bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–09–04
forty-one papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. A lysosomal regulatory circuit essential for the development and function of microglia.
  2. The endoplasmic reticulum contributes to lysosomal tubulation/sorting driven by LRRK2.
  3. Mid51/Fis1 mitochondrial oligomerization complex drives lysosomal untethering and network dynamics.
  4. A LRRK2/dLRRK-mediated lysosomal pathway that contributes to glial cell death and DA neuron survival.
  5. TRPML3 enhances drug resistance in non-small cell lung cancer cells by promoting Ca2+-mediated lysosomal trafficking.
  6. Loss of endosomal exchanger NHE6 leads to pathological changes in tau in human neurons.
  7. DEPDC5-dependent mTORC1 signaling mechanisms are critical for the anti-seizure effects of acute fasting.
  8. Identification of TMEM106B amyloid fibrils provides an updated view of TMEM106B biology in health and disease.
  9. Transport-dependent maturation of organelles in neurons.
  10. Cross-species efficacy of enzyme replacement therapy for CLN1 disease in mice and sheep.
  11. Genetic variations in GBA1 and LRRK2 genes: Biochemical and clinical consequences in Parkinson disease.
  12. Ait1 regulates TORC1 signaling and localization in budding yeast.
  13. Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events and slow maturation.
  14. Analyzing the impact of autotrophic and heterotrophic metabolism on the nutrient regulation of TOR.
  15. The trilateral interactions between mammalian target of rapamycin (mTOR) signaling, the circadian clock, and psychiatric disorders: an emerging model.
  16. The RNA-binding protein AUF1 facilitates Akt phosphorylation at the membrane.
  17. S6 kinase 1 at the central node of cell size and ageing.
  18. TFEB in Alzheimer's disease: From molecular mechanisms to therapeutic implications.
  19. Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.
  20. Life in lockdown: Orchestrating endoplasmic reticulum and lysosome homeostasis for quiescent cells.
  21. mTOR inhibition prevents coronary artery remodeling in a murine model of Kawasaki disease.
  22. Ubiquitination of phosphatidylethanolamine in organellar membranes.
  23. PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain.
  24. The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells.
  25. Generation of a novel disease model mouse for mucopolysaccharidosis type VI via c. 252T>C human ARSB mutation knock-in.
  26. Rab GTPases: The principal players in crafting the regulatory landscape of endosomal trafficking.
  27. HTT (huntingtin) and RAB7 co-migrate retrogradely on a signaling LAMP1-containing late endosome during axonal injury.
  28. Structural basis for activation of Arf1 at the Golgi complex.
  29. Huntington's disease phenotypes are improved via mTORC1 modulation by small molecule therapy.
  30. Dual contribution of the mTOR pathway and of the metabolism of amino acids in prostate cancer.
  31. VAMP4 regulates insulin levels by targeting secretory granules to lysosomes.
  32. V-ATPase inhibitor targets Ras-mutant cancers.
  33. SLC4A7 and mTORC1 raise nucleotide synthesis with bicarbonate.
  34. Structure of V-ATPase from citrus fruit.
  35. Identification of two pathways mediating protein targeting from ER to lipid droplets.
  36. To grow or not to grow under nutrient scarcity: Target of rapamycin-ethylene is the question.
  37. Regulation of membrane protein structure and function by their lipid nano-environment.
  38. Adipocytes control food intake and weight regain via Vacuolar-type H+ ATPase.
  39. GPHR-mediated acidification of the Golgi lumen is essential for cholesterol biosynthesis in the brain.
  40. The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins.
  41. Cathepsin D Drives the Formation of Hybrid Insulin Peptides Relevant to the Pathogenesis of Type 1 Diabetes.
  1. Sci Adv. 2022 Sep 02. 8(35): eabp8321
    A lysosomal regulatory circuit essential for the development and function of microglia.
    Harini Iyer, Kimberle Shen, Ana M Meireles, William S Talbot.
      As the primary phagocytic cells of the central nervous system, microglia exquisitely regulate their lysosomal activity to facilitate brain development and homeostasis. However, mechanisms that coordinate lysosomal activity with microglia development, chemotaxis, and function remain unclear. Here, we show that embryonic macrophages require the lysosomal guanosine triphosphatase (GTPase) RagA and the GTPase-activating protein Folliculin to colonize the brain in zebrafish. We demonstrate that embryonic macrophages in rraga mutants show increased expression of lysosomal genes but display significant down-regulation of immune- and chemotaxis-related genes. Furthermore, we find that RagA and Folliculin repress the key lysosomal transcription factor Tfeb and its homologs Tfe3a and Tfe3b in the macrophage lineage. Using RNA sequencing, we establish that Tfeb and Tfe3 are required for activation of lysosomal target genes under conditions of stress but not for basal expression of lysosomal pathways. Collectively, our data define a lysosomal regulatory circuit essential for macrophage development and function in vivo.
    DOI:  https://doi.org/10.1126/sciadv.abp8321
  2. Mol Biol Cell. 2022 Aug 31. mbcE22040139
    The endoplasmic reticulum contributes to lysosomal tubulation/sorting driven by LRRK2.
    Luis Bonet-Ponce, Mark R Cookson.
      Lysosomes are dynamic organelles that can remodel their membrane as an adaptive response to various cell signaling events including membrane damage. Recently, we have discovered that damaged lysosomes form and sort tubules into moving vesicles. We named this process LYTL for LYsosomal Tubulation/ sorting driven by LRRK2, as the Parkinson's disease protein LRRK2 promotes tubulation by recruiting the motor adaptor protein JIP4 to lysosomes via phosphorylated RAB proteins. Here we use spinning-disk microscopy combined with super-resolution to further characterize LYTL after membrane damage with LLOMe. We identified the endoplasmic reticulum (ER) colocalizing with sites of fission of lysosome-derived tubules. In addition, modifying the morphology of the ER by reducing ER tubules leads to a decrease in LYTL sorting suggesting that contact with tubular ER is necessary for lysosomal membrane sorting. Given the central roles of LRRK2 and lysosomal biology in PD, these discoveries are likely relevant to disease pathology and highlight interactions between organelles in this model. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-04-0139
  3. J Cell Biol. 2022 Oct 03. pii: e202206140. [Epub ahead of print]221(10):
    Mid51/Fis1 mitochondrial oligomerization complex drives lysosomal untethering and network dynamics.
    Yvette C Wong, Soojin Kim, Jasmine Cisneros, Catherine G Molakal, Pingping Song, Steven J Lubbe, Dimitri Krainc.
      Lysosomes are highly dynamic organelles implicated in multiple diseases. Using live super-resolution microscopy, we found that lysosomal tethering events rarely undergo lysosomal fusion, but rather untether over time to reorganize the lysosomal network. Inter-lysosomal untethering events are driven by a mitochondrial Mid51/Fis1 complex that undergoes coupled oligomerization on the outer mitochondrial membrane. Importantly, Fis1 oligomerization mediates TBC1D15 (Rab7-GAP) mitochondrial recruitment to drive inter-lysosomal untethering via Rab7 GTP hydrolysis. Moreover, inhibiting Fis1 oligomerization by either mutant Fis1 or a Mid51 oligomerization mutant potentially associated with Parkinson's disease prevents lysosomal untethering events, resulting in misregulated lysosomal network dynamics. In contrast, dominant optic atrophy-linked mutant Mid51, which does not inhibit Mid51/Fis1 coupled oligomerization, does not disrupt downstream lysosomal dynamics. As Fis1 conversely also regulates Mid51 oligomerization, our work further highlights an oligomeric Mid51/Fis1 mitochondrial complex that mechanistically couples together both Drp1 and Rab7 GTP hydrolysis machinery at mitochondria-lysosome contact sites. These findings have significant implications for organelle networks in cellular homeostasis and human disease.
