bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒03‒08
nineteen papers selected by
Viktor Korolchuk
Newcastle University

  1. Semin Cell Dev Biol. 2020 Feb 28. pii: S1084-9521(19)30136-3. [Epub ahead of print]
      The mechanistic (or mammalian) Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism. By integrating mitogenic signals, mTORC1-dependent phosphorylation of substrates dictates the balance between anabolic, pro-growth and catabolic, recycling processes in the cell. The discovery that amino acids activate mTORC1 by promoting its translocation to the lysosome was a fundamental advance in the understanding of mTORC1 signalling. It has since become clear that the lysosome-cytoplasm shuttling of mTORC1 represents just one layer of spatial control of this signalling pathway. This review will focus on exploring the subcellular localisation of mTORC1 and its regulators to multiple sites within the cell. We will discuss how these spatially distinct regions such as endoplasmic reticulum, plasma membrane and the endosomal pathway co-operate to transduce nutrient availability to mTORC1, allowing for tight control of cell growth.
    Keywords:  Amino acids; Autophagy; Endoplasmic reticulum; Endosome; Golgi; Lysosome; Rheb; Trafficking
  2. Nat Metab. 2020 Jan;2(1): 110-125
      High protein diets are commonly utilized for weight loss, yet have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.
  3. Mol Cell. 2020 Mar 05. pii: S1097-2765(19)30842-1. [Epub ahead of print]77(5): 1124-1142.e10
      The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.
    Keywords:  Parkin; USP30; Ub-clipping; deubiquitylating enzyme; mitophagyĆ; p97; quantitative proteomics; ubiquitin; ubiquitin ligase
  4. Autophagy. 2020 Mar 06.
      Oxidative stress underlies a number of pathological conditions, including cancer, neurodegeneration, and aging. Antioxidant-rich foods help maintain cellular redox homeostasis and mitigate oxidative stress, but the underlying mechanisms are not clear. For example, sulforaphane (SFN), an electrophilic compound that is enriched in cruciferous vegetables such as broccoli, is a potent inducer of cellular antioxidant responses. NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2), a transcriptional factor that controls the expression of multiple detoxifying enzymes through antioxidant response elements (AREs), is a proposed target of SFN. NFE2L2/NRF2 is a target gene of TFEB (transcription factor EB), a master regulator of autophagic and lysosomal functions, which we show here to be potently activated by SFN. SFN induces TFEB nuclear translocation via a Ca2+-dependent but MTOR (mechanistic target of rapamycin kinase)-independent mechanism through a moderate increase in reactive oxygen species (ROS). Activated TFEB then boosts the expression of genes required for autophagosome and lysosome biogenesis, which are known to facilitate the clearance of damaged mitochondria. Notably, TFEB activity is required for SFN-induced protection against both acute oxidant bursts and chronic oxidative stress. Hence, by simultaneously activating macroautophagy/autophagy and detoxifying pathways, natural compound SFN may trigger a self-defense cellular mechanism that can effectively mitigate oxidative stress commonly associated with many metabolic and age-related diseases.
    Keywords:  NFE2L2/NRF2; ROS; TFEB; autophagy; lysosome; sulforaphane
  5. Autophagy. 2020 Mar 04. 1-3
      Damaged mitochondria are selectively removed from the cell in a process termed mitophagy. This mitochondrial quality control mechanism is important for neuronal homeostasis, and mutations in pathway components are causative for Parkinson disease and amyotrophic lateral sclerosis (ALS). Here, we discuss our recent work using a novel mild induction paradigm to investigate the spatiotemporal dynamics of mitophagy in primary neurons. Using live-cell imaging, we find that mitophagy-associated proteins translocate to depolarized mitochondrial fragments. These mitophagic events were primarily localized to somatodendritic compartments, suggesting neuronal mitophagy is primarily a somal quality control mechanism. Damaged mitochondria were efficiently sequestered within autophagosomes, but lysosomal fusion or acidification was significantly delayed. Surprisingly, engulfed mitochondria persisted in non-acidified vesicular compartments for hours to days after initial damage. Expression of an ALS-associated mutation disrupted the membrane potential of the mitochondrial network, and oxidative stress exacerbated this effect. Importantly, our results highlight the slow kinetics of mitophagy and suggest that slow turnover of damaged mitochondria may increase neuronal susceptibility to neurodegeneration.
