bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021–07–18
forty-six papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.
  2. In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease.
  3. TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism.
  4. Early onset effects of single substrate accumulation recapitulate major features of LSD in patient-derived lysosomes.
  5. Purification of active human vacuolar ATPase in native lipid-containing nanodiscs.
  6. Lysosomal calcium and autophagy.
  7. Amino Acids in Autophagy: Regulation and Function.
  8. mTORC2: The other mTOR in autophagy regulation.
  9. The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt.
  10. A role for BDNF- and NMDAR-induced lysosomal recruitment of mTORC1 in the regulation of neuronal mTORC1 activity.
  11. The Fusion Between Autophagic Vesicles and Lysosomes.
  12. RAVE and Rabconnectin-3 Complexes as Signal Dependent Regulators of Organelle Acidification.
  13. Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR-v-ATPase axis.
  14. Adipocyte-specific deletion of Depdc5 exacerbates insulin resistance and adipose tissue inflammation in mice.
  15. The multifaceted role of cathepsins in liver disease.
  16. capCLIP: a new tool to probe translational control in human cells through capture and identification of the eIF4E-mRNA interactome.
  17. Depdc5 deficiency exacerbates alcohol-induced hepatic steatosis via suppression of PPARα pathway.
  18. Long-term running exercise alleviates cognitive dysfunction in APP/PSEN1 transgenic mice via enhancing brain lysosomal function.
  19. mTORC1 activity regulates post-translational modifications of glycine decarboxylase to modulate glycine metabolism and tumorigenesis.
  20. The inhibition of mammalian target of rapamycin (mTOR) in improving inflammatory response after traumatic brain injury.
  21. Identification of amino acid response element of SLC38A9 as an ATF4-binding site in porcine skeletal muscle cells.
  22. Loss of mTORC2 Activity in Neutrophils Impairs Fusion of Granules and Affects Cellular Metabolism Favoring Increased Bacterial Burden in Sepsis.
  23. AAV9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment.
  24. Tsc Gene Locus Disruption and Differences in Renal Epithelial Extracellular Vesicles.
  25. Golgi-58K can re-localize to late endosomes upon cellular uptake of PS-ASOs and facilitates endosomal release of ASOs.
  26. mTOR Repression in Response to Amino Acid Starvation Promotes ECM Degradation Through MT1-MMP Endocytosis Arrest.
  27. Regulation of Gene Expression by Amino Acids in Animal Cells.
  28. The role of mTORC1 activation in seizure-induced exacerbation of Alzheimer's disease.
  29. Inhibition of mTORC1 through ATF4-induced REDD1 and Sestrin2 expression by Metformin.
  30. Amino Acids in Cell Signaling: Regulation and Function.
  31. Combinatorial Approach Using Caenorhabditis elegans and Mammalian Systems for Aging Research.
  32. ATG9A protects the plasma membrane from programmed and incidental permeabilization.
  33. Viral model of maternal immune activation alters placental AMPK and mTORC1 signaling in rats.
  34. Inhibition of Rag GTPase signaling in mice suppresses B cell responses and lymphomagenesis with minimal detrimental trade-offs.
  35. Glaucoma in mucopolysaccharidoses.
  36. A multicenter open-label extension study of intrathecal heparan-N-sulfatase in patients with Sanfilippo syndrome type A.
  37. Exploring the Genotype-Phenotype Correlation in GBA-Parkinson Disease: Clinical Aspects, Biomarkers, and Potential Modifiers.
  38. Loss of Tsc1 in cerebellar Purkinje cells induces transcriptional and translation changes in FMRP target transcripts.
  39. Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.
  40. Prognostic significance and oncogene function of cathepsin A in hepatocellular carcinoma.
  41. Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation.
  42. SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis.
  43. Cancer metabolism: looking forward.
  44. Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake.
  45. TMEM97 ablation aggravates oxidant-induced retinal degeneration.
  46. Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis.
  1. Mol Cell. 2021 Jul 05. pii: S1097-2765(21)00497-4. [Epub ahead of print]
    An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress.
    Julian Nüchel, Marina Tauber, Janica L Nolte, Matthias Mörgelin, Clara Türk, Beate Eckes, Constantinos Demetriades, Markus Plomann.
      Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.
    Keywords:  ECM; GORASP2; GRASP55; Golgi; MMP2; Rapamycin; Tuberous Sclerosis Complex (TSC); cellular stress response; mTORC1; unconventional protein secretion (UPS)
    DOI:  https://doi.org/10.1016/j.molcel.2021.06.017
  2. Nat Commun. 2021 07 13. 12(1): 4291
    In utero adenine base editing corrects multi-organ pathology in a lethal lysosomal storage disease.
    Sourav K Bose, Brandon M White, Meghana V Kashyap, Apeksha Dave, Felix R De Bie, Haiying Li, Kshitiz Singh, Pallavi Menon, Tiankun Wang, Shiva Teerdhala, Vishal Swaminathan, Heather A Hartman, Sowmya Jayachandran, Prashant Chandrasekaran, Kiran Musunuru, Rajan Jain, David B Frank, Philip Zoltick, William H Peranteau.
      In utero base editing has the potential to correct disease-causing mutations before the onset of pathology. Mucopolysaccharidosis type I (MPS-IH, Hurler syndrome) is a lysosomal storage disease (LSD) affecting multiple organs, often leading to early postnatal cardiopulmonary demise. We assessed in utero adeno-associated virus serotype 9 (AAV9) delivery of an adenine base editor (ABE) targeting the Idua G→A (W392X) mutation in the MPS-IH mouse, corresponding to the common IDUA G→A (W402X) mutation in MPS-IH patients. Here we show efficient long-term W392X correction in hepatocytes and cardiomyocytes and low-level editing in the brain. In utero editing was associated with improved survival and amelioration of metabolic, musculoskeletal, and cardiac disease. This proof-of-concept study demonstrates the possibility of efficiently performing therapeutic base editing in multiple organs before birth via a clinically relevant delivery mechanism, highlighting the potential of this approach for MPS-IH and other genetic diseases.
    DOI:  https://doi.org/10.1038/s41467-021-24443-8
  3. Nat Commun. 2021 07 12. 12(1): 4245
    TSC2 regulates lysosome biogenesis via a non-canonical RAGC and TFEB-dependent mechanism.
    Nicola Alesi, Elie W Akl, Damir Khabibullin, Heng-Jia Liu, Anna S Nidhiry, Emma R Garner, Harilaos Filippakis, Hilaire C Lam, Wei Shi, Srinivas R Viswanathan, Manrico Morroni, Shawn M Ferguson, Elizabeth P Henske.
      Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, resulting in hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). Transcription factor EB (TFEB), a master regulator of lysosome biogenesis, is negatively regulated by mTORC1 through a RAG GTPase-dependent phosphorylation. Here we show that lysosomal biogenesis is increased in TSC-associated renal tumors, pulmonary lymphangioleiomyomatosis, kidneys from Tsc2+/- mice, and TSC1/2-deficient cells via a TFEB-dependent mechanism. Interestingly, in TSC1/2-deficient cells, TFEB is hypo-phosphorylated at mTORC1-dependent sites, indicating that mTORC1 is unable to phosphorylate TFEB in the absence of the TSC1/2 complex. Importantly, overexpression of folliculin (FLCN), a GTPase activating protein for RAGC, increases TFEB phosphorylation at the mTORC1 sites in TSC2-deficient cells. Overexpression of constitutively active RAGC is sufficient to relocalize TFEB to the cytoplasm. These findings establish the TSC proteins as critical regulators of lysosomal biogenesis via TFEB and RAGC and identify TFEB as a driver of the proliferation of TSC2-deficient cells.
    DOI:  https://doi.org/10.1038/s41467-021-24499-6
  4. iScience. 2021 Jul 23. 24(7): 102707
    Early onset effects of single substrate accumulation recapitulate major features of LSD in patient-derived lysosomes.
    Gianluca Scerra, Valeria De Pasquale, Luigi Michele Pavone, Maria Gabriella Caporaso, Andreas Mayer, Maurizio Renna, Massimo D'Agostino.
      Lysosome functions mainly rely on their ability to either degrade substrates or release them into the extracellular space. Lysosomal storage disorders (LSDs) are commonly characterized by a chronic lysosomal accumulation of different substrates, thereby causing lysosomal dysfunctions and secretion defects. However, the early effects of substrate accumulation on lysosomal homeostasis have not been analyzed so far. Here, we describe how the acute accumulation of a single substrate determines a rapid centripetal redistribution of the lysosomes, triggering their expansion and reducing their secretion, by limiting the motility of these organelles toward the plasma membrane. Moreover, we provide evidence that such defects could be explained by a trapping mechanism exerted by the extensive contacts between the enlarged lysosomes and the highly intertwined membrane structures of the endoplasmic reticulum which might represent a crucial biological cue ultimately leading to the clinically relevant secondary defects observed in the LSD experimental models and patients.
    Keywords:  Biochemistry; Biological sciences; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.102707
  5. J Biol Chem. 2021 Jul 13. pii: S0021-9258(21)00765-1. [Epub ahead of print] 100964
    Purification of active human vacuolar ATPase in native lipid-containing nanodiscs.
    Rebecca A Oot, Yeqi Yao, Morris F Manolson, Stephan Wilkens.
      Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to Concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme's isolated membrane subcomplex together with biosynthetic assembly factors CCDC115, TMEM199 and VMA21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform specific manner and establishes a platform for study of the assembly and regulation of the human holoenzyme.
