bims-auttor 2020-04-05 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2020–04–05
eleven papers selected by
Viktor Korolchuk, Newcastle University

The trehalose-6-phosphate phosphatase Tps2 regulates ATG8 transcription and autophagy in Saccharomyces cerevisiae.
Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites.
Mitophagy in the Pathogenesis of Liver Diseases.
PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72.
Mitochondrial fragmentation enables localized signaling required for cell repair.
Autophagy Regulation by the Translation Machinery and Its Implications in Cancer.
The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.
Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.
Emerging Role of the Autophagy/Lysosomal Degradative Pathway in Neurodevelopmental Disorders With Epilepsy.
Proteome and phosphoproteome analysis of brown adipocytes reveals that RICTOR loss dampens global insulin/AKT signaling.
Role and Mechanisms of Mitophagy in Liver Diseases.

Autophagy. 2020 Apr 02. 1-15

The trehalose-6-phosphate phosphatase Tps2 regulates ATG8 transcription and autophagy in Saccharomyces cerevisiae.

Bongkeun Kim, Yongook Lee, Hyojeong Choi, Won-Ki Huh.

  Macroautophagy/autophagy is an important catabolic process for maintaining cellular homeostasis by adapting to various stress conditions. Autophagy is mediated by a double-membrane autophagosome, which sequesters a portion of cytoplasmic components for delivery to the vacuole. Several autophagy-related (ATG) genes play crucial roles in autophagosome formation. The induction of ATG genes must be tightly regulated to maintain a proper autophagic activity, but their regulatory mechanisms are still largely unknown. Here, we report that the trehalose-6-phosphate phosphatase Tps2 functions as a positive regulator of autophagy in Saccharomyces cerevisiae. Cellular trehalose levels do not affect autophagy regulation by Tps2. Loss of Tps2 leads to impaired autophagic flux and reduced ATG8 expression under nitrogen starvation. In tps2Δ cells, Ume6 is predominantly dephosphorylated and represses ATG8 transcription by binding to its promoter region. Tps2 regulates nuclear translocation and activation of Rim15 kinase, a negative regulator of Ume6, by causing the dissociation of Rim15 from the 14-3-3 proteins Bmh1/2 under nitrogen starvation, suggesting that Rim15 mediates the function of Tps2 as a positive regulator of ATG8 induction. Furthermore, Tps2 plays a crucial role in the dephosphorylation of Ser1061 and Thr1075 residues of Rim15, which is important for controlling the dissociation of Rim15 from Bmh1/2 under nitrogen starvation. Together, our results reveal the role of Tps2 as a positive regulator of autophagy and provide new insight into the regulatory mechanisms of ATG gene expression.Abbreviations: ATG: autophagy-related; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; GFP: green fluorescent protein; PKA: protein kinase A; PtdIns3K: phosphatidylinositol 3-kinase; Rim15KI: kinase-inactive Rim15; Rim15-2A: Rim15S1061A,T1075A; TEM: transmission electron microscopy; TORC1: target of rapamycin complex 1.

Keywords:  ATG8; Rim15; Saccharomyces cerevisiae; Tps2; Ume6; autophagy; nitrogen starvation; trehalose

DOI:  https://doi.org/10.1080/15548627.2020.1746592
Nat Commun. 2020 Apr 03. 11(1): 1684

Kinase inhibition profiles as a tool to identify kinases for specific phosphorylation sites.

Nikolaus A Watson, Tyrell N Cartwright, Conor Lawless, Marcos Cámara-Donoso, Onur Sen, Kosuke Sako, Toru Hirota, Hiroshi Kimura, Jonathan M G Higgins.

There are thousands of known cellular phosphorylation sites, but the paucity of ways to identify kinases for particular phosphorylation events remains a major roadblock for understanding kinase signaling. To address this, we here develop a generally applicable method that exploits the large number of kinase inhibitors that have been profiled on near-kinome-wide panels of protein kinases. The inhibition profile for each kinase provides a fingerprint that allows identification of unknown kinases acting on target phosphosites in cell extracts. We validate the method on diverse known kinase-phosphosite pairs, including histone kinases, EGFR autophosphorylation, and Integrin β1 phosphorylation by Src-family kinases. We also use our approach to identify the previously unknown kinases responsible for phosphorylation of INCENP at a site within a commonly phosphorylated motif in mitosis (a non-canonical target of Cyclin B-Cdk1), and of BCL9L at S915 (PKA). We show that the method has clear advantages over in silico and genetic screening.

DOI: https://doi.org/10.1038/s41467-020-15428-0
Cells. 2020 Mar 30. pii: E831. [Epub ahead of print]9(4):

Mitophagy in the Pathogenesis of Liver Diseases.

