bims-lysosi 2022-10-23 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022‒10‒23
thirty-six papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes.
Zinc enhances autophagic flux and lysosomal function through transcription factor EB activation and V-ATPase assembly.
Neuronal Ganglioside and Glycosphingolipid (GSL) Metabolism and Disease : Cascades of Secondary Metabolic Errors Can Generate Complex Pathologies (in LSDs).
Lysosomal functions of progranulin and implications for treatment of frontotemporal dementia.
Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo.
Lysosomal Targeting of β-Cyclodextrin.
The critical role of the endolysosomal system in cerebral ischemia.
The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.
RagC GTPase regulates mTOR to promote chemoresistance in senescence-like HepG2 cells.
Gasdermin D maintains bone mass by rewiring the endo-lysosomal pathway of osteoclastic bone resorption.
SZT2 maintains hematopoietic stem cell homeostasis via nutrient-mediated mTORC1 regulation.
Inhibiting glutamine utilization creates a synthetic lethality for suppression of ATP citrate lyase in KRas-driven cancer cells.
Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics.
Inducible deletion of Raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation.
Resistance exercise enhances long-term mTORC1 sensitivity to leucine.
The intracellular Ca2+ channel TRPML3 is a PI3P effector that regulates autophagosome biogenesis.
Editorial: Mitochondrial and lysosomal dysfunction in neurodegenerative diseases: Molecular mechanisms and therapeutic strategies.
Angiotensin II regulates mitochondrial mTOR pathway activity dependent on acyl CoA synthetase 4 in adrenocortical cells.
Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.
Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole.
Proteomic analysis of s-acylated proteins in human retinal pigment epithelial cells and the role of palmitoylation of Niemann-Pick type C1 protein in cholesterol transport.
WASH activation controls endosomal recycling and EGFR and Hippo signaling during tumor-suppressive cell competition.
A second chance at yeast early endosomes.
Clathrin-associated AP-1 controls termination of STING signalling.
Glutamine synthetase limits b-catenin-mutated liver cancer growth by maintaining nitrogen homeostasis and suppressing mTORC1.
Targeting LRRK2 in Parkinson's disease.
Gangliosides and Cell Surface Ganglioside Metabolic Enzymes in the Nervous System.
LC3/GABARAP binding to fluid membranes is potentiated by ceramide.
Endocytosis triggers V-ATPase-SYK-mediated priming of cGAS activation and innate immune response.
Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense.
Characterization of a novel exonic deletion in the GALNS gene causing Morquio A syndrome.
What Is the Role of Organelle Membrane Contact Sites in Neurodegenerative Diseases?
Principles and functions of metabolic compartmentalization.
Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat.
Mechanism of an intramembrane chaperone for multipass membrane proteins.
Modelling membrane reshaping by staged polymerization of ESCRT-III filaments.

Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2205492119

Lysosomal positioning regulates Rab10 phosphorylation at LRRK2+ lysosomes.

Jillian H Kluss, Alexandra Beilina, Chad D Williamson, Patrick A Lewis, Mark R Cookson, Luis Bonet-Ponce.

  Genetic variation at the leucine-rich repeat kinase 2 (LRRK2) locus contributes to an enhanced risk of familial and sporadic Parkinson's disease. Previous data have demonstrated that recruitment to various membranes of the endolysosomal system results in LRRK2 activation. However, the mechanism(s) underlying LRRK2 activation at endolysosomal membranes and the cellular consequences of these events are still poorly understood. Here, we directed LRRK2 to lysosomes and early endosomes, triggering both LRRK2 autophosphorylation and phosphorylation of the direct LRRK2 substrates Rab10 and Rab12. However, when directed to the lysosomal membrane, pRab10 was restricted to perinuclear lysosomes, whereas pRab12 was visualized on both peripheral and perinuclear LRRK2+ lysosomes, suggesting that lysosomal positioning provides additional regulation of LRRK2-dependent Rab phosphorylation. Anterograde transport of lysosomes to the cell periphery by increasing the expression of ARL8B and SKIP or by knockdown of JIP4 blocked the recruitment and phosphorylation of Rab10 by LRRK2. The absence of pRab10 from the lysosomal membrane prevented the formation of a lysosomal tubulation and sorting process we previously named LYTL. Conversely, overexpression of RILP resulted in lysosomal clustering within the perinuclear area and increased LRRK2-dependent Rab10 recruitment and phosphorylation. The regulation of Rab10 phosphorylation in the perinuclear area depends on counteracting phosphatases, as the knockdown of phosphatase PPM1H significantly increased pRab10 signal and lysosomal tubulation in the perinuclear region. Our findings suggest that LRRK2 can be activated at multiple cellular membranes, including lysosomes, and that lysosomal positioning further provides the regulation of some Rab substrates likely via differential phosphatase activity or effector protein presence in nearby cellular compartments.

Keywords:  JIP4; LLOMe; LYTL; Parkinson's disease; kinase

DOI:  https://doi.org/10.1073/pnas.2205492119
Front Cell Neurosci. 2022 ;16 895750

Zinc enhances autophagic flux and lysosomal function through transcription factor EB activation and V-ATPase assembly.

Ki-Ryeong Kim, Sang Eun Park, Ji-Ye Hong, Jae-Young Koh, Dong-Hyung Cho, Jung Jin Hwang, Yang-Hee Kim.

  The stimulation of autophagy or lysosomes has been considered therapeutic for neurodegenerative disorders because the accumulation of misfolded proteins is commonly observed in the brains of individuals with these diseases. Although zinc is known to play critical roles in the functions of lysosomes and autophagy, the mechanism behind this regulatory relationship remains unclear. Therefore, in this study, we examined which mechanism is involved in zinc-mediated activation of autophagy and lysosome. Exposure to zinc at a sub-lethal concentration activated autophagy in a concentration-dependent manner in mRFP-GFP-LC3-expressing H4 glioma cells. Zinc also rescued the blocking of autophagic flux arrested by pharmaceutical de-acidification. Co-treatment with zinc attenuated the chloroquine (CQ)-induced increase in the number and size of mRFP-GFP-LC3 puncta in H4 cells and accumulation of p62 by CQ or ammonium chloride in both H4 and mouse cerebrocortical cultures. Zinc rapidly induced the expression of cathepsin B (CTSB) and cathepsin D (CTSD), representative lysosomal proteases in neurons, which appeared likely to be mediated by transcription factor EB (TFEB). We observed the translocation of TFEB from neurite to nucleus and the dephosphorylation of TFEB by zinc. The addition of cycloheximide, a chemical inhibitor of protein synthesis, inhibited the activity of CTSB and CTSD at 8 h after zinc exposure but not at 1 h, indicating that only late lysosomal activation was dependent on the synthesis of CTSB and CTSD proteins. At the very early time point, the activation of cathepsins was mediated by an increased assembly of V-ATPase on lysosomes and resultant lysosomal acidification. Finally, considering that P301L mutation in tau protein causes frontotemporal dementia through aggressive tau accumulation, we investigated whether zinc reduces the accumulation of protein aggregates in SK-N-BE(2)-C neuroblastoma cells expressing wild-type tau or mutant P301L-tau. Zinc markedly attenuated the levels of phosphorylated tau and total tau as well as p62 in both wild-type and mutant tau-overexpressing cells. We also observed that zinc was more effective than rapamycin at inducing TFEB-dependent CTSB and CTSD expression and V-ATPase-dependent lysosomal acidification and CTSB/CTSD activation. These results suggest that the regulation of zinc homeostasis could be a new approach for developing treatments for neurodegenerative diseases, including Alzheimer's and Parkinson's.