    Keywords:  ATP6V0a4; Vacuolar H(+)-ATPase; human; mass spectrometry; membrane protein; native lipid nanodisc; protein purification
    DOI:  https://doi.org/10.1016/j.jbc.2021.100964
  6. Int Rev Cell Mol Biol. 2021 ;pii: S1937-6448(21)00024-1. [Epub ahead of print]362 141-170
    Lysosomal calcium and autophagy.
    Diego L Medina.
      Lysosomal calcium is emerging as a modulator of autophagy and lysosomal compartment, an obligatory partner to complete the autophagic pathway. A variety of specific signals such as nutrient deprivation or oxidative stress can trigger lysosomal calcium-mediated nuclear translocation of the transcription factor EB (TFEB), a master regulator of global lysosomal function. Also, lysosomal calcium can promote the formation of autophagosome vesicles (AVs) by a mechanism that requires the production of the phosphoinositide PI3P by the VPS34 autophagic complex and the activation of the energy-sensing kinase AMPK. Additionally, lysosomal calcium plays a role in membrane fusion and fission events involved in cellular processes such as endocytic maturation, autophagosome-lysosome fusion, lysosomal exocytosis, and lysosomal reformation upon autophagy completion. Lysosomal calcium-dependent functions are defective in cellular and animal models of the non-selective cation channel TRPML1, whose mutations in humans cause the neurodegenerative lysosomal storage disease mucolipidosis type IV (MLIV). Lysosomal calcium is not only acting as a positive regulator of autophagy, but it is also responsible for turning-off this process through the reactivation of the mTOR kinase during prolonged starvation. More recently, it has been described the role of lysosomal calcium on an elegant sequence of intracellular signaling events such as membrane repair, lysophagy, and lysosomal biogenesis upon the induction of different grades of lysosomal membrane damage. Here, we will discuss these novel findings that re-define the importance of the lysosome and lysosomal calcium signaling at regulating cellular metabolism.
    Keywords:  Autophagy; Calcineurin; LSDs; Lysosomal calcium; Lysosome; MLIV; TFEB; TPCs; TRPML1; mTORC1
    DOI:  https://doi.org/10.1016/bs.ircmb.2021.03.002
  7. Adv Exp Med Biol. 2021 ;1332 51-66
    Amino Acids in Autophagy: Regulation and Function.
    James Z Shen, Guoyao Wu, Shaodong Guo.
      Autophagy is a dynamic process in which the eukaryotic cells break down intracellular components by lysosomal degradation. Under the normal condition, the basal level of autophagy removes damaged organelles, misfolded proteins, or protein aggregates to keep cells in a homeostatic condition. Deprivation of nutrients (e.g., removal of amino acids) stimulates autophagy activity, promoting lysosomal degradation and the recycling of cellular components for cell survival. Importantly, insulin and amino acids are two main inhibitors of autophagy. They both activate the mTOR complex 1 (mTORC1) signaling pathway to inhibit the autophagy upstream of the uncoordinated-51 like kinase 1/2 (ULK1/2) complex that triggers autophagosome formation. In particular, insulin activates mTORC1 via the PI3K class I-AKT pathway; while amino acids activate mTORC1 either through the PI3K class III (hVps34) pathway or through a variety of amino acid sensors located in the cytosol or lysosomal membrane. These amino acid sensors control the translocation of mTORC1 from the cytosol to the lysosomal surface where mTORC1 is activated by Rheb GTPase, therefore regulating autophagy and the lysosomal protein degradation.
    Keywords:  Amino acids; Arginine; Autophagosome; Autophagy; Calcium/calmodulin-dependent protein kinase kinase; Leucine; Lysosome; Mammalian target of rapamycin complex 1; Rag GTPase; Rheb
    DOI:  https://doi.org/10.1007/978-3-030-74180-8_4
  8. Aging Cell. 2021 Jul 12. e13431
    mTORC2: The other mTOR in autophagy regulation.
    Josué Ballesteros-Álvarez, Julie K Andersen.
      The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best-described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex-specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.
    Keywords:  AKT; FOXOs; SGK-1; autophagy; mTORC2
    DOI:  https://doi.org/10.1111/acel.13431
  9. J Biol Chem. 2021 Jul 07. pii: S0021-9258(21)00742-0. [Epub ahead of print] 100942
    The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt.
    Aaron Seth Tooley, Dubek Kazyken, Cagri Bodur, Ian E Gonzalez, Diane C Fingar.
      TBK1 responds to microbes to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) on S2159 to increase mTOR complex 1 (mTORC1) signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. While TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promote mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 co-immunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.
    Keywords:  Akt; TBK1; mTOR; mTORC2; phosphorylation
    DOI:  https://doi.org/10.1016/j.jbc.2021.100942
  10. Mol Brain. 2021 Jul 12. 14(1): 112
    A role for BDNF- and NMDAR-induced lysosomal recruitment of mTORC1 in the regulation of neuronal mTORC1 activity.
    Dany Khamsing, Solène Lebrun, Isabelle Fanget, Nathanaël Larochette, Christophe Tourain, Vincent de Sars, Maia Brunstein, Martin Oheim, Damien Carrel, François Darchen, Claire Desnos.
      Memory and long term potentiation require de novo protein synthesis. A key regulator of this process is mTORC1, a complex comprising the mTOR kinase. Growth factors activate mTORC1 via a pathway involving PI3-kinase, Akt, the TSC complex and the GTPase Rheb. In non-neuronal cells, translocation of mTORC1 to late endocytic compartments (LEs), where Rheb is enriched, is triggered by amino acids. However, the regulation of mTORC1 in neurons remains unclear. In mouse hippocampal neurons, we observed that BDNF and treatments activating NMDA receptors trigger a robust increase in mTORC1 activity. NMDA receptors activation induced a significant recruitment of mTOR onto lysosomes even in the absence of external amino acids, whereas mTORC1 was evenly distributed in neurons under resting conditions. NMDA receptor-induced mTOR translocation to LEs was partly dependent on the BDNF receptor TrkB, suggesting that BDNF contributes to the effect of NMDA receptors on mTORC1 translocation. In addition, the combination of Rheb overexpression and artificial mTORC1 targeting to LEs by means of a modified component of mTORC1 fused with a LE-targeting motif strongly activated mTOR. To gain spatial and temporal control over mTOR localization, we designed an optogenetic module based on light-sensitive dimerizers able to recruit mTOR on LEs. In cells expressing this optogenetic tool, mTOR was translocated to LEs upon photoactivation. In the absence of growth factor, this was not sufficient to activate mTORC1. In contrast, mTORC1 was potently activated by a combination of BDNF and photoactivation. The data demonstrate that two important triggers of synaptic plasticity, BDNF and NMDA receptors, synergistically power the two arms of the mTORC1 activation mechanism, i.e., mTORC1 translocation to LEs and Rheb activation. Moreover, they unmask a functional link between NMDA receptors and mTORC1 that could underlie the changes in the synaptic proteome associated with long-lasting changes in synaptic strength.
    Keywords:  BDNF; Endo-lysosomes; NMDA receptors; Optogenetics; Synaptic plasticity; mTOR
    DOI:  https://doi.org/10.1186/s13041-021-00820-8
  11. Adv Exp Med Biol. 2021 ;1208 55-66
    The Fusion Between Autophagic Vesicles and Lysosomes.
    Xiaoxia Liu, Qing Zhong.
      The autophagosome delivers engulfed substrates to the lysosome for degradation via membrane fusion between the autophagosome and the lysosome. The process of membrane fusion is highly conserved in evolution. It is widely accepted that membrane fusion in general is driven by the zippering of the SNARE complex to form a four-helix bundle. Besides SNAREs, other proteins are required to complete fusion efficiently, including tethering proteins, Rab GTPases, and SM proteins (Sec1/SM family proteins). This chapter will summarize the current knowledge of the key machinery involved in autophagosome-lysosome fusion, including autophagic SNAREs, involved ATG proteins, the HOPS complex, Rab GTPase, and other relevant aspects.
    DOI:  https://doi.org/10.1007/978-981-16-2830-6_4
  12. Front Cell Dev Biol. 2021 ;9 698190
    RAVE and Rabconnectin-3 Complexes as Signal Dependent Regulators of Organelle Acidification.
    Michael C Jaskolka, Samuel R Winkley, Patricia M Kane.
      The yeast RAVE (Regulator of H+-ATPase of Vacuolar and Endosomal membranes) complex and Rabconnectin-3 complexes of higher eukaryotes regulate acidification of organelles such as lysosomes and endosomes by catalyzing V-ATPase assembly. V-ATPases are highly conserved proton pumps consisting of a peripheral V1 subcomplex that contains the sites of ATP hydrolysis, attached to an integral membrane V o subcomplex that forms the transmembrane proton pore. Reversible disassembly of the V-ATPase is a conserved regulatory mechanism that occurs in response to multiple signals, serving to tune ATPase activity and compartment acidification to changing extracellular conditions. Signals such as glucose deprivation can induce release of V1 from Vo, which inhibits both ATPase activity and proton transport. Reassembly of V1 with Vo restores ATP-driven proton transport, but requires assistance of the RAVE or Rabconnectin-3 complexes. Glucose deprivation triggers V-ATPase disassembly in yeast and is accompanied by binding of RAVE to V1 subcomplexes. Upon glucose readdition, RAVE catalyzes both recruitment of V1 to the vacuolar membrane and its reassembly with Vo. The RAVE complex can be recruited to the vacuolar membrane by glucose in the absence of V1 subunits, indicating that the interaction between RAVE and the Vo membrane domain is glucose-sensitive. Yeast RAVE complexes also distinguish between organelle-specific isoforms of the Vo a-subunit and thus regulate distinct V-ATPase subpopulations. Rabconnectin-3 complexes in higher eukaryotes appear to be functionally equivalent to yeast RAVE. Originally isolated as a two-subunit complex from rat brain, the Rabconnectin-3 complex has regions of homology with yeast RAVE and was shown to interact with V-ATPase subunits and promote endosomal acidification. Current understanding of the structure and function of RAVE and Rabconnectin-3 complexes, their interactions with the V-ATPase, their role in signal-dependent modulation of organelle acidification, and their impact on downstream pathways will be discussed.