    Keywords:  Autophagy; lysosome; mitochondria; mitophagy; neurodegenerative diseases
  6. Cells. 2020 Feb 28. pii: E571. [Epub ahead of print]9(3):
      Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.
    Keywords:  C9orf72; SOD1; familial ALS; glutamate toxicity; induced pluripotent stem cells; protein aggregation; sporadic ALS
  7. J Mol Biol. 2020 Feb 28. pii: S0022-2836(20)30191-1. [Epub ahead of print]
      Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) constitute aggressive neurodegenerative pathologies that lead to the progressive degeneration of upper and lower motor neurons and of neocortical areas respectively. In the past decade, the identification of several genes that cause these disorders indicated that the two diseases overlap in a multifaceted spectrum of conditions. The autophagy-lysosome system has been identified as a main intersection for the onset and progression of neurodegeneration in ALS/FTD. Genetic evidence has revealed that several genes with a mechanistic role at different stages of the autophagy process are mutated in ALS/FTD patients. Moreover, the three main proteins aggregating in ALS/FTD, including in sporadic cases, are also targeted by autophagy and affect this process. Here, we examine the varied dysfunctions and degrees of involvement of the autophagy-lysosome system that have been discovered in ALS/FTD. We argue that these findings shed light on the pathological mechanisms in the ALS/FTD spectrum, and conclude that they have important consequences both for treatment options and for the basic bio-molecular understanding of how this process intersects with RNA metabolism, the other major cellular process reported to be dysfunctional in part of the ALS/FTD spectrum.
    Keywords:  ALS; Autophagy; FTD; TDP-43
  8. FEBS J. 2020 Mar 04.
      Autophagy plays a role in several human diseases, but each of the current methods to measure autophagy have significant drawbacks. ATG5 and ATG16L1 are regulators necessary for autophagy therefore, drugs which inhibit the interaction of these proteins may be therapeutically useful. To evaluate the interaction of ATG5 and ATG16L1 in cells, their cDNAs were fused to the coding sequences of SmBIT and LgBIT, two components of Nanoluc luciferase. This generated a luminescent signal when SmBIT and LgBIT interacted to form a functional luciferase as a result of their co-localization which was brought about by the binding of ATG5 and ATG16L1. The assay measures the interaction in real time and can be used in microplate format to allow for multiple experimental conditions to be assessed. The interaction of ATG5 and ATG16L1 is not significantly altered by inhibition of lysosomal function, or inhibitors of Ulk1, vps34 or mTORC1. Although there was constitutive interaction of ATG5 and ATG16L1 and luminescence was stimulated within 3 minutes, by up to 500%, when the cells are deprived of nutrients. When the nutrients are returned, the complex returns to its basal status equally rapidly. Sphingosine-1-Phosphate and CYM-5541 partially repressed the effects of nutrient starvation. Furthermore, we identified a small molecule inhibitor that interferes with the interaction of ATG5 and ATG16L1 in cells. This assay provides a novel tool for researchers to measure autophagy and can be potentially applied to many cell types.
    Keywords:  ATG16L1; ATG5; Autophagy; NanoBIT
  9. Cells. 2020 Mar 03. pii: E598. [Epub ahead of print]9(3):
      Mitochondrial dysfunction and failing mitochondrial quality control (MQC) are major determinants of aging. Far from being standalone organelles, mitochondria are intricately related with cellular other compartments, including lysosomes. The intimate relationship between mitochondria and lysosomes is reflected by the fact that lysosomal degradation of dysfunctional mitochondria is the final step of mitophagy. Inter-organelle membrane contact sites also allow bidirectional communication between mitochondria and lysosomes as part of nondegradative pathways. This interaction establishes a functional unit that regulates metabolic signaling, mitochondrial dynamics, and, hence, MQC. Contacts of mitochondria with the endoplasmic reticulum (ER) have also been described. ER-mitochondrial interactions are relevant to Ca2+ homeostasis, transfer of phospholipid precursors to mitochondria, and integration of apoptotic signaling. Many proteins involved in mitochondrial contact sites with other organelles also participate to degradative MQC pathways. Hence, a comprehensive assessment of mitochondrial dysfunction during aging requires a thorough evaluation of degradative and nondegradative inter-organelle pathways. Here, we present a geroscience overview on (1) degradative MQC pathways, (2) nondegradative processes involving inter-organelle tethering, (3) age-related changes in inter-organelle degradative and nondegradative pathways, and (4) relevance of MQC failure to inflammaging and age-related conditions, with a focus on Parkinson's disease as a prototypical geroscience condition.
