma cells via blocking the AKT-mTOR pathway.
Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector.
miR-141 exacerbates lung ischemia-reperfusion injury by targeting EGFR/β-catenin axis-mediated autophagy.
Piperine alleviates acute pancreatitis: A possible role for FAM134B and CCPG1 dependent ER-phagy.

Nat Commun. 2022 Aug 17. 13(1): 4836

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.

George K E Umanah, Leire Abalde-Atristain, Mohammed Repon Khan, Jaba Mitra, Mohamad Aasif Dar, Melissa Chang, Kavya Tangella, Amy McNamara, Samuel Bennett, Rong Chen, Vasudha Aggarwal, Marisol Cortes, Paul F Worley, Taekjip Ha, Ted M Dawson, Valina L Dawson.

The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.

DOI: https://doi.org/10.1038/s41467-022-32365-2
Autophagy. 2022 Aug 13. 1-3

The role of LC3B in autophagy as an RNA-binding protein.

Hyun Jung Hwang, Yoon Ki Kim.

  The hallmark of cellular events observed upon macroautophagic/autophagic induction is the conjugation of LC3B, one of the mammalian Atg8 homologs, with phosphatidylethanolamine. This conversion from LC3B-I (an unconjugated form) to LC3B-II (a conjugated form) is essential for phagophore expansion and formation of autophagosomes. Our recent study revealed that LC3B binds to RNAs with a preference for the consensus AAUAAA motif and recruits the CCR4-NOT deadenylase complex. Consequently, LC3B elicits rapid degradation of mRNAs, which we have termed as LC3B-mediated mRNA decay (LMD). LMD requires the conversion of LC3B-I to LC3B-II and occurs before the formation of autolysosomes. Furthermore, we identified PRMT1 mRNA, which encodes a protein that functions as a negative regulator of autophagy, as an LMD substrate. A failure of rapid degradation of PRMT1 mRNA via LMD results in inefficient autophagy. Thus, our study unravels an important role of LC3B in autophagy as an RNA-binding protein for efficient mRNA decay.

Keywords:  ATG8; CCR4-NOT deadenylase; LC3B; PRMT1; autophagy; mRNA decay

DOI:  https://doi.org/10.1080/15548627.2022.2111083
EMBO Rep. 2022 Aug 18. e54859

UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.

Jing Zheng, Yu Cao, Jun Yang, Hui Jiang.

  The hexameric AAA-ATPase valosin-containing protein (VCP) is essential for mitochondrial protein quality control. How VCP is recruited to mammalian mitochondria remains obscure. Here we report that UBXD8, an ER- and lipid droplet-localized VCP adaptor, also localizes to mitochondria and locally recruits VCP. UBXD8 associates with mitochondrial and ER ubiquitin E3 ligases and targets their substrates for degradation. Remarkably, both mitochondria- and ER-localized UBXD8 can degrade mitochondrial and ER substrates in cis and in trans. UBXD8 also associates with the TOM complex but is dispensable for translocation-associated degradation. UBXD8 knockout impairs the degradation of the pro-survival protein Mcl1 but surprisingly sensitizes cells to apoptosis and mitochondrial stresses. UBXD8 knockout also hyperactivates mitophagy. We identify pro-apoptotic BH3-only proteins Noxa, Bik, and Bnip3 as novel UBXD8 substrates and determine that UBXD8 inhibits apoptosis via degrading Noxa and restrains mitophagy via degrading Bnip3. Collectively, our characterizations reveal UBXD8 as the major mitochondrial adaptor of VCP and unveil its role in apoptosis and mitophagy regulation.

Keywords:  UBXD8; VCP; apoptosis; mitochondria-associated degradation; mitophagy

DOI:  https://doi.org/10.15252/embr.202254859
Biochem Biophys Res Commun. 2022 Aug 05. pii: S0006-291X(22)01109-3. [Epub ahead of print]625 167-173

PINK1-dependent and Parkin-independent mitophagy is involved in reprogramming of glycometabolism in pancreatic cancer cells.

Natsumi Miyazaki, Reika Shiratori, Taichi Oshima, Zhiheng Zhang, Robert Valencia, Joshua Kranrod, Liye Fang, John M Seubert, Kousei Ito, Shigeki Aoki.

  Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.

Keywords:  Glycometabolism; Mitophagy; PINK1; Pancreatic cancer; Parkin

DOI:  https://doi.org/10.1016/j.bbrc.2022.08.004
J Biol Chem. 2022 Aug 13. pii: S0021-9258(22)00822-5. [Epub ahead of print] 102379

Proximity labeling of endogenous RICTOR identifies mTOR Complex 2 regulation by ADP ribosylation factor ARF1.

Amelia K Luciano, Ekaterina Korobkina, Scott P Lyons, John A Haley, Shelagh Fluharty, Su Myung Jung, Arminja N Kettenbach, David A Guertin.

  Mechanistic Target of Rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2 interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain- and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.

Keywords:  ADP ribosylation factor (ARF); Akt/PKB; BioID2; RICTOR; cell signaling; growth factor; mTORC2; mammalian target of rapamycin (mTOR); proximity labeling

DOI:  https://doi.org/10.1016/j.jbc.2022.102379
Diabetes. 2022 Aug 19. pii: db220256. [Epub ahead of print]

TBK1-mTOR Signaling Attenuates Obesity-Linked Hyperglycemia and Insulin Resistance.

Cagri Bodur, Dubek Kazyken, Kezhen Huang, Aaron Seth Tooley, Kae Won Cho, Tammy M Barnes, Carey N Lumeng, Martin G Myers, Diane C Fingar.

The innate immune kinase TBK1 (TANK-binding kinase 1) responds to microbial-derived signals to initiate responses against viral and bacterial pathogens. More recent work implicates TBK1 in metabolism and tumorigenesis. The kinase mTOR (mechanistic target of rapamycin) integrates diverse environmental cues to control fundamental cellular processes. Our prior work demonstrated in cells that TBK1 phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 catalytic activity and signaling. Here we investigate a role for TBK1-mTOR signaling in control of glucose metabolism in vivo. We find that diet induced obese (DIO) but not lean mice bearing a whole-body "TBK1 resistant" Mtor S2159A knockin allele (MtorA/A) display exacerbated hyperglycemia and systemic insulin resistance with no change in energy balance. Mechanistically, Mtor S2159A knockin in DIO mice reduces mTORC1 and mTORC2 signaling in response to insulin and innate immune agonists, reduces anti-inflammatory gene expression in adipose tissue, and blunts anti-inflammatory macrophage M2 polarization, phenotypes shared by mice with tissue-specific inactivation of TBK1 or mTOR complexes. Tissues from DIO mice display elevated TBK1 activity and mTOR S2159 phosphorylation relative to lean mice. We propose a model whereby obesity-associated signals increase TBK1 activity and mTOR phosphorylation, which boosts mTORC1 and mTORC2 signaling in parallel to the insulin pathway, thereby attenuating insulin resistance to improve glycemic control during diet-induced obesity.