Keywords:  TFEB; V-ATPase; autophagy; cathepsin B; cathepsin D; lysosome; neurodegenerative disease; zinc

DOI:  https://doi.org/10.3389/fncel.2022.895750
Adv Neurobiol. 2023 ;29 333-390

Neuronal Ganglioside and Glycosphingolipid (GSL) Metabolism and Disease : Cascades of Secondary Metabolic Errors Can Generate Complex Pathologies (in LSDs).

Roger Sandhoff, Konrad Sandhoff.

  Glycosphingolipids (GSLs) are a diverse group of membrane components occurring mainly on the surfaces of mammalian cells. They and their metabolites have a role in intercellular communication, serving as versatile biochemical signals (Kaltner et al, Biochem J 476(18):2623-2655, 2019) and in many cellular pathways. Anionic GSLs, the sialic acid containing gangliosides (GGs), are essential constituents of neuronal cell surfaces, whereas anionic sulfatides are key components of myelin and myelin forming oligodendrocytes. The stepwise biosynthetic pathways of GSLs occur at and lead along the membranes of organellar surfaces of the secretory pathway. After formation of the hydrophobic ceramide membrane anchor of GSLs at the ER, membrane-spanning glycosyltransferases (GTs) of the Golgi and Trans-Golgi network generate cell type-specific GSL patterns for cellular surfaces. GSLs of the cellular plasma membrane can reach intra-lysosomal, i.e. luminal, vesicles (ILVs) by endocytic pathways for degradation. Soluble glycoproteins, the glycosidases, lipid binding and transfer proteins and acid ceramidase are needed for the lysosomal catabolism of GSLs at ILV-membrane surfaces. Inherited mutations triggering a functional loss of glycosylated lysosomal hydrolases and lipid binding proteins involved in GSL degradation cause a primary lysosomal accumulation of their non-degradable GSL substrates in lysosomal storage diseases (LSDs). Lipid binding proteins, the SAPs, and the various lipids of the ILV-membranes regulate GSL catabolism, but also primary storage compounds such as sphingomyelin (SM), cholesterol (Chol.), or chondroitin sulfate can effectively inhibit catabolic lysosomal pathways of GSLs. This causes cascades of metabolic errors, accumulating secondary lysosomal GSL- and GG- storage that can trigger a complex pathology (Breiden and Sandhoff, Int J Mol Sci 21(7):2566, 2020).

Keywords:  Alzheimer; Catabolism; Degradation; Development; Endosomal pathway; Frontal lobe dementia; Ganglio-series; Ganglioside; Genetic disease; Glycolipid; Glycosphingolipid; Glycosyltransferase; Hydrolase; Intra-lysosomal luminal vesicle (ILV); Lysosomal storage disease (LSD); Lysosome; Membrane-surface; Metabolism; Neurodegenerative disease; Neuron; Organelle; Parkinson; Receptor; Secondary storage; Secretory pathway; Sphingolipid-binding protein (SAP); Sphingolipid-transfer protein; Topology

DOI:  https://doi.org/10.1007/978-3-031-12390-0_12
Trends Cell Biol. 2022 Oct 13. pii: S0962-8924(22)00216-1. [Epub ahead of print]

Lysosomal functions of progranulin and implications for treatment of frontotemporal dementia.

Matthew J Simon, Todd Logan, Sarah L DeVos, Gilbert Di Paolo.

  Loss-of-function heterozygous mutations in GRN, the gene encoding progranulin (PGRN), were identified in patients with frontotemporal lobar degeneration (FTLD) almost two decades ago and are generally linked to reduced PGRN protein expression levels. Although initial characterization of PGRN function primarily focused on its role in extracellular signaling as a secreted protein, more recent studies revealed critical roles of PGRN in regulating lysosome function, including proteolysis and lipid degradation, consistent with its lysosomal localization. Emerging from these studies is the notion that PGRN regulates glucocerebrosidase activity via direct chaperone activities and via interaction with prosaposin (i.e., a key regulator of lysosomal sphingolipid-metabolizing enzymes), as well as with the anionic phospholipid bis(monoacylglycero)phosphate. This emerging lysosomal biology of PGRN identified novel and promising opportunities in therapeutic discovery as well as biomarker development.

Keywords:  GBA; lipofuscin; lysobisphosphatidic acid; lysosomal storage disorder; neuronal ceroid lipofuscinosis; saposin

DOI:  https://doi.org/10.1016/j.tcb.2022.09.006
Nat Commun. 2022 Oct 20. 13(1): 6212

Cross-linking of the endolysosomal system reveals potential flotillin structures and cargo.

Jasjot Singh, Hadeer Elhabashy, Pathma Muthukottiappan, Markus Stepath, Martin Eisenacher, Oliver Kohlbacher, Volkmar Gieselmann, Dominic Winter.

Lysosomes are well-established as the main cellular organelles for the degradation of macromolecules and emerging as regulatory centers of metabolism. They are of crucial importance for cellular homeostasis, which is exemplified by a plethora of disorders related to alterations in lysosomal function. In this context, protein complexes play a decisive role, regulating not only metabolic lysosomal processes but also lysosome biogenesis, transport, and interaction with other organelles. Using cross-linking mass spectrometry, we analyze lysosomes and early endosomes. Based on the identification of 5376 cross-links, we investigate protein-protein interactions and structures of lysosome- and endosome-related proteins. In particular, we present evidence for a tetrameric assembly of the lysosomal hydrolase PPT1 and a heterodimeric structure of FLOT1/FLOT2 at lysosomes and early endosomes. For FLOT1-/FLOT2-positive early endosomes, we identify >300 putative cargo proteins and confirm eleven substrates for flotillin-dependent endocytosis, including the latrophilin family of adhesion G protein-coupled receptors.

DOI: https://doi.org/10.1038/s41467-022-33951-0
Chemistry. 2022 Oct 20.

Lysosomal Targeting of β-Cyclodextrin.

Andrea Mascherpa, Nozomii Ishii, Ayelen Tayagui, Jiang Liu, Matthieu Sollogoub, Antony John Fairbanks.

  β-Cyclodextrin (β-CD) and derivatives are approved therapeutics in >30 clinical settings. β-CDs have also shown promise as therapeutics for treatment of some lysosomal storage disorders, such as Niemann-Pick disease type C, and other disease states which involve metabolite accumulation in the lysosome. In these cases, β-CD activity relies on transport to the lysosome, wherein it can bind hydrophobic substrate and effect extraction. The post-translational attachment of N-glycans terminated in mannose-6-phosphate (M6P) residues is the predominant method by which lysosomal enzymes are targeted to the lysosome. In this work we covalently attach a synthetic biantennary bis-M6P-terminated N-glycan to β-CD and study the effect of the added glycans in a mammalian cell line. The formation of a host guest complex with a Cy5 fluorophore allows study of both cellular internalisation and transport to the lysosome by fluorescence microscopy. Results indicate that the rates of both internalisation and lysosomal transport are increased by the attachment of M6P-glycans to β-CD, indicating that M6P-glycan conjugation may improve the therapeutic effectiveness of β-CD for the treatment of disorders involving hydrophobic metabolite accumulation in the lysosome.

