bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒04‒18
fifty-one papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Rab2 drives axonal transport of dense core vesicles and lysosomal organelles.
  2. Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells.
  3. Lysosome (Dys)function in Atherosclerosis-A Big Weight on the Shoulders of a Small Organelle.
  4. The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.
  5. Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.
  6. The lysosomal protein ABCD4 can transport vitamin B12 across liposomal membranes in vitro.
  7. A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage.
  8. Mini-Review Free Sialic Acid Storage Disorder: Progress and Promise.
  9. Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.
  10. Augmenting ATG14 alleviates atherosclerosis and inhibits inflammation via promotion of autophagosome-lysosome fusion in macrophages.
  11. Estradiol analogs attenuate autophagy, cell migration and invasion by direct and selective inhibition of TRPML1, independent of estrogen receptors.
  12. GM1 Gangliosidosis: Mechanisms and Management.
  13. Nuclear ErbB2 represses DEPTOR transcription to inhibit autophagy in breast cancer cells.
  14. How can we turn the PI3K/AKT/mTOR pathway down? Insights into inhibition and treatment of cancer.
  15. Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy.
  16. mTOR activation initiates renal cell carcinoma development by coordinating ERK and p38MAPK.
  17. Major Advances in Targeted Protein Degradation: PROTACs, LYTACs, and MADTACs.
  18. Destroy the Old to Build the New: Activity-Dependent Lysosomal Exocytosis in Neurons.
  19. Acetylated tau inhibits chaperone-mediated autophagy and promotes tau pathology propagation in mice.
  20. Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish.
  21. mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.
  22. Manipulation of EGFR-Induced Signaling for the Recruitment of Quiescent Neural Stem Cells in the Adult Mouse Forebrain.
  23. Examination of a blood-brain barrier targeting β-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis.
  24. Fusion and fission events regulate endosome maturation and viral escape.
  25. CRL4AMBRA1 is a master regulator of D-type cyclins.
  26. Phosphorylation of the LIR domain of SCOC modulates ATG8 binding affinity and specificity.
  27. Mechanisms of demyelination and neurodegeneration in globoid cell leukodystrophy.
  28. Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages.
  29. Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.
  30. Nuclear translocation of ASPL-TFE3 fusion protein creates favorable metabolism by mediating autophagy in translocation renal cell carcinoma.
  31. The Role of Autophagy in Skeletal Muscle Diseases.
  32. A model for a partnership of lipid transfer proteins and scramblases in membrane expansion and organelle biogenesis.
  33. mTOR as a senescence manipulation target: A forked road.
  34. Kog1/Raptor mediates metabolic rewiring during nutrient limitation by controlling SNF1/AMPK activity.
  35. ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.
  36. AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.
  37. The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.
  38. Food for thought: How cell adhesion coordinates nutrient sensing.
  39. TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.
  40. Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis.
  41. The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.
  42. Interorganelle communication, aging, and neurodegeneration.
  43. Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.
  44. A NOVEL NOX/PHOX-CD38-NAADP-TFEB AXIS IMPORTANT FOR MACROPHAGE ACTIVATION DURING BACTERIAL PHAGOCYTOSIS.
  45. SOD1 regulates ribosome biogenesis in KRAS mutant non-small cell lung cancer.
  46. The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains.
  47. Unconventional endocytic mechanisms.
  48. The mTOR/NF-κB Pathway Mediates Neuroinflammation and Synaptic Plasticity in Diabetic Encephalopathy.
  49. N- and C-terminal Gln3-Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization.
  50. Organelle degradation in the lens by PLAAT phospholipases.
  51. mTORC1 mediates fiber type-specific regulation of protein synthesis and muscle size during denervation.
  1. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00287-4. [Epub ahead of print]35(2): 108973
    Rab2 drives axonal transport of dense core vesicles and lysosomal organelles.
    Viktor Karlovich Lund, Matthew Domenic Lycas, Anders Schack, Rita Chan Andersen, Ulrik Gether, Ole Kjaerulff.
      Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. Here, we use Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. We also provide evidence that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. Our results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility.
    Keywords:  Arl8; Drosophila; Rab2; axonal transport; dense core vesicles; endosomes; lysosomes; membrane trafficking; neuropeptides; synaptogenesis
    DOI:  https://doi.org/10.1016/j.celrep.2021.108973
  2. Autophagy. 2021 Apr 14. 1-19
    Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells.
