bims-auttor 2020-05-24 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2020‒05‒24
thirty-two papers selected by
Viktor Korolchuk, Newcastle University

Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.
A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy.
A Healthy Heart and a Healthy Brain: Looking at Mitophagy.
Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex.
The aging lysosome: An essential catalyst for late-onset neurodegenerative diseases.
Mitochondrial phosphatase PGAM5 modulates cellular senescence by regulating mitochondrial dynamics.
The Dawn of Mitophagy: What Do We Know by Now?
Detection of LC3-Associated Phagocytosis (LAP).
Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.
Discovery of a potent SCAP degrader that ameliorates HFD-induced obesity, hyperlipidemia and insulin resistance via an autophagy-independent lysosomal pathway.
Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish.
β-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.
Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming.
A distinct inhibitory mechanism of the V-ATPase by Vibrio VopQ revealed by cryo-EM.
TFEB regulates murine liver cell fate during development and regeneration.
Regulation and function of V-ATPases in physiology and disease.
Synergies in exosomes and autophagy pathways for cellular homeostasis and metastasis of tumor cells.
EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.
Lactulose and Melibiose Inhibit α-Synuclein Aggregation and Up-Regulate Autophagy to Reduce Neuronal Vulnerability.
Copper - a novel stimulator of autophagy.
Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.
mTORC1-IRE1α pathway activation contributes to palmitate-elicited triglyceride secretion and cell death in hepatocytes.
Modeling Lysosomal Storage Diseases in the Zebrafish.
Autophagy-dependent mitochondrial function regulates osteoclast differentiation and maturation.
The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis.
AMPK-mediated formation of stress granules is required for dietary restriction-induced longevity in Caenorhabditis elegans.
Parkin on serine: a Parkinson disease gene suppresses serine synthesis in cancer.
A New Pathway Promotes Adaptation of Human Glioblastoma Cells to Glucose Starvation.
Compound K inhibits autophagy-mediated apoptosis induced by oxygen and glucose deprivation/reperfusion via regulating AMPK-mTOR pathway in neurons.
Split-TurboID enables contact-dependent proximity labeling in cells.
PINK1 and Parkin: the odd couple.
Targeting mTOR for fighting diseases: A revisited review of mTOR inhibitors.

Cell. 2020 May 18. pii: S0092-8674(20)30488-8. [Epub ahead of print]

Visualizing and Modulating Mitophagy for Therapeutic Studies of Neurodegeneration.

Hiroyuki Katayama, Hiroshi Hama, Koji Nagasawa, Hiroshi Kurokawa, Mayu Sugiyama, Ryoko Ando, Masaaki Funata, Nobuyo Yoshida, Misaki Homma, Takanori Nishimura, Megumu Takahashi, Yoko Ishida, Hiroyuki Hioki, Yoshiyuki Tsujihata, Atsushi Miyawaki.

  Dysfunctional mitochondria accumulate in many human diseases. Accordingly, mitophagy, which removes these mitochondria through lysosomal degradation, is attracting broad attention. Due to uncertainties in the operational principles of conventional mitophagy probes, however, the specificity and quantitativeness of their readouts are disputable. Thorough investigation of the behaviors and fates of fluorescent proteins inside and outside lysosomes enabled us to develop an indicator for mitophagy, mito-SRAI. Through strict control of its mitochondrial targeting, we were able to monitor mitophagy in fixed biological samples more reproducibly than before. Large-scale image-based high-throughput screening led to the discovery of a hit compound that induces selective mitophagy of damaged mitochondria. In a mouse model of Parkinsons disease, we found that dopaminergic neurons selectively failed to execute mitophagy that promoted their survival within lesions. These results show that mito-SRAI is an essential tool for quantitative studies of mitochondrial quality control.

Keywords:  FRET; Parkinsons disease; autophagy; fluorescent protein; high-throughput screening; lysosome; mitochondria; mitophagy

DOI:  https://doi.org/10.1016/j.cell.2020.04.025
J Cell Biol. 2020 Jul 06. pii: e201912098. [Epub ahead of print]219(7):

A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy.

Dorotea Fracchiolla, Chunmei Chang, James H Hurley, Sascha Martens.

Autophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic class III phosphatidylinositol-3 kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12-ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here, we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2 that in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3-kinase and LC3 lipidation reactions were performed simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to that seen in cellular autophagosome formation.

DOI: https://doi.org/10.1083/jcb.201912098
Front Cell Dev Biol. 2020 ;8 294

A Healthy Heart and a Healthy Brain: Looking at Mitophagy.

Hongke Luo, Ruohan Zhang, Judith Krigman, Allison McAdams, Serra Ozgen, Nuo Sun.

  Mitochondrial dysfunction is a hallmark of aging and is a major contributor to neurodegenerative diseases and various cardiovascular disorders. Mitophagy, a specialized autophagic pathway to remove damaged mitochondria, provides a critical mechanism to maintain mitochondrial quality. This function has been implicated in a tissue's ability to appropriately respond to metabolic and to bioenergetic stress, as well as to recover from mitochondrial damage. A global decline in mitophagic flux has been postulated to be linked to pathological alterations that occur in the heart and the brain as well as a general age-dependent decline in organ function. Cellular observation suggests multiple mechanistically distinct pathways converge upon and activate mitophagy. Over the past decade, additional molecular components within mitophagy have been discovered, including several disease-associated genes that are functionally implicated in mitophagy. However, the pathophysiological role of mitophagy, and how it is regulated within normal physiology or various disease states, is less well established. Here, we will review the evidence that a decline in mitophagy contributes to impaired mitochondrial homeostasis and may be particularly detrimental to postmitotic neurons and cardiomyocytes. We will discuss mitophagy's pathological significance in both neurodegenerative diseases and cardiovascular disorders. Additionally, signaling pathways regulating mitophagy are reviewed, with emphasis placed on how these pathways might contribute to disease progression. Understanding mitophagy's role in the mechanisms of disease pathogenesis should allow for the development of more efficient strategies to battle pathological conditions associated with mitochondrial dysfunction.