    DOI:  https://doi.org/10.1083/jcb.202206140
  4. Traffic. 2022 Aug 27.
    A LRRK2/dLRRK-mediated lysosomal pathway that contributes to glial cell death and DA neuron survival.
    Linfang Wang, Honglei Wang, Shuanglong Yi, Shiping Zhang, Margaret S Ho.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). A plethora of evidence has indicated a role for LRRK2 in endolysosomal trafficking in neurons, while LRRK2 function in glia, although highly expressed, remains largely unknown. Here we present evidence that LRRK2/dLRRK mediates a lysosomal pathway that contributes to glial cell death and the survival of dopaminergic (DA) neurons. LRRK2/dLRRK knockdown in the immortalized microglia or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, exhibit defective membrane permeability and reduced activity of the lysosome hydrolase Cathespin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, DA neurodegeneration, and locomotor deficits in an age-dependent manner. Taken together, these findings demonstrate a functional role of LRRK2/dLRRK in regulating the glial lysosomal pathway; deficits in lysosomal biogenesis and function linking to glial apoptosis potentially underlie the mechanism of DA neurodegeneration, providing insights on LRRK2/dLRRK function in normal and pathological brains.
    Keywords:  LRRK2/dLRRK; PD; glia; homeostasis; lysosome
    DOI:  https://doi.org/10.1111/tra.12866
  5. Biochem Biophys Res Commun. 2022 Aug 23. pii: S0006-291X(22)01181-0. [Epub ahead of print]627 152-159
    TRPML3 enhances drug resistance in non-small cell lung cancer cells by promoting Ca2+-mediated lysosomal trafficking.
    Mi Seong Kim, Sei Hoon Yang, Min Seuk Kim.
      Lysosomes are emerging as versatile signaling hubs that mediate numerous cellular processes, including the development of drug resistance in cancer cells. Transient receptor potential mucolipin 3 (TRPML3), an endolysosomal Ca2+-permeable channel, is implicated in regulating lysosomal trafficking during endocytosis and autophagy. However, the role of TRPML3 in cancer progression remains unclear. In this study, we focused on identifying key molecules that modulate exosomal release triggered by lysosomal exocytosis during the development of gefitinib resistance in non-small cell lung cancer (NSCLC). We found that the basal release of exosomes and lysosomal exocytosis is higher in the gefitinib-resistant NSCLC cell line HCC827/GR than in the gefitinib-sensitive NSCLC cell line HCC827. Notably, exosomal release and lysosomal exocytosis were associated with an increase in TRPML3 expression. Lysosomal Ca2+ release via TRPML3 was triggered by the gefitinib-mediated elevation of lysosomal pH. Furthermore, TRPML3 deficiency enhanced the gefitinib-mediated increase in sub-G0 cell population, reduction of cell proliferation, and poly (ADP-ribose) polymerase cleavage. These data demonstrated that TRPML3 is a promising modulator of drug resistance. By sensing the elevation of lysosomal pH, it mediates lysosomal Ca2+ release, lysosomal trafficking and exocytosis, and exosomal release. Taken together, our study is the first to report the autonomous defense mechanism developed in NSCLC cells against the small-molecule tyrosine kinase inhibitor gefitinib, leading to acquired drug resistance.
    Keywords:  Drug resistance; Gefitinib; Lysosome; Non-small cell lung cancer; Transient receptor potential channel
    DOI:  https://doi.org/10.1016/j.bbrc.2022.08.051
  6. Stem Cell Reports. 2022 Aug 17. pii: S2213-6711(22)00378-2. [Epub ahead of print]
    Loss of endosomal exchanger NHE6 leads to pathological changes in tau in human neurons.
    Marty A Fernandez, Fatmata Bah, Li Ma, YouJin Lee, Michael Schmidt, Elizabeth Welch, Eric M Morrow, Tracy L Young-Pearse.
      Disruption of endolysosomal and autophagy-lysosomal systems is increasingly implicated in neurodegeneration. Sodium-proton exchanger 6 (NHE6) contributes to the maintenance of proper endosomal pH, and loss-of function mutations in the X-linked NHE6 lead to Christianson syndrome (CS) in males. Neurodegenerative features of CS are increasingly recognized, with postmortem and clinical data implicating a role for tau. We generated cortical neurons from NHE6 knockout (KO) and isogenic wild-type control human induced pluripotent stem cells. We report elevated phosphorylated and sarkosyl-insoluble tau in NHE6 KO neurons. We demonstrate that NHE6 KO leads to lysosomal and autophagy dysfunction involving reduced lysosomal number and protease activity, diminished autophagic flux, and p62 accumulation. Finally, we show that treatment with trehalose or rapamycin, two enhancers of autophagy-lysosomal function, each partially rescue this tau phenotype. We provide insight into the neurodegenerative processes underlying NHE6 loss of function and into the broader role of the endosome-lysosome-autophagy network in neurodegeneration.
    Keywords:  Alzheimer disease and related dementias; Christianson syndrome; NHE6; SLC9A6; autophagy; endosome; iPSC; lysosome; tau; trehalose
    DOI:  https://doi.org/10.1016/j.stemcr.2022.08.001
  7. Cell Rep. 2022 Aug 30. pii: S2211-1247(22)01098-1. [Epub ahead of print]40(9): 111278
    DEPDC5-dependent mTORC1 signaling mechanisms are critical for the anti-seizure effects of acute fasting.
    Christopher J Yuskaitis, Jinita B Modasia, Sandra Schrötter, Leigh-Ana Rossitto, Karenna J Groff, Christopher Morici, Divakar S Mithal, Ram P Chakrabarty, Navdeep S Chandel, Brendan D Manning, Mustafa Sahin.
      Caloric restriction and acute fasting are known to reduce seizures but through unclear mechanisms. mTOR signaling has been suggested as a potential mechanism for seizure protection from fasting. We demonstrate that brain mTORC1 signaling is reduced after acute fasting of mice and that neuronal mTORC1 integrates GATOR1 complex-mediated amino acid and tuberous sclerosis complex (TSC)-mediated growth factor signaling. Neuronal mTORC1 is most sensitive to withdrawal of leucine, arginine, and glutamine, which are dependent on DEPDC5, a component of the GATOR1 complex. Metabolomic analysis reveals that Depdc5 neuronal-specific knockout mice are resistant to sensing significant fluctuations in brain amino acid levels after fasting. Depdc5 neuronal-specific knockout mice are resistant to the protective effects of fasting on seizures or seizure-induced death. These results establish that acute fasting reduces seizure susceptibility in a DEPDC5-dependent manner. Modulation of nutrients upstream of GATOR1 and mTORC1 could offer a rational therapeutic strategy for epilepsy treatment.
    Keywords:  CP: Metabolism; CP: Neuroscience; GATOR1; SUDEP; TSC; amino acids; cell signaling; epilepsy; fasting; mTOR; metabolomics; seizures
    DOI:  https://doi.org/10.1016/j.celrep.2022.111278
  8. Acta Neuropathol. 2022 Sep 02.
    Identification of TMEM106B amyloid fibrils provides an updated view of TMEM106B biology in health and disease.
    Jolien Perneel, Rosa Rademakers.