    Kazue Terasawa, Yuji Kato, Yuta Ikami, Kensaku Sakamoto, Kazumasa Ohtake, Seisuke Kusano, Yuri Tomabechi, Mutsuko Kukimoto-Niino, Mikako Shirouzu, Jun-Lin Guan, Toshihide Kobayashi, Takanori Iwata, Tetsuro Watabe, Shigeyuki Yokoyama, Miki Hara-Yokoyama.
      LAMP1 (lysosomal-associated membrane protein 1) and LAMP2 are the most abundant protein components of lysosome membranes. Both LAMPs have common structures consisting of a large lumenal domain composed of two domains (N-domain and C-domain, which are membrane-distal and -proximal, respectively), both with the β-prism fold, a transmembrane domain, and a short cytoplasmic tail. LAMP2 is involved in various aspects of autophagy, and reportedly forms high-molecular weight complexes at the lysosomal membrane. We previously showed that LAMP2 molecules coimmunoprecipitated with each other, but whether the homophilic interaction is direct or indirect has remained to be elucidated. In the present study, we demonstrated the direct homophilic interaction of mouse LAMP2A molecules, using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces. Specifically, the results suggested that LAMP2A molecules assemble by facing each other with one side of the β-prism (defined as side A) of the C-domains. The N-domain truncation, which increased the coimmunoprecipitation of LAMP2A molecules in our previous study, permitted the nonspecific involvement of both sides of the β-prism (side A and side B). Thus, the presence of the N-domain restricts the LAMP2A interactions to side A-specific. The truncation of LAMP2A impaired the recruitment of GAPDH (a CMA-substrate) fused to the HaloTag protein to the surface of late endosomes/lysosomes (LE/Lys) and affected a process that generates LE/Lys. The present study revealed that the homophilic interaction of LAMP2A is direct, and the side A-specific, homophilic interaction of LAMP2A is required for the functional aspects of LAMP2A.Abbreviations:Aloc-Lys: Nε-allyloxycarbonyl-l-lysine; CMA: chaperone-mediated autophagy; FFE: free-flow electrophoresis; GAPDH-HT: glyceraldehyde-3-phosphate dehydrogenase fused to HaloTag protein; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LBPA: lysobisphosphatidic acid; LE/Lys: late endosome/lysosomes; MEFs: mouse embryonic fibroblasts; pBpa: p-benzoyl- l-phenylalanine.
    Keywords:  Chaperone-mediated autophagy; GAPDH-HT; expanded genetic codes; lysosomes; photo-crosslinking; protein assembly
    DOI:  https://doi.org/10.1080/15548627.2021.1911017
  3. Front Cell Dev Biol. 2021 ;9 658995
    Lysosome (Dys)function in Atherosclerosis-A Big Weight on the Shoulders of a Small Organelle.
    André R A Marques, Cristiano Ramos, Gisela Machado-Oliveira, Otília V Vieira.
      Atherosclerosis is a progressive insidious chronic disease that underlies most of the cardiovascular pathologies, including myocardial infarction and ischemic stroke. The malfunctioning of the lysosomal compartment has a central role in the etiology and pathogenesis of atherosclerosis. Lysosomes are the degradative organelles of mammalian cells and process endogenous and exogenous substrates in a very efficient manner. Dysfunction of these organelles and consequent inefficient degradation of modified low-density lipoproteins (LDL) and apoptotic cells in atherosclerotic lesions have, therefore, numerous deleterious consequences for cellular homeostasis and disease progression. Lysosome dysfunction has been mostly studied in the context of the inherited lysosomal storage disorders (LSDs). However, over the last years it has become increasingly evident that the consequences of this phenomenon are more far-reaching, also influencing the progression of multiple acquired human pathologies, such as neurodegenerative diseases, cancer, and cardiovascular diseases (CVDs). During the formation of atherosclerotic plaques, the lysosomal compartment of the various cells constituting the arterial wall is under severe stress, due to the tremendous amounts of lipoproteins being processed by these cells. The uncontrolled uptake of modified lipoproteins by arterial phagocytic cells, namely macrophages and vascular smooth muscle cells (VSMCs), is the initial step that triggers the pathogenic cascade culminating in the formation of atheroma. These cells become pathogenic "foam cells," which are characterized by dysfunctional lipid-laden lysosomes. Here, we summarize the current knowledge regarding the origin and impact of the malfunctioning of the lysosomal compartment in plaque cells. We further analyze how the field of LSD research may contribute with some insights to the study of CVDs, particularly how therapeutic approaches that target the lysosomes in LSDs could be applied to hamper atherosclerosis progression and associated mortality.