Keywords:  autophagy; cardiovascular disorders; mitochondrial; mitophagy; neurodegenerative diseases

DOI:  https://doi.org/10.3389/fcell.2020.00294
Cell Rep. 2020 May 19. pii: S2211-1247(20)30617-3. [Epub ahead of print]31(7): 107664

Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex.

Laurensia Yuniati, Angela Lauriola, Manouk Gerritsen, Susana Abreu, Eric Ni, Chiara Tesoriero, Jacob O Onireti, Teck Yew Low, Albert J R Heck, Andrea Vettori, Timothy Cardozo, Daniele Guardavaccaro.

  Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.

Keywords:  ER three-way junction; Lunapark; cullin-RING ligases; endoplasmic reticulum; lysosome; mTORC1; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2020.107664
Biochim Biophys Acta Proteins Proteom. 2020 May 13. pii: S1570-9639(20)30090-X. [Epub ahead of print] 140443

The aging lysosome: An essential catalyst for late-onset neurodegenerative diseases.

Ralph A Nixon.

Lysosomes figure prominently in theories of aging as the proteolytic system most responsible for eliminating growing burdens of damaged proteins and organelles in aging neurons and other long lived cells. Newer evidence shows that diverse experimental measures known to extend lifespan in invertebrate aging models share the property of boosting lysosomal clearance of substrates through the autophagy pathway. Maintaining an optimal level of lysosome acidification is particularly crucial for these anti-aging effects. The exceptional dependence of neurons on fully functional lysosomes is reflected by the phenotypes seen in congenital lysosomal storage disorders, which commonly present as severe neurodevelopmental or neurodegenerative conditions even though lysosomal deficits are systemic. Similar connections are now being appreciated between risk for late age-onset neurodegenerative disorders and primary lysosomal deficits. In diseases such as Alzheimer's and Parkinson's, as in aging alone, primary lysosome dysfunction due to acidification impairment is emerging as a frequent theme, supported by the growing list of familial neurodegenerative disorders that involve primary vATPase dysfunction. The additional cellular roles played by intraluminal pH in sensing nutrient and stress and modulating cellular signaling have further expanded the possible ways that lysosomal pH dysregulation in aging and disease can disrupt neuronal function. Here, we consider the impact of cellular aging on lysosomes and how these changes may create the tipping point for disease emergence in major late-age onset neurodegenerative disorders.

DOI: https://doi.org/10.1016/j.bbapap.2020.140443
Nat Commun. 2020 May 21. 11(1): 2549

Mitochondrial phosphatase PGAM5 modulates cellular senescence by regulating mitochondrial dynamics.

Bo Yu, Jing Ma, Jing Li, Dazhi Wang, Zhigao Wang, Shusheng Wang.

Mitochondria undergo dynamic fusion/fission, biogenesis and mitophagy in response to stimuli or stresses. Disruption of mitochondrial homeostasis could lead to cell senescence, although the underlying mechanism remains unclear. We show that deletion of mitochondrial phosphatase PGAM5 leads to accelerated retinal pigment epithelial (RPE) senescence in vitro and in vivo. Mechanistically, PGAM5 is required for mitochondrial fission through dephosphorylating DRP1. PGAM5 deletion leads to increased mitochondrial fusion and decreased mitochondrial turnover. As results, cellular ATP and reactive oxygen species (ROS) levels are elevated, mTOR and IRF/IFN-β signaling pathways are enhanced, leading to cellular senescence. Overexpression of Drp1 K38A or S637A mutant phenocopies or rescues mTOR activation and senescence in PGAM5-/- cells, respectively. Young but not aging Pgam5-/- mice are resistant to sodium iodate-induced RPE cell death. Our studies establish a link between defective mitochondrial fission, cellular senescence and age-dependent oxidative stress response, which have implications in age-related diseases.

DOI: https://doi.org/10.1038/s41467-020-16312-7
Curr Neuropharmacol. 2020 May 22.

The Dawn of Mitophagy: What Do We Know by Now?

Dmitrii M Belousov, Elizaveta V Mikhaylenko, Vladimir N Chubarev, Vadim V Tarasov, Siva G Somasundaram, Cecil E Kirkland, Gjumrakch Aliev.

  Mitochondria are essential organelles for healthy eukaryotic cells. They produce energy-rich phosphate bond molecules (ATP) through oxidative phosphorylation using ionic gradients. The presence of mitophagy pathways in healthy cells enhances cell protection during mitochondrial damage. The PTEN-induced putative kinase 1 (PINK1)/Parkin-dependent pathway is the most studied for mitophage. In addition, there are other mechanisms leading to mitophagy (FKBP8, NIX, BNIP3, FUNDC1, BCL2L13). Each of these provide tethering of a mitochondrion to an autophagy apparatus via the interaction between receptor proteins (Optineurin, p62, NDP52, NBR1) or the proteins of the outer mitochondrial membrane with ATG9-like proteins (LC3A, LC3B, GABARAP, GABARAPL1, GATE16). Another pathogenesis of mitochondrial damage is mitochondrial depolarization. Reactive oxygen species (ROS) antioxidant responsive elements (AREs) along with antioxidant genes including pro-autophagic genes are all involved in mitochondrial depolarization. On the other hand, mammalian Target of Rapamycin Complex 1 (mTORC1) and AMP-dependent kinase (AMPK) are the major regulatory factors modulating mitophagy at the post-translational level. Protein-protein interactions are involved in controlling other mitophagy processes. The objective of the present review is to analyze research findings regarding the main pathways of mitophagy induction, recruitment of the autophagy machinery, and their regulations at the levels of transcription, post-translational modification and protein-protein interaction that appeared to be main target during the development and maturation of neurodegenerative disorders.

Keywords:  Mitochondria and mitophagy pathways; autophagy machinery; central nervous system disorders; factors modulating mitophagy at the post-translational level; healthy cells; mitochondria and post-translational modification; protein-protein interaction and mitophagy; risk factors

DOI:  https://doi.org/10.2174/1570159X18666200522202319
Curr Protoc Cell Biol. 2020 Jun;87(1): e104

Detection of LC3-Associated Phagocytosis (LAP).