Po-Yuan Ke.

  Autophagy is a catabolic process involving vacuolar sequestration of intracellular components and their targeting to lysosomes for degradation, thus supporting nutrient recycling and energy regeneration. Accumulating evidence indicates that in addition to being a bulk, nonselective degradation mechanism, autophagy may selectively eliminate damaged mitochondria to promote mitochondrial turnover, a process termed "mitophagy". Mitophagy sequesters dysfunctional mitochondria via ubiquitination and cargo receptor recognition and has emerged as an important event in the regulation of liver physiology. Recent studies have shown that mitophagy may participate in the pathogenesis of various liver diseases, such as liver injury, liver steatosis/fatty liver disease, hepatocellular carcinoma, viral hepatitis, and hepatic fibrosis. This review summarizes the current knowledge on the molecular regulations and functions of mitophagy in liver physiology and the roles of mitophagy in the development of liver-related diseases. Furthermore, the therapeutic implications of targeting hepatic mitophagy to design a new strategy to cure liver diseases are discussed.

Keywords:  autophagy; fibrosis; hepatitis; hepatocellular carcinoma; liver disease; liver injury; mitophagy; steatosis

DOI:  https://doi.org/10.3390/cells9040831
Biochem J. 2020 Mar 30. pii: BCJ20190664. [Epub ahead of print]

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72.

Sophie Vieweg, Katie Mulholland, Bastian Brauning, Nitin Kacharia, Yu-Chiang Lai, Rachel Toth, Pawan K Singh, Ilaria Volpi, Michael Sattler, Michael Groll, Aymelt Itzen, Miratul M K Muqit.

  Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson's disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2 in vitro. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 of recombinant nucleotide-bound Rab8A in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in transfected HEK293 cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B. The structures reveal that the phosphorylated SF3 motif does not induce any major changes, but may interfere with effector-Switch II interactions through intramolecular H-bond formation and/or charge effects with Arg79. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A indicating a potential crosstalk between PINK1-regulated mitochondrial homeostasis and LRRK2 signalling that requires further investigation in vivo.

Keywords:  GTPASE; LRRK2; MST3; PINK1; RAB; TAK1

DOI:  https://doi.org/10.1042/BCJ20190664
J Cell Biol. 2020 May 04. pii: e201909154. [Epub ahead of print]219(5):

Mitochondrial fragmentation enables localized signaling required for cell repair.

Adam Horn, Shreya Raavicharla, Sonna Shah, Dan Cox, Jyoti K Jaiswal.

Plasma membrane injury can cause lethal influx of calcium, but cells survive by mounting a polarized repair response targeted to the wound site. Mitochondrial signaling within seconds after injury enables this response. However, as mitochondria are distributed throughout the cell in an interconnected network, it is unclear how they generate a spatially restricted signal to repair the plasma membrane wound. Here we show that calcium influx and Drp1-mediated, rapid mitochondrial fission at the injury site help polarize the repair response. Fission of injury-proximal mitochondria allows for greater amplitude and duration of calcium increase in these mitochondria, allowing them to generate local redox signaling required for plasma membrane repair. Drp1 knockout cells and patient cells lacking the Drp1 adaptor protein MiD49 fail to undergo injury-triggered mitochondrial fission, preventing polarized mitochondrial calcium increase and plasma membrane repair. Although mitochondrial fission is considered to be an indicator of cell damage and death, our findings identify that mitochondrial fission generates localized signaling required for cell survival.

DOI: https://doi.org/10.1083/jcb.201909154
Front Oncol. 2020 ;10 322

Autophagy Regulation by the Translation Machinery and Its Implications in Cancer.

Pilar Sarah Acevo-Rodríguez, Giovanna Maldonado, Susana Castro-Obregón, Greco Hernández.

  Various metabolic pathways and molecular processes in the cell act intertwined, and dysregulating the interplay between some of them may lead to cancer. It is only recently that defects in the translation process, i.e., the synthesis of proteins by the ribosome using a messenger (m)RNA as a template and translation factors, have begun to gain strong attention as a cause of autophagy dysregulation with effects in different maladies, including cancer. Autophagy is an evolutionarily conserved catabolic process that degrades cytoplasmic elements in lysosomes. It maintains cellular homeostasis and preserves cell viability under various stress conditions, which is crucial for all eukaryotic cells. In this review, we discuss recent advances shedding light on the crosstalk between the translation and the autophagy machineries and its impact on tumorigenesis. We also summarize how this interaction is being the target for novel therapies to treat cancer.