    Keywords:  atherosclerosis; autophagy; lysosomal storage diseases; lysosome dysfunction; oxidized lipids
    DOI:  https://doi.org/10.3389/fcell.2021.658995
  4. Mol Cell. 2021 Apr 05. pii: S1097-2765(21)00226-4. [Epub ahead of print]
    The dynamic mechanism of 4E-BP1 recognition and phosphorylation by mTORC1.
    Raphael Böhm, Stefan Imseng, Roman P Jakob, Michael N Hall, Timm Maier, Sebastian Hiller.
      The activation of cap-dependent translation in eukaryotes requires multisite, hierarchical phosphorylation of 4E-BP by the 1 MDa kinase mammalian target of rapamycin complex 1 (mTORC1). To resolve the mechanism of this hierarchical phosphorylation at the atomic level, we monitored by NMR spectroscopy the interaction of intrinsically disordered 4E binding protein isoform 1 (4E-BP1) with the mTORC1 subunit regulatory-associated protein of mTOR (Raptor). The N-terminal RAIP motif and the C-terminal TOR signaling (TOS) motif of 4E-BP1 bind separate sites in Raptor, resulting in avidity-based tethering of 4E-BP1. This tethering orients the flexible central region of 4E-BP1 toward the mTORC1 kinase site for phosphorylation. The structural constraints imposed by the two tethering interactions, combined with phosphorylation-induced conformational switching of 4E-BP1, explain the hierarchy of 4E-BP1 phosphorylation by mTORC1. Furthermore, we demonstrate that mTORC1 recognizes both free and eIF4E-bound 4E-BP1, allowing rapid phosphorylation of the entire 4E-BP1 pool and efficient activation of translation. Finally, our findings provide a mechanistic explanation for the differential rapamycin sensitivity of the 4E-BP1 phosphorylation sites.
    Keywords:  NMR spectroscopy; atypical kinase; hierarchical phosphorylation; intrinsically disordered protein; kinetic modelling; mTOR signaling; multi‑site binding; protein dynamics; target of rapamycin; translational control
    DOI:  https://doi.org/10.1016/j.molcel.2021.03.031
  5. EMBO Mol Med. 2021 Apr 14. e13258
    Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.
    Reini E N van der Welle, Rebekah Jobling, Christian Burns, Paolo Sanza, Jan A van der Beek, Alfonso Fasano, Lan Chen, Fried J Zwartkruis, Susan Zwakenberg, Edward F Griffin, Corlinda Ten Brink, Tineke Veenendaal, Nalan Liv, Conny M A van Ravenswaaij-Arts, Henny H Lemmink, Rolph Pfundt, Susan Blaser, Carolina Sepulveda, Andres M Lozano, Grace Yoon, Teresa Santiago-Sim, Cedric S Asensio, Guy A Caldwell, Kim A Caldwell, David Chitayat, Judith Klumperman.
      Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662 * ; VPS41c.1423-2A>G and VPS41R662 * ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662 * abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.
    Keywords:  Autophagy; HOPS complex; TFEB/TFE3; lysosome-associated disorder; mTORC1
    DOI:  https://doi.org/10.15252/emmm.202013258
  6. J Biol Chem. 2021 Apr 09. pii: S0021-9258(21)00440-3. [Epub ahead of print] 100654
    The lysosomal protein ABCD4 can transport vitamin B12 across liposomal membranes in vitro.
    Katsuki Kitai, Kosuke Kawaguchi, Takenori Tomohiro, Masashi Morita, Takanori So, Tsuneo Imanaka.