Jennifer Martinez.

  Phagocytes, notably macrophages, are critical sentinels of their environment, patrolling for and eradicating unwanted components. The ability of cells to process extracellular cargo in an appropriate manner is important for both clearance of the cargo and eventual return to homeostasis. Although the evolutionarily conserved pathway of autophagy involves the degradation and recycling of unnecessary or dysfunctional cellular components during starvation, we now appreciate that the reach of autophagy extends beyond nutrient deprivation, notably including cellular quality control (e.g., mitophagy) and host defense against internalized pathogens (i.e., xenophagy). Despite being seemingly disparate, autophagic functions are unified as conserved mechanisms for containment and immunosuppression, suggesting an original immune function for autophagy. A recently described pathway called LC3-associated phagocytosis (LAP) marries the ancient concepts of phagocytosis and autophagy, revealing new ways in which the autophagy machinery, in a molecularly distinct pathway, contributes to the inflammatory response. In this article, protocols to detect LAP by electron microscopy, immunofluorescence, flow cytometry, and phagosome purification are described, allowing the user to detect multiple characteristics of LAP in both qualitative and quantitative manners. Published 2020. U.S. Government. Basic Protocol 1: Detection of LAP by electron microscopy Basic Protocol 2: Detection of LAP by confocal microscopy of LC3-GFP-expressing cells Alternate Protocol 1: Detection of LAP by confocal microscopy using immunofluorescence Basic Protocol 3: Detection of LAP using flow cytometry of LC3-GFP-expressing cells Alternate Protocol 2: Detection of LAP using antibody staining and flow cytometry Basic Protocol 4: Detection of LAP by western blot of purified LAPosomes.

Keywords:  LC3-associated phagocytosis (LAP); autophagy; confocal microscopy; electron microscopy; flow cytometry; phagosome purification

DOI:  https://doi.org/10.1002/cpcb.104
Nat Commun. 2020 May 22. 11(1): 2582

Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.

Huiming Yang, Su Yang, Liang Jing, Luoxiu Huang, Luxiao Chen, Xianxian Zhao, Weili Yang, Yongcheng Pan, Peng Yin, Zhaohui S Qin, Shihua Li, Xiao-Jiang Li.

Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.

DOI: https://doi.org/10.1038/s41467-020-16318-1
Autophagy. 2020 May 20. 1-22

Discovery of a potent SCAP degrader that ameliorates HFD-induced obesity, hyperlipidemia and insulin resistance via an autophagy-independent lysosomal pathway.

Zu-Guo Zheng, Si-Tong Zhu, Hui-Min Cheng, Xin Zhang, Gang Cheng, Pyone Myat Thu, Supeng Perry Wang, Hui-Jun Li, Ming Ding, Lei Qiang, Xiao-Wei Chen, Qing Zhong, Ping Li, Xiaojun Xu.

  SCAP (SREBF chaperone) regulates SREBFs (sterol regulatory element binding transcription factors) processing and stability, and, thus, becomes an emerging drug target to treat dyslipidemia and fatty liver disease. However, the current known SCAP inhibitors, such as oxysterols, induce endoplasmic reticulum (ER) stress and NR1H3/LXRα (nuclear receptor subfamily 1 group H member 3)-SREBF1/SREBP-1 c-mediated hepatic steatosis, which severely limited the clinical application of this inhibitor. In this study, we identified a small molecule, lycorine, which binds to SCAP, which suppressed the SREBF pathway without inducing ER stress or activating NR1H3. Mechanistically, lycorine promotes SCAP lysosomal degradation in a macroautophagy/autophagy-independent pathway, a mechanism completely distinct from current SCAP inhibitors. Furthermore, we determined that SQSTM1 captured SCAP after its exit from the ER. The interaction of SCAP and SQSTM1 requires the WD40 domain of SCAP and the TB domain of SQSTM1. Interestingly, lycorine triggers the lysosome translocation of SCAP independent of autophagy. We termed this novel protein degradation pathway as the SQSTM1-mediated autophagy-independent lysosomal degradation (SMAILD) pathway. In vivo, lycorine ameliorates high-fat diet-induced hyperlipidemia, hepatic steatosis, and insulin resistance in mice. Our study demonstrated that the inhibition of SCAP through the SMAILD pathway could be employed as a useful therapeutic strategy for treating metabolic diseases.ABBREVIATION: 25-OHD: 25-hydroxyvitamin D; 3-MA: 3-methyladenine; ABCG5: ATP binding cassette subfamily G member 5; ABCG8: ATP binding cassette subfamily G member 8; ACACA: acetyl-CoA carboxylase alpha; AEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AHI: anhydroicaritin; AKT/protein kinase B: AKT serine/threonine kinase; APOE: apolipoprotein E; ATF6: activating transcription factor 6; ATG: autophagy-related; BAT: brown adipose tissue; CD274/PD-L1: CD274 molecule; CETSA: cellular thermal shift assay; CMA: chaperone-mediated autophagy; COPII: cytoplasmic coat protein complex-II; CQ: chloroquine; DDIT3/CHOP: DNA damage inducible transcript 3; DNL: de novo lipogenesis; EE: energy expenditure; EGFR: epithelial growth factor receptor; eMI: endosomal microautophagy; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FADS2: fatty acid desaturase 2; FASN: fatty acid synthase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvate transaminase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSP90B1/GRP94: heat shock protein 90 beta family member 1; HSPA5/GRP78: heat hock protein family A (Hsp70) member 5; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INSIG1: insulin induced gene 1; LAMP2A: lysosomal associated membrane protein 2A; LDLR: low density lipoprotein receptor; LyTACs: lysosome targeting chimeras; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MBTPS1: membrane bound transcription factor peptidase, site 1; MEF: mouse embryonic fibroblast; MST: microscale thermophoresis; MTOR: mechanistic target of rapamycin kinase; MVK: mevalonate kinase; PROTAC: proteolysis targeting chimera; RQ: respiratory quotient; SCAP: SREBF chaperone; SCD1: stearoyl-coenzemy A desaturase 1; SMAILD: sequestosome 1 mediated autophagy-independent lysosomal degradation; SQSTM1: sequestosome 1; SREBF: sterol regulatory element binding transcription factor; TNFRSF10B/DR5: TNF receptor superfamily member 10b; TRAF6: TNF receptor associated factor 6; UPR: unfolded protein response; WAT: white adipose tissue; XBP1: X-box binding protein 1.