    Keywords:  DMXL2; RAVE = regulator of H+-ATPase of vacuoles and endosomes; Rabconnectin-3; V-ATPase; WDR7; endosome and lysosome; organelle acidification; vacuole
    DOI:  https://doi.org/10.3389/fcell.2021.698190
  13. Mol Metab. 2021 Jul 12. pii: S2212-8778(21)00138-1. [Epub ahead of print] 101293
    Specific amino acid supplementation rescues the heart from lipid overload-induced insulin resistance and contractile dysfunction by targeting the endosomal mTOR-v-ATPase axis.
    Shujin Wang, Francesco Schianchi, Dietbert Neumann, Li-Yen Wong, Aomin Sun, Frans A van Nieuwenhoven, Maurice P Zeegers, Agnieszka Strzelecka, Umare Col, Jan F C Glatz, Miranda Nabben, Joost J F P Luiken.
       OBJECTIVE: The diabetic heart is characterized by extensive lipid accumulation which often leads to cardiac contractile dysfunction. The underlying mechanism involves a pivotal role for vacuolar-type H+-ATPase (v-ATPase, functioning as endosomal/lysosomal proton pump). Specifically, lipid oversupply to the heart causes disassembly of v-ATPase and endosomal de-acidification. Endosomes are storage compartments for lipid transporter CD36. However, upon endosomal de-acidification, CD36 is expelled to translocate to the sarcolemma, thereby inducing myocardial lipid accumulation, insulin resistance and contractile dysfunction. Hence, v-ATPase assembly may be a suitable target for ameliorating diabetic cardiomyopathy. Another function of v-ATPase involves binding of anabolic master-regulator mTORC1 to endosomes, a prerequisite for activation of mTORC1 by amino acids (AAs). We examined whether the relationship between v-ATPase and mTORC1 also operates reciprocally, specifically whether AA induce v-ATPase reassembly in an mTORC1-dependent manner to prevent excess lipids from entering and damaging the heart.
    METHODS: Lipid overexposed rodent/human cardiomyocytes and high-fat diet-fed rats were treated with a specific cocktail of AAs (lysine/leucine/arginine). Then, v-ATPase assembly status/activity, cell surface CD36 content, myocellular lipid uptake/accumulation, insulin sensitivity and contractile function were measured. To elucidate underlying mechanisms, specific gene knockdown was employed, followed by subcellular fractionation and co-immunoprecipitation.
    RESULTS: In lipid-overexposed cardiomyocytes, lysine/leucine/arginine re-internalized CD36 to the endosomes, prevented/reversed lipid accumulation, and preserved/restored insulin sensitivity and contractile function. These beneficial AA actions required the mTORC1-v-ATPase axis, adaptor protein Ragulator and endosomal/lysosomal AA transporter SLC38A9, indicating an endosome-centric inside-out AA sensing mechanism. In high-fat diet-fed rats, lysine/leucine/arginine had similar beneficial actions at the myocellular level as in vitro in lipid-overexposed cardiomyocytes, and partially reversed cardiac hypertrophy.
    CONCLUSION: Specific AAs acting via v-ATPase re-assembly reduce cardiac lipid uptake raising the possibility for treatment in situations of lipid overload and associated insulin resistance.
    Keywords:  Contractile function; Diabetic heart; Endosomal CD36; Lipid-induced insulin resistance; Vacuolar H(+)-ATPase; mTORC1
    DOI:  https://doi.org/10.1016/j.molmet.2021.101293
  14. Biochem Biophys Res Commun. 2021 Jul 06. pii: S0006-291X(21)01027-5. [Epub ahead of print]569 118-124
    Adipocyte-specific deletion of Depdc5 exacerbates insulin resistance and adipose tissue inflammation in mice.
    Jinghong Zhang, Yiping Shi, Chenyan Yang, Bo Sun, Honghui Ma, Rong Huang, Zun Li, Mingyong Wang, Qingzhi Wang, Jie Ma, Xiwen Xiong.
      The mammalian target of rapamycin complex 1 (mTORC1) is a crucial regulator of adipogenesis and systemic energy metabolism. Its dysregulation leads to a diversity of metabolic diseases, including obesity and type 2 diabetes. DEP-domain containing 5 (DEPDC5) is a critical component of GATOR1 complex that functions as a key inhibitor of mTORC1. So far, its function in adipose tissue remains largely unknown. Herein we evaluated how persistent mTORC1 activation in adipocyte via Depdc5 knockout modulates adiposity in vivo. Our data indicated that adipocyte-specific knockout of Depdc5 in aged mice led to reduced visceral fat, aggravated insulin resistance and enhanced adipose tissue inflammation. Moreover, we found that Depdc5 ablation resulted in upregulation of adipose triglyceride lipase (ATGL) in adipocytes and elevated levels of serum free fatty acids (FFAs). Intriguingly, rapamycin treatment did not reverse insulin resistance but alleviated adipose tissue inflammation caused by Depdc5 deletion. Taken together, our findings revealed that mTORC1 activation caused by Depdc5 deletion promotes lipolysis process and further exacerbates insulin resistance and adipose tissue inflammation in mice.
    Keywords:  Adipocyte; Depdc5; Inflammation; Insulin resistance; mTORC1
    DOI:  https://doi.org/10.1016/j.bbrc.2021.07.003
  15. J Hepatol. 2021 Jul 06. pii: S0168-8278(21)01880-8. [Epub ahead of print]
    The multifaceted role of cathepsins in liver disease.
    Paloma Ruiz-Blázquez, Valeria Pistorio, María Fernández-Fernández, Anna Moles.
      Proteases are the most abundant enzyme gene family in vertebrates and execute essential functions in all living organisms. Their main role is to hydrolase the peptide bond within proteins, a process also called proteolysis. Contrary to the conventional paradigm, proteases are not only random catalytic devices, but can perform highly selective and targeted cleavage of specific substrates, finely modulating multiple essential cellular processes. Lysosomal protease cathepsins comprise three families of proteases with preferential activity within acidic cellular compartments but can also be found in other cellular locations. They can operate alone or as part of signalling cascades and regulatory circuits playing important roles in apoptosis, extracellular matrix remodelling, HSC activation, autophagy and metastasis, contributing to the initiation, development and progression of liver disease. This review comprehensively summarizes the current knowledge to date on the role and contribution of lysosomal cathepsins to liver disease with a particular emphasis in liver fibrosis, NAFLD and HCC.
    Keywords:  Cathepsin; Hepatocellular carcinoma; Liver fibrosis; Lysosome; NAFLD; Protease
    DOI:  https://doi.org/10.1016/j.jhep.2021.06.031
  16. Nucleic Acids Res. 2021 Jul 13. pii: gkab604. [Epub ahead of print]
    capCLIP: a new tool to probe translational control in human cells through capture and identification of the eIF4E-mRNA interactome.
    Kirk B Jensen, B Kate Dredge, John Toubia, Xin Jin, Valentina Iadevaia, Gregory J Goodall, Christopher G Proud.
      Translation of eukaryotic mRNAs begins with binding of their m7G cap to eIF4E, followed by recruitment of other translation initiation factor proteins. We describe capCLIP, a novel method to comprehensively capture and quantify the eIF4E (eukaryotic initiation factor 4E) 'cap-ome' and apply it to examine the biological consequences of eIF4E-cap binding in distinct cellular contexts. First, we use capCLIP to identify the eIF4E cap-omes in human cells with/without the mTORC1 (mechanistic target of rapamycin, complex 1) inhibitor rapamycin, there being an emerging consensus that rapamycin inhibits translation of TOP (terminal oligopyrimidine) mRNAs by displacing eIF4E from their caps. capCLIP reveals that the representation of TOP mRNAs in the cap-ome is indeed systematically reduced by rapamycin, thus validating our new methodology. capCLIP also refines the requirements for a functional TOP sequence. Second, we apply capCLIP to probe the consequences of phosphorylation of eIF4E. We show eIF4E phosphorylation reduces overall eIF4E-mRNA association and, strikingly, causes preferential dissociation of mRNAs with short 5'-UTRs. capCLIP is a valuable new tool to probe the function of eIF4E and of other cap-binding proteins such as eIF4E2/eIF4E3.
    DOI:  https://doi.org/10.1093/nar/gkab604
  17. Cell Death Dis. 2021 Jul 15. 12(7): 710
    Depdc5 deficiency exacerbates alcohol-induced hepatic steatosis via suppression of PPARα pathway.
    Lin Xu, Xinge Zhang, Yue Xin, Jie Ma, Chenyan Yang, Xi Zhang, Guoqing Hou, Xiaocheng Charlie Dong, Zhaoli Sun, Xiwen Xiong, Xuan Cao.