DOI: https://doi.org/10.2337/db22-0256
Autophagy. 2022 Aug 13.

Targeting Mitophagy to Promote Apoptosis is a Potential Therapeutic Strategy for Cancer.

Yancheng Tang, Liming Wang, Jiangjiang Qin, Ying-Ying Lu, Han-Ming Shen, Hu-Biao Chen.

Many anticancer agents exert cytotoxicity and trigger apoptosis through the induction of mitochondrial dysfunction. Mitophagy, as the key mitochondrial quality control mechanism, can remove damaged mitochondria in an effective and timely manner, which may result in drug resistance. Although the implication of mitophagy in neurodegenerative diseases has been extensively studied, the role and mechanism of mitophagy in tumorigenesis and cancer therapy are largely unknown. In a recent study, we found that the inhibition of PINK1-PRKN-mediated mitophagy can significantly enhance the anticancer efficacy of magnolol, a natural product with potential anticancer properties. On the one hand, magnolol can induce severe mitochondrial dysfunction, including mitochondrial depolarization, excessive mitochondrial fragmentation and the generation of mitochondrial ROS, leading to apoptosis. On the other hand, magnolol induces PINK1-PRKN-dependent mitophagy via activation of two rounds of feedforward amplification loops. The blockage of mitophagy through genetic or pharmacological approaches promotes rather than attenuates magnolol-induced cell death. Furthermore, inhibition of mitophagy by using distinct inhibitors targeting different mitophagic stages effectively enhances magnolol's anticancer efficacy in vivo. Taken together, our findings strongly indicate that manipulation of mitophagy in cancer treatment will be a promising therapeutic strategy for overcoming cancer drug resistance and improving the therapeutic efficacy of anticancer agents.

DOI: https://doi.org/10.1080/15548627.2022.2112830
Mitochondrion. 2022 Aug 16. pii: S1567-7249(22)00073-3. [Epub ahead of print]

Receptor-mediated mitophagy: An emerging therapeutic target in acute kidney injury.

Neha Dagar, Ajinath Kale, Stefanie Steiger, Hans-Joachim Anders, Anil Bhanudas Gaikwad.

  Acute kidney injury (AKI) is a global health concern associated with high morbidity and mortality. AKI etiology is linked to mitochondrial dysfunction along with oxidative stress and inflammation. The defective mitochondria are removed via mitophagy for maintaining cellular integrity. The main regulatory mechanisms of mitophagy in response to different stressors are Phosphatase and tensin homolog-induced kinase 1 (PINK1)/Parkin and receptor-mediated. Receptors like B-cell lymphoma 2/adenovirus E1B-interacting protein (BNIP3), BNIP3L, prohibitin2, tacrolimus (FK506)-binding protein8 (FKBP8), autophagy-beclin1-regulator1 (AMBRA1) and SMAD-ubiquitination regulatory factor1 (SMURF1), etc. participate in receptor-mediated mitophagy. In recent studies, receptor-mediated mitophagy showed protective effects in AKI. This review summarizes the evidence related to mitophagy in AKI and outlines the significance of receptor-mediated mitophagy modulation as a possible therapeutic approach in AKI.

Keywords:  Acute Kidney Injury; Mitochondria; Mitochondrial fusion/fission; Mitophagy; Mitophagy inducers; Receptor-mediated mitophagy

DOI:  https://doi.org/10.1016/j.mito.2022.08.004
Mol Cancer Ther. 2022 Aug 17. pii: MCT-21-1044. [Epub ahead of print]

Covalent JNK inhibitor, JNK-IN-8, suppresses tumor growth in triple-negative breast cancer by activating TFEB and TFE3 mediated lysosome biogenesis and autophagy.

Milad Soleimani, Alexander Somma, Tamer S Kaoud, Ria Goyal, Jorge Bustamante, Dennis C Wylie, Nisha Holay, Agnieszka P Looney, Uma Giri, Todd Triplett, Kevin N Dalby, Jeanne Kowalski, S Gail Eckhardt, Carla Van Den Berg.

The heterogeneity and aggressiveness of Triple-Negative Breast Cancer (TNBC) contribute to its early recurrence and metastasis. Despite substantial research to identify effective therapeutic targets, TNBC remains elusive in terms of improving patient outcomes. Here, we report that a covalent JNK inhibitor, JNK-IN-8, suppresses TNBC growth both in vitro and in vivo. JNK-IN-8 reduced colony formation, cell viability, and organoid growth in vitro and slowed patient-derived xenograft and syngeneic tumor growth in vivo. Cells treated with JNK-IN-8 exhibited large, cytoplasmic vacuoles with lysosomal markers. To examine the molecular mechanism of this phenotype, we looked at the master regulators of lysosome biogenesis and autophagy TFEB and TFE3. JNK-IN-8 inhibited TFEB phosphorylation and induced nuclear translocation of unphosphorylated TFEB and TFE3. This was accompanied by an upregulation of TFEB/TFE3 target genes associated with lysosome biogenesis and autophagy. Depletion of both TFEB and TFE3 diminished the JNK-IN-8-driven upregulation of lysosome biogenesis/autophagy markers. TFEB and TFE3 are phosphorylated by a number of kinases, including mTOR. JNK-IN-8 reduced phosphorylation of mTOR targets in a concentration-dependent manner. Knockout of JNK1 and/or JNK2 had no impact on TFEB/TFE3 activation or mTOR inhibition by JNK-IN-8, but inhibited colony formation. Similarly, re-expression of either wildtype or drug-nonbinding JNK (C116S) in JNK knockout cells did not reverse JNK-IN-8-induced TFEB dephosphorylation. In summary, JNK-IN-8 induced lysosome biogenesis and autophagy by activating TFEB/TFE3 via mTOR inhibition independently of JNK. Together, these findings demonstrate the efficacy of JNK-IN-8 as a targeted therapy for TNBC and reveal its novel lysosome- and autophagy-mediated mechanism of action.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-1044
Cell Biosci. 2022 Aug 14. 12(1): 131

Presenilin-1 F105C mutation leads to tau accumulation in human neurons via the Akt/mTORC1 signaling pathway.

Cheong-Meng Chong, Yuan Tan, Jiaqi Tong, Minjing Ke, Ke Zhang, Lingli Yan, Xiaotong Cen, Jia-Hong Lu, Guobing Chen, Huanxing Su, Dajiang Qin.