    Keywords:  biomarkers; exosomes; extracellular vesicles; geroprotective interventions; mitochondrial damage; mitochondrial dynamics; mitochondrial-derived vesicles; mitochondrial-lysosomal axis; mitophagy; neurodegeneration
  10. Sci Adv. 2020 02;6(8): eaay5154
      By hijacking endogenous E3 ligase to degrade protein targets via the ubiquitin-proteasome system, PROTACs (PRoteolysis TArgeting Chimeras) provide a new strategy to inhibit protein targets that were regarded as undruggable before. However, the catalytic nature of PROTAC potentially leads to uncontrolled degradation that causes systemic toxicity issues, limiting the application of PROTAC in the clinic. Here, we introduce a light-inducible switch on PROTACs, thereafter termed as opto-PROTAC, to enable the degradation of protein targets in a spatiotemporal manner. By adding a photolabile caging group on pomalidomide as a parental compound and two additional PROTACs, dBET1 and dALK, we demonstrated light-inducible protein degradation. These opto-PROTACs display no activities in the dark, while the restricted degradation can be induced at a specific time and rate by ultraviolet A irradiation. Our approach provides a generalizable platform for the development of light-controlled PROTACs and enables PROTAC to be a precision medicine.
  11. Front Mol Neurosci. 2020 ;13 19
      TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver's CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain's CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.
    Keywords:  TARDBP; amyotrophic lateral sclerosis; cellular model; chaperone mediated autophagy; lysosomal damage; protein aggregation
  12. Mol Cell. 2020 Mar 05. pii: S1097-2765(20)30105-2. [Epub ahead of print]77(5): 1107-1123.e10
      Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.
    Keywords:  Deubiquitinase; E3 Ubiquitin Ligase; Mitochondria; Mitochondrial Import; TOM complex; Ubiquitin System
  13. Front Cardiovasc Med. 2020 ;7 18
      Aging is associated with structural and functional changes in the heart and is a major risk factor in developing cardiovascular disease. Many recent studies have focused on increasing our understanding of the basis of aging at the cellular and molecular levels in various tissues, including the heart. It is known that there is an age-related decline in cellular quality control pathways such as autophagy and mitophagy, which leads to accumulation of potentially harmful cellular components in cardiac myocytes. There is evidence that diminished autophagy and mitophagy accelerate the aging process, while enhancement preserves cardiac homeostasis and extends life span. Here, we review the current knowledge of autophagy and mitophagy in aging and discuss how age-associated alterations in these processes contribute to cardiac aging and age-related cardiovascular diseases.
    Keywords:  PINK1; Parkin; aging; autophagy; heart; mitochondria; mitophagy; mitophagy receptors
  14. Biotechnol J. 2020 Mar 06. e1900267
      Imbalance between the main intracellular degradative, trafficking and intercellular shuttling pathways has been implicated in disease pathogenesis. Autophagy controls degradation of cellular components, while vesicular trafficking permits transport of material in and out of the cell. Emerging evidence has uncovered the extensive interconnectivity between these pathways, which is crucial to maintain organismal homeostasis. Thus, therapeutic intervention and drug development strategies targeting these processes, particularly in neurodegeneration, should account for this broad crosstalk, to maximize effectiveness. Here, we review recent findings underlining the highly dynamic nature of the crosstalk between autophagy, endosomal transport and secretion. Synergy of autophagy and endosomes for degradation, as well as, competition of autophagy and secretion are discussed. Perturbation of this crosstalk triggers pathology especially neurodegeneration. This article is protected by copyright. All rights reserved.
    Keywords:  Autophagy; Endosome; Exocytosis; Neurodegenerative disorder; Secretion
  15. Elife. 2020 Mar 02. pii: e51065. [Epub ahead of print]9
      Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly-defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae. The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HAts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires SSA family HSP70s and the HSP40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways.