      Alcohol-related liver disease (ALD), a condition caused by alcohol overconsumption, occurs in three stages of liver injury including steatosis, hepatitis, and cirrhosis. DEP domain-containing protein 5 (DEPDC5), a component of GAP activities towards Rags 1 (GATOR1) complex, is a repressor of amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In the current study, we found that aberrant activation of mTORC1 was likely attributed to the reduction of DEPDC5 in the livers of ethanol-fed mice or ALD patients. To further define the in vivo role of DEPDC5 in ALD development, we generated Depdc5 hepatocyte-specific knockout mouse model (Depdc5-LKO) in which mTORC1 pathway was constitutively activated through loss of the inhibitory effect of GATOR1. Hepatic Depdc5 ablation leads to mild hepatomegaly and liver injury and protects against diet-induced liver steatosis. In contrast, ethanol-fed Depdc5-LKO mice developed severe hepatic steatosis and inflammation. Pharmacological intervention with Torin 1 suppressed mTORC1 activity and remarkably ameliorated ethanol-induced hepatic steatosis and inflammation in both control and Depdc5-LKO mice. The pathological effect of sustained mTORC1 activity in ALD may be attributed to the suppression of peroxisome proliferator activated receptor α (PPARα), the master regulator of fatty acid oxidation in hepatocytes, because fenofibrate (PPARα agonist) treatment reverses ethanol-induced liver steatosis and inflammation in Depdc5-LKO mice. These findings provide novel insights into the in vivo role of hepatic DEPDC5 in the development of ALD.
    DOI:  https://doi.org/10.1038/s41419-021-03980-6
  18. Acta Pharmacol Sin. 2021 Jul 16.
    Long-term running exercise alleviates cognitive dysfunction in APP/PSEN1 transgenic mice via enhancing brain lysosomal function.
    Xue Wang, Yu-Ting Zhu, Yi Zhu, Yan-Ling Sun, Jun Huang, Zhe Li, Yan Wang, Jun-Chao Wu, Zheng-Hong Qin, Fang Lin.
      Amyloid-β peptide (Aβ) aggregation is the hallmark of Alzheimer's disease (AD). The imbalance between the production and clearance of Aβ results in the accumulation and aggregation of Aβ in the brain. Thus far, few drugs are available for AD treatment, but exercise has been recognized for its cognition-enhancing properties in AD patients. The underlying mechanisms remain unclear. Our recent study showed that long-term running exercise could activate the lysosomal function in the brains of mice. In this study, we investigated whether exercise could reduce Aβ accumulation by activating lysosomal function in APP/PSEN1 transgenic mice. Started at the age of 5 months, the mice were trained with a running wheel at the speed of 18 r/min, 40 min/d, 6 d/week for 5 months, and were killed at the end of the 10th month, then brain tissue was collected for biochemical analyses. The cognitive ability was assessed in the 9th month. We showed that long-term exercise significantly mitigated cognitive dysfunction in AD mice, accompanied by the enhanced lysosomal function and the clearance of Aβ in the brain. Exercise significantly promoted the nuclear translocation of transcription factor EB (TFEB), and increased the interaction between nuclear TFEB with AMPK-mediated acetyl-CoA synthetase 2, thus enhancing transcription of the genes associated with the biogenesis of lysosomes. Exercise also raised the levels of mature cathepsin D and cathepsin L, suggesting that more Aβ peptides could be degraded in the activated lysosomes. This study demonstrates that exercise may improve the cognitive dysfunction of AD by enhancing lysosomal function.
    Keywords:  ACSS2; APP/PSEN1 transgenic mice; Alzheimer’s disease; Aβ clearance; TFEB; exercise; lysosomal function
    DOI:  https://doi.org/10.1038/s41401-021-00720-6
  19. Nat Commun. 2021 07 09. 12(1): 4227
    mTORC1 activity regulates post-translational modifications of glycine decarboxylase to modulate glycine metabolism and tumorigenesis.
    Rui Liu, Lin-Wen Zeng, Rong Gong, Fanen Yuan, Hong-Bing Shu, Shu Li.
      Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.
    DOI:  https://doi.org/10.1038/s41467-021-24321-3
  20. J Cell Mol Med. 2021 Jul 10.
    The inhibition of mammalian target of rapamycin (mTOR) in improving inflammatory response after traumatic brain injury.
    Michela Campolo, Giovanna Casili, Marika Lanza, Alessia Filippone, Marika Cordaro, Alessio Ardizzone, Sarah Adriana Scuderi, Salvatore Cuzzocrea, Emanuela Esposito, Irene Paterniti.
      Traumatic brain injury (TBI) provokes primary and secondary damage on endothelium and brain parenchyma, leading neurons die rapidly by necrosis. The mammalian target of rapamycin signalling pathway (mTOR) manages numerous aspects of cellular growth, and it is up-regulated after moderate to severe traumatic brain injury (TBI). Currently, the significance of this increased signalling event for the recovery of brain function is unclear; therefore, we used two different selective inhibitors of mTOR activity to discover the functional role of mTOR inhibition in a mouse model of TBI performed by a controlled cortical impact injury (CCI). Treatment with KU0063794, a dual mTORC1 and mTORC2 inhibitor, and with rapamycin as well-known inhibitor of mTOR, was performed 1 and 4 hours subsequent to TBI. Results proved that mTOR inhibitors, especially KU0063794, significantly improved cognitive and motor recovery after TBI, reducing lesion volumes. Also, treatment with mTOR inhibitors ameliorated the neuroinflammation associated with TBI, showing a diminished neuronal death and astrogliosis after trauma. Our findings propose that the involvement of selective mTORC1/2 inhibitor may represent a therapeutic strategy to improve recovery after brain trauma.
    Keywords:  KU0063794; apoptosis; astrogliosis; behavioural performance; mTOR; mTORC1; mTORC2; neuroinflammation; neuronal death; rapamycin; traumatic brain injury
    DOI:  https://doi.org/10.1111/jcmm.16702
  21. Biochem Biophys Res Commun. 2021 Jul 08. pii: S0006-291X(21)01005-6. [Epub ahead of print]569 167-173
    Identification of amino acid response element of SLC38A9 as an ATF4-binding site in porcine skeletal muscle cells.
    Dan Wang, Changtong Guo, Xuebin Wan, Kai Guo, Hongdan Niu, Rong Zheng, Jin Chai, Siwen Jiang.
      Amino acids can affect protein synthesis by activating mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Amino acid transporters SLC38A9 on the lysosomal membrane not only transport amino acids, but also can sense amino acids and activate mTORC1 signaling pathway. Activating transcription factor 4 (ATF4) can promote the expression of amino acid transporters by binding with amino acid response element (AARE). In this study, two AAREs were found in the SLC38A9 promoter region of pig, and both of them bound to ATF4. The AARE in the first intron was located in the core promoter region of SLC38A9. ATF4 regulated mRNA expression level of SLC38A9 in porcine skeletal muscle cells. In the absence of amino acids, the expression of ATF4 decreased and the expression of SLC38A9 increased. After leucine addition, the expression levels of ATF4 and SLC38A9 increased. It suggested that in the absence of amino acids, the expression of SLC38A9 was increased via binding of ATF4 to AARE binding factors in SLC38A9 promoter fragment; after the addition of leucine, ATF4 was activated, resulting in the increase of SLC38A9 expression.
    Keywords:  Activating transcription factor 4; Amino acid response element; Amino acid transporter; Porcine skeletal muscle cells; SLC38A9
    DOI:  https://doi.org/10.1016/j.bbrc.2021.06.083
  22. J Immunol. 2021 Jul 14. pii: ji2000573. [Epub ahead of print]
    Loss of mTORC2 Activity in Neutrophils Impairs Fusion of Granules and Affects Cellular Metabolism Favoring Increased Bacterial Burden in Sepsis.
    Cristiane Naffah de Souza Breda, Leandro Carvalho Dantas Breda, Larissa Anastácio da Costa Carvalho, Mariane Tami Amano, Fernanda Fernandes Terra, Reinaldo Correia Silva, Matheus Garcia Fragas, Maria Fernanda Forni, Monique Thaís Costa Fonseca, Gabriela Venturini, Amanda Campelo Melo Feitosa, Bruno Ghirotto, Mario Costa Cruz, Flávia Franco Cunha, Aline Ignacio, Marcela Latância, Angela Castoldi, Vinícius Andrade-Oliveira, Eloisa Martins da Silva, Meire Ioshie Hiyane, Alexandre da Costa Pereira, William Festuccia, Flávia Carla Meotti, Niels Olsen Saraiva Câmara.
      Sepsis is a complex infectious syndrome in which neutrophil participation is crucial for patient survival. Neutrophils quickly sense and eliminate the pathogen by using different effector mechanisms controlled by metabolic processes. The mammalian target of rapamycin (mTOR) pathway is an important route for metabolic regulation, and its role in neutrophil metabolism has not been fully understood yet, especially the importance of mTOR complex 2 (mTORC2) in the neutrophil effector functions. In this study, we observed that the loss of Rictor (mTORC2 scaffold protein) in primary mouse-derived neutrophils affects their chemotaxis by fMLF and their microbial killing capacity, but not the phagocytic capacity. We found that the microbicidal capacity was impaired in Rictor-deleted neutrophils because of an improper fusion of granules, reducing the hypochlorous acid production. The loss of Rictor also led to metabolic alterations in isolated neutrophils, increasing aerobic glycolysis. Finally, myeloid-Rictor-deleted mice (LysMRic Δ/Δ) also showed an impairment of the microbicidal capacity, increasing the bacterial burden in the Escherichia coli sepsis model. Overall, our results highlight the importance of proper mTORC2 activation for neutrophil effector functions and metabolism during sepsis.