Keywords:  Autophagy; ER stress; SCAP; SQSTM1; SREBFs; lycorine

DOI:  https://doi.org/10.1080/15548627.2020.1757955
Elife. 2020 May 19. pii: e54935. [Epub ahead of print]9

Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish.

Mounir El Maï, Marta Marzullo, Inês Pimenta de Castro, Miguel Godinho Ferreira.

  Progressive telomere shortening during lifespan is associated with restriction of cell proliferation, genome instability and aging. Apoptosis and senescence are the two major outcomes upon irreversible cellular damage. Here, we show a transition of these two cell fates during aging of telomerase deficient zebrafish. In young telomerase mutants, proliferative tissues exhibit DNA damage and p53-dependent apoptosis, but no senescence. However, these tissues in older animals display loss of cellularity and senescence becomes predominant. Tissue alterations are accompanied by a pro-proliferative stimulus mediated by AKT signaling. Upon AKT activation, FoxO transcription factors are phosphorylated and translocated out of the nucleus. This results in reduced SOD2 expression causing an increase of ROS and mitochondrial dysfunction. These alterations induce p15/16 growth arrest and senescence. We propose that, upon telomere shortening, early apoptosis leads to cell depletion and insufficient compensatory proliferation. Following tissue damage, the mTOR/AKT is activated causing mitochondrial dysfunction and p15/16-dependent senescence.

Keywords:  AKT; aging; apoptosis; cell biology; p53; regenerative medicine; senescence; stem cells; telomeres; zebrafish

DOI:  https://doi.org/10.7554/eLife.54935
Proc Natl Acad Sci U S A. 2020 May 19. pii: 201913370. [Epub ahead of print]

β-Oxidation and autophagy are critical energy providers during acute glucose depletion in Saccharomyces cerevisiae.

Carmen A Weber, Karthik Sekar, Jeffrey H Tang, Philipp Warmer, Uwe Sauer, Karsten Weis.

  The ability to tolerate and thrive in diverse environments is paramount to all living organisms, and many organisms spend a large part of their lifetime in starvation. Upon acute glucose starvation, yeast cells undergo drastic physiological and metabolic changes and reestablish a constant-although lower-level of energy production within minutes. The molecules that are rapidly metabolized to fuel energy production under these conditions are unknown. Here, we combine metabolomics and genetics to characterize the cells' response to acute glucose depletion and identify pathways that ensure survival during starvation. We show that the ability to respire is essential for maintaining the energy status and to ensure viability during starvation. Measuring the cells' immediate metabolic response, we find that central metabolites drastically deplete and that the intracellular AMP-to-ATP ratio strongly increases within 20 to 30 s. Furthermore, we detect changes in both amino acid and lipid metabolite levels. Consistent with this, both bulk autophagy, a process that frees amino acids, and lipid degradation via β-oxidation contribute in parallel to energy maintenance upon acute starvation. In addition, both these pathways ensure long-term survival during starvation. Thus, our results identify bulk autophagy and β-oxidation as important energy providers during acute glucose starvation.

Keywords:  Saccharomyces cerevisiae; acute glucose starvation; lipidomics; metabolomics; β-oxidation

DOI:  https://doi.org/10.1073/pnas.1913370117
Biochem Biophys Res Commun. 2020 May 18. pii: S0006-291X(20)30610-0. [Epub ahead of print]

Generation of hepatocyte-like cells from human urinary epithelial cells and the role of autophagy during direct reprogramming.

Huijiao Wu, Cong Du, Fan Yang, Xiaofang Zheng, Dongbo Qiu, Qi Zhang, Wenjie Chen, Yan Xu.

  Somatic cells can be directly reprogrammed into other cell lineages, which holds great promise for regenerative medicine. However, low efficiency and obscure mechanism hinder the application of direct reprogramming. Here, we show that overexpressing the hepatic-specific transcription factors (TFs) HNF1α, FOXA3, and GATA4 was sufficient to convert human urinary epithelial cells (hUCs) into induced hepatocyte-like cells (iHeps). The obtained iHeps were confirmed to express various hepatocyte-specific genes with multiple mature hepatocyte functions. Moreover, autophagy-related genes P62, ULK1, BECN1, VPS34, and LC3B were upregulated in the early stage of reprogramming and knockout of P62 and BECN1 in hUCs with CRISPR/Cas9 technology increased the efficiency of direct reprogramming. Collectively, we established a non-invasive approach to convert hUCs into iHeps and provided a glimpse into the role of autophagy in this process.

Keywords:  Autophagy; Direct reprogramming; Human urinary epithelial cells; Induced hepatocyte-like cells

DOI:  https://doi.org/10.1016/j.bbrc.2020.03.119
Nat Struct Mol Biol. 2020 May 18.

A distinct inhibitory mechanism of the V-ATPase by Vibrio VopQ revealed by cryo-EM.

Wei Peng, Amanda K Casey, Jessie Fernandez, Emily M Carpinone, Kelly A Servage, Zhe Chen, Yang Li, Diana R Tomchick, Vincent J Starai, Kim Orth.

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show that VopQ arrests yeast growth in vivo by targeting the immature Vo subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.

DOI: https://doi.org/10.1038/s41594-020-0429-1
Nat Commun. 2020 May 18. 11(1): 2461

TFEB regulates murine liver cell fate during development and regeneration.

