bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒05‒15
seventy papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2022 May 09. 1-2
      The unique cellular organization and metabolic demands of neurons pose a challenge in the maintenance of neuronal homeostasis. A critical element in maintaining neuronal health and homeostasis is mitochondrial quality control via replacement and rejuvenation at the axon. Dysregulation of mitochondrial quality control mechanisms such as mitophagy has been implicated in neurodegenerative diseases including Parkinson disease and amyotrophic lateral sclerosis. To sustain mitophagy at the axon, a continuous supply of PINK1 is required; however, how do neurons maintain a steady supply of this protein at the distal axons? In the study highlighted here, Harbauer et al. show that axonal mitophagy is supported by local translation of Pink1 mRNA that is co-transported with mitochondria to the distal ends of the neuron. This neuronal-specific pathway provides a continuous supply of PINK1 to sustain mitophagy.
    Keywords:  Autophagy; mitochondria; neurodegeneration; neuron; stress
  2. FASEB J. 2022 May;36 Suppl 1
      The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. The complex is present at the plasma membrane, nucleus, lysosomes, and the outer mitochondrial membrane. Such spatial compartmentation has been suggested to enhance signaling efficiency and specificity. For instance, we recently discovered nuclear mTORC1 activity, which is distinctly regulated from the canonical lysosomal mTORC1 (Zhou et al., 2020). Previous studies have shown that mTOR is present at the outer mitochondrial membrane (OMM), but it is not clear whether mTORC1 is active at this location and what the functional consequences are. To investigate this, we targeted our FRET-based mTORC1 activity reporter, TORCAR (Zhou et al., 2015), to the OMM and probed the subcellular activity of mTORC1. We found that platelet-derived growth factor (PDGF) stimulation increases mTORC1 activity at the OMM in addition to at the lysosome and in the nucleus, whereas insulin specifically stimulates mTORC1 activity at the OMM without affecting the lysosomal and nuclear activities. We further dissected the regulation of mitochondrial mTORC1 activity and applied a novel approach of identifying new mTORC1 substrates. Elucidating the signaling events that lead to subcellular mTORC1 activity at mitochondria and its downstream functions will increase our understanding of the roles that mTORC1 may play in diseases associated with altered metabolism or mitochondrial dysfunction, such as diabetes and cancer.
  3. FASEB J. 2022 May;36 Suppl 1
      Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress-induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi-pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome-level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock-out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3-lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock-in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.
  4. Cells. 2022 Apr 27. pii: 1469. [Epub ahead of print]11(9):
      Myeloid cell leukemia-1 (Mcl-1) is a unique antiapoptotic Bcl-2 member that is critical for mitochondrial homeostasis. Recent studies have demonstrated that Mcl-1's functions extend beyond its traditional role in preventing apoptotic cell death. Specifically, data suggest that Mcl-1 plays a regulatory role in autophagy, an essential degradation pathway involved in recycling and eliminating dysfunctional organelles. Here, we investigated whether Mcl-1 regulates autophagy in the heart. We found that cardiac-specific overexpression of Mcl-1 had little effect on baseline autophagic activity but strongly suppressed starvation-induced autophagy. In contrast, Mcl-1 did not inhibit activation of autophagy during myocardial infarction or mitochondrial depolarization. Instead, overexpression of Mcl-1 increased the clearance of depolarized mitochondria by mitophagy independent of Parkin. The increase in mitophagy was partially mediated via Mcl-1's LC3-interacting regions and mutation of these sites significantly reduced Mcl-1-mediated mitochondrial clearance. We also found that Mcl-1 interacted with the mitophagy receptor Bnip3 and that the interaction was increased in response to mitochondrial stress. Overall, these findings suggest that Mcl-1 suppresses nonselective autophagy during nutrient limiting conditions, whereas it enhances selective autophagy of dysfunctional mitochondria by functioning as a mitophagy receptor.
    Keywords:  Bnip3; Mcl-1; autophagy; heart; mitochondria; mitophagy
  5. FASEB J. 2022 May;36 Suppl 1
      P21-activated serine/threonine Kinase 1 (PAK1) plays a critical role in cardiomyocyte survival under numerous stressful conditions. Autophagy is a cellular process that promotes homeostasis by removing and replacing antiquated or damaged cellular components. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria to maintain a pool of healthy mitochondria. We have previously demonstrated that PAK1 is essential for maintaining autophagy and mitophagy activities. However, the downstream mediators have not yet been discovered. In this study, we explored the mechanisms of PAK1-dependent autophagy and mitophagy by determining the protein expression levels of the major regulators of autophagy and mitophagy. H9c2 cardiac myoblasts were treated with siRNA to knockdown the expression of PAK1. Western blot analysis showed that PAK1 knockdown substantially reduced the protein expression levels of ATG5-12 complex, an essential promotor of autophagosome formation, TFEB, a master regulator of lysosome biogenesis and autophagy, and p62, an autophagy receptor for ubiquitinated cargos. All these changes are expected to reduce autophagy activity. In addition, PAK1 knockdown also reduced the expression of mitophagy receptor FUNDC1 and diminished the association of p62 with mitochondria, which coincided with reduced mitophagy activity. Collectively, these results suggest that the ability of PAK1 knockdown to inhibit autophagy and mitophagy is mediated by reduced expression levels of several important regulators of autophagy and/or mitophagy pathways. Future research is warranted to determine whether restoring the expression levels of these target genes can overcome the inhibition of autophagy and mitophagy by PAK1 deficiency.
  6. FASEB J. 2022 May;36 Suppl 1
      The conserved kinase mTOR (mechanistic target of rapamycin) regulates cell metabolism and promotes cell growth, proliferation, and survival in response to diverse environmental cues (e.g., nutrients; growth factors; hormones). mTOR forms the catalytic core of two multiprotein complexes, mTORC1 and mTORC2, which possess unique downstream targets and cellular functions. While mTORC1 and mTORC2 often respond to distinct upstream cues, they share a requirement for PI3K in their activation by growth factors. While many studies agree that amino acids activate mTORC1 but not mTORC2, several studies reported paradoxical activation of mTORC2 by amino acids. We noted that stimulating amino acid starved cells with a commercial mixture of amino acids increased mTORC2-dependent Akt S473 phosphorylation rapidly while re-feeding cells with complete DMEM containing amino acids failed to do so. Interestingly, we found the pH of the commercial amino acid mixture to be ~ pH 10. Upon controlling for pH, stimulating starved cells with amino acids at pH 10 but not 7.4 increased mTORC2 signaling. Moreover, DMEM at alkaline pH was sufficient to increase mTORC2 catalytic activity and signaling. Using a fluorescent pH-sensitive dye (cSNARF-1-AM) coupled to ratio-metric live cell imaging, we confirmed that alkaline extracellular pH (pHe) translated into a rapid increase in intracellular pH (pHi). Moreover, blunting this increase with a pharmacological inhibitor of an H+ transporter attenuated the increase in mTORC2 signaling by pHe. Alkaline pHi also activated AMPK, a canonical sensor of energetic stress that promotes mTORC2 signaling, as reported previously by us. Functionally, we found that alkaline pHi attenuated apoptosis caused by growth factor withdrawal through activation of AMPK-mTORC2 signaling. These results indicate that alkaline pHi augments mTORC2 signaling to promote cell survival, in part through AMPK. In the course of this work, we noted that pHi increased phosphorylation of several downstream targets of PI3K (e.g., Akt P-T308 and P-S473; S6K1 P-T389 and P-T229; PRAS40 P-T246; Tsc2 P-S939), suggesting that PI3K itself responds to changes in pHi. Indeed, alkaline pHi increased PI-3',4',5'-P3 levels in a manner sensitive to the PI3K inhibitor BYL-719. Thus, alkaline pHi elevates PI3K activity, which increases both mTORC1 and mTORC2 signaling. Mechanistically, we found that activation of PI3K by alkaline pHi induced dissociation of Tsc2 from lysosomal membranes, thereby relieving TSC-mediated suppression of Rheb, a mTORC1-activating GTPase. Functionally, we found that activation of PI3K by alkaline pHi increased mTORC1-mediated 4EBP1 phosphorylation, which initiates cap-dependent translation by eIF4E. Alkaline pHi also increased mTORC1-driven protein synthesis. Taken together, these findings reveal alkaline pHi as a previously unrecognized activator of PI3K-mTORC1/2 signaling that promotes protein synthesis and cell survival. As elevated pHi represents an under-appreciated hallmark of cancer cells, these findings suggest that by alkaline pHi sensing by the PI3K-mTOR axis and AMPK-mTORC2 axes may contribute to tumorigenesis.
  7. Cell Rep. 2022 May 10. pii: S2211-1247(22)00564-2. [Epub ahead of print]39(6): 110797
      The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.
    Keywords:  APEX2; CP: Cell biology; CP: Immunology; ER-mitochondria contact site; HIV-1; TRIM5α; ULK1 complex; autophagy; inflammation; mitochondrial metabolism; proteomics; tripartite motif
  8. FASEB J. 2022 May;36 Suppl 1
      In cancer, oncogene dependency is a phenomenon where a dominant driver oncogene promotes tumor cell proliferation and survival, and loss of this oncogene results in tumor cell death and, eventually, tumor regression. KRAS, a GTPase that regulates cell growth and proliferation, is an oncogene constitutively activated in over 90% of pancreatic cancer. However, clinically effective inhibitors of KRAS have been unsuccessful so efforts have been focused on identifying other potential targets associated with the KRAS signaling network. Spleen tyrosine kinase (SYK), which is expressed at high levels in KRAS-dependent pancreatic cancer cell lines, may be one of these targets. Our data indicate that SYK activates the mechanistic target of rapamycin kinase complex 1 (mTORC1), which promotes protein translation and cell growth. SYK activation also leads to decreased autolysosome count. In connecting SYK activation with increased mTORC1 activity and decreased autolysosome count, we hypothesis that the MiT/TFE transcription factors are involved. We propose that SYK inhibition in pancreatic cancer cells leads to reduced mTORC1 activity, which reduces phosphorylation of MiT/TFE transcription factors. The unphosphorylated MiT/TFE transcription factors may then enter the nucleus to activate genes for lysosomal biogenesis and autophagy. Autophagy is a process that recycles cellular macromolecules during nutrient deprivation by fusing autophagosomes and lysosomes, produced from lysosomal biogenesis, to generate autolysosomes. From our experiments, we were able to show that SYK inhibition blocks mTORC1-dependent phosphorylation of MITF and TFEB transcription factors, members of the MiT/TFE family. MITF and TFEB activation leads to increased autophagy due to autolysosomal biogenesis and accumulation. In summary, our studies of the SYK-mTORC1-autophagy pathway provide support to investigate SYK as a candidate therapeutic target for pancreatic cancer treatment.
  9. Adv Nutr. 2022 May 13. pii: nmac055. [Epub ahead of print]
      Mechanistic target of rapamycin complex 1 (mTORC1) is a multi-protein complex widely found in eukaryotes. It serves as a central signaling node to coordinate cell growth and metabolism by sensing diverse extracellular and intracellular inputs, including amino acid-, growth factor-, glucose-, and nucleotide-related signals. It is well documented that mTORC1 is recruited to the lysosomal surface, where it is activated and, accordingly, modulates downstream effectors involved in regulating protein, lipid, and glucose metabolism. mTORC1 is thus the central node for coordinating the storage and mobilization of nutrients and energy across various tissues. However, emerging evidence indicated that the overactivation of mTORC1 induced by nutritional disorders leads to the occurrence of a variety of metabolic diseases, including obesity and type 2 diabetes, as well as cancer, neurodegenerative disorders, and aging. That the mTORC1 pathway plays a crucial role in regulating the occurrence of metabolic diseases renders it a prime target for the development of effective therapeutic strategies. Here, we focus on recent advances in our understanding of the regulatory mechanisms underlying how mTORC1 integrates metabolic inputs as well as the role of mTORC1 in the regulation of nutritional and metabolic diseases.
    Keywords:  Metabolic diseases; Metabolism; Nutrient; Signal transduction; mTORC1
  10. EMBO J. 2022 May 13. e111424
      The lysosomal degradation pathway of autophagy depends on a set of evolutionarily conserved autophagy-related molecules (ATGs) bestowed with the power to direct membrane trafficking and biology. In this issue of EMBO Journal, Kakanj P et al reveal a surprising role for the autophagy machinery in cell fusion (Kakanj et al, 2022). Autophagy is physiologically required for cell syncytium formation through dismantling the lateral plasma membrane during wound healing, and unchecked autophagy can drive cell fusion in epithelial tissues without compromising epithelial integrity.