      Since the initial identification of TMEM106B as a risk factor for frontotemporal lobar degeneration (FTLD), multiple genetic studies have found TMEM106B variants to modulate disease risk in a variety of brain disorders and healthy aging. Neurodegenerative disorders are typically characterized by inclusions of misfolded proteins and since lysosomes are an important site for cellular debris clearance, lysosomal dysfunction has been closely linked to neurodegeneration. Consequently, many causal mutations or genetic risk variants implicated in neurodegenerative diseases encode proteins involved in endosomal-lysosomal function. As an integral lysosomal transmembrane protein, TMEM106B regulates several aspects of lysosomal function and multiple studies have shown that proper TMEM106B protein levels are crucial for maintaining lysosomal health. Yet, the precise function of TMEM106B at the lysosomal membrane is undetermined and it remains unclear how TMEM106B modulates disease risk. Unexpectedly, several independent groups recently showed that the C-terminal domain (AA120-254) of TMEM106B forms amyloid fibrils in the brain of patients with a diverse set of neurodegenerative conditions. The recognition that TMEM106B can form amyloid fibrils and is present across neurodegenerative diseases sheds new light on TMEM106B as a central player in neurodegeneration and brain health, but also raises important new questions. In this review, we summarize current knowledge and place a decade's worth of TMEM106B research into an exciting new perspective.
    Keywords:  Aggregation; Amyloid fibrils; Frontotemporal dementia; Lysosomal dysfunction; Progranulin; TMEM106B
    DOI:  https://doi.org/10.1007/s00401-022-02486-5
  9. Curr Opin Cell Biol. 2022 Aug 25. pii: S0955-0674(22)00074-6. [Epub ahead of print]78 102121
    Transport-dependent maturation of organelles in neurons.
    Amal Mathew, Sandhya P Koushika.
      Some organelles show a spatial gradient of maturation along the neuronal process where more mature organelles are found closer to the cell body. This gradient is set up by progressive maturation steps that are aided by differential organelle distribution as well as transport. Autophagosomes and endosomes mature as they acquire lysosomal membrane proteins and decrease their luminal pH as they are retrogradely transported towards the cell body. The acquisition of lysosomal proteins along the neuronal processes likely occurs through fusion or membrane exchange events with Golgi-derived donor transport carriers that are transported anterogradely from the cell body. The mechanisms by which endosomes and autophagosomes mature might be applicable to other organelles that are transported along neuronal processes. Defects in axonal transport may also contribute to the accumulation of immature organelles in neurons. Such accumulations have been seen in neurons of neurodegenerative models.
    DOI:  https://doi.org/10.1016/j.ceb.2022.102121
  10. J Clin Invest. 2022 Aug 30. pii: e163107. [Epub ahead of print]
    Cross-species efficacy of enzyme replacement therapy for CLN1 disease in mice and sheep.
    Hemanth R Nelvagal, Samantha L Eaton, Sophie H Wang, Elizabeth M Eultgen, Keigo Takahashi, Steven Q Le, Rachel Nesbitt, Joshua T Dearborn, Nicholas Siano, Ana C Puhl, Patricia I Dickson, Gerard Thompson, Fraser Murdoch, Paul M Brennan, Mark Gray, Stephen N Greenhalgh, Peter Tennant, Rachael Gregson, Eddie Clutton, James Nixon, Chris Proudfoot, Stefano Guido, Simon G Lillico, C Bruce A Whitelaw, Jui-Yun Lu, Sandra L Hofmann, Sean Ekins, Mark S Sands, Thomas M Wishart, Jonathan D Cooper.
      CLN1 disease is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by delivering monthly infusions of recombinant human PPT1 (rhPPT1) in PPT1-deficient mice (Cln1-/-), and CLN1R151X sheep to assess scale up for translation. In Cln1-/- mice, intracerebroventricular rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1 treated-mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, intracerebroventricular infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural magnetic resonance imaging and neuropathology. These findings demonstrate the feasibility and therapeutic efficacy of intracerebroventricular rhPPT1 enzyme replacement therapy. This represents a key step towards clinical testing of ERT in children with CLN1 disease and highlights the importance of a cross-species approach to developing a successful treatment strategy.
    Keywords:  Lysosomes; Monogenic diseases; Neurodegeneration; Neuroscience; Therapeutics
    DOI:  https://doi.org/10.1172/JCI163107
  11. Front Neurol. 2022 ;13 971252
    Genetic variations in GBA1 and LRRK2 genes: Biochemical and clinical consequences in Parkinson disease.
    Laura J Smith, Chiao-Yin Lee, Elisa Menozzi, Anthony H V Schapira.
      Variants in the GBA1 and LRRK2 genes are the most common genetic risk factors associated with Parkinson disease (PD). Both genes are associated with lysosomal and autophagic pathways, with the GBA1 gene encoding for the lysosomal enzyme, glucocerebrosidase (GCase) and the LRRK2 gene encoding for the leucine-rich repeat kinase 2 enzyme. GBA1-associated PD is characterized by earlier age at onset and more severe non-motor symptoms compared to sporadic PD. Mutations in the GBA1 gene can be stratified into severe, mild and risk variants depending on the clinical presentation of disease. Both a loss- and gain- of function hypothesis has been proposed for GBA1 variants and the functional consequences associated with each variant is often linked to mutation severity. On the other hand, LRRK2-associated PD is similar to sporadic PD, but with a more benign disease course. Mutations in the LRRK2 gene occur in several structural domains and affect phosphorylation of GTPases. Biochemical studies suggest a possible convergence of GBA1 and LRRK2 pathways, with double mutant carriers showing a milder phenotype compared to GBA1-associated PD. This review compares GBA1 and LRRK2-associated PD, and highlights possible genotype-phenotype associations for GBA1 and LRRK2 separately, based on biochemical consequences of single variants.
    Keywords:  GBA1; LRRK2; Parkinson's disease; glucocerebrosidase; lysosome
    DOI:  https://doi.org/10.3389/fneur.2022.971252
  12. Elife. 2022 Sep 01. pii: e68773. [Epub ahead of print]11
    Ait1 regulates TORC1 signaling and localization in budding yeast.
    Ryan L Wallace, Eric Lu, Xiangxia Luo, Andrew P Capaldi.
      The target of rapamycin complex I (TORC1) regulates cell growth and metabolism in eukaryotes. Previous studies have shown that nitrogen and amino acid signals activate TORC1 via the highly conserved small GTPases, Gtr1/2 (RagA/C in humans), and the GTPase activating complex SEAC/GATOR. However, it remains unclear if, and how, other proteins/pathways regulate TORC1 in simple eukaryotes like yeast. Here we report that the previously unstudied GPCR-like protein, Ait1, binds to TORC1-Gtr1/2 in Saccharomyces cerevisiae and holds TORC1 around the vacuole during log-phase growth. Then, during amino acid starvation, Ait1 inhibits TORC1 via Gtr1/2 using a loop that resembles the RagA/C binding domain in the human protein SLC38A9. Importantly, Ait1 is only found in the Saccharomycetaceae/codaceae, two closely related families of yeast that have lost the ancient TORC1 regulators Rheb and TSC1/2. Thus, the TORC1 circuit found in the Saccharomycetaceae/codaceae, and likely other simple eukaryotes, has undergone significant rewiring during evolution.
    Keywords:  S. cerevisiae; cell biology
    DOI:  https://doi.org/10.7554/eLife.68773
  13. Mol Biol Cell. 2022 Aug 31. mbcE22030111
    Spatiotemporal analysis of axonal autophagosome-lysosome dynamics reveals limited fusion events and slow maturation.
    Sydney E Cason, Saurabh S Mogre, Erika L F Holzbaur, Elena F Koslover.