Nunzia Pastore, Tuong Huynh, Niculin J Herz, Alessia Calcagni', Tiemo J Klisch, Lorenzo Brunetti, Kangho Ho Kim, Marco De Giorgi, Ayrea Hurley, Annamaria Carissimo, Margherita Mutarelli, Niya Aleksieva, Luca D'Orsi, William R Lagor, David D Moore, Carmine Settembre, Milton J Finegold, Stuart J Forbes, Andrea Ballabio.

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer.

DOI: https://doi.org/10.1038/s41467-020-16300-x
Biochim Biophys Acta Biomembr. 2020 May 15. pii: S0005-2736(20)30181-4. [Epub ahead of print] 183341

Regulation and function of V-ATPases in physiology and disease.

Michael P Collins, Michael Forgac.

  The vacuolar H+-ATPases (V-ATPases) are essential, ATP-dependent proton pumps present in a variety of eukaryotic cellular membranes. Intracellularly, V-ATPase-dependent acidification functions in such processes as membrane traffic, protein degradation, autophagy and the coupled transport of small molecules. V-ATPases at the plasma membrane of certain specialized cells function in such processes as bone resorption, sperm maturation and urinary acidification. V-ATPases also function in disease processes such as pathogen entry and cancer cell invasiveness, while defects in V-ATPase genes are associated with disorders such as osteopetrosis, renal tubular acidosis and neurodegenerative diseases. This review highlights recent advances in our understanding of V-ATPase structure, mechanism, function and regulation, with an emphasis on the signaling pathways controlling V-ATPase assembly in mammalian cells. The role of V-ATPases in cancer and other human pathologies, and the prospects for therapeutic intervention, are also discussed.

Keywords:  Acidification; Cancer metastasis; Nutrient sensing; Proton transport; Regulated assembly; Vacuolar ATPase

DOI:  https://doi.org/10.1016/j.bbamem.2020.183341
Cell Biosci. 2020 ;10 64

Synergies in exosomes and autophagy pathways for cellular homeostasis and metastasis of tumor cells.

Leila Salimi, Ali Akbari, Nassrollah Jabbari, Behnam Mojarad, Ali Vahhabi, Sławomir Szafert, Sadegh Asghari Kalashani, Hamid Soraya, Muhammad Nawaz, Jafar Rezaie.

  Background: Eukaryotic cells demonstrate two tightly linked vesicular transport systems, comprising intracellular vesicle transport and extracellular vesicle transport system. Intracellular transport vesicles can translocate biomolecules between compartments inside the cell, for example, proteins from the rough endoplasmic reticulum to the Golgi apparatus. Whereas, the secreted vesicles so-called extracellular vesicles facilitate the transport of biomolecules, for example, nucleic acids, proteins and lipids between cells. Vesicles can be formed during the process of endocytosis or/and autophagy and not only act as mediators of intra- and inter-cellular communication but also represent pathological conditions of cells or tissues.Methods: In this review, we searched articles in PubMed, published between 2000 and 2020, with following terms: autophagy, autophagocytosis, transport vesicles, lysosomes, endosomes, exocytosis, exosomes, alone or in different combinations. The biological functions that were selected based on relevancy to our topic include cellular homeostasis and tumorigenesis.
Results: The searched literature shows that there is a high degree of synergies between exosome biogenesis and autophagy, which encompass endocytosis and endosomes, lysosomes, exocytosis and exosomes, autophagocytosis, autophagosomes and amphisomes. These transport systems not only maintain cellular homeostasis but also operate synergically against fluctuations in the external and internal environment such as during tumorigenesis and metastasis. Additionally, exosomal and autophagic proteins may serve as cancer diagnosis approaches.
Conclusion: Exosomal and autophagy pathways play pivotal roles in homeostasis and metastasis of tumor cells. Understanding the crosstalk between endomembrane organelles and vesicular trafficking may expand our insight into cooperative functions of exosomal and autophagy pathways during disease progression and may help to develop effective therapies against lysosomal diseases including cancers and beyond.

Keywords:  Autophagosomes; Autophagy; Autophagy associated tumorigenesis; Autophagy-mediated exosomes; Cancer cell metastasis; Endosomes; Extracellular vesicles

DOI:  https://doi.org/10.1186/s13578-020-00426-y
Int J Mol Sci. 2020 May 14. pii: E3468. [Epub ahead of print]21(10):

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.

Marioara Chiritoiu, Gabriela N Chiritoiu, Cristian V A Munteanu, Florin Pastrama, N Erwin Ivessa, Stefana M Petrescu.

  Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein-protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

Keywords:  EDEM1; EDEM1 interaction network; ER-phagy; ERAD; autophagy; endoplasmic reticulum; intrinsically disordered region; mass spectrometry; protein degradation; protein quality control

DOI:  https://doi.org/10.3390/ijms21103468
Cells. 2020 May 16. pii: E1230. [Epub ahead of print]9(5):

Lactulose and Melibiose Inhibit α-Synuclein Aggregation and Up-Regulate Autophagy to Reduce Neuronal Vulnerability.

Chiung Mei Chen, Chih-Hsin Lin, Yih-Ru Wu, Chien-Yu Yen, Yu-Ting Huang, Jia-Lan Lin, Chung-Yin Lin, Wan-Ling Chen, Chih-Ying Chao, Guey-Jen Lee-Chen, Ming-Tsan Su, Kuo-Hsuan Chang.