  11. Acta Pharm Sin B. 2022 Mar;12(3): 1019-1040
      Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by memory loss and cognitive dysfunction. The accumulation of misfolded protein aggregates including amyloid beta (Aβ) peptides and microtubule associated protein tau (MAPT/tau) in neuronal cells are hallmarks of AD. So far, the exact underlying mechanisms for the aetiologies of AD have not been fully understood and the effective treatment for AD is limited. Autophagy is an evolutionarily conserved cellular catabolic process by which damaged cellular organelles and protein aggregates are degraded via lysosomes. Recently, there is accumulating evidence linking the impairment of the autophagy-lysosomal pathway with AD pathogenesis. Interestingly, the enhancement of autophagy to remove protein aggregates has been proposed as a promising therapeutic strategy for AD. Here, we first summarize the recent genetic, pathological and experimental studies regarding the impairment of the autophagy-lysosomal pathway in AD. We then describe the interplay between the autophagy-lysosomal pathway and two pathological proteins, Aβ and MAPT/tau, in AD. Finally, we discuss potential therapeutic strategies and small molecules that target the autophagy-lysosomal pathway for AD treatment both in animal models and in clinical trials. Overall, this article highlights the pivotal functions of the autophagy-lysosomal pathway in AD pathogenesis and potential druggable targets in the autophagy-lysosomal pathway for AD treatment.
    Keywords:  Alzheimer's disease (AD); Amyloid beta (Aβ) peptides; Autophagy; Autophagy enhancers; Autophagy–lysosomal pathway; MAPT/tau; Mitophagy; Neurodegenerative diseases
  12. Sci Rep. 2022 May 10. 12(1): 7652
      Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines, S. cerevisiae and C. elegans. LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances.
  13. FASEB J. 2022 May;36 Suppl 1
      Excessive inflammation underlies many human diseases, such as neurodegeneration, autoimmune disorders, cancer, and COVID-19. Thus, cellular mechanisms that control inflammation are of high therapeutic interest. Autophagy has been implicated in the suppression of inflammation, yet mechanistic links between autophagy and inflammation are not completely understood. Previous work demonstrated that loss of ATG9A, but not ATG5 or ATG7, increased inflammatory signaling through the STING-IRF3 cascade, suggesting that perhaps an autophagy-independent function of ATG9A regulates inflammation.1 Here, we show that loss of the essential basal autophagy regulators ATG9A and ATG101 sensitizes cells to dsDNA-induced IRF3 activation. Importantly, this effect was not observed with the loss of ATG5 or ATG7, suggesting that the canonical LC3-lipidation autophagy machinery is not required for suppression of IRF3 and inflammatory signaling.2 In an effort to understand the role of ATG9A and ATG101 in suppressing inflammatory signaling, we found that loss of ATG9A and ATG101, but not ATG5 or ATG7, caused an accumulation of ubiquitin-rich condensates. We also found that the accumulation of ubiquitin-rich condensates coincided with an overactivation of the ubiquitin-sensing, IRF3-targeted kinase TBK1. Importantly, we found that inhibiting the accumulation of ubiquitin-rich condensates via knock-out of p62/SQSTM1 abrogated the increased inflammatory signaling caused by loss of ATG9A. Together, our data suggest a model in which the loss of basal autophagy machinery causes an accumulation of ubiquitin-rich condensates, which, in turn, act as a platform for the aberrant activation of TBK1-mediated inflammatory signaling. We propose that these data have important implications for diseases of protein aggregation wherein similar ubiquitin-rich condensates form and aberrant inflammatory signaling plays a pathological role. REFERENCES: (1) Saitoh, T.; Fujita, N.; Hayashi, T.; Takahara, K.; Satoh, T.; Lee, H.; Matsunaga, K.; Kageyama, S.; Omori, H.; Noda, T.; Yamamoto, N.; Kawai, T.; Ishii, K.; Takeuchi, O.; Yoshimori, T.; Akira, S. Atg9a Controls DsDNA-Driven Dynamic Translocation of STING and the Innate Immune Response. PNAS 2009, 106 (49). (2) Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L. BioID Reveals an ATG9A Interaction with ATG13-ATG101 in the Degradation of P62/SQSTM1-ubiquitin Clusters. EMBO reports 2021, 22 (10).
  14. Int J Mol Sci. 2022 Apr 28. pii: 4899. [Epub ahead of print]23(9):
      Infectious diseases are a burden for aquaculture. Antigen processing and presentation (APP) to the immune effector cells that fight pathogens is key in the adaptive immune response. At the core of the adaptive immunity that appeared in lower vertebrates during evolution are the variable genes encoding the major histocompatibility complex (MHC). MHC class I molecules mainly present peptides processed in the cytosol by the proteasome and transported to the cell surface of all cells through secretory compartments. Professional antigen-presenting cells (pAPC) also express MHC class II molecules, which normally present peptides processed from exogenous antigens through lysosomal pathways. Autophagy is an intracellular self-degradation process that is conserved in all eukaryotes and is induced by starvation to contribute to cellular homeostasis. Self-digestion during autophagy mainly occurs by the fusion of autophagosomes, which engulf portions of cytosol and fuse with lysosomes (macroautophagy) or assisted by chaperones (chaperone-mediated autophagy, CMA) that deliver proteins to lysosomes. Thus, during self-degradation, antigens can be processed to be presented by the MHC to immune effector cells, thus, linking autophagy to APP. This review is focused on the essential components of the APP that are conserved in teleost fish and the increasing evidence related to the modulation of APP and autophagy during pathogen infection.
    Keywords:  LC3-Associated phagocytosis; MHC class II; antigen processing; antigen-presenting cell; bacteria; chaperone-mediated autophagy; macroautophagy; major histocompatibility complex MHC class I; vaccine; virus
  15. FASEB J. 2022 May;36 Suppl 1
      Macroautophagy (hereafter, autophagy) is a multi-step process through which cells degrade damaged or unutilized proteins or organelles (cargos) to maintain homeostasis. In the final step of autophagy, cargos are degraded by lysosomal enzymes, whose activities require an acidic environment. Intracellular Ca2+ has been shown to play diverse roles in autophagy. In many cases, Ca2+ signals are transduced into cellular activities by forming a complex with the ubiquitous Ca2+ -binding protein calmodulin (CaM), which then activates numerous target proteins, estimated to be over 300. However, CaM is not expressed sufficiently for all these targets. This generates a limiting condition in which CaM availability is competed for. Although several CaM-binding proteins have been implicated in autophagy, the role of CaM availability in autophagy is unknown, especially in basal condition when there are no stimulated increases in intracellular Ca2+ . Here we show that overexpressing CaM promotes autophagic flux in basal, unstimulated condition. Removal of amino acids triggers acute intracellular Ca2+ signals, and autophagic flux during this time frame is also significantly higher in CaM-overexpressing cells compared to the wildtype counterparts. Inhibiting CaM using various CaM antagonists is associated with increases in lysosomal pH and accumulation of LC3-II. Finally, buffering cellular free CaM using a high-affinity CaM-binding protein that can bind CaM at resting Ca2+ levels significantly increases basal lysosomal pH. These data indicate that CaM availability aids in maintaining optimal lysosomal pH, which contributes to the regulation of basal autophagy.
  16. J Exp Bot. 2022 May 13. 73(9): 2848-2858
      Autophagy is a catabolic process in which cytoplasmic components are delivered to vacuoles or lysosomes for degradation and nutrient recycling. Autophagy-mediated degradation of membrane lipids provides a source of fatty acids for the synthesis of energy-rich, storage lipid esters such as triacylglycerol (TAG). In eukaryotes, storage lipids are packaged into dynamic subcellular organelles, lipid droplets. In times of energy scarcity, lipid droplets can be degraded via autophagy in a process termed lipophagy to release fatty acids for energy production via fatty acid β-oxidation. On the other hand, emerging evidence suggests that lipid droplets are required for the efficient execution of autophagic processes. Here, we review recent advances in our understanding of metabolic interactions between autophagy and TAG storage, and discuss mechanisms of lipophagy. Free fatty acids are cytotoxic due to their detergent-like properties and their incorporation into lipid intermediates that are toxic at high levels. Thus, we also discuss how cells manage lipotoxic stresses during autophagy-mediated mobilization of fatty acids from lipid droplets and organellar membranes for energy generation.
    Keywords:  Autophagy; fatty acid; lipid droplet; lipid homeostasis; lipophagy; lipotoxicity
  17. Chem Biol Interact. 2022 May 09. pii: S0009-2797(22)00168-5. [Epub ahead of print] 109963
      4-Hydroxynonenal (4-HNE), the most toxic end-product of lipid peroxidation formed during oxidative stress, has been implicated in many diseases including neurodegenerative diseases, metabolic diseases, myocardial diseases, cancer and age-related diseases. 4-HNE can actively react with DNA, proteins and lipids, causing rapid cell death. The accumulation of 4-HNE leads to induction of autophagy, which clears damaged proteins and organelles. However, the underlying mechanism of 4-HNE-regulated autophagy is still not known. Transcriptional factor EB (TFEB) is a master regulator of lysosomal and autophagic functions, which we show here that TFEB is activated by 4-HNE. 4-HNE induces TFEB nuclear translocation and activated TFEB then upregulates the expression of genes required for autophagic and lysosomal biogenesis and function. Reactive oxygen species and Ca2+ are required in this process and TFEB activity is required for 4-HNE-mediated lysosomal function. Most importantly, genetic inhibition of TFEB (TFEB-KO) exacerbates 4-HNE-induced cell death, suggesting that TFEB is essential for cellular adaptive response to 4-HNE-induced cell damage. Hence, targeting TFEB to promote autophagic and lysosomal function may represent a promising approach to treat neurodegenerative and metabolic diseases in which 4-HNE accumulation has been implicated.
    Keywords:  4-Hydroxynonenal; Apoptosis; Lysosome; ROS; TFEB
  18. FASEB J. 2022 May;36 Suppl 1
      Autophagy is an essential cellular process by which cellular debris is collected and broken down in the lysosome. Cancer cells rely on autophagy for survival during chemotherapy treatment and periods of hypoxia within solid tumors. In this project, we investigate the ATG13-ATG101 protein complex, a sub-complex of the ULK1 initiatory complex whose regulatory role in autophagy is not completely understood. Our recent data demonstrate that an ULK1-independent ATG13-ATG101 complex is essential for the basal autophagic degradation of protein aggregates-a process known as aggrephagy. Furthermore, we found that the ULK1-independent ATG13-ATG101 complex traffics to condensates of poly-ubiquitinated protein aggregates where it colocalizes with the autophagy regulator ATG9A.1 Our current data suggest that this ATG13-ATG101 complex cooperates with ATG9A in the degradation of these protein aggregates. To further elucidate the function of the ATG13-ATG101 complex, we have developed a protein-fragment complementation assay using a novel split TurboID (BioID) system in which complementary halves of TurboID are fused to ATG13 and ATG101. Using this split BioID system, together with quantitative LC-MS/MS and mutants of ATG13 that cannot bind ULK1, we are identifying interactors that are unique to the ULK1-independent ATG13-ATG101 complex. Based on previous data, we hypothesize that the ATG13-ATG101 sub-complex participates in ATG9A trafficking pathways and recruitment of autophagy regulators to protein aggregate condensates. Thus, our project characterizes the first proximity interactome for the ATG13-ATG101 complex. 1. Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L., BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters. EMBO Rep2021, e51136.
  19. Front Plant Sci. 2022 ;13 866367
      Autophagy is a conserved intracellular trafficking pathway for bulk degradation and recycling of cellular components in eukaryotes. The hallmark of autophagy is the formation of double-membraned vesicles termed autophagosomes, which selectively or non-selectively pack up various macromolecules and organelles and deliver these cargoes into the vacuole/lysosome. Like all other membrane trafficking pathways, the observation of autophagy is largely dependent on marker lines. ATG8/LC3 is the only autophagy-related (ATG) protein that, through a covalent bond to phosphatidylethanolamine (PE), associates tightly with the isolation membrane/pre-autophagosomal structure (PAS), the growing phagophore, the mature autophagosome, and the autophagic bodies. Therefore, fluorescent protein (FP)-tagged ATG8 had been widely used for monitoring autophagosome formation and autophagic flux. In rice (Oryza sativa), FP-OsATG8 driven by Cauliflower mosaic virus (CaMV) 35S promoter had been used for imaging autophagosome and autophagic bodies. Here, we constructed three vectors carrying GFP-OsATG8a, driven by 35S, ubiquitin, and the endogenous ATG8a promoter, individually. Then, we compared them for their suitability in monitoring autophagy, by observing GFP-ATG8a puncta formation in transiently transformed rice protoplasts, and by tracking the autophagic flux with GFP-ATG8 cleavage assay in rice stable transgenic lines. GFP-Trap immunoprecipitation and mass spectrometry were also performed with the three marker lines to show that they can be used reliably for proteomic studies. We found out that the ubiquitin promoter is the best for protoplast imaging. Transgenic rice seedlings of the three marker lines showed comparable performance in autophagic flux measurement using the GFP-ATG8 cleavage assay. Surprisingly, the levels of GFP-ATG8a transcripts and protein contents were similar in all marker lines, indicating post-transcriptional regulation of the transgene expression by a yet unknown mechanism. These marker lines can serve as useful tools for autophagy studies in rice.