      Vitamin B12 (cobalamin) is an essential micronutrient for human health, and mutation and dysregulation of cobalamin metabolism are associated with serious diseases, such as methylmalonic aciduria and homocystinuria. Mutations in ABCD4 or LMBRD1, which encode the ATP-binding cassette (ABC) transporter ABCD4 and lysosomal membrane protein LMBD1, respectively, lead to errors in cobalamin metabolism, with the phenotype of a failure to release cobalamin from lysosomes. However, the mechanism of transport of cobalamin across the lysosomal membrane remains unknown. We previously demonstrated that LMBD1 is required for the translocation of ABCD4 from the endoplasmic reticulum to lysosomes. This suggests that ABCD4 performs an important function in lysosomal membrane cobalamin transport. In this study, we expressed human ABCD4 and LMBD1 in methylotrophic yeast and purified them. We prepared ABCD4 and/or LMBD1 containing liposomes loaded with cobalamin, and then quantified the release of cobalamin from the liposomes by reverse-phase HPLC. We observed that ABCD4 was able to transport cobalamin from the inside to the outside of liposomes dependent on its ATPase activity, and that LMBD1 exhibited no cobalamin transport activity. These results suggest that ABCD4 may be capable of transporting cobalamin from the lysosomal lumen to the cytosol. Furthermore, we examined a series of ABCD4 missense mutations to understand how these alterations impair cobalamin transport. Our findings give insight into the molecular mechanism of cobalamin transport by which ABCD4 involves and its importance in cobalamin deficiency.
    Keywords:  ABC transporter; cobalamin; lysosome; methylotrophic yeast; protein purification; proteoliposome; recombinant protein expression
    DOI:  https://doi.org/10.1016/j.jbc.2021.100654
  7. Elife. 2021 04 13. pii: e62653. [Epub ahead of print]10
    A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage.
    Madhuja Samaddar, Jérôme Goudeau, Melissa Sanchez, David H Hall, K Adam Bohnert, Maria Ingaramo, Cynthia Kenyon.
      Somatic cells age and die, but the germ-cell lineage is immortal. In Caenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.
    Keywords:  C. elegans; cell biology; genetic screen; germ lineage; lysosome acidification; oocyte maturation; proteostasis
    DOI:  https://doi.org/10.7554/eLife.62653
  8. Neurosci Lett. 2021 Apr 13. pii: S0304-3940(21)00274-3. [Epub ahead of print] 135896
    Mini-Review Free Sialic Acid Storage Disorder: Progress and Promise.
    Marjan Huizing, Mary E Hackbarth, David R Adams, Melissa Wasserstein, Marc C Patterson, Steven U Walkley, William A Gahl, , David R Adams, Kostantin Dobrenis, Jessica Foglio, William A Gahl, Bruno Gasnier, Mary Hackbarth, Marjan Huizing, Monkol Lek, May C V Malicdan, Liisa E Paavola, Marc C Patterson, Richard Reimer, Steven U Walkley, Melissa Wasserstein, Raymond Y Wang, Roberto Zoncu.
      Lysosomal free sialic acid storage disorder (FSASD) is an extremely rare, autosomal recessive, neurodegenerative, multisystemic disorder caused by defects in the lysosomal sialic acid membrane exporter SLC17A5 (sialin). SLC17A5 defects cause free sialic acid and some other acidic hexoses to accumulate in lysosomes, resulting in enlarged lysosomes in some cell types and 10-100-fold increased urinary excretion of free sialic acid. Clinical features of FSASD include coarse facial features, organomegaly, and progressive neurodegenerative symptoms with cognitive impairment, cerebellar ataxia and muscular hypotonia. Central hypomyelination with cerebellar atrophy and thinning of the corpus callosum are also prominent disease features. Around 200 FSASD cases are reported worldwide, with the clinical spectrum ranging from a severe infantile onset form, often lethal in early childhood, to a mild, less severe form with subjects living into adulthood, also called Salla disease. The pathobiology of FSASD remains poorly understood and FSASD is likely underdiagnosed. Known patients have experienced a diagnostic delay due to the rarity of the disorder, absence of routine urine sialic acid testing, and non-specific clinical symptoms, including developmental delay, ataxia and infantile hypomyelination. There is no approved therapy for FSASD. We initiated a multidisciplinary collaborative effort involving worldwide academic clinical and scientific FSASD experts, the National Institutes of Health (USA), and the FSASD patient advocacy group (Salla Treatment and Research [S.T.A.R.] Foundation) to overcome the scientific, clinical and financial challenges facing the development of new treatments for FSASD. We aim to collect data that incentivize industry to further develop, obtain approval for, and commercialize FSASD treatments. This review summarizes current aspects of FSASD diagnosis, prevalence, etiology, and disease models, as well as challenges on the path to therapeutic approaches for FSASD.