  Parkinson's disease (PD) is a neurodegenerative disease characterized by selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra (SN) and proteinaceous α-synuclein-positive Lewy bodies and Lewy neuritis. As a chemical chaperone to promote protein stability and an autophagy inducer to clear aggregate-prone proteins, a disaccharide trehalose has been reported to alleviate neurodegeneration in PD cells and mouse models. Its trehalase-indigestible analogs, lactulose and melibiose, also demonstrated potentials to reduce abnormal protein aggregation in spinocerebellar ataxia cell models. In this study, we showed the potential of lactulose and melibiose to inhibit α-synuclein aggregation using biochemical thioflavin T fluorescence, cryogenic transmission electron microscopy (cryo-TEM) and prokaryotic split Venus complementation assays. Lactulose and melibiose further reduced α-synuclein aggregation and associated oxidative stress, as well as protected cells against α-synuclein-induced neurotoxicity by up-regulating autophagy and nuclear factor, erythroid 2 like 2 (NRF2) pathway in DAergic neurons derived from SH-SY5Y cells over-expressing α-synuclein. Our findings strongly indicate the potential of lactulose and melibiose for mitigating PD neurodegeneration, offering new drug candidates for PD treatment.

Keywords:  Parkinson’s disease; autophagy inducer; lactulose; melibiose; α-synuclein aggregation inhibition

DOI:  https://doi.org/10.3390/cells9051230
Cell Stress. 2020 Apr 24. 4(5): 92-94

Copper - a novel stimulator of autophagy.

Hans Zischka, Guido Kroemer.

  Toxic copper accumulation causes Wilson disease, but trace amounts of copper are required for cellular and organismal survival. In a recent paper Tsang et al. (Nat Cell Biol, doi: 10.1038/s41556-020-0481-4) demonstrate that copper binds with high affinity to a designated interaction site in the pro-autophagic kinases ULK1 and ULK2. Chelation of copper or genetic deletion of this copper-binding site inhibits autophagy and hence reduces the fitness of KRAS-induced cancers. These findings suggest that copper chelation might constitute a novel therapeutic intervention on autophagy-dependent malignancies.

Keywords:  MEK1; ULK1; ULK2; Wilson disease; autophagy; cancer; copper

DOI:  https://doi.org/10.15698/cst2020.05.218
Commun Biol. 2020 May 22. 3(1): 253

Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.

Lena Reimann, Anja N Schwäble, Anna L Fricke, Wignand W D Mühlhäuser, Yvonne Leber, Keerthika Lohanadan, Martin G Puchinger, Sascha Schäuble, Erik Faessler, Heike Wiese, Christa Reichenbach, Bettina Knapp, Christian D Peikert, Friedel Drepper, Udo Hahn, Clemens Kreutz, Peter F M van der Ven, Gerald Radziwill, Kristina Djinović-Carugo, Dieter O Fürst, Bettina Warscheid.

The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig-like domain 20 of FLNc, is doubly phosphorylated. The protein kinases responsible for this dual-site phosphorylation are Akt and PKCα. Proximity proteomics and interaction analysis identified filamin A-interacting protein 1 (FILIP1) as direct FLNc binding partner. FILIP1 binding induces filamin degradation, thereby negatively regulating its function. Here, dual-site phosphorylation of FLNc not only reduces FILIP1 binding, providing a mechanism to shield FLNc from FILIP1-mediated degradation, but also enables fast dynamics of FLNc necessary for its function as signaling adaptor in cross-striated muscle cells.

DOI: https://doi.org/10.1038/s42003-020-0982-5
Exp Biol Med (Maywood). 2020 May 21. 1535370220928276

mTORC1-IRE1α pathway activation contributes to palmitate-elicited triglyceride secretion and cell death in hepatocytes.

Jun Wang, Yingli Chen, Qing Song, Alexandra Griffiths, Zhenyuan Song.

  IMPACT STATEMENT: Lipotoxicity induced by saturated fatty acids (SFA) plays a pivotal role in the pathogenesis of a variety of obesity-related metabolic disorders; however, the exact mechanism(s) underlying lipotoxicity development remains elusive. The liver plays a central role in regulating intrahepatic and circulatory lipid homeostasis. In the current study, we identified that mammalian target of rapamycin complex 1 (mTORC1) activation plays an important role in regulating the detrimental effects of SFA palmitate in hepatocytes, in specific cell death, and TG overproduction. Furthermore, our data confirmed that palmitate-induced mTORC1 activation is attributable to its stimulatory effect on IRE1α, one of three canonical pathways activated during ER stress. Importantly, IRE1α inhibition prevented palmitate-triggered cell death and TG overproduction, suggesting mTORC1-IRE1α pathway is mechanistically implicated in palmitate lipotoxicity. The data obtained in the current investigation support future study to explore the therapeutic potential of targeting the mTORC1-IRE1α pathway as a novel clinical strategy for the treatment of metabolic disorders involving lipotoxicity.

Keywords:  ER stress; IRE1α; Lipotoxicity; SCD-1; mTORC1; palmitate

DOI:  https://doi.org/10.1177/1535370220928276
Front Mol Biosci. 2020 ;7 82

Modeling Lysosomal Storage Diseases in the Zebrafish.

T Zhang, R T Peterson.

  Lysosomal storage diseases (LSDs) are a family of 70 metabolic disorders characterized by mutations in lysosomal proteins that lead to storage material accumulation, multiple-organ pathologies that often involve neurodegeneration, and early mortality in a significant number of patients. Along with the necessity for more effective therapies, there exists an unmet need for further understanding of disease etiology, which could uncover novel pathways and drug targets. Over the past few decades, the growth in knowledge of disease-associated pathways has been facilitated by studies in model organisms, as advancements in mutagenesis techniques markedly improved the efficiency of model generation in mammalian and non-mammalian systems. In this review we highlight non-mammalian models of LSDs, focusing specifically on the zebrafish, a vertebrate model organism that shares remarkable genetic and metabolic similarities with mammals while also conferring unique advantages such as optical transparency and amenability toward high-throughput applications. We examine published zebrafish LSD models and their reported phenotypes, address organism-specific advantages and limitations, and discuss recent technological innovations that could provide potential solutions.

Keywords:  CRISPR-Cas9; Lysosomal storage disease; chemical screening; genetics; metabolism; zebrafish

DOI:  https://doi.org/10.3389/fmolb.2020.00082
Biochem Biophys Res Commun. 2020 May 16. pii: S0006-291X(20)30900-1. [Epub ahead of print]

Autophagy-dependent mitochondrial function regulates osteoclast differentiation and maturation.