Keywords:  Fluorescent probes; carbohydrates; cyclodextrins; lysosome; mannose-6-phosphate

DOI:  https://doi.org/10.1002/chem.202203252
Neural Regen Res. 2023 May;18(5): 983-990

The critical role of the endolysosomal system in cerebral ischemia.

Hui-Yi Zhang, Ye Tian, Han-Yan Shi, Ya Cai, Ying Xu.

  Cerebral ischemia is a serious disease that triggers sequential pathological mechanisms, leading to significant morbidity and mortality. Although most studies to date have typically focused on the lysosome, a single organelle, current evidence supports that the function of lysosomes cannot be separated from that of the endolysosomal system as a whole. The associated membrane fusion functions of this system play a crucial role in the biodegradation of cerebral ischemia-related products. Here, we review the regulation of and the changes that occur in the endolysosomal system after cerebral ischemia, focusing on the latest research progress on membrane fusion function. Numerous proteins, including N-ethylmaleimide-sensitive factor and lysosomal potassium channel transmembrane protein 175, regulate the function of this system. However, these proteins are abnormally expressed after cerebral ischemic injury, which disrupts the normal fusion function of membranes within the endolysosomal system and that between autophagosomes and lysosomes. This results in impaired "maturation" of the endolysosomal system and the collapse of energy metabolism balance and protein homeostasis maintained by the autophagy-lysosomal pathway. Autophagy is the final step in the endolysosomal pathway and contributes to maintaining the dynamic balance of the system. The process of autophagosome-lysosome fusion is a necessary part of autophagy and plays a crucial role in maintaining energy homeostasis and clearing aging proteins. We believe that, in cerebral ischemic injury, the endolysosomal system should be considered as a whole rather than focusing on the lysosome. Understanding how this dynamic system is regulated will provide new ideas for the treatment of cerebral ischemia.

Keywords:  autophagy; biodegradation; brain injury; chaperone-mediated autophagy; endolysosomal system; fusion; hypoxia-ischemia; brain; mitophagy; N-ethylmaleimide-sensitive protein; TMEM175

DOI:  https://doi.org/10.4103/1673-5374.355745
Mol Ther. 2022 Oct 18. pii: S1525-0016(22)00613-X. [Epub ahead of print]

The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.

Antonio Monaco, Veronica Maffia, Nicolina Cristina Sorrentino, Irene Sambri, Yulia Ezhova, Teresa Giuliano, Vincenzo Cacace, Edoardo Nusco, Maria De Risi, Elvira De Leonibus, Thomas Schrader, Frank-Gerrit Klärner, Gal Bitan, Alessandro Fraldi.

DOI: https://doi.org/10.1016/j.ymthe.2022.10.001
Front Physiol. 2022 ;13 949737

RagC GTPase regulates mTOR to promote chemoresistance in senescence-like HepG2 cells.

Wei Jiang, Zhenglin Ou, Qin Zhu, Hongyan Zai.

  Radiotherapy and chemotherapy can arrest cancer cells in a senescence-like state, which can lead to therapy resistance and cancer relapse. mTOR is hyperactivated in senescent cells but the mechanisms remain unclear. In this study, we examine the roles of several mTOR-regulated GTPases in senescence-like liver cancer cells and the mechanisms in drug resistance. We show that although RagC, Rheb, Rab1A, Rab5 and Arf1 GTPases were required for optimal mTOR activation in proliferating HepG2 cells, only RagC and Rheb are required in the senescence-like counterparts. Consistently, the drug resistance of the senescence-like HepG2 can be reduced by knocking down RagC and Rheb but not the other GTPases. Autophagic and lysosomal activity were increased in senescence-like cells; pharmacological inhibition of autophagy-lysosome decreased mTOR activity and preferentially sensitized senescence-like HepG2 cells to chemotherapy drugs including trametinib, cisplatin, and doxorubicin. In liver cancer patients, expression of RagC and Rheb but not other GTPases examined was associated with unfavorable prognosis. Our study therefore has defined a key role of Rag-Rheb GTPase in mediating mTOR activation and drug resistance in senescence-like HepG2 cells, which could have important implications in developing second-line treatments for liver cancer patients.

Keywords:  GTPase; chemoresistance; liver cancer; mTOR; senescence

DOI:  https://doi.org/10.3389/fphys.2022.949737
Dev Cell. 2022 Oct 10. pii: S1534-5807(22)00683-9. [Epub ahead of print]

Gasdermin D maintains bone mass by rewiring the endo-lysosomal pathway of osteoclastic bone resorption.

Mobai Li, Dehang Yang, Huige Yan, Zhibin Tang, Danlu Jiang, Jian Zhang, Zhexu Chi, Wanyun Nie, Wenxuan Zhen, Weiwei Yu, Sheng Chen, Zhen Wang, Qianzhou Yu, Xue Zhang, Fan Yang, Shunwu Fan, Xianfeng Lin, Di Wang.

  Gasdermin D (GSDMD)-mediated pyroptosis induces immunogenic cell death and promotes inflammation. However, the functions of GSDMD in tissue homeostasis remain unclear. Here, we identify a physiological function of GSDMD in osteoclasts via a non-lytic p20-generated protein, which prevents bone loss to maintain bone homeostasis. In the late stage of RANKL-induced osteoclastogenesis, GSDMD underwent cleavage, which is dependent on RIPK1 and caspase-8/-3, to yield this p20 product. Gsdmd-deficient osteoclasts showed normal differentiation but enhanced bone resorption with excessive lysosomal activity. Mice with complete or myeloid-specific Gsdmd deletion exhibited increased trabecular bone loss and more severe aging/ovariectomy-induced osteoporosis. GSDMD p20 was preferentially localized to early endosomes and limited endo-lysosomal trafficking and maturation, relying on its oligomerization and control of phosphoinositide conversion by binding to phosphatidylinositol 3-phosphate (PI(3)P). We have thus identified an anti-osteoclastic function of GSDMD as a checkpoint for lysosomal maturation and secretion and linked this to bone homeostasis and endosome-lysosome biology.

Keywords:  endosome; gasdermin D; lysosome; osteoclast; tissue homeostasis

DOI:  https://doi.org/10.1016/j.devcel.2022.09.013
J Clin Invest. 2022 Oct 17. pii: e146272. [Epub ahead of print]132(20):

SZT2 maintains hematopoietic stem cell homeostasis via nutrient-mediated mTORC1 regulation.

Na Yin, Gang Jin, Yuying Ma, Hanfei Zhao, Guangyue Zhang, Ming O Li, Min Peng.