   BACKGROUND: The mammalian target of rapamycin (mTOR) plays a critical role in controlling cellular homeostasis, and its dysregulation has been implicated in Alzheimer's disease (AD). Presenilin-1 (PS1) mutations account for the most common causes of familial Alzheimer's disease (FAD); however, whether PS1 mutation causes mTOR dysregulation in human neurons remains a key unresolved issue.
METHODS: We generated heterozygotes and homozygotes of PS1 F105C knock-in mutation in human induced pluripotent stem cells (iPSCs) via CRISPR/Cas9/piggyback-based gene editing and differentiated them into human neurons. Secreted Aβ and tau accumulation were determined by ELISA assay, immunofluorescence staining, and western blotting analysis. mTOR signaling was evaluated by western blotting analysis, immunofluorescence staining, and co-immunoprecipitation. Autophagy/lysosome activities were determined by LC3-based assay, LysoTracker Red staining, and DQ-Red BSA staining.
RESULTS: Through comparison among these isogenic neurons, PS1 F105C mutant neurons exhibited elevated Aβ and tau accumulation. In addition, we found that the response of mTORC1 to starvation decreases in PS1 F105C mutant neurons. The Akt/mTORC1/p70S6K signaling pathway remained active upon EBSS starvation, leading to the co-localization of the vast majority of mTOR with lysosomes. Consistently, PS1 F105C neurons displayed a significant decline in starvation-induced autophagy. Notably, Torin1, a mTOR inhibitor, could efficiently reduce prominent tau pathology that occurred in PS1 F105C neurons.
CONCLUSION: We demonstrate that Chinese PS1 F105C mutation causes dysregulation of mTORC1 signaling, contributing to tau accumulation in human neurons. This study on inherited FAD PS1 mutation provides unprecedented insights into our understanding of the molecular mechanisms of AD. It supports that pharmaceutical blocking of mTOR is a promising therapeutic strategy for the treatment of AD.

Keywords:  CRISPR/Cas9; Presenilin-1; iPSC; mTOR

DOI:  https://doi.org/10.1186/s13578-022-00874-8
Anal Chem. 2022 Aug 14.

PET- and ICT-Based Ratiometric Probe: An Unusual Phenomenon of Morpholine-Conjugated Fluorophore for Mitochondrial pH Mapping during Mitophagy.

Subrata Munan, Mudassar Ali, Rashmi Yadav, Koyeli Mapa, Animesh Samanta.

Mitochondrial functions are heavily influenced by acid-base homeostasis. Hence, elucidation of the mitochondrial pH is essential in living cells, and its alterations during pathologies is an interesting question to be addressed. Small molecular fluorescent probes are progressively applied to quantify the mitochondrial pH by fluorescence imaging. Herein, we designed a unique small molecular fluorescent probe, PM-Mor-OH, based on the lipophilic morpholine ligand-conjugated pyridinium derivative of "IndiFluors". The morpholine-conjugated fluorescent probe usually localized the lysosome. However, herein, we observed unusual phenomena of morpholine-tagged PM-Mor-OH that localized mitochondria explicitly. The morpholine ligand also plays a pivotal role in tuning optical properties via photoinduced electron transfer (PET) during internal pH alteration (ΔpHi). In the mitophagy process, lysosomes engulf damaged mitochondria, leading to ΔpHi, which can be monitored using our probe. It exhibited "ratiometric" emission at single wavelength excitation (ex. 488) and is suitable for monitoring and quantifying the ΔpHi using confocal microscope high-resolution image analysis during mitophagy. The bathochromic emission shifts due to intramolecular charge transfer (ICT) in basic pH were well explained by the time-dependent density functional theory (TD-DFT/PCM). Similarly, the change in the emission ratio (green/red) with pH variations was also validated by the PET process. In addition, PM-Mor-OH can quantify the pH change during oxidative stress induced by rapamycin, mutant A53T α-synuclein-mediated protein misfolding stress in mitochondria, and during starvation. Rapamycin-induced mitophagy was further elucidated by the translocation of mCherry Parkin to damaged mitochondria, which well correlates with our probe. Thus, PM-Mito-OH is a valuable probe for visualizing mitophagy and can act as a suitable tool for the diagnosis of mitochondrial diseases.

DOI: https://doi.org/10.1021/acs.analchem.2c02177
J Immunol. 2022 Aug 19. pii: ji2200280. [Epub ahead of print]

Conserved Role of mTORC1 Signaling in B Cell Immunity in Teleost Fish.

Jia-Feng Cao, Li-Guo Ding, Qing-Chao Wang, Guang-Kun Han, Da-Cheng Qin, Gao-Feng Cheng, Zhao-Ran Dong, Qing-Jiang Mu, Wei-Guang Kong, Xia Liu, Yong-Yao Yu, Zhen Xu.

Mammalian studies have demonstrated that B cell immune responses are regulated by mechanistic target of rapamycin complex 1 (mTORC1) signaling. Teleost fish represent the oldest living bony vertebrates that contain bona fide B cells. So far, whether the regulatory mechanism of mTORC1 signaling in B cells occurred in teleost fish is still unknown. In this study, we developed a fish model by using rapamycin (RAPA) treatment to inhibit mTORC1 signaling and demonstrated the role of mTORC1 signaling in teleost B cells. In support, we found inhibition of mTORC1 signaling by RAPA decreased the phagocytic capacity, proliferation, and Ig production of B cells. Critically, Flavobacterium columnare induced specific IgM binding in serum, and these titers were significantly inhibited by RAPA treatment, thus decreasing Ab-mediated agglutination of F. columnare and significantly increasing the susceptibility of fish upon F. columnare reinfection. Collectively, our findings elucidated that the mTORC1 pathway is evolutionarily conserved in regulating B cell responses, thus providing a new point for understanding the B cells functions in teleost fish.

DOI: https://doi.org/10.4049/jimmunol.2200280
J Cell Sci. 2022 Aug 17. pii: jcs.260378. [Epub ahead of print]

TORC1 and PKA activity towards ribosome biogenesis oscillates in synchrony with the budding yeast cell cycle.

Paolo Guerra, Luc-Alban P E Vuillemenot, Yulan B van Oppen, Marije Been, Andreas Milias-Argeitis.

  Recent studies have revealed that the growth rate of budding yeast and mammalian cells varies during the cell cycle. By linking a multitude of signals to cell growth, the highly conserved Target of Rapamycin Complex 1 (TORC1) and Protein Kinase A (PKA) pathways are prime candidates for mediating the dynamic coupling between growth and division. However, measurements of TORC1 and PKA activity during the cell cycle are still lacking. Following the localization dynamics of two TORC1 and PKA targets via time-lapse microscopy in hundreds of yeast cells, we found that the activity of these pathways towards ribosome biogenesis fluctuates in synchrony with the cell cycle even under constant external conditions. Mutations of upstream TORC1 and PKA regulators suggested that internal metabolic signals partially mediate these activity changes. Our study reveals a new aspect of TORC1 and PKA signaling, which will be important for understanding growth regulation during the cell cycle.

Keywords:  Cell growth; PKA Signaling; Ribosome biogenesis; Single-cell microscopy; TOR signaling; TORC1

DOI:  https://doi.org/10.1242/jcs.260378
Gene. 2022 Aug 16. pii: S0378-1119(22)00640-0. [Epub ahead of print] 146821

Pan-cancer gene expression analysis: Identification of deregulated autophagy genes and drugs to target them.