      Macroautophagy is a homeostatic process required to clear cellular waste. Neuronal autophagosomes form constitutively in the distal tip of the axon and are actively transported toward the soma, with cargo degradation initiated en route. Cargo turnover requires autophagosomes to fuse with lysosomes to acquire degradative enzymes; however, directly imaging these fusion events in the axon is impractical. Here we use a quantitative model, parameterized and validated using data from primary hippocampal neurons, to explore the autophagosome maturation process. We demonstrate that retrograde autophagosome motility is independent from fusion, and that most autophagosomes fuse with only a few lysosomes during axonal transport. Our results indicate breakdown of the inner autophagosomal membrane is much slower in neurons than in non-neuronal cell types, highlighting the importance of this late maturation step. Together, rigorous quantitative measurements and mathematical modeling elucidate the dynamics of autophagosome-lysosome interaction and autophagosomal maturation in the axon. [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-03-0111
  14. New Phytol. 2022 Sep 02.
    Analyzing the impact of autotrophic and heterotrophic metabolism on the nutrient regulation of TOR.
    Manuel J Mallén-Ponce, María Esther Pérez-Pérez, José L Crespo.
      The target of rapamycin (TOR) protein kinase is a master regulator of cell growth in all eukaryotes, from unicellular yeast and algae to multicellular animals and plants. TOR balances the synthesis and degradation of proteins, lipids, carbohydrates and nucleic acids in response to nutrients, growth factors and cellular energy to promote cell growth. Among nutrients, amino acids and glucose are central regulators of TOR activity in evolutionary distant eukaryotes such as mammals, plants and algae. However, these organisms obtain the nutrients through totally different metabolic processes. While photosynthetic eukaryotes can use atmospheric CO2 as the sole carbon source for all reactions in the cell, heterotrophic organisms get nutrients from other sources of organic carbon including glucose. Here, we discuss the impact of autotrophic and heterotrophic metabolism on the nutrient regulation of TOR, focusing on the role of amino acids and carbon sources upstream of this signaling pathway.
    Keywords:  CO2; amino acid; carbon; glucose; nutrient; target of rapamycin (TOR)
    DOI:  https://doi.org/10.1111/nph.18450
  15. Transl Psychiatry. 2022 Aug 31. 12(1): 355
    The trilateral interactions between mammalian target of rapamycin (mTOR) signaling, the circadian clock, and psychiatric disorders: an emerging model.
    Rubal Singla, Abhishek Mishra, Ruifeng Cao.
      Circadian (~24 h) rhythms in physiology and behavior are evolutionarily conserved and found in almost all living organisms. The rhythms are endogenously driven by daily oscillatory activities of so-called "clock genes/proteins", which are widely distributed throughout the mammalian brain. Mammalian (mechanistic) target of rapamycin (mTOR) signaling is a fundamental intracellular signal transduction cascade that controls important neuronal processes including neurodevelopment, synaptic plasticity, metabolism, and aging. Dysregulation of the mTOR pathway is associated with psychiatric disorders including autism spectrum disorders (ASD) and mood disorders (MD), in which patients often exhibit disrupted daily physiological rhythms and abnormal circadian gene expression in the brain. Recent work has found that the activities of mTOR signaling are temporally controlled by the circadian clock and exhibit robust circadian oscillations in multiple systems. In the meantime, mTOR signaling regulates fundamental properties of the central and peripheral circadian clocks, including period length, entrainment, and synchronization. Whereas the underlying mechanisms remain to be fully elucidated, increasing clinical and preclinical evidence support significant crosstalk between mTOR signaling, the circadian clock, and psychiatric disorders. Here, we review recent progress in understanding the trilateral interactions and propose an "interaction triangle" model between mTOR signaling, the circadian clock, and psychiatric disorders (focusing on ASD and MD).
    DOI:  https://doi.org/10.1038/s41398-022-02120-8
  16. J Biol Chem. 2022 Aug 27. pii: S0021-9258(22)00880-8. [Epub ahead of print] 102437
    The RNA-binding protein AUF1 facilitates Akt phosphorylation at the membrane.
    Mei-Ling Li, Aparna Ragupathi, Nikhil Patel, Tatiana Hernandez, Jedrick Magsino, Guy Werlen, Gary Brewer, Estela Jacinto.
      mTOR, which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. We determined that AUF1 is required for phosphorylation of Akt at Thr308, Thr450, and Ser473, and that AUF1 also mediates phosphorylation of the mTORC2-modulated metabolic enzyme GFAT1 at Ser243. Additionally, AUF1 immunoprecipitation followed by qRT-PCR revealed that the mRNAs of Akt, GFAT1, and the mTORC2 component SIN1 associate with AUF1. Furthermore, expression of the p40 and p45, but not the p37 or p42, isoforms of AUF1 specifically mediate Akt phosphorylation. In the absence of AUF1, subcellular fractionation indicated that Akt fails to localize to the membrane. However, ectopic expression of a membrane-targeted allele of Akt is sufficient to allow Akt-Ser473 phosphorylation despite AUF1 depletion. Finally, conditions that enhance mTORC2 signaling, such as acute glutamine withdrawal augment AUF1 phosphorylation while mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in promoting membrane localization of Akt to facilitate its phosphorylation on this cellular compartment. Targeting AUF1 could have therapeutic benefit for cancers with upregulated mTORC2/Akt signaling.
    Keywords:  AUF1; Akt; RNA binding protein; glutamine; hnRNP D; mTOR; mTORC2
    DOI:  https://doi.org/10.1016/j.jbc.2022.102437
  17. Front Cell Dev Biol. 2022 ;10 949196
    S6 kinase 1 at the central node of cell size and ageing.
    Stefano Fumagalli, Mario Pende.
      Genetic evidence in living organisms from yeast to plants and animals, including humans, unquestionably identifies the Target Of Rapamycin kinase (TOR or mTOR for mammalian/mechanistic) signal transduction pathway as a master regulator of growth through the control of cell size and cell number. Among the mTOR targets, the activation of p70 S6 kinase 1 (S6K1) is exquisitely sensitive to nutrient availability and rapamycin inhibition. Of note, in vivo analysis of mutant flies and mice reveals that S6K1 predominantly regulates cell size versus cell proliferation. Here we review the putative mechanisms of S6K1 action on cell size by considering the main functional categories of S6K1 targets: substrates involved in nucleic acid and protein synthesis, fat mass accumulation, retrograde control of insulin action, senescence program and cytoskeleton organization. We discuss how S6K1 may be involved in the observed interconnection between cell size, regenerative and ageing responses.
    Keywords:  ageing; growth; mTOR; nutrition; senescence; signal transduction
    DOI:  https://doi.org/10.3389/fcell.2022.949196
  18. Neurobiol Dis. 2022 Aug 25. pii: S0969-9961(22)00247-9. [Epub ahead of print] 105855
    TFEB in Alzheimer's disease: From molecular mechanisms to therapeutic implications.
    Zhongya Gu, Huan Cao, Chengchao Zuo, Yaqi Huang, Jinfeng Miao, Yu Song, Yuyan Yang, Liudi Zhu, Furong Wang.
      Alzheimer's disease (AD), an age-dependent neurodegenerative disorder, is the most prevalent neurodegenerative disease worldwide. The primary pathological hallmarks of AD are the deposition of β-amyloid plaques and neurofibrillary tangles. Autophagy, a pathway of clearing damaged organelles, macromolecular aggregates, and long-lived proteins via lysosomal degradation, has emerged as critical for proteostasis in the central nervous system (CNS). Studies have demonstrated that defective autophagy is strongly implicated in AD pathogenesis. Transcription factor EB (TFEB), a master transcriptional regulator of autophagy, enhances the expression of related genes that control autophagosome formation, lysosome function, and autophagic flux. The study of TFEB has greatly increased over the last decade, and the dysfunction of TFEB has been reported to be strongly associated with the pathogenesis of many neurodegenerative disorders, including AD. Here, we delineate the basic understanding of TFEB dysregulation involved in AD pathogenesis, highlighting the existing work that has been conducted on TFEB-mediated autophagy in neurons and other nonneuronal cells in the CNS. Additionally, we summarize the small molecule compounds that target TFEB-regulated autophagy involved in AD therapy. Our review may yield new insights into therapeutic approaches by targeting TFEB and provide a broadly applicable basis for the clinical treatment of AD.