  The mTORC1 pathway coordinates nutrient and growth factor signals to maintain organismal homeostasis. Whether nutrient signaling to mTORC1 regulates stem cell function remains unknown. Here, we show that SZT2 - a protein required for mTORC1 downregulation upon nutrient deprivation - is critical for hematopoietic stem cell (HSC) homeostasis. Ablation of SZT2 in HSCs decreased the reserve and impaired the repopulating capacity of HSCs. Furthermore, ablation of both SZT2 and TSC1 - 2 repressors of mTORC1 on the nutrient and growth factor arms, respectively - led to rapid HSC depletion, pancytopenia, and premature death of the mice. Mechanistically, loss of either SZT2 or TSC1 in HSCs led to only mild elevation of mTORC1 activity and reactive oxygen species (ROS) production. Loss of both SZT2 and TSC1, on the other hand, simultaneously produced a dramatic synergistic effect, with an approximately 10-fold increase of mTORC1 activity and approximately 100-fold increase of ROS production, which rapidly depleted HSCs. These data demonstrate a critical role of nutrient mTORC1 signaling in HSC homeostasis and uncover a strong synergistic effect between nutrient- and growth factor-mediated mTORC1 regulation in stem cells.

Keywords:  Amino acid metabolism; Bone marrow transplantation; Hematology; Hematopoietic stem cells; Metabolism

DOI:  https://doi.org/10.1172/JCI146272
PLoS One. 2022 ;17(10): e0276579

Inhibiting glutamine utilization creates a synthetic lethality for suppression of ATP citrate lyase in KRas-driven cancer cells.

Ahmet Hatipoglu, Deepak Menon, Talia Levy, Maria A Frias, David A Foster.

Metabolic reprogramming is now considered a hallmark of cancer cells. KRas-driven cancer cells use glutaminolysis to generate the tricarboxylic acid cycle intermediate α-ketoglutarate via a transamination reaction between glutamate and oxaloacetate. We reported previously that exogenously supplied unsaturated fatty acids could be used to synthesize phosphatidic acid-a lipid second messenger that activates both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2). A key target of mTORC2 is Akt-a kinase that promotes survival and regulates cell metabolism. We report here that mono-unsaturated oleic acid stimulates the phosphorylation of ATP citrate lyase (ACLY) at the Akt phosphorylation site at S455 in an mTORC2 dependent manner. Inhibition of ACLY in KRas-driven cancer cells in the absence of serum resulted in loss of cell viability. We examined the impact of glutamine (Gln) deprivation in combination with inhibition of ACLY on the viability of KRas-driven cancer cells. While Gln deprivation was somewhat toxic to KRas-driven cancer cells by itself, addition of the ACLY inhibitor SB-204990 increased the loss of cell viability. However, the transaminase inhibitor aminooxyacetate was minimally toxic and the combination of SB-204990 and aminooxtacetate led to significant loss of cell viability and strong cleavage of poly-ADP ribose polymerase-indicating apoptotic cell death. This effect was not observed in MCF7 breast cancer cells that do not have a KRas mutation or in BJ-hTERT human fibroblasts which have no oncogenic mutation. These data reveal a synthetic lethality between inhibition of glutamate oxaloacetate transaminase and ACLY inhibition that is specific for KRas-driven cancer cells and the apparent metabolic reprogramming induced by activating mutations to KRas.

DOI: https://doi.org/10.1371/journal.pone.0276579
Nat Commun. 2022 Oct 16. 13(1): 6112

Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics.

Hankum Park, Frances V Hundley, Qing Yu, Katherine A Overmyer, Dain R Brademan, Lia Serrano, Joao A Paulo, Julia C Paoli, Sharan Swarup, Joshua J Coon, Steven P Gygi, J Wade Harper.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by β and γ-Secretases, which produce amyloidogenic Aβ species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

DOI: https://doi.org/10.1038/s41467-022-33881-x
J Physiol. 2022 Oct 18.

Inducible deletion of Raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation.

Martina Baraldo, Sabrina Zorzato, Achille Homère Tchampda Dondjang, Alessia Geremia, Leonardo Nogara, Ana Georgia Dumitras, Marta Canato, Lorenzo Marcucci, Hendrik Nolte, Bert Blaauw.

  KEY POINTS: Skeletal muscle wasting and weakness have been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mTOR signaling Mammalian Target of Rapamycin (mTOR) plays a crucial role in the maintenance of muscle mass and functionality We found that the loss of both mTOR and Raptor results in contractile abnormalities, with severe muscle weakness and delayed relaxation following tetanic stimulation These results are associated with alterations in the expression of genes involved in sarcomere organization and calcium handling, and with an impairment in calcium reuptake after contraction Taken together, these results reveal a mechanistic insight into the role of mTOR in muscle contractility ABSTRACT: Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mTOR signaling. Here we wanted to better elucidate the functional role of mTOR on muscle contractility. Most loss of function studies for mTOR signaling have used the drug rapamycin to inhibit some of the signaling downstream of mTOR. However, as rapamycin does not completely inhibit all mTOR signaling, we generated a double k.o. for mTOR and for the scaffold protein of mTORC1, Raptor, in skeletal muscle. We found that dk.o. mice results in a more severe phenotype compared to Raptor or mTOR deletion alone. Indeed, they display muscle weakness, increased fiber denervation, and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibers and changes in the expression levels of calcium-related genes, like Serca1 and Casq1. Indeed, dk.o. mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes are linked to sarcomere organization, like Tcap and Fhod3. These results suggest a key role for mTOR signaling in maintaining a proper fiber relaxation in skeletal muscle. Abstract figure legend This article is protected by copyright. All rights reserved.

Keywords:  Raptor; calcium; mTOR; muscle force; relaxation; skeletal muscle

DOI:  https://doi.org/10.1113/JP283686
Mol Metab. 2022 Oct 14. pii: S2212-8778(22)00184-3. [Epub ahead of print] 101615

Resistance exercise enhances long-term mTORC1 sensitivity to leucine.

Gommaar D'Hulst, Evi Masschelein, Katrien De Bock.

  OBJECTIVE: Exercise enhances the sensitivity of mammalian target of rapamycin complex 1 (mTORC1) to amino acids, in particular leucine. How long this enhanced sensitivity lasts, and which mechanisms control enhanced leucine-mediated mTORC1 activation following exercise is currently unknown.METHODS: C57BL/6J mice were exercised for one night in a resistance-braked running wheel after a 12-day acclimatization period. Mice were gavaged with a submaximal dose of L-leucine or saline acutely or 48 hours after exercise cessation, following 3 h food withdrawal. Muscles were excised 30 min after leucine administration. To study the contribution of mTORC1, we repeated those experiments but blocked mTORC1 activation using rapamycin immediately before the overnight running bout and one hour before the first dose of leucine. mTORC1 signaling, muscle protein synthesis and amino acid sensing machinery were assessed using immunoblot and qPCR. Leucine uptake was measured using L-[14C(U)]-leucine tracer labeling.
RESULTS: When compared to sedentary conditions, leucine supplementation more potently activated mTORC1 and protein synthesis in acutely exercised muscle. This effect was observed in m. soleus but not in m. tibialis anterior nor m. plantaris. The synergistic effect in m. soleus was long-lasting as key downstream markers of mTORC1 as well as protein synthesis remained higher when leucine was administered 48 h after exercise. We found that exercise enhanced the expression of amino acid transporters and promoted uptake of leucine into the muscle, leading to higher free intramuscular leucine levels. This coincided with increased expression of activating transcription factor 4 (ATF4), a main transcriptional regulator of amino acid uptake and metabolism, and downstream activation of amino acid genes as well as leucyl-tRNA synthetase (LARS), a putative leucine sensor. Finally, blocking mTORC1 using rapamycin did not reduce expression and activation of ATF4, suggesting that the latter does not act downstream of mTORC1. Rather, we found a robust increase in eukaryotic initiation factor 2α (eIF2α) phosphorylation, suggesting that the integrated stress response pathway, rather than exercise-induced mTORC1 activation, drives long-term ATF4 expression in skeletal muscle after exercise.
CONCLUSIONS: The enhanced sensitivity of mTORC1 to leucine is maintained at least 48 h after exercise. This shows that the anabolic window of opportunity for protein ingestion is not restricted to the first hours immediately following exercise. Increased mTORC1 sensitivity to leucine coincided with enhanced leucine influx into muscle and higher expression of genes involved in leucine sensing and amino acid metabolism. Also, exercise induced an increase in ATF4 protein expression. Altogether, these data suggest that muscular contractions switch on a coordinated program to enhance amino acid uptake as well as intramuscular sensing of key amino acids involved in mTORC1 activation and the stimulation of muscle protein synthesis.