    Keywords:  Infantile Sialic Acid Storage Disorder; N-acetylneuraminic acid; SLC17A5; Salla disease; hypomyelination; lysosomal membrane transporter; sialic acid
    DOI:  https://doi.org/10.1016/j.neulet.2021.135896
  9. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00314-4. [Epub ahead of print]35(2): 109000
    Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.
    Frida Sommer, Vincenzo Torraca, Yufei Xie, Aliede E In 't Veld, Joost Willemse, Annemarie H Meijer.
      Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.
    Keywords:  chemotaxis; infection; inflammation; lysosome; macrophage; mycobacteria; vesicle; zebrafish
    DOI:  https://doi.org/10.1016/j.celrep.2021.109000
  10. Autophagy. 2021 Apr 14.
    Augmenting ATG14 alleviates atherosclerosis and inhibits inflammation via promotion of autophagosome-lysosome fusion in macrophages.
    Hui Zhang, Song Ge, Buqing Ni, Keshuai He, Pengcheng Zhu, Xiaohong Wu, Yongfeng Shao.
      Dysfunction of macroautophagy/autophagy in macrophages contributes to atherosclerosis. Impaired autophagy-lysosomal degradation system leads to lipid accumulation, facilitating atherosclerotic plaque. ATG14 is an essential regulator for the fusion of autophagosomes with lysosomes. Whether ATG14 plays a role in macrophage autophagy dysfunction in atherosclerosis is unknown. To investigate the effects of ATG14 on macrophage autophagy, human atherosclerotic plaque, apoe-/- mice and cultured mouse macrophages were evaluated. Overexpression of ATG14 by adenovirus was used to reveal its function in autophagy, inflammation and atherosclerotic plaque formation. Results showed that impaired autophagy function with reduction of ATG14 expression existed in macrophages of human and mouse atherosclerotic plaques. Ox-LDL impaired autophagosome-lysosome fusion with reduction of ATG14 expression in macrophages. Overexpression of ATG14 in macrophages enhanced fusion of autophagosomes with lysosomes and promoted lipid degradation, decreasing Ox-LDL-induced apoptosis and inflammatory response. Augmenting ATG14 expression reversed the autophagy dysfunction in macrophages of apoe-/- mice plaque, blunted SQSTM1/p62 accumulation, inhibited inflammation, and upregulated the population of Treg cells, resulting in alleviating atherosclerotic lesions.
    Keywords:  ATG14; atherosclerosis; autophagy; inflammation; lipid degradation
    DOI:  https://doi.org/10.1080/15548627.2021.1909833
  11. Sci Rep. 2021 Apr 15. 11(1): 8313
    Estradiol analogs attenuate autophagy, cell migration and invasion by direct and selective inhibition of TRPML1, independent of estrogen receptors.
    Philipp Rühl, Anna Scotto Rosato, Nicole Urban, Susanne Gerndt, Rachel Tang, Carla Abrahamian, Charlotte Leser, Jiansong Sheng, Archana Jha, Günter Vollmer, Michael Schaefer, Franz Bracher, Christian Grimm.
      The cation channel TRPML1 is an important regulator of lysosomal function and autophagy. Loss of TRPML1 is associated with neurodegeneration and lysosomal storage disease, while temporary inhibition of this ion channel has been proposed to be beneficial in cancer therapy. Currently available TRPML1 channel inhibitors are not TRPML isoform selective and block at least two of the three human isoforms. We have now identified the first highly potent and isoform-selective TRPML1 antagonist, the steroid 17β-estradiol methyl ether (EDME). Two analogs of EDME, PRU-10 and PRU-12, characterized by their reduced activity at the estrogen receptor, have been identified through systematic chemical modification of the lead structure. EDME and its analogs, besides being promising new small molecule tool compounds for the investigation of TRPML1, selectively affect key features of TRPML1 function: autophagy induction and transcription factor EB (TFEB) translocation. In addition, they act as inhibitors of triple-negative breast cancer cell migration and invasion.
    DOI:  https://doi.org/10.1038/s41598-021-87817-4
  12. Appl Clin Genet. 2021 ;14 209-233
    GM1 Gangliosidosis: Mechanisms and Management.
    Allisandra K Rha, Anne S Maguire, Douglas R Martin.