    Keywords:  Alzheimer's disease; Autophagy; Aβ plaque; Lysosome; Tau phosphorylation; Transcription factor EB
    DOI:  https://doi.org/10.1016/j.nbd.2022.105855
  19. Pancreatology. 2022 Aug 23. pii: S1424-3903(22)00482-3. [Epub ahead of print]
    Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.
    Wenting Chen, Mai Imasaka, Hideaki Iwama, Hiroshi Nishiura, Masaki Ohmuraya.
       BACKGROUND: Premature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP.
    METHODS: AP was induced by 7 hourly intraperitoneal injections of cerulein (50 μg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (CtsbΔpan), Ctsl knockout (CtslΔpan), and Ctsb;Ctsl double-knockout (CtsbΔpan;CtslΔpan) mice. Pancreatic samples were collected and analyzed by histology, immunohistochemistry, real-time PCR, and immunoblots. Trypsin activity was measured in pancreatic homogenates. Peripheral blood was collected, and serum amylase activity was measured.
    RESULTS: Double deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. CtsbΔpan;CtslΔpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, CtsbΔpan, and CtslΔpan mice.
    CONCLUSION: Double deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.
    Keywords:  Cysteine proteases; Inflammation; Knockout mice; Lysosomal proteins
    DOI:  https://doi.org/10.1016/j.pan.2022.08.011
  20. Mol Cell. 2022 Aug 25. pii: S1097-2765(22)00758-4. [Epub ahead of print]
    Life in lockdown: Orchestrating endoplasmic reticulum and lysosome homeostasis for quiescent cells.
    Andrew Murley, Kevin Wickham, Andrew Dillin.
      Cellular quiescence-reversible exit from the cell cycle-is an important feature of many cell types important for organismal health. Quiescent cells activate protective mechanisms that allow their persistence in the absence of growth and division for long periods of time. Aging and cellular dysfunction compromise the survival and re-activation of quiescent cells over time. Counteracting this decline are two interconnected organelles that lie at opposite ends of the secretory pathway: the endoplasmic reticulum and lysosomes. In this review, we highlight recent studies exploring the roles of these two organelles in quiescent cells from diverse contexts and speculate on potential other roles they may play, such as through organelle contact sites. Finally, we discuss emerging models of cellular quiescence, utilizing new cell culture systems and model organisms, that are suited to the mechanistic investigation of the functions of these organelles in quiescent cells.
    Keywords:  ER; aging; lysosome; quiescence; stem cells
    DOI:  https://doi.org/10.1016/j.molcel.2022.08.005
  21. Arthritis Rheumatol. 2022 Sep 03.
    mTOR inhibition prevents coronary artery remodeling in a murine model of Kawasaki disease.
    Angus T Stock, Sarah Parsons, Damian B D'Silva, Jacinta A Hansen, Varun J Sharma, Fiona James, Graham Starkey, Rohit D'Costa, Claire L Gordon, Ian P Wicks.
       OBJECTIVE: Remodeling of the coronary arteries is a common feature in severe cases of Kawasaki Disease (KD). This pathology is driven by the dysregulated proliferation of vascular fibroblasts which can lead to coronary artery aneurysms, stenosis and myocardial ischemia. We therefore investigated whether inhibiting fibroblast proliferation might be an effective therapeutic strategy to prevent coronary artery remodeling in KD.
    METHOD: We used a murine model of KD (induced by the injection of the Candida albicans water soluble complex; CAWS) and analysis of patient samples to evaluate potential anti-fibrotic therapies for KD.
    RESULTS: We identified the mTOR pathway as a potential therapeutic target in KD. The mTOR inhibitor rapamycin potently inhibited cardiac fibroblast proliferation in vitro, and vascular fibroblasts upregulated mTOR kinase signaling in vivo during the CAWS mouse model of KD. We evaluated the in vivo efficacy of mTOR inhibition and found that the therapeutic administration of rapamycin reduced vascular fibrosis and intimal hyperplasia of the coronary arteries in CAWS injected mice. Furthermore, the analysis of cardiac tissue from KD fatalities revealed that vascular fibroblasts localizing with inflamed coronary arteries upregulate mTOR signaling, confirming that the mTOR pathway is active in human KD.
    CONCLUSIONS: Our findings demonstrate that mTOR signaling contributes to coronary artery remodeling in KD, and that targeting this pathway offers a potential therapeutic strategy to prevent or restrict this pathology in high-risk KD patients. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1002/art.42340
  22. Mol Cell. 2022 Aug 23. pii: S1097-2765(22)00761-4. [Epub ahead of print]
    Ubiquitination of phosphatidylethanolamine in organellar membranes.
    Jun-Ichi Sakamaki, Koji L Ode, Yoshitaka Kurikawa, Hiroki R Ueda, Hayashi Yamamoto, Noboru Mizushima.
      The covalent conjugation of ubiquitin family proteins is a widespread post-translational protein modification. In the ubiquitin family, the ATG8 subfamily is exceptional because it is conjugated mainly to phospholipids. However, it remains unknown whether other ubiquitin family proteins are also conjugated to phospholipids. Here, we report that ubiquitin is conjugated to phospholipids, mainly phosphatidylethanolamine (PE), in yeast and mammalian cells. Ubiquitinated PE (Ub-PE) accumulates at endosomes and the vacuole (or lysosomes), and its level increases during starvation. Ub-PE is also found in baculoviruses. In yeast, PE ubiquitination is catalyzed by the canonical ubiquitin system enzymes Uba1 (E1), Ubc4/5 (E2), and Tul1 (E3) and is reversed by Doa4. Liposomes containing Ub-PE recruit the ESCRT components Vps27-Hse1 and Vps23 in vitro. Ubiquitin-like NEDD8 and ISG15 are also conjugated to phospholipids. These findings suggest that the conjugation to membrane phospholipids is not specific to ATG8 but is a general feature of the ubiquitin family.
    Keywords:  Doa4; Tul1; endosome; lysosome; phosphatidylethanolamine; phospholipids; ubiquitin; ubiquitin-like proteins; vacuole
    DOI:  https://doi.org/10.1016/j.molcel.2022.08.008
  23. Biochem J. 2022 Aug 30. pii: BCJ20220308. [Epub ahead of print]
    PKC isoforms activate LRRK1 kinase by phosphorylating conserved residues (Ser1064, Ser1074 and Thr1075) within the CORB GTPase domain.
    Asad U Malik, Athanasios Karapetsas, Raja S Nirujogi, Deep Chatterjee, Toan K Phung, Melanie Wightman, Robert Gourlay, Nicholas A Morrice, Sebastian Mathea, Stefan Knapp, Dario R Alessi.