Keywords:  ATF4; exercise; leucine; mTOR; sensitivity

DOI:  https://doi.org/10.1016/j.molmet.2022.101615
Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2200085119

The intracellular Ca2+ channel TRPML3 is a PI3P effector that regulates autophagosome biogenesis.

So Woon Kim, Mi Kyung Kim, Seokwoo Hong, Areum Choi, Jee Hye Choi, Shmuel Muallem, Insuk So, Dongki Yang, Hyun Jin Kim.

  Autophagy is a multiple fusion event, initiating with autophagosome formation and culminating with fusion with endo-lysosomes in a Ca2+-dependent manner. The source of Ca2+ and the molecular mechanism by which Ca2+ is provided for this process are not known. The intracellular Ca2+ permeable channel transient receptor potential mucolipin 3 (TRPML3) localizes in the autophagosome and interacts with the mammalian autophagy-related protein 8 (ATG8) homolog GATE16. Here, we show that lipid-regulated TRPML3 is the Ca2+ release channel in the phagophore that provides the Ca2+ necessary for autophagy progress. We generated a TRPML3-GCaMP6 fusion protein as a targeted reporter of TRPML3 compartment localization and channel function. Notably, TRPML3-GCaMP6 localized in the phagophores, the level of which increased in response to nutrient starvation. Importantly, phosphatidylinositol-3-phosphate (PI3P), an essential lipid for autophagosome formation, is a selective regulator of TRPML3. TRPML3 interacted with PI3P, which is a direct activator of TRPML3 current and Ca2+ release from the phagophore, to promote and increase autophagy. Inhibition of TRPML3 suppressed autophagy even in the presence of excess PI3P, while activation of TRPML3 reversed the autophagy inhibition caused by blocking PI3P. Moreover, disruption of the TRPML3-PI3P interaction abolished both TRPML3 activation by PI3P and the increase in autophagy. Taken together, these results reveal that TRPML3 is a downstream effector of PI3P and a key regulator of autophagy. Activation of TRPML3 by PI3P is the critical step providing Ca2+ from the phagophore for the fusion process, which is essential for autophagosome biogenesis.

Keywords:  Ca2+ channel; GCaMP6; PI3P; TRPML3; autophagy

DOI:  https://doi.org/10.1073/pnas.2200085119
Front Aging Neurosci. 2022 ;14 1029440

Editorial: Mitochondrial and lysosomal dysfunction in neurodegenerative diseases: Molecular mechanisms and therapeutic strategies.

Chen Xiang, Xiao Yousheng, Zhu Xiaolei, Yang Wenjie, Pu Jiali, Pei Zhong.



Keywords:  lysosomal dysfunction; mitochondrial dysfunction; molecular mechanism; neurodegenerative disease; therapeutic

DOI:  https://doi.org/10.3389/fnagi.2022.1029440
Endocrinology. 2022 Oct 18. pii: bqac170. [Epub ahead of print]

Angiotensin II regulates mitochondrial mTOR pathway activity dependent on acyl CoA synthetase 4 in adrenocortical cells.

Katia E Helfenberger, Giuliana F Argentino, Yanina Benzo, Lucía Herrera, Paola Finocchietto, Cecilia Poderoso.

  Two well-known protein complexes in mammalian cells, mTOR type 1 and type 2 (mTORC1/2) are involved in several cellular processes such as protein synthesis, cell proliferation and commonly dysregulated in cancer. An acyl-CoA synthetase type 4 (ACSL4) is one of the most recently mTORC1/2 regulators described, in breast cancer cells. The expression of ACSL4 is hormone-regulated in adrenocortical cells and required for steroid biosynthesis. mTORC1/2 have been reported to be crucial in the proliferation of human adrenocortical tumor cells H295R and interestingly reported at several subcellular locations, which has brought cell biology to the vanguard of the mTOR signaling field. In the present work, we study the regulation of mTORC1/2 activation by angiotensin II (Ang II) -the trophic hormone for adrenocortical cells-, the subcellular localization of mTORC1/2 signaling proteins and the role of ACSL4 in the regulation of this pathway, in H295R cells. Ang II promotes activation by phosphorylation of mTORC1/2 pathway proteins in a time-dependent manner. Mitochondrial pools of ribosomal protein S6, Akt in threonine 308 and serine 473 and Rictor are phosphorylated and activated. Glycogen synthase kinase type 3 (GSK3) is phosphorylated and inactivated in mitochondria, favoring mTORC1 activation. Epidermal growth factor, a classic mTORC1/2 activator, promoted unique activation kinetics of mTORC1/2 pathway, except for Akt phosphorylation. Here, we demonstrate that ACSL4 is necessary for mTORC1/2 effectors phosphorylation and H295R proliferation, triggered by Ang II. Ang II promotes activation of mitochondrial mTORC1/2 signaling proteins, through ACSL4, with a direct impact on adrenocortical cellular proliferation.

Keywords:  acyl-CoA synthetase type 4; adrenocortical human cells; angiotensin II; compartmentalization; mTORC proteins; mitochondria

DOI:  https://doi.org/10.1210/endocr/bqac170
J Clin Invest. 2022 Oct 20. pii: e160766. [Epub ahead of print]

Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2.

Guoli Shi, Abhilash I Chiramel, Tiansheng Li, Kin Kui Lai, Adam D Kenney, Ashley Zani, Adrian C Eddy, Saliha Majdoul, Lizhi Zhang, Tirhas Dempsey, Paul A Beare, Swagata Kar, Jonathan W Yewdell, Sonja M Best, Jacob S Yount, Alex A Compton.

  SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.

Keywords:  Autophagy; COVID-19; Innate immunity; Lysosomes

DOI:  https://doi.org/10.1172/JCI160766
J Cell Biol. 2022 Dec 05. pii: e202203139. [Epub ahead of print]221(12):

Plant autophagosomes mature into amphisomes prior to their delivery to the central vacuole.