Sree Karani Kondapuram, Mohane Selvaraj Coumar.

  Identifying suitable deregulated targets in autophagy pathway is essential for developing autophagy modulating cancer therapies. With this aim, we systematically analyzed the expression levels of genes that contribute to the execution of autophagy in 21 cancers. Deregulated genes for 21 cancers were analyzed using the level 3 mRNA data from TCGAbiolinks. A total of 574 autophagy genes were mapped to the deregulated genes across 21 cancers. PPI network, cluster analysis, gene enrichment, gene ontology, KEGG pathway, patient survival, protein expression and cMap analysis were performed. Among the autophagy genes, 260 were upregulated, and 43 were downregulated across pan-cancer. The upregulated autophagy genes - CDKN2A and BIRC5 - were the most frequent signatures in cancers and could be universal cancer biomarkers. Significant involvement of autophagy process was found in 8 cancers (CHOL, HNSC, GBM, KICH, KIRC, KIRP, LIHC and SARC). Fifteen autophagy hub genes (ATP6V0C, BIRC5, HDAC1, IL4, ITGB1, ITGB4, MAPK3, mTOR, cMYC, PTK2, SRC, TCIRG1, TP63, TP73 and ULK1) were found to be linked with patients survival and also expressed in cancer patients tissue samples, making them as potential drug targets for these cancers. The deregulated autophagy genes were further used to identify drugs Losartan, BMS-345541, Embelin, Abexinostat, Panobinostat, Vorinostat, PD-184352, PP-1, XMD-1150, Triplotide, Doxorubicin and Ouabain, which could target one or more autophagy hub genes. Overall, our findings shed light on the most frequent cancer-associated autophagy genes, potential autophagy targets and molecules for cancer treatment. These findings can accelerate autophagy modulation in cancer therapy.

Keywords:  Autophagy; Biomarker; Drug-target network; Gene expression analysis; Pan-cancer; protein-protein interaction network

DOI:  https://doi.org/10.1016/j.gene.2022.146821
New Phytol. 2022 Aug 17.

Plant UVRAG interacts with ATG14 to regulate autophagosome maturation and geminivirus infection.

Yan Wang, Jinlin Li, Jingran Wang, Ping Han, Shulei Miao, Xiyin Zheng, Meng Han, Xueqi Shen, Huangai Li, Ming Wu, Yiguo Hong, Yule Liu.

  Autophagy is an essential degradation pathway that assists eukaryote survival under multiple stress conditions. Autophagosomes engulfing cargoes accomplish degradation only when they have matured through fusing with lysosomes or vacuoles. However, the molecular machinery mediating autophagosome maturation in plants remains unknown. Using combined approaches of mass spectrometry, biochemistry, reverse genetics and microscopy, we uncover that, UVRAG, a subunit of the class III phosphatidylinositol 3-kinase complexes in Nicotiana benthamiana, plays an essential role in autophagsome maturation via ATG14-assisted recruitment to autophagosomes and facilitating RAB7 activation. An interaction between N. benthamiana UVRAG and ATG14 is observed in vitro and in vivo, which strikingly differs from their mutually exclusive appearance in different PI3KC3 complexes in yeast and mammals. This interaction increases localization of UVRAG on autophagosomes and enables convergence of autophagic and late endosomal structures, where they contribute to fusions between these two types of organelles by recruiting the essential membrane fusion factors RAB7 GTPase and HOPS complex. In addition, we uncover a joint contribution of ATG14 and UVRAG to geminiviral infection, beyond autophagy. Our study provides insights into mechanisms of autophagosome maturation in plants and expands understanding of organizations and roles of the PI3KC3 complexes.

Keywords:  Nicotiana benthamiana; RAB7 GTPase; UVRAG; ATG14; autophagosome maturation; geminivirus; plant PI3K complex; vacuolar trafficking

DOI:  https://doi.org/10.1111/nph.18437
Mol Cell Endocrinol. 2022 Aug 15. pii: S0303-7207(22)00201-5. [Epub ahead of print] 111753

Oleate restores altered autophagic flux to rescue palmitate lipotoxicity in hypothalamic neurons.

Wenyuan He, Andy Tran, Chuck T Chen, Neruja Loganthan, Richard P Bazinet, Denise D Belsham.

  Accumulation of excess lipids in non-adipose tissues, such as the hypothalamus, is termed lipotoxicity and causative of free fatty acid-mediated pathology in metabolic disease. This study aimed to elucidate the molecular mechanisms behind oleate (OA)- and palmitate (PA)-mediated changes in hypothalamic neurons. Using the well-characterized hypothalamic neuronal cell model, mHypoE-46, we assessed gene changes through qRT-PCR, cell death with quantitative imaging, PA metabolism using stable isotope labeling, and cellular mechanisms using pharmacological modulation of lipid metabolism and autophagic flux. Palmitate (PA) disrupts gene expression, including Npy, Grp78, and Il-6 mRNA in mHypoE-46 hypothalamic neurons. Blocking PA metabolism using triacsin-C prevented the increase of these genes, implying that these changes depend on PA intracellular metabolism. Co-incubation with oleate (OA) is also potently protective and prevents cell death induced by increasing concentrations of PA. However, OA does not decrease 13C-PA incorporation into diacylglycerol and phospholipids. Remarkably, OA can reverse PA toxicity even after significant PA metabolism and cellular impairment. OA can restore PA-mediated impairment of autophagy to prevent or reverse the accumulation of PA metabolites through lysosomal degradation, and not through other reported mechanisms. The autophagic flux inhibitor chloroquine mimics PA toxicity by upregulating autophagy-related genes, Npy, Grp78, and Il-6, an effect partially reversed by OA. Chloroquine also prevented the OA defense against PA toxicity, whereas the autophagy inducer rapamycin provided some protection. Thus, PA impairment of autophagic flux significantly contributes to its lipotoxicity, and OA-mediated protection requires functional autophagy. Overall, our results suggest that impairment of autophagy contributes to hypothalamic lipotoxicity.

Keywords:  Autophagy; Hypothalamus; Lipid metabolism; Lipotoxicity; Oleate; Palmitate

DOI:  https://doi.org/10.1016/j.mce.2022.111753
Sci Rep. 2022 Aug 17. 12(1): 13983

Human milk oligosaccharide 2'-fucosyllactose promotes melanin degradation via the autophagic AMPK-ULK1 signaling axis.

Hyojin Heo, Byungsun Cha, Dongmin Jang, Chaewon Park, Gunwoo Park, Byeong-Mun Kwak, Bum-Ho Bin, Ji-Hwan Park, Mi-Gi Lee.