    DOI:  https://doi.org/10.4049/jimmunol.2000573
  23. J Virol. 2021 Jul 14. JVI0084321
    AAV9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment.
    Judit J Penzes, Paul Chipman, Nilakshee Bhattacharya, Allison Zeher, Rick Huang, Robert McKenna, Mavis Agbandje-McKenna.
      Adeno-associated viruses (AAVs) are small non-enveloped ssDNA viruses, that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enables the externalization of the capsid protein (VP) N-termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0) and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, both in the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N-termini, presumably in prelude to the externalization of VP1u at pH 4.0, that is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N-terminus externalization. Importance There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1-3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and used as gene therapy vector. This is part because, AAV9 is capable of crossing the blood brain barrier as well as readily transduces a wide array of tissues, including the central nervous system. In this study we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N-termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering pH.
    DOI:  https://doi.org/10.1128/JVI.00843-21
  24. Front Physiol. 2021 ;12 630933
    Tsc Gene Locus Disruption and Differences in Renal Epithelial Extracellular Vesicles.
    Prashant Kumar, Fahad Zadjali, Ying Yao, Brian Siroky, Aristotelis Astrinidis, Kenneth W Gross, John J Bissler.
      In tuberous sclerosis complex (TSC), Tsc2 mutations are associated with more severe disease manifestations than Tsc1 mutations and the role of extracellular vesicles (EVs) in this context is not yet studied. We report a comparative analysis of EVs derived from isogenic renal cells except for Tsc1 or Tsc2 gene status and hypothesized that in spite of having similar physical characteristics, EVs modulate signaling pathways differently, thus leading to TSC heterogenicity. We used mouse inner medullary collecting duct (mIMCD3) cells with the Tsc1 (T1G cells) or Tsc2 (T2J cells) gene disrupted by CRISPR/CAS9. EVs were isolated from the cell culture media by size-exclusion column chromatography followed by detailed physical and chemical characterization. Physical characterization of EVs was accessed by tunable resistive pulse sensing and dynamic light scattering, revealing similar average sizes and zeta potentials (at pH 7.4) for EVs from mIMCD3 (123.5 ± 5.7 nm and -16.3 ± 2.1 mV), T1G cells (131.5 ± 8.3 nm and -19.8 ± 2.7 mV), and T2J cells (127.3 ± 4.9 nm and -20.2 ± 2.1 mV). EVs derived from parental mIMCD3 cells and both mutated cell lines were heterogeneous (>90% of EVs < 150 nm) in nature. Immunoblotting detected cilial Hedgehog signaling protein Arl13b; intercellular proteins TSG101 and Alix; and transmembrane proteins CD63, CD9, and CD81. Compared to Tsc2 deletion, Tsc1 deletion cells had reduced EV production and release rates. EVs from Tsc1 mutant cells altered mTORC1, autophagy, and β-catenin pathways differently than EVs from Tsc2-mutated cells. Quantitative PCR analysis revealed the down regulation of miR-212a-3p and miR-99a-5p in EVs from Tsc2-mutated cells compared to EVs from Tsc1-mutant cells. Thus, EV-derived miR-212-3p and mIR-99a-5p axes may represent therapeutic targets or biomarkers for TSC disease.
    Keywords:  autophagy; cell signaling; extracellular vesicles; miRNA; tuberous sclerosis complex
    DOI:  https://doi.org/10.3389/fphys.2021.630933
  25. Nucleic Acids Res. 2021 Jul 09. pii: gkab599. [Epub ahead of print]
    Golgi-58K can re-localize to late endosomes upon cellular uptake of PS-ASOs and facilitates endosomal release of ASOs.
    Xue-Hai Liang, Joshua G Nichols, Cheryl Li De Hoyos, Hong Sun, Lingdi Zhang, Stanley T Crooke.
      Phosphorothioate (PS) modified antisense oligonucleotide (ASO) drugs can trigger RNase H1 cleavage of cellular target RNAs to modulate gene expression. Internalized PS-ASOs must be released from membraned endosomal organelles, a rate limiting step that is not well understood. Recently we found that M6PR transport between Golgi and late endosomes facilitates productive release of PS-ASOs, raising the possibility that Golgi-mediated transport may play important roles in PS-ASO activity. Here we further evaluated the involvement of Golgi in PS-ASO activity by examining additional Golgi proteins. Reduction of certain Golgi proteins, including Golgi-58K, GCC1 and TGN46, decreased PS-ASO activity, without substantial effects on Golgi integrity. Upon PS-ASO cellular uptake, Golgi-58K was recruited to late endosomes where it colocalized with PS-ASOs. Reduction of Golgi-58K caused slower PS-ASO release from late endosomes, decreased GCC2 late endosome relocalization, and led to slower retrograde transport of M6PR from late endosomes to trans-Golgi. Late endosome relocalization of Golgi-58K requires Hsc70, and is most likely mediated by PS-ASO-protein interactions. Together, these results suggest a novel function of Golgi-58K in mediating Golgi-endosome transport and indicate that the Golgi apparatus plays an important role in endosomal release of PS-ASO, ensuring antisense activity.
    DOI:  https://doi.org/10.1093/nar/gkab599
  26. Adv Sci (Weinh). 2021 Jul 11. e2101614
    mTOR Repression in Response to Amino Acid Starvation Promotes ECM Degradation Through MT1-MMP Endocytosis Arrest.
    Cecilia Colombero, David Remy, Sandra Antoine-Bally, Anne-Sophie Macé, Pedro Monteiro, Nadia ElKhatib, Margot Fournier, Ahmed Dahmani, Elodie Montaudon, Guillaume Montagnac, Elisabetta Marangoni, Philippe Chavrier.
      Under conditions of starvation, normal and tumor epithelial cells can rewire their metabolism toward the consumption of extracellular proteins, including extracellular matrix-derived components as nutrient sources. The mechanism of pericellular matrix degradation by starved cells has been largely overlooked. Here it is shown that matrix degradation by breast and pancreatic tumor cells and patient-derived xenograft explants increases by one order of magnitude upon amino acid and growth factor deprivation. In addition, it is found that collagenolysis requires the invadopodia components, TKS5, and the transmembrane metalloproteinase, MT1-MMP, which are key to the tumor invasion program. Increased collagenolysis is controlled by mTOR repression upon nutrient depletion or pharmacological inhibition by rapamycin. The results reveal that starvation hampers clathrin-mediated endocytosis, resulting in MT1-MMP accumulation in arrested clathrin-coated pits. The study uncovers a new mechanism whereby mTOR repression in starved cells leads to the repurposing of abundant plasma membrane clathrin-coated pits into robust ECM-degradative assemblies.
    Keywords:  MT1-MMP; breast cancer; clathrin-mediated endocytosis; extracellular matrix; invadopodia; mTOR; starvation
    DOI:  https://doi.org/10.1002/advs.202101614
  27. Adv Exp Med Biol. 2021 ;1332 1-15
    Regulation of Gene Expression by Amino Acids in Animal Cells.
    Nirvay Sah, Guoyao Wu, Fuller W Bazer.
      Amino acids have pleiotropic roles in animal biology including protein and glucose synthesis, cellular metabolism, antioxidant reactions, immune enhancers, and inducers or suppressors of gene expression. Recent studies have revealed important roles of amino acids in the regulation of gene expression in animals. Discoveries of cellular amino acid sensors and their mechanistic pathways have broadened our understanding of how the body responds to the deprivation of nutrients and amino acids in particular. Alterations in concentrations of extracellular amino acids can modulate transcription, translation, posttranscriptional modifications, and epigenetic regulation of genes and proteins. Cells have intracellular amino acid sensors, for example, Sestrin2 for leucine and CASTOR2 for arginine, that respond to sufficiency or deficiency in amino acids, thereby inhibiting or activating downstream signals for gene expression, respectively. The sufficiency of an amino acid in cells ensures its binding to cognate sensors and suppression of inhibitors of MTOR, leading to increased global protein synthesis. On the other hand, deprivation of amino acids activates the amino acid response pathway (GCN2-eIF2a-ATF4), leading to increased selective translation of the activating transcription factor 4 (ATF4). Deficiency of an amino acid itself or via the action of ATF4 suppression of MTORC1 activity limits global protein synthesis. ATF4, in response to low concentrations of cellular amino acids, mediates the transcription of groups of genes such as those for amino acid transport and biosynthesis (ASNS, CAT-1, SNAT2), autophagy (ATG3, ATG10, ATG12), and serine-glycine synthesis (PHGDH, PSAT1, PSPH, MTHFD2). Long-term amino acid starvation has a pronounced effect on cells: suppressed expression and translation of genes required for normal cell growth and metabolism and enhanced expression of genes required for cell adaptation and survival. Levels of amino acids also affect the posttranslational modifications of proteins through mechanisms such as acetylation, ADP-ribosylation, disulfide bond formation, glutamylation, and hydroxylation.
    Keywords:  ATF4; Amino acids; Gene expression; MTOR; Protein synthesis
    DOI:  https://doi.org/10.1007/978-3-030-74180-8_1
  28. Brain. 2021 Jul 15. pii: awab268. [Epub ahead of print]
    The role of mTORC1 activation in seizure-induced exacerbation of Alzheimer's disease.