    Keywords:  ATG8; autophagic flux; autophagy; post-transcriptional regulation; rice
  20. FASEB J. 2022 May;36 Suppl 1
      Autophagy is a cellular digestion process that contributes to cellular homeostasis by the elimination of proteins and damaged organelles. Impaired autophagy has been implicated in aging-related disorders and in neurodegenerative diseases, including motor neuron disorders. Therefore, upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue at low concentrations, activates autophagy in rat glioma and human neuroblastoma cells in vitro, as well as in hippocampus and cortex after in vivo administration. With the overall goal of using LK to induce autophagy flux to prevent age-related motor neuron dysfunction, we recently tested the effects of two lanthionine ketenamine phosphonates (LK-Ps) and one lanthionine ketenamine ethyl (ester) phosphonate (LKE-P) on autophagy modulation in a mouse motor neuron-like hybrid cell line (NSC-34). For fluorescence visualization of autophagy flux, NSC-34 cells were transfected with mCherry-GFP-LC3 plasmid. Motor neurons treated with LK analogs displayed increased autophagy flux measured by the ratio of mCherry area (lysosomes) over GFP area (autophagosomes that have not fused with lysosomes) compared to control cells. There results were corroborated by increased LC3-II/LC3-I ratio as measured by protein expression using Western blot; and presence of autophagic vacuoles identified using transmission electron microscopy. In conclusion, LK analogs constitute a promising tool to induce autophagy flux in motor neurons, which may serve to prevent age-related motor axon withdrawal and muscle denervation.
  21. J Cell Mol Med. 2022 May 13.
      Autophagy is designated as a biological recycling process to maintain cellular homeostasis by the sequestration of damaged proteins and organelles in plasma and cargo delivery to lysosomes for degradation and reclamation. This organelle recycling process promotes chondrocyte homeostasis and has been previously implicated in osteoarthritis (OA). Autophagy is widely involved in regulating chondrocyte degeneration markers such as MMPs, ADAMSTs and Col10 in chondrocytes. The critical autophagy-related (ATG) proteins have now been considered the protective factor against late-onset OA. The current research field proposes that the autophagic pathway is closely related to chondrocyte activity. However, the mechanism is complex yet needs precise elaboration. This review concluded that FoxO1, a forkhead O family protein, which is a decisive mediator of autophagy, facilitates the pathological process of osteoarthritis. Diverse mechanisms regulate the activity of FoxO1 and promote the initiation of autophagy, including the prominent AMPK and Sirt-2 cellular pathways. FoxO1 transactive is regulated by phosphorylation and acetylation processes, which modulates the downstream ATGs expression. Furthermore, FoxO1 induces autophagy by directly interacting with ATGs proteins, which control the formation of autophagosomes and lysosomes fusion. This review will discuss cutting-edge evidence that the FoxO-autophagy pathway plays an essential regulator in the pathogenesis of osteoarthritis.
    Keywords:  FoxO1; autophagy; chondrocytes; osteoarthritis
  22. FASEB J. 2022 May;36 Suppl 1
      mTOR, which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. AUF1 is required for Akt phosphorylation. It also mediates phosphorylation of the mTORC2-modulated metabolic enzyme GFAT1 at Ser243. Reciprocally, mTORC2 could also modulate AUF1. Conditions that enhance mTORC2 signaling, such as serum restimulation or acute glutamine withdrawal augments AUF1 phosphorylation while mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in mTORC2/Akt signaling. Targeting AUF1 could serve as a novel treatment strategy for cancers with upregulated mTORC2/Akt signaling.
  23. FASEB J. 2022 May;36 Suppl 1
      Akin to organic nutrients, such as oxygen, lipids, amino acids, and carbohydrates, the transition metal copper (Cu) is an essential dietary nutrient for normal physiology and development. Decades of research highlight the physiological and disease associated consequences of disrupting homeostatic mechanisms that ensure proper Cu acquisition, storage, and distribution to Cu-dependent enzymes. However, phenotypes associated with alterations in Cu availability cannot be fully explained by the limited number of enzymes that traditionally harness the redox potential of Cu as a catalytic cofactor. Recent discoveries in Cu biology have revealed direct Cu binding at non-catalytic sites within signaling molecules that modulate cell proliferation via the protein kinases MEK1/2, lipid metabolism via the phosphodiesterase PDE3B, and nutrient recycling via the autophagic kinases ULK1/2. The emergence of this new paradigm in nutrient sensing and protein regulation has established that Cu is a critical mediator of intracellular signaling, provided evidence for the existence of molecular mechanisms for sensing changes in Cu abundance, and expanded the contribution of Cu to cellular processes necessary for adaptation to nutrient scarcity. Our presentation will focus on the intersections between Cu homeostasis, nutrient signaling, and metabolism by examining the interplay between mechanisms of Cu-sensing necessary for cellular energy homeostasis and evaluating the necessity of Cu for metabolic flexibility under nutrient and oxygen stress. We will present novel findings on Cu-controlled autophagy-lysosomal biogenesis and function, and interconnectivity between mitochondrial Cu transport and cytosolic nutrient sensing signaling pathways necessary for metabolism. These studies increase our fundamental knowledge of the molecular and cellular features of Cu-dependent enzymes and cellular processes and enable therapeutic targeting of Cu-dependent disease vulnerabilities.
  24. Curr Opin Cell Biol. 2022 May 06. pii: S0955-0674(22)00030-8. [Epub ahead of print]76 102084
      Autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is responsible for the degradation of ER portions by lysosomes. ER-phagy is induced in both physiological and stress conditions to maintain ER homeostasis and protein quality control. ER-phagy receptors and their interactors are key regulators of this process. Transcriptional and post-translational regulation of ER-phagy receptors have emerged as critical mechanisms for the modulation of ER-phagy, providing the first hints to understand how this process responds to the cellular needs. Here, we concisely review the main mechanisms regulating ER-phagy receptors and discuss their potential implications in diseases.
  25. Proc Natl Acad Sci U S A. 2022 May 24. 119(21): e2202016119
      SignificanceAutophagy defects are a risk factor for inflammatory bowel diseases (IBDs), but the mechanism remains unknown. We show here that conditional whole-body deletion of Atg5 or Fip200, but not Atg7, is lethal due to loss of ileum stem cells and barrier function likely caused by different kinetics of autophagy loss, which was rescued by slow deletion. Specific autophagy loss in PDGFRα+ mesenchymal cells (PMCs) resulted in loss of Wnt signaling responsible for failed stem cell renewal. We also observed depletion of aspartate and nucleotides throughout the ileum. Our results illustrate that autophagy is required for PMC metabolism and survival necessary to sustain intestinal stem cells and mouse survival, and failure to maintain PMCs through autophagy contributes to IBD.
    Keywords:  IBD; autophagy; intestine; metabolism; stem cells
  26. FASEB J. 2022 May;36 Suppl 1
      The Moeller Molecular Modeling Team in conjunction with Dr. Jiajie Diao at the University of Cincinnati Medical College used 3D modeling and printing technology to study the vital role of the focal adhesion kinase family interacting protein of 200 kD (FIP200) in the autophagy process. The cellular process of autophagy is enabled by formation of autophagosomes, a double membraned structure that isolates substrates designated by the cell to be eliminated by autophagy. FIP200 is a 200 kD subunit of the ULK1 complex which facilitates the formation of phagophores, the first step in autophagosome formation. An ULK1 protein kinase is necessary for the activation of the ULK1 complex during autophagosome formation. The positively charged "claw domain" area at the C-terminal region of FIP200 interacts with p62, a receptor protein, which delivers ubiquitinated cargo proteins bound to LC3B, a central regulator for autophagosome formation. After the binding of these proteins, the autophagosome forms around the ubiquitinated cargo and continues to the next step of the autophagy process.
  27. FASEB J. 2022 May;36 Suppl 1
      OBJECTIVE AND HYPOTHESIS: The importance of endothelial cell (EC) autophagy to vascular homeostasis in the context of health and disease is evolving. Earlier we reported that intact EC autophagy is requisite to maintain shear-stress-induced nitric oxide (NO) generation via glycolysis-dependent purinergic signaling to eNOS. Here we illustrate the translational and functional significance of these findings.METHODS AND RESULTS: First, we assessed translational relevance using older male humans and mice that exhibit blunted EC autophagy and impaired arterial function vs. adult controls. Active hyperemia evoked by rhythmic handgrip exercise elevated radial artery shear rate similarly from baseline in adult (23±1 y) and older (68±2 y) subjects for 60-min. Compared to baseline, indexes of autophagy initiation, p-eNOSS1177 activation, and NO generation, occurred in radial artery ECs obtained from adult but not older volunteers. Regarding mice, indexes of autophagy and p-eNOSS1177 activation were robust in ECs from adult (7 ± 1 months) but not older (23 ± 1 months) animals that completed 60-min treadmill-running. Further, results concerning the extracellular acidification rate (ECAR; Seahorse Bioanalyzer) indicate glycolysis and glycolytic capacity were elevated in response to 20 dyn/cm2 laminar shear stress x 45-min in primary arterial ECs obtained from adult but not older mice. We next questioned whether the inability to initiate EC autophagy, glycolysis, and p-eNOSS1177 precipitates dysfunction in arteries from older vs. adult mice. Compromised intraluminal flow-mediated vasodilation displayed by arteries from older vs. adult mice was recapitulated in vessels from adult mice by : (i) NO synthase inhibition; (ii) acute autophagy impairment using 3-methyladenine (3-MA); (iii) EC Atg3 depletion (Atg3EC-/- mice); (iv) purinergic 2Y1 -receptor (P2Y1 -R) blockade; and (v) germline depletion of P2Y1 -Rs. Importantly, P2Y1 -R activation using 2-methylthio-ADP (2-Me-ADP) improved vasodilatory capacity in arteries from : (i) adult mice treated with 3-MA; (ii) adult Atg3EC-/- mice; and (iii) older animals with repressed EC autophagy.
    CONCLUSIONS: Arterial dysfunction concurrent with pharmacological, genetic, and age-associated EC autophagy disruption is improved by activating P2Y1 -Rs.
  28. Proc Natl Acad Sci U S A. 2022 May 17. 119(20): e2123261119
      SignificanceThe mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is frequently elevated in human disease, including cancer, type 2 diabetes, metabolic disorders, and neurodegeneration. We identify SNAT7 as an important regulator of mTORC1. We believe this research will provide valuable insight about mTORC1 biology and may uncover novel therapeutic targets for patients.
    Keywords:  SNAT7; mTOR; macropinocytosis
  29. FASEB J. 2022 May;36 Suppl 1
      Autophagy is the cellular degradation process in which cellular contents are encapsulated by double-membrane vesicles, autophagosomes, and delivered to the vacuole to be degraded and recycled. This process is important for cell health and homeostasis. It involves the upregulation of many autophagy-related proteins, including Atg3 and Atg10. We are investigating the role of these proteins in the model system S. Cerevisiae is to measure the size and number of autophagosomes formed when insufficient amounts of that protein are available. Atg8 levels are known to affect only autophagosome size. However, we have found that levels of Atg7, the most upstream protein in the Atg8-PE conjugation pathway, affect both autophagosome size and number. Atg3 and Atg10 are intermediate proteins involved downstream of Atg7 and upstream of Atg8. Atg3 directly conjugates Atg8 to PE, while Atg10 conjugates Atg12 to Atg5, and the resulting Atg12-5 complex stimulates the activity of Atg3. It is possible that the Atg12-5 complex has additional roles independent of Atg8 that explain the effect of Atg7 levels on autophagosome number. If so, we would expect that Atg3 activity would correlate only with autophagosome size, while Atg10 might control size and number. We are generating Atg3 and Atg10 active site mutants and testing the functionality of these mutants by performing western blots and enzymatic assays. Mutants that give a partial loss of autophagic activity will be selected for later measurement of autophagosome size and number.
  30. Aging Cell. 2022 May 10. e13616
      Accumulation of oxidative stress is highly intertwined with aging process and contributes to aging-related diseases, such as neurodegenerative diseases. Deciphering the molecular machinery that regulates oxidative stress is fundamental to further uncovering the pathogenesis of these diseases. Chaperone-mediated autophagy (CMA), a highly selective lysosome-dependent degradation process, has been proven to be an important maintainer of cellular homeostasis through multiple mechanisms, one of which is the attenuation of oxidative stress. However, the specific mechanisms underlying this antioxidative action of CMA are not fully understood. In this study, we found that CMA directly degrades Kelch-like ECH-associated protein 1 (Keap1), an adaptor of E3 ligase complex that promotes the degradation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is a master transcriptional regulator in antioxidative response. Activated CMA induced by prolonged oxidative stress led to an increase in Nrf2 level by effectively degrading Keap1, contributing to Nrf2 nuclear translocation and the expression of multiple downstream antioxidative genes. Meanwhile, together with previous study showing that Nrf2 can also transcriptionally regulate LAMP2A, the rate-limiting factor of CMA process, we reveal a feed-forward loop between CMA and Nrf2. Our study identifies CMA as a previously unrecognized regulator of Keap1-Nrf2 pathway and reinforces the antioxidative role of CMA.
    Keywords:  6-OHDA; CMA; Keap1-Nrf2 pathway; oxidative stress
  31. Cell Death Dis. 2022 May 09. 13(5): 444
      Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death. Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases, such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and its role in cardiovascular disease.
  32. EMBO J. 2022 May 10. e110031
      Autophagy is a cellular degradative pathway that plays diverse roles in maintaining cellular homeostasis. Cellular stress caused by starvation, organelle damage, or proteotoxic aggregates can increase autophagy, which uses the degradative capacity of lysosomal enzymes to mitigate intracellular stresses. Early studies have shown a role for autophagy in the suppression of tumorigenesis. However, work in genetically engineered mouse models and in vitro cell studies have now shown that autophagy can be either cancer-promoting or inhibiting. Here, we summarize the effects of autophagy on cancer initiation, progression, immune infiltration, and metabolism. We also discuss the efforts to pharmacologically target autophagy in the clinic and highlight future areas for exploration.