There is still an unmet need for development of safer antimelanogenic or melanin-degrading agents for skin hyperpigmentation, induced by intrinsic or extrinsic factors including aging or ultraviolet irradiation. Owing to the relatively low cytotoxicity compared with other chemical materials, several studies have explored the role of 2'-fucosyllactose (2'-FL), the most dominant component of human milk oligosaccharides. Here, we showed that 2'-FL reduced melanin levels in both melanocytic cells and a human skin equivalent three-dimensional in vitro model. Regarding the cellular and molecular mechanism, 2'-FL induced LC3I conversion into LC3II, an autophagy activation marker, followed by the formation of LC3II+/PMEL+ autophagosomes. Comparative transcriptome analysis provided a comprehensive understanding for the up- and downstream cellular processes and signaling pathways of the AMPK-ULK1 signaling axis triggered by 2'-FL treatment. Moreover, 2'-FL activated the phosphorylation of AMPK at Thr172 and of ULK1 at Ser555, which were readily reversed in the presence of dorsomorphin, a specific AMPK inhibitor, with consequent reduction of the 2'-FL-mediated hypopigmentation. Taken together, these findings demonstrate that 2'-FL promotes melanin degradation by inducing autophagy through the AMPK-ULK1 axis. Hence, 2'-FL may represent a new natural melanin-degrading agent for hyperpigmentation.

DOI: https://doi.org/10.1038/s41598-022-17896-4
Microb Cell. 2022 Aug 01. 9(8): 145-157

Up-regulation of Osh6 boosts an anti-aging membrane trafficking pathway toward vacuoles.

Ilham Kadhim, Nazneen Begum, William King, Licheng Xu, Fusheng Tang.

  Members of the family of oxysterol-binding proteins mediate non-vesicular lipid transport between membranes and contribute to longevity in different manners. We previously found that a 2-fold up-regulation of Osh6, one of seven yeast oxysterol-binding proteins, remedies vacuolar morphology defects in mid-aged cells, partly down-regulates the target of rapamycin complex 1 (TORC1), and increases the replicative lifespan. At the molecular level, Osh6 transports phosphatidylserine (PS) and phosphatidylinositol-4-phosphate (PI4P) between the endoplasmic reticulum (ER) and the plasma membrane (PM). To decipher how an ER-PM working protein controls vacuolar morphology, we tested genetic interactions between OSH6 and DRS2, whose protein flips PS from the lumen to the cytosolic side of the Golgi, the organelle between ER and vacuoles in many pathways. Up-regulated OSH6 complemented vacuolar morphology of drs2Δ and enriched PI4P on the Golgi, indicating that Osh6 also works on the Golgi. This altered PI4P-enrichment led to a delay in the secretion of the proton ATPase Pma1 to the PM and a rerouting of Pma1 to vacuoles in a manner dependent on the trans-Golgi network (TGN) to late endosome (LE) trafficking pathway. Since the TGN-LE pathway controls endosomal and vacuolar TORC1, it may be the anti-aging pathway boosted by up-regulated Osh6.

Keywords:  Golgi; Osh6; PI4P; longevity; vacuole

DOI:  https://doi.org/10.15698/mic2022.08.783
Autophagy. 2022 Aug 18. 1-18

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.

Xiangyu Zhang, Santosh Kumar Misra, Parikshit Moitra, Xiuli Zhang, Se-Jin Jeong, Jeremiah Stitham, Astrid Rodriguez-Velez, Arick Park, Yu-Sheng Yeh, William E Gillanders, Daping Fan, Abhinav Diwan, Jaehyung Cho, Slava Epelman, Irfan J Lodhi, Dipanjan Pan, Babak Razani.

  Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1.

Keywords:  Acidic nanoparticles; PLGA; atherosclerosis; lysosomal dysfunction; macrophage

DOI:  https://doi.org/10.1080/15548627.2022.2108252
mBio. 2022 Aug 17. e0195622

Histone Acetyltransferase CfGcn5-Mediated Autophagy Governs the Pathogenicity of Colletotrichum fructicola.

Shengpei Zhang, Yuan Guo, Sizheng Li, He Li.

  Camellia oleifera is a woody edible-oil plant in China, and anthracnose occurs wherever it is grown, causing serious losses each year. We previously identified that the histone acetyltransferase CfGcn5 orchestrates growth, development, and pathogenicity in Colletotrichum fructicola, the major causal agent of anthracnose on C. oleifera. To elucidate the underlying mechanism, we conducted a transcriptome analysis and found that CfGcn5 is mainly involved in ribosomes, catalytic and metabolic processes, primary metabolism, and autophagy. In addition, we provided evidence showing that CfGcn5 serves as an autophagy repressor to mediate the expression of many autophagy-related genes (ATG) and undergoes degradation during autophagy. Moreover, we found that the CfATG8 and CfATG9 gene-deletion mutants had defects in mitosis and autophagy, resulting in their decreased appressoria formation rates and lower turgor pressure. These combined effects caused the failure of their appressoria functions and caused defects on their pathogenicity, revealing the importance of autophagy in pathogenicity. Taken together, our study illustrates that the autophagy repressor CfGcn5 undergoes degradation in order to regulate autophagy-dependent pathogenicity in C. fructicola. IMPORTANCE Colletotrichum spp. is ranked in the top 10 plant fungal pathogens and serves as a model for the study of hemibiotrophic pathogens, but its molecular mechanisms of pathogenesis remain largely unknown. Among species of Colletotrichum, C. fructicola causes anthracnose disease on more than 50 plants, such as pears, apples, and the important, edible-oil plant Camellia oleifera. We previously identified that the histone acetyltransferase CfGcn5 regulates growth, development, and pathogenicity in C. fructicola. To explore the underlying mechanisms, we performed comparative transcriptomic studies and found that CfGcn5 regulates global gene expression, including multiple autophagy-related genes (ATG genes). We revealed that CfGcn5 is an autophagy repressor that undergoes degradation during autophagy to govern pathogenicity. We also showed that the autophagy-related proteins CfAtg8 and CfAtg9 are required for full pathogenicity due to their regulatory functions in mitosis and autophagy. Our findings are important because we provide the first comprehensive characterization of autophagy as well as the relationship between acetylation and autophagy functioning in the pathogenesis of Colletotrichum spp., which might offer new potential targets for the management of anthracnose disease.

Keywords:  C. fructicola; autophagy; degradation; histone acetyltransferase; pathogenicity

DOI:  https://doi.org/10.1128/mbio.01956-22
Nucleic Acids Res. 2022 Aug 18. pii: gkac690. [Epub ahead of print]

Regulation of nuclear DNA damage response by mitochondrial morphofunctional pathway.

Nguyen Thi Kim Oanh, Ho-Soo Lee, Yong-Hyun Kim, Sunwoo Min, Yeon-Ji Park, June Heo, Yong-Yea Park, Won-Chung Lim, Hyeseong Cho.