      Leucine-rich-repeat-kinase 1 (LRRK1) and its homologue LRRK2 are multidomain kinases possessing a ROC-CORA-CORB containing GTPase domain and phosphorylate distinct Rab proteins. LRRK1 loss of function mutations cause the bone disorder osteosclerotic metaphyseal dysplasia, whereas LRRK2 missense mutations that enhance kinase activity cause Parkinson's disease. Previous work suggested that LRRK1 but not LRRK2, is activated via a Protein Kinase C (PKC)-dependent mechanism. Here we demonstrate that phosphorylation and activation of LRRK1 in HEK293 cells is blocked by PKC inhibitors including LXS-196 (Darovasertib), a compound that has entered clinical trials. We show multiple PKC isoforms phosphorylate and activate recombinant LRRK1 in a manner reversed by phosphatase treatment. PKCα unexpectedly does not activate LRRK1 by phosphorylating the kinase domain, but instead phosphorylates a cluster of conserved residues (Ser1064, Ser1074 and Thr1075) located within a region of the CORB domain of the GTPase domain. These residues are positioned at the equivalent region of the LRRK2 DK helix reported to stabilize the kinase domain αC-helix in the active conformation. Thr1075 represents an optimal PKC site phosphorylation motif and its mutation to Ala, blocked PKC-mediated activation of LRRK1. A triple Glu mutation of Ser1064/Ser1074/Thr1075 to mimic phosphorylation, enhanced LRRK1 kinase activity ~3-fold. From analysis of available structures, we postulate that phosphorylation of Ser1064, Ser1074 and Thr1075 activates LRRK1 by promoting interaction and stabilization of the aC-helix on the kinase domain. This study provides new fundamental insights into the mechanism controlling LRRK1 activity and reveals a novel unexpected activation mechanism.
    Keywords:  GTPases; RAB GTPases; leucine rich repeat kinase; protein kinase C
    DOI:  https://doi.org/10.1042/BCJ20220308
  24. EMBO J. 2022 Aug 29. e111161
    The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells.
    Cynthia López-Haber, Daniel J Netting, Zachary Hutchins, Xianghui Ma, Kathryn E Hamilton, Adriana R Mantegazza.
      Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1β production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1β production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1β levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.
    Keywords:  SLC15A4; dendritic cells; inflammasomes; mTORC1; phagocytosis
    DOI:  https://doi.org/10.15252/embj.2022111161
  25. Biochem Biophys Rep. 2022 Sep;31 101321
    Generation of a novel disease model mouse for mucopolysaccharidosis type VI via c. 252T>C human ARSB mutation knock-in.
    Kosuke Hosoba.
      Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a mutation in the ARSB gene, which encodes arylsulfatase B (ARSB), and is characterized by glycosaminoglycan accumulation. Some pathogenic mutations have been identified in or near the substrate-binding pocket of ARSB, whereas many missense mutations present far from the substrate-binding pocket. Each MPS VI patient shows different severity of clinical symptoms. To understand the relationship between mutation patterns and the severity of MPS VI clinical symptoms, mutations located far from the substrate-binding pocket must be investigated using mutation knock-in mice. Here, I generated a knock-in mouse model of human ARSB Y85H mutation identified in Japanese MPS VI patients using a CRISPR-Cas9-mediated approach. The generated mouse model exhibited phenotypes similar to those of MPS VI patients, including facial features, mucopolysaccharide accumulation, and smaller body size, suggesting that this mouse will be a valuable model for understanding MPS VI pathology.
    Keywords:  Arylsulfatase B; CRISPR-Cas9 system; Disease model; Mucopolysaccharidosis type VI
    DOI:  https://doi.org/10.1016/j.bbrep.2022.101321
  26. Comput Struct Biotechnol J. 2022 ;20 4464-4472
    Rab GTPases: The principal players in crafting the regulatory landscape of endosomal trafficking.
    Jing Zhang, Zongyan Jiang, Anbing Shi.
      After endocytosis, diverse cargos are sorted into endosomes and directed to various destinations, including extracellular macromolecules, membrane lipids, and membrane proteins. Some cargos are returned to the plasma membrane via endocytic recycling. In contrast, others are delivered to the Golgi apparatus through the retrograde pathway, while the rest are transported to late endosomes and eventually to lysosomes for degradation. Rab GTPases are major regulators that ensure cargos are delivered to their proper destinations. Rabs are localized to distinct endosomes and play predominant roles in membrane budding, vesicle formation and motility, vesicle tethering, and vesicle fusion by recruiting effectors. The cascades between Rabs via shared effectors or the recruitment of Rab activators provide an additional layer of spatiotemporal regulation of endocytic trafficking. Notably, several recent studies have indicated that disorders of Rab-mediated endocytic transports are closely associated with diseases such as immunodeficiency, cancer, and neurological disorders.
    DOI:  https://doi.org/10.1016/j.csbj.2022.08.016
  27. Autophagy. 2022 Sep 01.
    HTT (huntingtin) and RAB7 co-migrate retrogradely on a signaling LAMP1-containing late endosome during axonal injury.
    Thomas J Krzystek, Joseph A White, Rasika Rathnayake, Layne Thurston, Hayley Hoffmar-Glennon, Yichen Li, Shermali Gunawardena.
      HTT (huntingtin) is a 350-kDa protein of unknown function. While HTT moves bidirectionally within axons and HTT loss/reduction causes axonal transport defects, the identity of cargo-containing vesicles that HTT helps move remain elusive. Previously, we found an axonal retrogradely moving HTT-Rab7 vesicle complex; however, its biological relevance is unclear. Using Drosophila genetics, in vivo microscopy, membrane isolation and pharmacological inhibition, here we identified that adaptors Hip1 and Rilpl aid the retrograde motility of LAMP1-containing HTT-Rab7 late endosomes, not autophagosomes. Reduction of Syx17 and chloroquine- or bafilomycin A1-mediated pharmacological inhibition, but not reduction of Atg5, disrupted the in vivo motility of these vesicles. Further, because HTT-Rab7 vesicles colocalized with long-distance signaling components (BMP signaling: tkv-wit, injury: wnd) and move in a retrograde direction after Drosophila nerve crush, we propose that these vesicles likely traffic damage signals following axonal injury. Together, our findings support a previously unknown role for HTT in the retrograde movement of a Rab7-LAMP1-containing signaling late endosome.
    Keywords:  Axonal injury; Rab7; axonal transport; drosophila; endolysosomes; huntingtin; late endosome; signaling endosome
    DOI:  https://doi.org/10.1080/15548627.2022.2119351
  28. Cell Rep. 2022 Aug 30. pii: S2211-1247(22)01102-0. [Epub ahead of print]40(9): 111282
    Structural basis for activation of Arf1 at the Golgi complex.
    Arnold J Muccini, Margaret A Gustafson, J Christopher Fromme.
      The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.
    Keywords:  ARF1; CP: Cell biology; GTPase; Golgi; cryoEM; membrane; membrane insertion
    DOI:  https://doi.org/10.1016/j.celrep.2022.111282
  29. PLoS One. 2022 ;17(8): e0273710
    Huntington's disease phenotypes are improved via mTORC1 modulation by small molecule therapy.
    Sophie St-Cyr, Daniel D Child, Emilie Giaime, Alicia R Smith, Christine J Pascua, Seung Hahm, Eddine Saiah, Beverly L Davidson.
      Huntington's Disease (HD) is a dominantly inherited neurodegenerative disease for which the major causes of mortality are neurodegeneration-associated aspiration pneumonia followed by cardiac failure. mTORC1 pathway perturbations are present in HD models and human tissues. Amelioration of mTORC1 deficits by genetic modulation improves disease phenotypes in HD models, is not a viable therapeutic strategy. Here, we assessed a novel small molecule mTORC1 pathway activator, NV-5297, for its improvement of the disease phenotypes in the N171-82Q HD mouse model. Oral dosing of NV-5297 over 6 weeks activated mTORC1, increased striatal volume, improved motor learning and heart contractility. Further, the heart contractility, heart fibrosis, and survival were improved in response to the cardiac stressor isoprenaline when compared to vehicle-treated mice. Cummulatively, these data support mTORC1 activation as a therapeutic target in HD and consolidates NV-5297 as a promising drug candidate for treating central and peripheral HD phenotypes and, more generally, mTORC1-deficit related diseases.