    Sarah Gourmaud, David A Stewart, David J Irwin, Nicholas Roberts, Aaron J Barbour, Grace Eberwine, William T O'Brien, Robert Vassar, Delia M Talos, Frances E Jensen.
      Seizure risk is 10-fold higher in Alzheimer's disease patients than the general population, yet the mechanisms underlying this susceptibility and the effects of seizures on Alzheimer's disease are poorly understood. To elucidate our proposed bidirectional relationship between Alzheimer's disease and seizures, we studied Alzheimer's disease human brain samples (n = 34) and found that patients with a history of seizures (n = 14) had increased β-amyloid and tau pathology, and upregulation of the mechanistic target of rapamycin (mTOR) pathway compared to cases without known seizure history (n = 20). To establish whether seizures could accelerate Alzheimer's disease progression, we induced chronic hyperexcitability in the 5XFAD Alzheimer's disease mouse model by kindling with the chemoconvulsant pentylenetetrazol and observed that 5XFAD mice displayed higher seizure severity compared to wild type. Furthermore, kindled seizures exacerbated later cognitive impairment, Alzheimer's disease neuropathology and mTORC1 activation. Finally, we demonstrate that administration of the mTOR inhibitor rapamycin following kindled seizures rescued enhanced remote and long-term memory deficits associated with earlier kindling and prevented the seizure-induced increases in Alzheimer's disease neuropathology. These data demonstrate an important link between chronic hyperexcitability and progressive Alzheimer's disease pathology and suggest a mechanism whereby rapamycin may serve as an adjunct therapy to attenuate Alzheimer's disease progression.
    Keywords:  Alzheimer’s disease; cognition; mTOR; neuropathology; seizures
    DOI:  https://doi.org/10.1093/brain/awab268
  29. BMC Cancer. 2021 Jul 12. 21(1): 803
    Inhibition of mTORC1 through ATF4-induced REDD1 and Sestrin2 expression by Metformin.
    Se-Kyeong Jang, Sung-Eun Hong, Da-Hee Lee, Ji-Young Kim, Ji Yea Kim, Sang-Kyu Ye, Jungil Hong, In-Chul Park, Hyeon-Ok Jin.
       BACKGROUND: Although the major anticancer effect of metformin involves AMPK-dependent or AMPK-independent mTORC1 inhibition, the mechanisms of action are still not fully understood.
    METHODS: To investigate the molecular mechanisms underlying the effect of metformin on the mTORC1 inhibition, MTT assay, RT-PCR, and western blot analysis were performed.
    RESULTS: Metformin induced the expression of ATF4, REDD1, and Sestrin2 concomitant with its inhibition of mTORC1 activity. Treatment with REDD1 or Sestrin2 siRNA reversed the mTORC1 inhibition induced by metformin, indicating that REDD1 and Sestrin2 are important for the inhibition of mTORC1 triggered by metformin treatment. Moreover, REDD1- and Sestrin2-mediated mTORC1 inhibition in response to metformin was independent of AMPK activation. Additionally, lapatinib enhances cell sensitivity to metformin, and knockdown of REDD1 and Sestrin2 decreased cell sensitivity to metformin and lapatinib.
    CONCLUSIONS: ATF4-induced REDD1 and Sestrin2 expression in response to metformin plays an important role in mTORC1 inhibition independent of AMPK activation, and this signalling pathway could have therapeutic value.
    Keywords:  AMPK; Metformin; REDD1; Sestrin2; mTORC1
    DOI:  https://doi.org/10.1186/s12885-021-08346-x
  30. Adv Exp Med Biol. 2021 ;1332 17-33
    Amino Acids in Cell Signaling: Regulation and Function.
    Sudikshya Paudel, Guoyao Wu, Xiaoqiu Wang.
      Amino acids are the main building blocks for life. Aside from their roles in composing proteins, functional amino acids and their metabolites play regulatory roles in key metabolic cascades, gene expressions, and cell-to-cell communication via a variety of cell signaling pathways. These metabolic networks are necessary for maintenance, growth, reproduction, and immunity in humans and animals. These amino acids include, but are not limited to, arginine, glutamine, glutamate, glycine, leucine, proline, and tryptophan. We will discuss these functional amino acids in cell signaling pathways in mammals with a particular emphasis on mTORC1, AMPK, and MAPK pathways for protein synthesis, nutrient sensing, and anti-inflammatory responses, as well as cell survival, growth, and development.
    Keywords:  AMPK; Functional amino acids; MAPK; Sensor; Transceptor; mTORC1
    DOI:  https://doi.org/10.1007/978-3-030-74180-8_2
  31. Mol Cells. 2021 Jul 12.
    Combinatorial Approach Using Caenorhabditis elegans and Mammalian Systems for Aging Research.
    Gee-Yoon Lee, Jooyeon Sohn, Seung-Jae V Lee.
      Aging is associated with functional and structural declines in organisms over time. Organisms as diverse as the nematode Caenorhabditis elegans and mammals share signaling pathways that regulate aging and lifespan. In this review, we discuss recent combinatorial approach to aging research employing C. elegans and mammalian systems that have contributed to our understanding of evolutionarily conserved aging-regulating pathways. The topics covered here include insulin/IGF-1, mechanistic target of rapamycin (mTOR), and sirtuin signaling pathways; dietary restriction; autophagy; mitochondria; and the nervous system. A combinatorial approach employing high-throughput, rapid C. elegans systems, and human model mammalian systems is likely to continue providing mechanistic insights into aging biology and will help develop therapeutics against age-associated disorders.
    Keywords:  Caenorhabditis elegans; aging; combinatorial approach; lifespan; mammal
    DOI:  https://doi.org/10.14348/molcells.2021.0080
  32. Nat Cell Biol. 2021 Jul 12.
    ATG9A protects the plasma membrane from programmed and incidental permeabilization.
    Aurore Claude-Taupin, Jingyue Jia, Zambarlal Bhujabal, Meriem Garfa-Traoré, Suresh Kumar, Gustavo Peixoto Duarte da Silva, Ruheena Javed, Yuexi Gu, Lee Allers, Ryan Peters, Fulong Wang, Luciana Jesus da Costa, Sandeep Pallikkuth, Keith A Lidke, Mario Mauthe, Pauline Verlhac, Yasuo Uchiyama, Michelle Salemi, Brett Phinney, Sharon A Tooze, Muriel C Mari, Terje Johansen, Fulvio Reggiori, Vojo Deretic.
      The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.
    DOI:  https://doi.org/10.1038/s41556-021-00706-w
  33. Placenta. 2021 Jul 08. pii: S0143-4004(21)00197-1. [Epub ahead of print]112 36-44
    Viral model of maternal immune activation alters placental AMPK and mTORC1 signaling in rats.
    Eliza R McColl, Micheline Piquette-Miller.
       INTRODUCTION: Maternal immune activation (MIA) is associated with neurodevelopmental disorders in offspring. We previously demonstrated that poly(I:C)-mediated MIA alters placental and fetal brain amino acid transporter expression in rats, which could potentially play a role in altered neurodevelopment; however, the mechanism(s) underlying these changes in amino acid transporter expression remain unknown. The objective of the current study was to investigate the mechanism(s) underlying poly(I:C)-mediated changes in the expression of the amino acid transporters in the placenta.
    METHODS: Pregnant rats received poly(I:C) on gestational day 14 and placentas were collected 6 h later. Mass spectrometry-based proteomics of placentas was performed followed by pathway enrichment analysis. Activation of mTORC1 and its upstream regulator, AMPK, was investigated using immunoblotting. Finally, the role of mTORC1 and AMPK in regulating the expression and localization of the amino acid transporters EAAT2 and ASCT1 was investigated in the human choriocarcinoma cell line JAR.
    RESULTS: The impact of poly(I:C) on the placental proteome was highly sexually dimorphic. While proteomics-based pathway enrichment analysis indicated enrichment of mTOR signaling in male placentas only, further investigation revealed inhibition of mTORC1 in both male and female placentas in addition to activation of AMPK. In vitro, activation of AMPK and inhibition of mTORC1 decreased membrane localization of EAAT2 and ASCT1.
    DISCUSSION: Poly(I:C)-mediated MIA activates AMPK and inhibits mTORC1 in rat placenta, both of which decrease expression and membrane localization of EAAT2 and ASCT1 in vitro. Thus, AMPK/mTORC1 signaling could be a novel treatment target for alleviating MIA-mediated changes in placental amino acid transport.
    Keywords:  Amino acid transporters; Inflammation; Maternal immune activation; Neurodevelopment; Placenta; Pregnancy
    DOI:  https://doi.org/10.1016/j.placenta.2021.07.002
  34. Cell Rep. 2021 Jul 13. pii: S2211-1247(21)00770-1. [Epub ahead of print]36(2): 109372
    Inhibition of Rag GTPase signaling in mice suppresses B cell responses and lymphomagenesis with minimal detrimental trade-offs.
    Ana Ortega-Molina, Cristina Lebrero-Fernández, Alba Sanz, Nerea Deleyto-Seldas, Ana Belén Plata-Gómez, Camino Menéndez, Osvaldo Graña-Castro, Eduardo Caleiras, Alejo Efeyan.