Keywords:  ATG; PERK; autophagy; cancer; eIF2alpha; endoplasmic reticulum; mTOR; translation initiation

DOI:  https://doi.org/10.3389/fonc.2020.00322
Neuron. 2020 Mar 23. pii: S0896-6273(20)30191-4. [Epub ahead of print]

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

Valeria Gerbino, Esther Kaunga, Junqiang Ye, Daniele Canzio, Sean O'Keeffe, Noam D Rudnick, Paolo Guarnieri, Cathleen M Lutz, Tom Maniatis.

  DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.

Keywords:  ALS; Amyotrophic Lateral Sclerosis; SOD1 mouse model; TBK1; glia; interferon response; motor neuron autophagy; neuroinflammation; selective autophagy

DOI:  https://doi.org/10.1016/j.neuron.2020.03.005
Cells. 2020 Mar 27. pii: E815. [Epub ahead of print]9(4):

Redox Modifications of Proteins of the Mitochondrial Fusion and Fission Machinery.

Christina Wolf, Víctor López Del Amo, Sabine Arndt, Diones Bueno, Stefan Tenzer, Eva-Maria Hanschmann, Carsten Berndt, Axel Methner.

  Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins that are conserved in phylogenetically distant organisms and which represent potential sites of posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin, which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1 are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.

Keywords:  fission; fusion; metabolism; mitochondria; redox; thiol switch

DOI:  https://doi.org/10.3390/cells9040815
Front Cell Neurosci. 2020 ;14 39

Emerging Role of the Autophagy/Lysosomal Degradative Pathway in Neurodevelopmental Disorders With Epilepsy.

Anna Fassio, Antonio Falace, Alessandro Esposito, Davide Aprile, Renzo Guerrini, Fabio Benfenati.

  Autophagy is a highly conserved degradative process that conveys dysfunctional proteins, lipids, and organelles to lysosomes for degradation. The post-mitotic nature, complex and highly polarized morphology, and high degree of specialization of neurons make an efficient autophagy essential for their homeostasis and survival. Dysfunctional autophagy occurs in aging and neurodegenerative diseases, and autophagy at synaptic sites seems to play a crucial role in neurodegeneration. Moreover, a role of autophagy is emerging for neural development, synaptogenesis, and the establishment of a correct connectivity. Thus, it is not surprising that defective autophagy has been demonstrated in a spectrum of neurodevelopmental disorders, often associated with early-onset epilepsy. Here, we discuss the multiple roles of autophagy in neurons and the recent experimental evidence linking neurodevelopmental disorders with epilepsy to genes coding for autophagic/lysosomal system-related proteins and envisage possible pathophysiological mechanisms ranging from synaptic dysfunction to neuronal death.

Keywords:  autophagy; epilepsy; lysosome; neuron development; synapse

DOI:  https://doi.org/10.3389/fncel.2020.00039
Mol Cell Proteomics. 2020 Mar 31. pii: mcp.RA120.001946. [Epub ahead of print]

Proteome and phosphoproteome analysis of brown adipocytes reveals that RICTOR loss dampens global insulin/AKT signaling.

Samuel W Entwisle, Camila Martinez Calejman, Anthony S Valente, Robert T Lawrence, Chien-Min Hung, David A Guertin, Judit Villen.

  Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-minute time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation due to chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.

Keywords:  Phosphoproteome; Phosphorylation; Signal Transduction*; Signaling Circuits*; Targeted mass spectrometry; adipocytes; insulin signaling; mTOR

DOI:  https://doi.org/10.1074/mcp.RA120.001946
Cells. 2020 Mar 31. pii: E837. [Epub ahead of print]9(4):

Role and Mechanisms of Mitophagy in Liver Diseases.

Xiaowen Ma, Tara McKeen, Jianhua Zhang, Wen-Xing Ding.

  The mitochondrion is an organelle that plays a vital role in the regulation of hepatic cellular redox, lipid metabolism, and cell death. Mitochondrial dysfunction is associated with both acute and chronic liver diseases with emerging evidence indicating that mitophagy, a selective form of autophagy for damaged/excessive mitochondria, plays a key role in the liver's physiology and pathophysiology. This review will focus on mitochondrial dynamics, mitophagy regulation, and their roles in various liver diseases (alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, hepatic ischemia-reperfusion injury, viral hepatitis, and cancer) with the hope that a better understanding of the molecular events and signaling pathways in mitophagy regulation will help identify promising targets for the future treatment of liver diseases.

Keywords:  NAFLD; Parkin; Pink1; alcohol; autophagy; mitochondria

DOI:  https://doi.org/10.3390/cells9040837