    DOI:  https://doi.org/10.1371/journal.pone.0273710
  30. Cell Oncol (Dordr). 2022 Aug 29.
    Dual contribution of the mTOR pathway and of the metabolism of amino acids in prostate cancer.
    Alejandro Schcolnik-Cabrera, Daniel Juárez-López.
       BACKGROUND: Prostate cancer is the leading cause of cancer in men, and its incidence increases with age. Among other risk factors, pre-existing metabolic diseases have been recently linked with prostate cancer, and our current knowledge recognizes prostate cancer as a condition with important metabolic anomalies as well. In malignancies, metabolic disorders are commonly associated with aberrations in mTOR, which is the master regulator of protein synthesis and energetic homeostasis. Although there are reports demonstrating the high dependency of prostate cancer cells for lipid derivatives and even for carbohydrates, the understanding regarding amino acids, and the relationship with the mTOR pathway ultimately resulting in metabolic aberrations, is still scarce.
    CONCLUSIONS AND PERSPECTIVES: In this review, we briefly provide evidence supporting prostate cancer as a metabolic disease, and discuss what is known about mTOR signaling and prostate cancer. Next, we emphasized on the amino acids glutamine, leucine, serine, glycine, sarcosine, proline and arginine, commonly related to prostate cancer, to explore the alterations in their regulatory pathways and to link them with the associated metabolic reprogramming events seen in prostate cancer. Finally, we display potential therapeutic strategies for targeting mTOR and the referred amino acids, as experimental approaches to selectively attack prostate cancer cells.
    Keywords:  Amino acids; Cancer metabolism; Prostate cancer; mTOR
    DOI:  https://doi.org/10.1007/s13402-022-00706-4
  31. J Cell Biol. 2022 Oct 03. pii: e202110164. [Epub ahead of print]221(10):
    VAMP4 regulates insulin levels by targeting secretory granules to lysosomes.
    Min Li, Fengping Feng, Han Feng, Pengkai Hu, Yanhong Xue, Tao Xu, Eli Song.
      Insulin levels are essential for the maintenance of glucose homeostasis, and deviations lead to pathoglycemia or diabetes. However, the metabolic mechanism controlling insulin quantity and quality is poorly understood. In pancreatic β cells, insulin homeostasis and release are tightly governed by insulin secretory granule (ISG) trafficking, but the required regulators and mechanisms are largely unknown. Here, we identified that VAMP4 controlled the insulin levels in response to glucose challenge. VAMP4 deficiency led to increased blood insulin levels and hyperresponsiveness to glucose. In β cells, VAMP4 is packaged into immature ISGs (iISGs) at trans-Golgi networks and subsequently resorted to clathrin-coated vesicles during granule maturation. VAMP4-positive iISGs and resorted vesicles then fuse with lysosomes facilitated by a SNARE complex consisting of VAMP4, STX7, STX8, and VTI1B, which ensures the breakdown of excess (pro)insulin and obsolete materials and thus maintenance of intracellular insulin homeostasis. Thus, VAMP4 is a key factor regulating the insulin levels and a potential target for the treatment of diabetes.
    DOI:  https://doi.org/10.1083/jcb.202110164
  32. Nat Rev Drug Discov. 2022 Sep 02.
    V-ATPase inhibitor targets Ras-mutant cancers.
    Sarah Crunkhorn.
      
    DOI:  https://doi.org/10.1038/d41573-022-00151-w
  33. Mol Cell. 2022 Sep 01. pii: S1097-2765(22)00763-8. [Epub ahead of print]82(17): 3121-3123
    SLC4A7 and mTORC1 raise nucleotide synthesis with bicarbonate.
    Jessica C Koe, Keeley G Hewton, Seth J Parker.
      In this issue of Molecular Cell, Ali et al. (2022) show that bicarbonate uptake by SLC4A7 fuels de novo nucleotide synthesis and cell proliferation and is regulated by mTORC1.
    DOI:  https://doi.org/10.1016/j.molcel.2022.08.010
  34. Structure. 2022 Aug 16. pii: S0969-2126(22)00275-1. [Epub ahead of print]
    Structure of V-ATPase from citrus fruit.
    Yong Zi Tan, Kristine A Keon, Rana Abdelaziz, Peter Imming, Waltraud Schulze, Karin Schumacher, John L Rubinstein.
      We used the Legionella pneumophila effector SidK to affinity purify the endogenous vacuolar-type ATPases (V-ATPases) from lemon fruit. The preparation was sufficient for cryoelectron microscopy, allowing structure determination of the enzyme in two rotational states. The structure defines the ATP:H+ ratio of the enzyme, demonstrating that it can establish a maximum ΔpH of ∼3, which is insufficient to maintain the low pH observed in the vacuoles of juice sac cells in lemons and other citrus fruit. Compared with yeast and mammalian enzymes, the membrane region of the plant V-ATPase lacks subunit f and possesses an unusual configuration of transmembrane α helices. Subunit H, which inhibits ATP hydrolysis in the isolated catalytic region of V-ATPase, adopts two different conformations in the intact complex, hinting at a role in modulating activity in the intact enzyme.
    Keywords:  V-ATPase; citrus; cryo-EM; lemon; membrane; plant; protein; structure
    DOI:  https://doi.org/10.1016/j.str.2022.07.006
  35. Nat Cell Biol. 2022 Sep 01.
    Identification of two pathways mediating protein targeting from ER to lipid droplets.
    Jiunn Song, Arda Mizrak, Chia-Wei Lee, Marcelo Cicconet, Zon Weng Lai, Wei-Chun Tang, Chieh-Han Lu, Stephanie E Mohr, Robert V Farese, Tobias C Walther.
      Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD-ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.
    DOI:  https://doi.org/10.1038/s41556-022-00974-0
  36. Front Plant Sci. 2022 ;13 968665
    To grow or not to grow under nutrient scarcity: Target of rapamycin-ethylene is the question.
    María José García, Macarena Angulo, Carlos Lucena, Rafael Pérez-Vicente, Francisco Javier Romera.
      To cope with nutrient scarcity, plants generally follow two main complementary strategies. On the one hand, they can slow down growing, mainly shoot growth, to diminish the demand of nutrients. We can call this strategy as "stop growing." On the other hand, plants can develop different physiological and morphological responses, mainly in their roots, aimed to facilitate the acquisition of nutrients. We can call this second strategy as "searching for nutrients." Both strategies are compatible and can function simultaneously but the interconnection between them is not yet well-known. In relation to the "stop growing" strategy, it is known that the TOR (Target Of Rapamycin) system is a central regulator of growth in response to nutrients in eukaryotic cells. TOR is a protein complex with kinase activity that promotes protein synthesis and growth while some SnRK (Sucrose non-fermenting 1-Related protein Kinases) and GCN (General Control Non-derepressible) kinases act antagonistically. It is also known that some SnRKs and GCNs are activated by nutrient deficiencies while TOR is active under nutrient sufficiency. In relation to the "searching for nutrients" strategy, it is known that the plant hormone ethylene participates in the activation of many nutrient deficiency responses. In this Mini Review, we discuss the possible role of ethylene as the hub connecting the "stop growing" strategy and the "searching for nutrients" strategy since very recent results also suggest a clear relationship of ethylene with the TOR system.