    Keywords:  ATG; autophagy; cancer; chloroquine; metabolism
  33. FASEB J. 2022 May;36 Suppl 1
      Autophagy is an essential cellular process regulated by intracellular calcium signals, which play an important role in autophagic activation during metabolic changes. Calcium transporters are present in mitochondria, an organelle able to uptake and release calcium ions, thus participating in cellular calcium signaling. Uptake by mitochondria is mediated by the mitochondrial calcium uniporter (MCU), while extrusion occurs through the mitochondrial sodium/lithium/calcium exchanger (NCLX). The aim of this work was to investigate how MCU and NCLX affect autophagy. To do so, we evaluated makers of autophagic activity in murine Aml-12 hepatic cells transfected with siRNAs targeting either MCU or NCLX expression, leading to genetic knockdown (KD). Additionally, we investigated the autophagic response to serum/amino acid starvation and rapamycin (mTORC1 inhibitor) treatment in Aml-12 cells with NCLX KD or pharmacological inhibition by CGP37157 (CGP). Using a cell line stably expressing the autophagic probe LC3-GFP-mCherry, we observed that NCLX KD leads to impaired autophagosome formation under basal conditions. Curiously, this effect was associated with a significant decrease in mRNA expression of LC3A and LC3B genes, while the expression of other autophagy-related genes, such as TFEB, ATG5, ATG12, and ATG7, was upregulated or unchanged. Conversely, MCU KD led to an apparent increase in autophagosome and autolysosome numbers, indicating enhanced autophagic activity. Interestingly, MCU KD also led to decreased expression of LC3A, but not LC3B. The expression of TFEB, ATG12, ATG5, and ATG7 were decreased or unchanged by MCU KD. After autophagic stimulation by serum/amino acid starvation, the levels of LC3 II were lower in NCLX KD cells compared to negative control in the presence or absence of bafilomycin A1, which indicates a reduction of autophagic flux. The levels of LC3 I were significantly lower in NCLX KD cells under basal and stimulated conditions, corroborating the decreased LC3 mRNA levels observed. Importantly, these effects were also observed using CGP to inhibit NCLX. The reduction of autophagic flux by NCLX KD or CGP was not observed in cells treated with rapamycin. We also measured the levels of phosphorylated 4E-BP1 as an indication of mTORC1 activity. As expected, serum/amino acid starvation and rapamycin decreased the levels of p-4E-BP1; however, this decrease was modulated by CGP only in starved cells, indicating that NCLX may affect mTORC1 activity in an upstream pathway. In conclusion, we show that mitochondrial calcium transporters are novel autophagy-regulating pathways: MCU modulates autophagic activation under basal conditions, while NCLX maintains autophagic activity under basal and stimulated conditions.
  34. FASEB J. 2022 May;36 Suppl 1
      mTORC1 controls cellular processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by Ragulator. The Rag GTPases receive amino acid signals from upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binding to the Rag GTPases occurs via either of two modes: an inhibitory mode that has low enzymatic activity but high affinity, and a GAP mode that has high enzymatic activity but low affinity. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both sites simultaneously. These structural models, together with the spatial constraints from the lysosomal membrane, reveal how GATOR1 coordinates the nucleotide loading states of both Rag subunits to transmit amino acid signals.
  35. Int J Mol Sci. 2022 Apr 26. pii: 4793. [Epub ahead of print]23(9):
      Autophagy is an evolutionarily conserved catabolic process in eukaryotic cells, by which the superfluous or damaged cytoplasmic components can be delivered into vacuoles or lysosomes for degradation and recycling. Two decades of autophagy research in plants uncovers the important roles of autophagy during diverse biological processes, including development, metabolism, and various stress responses. Additionally, molecular machineries contributing to plant autophagy onset and regulation have also gradually come into people's sights. With the advancement of our knowledge of autophagy from model plants, autophagy research has expanded to include crops in recent years, for a better understanding of autophagy engagement in crop biology and its potentials in improving agricultural performance. In this review, we summarize the current research progress of autophagy in crops and discuss the autophagy-related approaches for potential agronomic trait improvement in crop plants.
    Keywords:  abiotic stress; agronomic trait; autophagy; autophagy manipulation; biotic stress; crops; nutrient recycling and remobilization; yield
  36. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: The intestinal epithelium is a highly proliferative tissue that undergoes complete turnover in 3 to 5 days. The presence of intestinal stem cells expressing the canonical Wnt target gene Lgr5, known as active intestinal stem cells (a-ISCs), are responsible for generating daughter cells that differentiate into specialized epithelial cells to carryout various functions in the intestine. The proliferative nature of the intestinal epithelium renders it susceptible to DNA damage-inducing injury, such as high-dose irradiation and chemotherapy. Although a-ISCs and early daughter cells are killed by these insults, a radio-resistant population of facultative intestinal stem cells (f-ISCs) can regenerate lost a-ISCs to restore homeostasis following injury. The functional characteristics of f-ISCs are becoming increasingly understood, however, whether any epithelial cell can act as an f-ISC is less clear. One reason for this ambiguity is a heavy reliance on Cre-driven reporter mouse models with low recombination efficiency. Our objective was to find a functional marker to identify f-ISCs based on cellular state rather than gene expression. The autophagy pathway has been shown to protect against DNA damage and irradiation-induced apoptosis in the intestinal epithelium. Furthermore, recent studies demonstrate a requirement for the autophagy pathway during cellular de-differentiation in both gastric chief and pancreatic acinar cells following metaplasia-inducing injury. Given that autophagy plays roles in both radio-resistance and cellular plasticity, two integral features of f-ISCs, we hypothesized that autophagic activity could serve to identify cells with f-ISC capacity.METHODS AND RESULTS: Using the autophagic vesicle tracer dye CytoID combined with fluorescence-activated cell sorting, we demonstrate that intestinal epithelial cells with high levels of CytoID exhibit increased organoid-formation efficiency (OFE) compared to CytoID 'low' cells. Single cell sequencing reveals that the CytoID high population is largely composed of secretory cells including Paneth, Goblet, Tuft, and Enteroendocrine cells, whereas the CytoID low population consisted mainly of a-ISCs and absorptive Enterocytes. Using reporter mice and antibodies against cell surface receptors, we observe that CytoID can identify cells with high OFE within Enteroendocrine, Paneth, and Goblet cell lineages. Finally, we demonstrate that autophagy is required for the enhanced organoid-formation observed in CytoID high cells by plating these cells in the presence of the lysosomal inhibitor Bafilomycin A1.
    CONCLUSIONS: Our new data support the notion that epithelial cells in a high autophagic state are biased to secretory lineages and that autophagy status functions as a lineage agnostic marker of f-ISCs. Furthermore, our data suggests that autophagy is required for organoid formation in these lineages.
  37. FASEB J. 2022 May;36 Suppl 1
      The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization and is a crucial factor regulating organelle shape and biogenesis. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that generates negative membrane curvature, has many cellular roles, including regulating mitochondrial architecture and lipidation of the autophagy factor LC3. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which canonically operates on the mitochondrial inner membrane, unexpectedly also localizes to the ER. However, it has been unclear what the role of ER-localized Psd1 is. We now resolve this mystery and demonstrate that ER-localized Psd1 is a critical regulator of lipid droplet (LD) biogenesis at ER subdomains. While it is constitutively targeted to the ER membrane, Psd1 transiently concentrates on the ER to LD attachment sites specifically during stimulated LD biogenesis. Using an inducible LD biogenesis assay, we demonstrate that ER-localized Psd1 is required for normal LD formation. Further, we identify a LD binding motif on Psd1 and show that this motif is required for Psd1 to influence LD morphogenesis. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in fission yeast, though occurs through a distinct targeting mechanism than in budding yeast. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs are organized and shaped by the spatial positioning of a single PE synthesis enzyme.
  38. FASEB J. 2022 May;36 Suppl 1
      Akt1 and Akt2 are the main protein kinase B (Akt) isoforms expressed in the mammalian heart. Tamoxifen(OHTx) -inducible, cardiomyocyte-specific Akt1/ Akt2 double knockout mice (iCM-Akt12) show progressive cardiac atrophy and loss of contractile function leading to terminal heart failure 23.9 ± 2 days after first OHTx injection. TUNEL staining of iCM-Akt12 hearts revealed that cardiomyocyte apoptosis did not contribute substantially to cardiac atrophy. Rather, cellular atrophy caused the loss of cardiac mass. The cellular area (a), width (w), and length (l) declined between day 9 and day 14 [-18 (a)/-18 (w)/-9% (l)] as compared to WT controls. This size reduction was retarded between d14 and d21 (-20 (a)/-24 (w)/-8% (l). In vivo 31 P-imaging identified a progressive energetic deficiency of iCM-Akt12 hearts on d15 and d20 after OHTx injection. Further analyses showed that in the early phase up to day 14 increased autophagic activity and reduced de novo protein synthesis seemed to cause cellular atrophy. As expected, deletion of Akt led to impaired mTORC1 signaling indicated by reduced phosphorylation of S6K, RPS6, 4E-BP1, all involved in translation. Concomitantly, we measured reduced protein synthesis. Autophagy was increased in iCM-Akt12 hearts, as shown by increased LC3-II/I ratio and higher expression of autophagic genes. From day 14 after KO induction (late phase), protein synthesis appeared to increase and mTORC1 substrates involved in translation (S6K, RPS6 and 4E-BP1) were higher phosphorylated. Of note, also a higher phosphorylation of the inhibitory mTORC1 target site in the central autophagy kinase Ulk1 (S757) was found. Moreover, autophagy appeared to be disturbed, as p62 accumulated. Despite increasing energetic depletion, the cellular energy sensor AMPK was not activated in iCM-Akt12 hearts. Rather, AMPK was inhibited by phosphorylation of the α-subunit on serine 485/491. The S485/491 phosphorylation has been attributed to several kinases (Akt, PKD1, S6K, PKC, PKA). Akt deletion led to a pronounced hyperactivation of Pdk1 signaling upstream of Akt, causing an activation of protein kinases regulated by Pdk1 (S6K, PKA, PKC, PKD). This was demonstrated by increased phosphorylation of specific kinase substrates and consensus motifs in late iCM-Akt12 hearts and might promote the AMPK inactivation. To test to what extent a non-inhibitable AMPK might attenuate the fatal phenotype of cardiac Akt loss, AAV-mediated infection of iCM-Akt12 mice with a phospho-defective AMPKα2 S491A mutant prior to KO induction was performed. This led to an increased survival and improved heart function, demonstrating that the AMPK inhibition contributes to the lethal phenotype. In conclusion, loss of Akt signaling leads to a widespread dysregulation of intracellular signal transduction. Whereas enhanced autophagy and inhibition of mTORC1 are direct consequences of Akt deletion, the later reactivation of mTORC1 and inhibition of AMPK causes attenuation of autophagy, increased translation, deceleration of cellular atrophy, worsenes energetic shortage, and aggravates cardiac dysfunction.
  39. FASEB J. 2022 May;36 Suppl 1
      Thirty-eight-negative kinase 1 (TNK1) is a poorly understood member of the ACK family of non-receptor tyrosine kinases. TNK1 has been linked to oncogenic activity. However, the biological function of TNK1 remains unclear. Recently, we discovered the presence of a functional ubiquitin association (UBA) domain on the C-terminus of TNK1, which is an unusual feature on a kinase. Previously, we demonstrated that the TNK1 UBA domain binds to poly-ubiquitin with high affinity and with no preference for length or linkage type (1). Interestingly, deletion of the UBA domain (TNK1 ΔUBA) affects TNK1 intracellular localization and decreases its phospho-substrates network. Based on these data, we hypothesized that the UBA domain homes TNK1 to substrates. To test this hypothesis, we used quantitative phospho-tyrosine proteomics in pro-B cells transformed with either TNK1 or TNK1 ΔUBA to identify UBA-dependent TNK1 substrates. Among the putative UBA-dependent substrates, we identified phospho-Y235 of TANK-binding kinase 1 (TBK1). TBK1 is an important serine/threonine protein kinase involved in the regulation of inflammation, autophagy, and NF-kB signaling. Through confocal microscopy, we demonstrated a colocalization between TNK1 and TBK1 at p62-positive phase-separated clusters of ubiquitin-referred to here as ubiquitin condensates. We found that TNK1 localizes to ubiquitin condensates in a UBA-dependent manner. Furthermore, our data suggest that TBK1 is inhibited by phosphorylation at Y235, as phospho-null mutation at Y235 (Y235F) significantly increases phosphorylation of the TBK1 substrates p62 and OPTN. We also found that CRISPR/Cas9 deletion of TNK1 increased TBK1 activity and phosphorylation of p62 and OPTN. These data suggest that TNK1 negatively regulates TBK1 activity. Intriguingly, we also identified an enrichment of other putative UBA-dependent TNK1 substrates in the NF-kB signaling pathway, suggesting that the UBA domain tethers TNK1 to multiple substrates in this pathway, perhaps as a means to dampen NF-kB signaling. Our current work focuses on the biological impact of these phosphorylations on NF-kB activity, inflammatory signaling, and autophagy. 1. Chan, TY., Egbert, C.M. et al. TNK1 is a ubiquitin-binding and 14-3-3-regulated kinase that can be targeted to block tumor growth. Nat Commun 12, 5337 (2021).