      B lymphocytes are exquisitely sensitive to fluctuations in nutrient signaling by the Rag GTPases, and 15% of follicular lymphomas (FLs) harbor activating mutations in RRAGC. Hence, a potential therapeutic approach against malignant B cells is to inhibit Rag GTPase signaling, but because such inhibitors are still to be developed, efficacy and safety remain unknown. We generated knockin mice expressing a hypomorphic variant of RagC (Q119L); RagCQ119L/+ mice are viable and show attenuated nutrient signaling. B lymphocyte activation is cell-intrinsically impaired in RagCQ119L/+ mice, which also show significant suppression of genetically induced lymphomagenesis and autoimmunity. Surprisingly, no overt systemic trade-offs or phenotypic alterations caused by partial suppression of nutrient signaling are seen in other organs, and RagCQ119L/+ mice show normal longevity and normal age-dependent health decline. These results support the efficacy and safety of moderate inhibition of nutrient signaling against pathological B cells.
    Keywords:  B cell lymphoma; RRAGC; Rag GTPase; aging; cell growth; germinal center; longevity; lymphocytes; mTOR; nutrient signaling
    DOI:  https://doi.org/10.1016/j.celrep.2021.109372
  35. Orphanet J Rare Dis. 2021 Jul 15. 16(1): 312
    Glaucoma in mucopolysaccharidoses.
    Weijing Kong, Jing Zhang, Cheng Lu, Yingxue Ding, Yan Meng.
      Mucopolysaccharidoses are a group of lysosomal storage disorders that are caused by deficiency of enzymes involved in glycosaminoglycans degradation. Due to low prevalence and high childhood mortality, researches on mucopolysaccharidoses were mainly focused on the fatal manifestations. With the development of treatments, more and more mucopolysaccharidoses patients were treated by approved therapies, thereby getting prolonged life span and improved quality of life. Abnormal accumulation of glycosaminoglycans in the eye may block trabecular meshwork, thicken sclera and change mechanical behavior of lamina cribrosa, which, by increasing intraocular pressure and damaging optic nerve, could cause glaucoma. Glaucoma was the leading cause of irreversible blindness worldwide, but it was rarely reported in mucopolysaccharidoses patients. Although non-fatal, it seriously affected quality of life. Prevalence of glaucoma in mucopolysaccharidoses patients (ranged from 2.1 to 12.5%) indicated that glaucoma in patients with mucopolysaccharidoses was worthy of attention and further study, thereby improving the quality of life for MPSs patients.
    Keywords:  Glaucoma; Glycosaminoglycans; Lysosomal storage disorders; Mucopolysaccharidoses; Mucopolysaccharidosis; Rare disease
    DOI:  https://doi.org/10.1186/s13023-021-01935-w
  36. Mol Genet Metab. 2021 Jul 07. pii: S1096-7192(21)00743-5. [Epub ahead of print]
    A multicenter open-label extension study of intrathecal heparan-N-sulfatase in patients with Sanfilippo syndrome type A.
    Frits A Wijburg, Chester B Whitley, Joseph Muenzer, Serena Gasperini, Mireia Del Toro, Nicole Muschol, Maureen Cleary, Caroline Sevin, Elsa Shapiro, David Alexanderian.
      Sanfilippo syndrome type A (mucopolysaccharidosis type IIIA) is a rare autosomal recessive lysosomal disorder characterized by deficient heparan-N-sulfatase (HNS) activity, and subsequent accumulation of heparan sulfate, especially in the central nervous system. The disease is associated with progressive neurodegeneration in early childhood. For this open-label extension study of a phase 2b clinical trial, we report on safety and cognitive decline in patients receiving intrathecal (IT) administration of recombinant human HNS (rhHNS). Of 21 patients who completed the phase 2b study, 17 continued in the open-label extension. Patients receiving rhHNS IT 45 mg continued to receive the same treatment regimen (i.e., every 2 weeks or every 4 weeks) throughout the extension. Patients receiving no treatment in the phase 2b study were re-randomized to the treatment groups. Neurocognition was assessed using the Bayley Scales of Infant and Toddler Development®, Third Edition (BSID-III). Adverse events were recorded over the duration of the treatment period. Cognitive decline was observed in most patients in both treatment groups; however, improvements in BSID-III development quotient score were observed for two patients, in receptive and expressive communication scores for three patients each, in fine motor skills for one patient, and in gross motor skills for six patients. Treatment-emergent adverse events that occurred with rhHNS IT were mostly mild, none led to study discontinuation, and there were no deaths. The extension study was terminated early as the primary endpoints of the phase 2b study were not met, and no statistical analyses were carried out. Although cognitive decline was apparent in most patients, improvements were observed in a small group of patients. Greater declines were observed in patients at the higher end of the age range, suggesting earlier intervention may increase the possibility of a response to treatment. rhHNS IT treatment remained generally well tolerated up to 96 weeks.
    Keywords:  Enzyme replacement therapy; Lysosomal storage disease; Mucopolysaccharidosis type IIIA; Recombinant human heparan-N-sulfatase; Sanfilippo syndrome type A
    DOI:  https://doi.org/10.1016/j.ymgme.2021.07.001
  37. Front Neurol. 2021 ;12 694764
    Exploring the Genotype-Phenotype Correlation in GBA-Parkinson Disease: Clinical Aspects, Biomarkers, and Potential Modifiers.
    Elisa Menozzi, Anthony H V Schapira.
      Variants in the glucocerebrosidase (GBA) gene are the most common genetic risk factor for Parkinson disease (PD). These include pathogenic variants causing Gaucher disease (GD) (divided into "severe," "mild," or "complex"-resulting from recombinant alleles-based on the phenotypic effects in GD) and "risk" variants, which are not associated with GD but nevertheless confer increased risk of PD. As a group, GBA-PD patients have more severe motor and nonmotor symptoms, faster disease progression, and reduced survival compared with noncarriers. However, different GBA variants impact variably on clinical phenotype. In the heterozygous state, "complex" and "severe" variants are associated with a more aggressive and rapidly progressive disease. Conversely, "mild" and "risk" variants portend a more benign course. Homozygous or compound heterozygous carriers usually display severe phenotypes, akin to heterozygous "complex" or "severe" variants carriers. This article reviews genotype-phenotype correlations in GBA-PD, focusing on clinical and nonclinical aspects (neuroimaging and biochemical markers), and explores other disease modifiers that deserve consideration in the characterization of these patients.
    Keywords:  GBA; Parkinson's; biomarker; genotype-phenotype; glucocerebrosidase
    DOI:  https://doi.org/10.3389/fneur.2021.694764
  38. Elife. 2021 Jul 14. pii: e67399. [Epub ahead of print]10
    Loss of Tsc1 in cerebellar Purkinje cells induces transcriptional and translation changes in FMRP target transcripts.
    Jasbir Singh Dalal, Kellen Diamond Winden, Catherine Lourdes Salussolia, Maria Sundberg, Achint Singh, Truc Thanh Pham, Pingzhu Zhou, William T Pu, Meghan T Miller, Mustafa Sahin.
      Tuberous sclerosis complex (TSC) is a genetic disorder that is associated with multiple neurological manifestations. Previously, we demonstrated that Tsc1 loss in cerebellar Purkinje cells (PCs) can cause altered social behavior in mice. Here, we performed detailed transcriptional and translational analyses of Tsc1-deficient PCs to understand the molecular alterations in these cells. We found that target transcripts of the Fragile X Mental Retardation Protein (FMRP) are reduced in mutant PCs with evidence of increased degradation. Surprisingly, we observed unchanged ribosomal binding for many of these genes using translating ribosome affinity purification. Finally, we found that multiple FMRP targets, including SHANK2, were reduced, suggesting that compensatory increases in ribosomal binding efficiency may be unable to overcome reduced transcript levels. These data further implicate dysfunction of FMRP and its targets in TSC and suggest that treatments aimed at restoring the function of these pathways may be beneficial.
    Keywords:  FMRP; Tuberous Sclerosis; autism; mTOR; mouse; neuroscience; ribosome; translation
    DOI:  https://doi.org/10.7554/eLife.67399
  39. Sci Rep. 2021 Jul 14. 11(1): 14486
    Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.
    Michael C Babcock, Christina R Mikulka, Bing Wang, Sanjay Chandriani, Sundeep Chandra, Yue Xu, Katherine Webster, Ying Feng, Hemanth R Nelvagal, Alex Giaramita, Bryan K Yip, Melanie Lo, Xuntian Jiang, Qi Chao, Josh C Woloszynek, Yuqiao Shen, Shripad Bhagwat, Mark S Sands, Brett E Crawford.
      Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.
    DOI:  https://doi.org/10.1038/s41598-021-93601-1
  40. Sci Rep. 2021 Jul 16. 11(1): 14611
    Prognostic significance and oncogene function of cathepsin A in hepatocellular carcinoma.
    Huaxiang Wang, Fengfeng Xu, Fang Yang, Lizhi Lv, Yi Jiang.
      Cathepsin A (CTSA) is a lysosomal protease that regulates galactoside metabolism. The previous study has shown CTSA is abnormally expressed in various types of cancer. However, rarely the previous study has addressed the role of CTSA in hepatocellular carcinoma (HCC) and its prognostic value. To study the clinical value and potential function of CTSA in HCC, datasets from the Cancer Genome Atlas (TCGA) database and a 136 HCC patient cohort were analyzed. CTSA expression was found to be significantly higher in HCC patients compared with normal liver tissues, which was supported by immunohistochemistry (IHC) validation. Both gene ontology (GO) and The Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that CTSA co-expressed genes were involved in ATP hydrolysis coupled proton transport, carbohydrate metabolic process, lysosome organization, oxidative phosphorylation, other glycan degradation, etc. Survival analysis showed a significant reduction both in overall survival (OS) and recurrence-free survival (RFS) of patients with high CTSA expression from both the TCGA HCC cohort and 136 patients with the HCC cohort. Furthermore, CTSA overexpression has diagnostic value in distinguishing between HCC and normal liver tissue [Area under curve (AUC) = 0.864]. Moreover, Gene set enrichment analysis (GSEA) showed that CTSA expression correlated with the oxidative phosphorylation, proteasome, and lysosome, etc. in HCC tissues. These findings demonstrate that CTSA may as a potential diagnostic and prognostic biomarker in HCC.