Jierui Zhao, Mai Thu Bui, Juncai Ma, Fabian Künzl, Lorenzo Picchianti, Juan Carlos De La Concepcion, Yixuan Chen, Sofia Petsangouraki, Azadeh Mohseni, Marta García-Leon, Marta Salas Gomez, Caterina Giannini, Dubois Gwennogan, Roksolana Kobylinska, Marion Clavel, Swen Schellmann, Yvon Jaillais, Jiri Friml, Byung-Ho Kang, Yasin Dagdas.

Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been extensively studied, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. Here, we have identified an autophagy adaptor, CFS1, that directly interacts with the autophagosome marker ATG8 and localizes on both membranes of the autophagosome. Autophagosomes form normally in Arabidopsis thaliana cfs1 mutants, but their delivery to the vacuole is disrupted. CFS1's function is evolutionarily conserved in plants, as it also localizes to the autophagosomes and plays a role in autophagic flux in the liverwort Marchantia polymorpha. CFS1 regulates autophagic flux by bridging autophagosomes with the multivesicular body-localized ESCRT-I component VPS23A, leading to the formation of amphisomes. Similar to CFS1-ATG8 interaction, disrupting the CFS1-VPS23A interaction blocks autophagic flux and renders plants sensitive to nitrogen starvation. Altogether, our results reveal a conserved vacuolar sorting hub that regulates autophagic flux in plants.

DOI: https://doi.org/10.1083/jcb.202203139
Front Aging Neurosci. 2022 ;14 965943

Proteomic analysis of s-acylated proteins in human retinal pigment epithelial cells and the role of palmitoylation of Niemann-Pick type C1 protein in cholesterol transport.

Jia Kai Li, Yu Qing Rao, Siew Kwan Koh, Peiquan Zhao, Lei Zhou, Jing Li.

  Palmitoylation is a dynamic process that regulates the activity of the modified proteins. Retinal pigment epithelial (RPE) cells play pivotal roles in the visual cycle and maintaining healthy photoreceptor cells. Dysfunctional RPE cells are often associated with degenerative retinal diseases. The aim of the study was to identify potentially palmitoylated proteins in human RPE cells. By using the detergent-resistant membrane, we found 312 potentially palmitoylated peptides which corresponded to 192 proteins in RPE cells, including 55 new candidate proteins which were not reported before. Gene enrichment analysis highlighted significant enrichment of palmitoylated proteins in cell-matrix adhesion, cell-cell recognition, protein cellular localization, and translation, among others. We further studied the effect of 3 potential palmitoylation sites (Cys 799, 900, and 816) of Niemann-Pick type C1 protein (NPC1) on cholesterol accumulation. We found that mutation of any single Cys alone had no significant effect on intracellular cholesterol accumulation while simultaneous mutation of Cys799 and 800 caused significant cholesterol accumulation in the late endosome. No further cholesterol accumulation was observed by adding another mutation at Cys 816. However, the mutation did not alter the cellular localization of the protein. Conclusion: PRE cells have an abundant number of palmitoylated proteins which are involved in cellular processes critical to visual function. The palmitoylation at Cys799 and 800 was needed for cholesterol export, but not the intracellular localization of NPC1.

Keywords:  Niemann-Pick type C1 protein; acyl-biotin exchange (ABE); cholesterol transport; palmitoylation; protein post-translational modification; retinal pigment epithelial cells

DOI:  https://doi.org/10.3389/fnagi.2022.965943
Nat Commun. 2022 Oct 21. 13(1): 6243

WASH activation controls endosomal recycling and EGFR and Hippo signaling during tumor-suppressive cell competition.

Dan Liu, Vasilios Tsarouhas, Christos Samakovlis.

Cell competition is a conserved homeostatic mechanism whereby epithelial cells eliminate neighbors with lower fitness. Cell communication at the interface of wild-type "winner" cells and polarity-deficient (scrib-/-) "losers" is established through Sas-mediated Ptp10D activation in polarity-deficient cells. This tumor-suppressive cell competition restrains EGFR and Hippo signaling and enables Eiger-JNK mediated apoptosis in scrib-/- clones. Here, we show that the activation state of the endosomal actin regulator WASH is a central node linking EGFR and Hippo signaling activation. The tyrosine kinase Btk29A and its substrate WASH are required downstream of Ptp10D for "loser" cell elimination. Constitutively active, phosphomimetic WASH is sufficient to induce both EGFR and Yki activation leading to overgrowth. On the mechanistic level we show that Ptp10D is recycled by the WASH/retromer complex, while EGFR is recycled by the WASH/retriever complex. Constitutive WASH activation selectively interferes with retromer function leading to Ptp10D mistargeting while promoting EGFR recycling and signaling activation. Phospho-WASH also activates aberrant Arp2/3 actin polymerization, leading to cytoskeletal imbalance, Yki activation and reduced apoptosis. Selective manipulation of WASH phosphorylation on sorting endosomes may restrict epithelial tumorous growth.

DOI: https://doi.org/10.1038/s41467-022-34067-1
J Cell Biol. 2022 11 07. pii: e202210014. [Epub ahead of print]221(11):

A second chance at yeast early endosomes.

Allyson F O'Donnell.

Post-endocytic recycling in yeast has been posited to transit solely through the Golgi, raising the possibility that yeast lack early endosomes. In this issue, Laidlaw and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109137) describe a yeast endosomal recycling pathway that gives proteins a second chance to return to the plasma membrane.

DOI: https://doi.org/10.1083/jcb.202210014
Nature. 2022 Oct 19.

Clathrin-associated AP-1 controls termination of STING signalling.

Ying Liu, Pengbiao Xu, Sophie Rivara, Chong Liu, Jonathan Ricci, Xuefeng Ren, James H Hurley, Andrea Ablasser.

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

DOI: https://doi.org/10.1038/s41586-022-05354-0
J Clin Invest. 2022 Oct 18. pii: e161408. [Epub ahead of print]

Glutamine synthetase limits b-catenin-mutated liver cancer growth by maintaining nitrogen homeostasis and suppressing mTORC1.

Weiwei Dai, Jianliang Shen, Junrong Yan, Alex J Bott, Sara Maimouni, Heineken Q Daguplo, Yujue Wang, Khoosheh Khayati, Jessie Yanxiang Guo, Lanjing Zhang, Yongbo Wang, Alexander Valvezan, Wen-Xing Ding, Xin Chen, Xiaoyang Su, Shenglan Gao, Wei-Xing Zong.

  Glutamine synthetase (GS) catalyzes de novo synthesis of glutamine that facilitates cancer cell growth. In the liver, GS functions next to the urea cycle to remove ammonia waste. As dysregulated urea cycle is implicated in cancer development, the impact of GS' ammonia clearance function has not been explored in cancer. Here we show that, oncogenic activation of beta-catenin led to decreased urea cycle and elevated ammonia waste burden. While beta-catenin induced the expression of GS, which is thought to be cancer-promoting, surprisingly, genetic ablation of hepatic GS accelerated the onset of liver tumors in several mouse models that involved β-catenin activation. Mechanistically, GS ablation exacerbated hyperammonemia and facilitated the production of glutamate-derived non-essential amino acids (NEAAs), which subsequently stimulated mTORC1. Pharmacological and genetic inhibition of mTORC1 and glutamic transaminases suppressed tumorigenesis facilitated by GS ablation. While HCC patients, especially those with CTNNB1 mutations, have an overall defective urea cycle and increased expression of GS, there exists a subset of patients with low GS expression that is associated with mTORC1 hyperactivation. Therefore, GS-mediated ammonia clearance serves as a tumor-suppressing mechanism in livers that harbor β-catenin activation mutations and a compromised urea cycle.