  40. Int J Biol Sci. 2022 ;18(7): 2684-2702
      Macroautophagy/autophagy is the process of self-digestion through the lysosomes; it disassembles unnecessary or dysfunctional long-lived proteins and damaged organelles for the recycling of biomacromolecules. Unfortunately, cancer cells can hijack this mechanism to survive under metabolic stress or develop drug resistance during chemotherapy. Increasing evidence indicates that the combination of autophagy inhibition and chemotherapy is a promising cancer treatment strategy. However, effective autophagy inhibitors with satisfied potency, bioavailability, and clearly-defined drug targets are still rare. Here, we report the identification of a potent autophagy inhibitor toosendanin which can effectively block autophagosome maturation, causing the accumulation of autophagy substrates in multiple cancer cells. Toosendanin did not inhibit the fusion process between autophagosome and lysosome but elevated lysosomal pH and impaired lysosomal enzymes activity. Using rat liver lysosome fraction and purified yeast V-ATPase, we found that toosendanin directly inhibited V-ATPase activity. By applying cellular thermal shift assay (CETSA), immunoprecipitation-coupled LC-MS/MS analysis, and biotin-toosendanin pull-down assay, we confirmed the direct binding between toosendanin and V-ATPase. Furthermore, toosendanin blocked chemotherapy-induced protective autophagy in cultured cancer cells and xenograft tumor tissues to significantly enhance anti-cancer activity. These results suggest that toosendanin has the potential to be developed into an anti-cancer drug by blocking chemotherapy-induced protective autophagy.
    Keywords:  V-ATPase inhibitor; anti-cancer effect; autophagy inhibitor; protective autophagy; toosendanin
  41. FASEB J. 2022 May;36 Suppl 1
      Prolonged muscle disuse is accompanied by a phenotypic shift characterized by declines in mitochondrial content and function within skeletal muscle. This loss in mitochondrial content can be partially attributed to elevations in various catabolic processes that occur with atrophy. One of these is termed mitophagy, a selective form of cellular recycling (autophagy) whereby dysfunctional mitochondria are degraded via lysosomes. TFEB and TFE3 are key transcription factors that regulate lysosomes by activating the expression of various lysosomal genes. Loss of TFE3 has been associated with depressed levels of autophagy and with mitochondrial dysfunction. It is speculated that these functional impairments are due to diminished clearance via the lysosomes, however to date this has not been examined. We hypothesized that the loss of TFE3 would amplify the mitochondrial dysfunction associated with muscle disuse, while paradoxically preserving muscle mass and mitochondrial content. Using a severe model of muscle disuse, sciatic denervation, we observed a 10% loss in hindlimb muscle mass within 7 days of denervation. In the absence of TFE3, a trend for muscle preservation was observed, as these animals lost 20% less muscle mass than WT counterparts. Reduced rates of respiration supported by complex I and II were observed irrespective of genotype, concurrent with elevated ROS emissions, suggesting an impaired oxidative capacity following 7 days of denervation. Surprisingly, TFE3 KO animals did exhibit lower levels of oxidative stress basally, but this too increased following 7 days of denervation, and taken relative to baseline the fold induction of ROS emission was 3 times greater than WT animals. Higher levels of mitophagy flux were observed in the absence of TFE3 basally, which supports the observed 35% decline in mitochondrial content as measured by COX activity, as well as the lower levels of ROS emissions. Following only 1 day of denervation, increases in mitophagy flux were observed in WT animals, while the response in KO animals was clearly attenuated. In the WT animals, denervation led to a 30% reduction in mitochondrial content, however no change was observed in the absence of TFE3. Finally, increases in a number of autophagy-related markers such Beclin-1 and ATG7 were observed following denervation irrespective of genotype. However, the mature form of Cathepsin B, a hydrolytic enzyme, was markedly reduced by 55% in the absence of TFE3 and did not increase to the same extent as WT following 7 days of denervation suggesting an impairment in lysosomal function. Together, our results suggest that TFE3 exerts multiple roles in skeletal muscle plasticity, as a partial mediator of muscle mass, and in the control of lysosomal function and mitophagy in response to the acute stress of muscle disuse.
  42. FASEB J. 2022 May;36 Suppl 1
      Alternative polyadenylation (APA), an RNA processing mechanism that results in mRNA with distinct 3' termini, is a rapidly expanding area of research that in recent studies has been linked to the mechanistic target of rapamycin (mTOR) signaling pathway, a key regulatory pathway in physiology and metabolism. Despite the recent implications of APA in mTOR signaling, the mechanistic link between mTOR signaling and APA remains poorly understood. We previously leveraged our cTag-PAPERCLIP technique to generate a dataset of in vivo APA shifts following neuronal mTOR induction and identified TRIM9, an E3 ubiquitin ligase with a role in neurodevelopment, as a gene of interest. In this study, we further characterized the regulation of the mTOR-induced TRIM9 APA shift observed in mouse neurons in vivo. Further study of the regulation of TRIM9 APA by the core protein complexes of the cleavage and polyadenylation (CP) machinery revealed CSPF6, a component of the CFIm complex, as essential for physiological regulation of TRIM9 isoforms, with loss of CPSF6 leading to an enrichment of the distal TRIM9 isoform. Additional study into the 3'UTR sequence elements of TRIM9 isoforms revealed multiple UGUA sequence motifs, the binding sequence element of the CFIm complex, upstream of the TRIM9 proximal polyA site (PAS). In order to identify the key sequence elements essential for CPSF6-mediated regulation of the proximal TRIM9 PAS, we developed a RT-qPCR PAS competition assay to quantify sequence-mediated usage of PASs. Utilizing this assay, we assessed usage of the TRIM9 proximal PAS in both the absence and the presence of CPSF6. Additionally, we generated constructs containing mutated UGUA sequences in order to ascertain the importance of the UGUA motif to TRIM9 proximal PAS usage. We found that loss of CPSF6 leads to reduced usage of the TRIM9 proximal PAS. Furthermore, mutation of a twin UGUA motif (UGUACUGUA) lead to a reduction in TRIM9 proximal PAS usage. Our results demonstrate a direct role of CPSF6 and identify a key cis-acting motif in promoting TRIM9 proximal PAS usage. Furthermore, our results also suggest a possible link between neurological disorders with mTOR pathway dysregulation ("mTORopathies") and neurodevelopment through TRIM9.
  43. FASEB J. 2022 May;36 Suppl 1
      BACKGROUNDS: Liver has an innate capacity to regenerate following partial hepatectomy (PH). Pathways including the mTORC1 and the Wnt/β-catenin pathways have been shown to become activated after PH to enable liver regeneration (LR). However, how one pathway could compensate during deficiency of another is less well understood. Here, we report discovery and characterization of a compensatory interplay between mTOR and Wnt signaling that enables LR following PH when mTORC1 pathway is interrupted genetically.METHODS: We generated hepatocyte-specific Raptor knockout (Raptor∆HC ) mice by delivering AAV8-TBG-Cre to 6-week-old Raptorflox/flox mice. Two weeks after AAV8 injection, animals were either harvested at baseline, or subjected to partial hepatectomy (PH) for analysis to address changes and compensations during the process of LR.
    RESULTS: Acute deletion of hepatocyte Raptor abolished mTORC1 activity and led to compensatory activation of mTORC2 and its downstream target p-AKT (S473). Raptor∆HC mice had baseline liver injury as shown by elevated serum levels of ALT, AST, and ALP. Raptor∆HC mice exhibited decreased levels of pericentral enzyme (Glul, Cyp2e1), periportal enzyme (G6pc), and main secretory proteins (Alb, Trf, Ttr), suggesting global impairment of metabolic, synthetic, and secretory functions. These mice were also under metabolic stress as seen by ER whorl formation, mitochondrial swelling, and increased autophagy, by transmission electron microscopy (TEM). There was an increase in cell death seen as enhanced apoptotic body formation by TUNEL and cleaved caspase-3 staining and an associated increase in macrophage infiltration in the immediate proximity of dying hepatocytes. An attempt at compensatory liver regeneration was concurrently evident through activation of the Wnt/β-catenin pathway leading to enhanced Cyclin D1, Ki67, Axin2 and c-Myc. β-Catenin protein increased 5-fold in Raptor∆HC mice and appears to be due to both increased gene transcription and decreased phosphorylation-mediated degradation. Further, stabilization of β-catenin protein appears to be due to both cell-extrinsic mechanism seen as increased p-LPR6 (S1490) suggesting Wnt stimulation, and cell-intrinsic mechanism seen as decreased GSK3β activity which was phosphorylated and inhibited by compensatory elevation of AKT activity in the Raptor∆HC mice. Despite activation of the Wnt pathways, hepatocytes failed to finish cell cycle seen as an overall decrease in BrdU incorporation after a 14-day continuous pulse. Indeed, when Raptor∆HC mice were subjected to PH, they all died within 48h.
    CONCLUSIONS: mTORC1 activity is essential for homeostatic liver functions and for LR after PH. Stabilization of β-catenin due to multiple mechanisms including Wnt secretion likely from macrophages, secondary to AKT mediated GSK3β inhibition and via de novo β-catenin transcription, attempts to compensate to maintain homeostasis in the Raptor∆HC mice.
  44. Exp Mol Med. 2022 May 10.
      Leukemia is caused by the malignant clonal expansion of hematopoietic stem cells, and in adults, the most common type of leukemia is acute myeloid leukemia (AML). Autophagy inhibitors are often used in preclinical and clinical models in leukemia therapy. However, clinically available autophagy inhibitors and their efficacy are very limited. More effective and safer autophagy inhibitors are urgently needed for leukemia therapy. In a previous study, we showed that ΔA146Ply, a mutant of pneumolysin that lacks hemolytic activity, inhibited autophagy of triple-negative breast cancer cells by activating mannose receptor (MR) and toll-like receptor 4 (TLR4) and that tumor-bearing mice tolerated ΔA146Ply well. Whether this agent affects AML cells expressing TLR4 and MR and the related mechanisms remain to be determined. In this study, we found that ΔA146Ply inhibited autophagy and induced apoptosis in AML cells. A mechanistic study showed that ΔA146Ply inhibited autophagy by activating mammalian target of rapamycin signaling and induced apoptosis by inhibiting autophagy. ΔA146Ply also inhibited autophagy and induced apoptosis in a mouse model of AML. Furthermore, the combination of ΔA146Ply and chloroquine synergistically inhibited autophagy and induced apoptosis in vitro and in vivo. Overall, this study provides an alternative effective autophagy inhibitor that may be used for leukemia therapy.
  45. Cell Rep. 2022 May 10. pii: S2211-1247(22)00563-0. [Epub ahead of print]39(6): 110796
      Malignant tumors exhibit altered metabolism resulting in a highly acidic extracellular microenvironment. Here, we show that cytoplasmic lipid droplet (LD) accumulation, indicative of a lipogenic phenotype, is a cellular adaption to extracellular acidity. LD marker PLIN2 is strongly associated with poor overall survival in breast cancer patients. Acid-induced LD accumulation is triggered by activation of the acid-sensing G-protein-coupled receptor (GPCR) OGR1, which is expressed highly in breast tumors. OGR1 depletion inhibits acid-induced lipid accumulation, while activation by a synthetic agonist triggers LD formation. Inhibition of OGR1 downstream signaling abrogates the lipogenic phenotype, which can be rescued with OGR1 ectopic expression. OGR1-depleted cells show growth inhibition under acidic growth conditions in vitro and tumor formation in vivo. Isotope tracing shows that the source of lipid precursors is primarily autophagy-derived ketogenic amino acids. OGR1-depleted cells are defective in endoplasmic reticulum stress response and autophagy and hence fail to accumulate LDs affecting survival under acidic stress.
    Keywords:  CP: Cancer; ER stress; OGR1/GPR68; acid-sensing GPCR; acidosis; adiposomes; autophagy; lipid droplets; lipid metabolism; lipogenesis; metabolic adaptation
  46. Cells. 2022 May 03. pii: 1535. [Epub ahead of print]11(9):
      Apart from a well-known role in the innate immune system, lipocalin 2 (Lcn2) has been recently characterized as a critical regulator of thermogenesis and lipid metabolism. However, the physiological mechanism through which Lcn2 regulates cellular metabolism and thermogenesis in adipocytes remains unknown. We found that Lcn2 expression and secretion are significantly upregulated by arachidonic acid (AA) and mTORC1 inhibition in differentiated inguinal adipocytes. AA-induced Lcn2 expression and secretion correlate with the inflammatory NFkB activation. Lcn2 deficiency leads to the upregulation of cyclooxygenase-2 (COX2) expression, as well as increased biosynthesis and secretion of prostaglandins (PGs), particularly PGE2 and PGD2, induced by AA in adipocytes. Furthermore, Lcn2 deficiency affects the mTOR signaling regulation of thermogenic gene expression, lipogenesis, and lipolysis. The loss of Lcn2 dismisses the effect of mTORC1 inhibition by rapamycin on COX2, thermogenesis genes, lipogenesis, and lipolysis, but has no impact on p70 S6Kinase-ULK1 activation in Lcn2-deficient adipocytes. We conclude that Lcn2 converges the COX2-PGE2 and mTOR signaling pathways in the regulation of thermogenesis and lipid metabolism in adipocytes.