      The lysosomal storage disorder, GM1 gangliosidosis (GM1), is a neurodegenerative condition resulting from deficiency of the enzyme β-galactosidase (β-gal). Mutation of the GLB1 gene, which codes for β-gal, prevents cleavage of the terminal β-1,4-linked galactose residue from GM1 ganglioside. Subsequent accumulation of GM1 ganglioside and other substrates in the lysosome impairs cell physiology and precipitates dysfunction of the nervous system. Beyond palliative and supportive care, no FDA-approved treatments exist for GM1 patients. Researchers are critically evaluating the efficacy of substrate reduction therapy, pharmacological chaperones, enzyme replacement therapy, stem cell transplantation, and gene therapy for GM1. A Phase I/II clinical trial for GM1 children is ongoing to evaluate the safety and efficacy of adeno-associated virus-mediated GLB1 delivery by intravenous injection, providing patients and families with hope for the future.
    Keywords:  GLB1; GM1 gangliosidosis; LSD; biomarkers; chaperone; gene therapy
    DOI:  https://doi.org/10.2147/TACG.S206076
  13. Cell Death Dis. 2021 Apr 14. 12(4): 397
    Nuclear ErbB2 represses DEPTOR transcription to inhibit autophagy in breast cancer cells.
    Yanli Bi, Longyuan Gong, Pengyuan Liu, Xiufang Xiong, Yongchao Zhao.
      ErbB2, a classical receptor tyrosine kinase, is frequently overexpressed in breast cancer cells. Although the role of ErbB2 in the transmission of extracellular signals to intracellular matrix has been widely studied, the functions of nuclear ErbB2 remain largely elusive. Here, we report a novel function of nuclear ErbB2 in repressing the transcription of DEPTOR, a direct inhibitor of mTOR. Nuclear ErbB2 directly binds to the consensus binding sequence in the DEPTOR promoter to repress its transcription. The kinase activity of ErbB2 is required for its nuclear translocation and transcriptional repression of DEPTOR. Moreover, the repressed DEPTOR by nuclear ErbB2 inhibits the induction of autophagy by activating mTORC1. Thus, our study reveals a novel mechanism for autophagy regulation by functional ErbB2, which translocates to the nucleus and acts as a transcriptional regulator to suppress DEPTOR transcription, leading to activation of the PI3K/AKT/mTOR pathway to inhibit autophagy.
    DOI:  https://doi.org/10.1038/s41419-021-03686-9
  14. Expert Rev Anticancer Ther. 2021 Apr 15.
    How can we turn the PI3K/AKT/mTOR pathway down? Insights into inhibition and treatment of cancer.
    Said M Afify, Aung Ko Ko Oo, Ghmkin Hassan, Akimasa Seno, Masaharu Seno.
      INTRODUCTION: The phosphatidylinositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway is a fundamental regulator of cell proliferation and survival. Dysregulation in this pathway leads to the development of cancer. Accumulating evidence indicates that dysregulation in this pathway is involved in cancer initiation, progression, and recurrence. However, the pathway consists of various signal transducing factors related with cellular events, such as transformation, tumorigenesis, cancer progression, and drug resistance. Therefore, it is very important to determine the targets in this pathway for cancer therapy. Although many drugs inhibiting this signaling pathway are in clinical trials or have been approved for treating solid tumors and hematologic malignancies, further understanding of the signaling mechanism is required to achieve better therapeutic efficacy.AREAS COVERED: In this review, we have describe the PI3K/AKT/mTOR pathway in detail, along with its critical role in cancer stem cells, for identifying potential therapeutic targets. We also summarize the recent developments in different types of signaling inhibitors.
    EXPERT OPINION: Downregulation of the PI3K/AKT/mTOR pathway is very important for treating all types of cancers. Thus, further studies are required to establish novel prognostic factors to support the current progress in cancer treatment with emphasis on this pathway.
    Keywords:  4E-BP; AKT/PKB; HIF1-alpha; PI3K; RHEV; S6 kinase; Signaling pathway; TSC1/2; dual inhibitors; mTOR
    DOI:  https://doi.org/10.1080/14737140.2021.1918001
  15. Mol Cell Biochem. 2021 Apr 17.
    Upregulated SOCC and IP3R calcium channels and subsequent elevated cytoplasmic calcium signaling promote nonalcoholic fatty liver disease by inhibiting autophagy.
    Lin Zhang, Yifan Zhang, Yuanqing Jiang, Xiaobing Dou, Songtao Li, Hui Chai, Qianyu Qian, Miaojuan Wang.