Keywords:  Hepatology; Liver cancer; Metabolism

DOI:  https://doi.org/10.1172/JCI161408
Cell Rep Med. 2022 Oct 18. pii: S2666-3791(22)00333-0. [Epub ahead of print]3(10): 100778

Targeting LRRK2 in Parkinson's disease.

Bin Xiao, Eng-King Tan.

Jennings et al.1 reported that LRRK2 inhibitors can reduce kinase activity and improve lysosomal function with minor adverse effects in both animal models and human subjects. The findings provide proof of principle for LRRK2 inhibitor as a Parkinson's disease therapeutic option.

DOI: https://doi.org/10.1016/j.xcrm.2022.100778
Adv Neurobiol. 2023 ;29 305-332

Gangliosides and Cell Surface Ganglioside Metabolic Enzymes in the Nervous System.

Massimo Aureli, Laura Mauri, Emma Veronica Carsana, Dorina Dobi, Silvia Breviario, Giulia Lunghi, Sandro Sonnino.

  Gangliosides are a large group of complex lipids found predominantly in the outer layer of the plasma membrane of cells, particularly abundant in nerve endings. Their half-life in the nervous system is short, and their membrane composition and content are strictly connected to their metabolism. The neobiosynthesis of gangliosides starts in the endoplasmic reticulum and is completed in the Golgi apparatus, whereas catabolism occurs primarily in lysosomes. However, the final content of gangliosides in the plasma membrane is defined by other cellular processes.This chapter will discuss structural changes in the oligosaccharide chains of gangliosides, induced by the activity of plasma membrane-associated glycohydrolases and glycosyltransferases. Some of the plasma membrane enzymes originate from fusion processes between intracellular fractions and the plasma membrane, while, others display a different structure. Several of these plasma membrane enzymes have been characterized and some of them seem to have a specific role in the nervous system.

Keywords:  Central nervous system; Gangliosides; Glycohydrolases; Glycosphingolipids; Neurodegeneration; Neuronal differentiation; Sphingolipid metabolism

DOI:  https://doi.org/10.1007/978-3-031-12390-0_11
Autophagy. 2022 Oct 17.

LC3/GABARAP binding to fluid membranes is potentiated by ceramide.

Yaiza R Varela, Alicia Alonso.

  LC3/GABARAP constitute a macroautophagy/autophagy-related protein family derived from yeast Atg8. The involvement of specific lipids in LC3/GABARAP function is poorly understood. Exploring the interaction of LC3/GABARAP proteins with phosphatidylcholine- or sphingomyelin-based bilayers has revealed that cardiolipin is essential for the protein-bilayer interaction, and that ceramide markedly increases binding. Giant unilamellar vesicles examined under confocal fluorescence microscopy reveal that ceramide segregates laterally into very rigid domains, while GABARAP binds only the more fluid regions, suggesting that the enhancing role of ceramide is exerted by the minority of ceramide molecules dispersed in the fluid phase.

Keywords:  LC3/GABARAP; autophagy proteins; cardiolipin; ceramide; mitochondria

DOI:  https://doi.org/10.1080/15548627.2022.2136821
Proc Natl Acad Sci U S A. 2022 Oct 25. 119(43): e2207280119

Endocytosis triggers V-ATPase-SYK-mediated priming of cGAS activation and innate immune response.

Yu-Lin Yang, Li-Bo Cao, Wen-Rui He, Li Zhong, Yi Guo, Qing Yang, Hong-Bing Shu, Ming-Ming Hu.

  The current view of nucleic acid-mediated innate immunity is that binding of intracellular sensors to nucleic acids is sufficient for their activation. Here, we report that endocytosis of virus or foreign DNA initiates a priming signal for the DNA sensor cyclic GMP-AMP synthase (cGAS)-mediated innate immune response. Mechanistically, viral infection or foreign DNA transfection triggers recruitment of the spleen tyrosine kinase (SYK) and cGAS to the endosomal vacuolar H+ pump (V-ATPase), where SYK is activated and then phosphorylates human cGASY214/215 (mouse cGasY200/201) to prime its activation. Upon binding to DNA, the primed cGAS initiates robust cGAMP production and mediator of IRF3 activation/stimulator of interferon genes-dependent innate immune response. Consistently, blocking the V-ATPase-SYK axis impairs DNA virus- and transfected DNA-induced cGAMP production and expression of antiviral genes. Our findings reveal that V-ATPase-SYK-mediated tyrosine phosphorylation of cGAS following endocytosis of virus or other cargos serves as a priming signal for cGAS activation and innate immune response.

Keywords:  DNA sensor cGAS; antiviral innate immunity; phosphorylation modification; signal transduction

DOI:  https://doi.org/10.1073/pnas.2207280119
Front Immunol. 2022 ;13 946929

Mycobacterial acyl carrier protein suppresses TFEB activation and upregulates miR-155 to inhibit host defense.

Seungwha Paik, Kyeong Tae Kim, In Soo Kim, Young Jae Kim, Hyeon Ji Kim, Seunga Choi, Hwa-Jung Kim, Eun-Kyeong Jo.

  Mycobacterial acyl carrier protein (AcpM; Rv2244), a key protein involved in Mycobacterium tuberculosis (Mtb) mycolic acid production, has been shown to suppress host cell death during mycobacterial infection. This study reports that mycobacterial AcpM works as an effector to subvert host defense and promote bacterial growth by increasing microRNA (miRNA)-155-5p expression. In murine bone marrow-derived macrophages (BMDMs), AcpM protein prevented transcription factor EB (TFEB) from translocating to the nucleus in BMDMs, which likely inhibited transcriptional activation of several autophagy and lysosomal genes. Although AcpM did not suppress autophagic flux in BMDMs, AcpM reduced Mtb and LAMP1 co-localization indicating that AcpM inhibits phagolysosomal fusion during Mtb infection. Mechanistically, AcpM boosted the Akt-mTOR pathway in BMDMs by upregulating miRNA-155-5p, a SHIP1-targeting miRNA. When miRNA-155-5p expression was inhibited in BMDMs, AcpM-induced increased intracellular survival of Mtb was suppressed. In addition, AcpM overexpression significantly reduced mycobacterial clearance in C3HeB/FeJ mice infected with recombinant M. smegmatis strains. Collectively, our findings point to AcpM as a novel mycobacterial effector to regulate antimicrobial host defense and a potential new therapeutic target for Mtb infection.

Keywords:  Mycobacterium tuberculosis; acyl carrier protein; bone marrow-derived macrophages; microRNA-155-5p; phagosome-lysosome fusion; transcription factor EB

DOI:  https://doi.org/10.3389/fimmu.2022.946929
Mol Genet Metab Rep. 2022 Dec;33 100920

Characterization of a novel exonic deletion in the GALNS gene causing Morquio A syndrome.