    Keywords:  adipocyte; lipocalin 2; mTOR signaling; prostaglandin
  47. Cells. 2022 Apr 30. pii: 1503. [Epub ahead of print]11(9):
      Mesenchymal stem cells (MSC) have emerged as a promising tool to treat inflammatory diseases, such as inflammatory bowel disease (IBD), due to their immunoregulatory properties. Frequently, IBD is modeled in mice by using dextran sulfate sodium (DSS)-induced colitis. Recently, the modulation of autophagy in MSC has been suggested as a novel strategy to improve MSC-based immunotherapy. Hence, we investigated a possible role of Pacer, a novel autophagy enhancer, in regulating the immunosuppressive function of MSC in the context of DSS-induced colitis. We found that Pacer is upregulated upon stimulation with the pro-inflammatory cytokine TNFα, the main cytokine released in the inflammatory environment of IBD. By modulating Pacer expression in MSC, we found that Pacer plays an important role in regulating the autophagy pathway in this cell type in response to TNFα stimulation, as well as in regulating the immunosuppressive ability of MSC toward T-cell proliferation. Furthermore, increased expression of Pacer in MSC enhanced their ability to ameliorate the symptoms of DSS-induced colitis in mice. Our results support previous findings that autophagy regulates the therapeutic potential of MSC and suggest that the augmentation of autophagic capacity in MSC by increasing Pacer levels may have therapeutic implications for IBD.
    Keywords:  KIAA0226L; PACER; RUBCNL; autophagy; colitis; inflammatory bowel disease; mesenchymal stem cells; therapy
  48. FASEB J. 2022 May;36 Suppl 1
      In response to stress, cells rapidly adjust gene expression, translation, and protein levels to mitigate damage and initiate repair pathways. The Cdk8 Kinase Module (CKM) is a highly conserved, detachable unit of the Mediator complex that plays a vital role in regulating transcription and communicating stress signals from the nucleus to other organelles. Previously, our laboratory has shown that the scaffold protein within the CKM, Med13, is exported from the nucleus and degraded via a selective autophagy pathway following nitrogen starvation. In periods of starvation, cells attenuate translation and biosynthetic pathways to reserve nutrient pools. To rapidly adapt to starvation, cytosolic ribonucleoprotein (RNP) granules such as processing bodies (P-bodies) tightly regulate mRNA accessibility and translation. Here we show that Med13 plays a dual role in promoting P-body assembly. In physiological conditions, Med13 functions as a positive transcriptional regulator of P-body formation as the deletion of MED13 significantly decreases the quantity of P-bodies. In addition, during stress Med13 co-localizes with the P-body marker, Edc3, suggesting that Med13 may also play a scaffolding role within these membrane-less organelles. Taken together, these data demonstrate the duality of Med13 as a transcriptional regulator of P-bodies in unstressed conditions and a scaffolding component during stress. The nutrient sensing kinase, Ksp1, plays an important role in mediating P-body localization of Med13 and other mRNA binding proteins following nitrogen starvation. These data demonstrate a novel cytosolic role for Med13 and expand the known proteome of P-bodies.
  49. Autophagy. 2022 May 10.
      Within the thymus, thymic epithelial cells (TECs) provide dedicated thymic stroma microenvironments for T cell development. Because TEC functionality is sensitive to aging and cytoablative therapies, unravelling the molecular elements that coordinate their thymopoietic role has fundamental and clinical implications. Particularly, the selection of CD4 T cells depends on interactions between TCRs expressed on T cell precursors and self-peptides:MHC II complexes presented by cortical TECs (cTECs). Although the macroautophagy/autophagy-lysosomal protein degradation pathway is implicated in CD4 T cell selection, the molecular mechanism that controls the generation of selecting MHC II ligands remains elusive. LAMP2 (lysosomal-associated membrane protein 2) is a well-recognized mediator of autolysosome (AL) maturation. We showed that LAMP2 is highly expressed in cTECs. Notably, genetic inactivation of Lamp2 in thymic stromal cells specifically impaired the development of CD4 T cells that completed positive selection, without misdirecting MHC II-restricted cells into the CD8 lineage. Mechanistically, defects in autophagy in lamp2-deficient cTECs were linked to alterations in MHC II processing, which was associated with a marked reduction in CD4 TCR repertoire diversity selected within the lamp2-deficient thymic stroma. Together, our findings suggest that LAMP2 interconnects the autophagy-lysosomal axis and the processing of selecting self-peptides:MHC II complexes in cTECs, underling its implications for the generation of a broad CD4 TCR repertoire.
    Keywords:  Autophagy; LAMP2; TCR repertoire; thymic selection; thymus
  50. FASEB J. 2022 May;36 Suppl 1
      Aging represents the progressive deterioration in the efficacy of cellular survival mechanisms, including the important mechanism of autophagy. Deficiencies in autophagic function, which occur with increasing age, may contribute to the development of many diseases, including neurodegeneration, cardiovascular diseases, and the development of various cancers. Thus, it is imperative to understand the regulation of autophagy in the context of human physiology. Of note, it is suggested that engaging in exercise represents an optimal strategy to improve autophagic function, which may help explain the many health benefits associated with regular exercise. During acute cellular stress, such as exercise, autophagy degrades misfolded or damaged proteins to provide the base constituents (i.e. amino acids) for cellular energy production. Although, until the past decade, few studies have examined the autophagic response to exercise in humans. We have recently demonstrated that exercise-induced autophagy is intensity-dependent, with greater intensities required to elicit an increase in autophagic flux, as indicated by elevated levels of microtubule-associated protein 1 light chain 3 beta (LC3-II). However, no known studies have evaluated the autophagic response to maximal exercise in humans, and it is unknown if this response is altered by aging. Therefore, we aimed to examine whether autophagy would increase in response to an acute (<15 min) incremental maximal exercise bout in peripheral blood mononuclear cells (PBMCs), and if this response would be altered in older adults. We evaluated the hypothesis that autophagy would increase in response to maximal exercise and that young adults would display greater elevations in autophagy than their older counterparts. To test this hypothesis, PBMCs were collected from 8 young (mean [SD], 20 [2.1] years; 4 women, 4 men) and 8 older (66 [5.5] years; 4 women, 4 men) adults before and immediately after a graded maximal exercise test performed on a semi-recumbent cycle ergometer. The autophagic marker LC3-II was assessed by Western blotting, which was normalized to β-actin (an internal loading control) and reported as a fold change relative to its respective baseline. Data were analyzed using unpaired t-tests with an alpha set at 0.05. Peak oxygen consumption (VO2peak ) was significantly higher in young (41.6 [10.3] ml/kg/min) compared to older (30.3 [6.6] ml/kg/min) adults (p=0.02). Immediately following the maximal exercise bout, an increase in LC3-II was observed in both young (1.64 [0.45], p=0.005) and older (1.21, [0.24] p=0.048) adults. When comparing between groups, the relative increase in LC3-II was significantly greater (+27%) in young compared to older adults (p=0.03). Taken together, our preliminary findings show that autophagy is elevated in response to maximal exercise in young and older adults, albeit to a lesser extent in older adults, suggesting an impaired ability to respond to cellular stress. Further research is necessary to understand the mechanisms underlying age-related impairments in autophagy in older adults and if these responses can be restored.
  51. J Cell Biol. 2022 Jun 06. pii: e202112081. [Epub ahead of print]221(6):
      β-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3-nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.
  52. FASEB J. 2022 May;36 Suppl 1
      Insulin is a peptide hormone produced in the pancreas that is crucial in regulating systemic glucose homeostasis and energy balance. Diabetes mellitus is an endocrine disease that can be broken down into two subtypes. Type I diabetes is low to non-measurable amounts of insulin being produced by the pancreas consequent to cellular damage. Type II diabetes is caused by insulin resistance and later, decreased insulin secretion. Both conditions lead to hyperglycemia and impaired insulin signaling throughout the body if left untreated. Numerous studies have shown that insulin signaling controls several metabolic pathways in peripheral insulin-sensitive tissues, such as liver, adipose tissue, and muscle. Among them, autophagy is one of the most potent pathways suppressed by insulin. Autophagy is a molecular mechanism by which cells recycle the damaged or outdated components, including proteins and lipids, to alleviate cellular stresses. In addition, during the fasting state, autophagy acts as a "metabolic rescuing" pathway to degrade non-essential proteins, providing free amino acids for the synthesis of essential proteins. Conversely, when nutrients are abundant postprandially, insulin potently suppresses this "self-eating" process in cells. While studies have demonstrated the important role of insulin signaling on the autophagic pathway in peripheral tissues, whether insulin signaling contributes to the regulation of the autophagic pathway in the central nervous system remains elusive. Here, we investigate the role of insulin stimulation on autophagy in mouse astrocytes, the most abundant glial cells in the brain. We show that following insulin stimulation (100 nM, 6 h), the transcription of the critical genes involved in autophagic pathway, including p62, Ulk1/2, several Atg genes, are all dramatically decreased. Consistently, the protein levels of p62, Ulk1/2, as well as LC3, are all reduced following insulin stimulation, indicating suppression of the autophagic process. The effect of insulin signaling on the transcriptional regulation is potent, since 1 nM of insulin is sufficient to suppress many of the critical autophagic genes. To confirm whether insulin suppresses cellular autophagy in astrocytes through insulin receptors, we assessed the expression of autophagic genes following insulin stimulation in both wild-type (WT) and insulin receptor knockout (IRKO) astrocytes. Compared with WT astrocytes, insulin partially suppresses the expression of autophagic genes in IRKO astrocytes, indicating insulin also can signal through the IGF-1 receptor in astrocytes. In summary, insulin signaling potently suppresses the autophagic process in astrocytes. This connection between autophagy gene transcription and insulin levels may be an important mechanism for the CNS-related complications in diabetic patients.
  53. FASEB J. 2022 May;36 Suppl 1
      Directed cell migration is an important biological process that is necessary for the embryonic development, maintenance of multicellular organisms and immune response to external stimuli. Dysregulation of cell migration is implicated in the onset and progression of diseases including cardiovascular diseases and cancer metastasis. Many studies have identified genes and proteins that are important for directed cell migration, but the process remains incompletely understood. The mechanistic Target of Rapamycin Complex 2 (mTORC2) is a complex of proteins that have been identified as important players in cell migration. mTORC2 is known to have a role in regulating F-actin organization and actomyosin (MyoII). Moreover, there is increasing evidence that shows a correlation between mTORC2 and cancer cell migration and invasion. Despite this knowledge, the role and regulation of mTORC2 is not fully understood. Recently, we found that Rap1, a member of the Ras superfamily of small GTPases with conserved roles in cytoskeleton remodeling and cell adhesion, is a conserved binding partner of mTORC2 component RIP3/SIN1. We previously showed that Rap1 positively regulates mTORC2 activation in response to the chemoattractant cAMP in the model organism Dictyostelium discoideum. Moreover, we recently found that expression of a constitutively active Rap1 mutant (Rap1CA) potentiates the insulin-induced activation of mTORC2 in human HEK293 cells. We then undertook a study to investigate if Rap1 regulation of mTORC2 is part of a mechanism involved in regulating human cell migration. We observed that basal mTORC2 activity, as well as that induced by several potential promigratory signals, including insulin, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and lysophosphatidic acid (LPA), was increased by the overexpression of wild-type Rap1 or Rap1CA. Therefore, these observations suggest that Rap1 plays a role in positively regulating mTORC2 activation in HEK293 cells in response to stimulation of promigratory signals, similar to our previous observations made in Dictyostelium. Ongoing studies now aim at determining how Rap1 regulates the function of mTORC2 in cell migration.
  54. Fish Shellfish Immunol. 2022 May 05. pii: S1050-4648(22)00235-2. [Epub ahead of print]125 48-53
      PLAAT1 belongs to the PLAAT family and plays regulatory roles in cell growth, tumor suppression and phospholipid metabolism. However, whether PLAAT1 is involved in p53 mediated signaling has not been investigated. Here, we report that PLAAT1 promotes degradation of p53 in zebrafish. We found that the plaat1 gene was constitutively expressed in tissues including liver, kidney, spleen, intestine, eye and brain, with relative higher expression levels detected in the brain and eye. Overexpression of plaat1 led to inhibition of p53 and tnfα mRNA expression. Furthermore, it was shown that PLAAT1 interacted with p53 to facilitate p53 degradation via autophagy-lysosome dependent pathway. Our work indicates that PLAAT1 is involved in the interplay between p53 mediated cellular responses and autophagy.