Shigeki Aoki, Kengo Shimizu, Kousei Ito.

  Bone homeostasis is maintained by bone remodeling, which involves continuous bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation of bone turnover, caused by osteoclast overactivation, causes destructive bone diseases. However, the mechanisms underlying the maintenance of osteoclast differentiation and activation are unclear. Herein, we examined the role of autophagy in the maintenance of osteoclast differentiation and maturation. We used in vitro and in vivo assays to evaluate relationships between mitochondrial activity and autophagy during osteoclast differentiation and maturation. Our results indicate that autophagy was enhanced during osteoclast differentiation and maturation, and autophagic activity was positively correlated with osteoclast activity and survival. Maintenance of mitochondrial function, which is critical during osteoclast differentiation and maturation, was controlled by autophagy. Continuous exposure of osteoclasts to glucocorticoids upregulated autophagic processes. Treatment with the autophagic inhibitor chloroquine suppressed prolonged survival of activated osteoclasts and attenuated excessive osteoclast activity. Our study shows that autophagy-dependent mitochondrial function plays an important role in osteoclast differentiation and maturation. Elucidating the mechanisms regulating autophagic activity in osteoclasts, and developing bone-tissue-specific inhibitors of autophagy, will lead to improved understanding of the pathologies involved in destructive bone diseases.

Keywords:  Autophagy; Bone remodeling; Mitochondria; Osteoclasts; Osteoporosis; Steroids

DOI:  https://doi.org/10.1016/j.bbrc.2020.04.155
J Biol Chem. 2020 May 19. pii: jbc.RA120.012979. [Epub ahead of print]

The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis.

Xin Jin, Jianling Xie, Michael Zabolocki, Xuemin Wang, Tao Jiang, Dong Wang, Laurent Desaubry, Cedric Bardy, Christopher G Proud.

  Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely due to activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival.

Keywords:  calcium; cancer; cell death; eEF2; endoplasmic reticulum stress (ER stress); eukaryotic initiation factor 2 (eIF2); eukaryotic translation initiation; prohibitin; protein synthesis; translation elongation factor

DOI:  https://doi.org/10.1074/jbc.RA120.012979
Aging Cell. 2020 May 20. e13157

AMPK-mediated formation of stress granules is required for dietary restriction-induced longevity in Caenorhabditis elegans.

Chen-Ting Kuo, Guan-Ting You, Ying-Jie Jian, Ting-Shin Chen, Yu-Chen Siao, Ao-Lin Hsu, Tsui-Ting Ching.

  Stress granules (SGs) are nonmembranous organelles that are dynamically assembled and disassembled in response to various stressors. Under stressed conditions, polyadenylated mRNAs and translation factors are sequestrated in SGs to promote global repression of protein synthesis. It has been previously demonstrated that SG formation enhances cell survival and stress resistance. However, the physiological role of SGs in organismal aging and longevity regulation remains unclear. In this study, we used TIAR-1::GFP and GTBP-1::GFP as markers to monitor the formation of SGs in Caenorhabditis elegans. We found that, in addition to acute heat stress, SG formation could also be triggered by dietary changes, such as starvation and dietary restriction (DR). We found that HSF-1 is required for the SG formation in response to acute heat shock and starvation but not DR, whereas the AMPK-eEF2K signaling is required for starvation and DR-induced SG formation but not heat shock. Moreover, our data suggest that this AMPK-eEF2K pathway-mediated SG formation is required for lifespan extension by DR, but dispensable for the longevity by reduced insulin/IGF-1 signaling. Collectively, our findings unveil a novel role of SG formation in DR-induced longevity.

Keywords:  AMPK; HSF-1; dietary restriction; heat shock; longevity; stress granule

DOI:  https://doi.org/10.1111/acel.13157
J Clin Invest. 2020 May 18. pii: 137411. [Epub ahead of print]

Parkin on serine: a Parkinson disease gene suppresses serine synthesis in cancer.

W Brian Dalton.

Phosphoglycerate dehydrogenase (PHGDH) catalyzes the first step in the synthesis of the amino acid serine, important for protein synthesis, one-carbon metabolism, lipid production, redox homeostasis, and other key processes of normal and cancer metabolism. While PHGDH is often overexpressed in cancer cells, how it is regulated has been unclear. In this issue of the JCI, Liu and colleagues describe a new aspect of PHGDH regulation, demonstrating that the Parkinson disease gene and tumor suppressor Parkin bound and ubiquitinated PHGDH. Parkin promoted PHGDH degradation, suppressed serine synthesis, and inhibited tumor growth in human cancer cell line xenografts. Conversely, inactivation of Parkin not only accelerated tumor growth, but also sensitized tumors to small molecule inhibitors of PHGDH. These results offer a new link between Parkin and the serine synthesis pathway, and they bear translational potential that warrants further study in Parkin-deficient human cancers.

DOI: https://doi.org/10.1172/JCI137411
Cells. 2020 May 18. pii: E1249. [Epub ahead of print]9(5):

A New Pathway Promotes Adaptation of Human Glioblastoma Cells to Glucose Starvation.

Alberto Azzalin, Francesca Brambilla, Eloisa Arbustini, Katia Basello, Attilio Speciani, Pierluigi Mauri, Paola Bezzi, Lorenzo Magrassi.

  Adaptation of glioblastoma to caloric restriction induces compensatory changes in tumor metabolism that are incompletely known. Here we show that in human glioblastoma cells maintained in exhausted medium, SHC adaptor protein 3 (SHC3) increases due to down-regulation of SHC3 protein degradation. This effect is reversed by glucose addition and is not present in normal astrocytes. Increased SHC3 levels are associated to increased glucose uptake mediated by changes in membrane trafficking of glucose transporters of the solute carrier 2A superfamily (GLUT/SLC2A). We found that the effects on vesicle trafficking are mediated by SHC3 interactions with adaptor protein complex 1 and 2 (AP), BMP-2-inducible protein kinase and a fraction of poly ADP-ribose polymerase 1 (PARP1) associated to vesicles containing GLUT/SLC2As. In glioblastoma cells, PARP1 inhibitor veliparib mimics glucose starvation in enhancing glucose uptake. Furthermore, cytosol extracted from glioblastoma cells inhibits PARP1 enzymatic activity in vitro while immunodepletion of SHC3 from the cytosol significantly relieves this inhibition. The identification of a new pathway controlling glucose uptake in high grade gliomas represents an opportunity for repositioning existing drugs and designing new ones.