Cells are constantly challenged by genotoxic stresses that can lead to genome instability. The integrity of the nuclear genome is preserved by the DNA damage response (DDR) and repair. Additionally, these stresses can induce mitochondria to transiently hyperfuse; however, it remains unclear whether canonical DDR is linked to these mitochondrial morphological changes. Here, we report that the abolition of mitochondrial fusion causes a substantial defect in the ATM-mediated DDR signaling. This deficiency is overcome by the restoration of mitochondria fusion. In cells with fragmented mitochondria, genotoxic stress-induced activation of JNK and its translocation to DNA lesion are lost. Importantly, the mitochondrial fusion machinery of MFN1/MFN2 associates with Sab (SH3BP5) and JNK, and these interactions are indispensable for the Sab-mediated activation of JNK and the ATM-mediated DDR signaling. Accordingly, the formation of BRCA1 and 53BP1 foci, as well as homology and end-joining repair are impaired in cells with fragmented mitochondria. Together, these data show that mitochondrial fusion-dependent JNK signaling is essential for the DDR, providing vital insight into the integration of nuclear and cytoplasmic stress signals.

DOI: https://doi.org/10.1093/nar/gkac690
Vet Res. 2022 Aug 17. 53(1): 64

The DHAV-1 protein VP1 interacts with PI3KC3 to induce autophagy through the PI3KC3 complex.

Juan Li, Mingshu Wang, Shan Zhou, Anchun Cheng, Xuming Ou, Di Sun, Ying Wu, Qiao Yang, Qun Gao, Juan Huang, Bin Tian, Sai Mao, Shaqiu Zhang, Xinxin Zhao, Renyong Jia, Mafeng Liu, Dekang Zhu, Shun Chen, Yunya Liu, Yanling Yu, Ling Zhang, Leichang Pan.

  Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens responsible for death in ducklings. Autophagy is a catabolic process that maintains cellular homeostasis, and the PI3KC3 protein plays an important role in the initiation of autophagy. DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) but the molecular mechanism between it and autophagy has not been reported. First, we determined that DHAV-1 infection induces autophagy in DEFs and that autophagy induction is dependent on the integrity of viral proteins by infecting DEFs with UV-inactivated or heat-inactivated DHAV-1. Then, in experiments using the pharmacological autophagy inducer rapamycin and the autophagy inhibitor chloroquine, autophagy inhibition was shown to reduce intracellular and extracellular DHAV-1 genome copies and viral titres. These results suggest that autophagy activated by DHAV-1 infection in DEFs affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of the DHAV-1 structural proteins VP0, VP3, and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEFs but not the full autophagic flux. Finally, we found that VP1 promotes protein expression of PI3KC3 and Beclin1 by western blot experiments and that VP1 interacts with PI3KC3 by co-immunoprecipitation experiments; moreover, 3-MA-induced knockdown of PI3KC3 inhibited VP1 protein-induced autophagy in DEFs. In conclusion, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEFs.

Keywords:  Beclin1; Duck hepatitis A virus type 1; PI3KC3; VP1; autophagy

DOI:  https://doi.org/10.1186/s13567-022-01081-6
Elife. 2022 Aug 17. pii: e78069. [Epub ahead of print]11

Biphasic regulation of osteoblast development via the ERK MAPK-mTOR pathway.

Jung-Min Kim, Yeon-Suk Yang, Jaehyoung Hong, Sachin Chaugule, Hyonho Chun, Marjolein C H van der Meulen, Ren Xu, Matthew B Greenblatt, Jae-Hyuck Shim.

  Emerging evidence supports that osteogenic differentiation of skeletal progenitors is a key determinant of overall bone formation and bone mass. Despite extensive studies showing the function of mitogen-activated protein kinases (MAPKs) in osteoblast differentiation, none of these studies show in vivo evidence of a role for MAPKs in osteoblast maturation subsequent to lineage commitment. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Map2k1 (MEK1) and Map2k2 (MEK2), in mature osteoblasts and osteocytes, markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts. also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partially reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1.

Keywords:  medicine; mouse

DOI:  https://doi.org/10.7554/eLife.78069
Autophagy. 2022 Aug 18.

Growth factor receptor signaling induces mitophagy through epitranscriptomic regulation.

Deguan Lv, Kailin Yang, Jeremy N Rich.

  Aberrant growth factor receptor signaling is among the most common oncogenic drivers in cancer biology. Receptor signaling classically induces cancer growth through signaling cascades that mediate effects largely through transcriptional control. Recently, post-transcriptional RNA modifications, collectively designated as epitranscriptomics, have emerged as a critical layer of dysregulation in cancer biology. We recently reported that PDGFR (platelet derived growth factor receptor) activity in cancer stem cells (CSCs) derived from glioblastoma patients display increased post-transcriptional mRNA methylation (N6-methyladenosine [m6A]), which promotes CSC maintenance through regulation of mitophagy. Specifically, PDGF-PDGFRB signaling upregulates expression of the m6A methyltransferase METTL3, which then decorates the mitophagy regulator OPTN (optineurin) mRNA with m6A, thereby promoting OPTN mRNA degradation. Glioblastomas express lower levels of OPTN than normal brain, and forced expression of OPTN reduces tumor growth, supporting a tumor suppressive role for OPTN. Pharmacological targeting of METTL3 with PDGFR or activation of mitophagy demonstrate a combinatorial benefit. Collectively, our results suggest that upstream regulation of mitophagy in lethal cancers is mediated through growth factor receptor control of post-transcriptional RNA regulation, offering novel therapeutic paradigms.

Keywords:  Cancer stem cell; METTL3; N6-methyladenosine (m6A); OPTN; PDGF; PDGFR; glioblastoma; mitophagy; optineurin

DOI:  https://doi.org/10.1080/15548627.2022.2114765
Mol Biol Cell. 2022 Aug 17. mbcE21090473

AP-4 regulates neuronal lysosome composition, function, and transport via regulating export of critical lysosome receptor proteins at the trans-Golgi network.

Piyali Majumder, Daisy Edmison, Catherine Rodger, Sruchi Patel, Evan Reid, Swetha Gowrishankar.

The adaptor protein complex-4 or AP-4 is known to mediate autophagosome maturation through regulating sorting of transmembrane cargo such as ATG9A at the Golgi. There is a need to understand AP-4 function in neurons, as mutations in any of its four subunits cause a complex form of hereditary spastic paraplegia (HSP) with intellectual disability. While AP-4 has been implicated in regulating trafficking and distribution of cargo such as ATG9A and APP, little is known about its effect on neuronal lysosomal protein traffic, lysosome biogenesis and function. In this study, we demonstrate that in human iPSC-derived neurons AP-4 regulates lysosome composition, function and transport via regulating export of critical lysosomal receptors, including Sortilin 1, from the trans-Golgi network to endo-lysosomes. Additionally, loss of AP-4 causes endo-lysosomes to stall and build up in axonal swellings potentially through reduced recruitment of retrograde transport machinery to the organelle. These findings of axonal lysosome build-up are highly reminiscent of those observed in Alzheimer's disease as well as in neurons modelling the most common form of HSP, caused by spastin mutations. Our findings implicate AP-4 as a critical regulator of neuronal lysosome biogenesis and altered lysosome function and axonal endo-lysosome transport as an underlying defect in AP-4 deficient HSP. Additionally, our results also demonstrate the utility of the human i3Neuronal model system in investigating neuronal phenotypes observed in AP-4 deficient mice and/or the human AP-4 deficiency syndrome. [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E21-09-0473
Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01012-9. [Epub ahead of print]40(7): 111195

Nondegradable ubiquitinated ATG9A organizes Golgi integrity and dynamics upon stresses.