    Keywords:  Apoptosis; Autophagy; Phospholipase A/acyltransferases; Protein interaction; Zebrafish; p53
  55. PLoS Pathog. 2022 May 09. 18(5): e1010506
      Viruses can hijack autophagosomes as the nonlytic release vehicles in cultured host cells. However, how autophagosome-mediated viral spread occurs in infected host tissues or organs in vivo remains poorly understood. Here, we report that an important rice reovirus, rice gall dwarf virus (RGDV) hijacks autophagosomes to traverse multiple insect membrane barriers in the midgut and salivary gland of leafhopper vector to enhance viral spread. Such virus-containing double-membraned autophagosomes are prevented from degradation, resulting in increased viral propagation. Mechanistically, viral nonstructural protein Pns11 induces autophagy and embeds itself in the autophagosome membranes. The autophagy-related protein 5 (ATG5)-ATG12 conjugation is essential for initial autophagosome membrane biogenesis. RGDV Pns11 specifically interacts with ATG5, both in vitro and in vivo. Silencing of ATG5 or Pns11 expression suppresses ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, Pns11 could directly recruit ATG5-ATG12 conjugation to induce the formation of autophagosomes, facilitating viral spread within the insect bodies. Furthermore, Pns11 potentially blocks autophagosome degradation by directly targeting and mediating the reduced expression of N-glycosylated Lamp1 on lysosomal membranes. Taken together, these results highlight how RGDV remodels autophagosomes to benefit viral propagation in its insect vector.
  56. FASEB J. 2022 May;36 Suppl 1
      RATIONALE: The loss of skeletal muscle function can have dramatic health consequence. Skeletal muscle dysfunction are found in a wide range of clinical conditions, including cancer cachexia, starvation, sepsis, and aging. Autophagy is an essential catabolic process that remove damaged cytosolic components. Accumulating evidence indicates that insufficient or excessive autophagy can contribute to the development of skeletal muscle atrophy. We recently identified a novel FoxO-dependent gene, which we named MYTHO, with the potential to regulate autophagy in skeletal muscles. However, to date, the roles that MYTHO plays in skeletal muscles remain unknown. In this study, we evaluated the functional importance of MYTHO in skeletal muscle structure and function.METHODS: To study the functional importance of MYTHO in skeletal muscle mass and function, we used a combination of genetic strategies in mice to delete or overexpress MYTHO.
    RESULTS: MYTHO expression is induced in various muscle wasting conditions, including starvation, denervation, cancer cachexia, and sepsis. Using different genetics approaches, we confirmed that MYTHO is an important regulator of autophagy in skeletal muscle. Importantly, we also found that MYTHO knockdown in skeletal muscles caused various myopathic features, including muscle weakness, accumulation of tubular aggregates, a shift in myofiber type composition and nuclei mispositioning in myofibers. Using animals with muscle specific Atg7 deletion (a model of autophagy inactivation), we demonstrated that the pathological features associated with MYTHO knockdown are independent of its role in regulating autophagy.
    CONCLUSION: We conclude that MYTHO is a central player in regulating skeletal muscle mass and integrity.
  57. FASEB J. 2022 May;36 Suppl 1
      INTRODUCTION: Musculoskeletal disorders of the masticatory system impact the quality of life and have a high cost of diagnosis and treatment. Masseter muscle hyperactivity is a cause of pain in the chewing apparatus. Botulinum toxin type A (BoNTA) injection is widely used to induce paralysis of the masseter muscle, thereby decreasing impaired muscle activity. However, our laboratory has described in a preclinical model that the injection of BoNTA not only induces paralysis, but also muscle atrophy, which subsequently decreases bone quality. However, it is unknown whether apoptosis or autophagy mechanisms could contribute to muscle atrophy.AIM: To evaluate the induction of apoptosis and autophagy in BoNTA-injected masseter muscle of adult mice.
    METHODOLOGY: Unilateral injection of BoNTA (0.2U/10µl) in the masseter muscle was performed in adult BalbC mice (approved by IACUC-Universidad de Chile, #21446-ODO-UCH). Apoptosis and autophagy markers were evaluated in masseter muscles by immunoblot at 2-7d post-injection and immunofluorescence at 7d post-injection. Autophagy activity was blocked by i.p injection of chloroquine. The data were evaluated with t-test, one-way ANOVA test, Mann-Whitney test, or Kruskal-Wallis test, as appropriate. The results were expressed as mean ±SEM (n=4-8; p <0.05).
    RESULTS: Unilateral injection of BoNTA did not change the relative levels of apoptosis-related proteins like cleaved Caspase 3, PARP, and AIF. There was a significant increase in protein levels related to autophagy, such as lipidated LC3 (1.86-fold), non-lipidated LC3 (1.66-fold), and P62 (1.24-fold) at 7 d post-injection. Also, there was a strong punctuated stain for LC3 in histological sections of masseter muscle 7d post injection, suggesting autophagic vesicles. However, the injection of chloroquine for blocking the autophagy flux did not improve the accumulation of autophagy proteins in masseter muscle evoked by BoNTA in the induction.
    CONCLUSIONS: The atrophy of masseter muscle evoked by BoNTA injection is not related to autophagy- or apoptosis-induction. The increase in autophagy markers after BoNTA injection, with no further increase after autophagic flux blockade, suggests that BoNTA may be blocking autophagy in the masseter muscle, perhaps favoring a proteasomal pathway of muscle atrophy.
  58. FEBS Lett. 2022 May 09.
      Profilin regulates actin polymerization, and its balanced expression is required for cellular growth and development. Most tumors have compromised profilin expression, and its overexpression in MDA MB-231 breast cancer cells has been reported to activate AMP-activated protein kinase α (AMPKα), an energy-sensing molecule that affects various cellular processes including autophagy. The present study aims to explore the role of profilin in inducing autophagy. We employed all-trans retinoic acid (ATRA) as an inducer of profilin expression and showed that profilin induces autophagy through mTOR inhibition, autophagy-activating kinase ULK1 upregulation, and AMPK stabilization as well as its activation. Furthermore, evidence from our study indicates physical interaction between profilin and AMPK, which results in AMPK stabilization and induction of prolonged autophagy, thereby leading to apoptosis. This study uncovers a novel mechanism that induces autophagy in triple-negative breast cancer cells.
    Keywords:  AMP-activated protein kinase (AMPK); Autophagy; LAMP 2; LC3; Profilin; Retinoic acid (ATRA)
  59. FASEB J. 2022 May;36 Suppl 1
      The endo-lysosomal pathway plays an important role in pathogen clearance and both bacteria and viruses have evolved complex mechanisms to evade this host system. Here, we describe a novel aspect of coronaviral infection, whereby the master transcriptional regulator of lysosome biogenesis - TFEB - is targeted for proteasomal-mediated degradation upon viral infection. Through mass spectrometry analysis and an unbiased siRNA screen, we identify that TFEB protein stability is coordinately regulated by the E3 ubiquitin ligase subunit DCAF7 and the PAK2 kinase. In particular, viral infection triggers marked PAK2 activation, which in turn, phosphorylates and primes TFEB for ubiquitin-mediated protein degradation. Deletion of either DCAF7 or PAK2 blocks viral-mediated TFEB degradation and protects against viral-induced cytopathic effects. We further derive a series of small molecules that interfere with the DCAF7-TFEB interaction. These agents inhibit viral-triggered TFEB degradation and demonstrate broad anti-viral activities including attenuating in vivo SARS-CoV-2 infection. Together, these results delineate a viral-triggered pathway that disables the endogenous cellular system that maintains lysosomal function and suggest that small molecule inhibitors of the E3 ubiquitin ligase DCAF7 represent a novel class of endo-lysosomal, host-directed, anti-viral therapies.
  60. FASEB J. 2022 May;36 Suppl 1
      Neuronal autophagy controls the quality of cytoplasmic proteins through degradation of important synaptic proteins and modulates synaptic organization and morphogenesis (1). Evidence has shown that G protein coupled receptors are direct sensors regulating the autophagic machinery (2) and opioid receptors regulate neuronal plasticity and neurotransmission with as yet unclarified mechanism (3,4). Using in vitro and in vivo studies, we demonstrated that κ-opioid receptor (κ-OR) agonists induce autophagy via a PTX-sensitive G protein manner and identified the downstream components involved (5). Our molecular analysis also revealed a κ-OR-driven upregulation of becn1 gene through ERK1,2-dependent activation of the transcription factor CREB in neuronal cells. Moreover, our studies demonstrated that sub-chronic U50,488H administration in mice causes profound increases of specific autophagic markers exclusively in the hippocampus with a concomitant decrease of several pre- and post-synaptic proteins such as spinophilin, PSD-95 and SNAP25. Finally, using acute stress, a stimulus known to increase the levels of the endogenous κ-OR ligand dynorphin, we are demonstrating that administration of the κ-ΟR selective antagonist, nor-binaltorhimine, blocks the induction of autophagy and the stress-evoked reduction of synaptic proteins in the hippocampus. These findings provide novel insights about the essential role of autophagic machinery into the mechanisms through which κ-OR and its signaling regulates brain plasticity. REFERENCES: 1. Nikoletopoulou V, Papandreou ME, Tavernarakis N (2015) Cell Death Differ 22: 398-407 2. Wauson EM, Dbouk HA, Ghosh AB, Cobb MH (2014) Trends Endocrinol Metab 25: 274-2823. 3. Georganta EM, Tsoutsi L, Gaitanou M, Georgoussi Z (2013) J Neurochem 127: 329-341 4. Kibaly C, Xu C, Cahill CM, Evans CJ, Law PY (2019) Nat Rev Neurosci 20: 5-18 5. Georgoussi Z and Karoussiotis C (2019) FASEB J. Supplement Issue Vol.33.
  61. Clin Transl Med. 2022 May;12(5): e852
      BACKGROUND: Glutaminolysis is a critical metabolic process that promotes cancer cell proliferation, including hepatocellular carcinoma (HCC). Delineating the molecular control of glutaminolysis could identify novel targets to ameliorate this oncogenic metabolic pathway. Here, we evaluated the role of general control of amino acid synthesis 5 like 1 (GCN5L1), a regulator of mitochondrial protein acetylation, in modulating the acetylation and activity of glutaminase to regulate HCC development.METHODS: Cell proliferation was determined by MTT, 2D and soft agar clone formation assays and orthotopic tumour assays in nude mice. GLS1/2 acetylation and activities were measured in cells and tumours to analyse the correlation with GCN5L1 expression and mTORC1 activation.
    RESULTS: Hepatic GCN5L1 ablation in mice markedly increased diethylnitrosamine (DEN)-induced HCC, and conversely, the transduction of mitochondrial-restricted GCN5L1 protected wild-type mice against HCC progression in response to DEN and carbon tetrachloride (CCl4 ) exposure. GCN5L1-depleted HepG2 hepatocytes enhanced tumour growth in athymic nude mice. Mechanistically, GCN5L1 depletion promoted cell proliferation through mTORC1 activation. Interestingly, liver-enriched glutaminase 2 (GLS2) appears to play a greater role than ubiquitous and canonical tumour-enriched glutaminase 1 (GLS1) in promoting murine HCC. Concurrently, GCN5L1 promotes acetylation and inactivation of both isoforms and increases enzyme oligomerisation. In human HCC tumours compared to adjacent tissue, there were variable levels of mTORC1 activation, GCN5L1 levels and glutaminase activity. Interestingly, the levels of GCN5L1 inversely correlated with mTORC1 activity and glutaminase activity in these tumours.
    CONCLUSIONS: Our study identified that glutaminase activity, rather than GLS1 or GLS2 expression, is the key factor in HCC development that activates mTORC1 and promotes HCC. In the Kaplan-Meier analysis of liver cancer, we found that HCC patients with high GCN5L1 expression survived longer than those with low GCN5L1 expression. Collectively, GCN5L1 functions as a tumour regulator by modulating glutaminase acetylation and activity in the development of HCC.
    Keywords:  GCN5L1; HCC; glutaminase; mTORC1; mitochondria acetylation
  62. FASEB J. 2022 May;36 Suppl 1
      The electrogenic sodium bicarbonate co-transporter 1 (NBCe1, Slc4a4) is a key pH-regulating membrane protein and is widely expressed in kidney, pancreas, cornea, heart and brain. Phosphorylation in several residues can affect the surface expression and the activity of the transporter. NBCe1 has been found to be phosphorylated in several residues among them in Ser65 where SPAK inhibits its activity and in Thr49 required for regulation of NBCe1 by IRBIT and SPAK. Moreover, IRBIT regulates the activity of NBCe1 by controlling the phosphorylation status of Ser232 , Ser233 and Ser235 . The aim of this study is to identify novel phosphorylation sites and examine their putative contribution in the transport activity of NBCe1. Phosphoproteomic analysis in mouse cortical astrocytes revealed constitutively phosphorylated NBCe1 at Ser245 and Ser255-257 . Mutational analysis in Hela cells and intracellular H+ recordings using pH-sensitive fluorescent dye (BCECF) showed that the activity of NBCe1 is regulated by the phosphorylation of these residues, phospho-Ser245 significantly reduced the activity of NBCe1 whereas phospho-Ser255-257 led to increased NBCe1 activity. Additionally, in the presence of the mTOR inhibitor rapamycin or the mTOR activator 3BDO our data demonstrated that mTOR signaling pathway regulates the phosphorylation of the Ser245 and Ser255-257 . Our findings reveal phosphorylation at Ser245 and Ser255-257 as novel regulators of NBCe1 transport activity. We suggest that mTOR pathway is capable to fine tune NBCe1 activity, an event with putative implications during pathophysiological conditions.