    Keywords:  ethylene; nutrient deficiency responses; nutrient scarcity; plant growth; target of rapamycin (TOR)
    DOI:  https://doi.org/10.3389/fpls.2022.968665
  37. Nat Rev Mol Cell Biol. 2022 Sep 02.
    Regulation of membrane protein structure and function by their lipid nano-environment.
    Ilya Levental, Ed Lyman.
      Transmembrane proteins comprise ~30% of the mammalian proteome, mediating metabolism, signalling, transport and many other functions required for cellular life. The microenvironment of integral membrane proteins (IMPs) is intrinsically different from that of cytoplasmic proteins, with IMPs solvated by a compositionally and biophysically complex lipid matrix. These solvating lipids affect protein structure and function in a variety of ways, from stereospecific, high-affinity protein-lipid interactions to modulation by bulk membrane properties. Specific examples of functional modulation of IMPs by their solvating membranes have been reported for various transporters, channels and signal receptors; however, generalizable mechanistic principles governing IMP regulation by lipid environments are neither widely appreciated nor completely understood. Here, we review recent insights into the inter-relationships between complex lipidomes of mammalian membranes, the membrane physicochemical properties resulting from such lipid collectives, and the regulation of IMPs by either or both. The recent proliferation of high-resolution methods to study such lipid-protein interactions has led to generalizable insights, which we synthesize into a general framework termed the 'functional paralipidome' to understand the mutual regulation between membrane proteins and their surrounding lipid microenvironments.
    DOI:  https://doi.org/10.1038/s41580-022-00524-4
  38. Nat Commun. 2022 Aug 30. 13(1): 5092
    Adipocytes control food intake and weight regain via Vacuolar-type H+ ATPase.
    Rizaldy C Zapata, Maria Carretero, Felipe Castellani Gomes Reis, Besma S Chaudry, Jachelle Ofrecio, Dinghong Zhang, Roman Sasik, Theodore Ciaraldi, Michael Petrascheck, Olivia Osborn.
      Energy metabolism becomes dysregulated in individuals with obesity and many of these changes persist after weight loss and likely play a role in weight regain. In these studies, we use a mouse model of diet-induced obesity and weight loss to study the transcriptional memory of obesity. We found that the 'metabolic memory' of obesity is predominantly localized in adipocytes. Utilizing a C. elegans-based food intake assay, we identify 'metabolic memory' genes that play a role in food intake regulation. We show that expression of ATP6v0a1, a subunit of V-ATPase, is significantly induced in both obese mouse and human adipocytes that persists after weight loss. C. elegans mutants deficient in Atp6v0A1/unc32 eat less than WT controls. Adipocyte-specific Atp6v0a1 knockout mice have reduced food intake and gain less weight in response to HFD. Pharmacological disruption of V-ATPase assembly leads to decreased food intake and less weight re-gain. In summary, using a series of genetic tools from invertebrates to vertebrates, we identify ATP6v0a1 as a regulator of peripheral metabolic memory, providing a potential target for regulation of food intake, weight loss maintenance and the treatment of obesity.
    DOI:  https://doi.org/10.1038/s41467-022-32764-5
  39. FEBS Lett. 2022 Sep 03.
    GPHR-mediated acidification of the Golgi lumen is essential for cholesterol biosynthesis in the brain.
    Yu-Shin Sou, Junji Yamaguchi, Hiroshi Kameda, Keisuke Masuda, Yusuke Maeda, Yasuo Uchiyama, Masato Koike.
      The Golgi pH regulator (GPHR) is essential for maintaining the function and morphology of the Golgi apparatus through the regulation of luminal acidic pH. Abnormal morphology of the Golgi apparatus is associated with neurodegenerative diseases. Here, we found that knockout of GPHR in the mouse brain led to morphological changes in the Golgi apparatus and neurodegeneration, which included brain atrophy, neuronal cell death, and gliosis. Furthermore, in the GPHR knockout mouse brain, transcriptional activity of sterol regulatory element-binding protein 2 (SREBP2) decreased, resulting in a reduction in cholesterol levels. GPHR-deficient cells exhibited suppressed neurite outgrowth, which was recovered by exogenous expression of the active form of SREBP2. Our results show that GPHR-mediated luminal acidification of the Golgi apparatus maintains proper cholesterol levels and, thereby, neuronal morphology.
    Keywords:  GPHR; Golgi apparatus; Golgi fragmentation; Golgi pH; SREBP2; cholesterol
    DOI:  https://doi.org/10.1002/1873-3468.14491
  40. FEBS J. 2022 Sep 01.
    The intramembrane proteases SPPL2a and SPPL2b regulate the homeostasis of selected SNARE proteins.
    Moritz Ballin, Wolfram Griep, Mehul Patel, Martin Karl, Torben Mentrup, Jhon Rivera-Monroy, Brian Foo, Blanche Schwappach, Bernd Schröder.
      Signal peptide peptidase (SPP) and SPP-like (SPPL) aspartyl intramembrane proteases are known to contribute to a sequential processing of type II-oriented membrane proteins referred to as regulated intramembrane proteolysis. The ER-resident family members SPP and SPPL2c were shown to also cleave tail-anchored proteins, including selected SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitating membrane fusion events. Here, we analysed whether the related SPPL2a and SPPL2b proteases, which localise to the endocytic or late secretory pathway, are also able to process SNARE proteins. Therefore, we screened 18 SNARE proteins for cleavage by SPPL2a and SPPL2b based on cellular co-expression assays, of which the proteins VAMP1, VAMP2, VAMP3 and VAMP4 were processed by SPPL2a/b demonstrating the capability of these two proteases to proteolyse tail-anchored-proteins. Cleavage of the four SNARE proteins was scrutinised at the endogenous level upon SPPL2a/b inhibition in different cell lines as well as by analysing VAMP1-4 levels in tissues and primary cells of SPPL2a/b double-deficient (dKO) mice. Loss of SPPL2a/b activity resulted in an accumulation of VAMP1-4 in a cell type- and tissue-dependent manner identifying these proteins as SPPL2a/b substrates validated in vivo. Therefore, we propose that SPPL2a/b control cellular levels of VAMP1-4 by initiating the degradation of these proteins, which might impact on cellular trafficking.
    Keywords:  Intramembrane Proteolysis; Membrane trafficking; Protein degradation; SNARE protein; Signal peptide peptidase-like proteases
    DOI:  https://doi.org/10.1111/febs.16610
  41. Diabetes. 2022 Aug 30. pii: db220303. [Epub ahead of print]
    Cathepsin D Drives the Formation of Hybrid Insulin Peptides Relevant to the Pathogenesis of Type 1 Diabetes.
    Samantha A Crawford, Timothy A Wiles, Janet M Wenzlau, Roger L Powell, Gene Barbour, Mylinh Dang, Jason Groegler, Jessie M Barra, KaLia S Burnette, Anita C Hohenstein, Rocky L Baker, Hubert M Tse, Kathryn Haskins, Thomas Delong.
      Hybrid insulin peptides (HIPs) form in pancreatic beta-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs have been identified in pancreatic islets by mass spectrometry and are targeted by CD4 T cells in patients with Type 1 Diabetes (T1D), as well as by pathogenic CD4 T cell clones in non-obese diabetic (NOD) mice. The mechanism of HIP formation is currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient in cathepsin L (CatL), another protease implicated in the formation of disease-relevant HIPs, contain elevated levels of HIPs, indicating a role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow down the autoimmune destruction of beta-cells mediated by HIP-reactive CD4 T cells in T1D.
    DOI:  https://doi.org/10.2337/db22-0303
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