    DOI:  https://doi.org/10.1038/s41598-021-93998-9
  41. EMBO J. 2021 Jul 16. e107480
    Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation.
    Jialiang Shao, Tiezhu Shi, Hua Yu, Yufeng Ding, Liping Li, Xiang Wang, Xiongjun Wang.
      The mTORC1 pathway plays key roles in regulating various biological processes, including sensing amino acid deprivation and driving expression of ribosomal protein (RP)-coding genes. In this study, we observed that depletion of glutamate dehydrogenase 1 (GDH1), an enzyme that converts glutamate to α-ketoglutarate (αKG), confers resistance to amino acid deprivation on kidney renal clear cell carcinoma (KIRC) cells. Mechanistically, under conditions of adequate nutrition, GDH1 maintains RP gene expression in a manner dependent on its enzymatic activity. Following amino acid deprivation or mTORC1 inhibition, GDH1 translocates from mitochondria to the cytoplasm, where it becomes ubiquitinated and degraded via the E3 ligase RNF213. GDH1 degradation reduces intracellular αKG levels by more than half and decreases the activity of αKG-dependent lysine demethylases (KDMs). Reduced KDM activity in turn leads to increased histone H3 lysine 9 and 27 methylation, further suppressing RP gene expression and preserving nutrition to support cell survival. In summary, our study exemplifies an economical and efficient strategy of solid tumor cells for coping with amino acid deficiency, which might in the future be targeted to block renal carcinoma progression.
    Keywords:  GDH1; amino acid deprivation; kidney cancer; ribosomes; αKG
    DOI:  https://doi.org/10.15252/embj.2020107480
  42. Cell Rep. 2021 Jul 13. pii: S2211-1247(21)00721-X. [Epub ahead of print]36(2): 109345
    SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis.
    Elma Zaganjor, Haejin Yoon, Jessica B Spinelli, Elizabeth R Nunn, Gaëlle Laurent, Paulina Keskinidis, Suganja Sivaloganathan, Shakchhi Joshi, Giulia Notarangelo, Stacy Mulei, Mathew T Chvasta, Sarah A Tucker, Krystle Kalafut, Robert A H van de Ven, Clary B Clish, Marcia C Haigis.
      Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytes capable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolic drivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumption of numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and promotes peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of adipogenesis. In early adipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcrotonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxylation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adipose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes, suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. In summary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolic events that stimulate transcriptional reprogramming.
    Keywords:  BCAA catabolism; MCCC; PPARg; SIRT4; adipogenesis; amino acids; differentiation; sirtuin
    DOI:  https://doi.org/10.1016/j.celrep.2021.109345
  43. Nat Rev Cancer. 2021 Jul 16.
    Cancer metabolism: looking forward.
    Inmaculada Martínez-Reyes, Navdeep S Chandel.
      Tumour initiation and progression requires the metabolic reprogramming of cancer cells. Cancer cells autonomously alter their flux through various metabolic pathways in order to meet the increased bioenergetic and biosynthetic demand as well as mitigate oxidative stress required for cancer cell proliferation and survival. Cancer driver mutations coupled with environmental nutrient availability control flux through these metabolic pathways. Metabolites, when aberrantly accumulated, can also promote tumorigenesis. The development and application of new technologies over the last few decades has not only revealed the heterogeneity and plasticity of tumours but also allowed us to uncover new metabolic pathways involved in supporting tumour growth. The tumour microenvironment (TME), which can be depleted of certain nutrients, forces cancer cells to adapt by inducing nutrient scavenging mechanisms to sustain cancer cell proliferation. There is growing appreciation that the metabolism of cell types other than cancer cells within the TME, including endothelial cells, fibroblasts and immune cells, can modulate tumour progression. Because metastases are a major cause of death of patients with cancer, efforts are underway to understand how metabolism is harnessed by metastatic cells. Additionally, there is a new interest in exploiting cancer genetic analysis for patient stratification and/or dietary interventions in combination with therapies that target metabolism. In this Perspective, we highlight these main themes that are currently under investigation in the context of in vivo tumour metabolism, specifically emphasizing questions that remain unanswered.
    DOI:  https://doi.org/10.1038/s41568-021-00378-6
  44. Sci Adv. 2021 Jul;pii: eabg3188. [Epub ahead of print]7(29):
    Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake.
    Miaoqing Hu, Fan Yang, Yawen Huang, Xin You, Desheng Liu, Shan Sun, Sen-Fang Sui.
      Niemann-Pick C1-like 1 (NPC1L1) protein plays a central role in the intestinal cholesterol absorption and is the target of a drug, ezetimibe, which inhibits NPC1L1 to reduce cholesterol absorption. Here, we present cryo-electron microscopy structures of human NPC1L1 in apo state, cholesterol-enriched state, and ezetimibe-bound state to reveal molecular details of NPC1L1-mediated cholesterol uptake and ezetimibe inhibition. Comparison of these structures reveals that the sterol-sensing domain (SSD) could respond to the cholesterol level alteration by binding different number of cholesterol molecules. Upon increasing cholesterol level, SSD binds more cholesterol molecules, which, in turn, triggers the formation of a stable structural cluster in SSD, while binding of ezetimibe causes the deformation of the SSD and destroys the structural cluster, leading to the inhibition of NPC1L1 function. These results provide insights into mechanisms of NPC1L1 function and ezetimibe action and are of great significance for the development of new cholesterol absorption inhibitors.
    DOI:  https://doi.org/10.1126/sciadv.abg3188
  45. Cell Signal. 2021 Jul 07. pii: S0898-6568(21)00167-4. [Epub ahead of print] 110078
    TMEM97 ablation aggravates oxidant-induced retinal degeneration.
    Hongtao Shen, Jing Li, Tyler Heisler-Taylor, Ryan Makin, Huan Yang, Timur A Mavlyutov, Bradley Gelfand, Colleen M Cebulla, Lian-Wang Guo.
      The retinal pigment epithelium (RPE) is critical to the survival of the overlying photoreceptors. Subject to light exposure and active metabolism, the RPE and photoreceptors are particularly susceptible to oxidative damage that plays an important part in age-related macular degeneration (AMD). Recent meta-analyses identified TMEM97 as a new putative AMD risk locus, though it is yet to be functionally verified. The role of TMEM97 in the retina and RPE is not known. Here we investigated TMEM97 function using the sodium iodate model of oxidant-induced retinal degeneration in TMEM97 knockout (KO) mice. We found markedly increased reactive oxygen species (ROS) and loss of photoreceptos in TMEM97 KO mouse retinas relative to wild type (WT) controls. In vitro, sodium iodate treatment of CRISPR-mediated TMEM97 KO RPE cells resulted in diminished abundance of the master antioxidant transcription factor NRF2 and its target gene product SOD2, the mitochondrial superoxide dismutase, as well as elevated ROS and apoptosis markers. Moreover, TMEM97 KO affected proteins key to mitochondrial and lysosomal stability and impeded autophagy flux. These findings suggest that the absence of TMEM97 in RPE cells disturbs redox-balancing systems, thereby heightening oxidative stress. As TMEM97 is a druggable target, this study may inspire interest in basic and translational research in the context of retinal degeneration.
    Keywords:  Lysosome; Mitochondria; Oxidative stress; Photoreceptor; RPE; Retina; TMEM97
    DOI:  https://doi.org/10.1016/j.cellsig.2021.110078
  46. Elife. 2021 Jul 13. pii: e66942. [Epub ahead of print]10
    Akt phosphorylates insulin receptor substrate to limit PI3K-mediated PIP3 synthesis.
    Alison L Kearney, Dougall M Norris, Milad Ghomlaghi, Martin Kin Lok Wong, Sean J Humphrey, Luke Carroll, Guang Yang, Kristen C Cooke, Pengyi Yang, Thomas A Geddes, Sungyoung Shin, Daniel J Fazakerley, Lan K Nguyen, David E James, James G Burchfield.
      The phosphoinositide 3-kinase (PI3K)-Akt network is tightly controlled by feedback mechanisms that regulate signal flow and ensure signal fidelity. A rapid overshoot in insulin-stimulated recruitment of Akt to the plasma membrane has previously been reported, which is indicative of negative feedback operating on acute timescales. Here, we show that Akt itself engages this negative feedback by phosphorylating insulin receptor substrate (IRS) 1 and 2 on a number of residues. Phosphorylation results in the depletion of plasma membrane-localised IRS1/2, reducing the pool available for interaction with the insulin receptor. Together these events limit plasma membrane-associated PI3K and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) synthesis. We identified two Akt-dependent phosphorylation sites in IRS2 at S306 (S303 in mouse) and S577 (S573 in mouse) that are key drivers of this negative feedback. These findings establish a novel mechanism by which the kinase Akt acutely controls PIP3 abundance, through post-translational modification of the IRS scaffold.
    Keywords:  Akt; PI3K; cell biology; computational biology; human; insulin; mouse; phosphorylation; plasma membrane; signal transduction; systems biology
    DOI:  https://doi.org/10.7554/eLife.66942
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