  63. FASEB J. 2022 May;36 Suppl 1
      Esophageal squamous epithelium comprises a basal layer of proliferative cells that differentiate in suprabasal layers before luminal desquamation, facilitating tissue renewal. Controversy exists as to whether the esophageal basal cells are functionally homogenous, with each cell having equal potential to proliferate and differentiate, or heterogenous, with subsets of basal cells harboring differential proliferation and differentiation kinetics. To examine the relationship between the stemness/proliferation axis and autophagy in esophageal basal cells, we stained primary basal cells from wild type mice with the autophagic vesicle (AVs)-identifying dye Cyto-ID. AV-high and AV-low basal esophageal epithelial cell fractions were sorted by fluorescence-activated cell sorting (Figure 1A) and stem/progenitor function was assessed by organoid formation assay and passaging. Upon initial plating, organoid formation rate (OFR) was decreased in Cyto-IDhigh basal cells(0.8%±0.11) as compared to their Cyto-IDlow counterparts (10.0%±0.06; n=3; p<0.001) (Figure 1B). However, passaging revealed that OFR decreased in Cyto-IDlow cells (0.36% for passage 2) while increasing in Cyto-IDhigh cells (14.88% for passage 4) (Figure 1B). Additionally, while Cyto-IDlow basal cells maintained spherical morphology upon passaging, structures reminiscent of bud-like intestinal organoids with enhanced self-renewal capacity emerged in Cyto-IDhigh cells (Figure 1C). These findings suggest that cells with high AV content may serve as a quiescent stem cell population. To examine the gene expression profiles of Cyto-IDlow and Cyto-IDhigh cells, we performed RNA-Seq and identified >7000 differentially expressed genes (p<0.05, log2 fold-change= 2) (Figure 1D). Ingenuity pathway analysis revealed reduction in Cell Cycle-associated genes in Cyto-IDhigh cells and propidium iodide flow cytometry confirmed impaired cell cycle progression with significant depletion of the S and G2/M fractions in Cyto-IDhigh cells (S: 0.5 %; G2/M: 2.2%) as compared to Cyto-IDlow cells (S: 3.0%; G2/M: 8.3%, n=3, p=<0.05). Finally, we utilized K5-CreERT2+/- ; Atg7flox/flox mice with tamoxifen-inducible autophagy impairment in squamous epithelium to evaluate the functional consequences of autophagy depletion upon esophageal epithelial tissue architecture. Atg7 depletion in vivo resulted in hyperkeratinization, consistent with autophagy limiting cell proliferation of esophageal keratinocytes and supporting a functional role for autophagy in regulating stem cell biology and cell fate determination in the esophagus. These studies identify a novel quiescent/slow-cycling stem-like population in esophageal epithelium marked by high AV content. Future studies will define the detailed molecular mechanisms through which autophagy supports stemness in esophageal epithelium and determine the functional contribution of autophagy to the pathogenesis of widely prevalent esophageal diseases.
  64. Int J Mol Sci. 2022 Apr 23. pii: 4684. [Epub ahead of print]23(9):
      Cellular trafficking through the endosomal-lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal-lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.
    Keywords:  Arrestins; Dlg; Drosophila testis; EGFR; JNK; Lgl; Notch; PAR complex; Rab proteins; SOP; Saccharomyces cerevisiae; Scrib; TORC1; autophagy; cell trafficking; endocytosis; intestine; lysosome; mTOR; nephrocytes; polarity; signaling regulation; squamous epithelia
  65. Autophagy. 2022 May 09.
      A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex towards the site of autophagosome assembly near the vacuole.
    Keywords:  Actin assembly; End3-Pan1; Myo3/Myo5; Osh2/Osh3; Scs2/Scs22; contact sites; endocytic pits; endoplasmic reticulum; reticulophagy
  66. FASEB J. 2022 May;36 Suppl 1
      Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is one of the three clinical subtypes of breast cancer and is defined by having HER2 gene amplification that coincides with HER2 protein overexpression. HER2 is amplified in 15-30% of breast cancers and overexpression of this gene is a predictor of survival in breast cancer patients. HER2-targeted therapies have been successful in treating HER2+ breast cancer; however, over time, HER2+ breast cancer can develop resistance to these therapies. Therefore, there is an urgent need to establish novel targeted agents. One proposed method of resistance to these HER2 agents is dysregulation of autophagy where cancer cells can shift to a cytoprotective autophagy phenotype to avoid cell death. Previous work in our lab established that Hormonally Upregulated Neu-associated kinase (HUNK) is a Serine/Threonine (S/T) protein kinase that is overexpressed in HER2+ breast cancer and is responsible for promoting autophagy, thereby leading to therapeutic resistance. Our previous work shows that HUNK phosphorylates an autophagy protein, Rubicon, in the N-terminal domain at S92, promoting autophagy in 293T cells. However, we have yet to establish a role for this phosphorylation site within HER2+ breast cancer cells. Therefore, the objective of this study is to test whether Rubicon S92 phosphorylation plays a role in promoting tumorigenesis in HER2+ breast cancer. We generated multiple scientific tools: a phosphoserine antibody at S92 in Rubicon, a phospho-mimetic (S92A) mutant, and a phospho-deficient (S92D) mutant. We have established an autophagy phenotype within a HER2/neu model; mouse mammary tumor virus driven neu(rodent form of HER2) expressing mammary tumor cells (MMTV-neu) genetically engineered to be HUNK wild type (WT) or HUNK knockout (KO). We performed an endogenous Rubicon immunoprecipitation (IP) and found that KO cells are deficient in S92 phosphorylation alongside with a lack of autophagy response, measured using LC3-II western blot. These findings confirm that HUNK is required for Rubicon S92 phosphorylation in a genetically tractable model. Utilizing these cell lines alongside the phospho-mimetic and phospho-deficient Rubicon mutants will allow us to determine the role that HUNK plays in promoting tumorigenesis within HER2+ breast cancer in-vitro and in-vivo.
  67. Commun Biol. 2022 May 12. 5(1): 453
      Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1-8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson's disease.
  68. FASEB J. 2022 May;36 Suppl 1
      The O-GlcNAc post-translational modification is reversibly added onto intracellular proteins. It is a unique glucose rheostat for cell signaling relying on the availability of UDP-GlcNAc, itself reflecting extracellular glucose. With more than 7000 human targets identified to date, O-GlcNAcylation regulates numerous physiological processes such as cell cycle, transcriptional/translational regulation, protein localization or degradation, and development. Therefore, O-GlcNAcylation is a molecular bridge between dietary glucose level and proper signaling regulation. Using cellular and mouse models, our lab has previously delved into the consequences of hyper-O-GlcNAcylation in the brain. Among phenotypes of obesity and growth defects, the pituitary gland of these mice was generally delayed in development. A critical aspect of pituitary's ontogeny is the transient expression of the homeobox protein OTX2, an O-GlcNAcylated protein. However, the function of O-GlcNAcylation in regulating OTX2 has not been investigated. Interestingly, in hyper-O-GlcNAcylated mouse embryonic stem cells, this transcription factor's expression increased, suggesting that O-GlcNAc plays a role in OTX2 stability/degradation. Thus, we hypothesized that OTX2 is degraded by the proteasome and O-GlcNAc cycling regulates its timely degradation. Using a combination of proteasome inhibition (MG-132 mediated) and O-GlcNAc increase (Thiamet-G, TG), we demonstrated that endogenous OTX2 was indeed degraded by the proteasome. Interestingly, increased O-GlcNAc levels caused further stabilization of OTX2, additional to the one achieved with proteasome inhibition alone. This suggested that OTX2 utilizes another degradation pathway, possibly autophagy. We showed that the macroautophagy inhibitor Chloroquine (CQ) prevented OTX2 degradation, and the addition of TG did not further stabilize the protein. This confirmed that OTX2 has two degradation modes, and that O-GlcNAcylation is involved in the autophagy-mediated degradation of this protein. However, we believe that autophagic degradation of OTX2 only occurs when this homeobox protein is abnormally overexpressed, such as in Medulloblastoma. Similar crosstalk between proteasome and autophagy has been demonstrated by others for developmental transcription factors like SOX2 and OCT4, following proteasome inhibition. We performed mass spectrometry site-mapping of OTX2, and identified three O-GlcNAc sites in the central domain of OTX2, likely involved in its autophagy-mediated degradation. To summarize, this study highlights the O-GlcNAc modification as a nutrient-dependent sensor that regulates the homeobox protein OTX2 during pituitary and brain development. Like many homeobox proteins, OTX2 level needs to be tightly regulated for proper patterning and development, and its deregulation amongst other proteins is a major driver of Medulloblastoma. Therefore, we foresee that O-GlcNAcylated OTX2 may play a major role in Medulloblastoma pathogenesis.
  69. FASEB J. 2022 May;36 Suppl 1
      The Sestrins (Sesn), a family of stress-response proteins (Sesn1-3), coordinate metabolism and protein synthesis by affecting Mechanistic Target of Rapamycin in Complex 1 (mTORC1) activation. Prior research demonstrates a role for Sesns in the regulation of mTORC1 by amino acids, but mechanisms regulating glucose-induced stimulation of mTORC1 are undefined. Initially, glucose deprivation in C2C12 myotubes was found to attenuate mTORC1 activation, while glucose resupplementation thereafter stimulated mTORC1 activation. Similarly, rats that were fasted overnight and then given an oral gavage of glucose showed mTORC1 activation in the tibialis anterior muscle (TA) compared to saline-gavaged rats. To elucidate the role of Sesns in affecting mTORC1 activation, HEK293T wild-type (WT) and Sesn-ablated cells (SesnTKO) were incubated in Dulbecco's Modified Eagle Medium (DMEM) containing (CM) or lacking glucose (-Glu) or DMEM lacking glucose followed by glucose readdition (GluAB). In WT, but not SesnTKO cells, GluAB activated mTORC1 showing that Sesns are necessary for glucose-induced activation of mTORC1. To determine potential mechanisms, plasmids expressing FLAG-Sesn1, 2, or 3 or a control protein were transfected into HEK293T cells. Cells were treated similarly to the above media conditions, harvested, immunoprecipitated using FLAG beads, and probed for protein interactions via western blot. Interestingly, Hexokinase2 (HK2), but not HK1, was associated with Sesn1-3, and the interaction was more prominent in -Glu compared to CM and GluAB. Based on this finding and previous studies showing Sesn3 affects glucose metabolism, we hypothesized Sesn3 attenuates glucose-induced mTORC1 activation. To characterize this interaction, we performed similar immunoprecipitations; however, in CM and -Glu cells, glucose was added directly to cell lysates to determine if glucose directly altered the interaction between Sesn3 and HK2. Glucose addition to lysates did not affect the interaction suggesting glucose does not directly modify the interaction. We also found that the interaction is particularly influenced by glucose as individual resupplementation of leucine, fructose, sodium pyruvate, nor mannitol following glucose deprivation dissociated the complex. To illustrate in vivo reproducibility, plasmids expressing FLAG-Sesn3 or Green fluorescent protein (GFP) were transfected into either TA of 12 rats. Rats were then given a glucose or saline oral gavage, and then both TAs were excised and analyzed via western blot. Notably, glucose-induced activation of mTORC1 occurred in the GFP-leg of glucose-gavaged animals; however, the Sesn3-leg of glucose-gavaged animals showed activation similar to saline-gavaged animals suggesting Sesn3 attenuates glucose-induced mTORC1 activation. These data demonstrate Sesn3 affects glucose-induced stimulation of mTORC1 through a possible mechanism involving HK2. Importantly, the precise mechanism for how Sesn3 affects mTORC1 warrants further investigation.
  70. FASEB J. 2022 May;36 Suppl 1
      The beneficial effects of exercise on skeletal muscle health and function are well-established. The intracellular signaling that mediates exercise's effects on muscle are undoubtedly complex and are not entirely understood. Nonetheless, increasing adenosine monophosphate (AMP) levels during muscle contraction signals a declining energy charge in the cell and this certainly drives many of the beneficial adaptations of exercise. AMP-activated protein kinase (AMPK) is one of AMP's best-defined targets and has been described as the cell's master energy regulator. Because it is activated by exercise, AMPK has gained recent attention as an attractive pharmacological target in harnessing at least some of the benefits of exercise for those who cannot exercise due to old age or other disorders. The prodrug AICAR effectively activates AMPK as it is taken up into the cell and phosphorylated to form ZMP, an AMP mimetic. However, AICAR's therapeutic potential is severely limited by its poor pharmacokinetics. Here, we describe a novel approach to ZMP delivery in a novel prodrug (Prodrug-39; P39). We found that similar to AICAR, P39 concentrations above ~400 μM effectively achieved significant AMPK activation and glycogen depletion. Surprisingly, given AMPK's well-documented inhibition of the mechanistic target of rapamycin (mTORC1), P39 was also effective at stimulating p70S6k and rpS6 phosphorylation concentrations as low as 10 uM and early in the timecourse after administration of P39 at higher concentrations. Given this evidence, we conclude that Prodrug-39 is indeed an effective AMPK activator and may also have some utility in mTOR activation at lower concentrations.