Qian Luo, Qiangqiang Liu, Hongcheng Cheng, Jiale Wang, Tian Zhao, Jiaojiao Zhang, Chenglong Mu, Yuanyuan Meng, Linbo Chen, Changqian Zhou, Hong Lei, Jianyu Yang, Guo Chen, Yanjun Li, Leiting Pan, Quan Chen, Yushan Zhu.

  ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.

Keywords:  ATG9A; CP: Molecular biology; Golgi dynamics; Golgi stress response; ubiquitination

DOI:  https://doi.org/10.1016/j.celrep.2022.111195
Stem Cells Transl Med. 2022 Aug 18. pii: szac049. [Epub ahead of print]

METTL14 Regulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells via Inducing Autophagy Through m6A/IGF2BPs/Beclin-1 Signal Axis.

Mingyu He, Hong Lei, Xiaoqi He, Ying Liu, Ao Wang, Zijing Ren, Xiaoyan Liu, Gege Yan, Wenbo Wang, Yang Wang, Guanghui Li, Tong Wang, Jiaying Pu, Zhihua Shen, Yanquan Wang, Jiajie Xie, Weijie Du, Ye Yuan, Lei Yang.

  The development of osteoporosis is often accompanied by autophagy disturbance, which also causes new osteoblast defects from bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are still not fully understood. Methyltransferase-like 14 (METTL14) is the main enzyme for N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, and it has been implicated in many bioprocesses. Herein, we demonstrate that METTL14 plays a critical role in autophagy induction and hinders osteoporosis process whose expression is decreased both in human osteoporosis bone tissue and ovariectomy (OVX) mice. In vivo, METTL14+/- knockdown mice exhibit elevated bone loss and impaired autophagy similar to the OVX mice, while overexpression of METTL14 significantly promotes bone formation and inhibits the progression of osteoporosis caused by OVX surgery. In vitro, METTL14 overexpression significantly enhances the osteogenic differentiation ability of BMSCs through regulating the expression of beclin-1 depending on m6A modification and inducing autophagy; the opposite is true with METTL14 silencing. Subsequently, m6A-binding proteins IGF2BP1/2/3 recognize m6A-methylated beclin-1 mRNA and promote its translation via mediating RNA stabilization. Furthermore, METTL14 negatively regulates osteoclast differentiation. Collectively, our study reveals the METTL14/IGF2BPs/beclin-1 signal axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14-mediated m6A modification in osteoporosis.

Keywords:  METTL14; autophagy; bone marrow mesenchymal stem cells; m6A RNA methylation; osteogenic differentiation

DOI:  https://doi.org/10.1093/stcltm/szac049
Free Radic Biol Med. 2022 Aug 16. pii: S0891-5849(22)00547-0. [Epub ahead of print]

MTP18 inhibition triggers mitochondrial hyperfusion to induce apoptosis through ROS-mediated lysosomal membrane permeabilization-dependent pathway in oral cancer.

Debasna Pritimanjari Panigrahi, Srimanta Patra, Bishnu Prasad Behera, Pradyota Kumar Behera, Shankargouda Patil, Birija Sankar Patro, Laxmidhar Rout, Itisam Sarangi, Sujit Kumar Bhutia.

  Although stress-induced mitochondrial hyperfusion (SIMH) exerts a protective role in aiding cell survival, in the absence of mitochondrial fission, SIMH drives oxidative stress-related induction of apoptosis. In this study, our data showed that MTP18, a mitochondrial fission-promoting protein expression, was increased in oral cancer. We have screened and identified S28, a novel inhibitor of MTP18, which was found to induce SIMH and subsequently trigger apoptosis. Interestingly, it inhibited MTP18-mediated mitochondrial fission, as shown by a decrease in p-Drp1 along with increased Mfn1 expression in oral cancer cells. Moreover, S28 induced autophagy but not mitophagy due to the trouble in engulfment of hypoperfused mitochondria. Interestingly, S28-mediated SIMH resulted in the loss of mitochondrial membrane potential, leading to the consequent generation of mitochondrial superoxide to induce intrinsic apoptosis. Mechanistically, S28-induced mitochondrial superoxide caused lysosomal membrane permeabilization (LMP), resulting in decreased lysosomal pH, which impaired autophagosome-lysosome fusion. In this setting, it showed that overexpression of MTP18 resulted in mitochondrial fission leading to mitophagy and inhibition of ROS-mediated LMP and apoptosis. Further, S28, in combination with FDA-approved anticancer drugs, exhibited higher apoptotic activity and decreased cell viability, suggesting the MTP18 inhibition combined with the anticancer drug could have greater efficacy against cancer.

Keywords:  Apoptosis; Hyperfused mitochondria; Lysosomal membrane permeabilization; MTP18; Mitochondrial superoxide

DOI:  https://doi.org/10.1016/j.freeradbiomed.2022.08.019
Med Oncol. 2022 Aug 16. 39(11): 167

Matrine induces autophagy in human neuroblastoma cells via blocking the AKT-mTOR pathway.

Nanjing Liu, Chunmei Yang, Li Yang, Ting Li, Maoyuan Gong, Haobiao Wang, Jun Zhang, Hui Zhao, Lin Zou, Xiaoyan He.

  Neuroblastoma (NB) is one of the most common malignant solid tumors in children. Despite significant advances in the treatment strategy, the long-term survival rate of NB patients is only 50%. Developing new agents for NB patients deserves attention. Recent research indicates that matrine, a natural quinolizidine alkaloid component extracted from the traditional Chinese medicine Sophora root, is widely used for various diseases, including antitumor effects against a variety of cancers. However, the effect of matrine on NB is unknown. Herein, we found that matrine exerted antiproliferative activity in human NB cells in dose- and time-dependent manner. Matrine triggered autophagy in NB cells by blocking the AKT-mTOR signaling pathway and suppressing the phosphorylation of AKT and mTOR. 3-Methyladenine (3-MA), a PI3K inhibitor, protected against matrine-induced inhibition of cell proliferation, further supporting that the antitumor activity of matrine was at least partly autophagy-dependent. In vivo, matrine reduced tumor growth of SK-N-DZ cells in a dose-dependent manner. Matrine treatment significantly declined the phosphorylation of AKT and mTOR and enhanced the LC3 II/GAPDH ratio in NB xenografts. Altogether, our work uncovered the molecular mechanism underlying matrine-induced autophagy in NB and provided implications for matrine as a potential therapeutic agent against NB.