    Keywords:  S. cerevisiae; cell biology
  16. Elife. 2020 Mar 03. pii: e51440. [Epub ahead of print]9
      Although mTOR signaling is known as a broad regulator of cell growth and proliferation, in neurons it regulates synaptic transmission, which is thought to be a major mechanism through which altered mTOR signaling leads to neurological disease. Although previous studies have delineated postsynaptic roles for mTOR, whether it regulates presynaptic function is largely unknown. Moreover, the mTOR kinase operates in two complexes, mTORC1 and mTORC2, suggesting that mTOR's role in synaptic transmission may be complex-specific. To better understand their roles in synaptic transmission, we genetically inactivated mTORC1 or mTORC2 in cultured mouse glutamatergic hippocampal neurons. Inactivation of either complex reduced neuron growth and evoked EPSCs (eEPSCs), however, the effects of mTORC1 on eEPSCs were postsynaptic and the effects of mTORC2 were presynaptic. Despite postsynaptic inhibition of evoked release, mTORC1 inactivation enhanced spontaneous vesicle fusion and replenishment, suggesting that mTORC1 and mTORC2 differentially modulate postsynaptic responsiveness and presynaptic release to optimize glutamatergic synaptic transmission.
    Keywords:  molecular biophysics; mouse; neuroscience; structural biology
  17. Traffic. 2020 Mar 06.
      The human Niemann-Pick C1 (NPC1) gene encoding a 1278 AMino acid protein is very heterogeneous. While some variants represent benign polymorphisms, NPC disease carriers and patients may possess rare variants, whose functional importance remains unknown. An NPC1 cDNA construct known as NPC1 wild-type variant (WT-V), distributed between laboratories and used as a WT control in several studies, also contains changes regarding specific amino acids compared to the NPC1 Genbank reference sequence. To improve the dissection of subtle functional differences, we generated human cells stably expressing NPC1 variants from the AAVS1 safe-harbor locus on an NPC1-null background engineered by CRISPR/Cas9 editing. We then employed high-content imaging with automated image analysis to quantitatively assess LDL-induced, time-dependent changes in lysosomal cholesterol content and lipid droplet formation. Our results indicate that the L472P change present in NPC1 WT-V compromises NPC1 functionality in lysosomal cholesterol export. All-atom molecular dynamics simulations suggest that the L472P change alters the relative position of the NPC1 middle and the C-terminal luminal domains, disrupting the recently characterized cholesterol efflux tunnel. These results reveal functional defects in NPC1 WT-V and highlight the strength of simulations and quantitative imaging upon stable protein expression in elucidating subtle differences in protein function. This article is protected by copyright. All rights reserved.
    Keywords:  Niemann-Pick C1; cholesterol transport; gene variants; late endosomes; lipid droplets; lysosomal storage diseases
  18. EMBO Rep. 2020 Mar 05. e48791
      PI3K-Akt-FoxO-mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K-Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63-linked polyubiquitin chains on Akt to restrict PI3K-Akt-FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K-Akt-FoxO signaling, and glucose uptake during fasting. Co-immunoprecipitation and mass spectrometry identified disabled homolog-2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1-TSC1-PHLPP1 complex, and for PI3K-Akt-FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR-mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1-TSC1-PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low.
    Keywords:  Dab2; PI3K-Akt signaling; USP1; fasting; muscle atrophy
  19. Neuron. 2020 Feb 18. pii: S0896-6273(20)30065-9. [Epub ahead of print]
      Autism is characterized by repetitive behaviors, impaired social interactions, and communication deficits. It is a prevalent neurodevelopmental disorder, and available treatments offer little benefit. Here, we show that genetically reducing the protein tau prevents behavioral signs of autism in two mouse models simulating distinct causes of this condition. Similar to a proportion of people with autism, both models have epilepsy, abnormally enlarged brains, and overactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B)/ mammalian target of rapamycin (mTOR) signaling pathway. All of these abnormalities were prevented or markedly diminished by partial or complete genetic removal of tau. We identify disinhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative PI3K regulator that tau controls, as a plausible mechanism and demonstrate that tau interacts with PTEN via tau's proline-rich domain. Our findings suggest an enabling role of tau in the pathogenesis of autism and identify tau reduction as a potential therapeutic strategy for some of the disorders that cause this condition.
    Keywords:  Akt; Cntnap2; PI3 kinase; PTEN; Scn1a; Shank3; autism spectrum disorders; mTOR; megalencephaly; tau