Kathryn DeLong, Annette Feigenbaum, Laura Pollard, Andrew Lay, Timothy Wood.

  Mucopolysaccharidosis IVA or Morquio A syndrome is a rare lysosomal storage disorder caused by N-acetylgalactosamine-6-sulfatase deficiency. A diagnosis can be provided by the identification of reduced N-acetylgalactosamine-6-sulfatase activity as well as detection of compound heterozygous or homozygous pathogenic variants in GALNS. We present a case of two sisters of healthy non-consanguineous parents with a severe classical phenotype of Morquio A syndrome. Both patients were found to carry a novel homozygous deletion of exon 9, which was initially suspected by next generation sequencing (NGS) due to lack of coverage, but could not be confirmed by this methodology. Therefore, an allele specific polymerase chain reaction assay was designed to confirm the exon 9 deletion and determine the precise deletion breakpoints (c.899-397_1003-1862del) for our patients. Recognizing limitations of molecular testing is important to ensure proper diagnosis and subsequent treatment for individuals with Morquio A syndrome.

Keywords:  GALNS; Homozygous deletion; Lysosomal storage disorder; MPS IVA; Morquio A syndrome

DOI:  https://doi.org/10.1016/j.ymgmr.2022.100920
Neurology. 2022 Oct 18. 99(16): 703-710

What Is the Role of Organelle Membrane Contact Sites in Neurodegenerative Diseases?

Eduardo Benarroch.

DOI: https://doi.org/10.1212/WNL.0000000000201354
Nat Metab. 2022 Oct;4(10): 1232-1244

Principles and functions of metabolic compartmentalization.

Liron Bar-Peled, Nora Kory.

Metabolism has historically been studied at the levels of whole cells, whole tissues and whole organisms. As a result, our understanding of how compartmentalization-the spatial and temporal separation of pathways and components-shapes organismal metabolism remains limited. At its essence, metabolic compartmentalization fulfils three important functions or 'pillars': establishing unique chemical environments, providing protection from reactive metabolites and enabling the regulation of metabolic pathways. However, how these pillars are established, regulated and maintained at both the cellular and systemic levels remains unclear. Here we discuss how the three pillars are established, maintained and regulated within the cell and discuss the consequences of dysregulation of metabolic compartmentalization in human disease. Organelles are increasingly emerging as 'command-and-control centres' and the increased understanding of metabolic compartmentalization is revealing new aspects of metabolic homeostasis, with this knowledge being translated into therapies for the treatment of cancer and certain neurodegenerative diseases.

DOI: https://doi.org/10.1038/s42255-022-00645-2
Sci Adv. 2022 Oct 21. 8(42): eadd3914

Self-assembly and structure of a clathrin-independent AP-1:Arf1 tubular membrane coat.

Richard M Hooy, Yuichiro Iwamoto, Dan A Tudorica, Xuefeng Ren, James H Hurley.

The adaptor protein (AP) complexes not only form the inner layer of clathrin coats but also have clathrin-independent roles in membrane traffic whose mechanisms are unknown. HIV-1 Nef hijacks AP-1 to sequester major histocompatibility complex class I (MHC-I), evading immune detection. We found that AP-1:Arf1:Nef:MHC-I forms a coat on tubulated membranes without clathrin and determined its structure. The coat assembles via Arf1 dimer interfaces. AP-1-positive tubules are enriched in cells upon clathrin knockdown. Nef localizes preferentially to AP-1 tubules in cells, explaining how Nef sequesters MHC-I. Coat contact residues are conserved across Arf isoforms and the Arf-dependent AP complexes AP-1, AP-3, and AP-4. Thus, AP complexes can self-assemble with Arf1 into tubular coats without clathrin or other scaffolding factors. The AP-1:Arf1 coat defines the structural basis of a broader class of tubulovesicular membrane coats as an intermediate in clathrin vesicle formation from internal membranes and as an MHC-I sequestration mechanism in HIV-1 infection.

DOI: https://doi.org/10.1126/sciadv.add3914
Nature. 2022 Oct 19.

Mechanism of an intramembrane chaperone for multipass membrane proteins.

Luka Smalinskaitė, Min Kyung Kim, Aaron J O Lewis, Robert J Keenan, Ramanujan S Hegde.

Multipass membrane proteins play numerous roles in biology and include receptors, transporters, ion channels and enzymes1,2. How multipass proteins are co-translationally inserted and folded at the endoplasmic reticulum is not well understood2. The prevailing model posits that each transmembrane domain (TMD) of a multipass protein successively passes into the lipid bilayer through a front-side lateral gate of the Sec61 protein translocation channel3-9. The PAT complex, an intramembrane chaperone comprising Asterix and CCDC47, engages early TMDs of multipass proteins to promote their biogenesis by an unknown mechanism10. Here, biochemical and structural analysis of intermediates during multipass protein biogenesis showed that the nascent chain is not engaged with Sec61, which is occluded and latched closed by CCDC47. Instead, Asterix binds to and redirects the substrate to a location behind Sec61, where the PAT complex contributes to a multipass translocon surrounding a semi-enclosed, lipid-filled cavity11. Detection of multiple TMDs in this cavity after their emergence from the ribosome suggests that multipass proteins insert and fold behind Sec61. Accordingly, biogenesis of several multipass proteins was unimpeded by inhibitors of the Sec61 lateral gate. These findings elucidate the mechanism of an intramembrane chaperone and suggest a new framework for multipass membrane protein biogenesis at the endopasmic reticulum.

DOI: https://doi.org/10.1038/s41586-022-05336-2
PLoS Comput Biol. 2022 Oct 17. 18(10): e1010586

Modelling membrane reshaping by staged polymerization of ESCRT-III filaments.

Xiuyun Jiang, Lena Harker-Kirschneck, Christian Vanhille-Campos, Anna-Katharina Pfitzner, Elene Lominadze, Aurélien Roux, Buzz Baum, Anđela Šarić.

ESCRT-III filaments are composite cytoskeletal polymers that can constrict and cut cell membranes from the inside of the membrane neck. Membrane-bound ESCRT-III filaments undergo a series of dramatic composition and geometry changes in the presence of an ATP-consuming Vps4 enzyme, which causes stepwise changes in the membrane morphology. We set out to understand the physical mechanisms involved in translating the changes in ESCRT-III polymer composition into membrane deformation. We have built a coarse-grained model in which ESCRT-III polymers of different geometries and mechanical properties are allowed to copolymerise and bind to a deformable membrane. By modelling ATP-driven stepwise depolymerisation of specific polymers, we identify mechanical regimes in which changes in filament composition trigger the associated membrane transition from a flat to a buckled state, and then to a tubule state that eventually undergoes scission to release a small cargo-loaded vesicle. We then characterise how the location and kinetics of polymer loss affects the extent of membrane deformation and the efficiency of membrane neck scission. Our results identify the near-minimal mechanical conditions for the operation of shape-shifting composite polymers that sever membrane necks.

DOI: https://doi.org/10.1371/journal.pcbi.1010586