Keywords:  Autophagy; Cell proliferation; Matrine; Neuroblastoma

DOI:  https://doi.org/10.1007/s12032-022-01762-4
Cell Death Discov. 2022 Aug 13. 8(1): 357

Elucidation of an mTORC2-PKC-NRF2 pathway that sustains the ATF4 stress response and identification of Sirt5 as a key ATF4 effector.

Ruizhi Li, Kristin F Wilson, Richard A Cerione.

Proliferating cancer cells are dependent on glutamine metabolism for survival when challenged with oxidative stresses caused by reactive oxygen species, hypoxia, nutrient deprivation and matrix detachment. ATF4, a key stress responsive transcription factor, is essential for cancer cells to sustain glutamine metabolism when challenged with these various types of stress. While it is well documented how the ATF4 transcript is translated into protein as a stress response, an important question concerns how the ATF4 message levels are sustained to enable cancer cells to survive the challenges of nutrient deprivation and damaging reactive oxygen species. Here, we now identify the pathway in triple negative breast cancer cells that provides a sustained ATF4 response and enables their survival when encountering these challenges. This signaling pathway starts with mTORC2, which upon sensing cellular stresses arising from glutamine deprivation or an acute inhibition of glutamine metabolism, initiates a cascade of events that triggers an increase in ATF4 transcription. Surprisingly, this signaling pathway is not dependent on AKT activation, but rather requires the mTORC2 target, PKC, which activates the transcription factor Nrf2 that then induces ATF4 expression. Additionally, we identify a sirtuin family member, the NAD+-dependent de-succinylase Sirt5, as a key transcriptional target for ATF4 that promotes cancer cell survival during metabolic stress. Sirt5 plays fundamental roles in supporting cancer cell metabolism by regulating various enzymatic activities and by protecting an enzyme essential for glutaminolysis, glutaminase C (GAC), from degradation. We demonstrate that ectopic expression of Sirt5 compensates for knockdowns of ATF4 in cells exposed to glutamine deprivation-induced stress. These findings provide important new insights into the signaling cues that lead to sustained ATF4 expression as a general stress-induced regulator of glutamine metabolism, as well as highlight Sirt5 an essential effector of the ATF4 response to metabolic stress.

DOI: https://doi.org/10.1038/s41420-022-01156-5
Aging (Albany NY). 2022 Aug 16. 14(undefined):

miR-141 exacerbates lung ischemia-reperfusion injury by targeting EGFR/β-catenin axis-mediated autophagy.

Miao Yang, Xiaomei Ling, Jinfang Xiao.

  Some microRNAs (miRNAs) play important roles in lung ischemia-reperfusion injury (LIRI) injury. Here, this study aimed to examine whether miR-141 was related to lung ischemia-reperfusion injury (IRI) via regulating autophagy and the epidermal growth factor receptor (EGFR), and to explore the underlying signal transduction pathways. To this end, we constructed the LIRI cell model and mouse models, separately. According to RT-qPCR and Western blotting (WB) analysis results, miR-141 up-regulation together with β-catenin and EGFR down-regulation within mouse pulmonary microvascular endothelial cells (PMVECs) or lung tissues was related to lung IRI. Besides, we conducted dual-luciferase reporter assay, which suggested the binding of EGFR to miR-141. In addition, we carried out TUNEL staining, HE staining, and flow cytometric analysis to assess the apoptosis of PMVECs and the injury to mouse lung tissues. Furthermore, we performed light-chain immunofluorescence assay to examine autophagosomes within PMVECs. According to our results, miR-141 suppressed β-catenin level through reducing EGFR level. Besides, the miR-141/EGFR/β-catenin axis enhanced autophagy to aggravate LIRI. To sum up, miR-141 suppresses EGFR expression to inhibit β-catenin level, which subsequently aggravates autophagy and complicates LIRI. The above results offer the candidate therapeutic target for the treatment of lung IRI.

Keywords:  EGFR; autophagy; lung ischemia reperfusion injury; microRNA-141 (miR-141); β-catenin

DOI:  https://doi.org/10.18632/aging.204137
Phytomedicine. 2022 Jul 28. pii: S0944-7113(22)00440-8. [Epub ahead of print]105 154361

Piperine alleviates acute pancreatitis: A possible role for FAM134B and CCPG1 dependent ER-phagy.

Weiguo Huang, Jie Zhang, Wenzhang Jin, Jintao Yang, Guanzhen Yu, Hongqi Shi, Keqing Shi.

   BACKGROUND: Acute pancreatitis was a common acute abdominal disease characterized by pancreatic acinar cell death and inflammation. Endoplasmic reticulum autophagy (ER-phagy) coud maintain cell homeostasis by degrading redundant and disordered endoplasmic reticulum and FAM134B and CCPG1 was main ER-phagy receptors. As a natural alkaloid, piperin is found in black pepper and has anti-inflammatory properties, whose effect on ER-phagy in pancreatitis has not been studied.
PURPOSE: The objective of this study was to demonstrate the pivotal role of FAM134B and CCPG1 dependent ER-phagy for alleviating acute pancreatitis and explore the molecular mechanism of piperine in alleviating acute pancreatitis.
METHOD: In this study we investigated the role of ER-phagy in acute pancreatitis and whether piperine could alleviate pancreatitis through ER-phagy regulation. We first detected endoplasmic reticulum stress (ER-stress) and ER-phagy in different degrees of acute pancreatitis. Then we used ER-stress and autophagy regulators to explore the relationship between ER-stress and ER-phagy in acute pancreatitis and their regulation of cell death. Through using FAM134B-/- and CCPG1-/-, we investigated the mechanism of piperine in the treatment of acute pancreatitis.
RESULTS: In this study, we confirmed that with the progression of acute pancreatitis, the pancreatic endoplasmic reticulum stress increased continuously, but the ER-phagy increased first and then was inhibited. Meanwhile, in acute pancreatitis, ER-stress and ER-phagy interacted: endoplasmic reticulum stress can induce ER-phagy, but serious ER-stress would inhibit ER-phagy; and ER-phagy could alleviate ER-stress. Next, we found that piperine reduced ER-stress by enhancing FAM134B and CCPG1 dependent ER-phagy, thereby alleviating pancreatic injury.
CONCLUSION: Impaired ER-phagy was both a cause and a consequence of ER-stress in AP mice, which contributed to the transition from AP to SAP. Piperine targeting ER-phagy provided a new insight into the pharmacological mechanism of piperine in treating AP.

Keywords:  Acute pancreatitis; CCPG1; ER-phagy; FAM134B; Piperine

DOI:  https://doi.org/10.1016/j.phymed.2022.154361