Keywords:  GLUT/SLC2A; PARP1; SHC3; aerobic glycolysis; glioblastoma cells

DOI:  https://doi.org/10.3390/cells9051249
Life Sci. 2020 May 13. pii: S0024-3205(20)30542-7. [Epub ahead of print] 117793

Compound K inhibits autophagy-mediated apoptosis induced by oxygen and glucose deprivation/reperfusion via regulating AMPK-mTOR pathway in neurons.

Qingxia Huang, Tingting Lou, Manying Wang, Linyuan Xue, Jing Lu, He Zhang, Zepeng Zhang, Hui Wang, Chenxu Jing, Daqing Zhao, Liwei Sun, Xiangyan Li.

  AIMS: Oxygen and glucose deprivation and reperfusion (OGD/R) injury contributes to the pathophysiology after ischemic stroke, which needs to urgently develop treatment strategies. Previous studies have demonstrated that autophagy in reperfusion period exerted adverse effects on the cerebral ischemic injury. Ginsenoside monomer compound K (CK) is the main intestinal metabolite of ginseng that exerts the pharmacological activities and has a protective effect against cerebral OGD/R injury. However, the specific molecular mechanism of CK protects against OGD/R injury in neurons is still unclear.MATERIALS AND METHODS: In this study, cell viability, reactive oxygen species (ROS) generation, Ca2+ overload, mitochondrial membrane potential depolarization, autophagy and apoptosis were investigated in OGD/R-induced neuronal cells injury after pretreatment with CK and in combination with BML-275 or rapamycin.
KEY FINDINGS: Our study found that pretreatment with CK protected neurons against OGD/R injury by increasing cell viability and decreasing the ROS generation, mitochondrial damage, and Ca2+ overload. Moreover, CK cut down autophagy-mediated apoptosis via promoting the process of forming autophagosomes into phagocytic precursors. Furthermore, our study clarified the neuroprotective of CK against OGD/R-induced neural autophagy and apoptosis through the regulation of the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway.
SIGNIFICANCE: Taken together, our study provides credible experimental evidence and explains the potential molecular mechanism of CK as one of the main bioactive ingredients of ginseng for the treatment of cerebral ischemia/reperfusion injury.

Keywords:  AMPK-mTOR pathway; Autophagy; Compound K; Neuron; Oxygen and glucose deprivation/reperfusion

DOI:  https://doi.org/10.1016/j.lfs.2020.117793
Proc Natl Acad Sci U S A. 2020 May 18. pii: 201919528. [Epub ahead of print]

Split-TurboID enables contact-dependent proximity labeling in cells.

Kelvin F Cho, Tess C Branon, Sanjana Rajeev, Tanya Svinkina, Namrata D Udeshi, Themis Thoudam, Chulhwan Kwak, Hyun-Woo Rhee, In-Kyu Lee, Steven A Carr, Alice Y Ting.

  Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.

Keywords:  ER–mitochondria contacts; proximity labeling; split-TurboID

DOI:  https://doi.org/10.1073/pnas.1919528117
Neurosci Res. 2020 May 14. pii: S0168-0102(20)30309-6. [Epub ahead of print]

PINK1 and Parkin: the odd couple.

Edgar Djaha Yoboue, Enza Maria Valente.

  In 2004, PINK1 was established as a gene linked to early onset of autosomal recessive juvenile Parkinsonism. Since then, tremendous efforts allowed involving the gene product in diverse events but with a strong focus on its partnership with the protein Parkin for the degradation of damaged mitochondria through mitophagy. Yet, it is now clear that the importance of PINK1 encompasses a wider spectrum of intracellular processes. In this minireview, we highlight some of the PINK1 interplays and recent advances, including its growing involvement in immunity and also its emerging place in this era of mitochondria-organelles contact sites.

Keywords:  MAMs; PINK1; Parkin; Parkinson; mitochondria; mitophagy; ubiquitin

DOI:  https://doi.org/10.1016/j.neures.2020.04.007
Eur J Med Chem. 2020 May 04. pii: S0223-5234(20)30361-5. [Epub ahead of print]199 112391

Targeting mTOR for fighting diseases: A revisited review of mTOR inhibitors.

Tian Xu, Dejuan Sun, Yi Chen, Liang Ouyang.

  mTOR (mammalian target of rapamycin), which is a serine/threonine kinase, has been well-established as being closely correlated with the occurrence of various human diseases, such as tumors and neurodegenerative diseases. Inhibition of the mTOR signaling pathways may effectively block the abnormal signal transduction of various growth factors and thereby block the occurrence and development of diseases. Of note, first-generation mTOR inhibitors are mainly reported to be rapamycin and its derivatives and second-generation mTOR inhibitors that consist of several ATP-competitive kinase inhibitors. Interestingly, the third-generation mTOR inhibitor, RapaLink-1, mediates rapamycin and mTOR kinase inhibitors via the same molecule and shows much higher efficiency. In addition, there are many mTOR inhibitors that have potential therapeutic effects for tumors and other types of diseases. Thus, we focus on summarizing the basic structures of mTOR and its complexes, some key upstream and downstream signaling pathways, and the structures and characteristics of the three generations of mTOR inhibitors in disease. Together, these findings may provide a better understanding of mTOR-regulated mechanisms and their inhibitors for fighting human diseases in the near future.

Keywords:  Human disease; Rapamycin; mTOR; mTOR inhibitor; mTOR pathway

DOI:  https://doi.org/10.1016/j.ejmech.2020.112391