      Nonalcoholic fatty liver disease (NAFLD) is related to elevated cytoplasmic calcium signaling in hepatocytes, which may be mediated by store-operated calcium channel (SOCC) and inositol triphosphate receptor (IP3R). However, the regulatory effect of calcium signaling on lipid accumulation and degeneration in hepatocytes and the underlying molecular mechanism remain unknown. Autophagy inhibition promotes lipid accumulation and steatosis in hepatocytes. However, the association between elevated calcium signaling and autophagy inhibition in hepatocytes and its effect on hepatocyte fatty lesions remain unclear. Here, we established a mouse hepatocyte fatty gradient model using oleic acid. SOCC and IP3R channel opening and cytoplasmic calcium levels gradually increased with the hepatocyte pimelosis degree, whereas autophagy gradually decreased. We also established an optimal oleic acid (OOA) hepatocyte model, observing significantly increased SOCC and IP3R channel opening and calcium influx alongside significantly decreased autophagy and aggravated cellular fatty lesion. Calcium channel blockers (CCBs) and calcium channel gene silencing reagents (CCGSRs), respectively, reversed these effects, indicating that elevated cytoplasmic calcium signaling promotes NAFLD occurrence and the development by inhibiting hepatocyte autophagy. In the OOA model, upregulated extracellular regulated protein kinases 1/2 (ERK1/2), which can be regulated by SOCC and IP3R proteins transient receptor potential canonical 1 (TRPC1)/IP3R with elevated cytoplasmic calcium signaling, over-inhibited forkhead/winged helix O (FOXO) signaling and over-activated mammalian target of rapamycin complex 1 (mTORC1) signaling. Over-inhibited FOXO signaling significantly downregulated autophagy-related gene 12, which inhibits autophagosome maturation, while over-activated mTORC1 signaling over-inactivated Unc-51 like autophagy activating kinase 1, which inhibits preautophagosome formation. CCBs and CCGSRs recovered autophagy by significantly downregulating ERK1/2 to block abnormal changes in FOXO and mTORC1 signaling. Our findings indicate that upregulated SOCC and IP3R channels and subsequent elevated cytoplasmic calcium signaling in hepatocyte fatty lesions inhibits hepatocyte autophagy through (TRPC1/IP3R)/ERK/(FOXO/mTORC1) signaling pathways, causes lipid accumulation and degeneration in hepatocytes, and promotes NAFLD occurrence and development.
    Keywords:  (TRPC1/IP3R)/ERK/(FOXO/MTORC1) signaling; Calcium signaling; Hepatocyte autophagy; NAFLD; SOCC/IP3R channel
    DOI:  https://doi.org/10.1007/s11010-021-04150-0
  16. Cancer Res. 2021 Apr 16. pii: canres.3979.2020. [Epub ahead of print]
    mTOR activation initiates renal cell carcinoma development by coordinating ERK and p38MAPK.
    Hongguang Wu, Dan He, Soma Biswas, Md Shafiquzzaman, Xin Zhou, Jean Charron, Yibin Wang, Bijaya K Nayak, Samy L Habib, Huijuan Liu, Baojie Li.
      Renal cell carcinoma (RCC) mainly originates from renal proximal tubules. Intriguingly, disruption of genes frequently mutated in human RCC samples thus far has only generated RCC originated from other renal tubule parts in mouse models. This hampers our understanding of the pathogenesis of RCC. Here we show that mTOR signaling, often activated in RCC samples, initiates RCC development from renal proximal tubules. Ablation of Tsc1, encoding an mTOR suppressor, in proximal tubule cells led to multiple precancerous renal cysts. mTOR activation increased MEK1 expression and ERK activation, and Mek1 ablation or inhibition diminished cyst formation in Tsc1-deficient mice. mTOR activation also increased MKK6 expression and p38MAPK activation, and ablation of the p38α-encoding gene further enhanced cyst formation and led to RCC with clear cell RCC features. Mechanistically, Tsc1 deletion induced p53 and p16 expression in a p38MAPK-dependent manner, and deleting Tsc1 and Trp53 or Cdkn2a (encoding p16) enhanced renal cell carcinogenesis. Thus, mTOR activation in combination with inactivation of the p38MAPK-p53/p16 pathway drives RCC development from renal proximal tubules. Moreover, this study uncovers previously unidentified mechanisms by which mTOR controls cell proliferation and suggests the MEK-ERK axis to be a potential target for treatment of RCC.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-20-3979
  17. J Biol Chem. 2021 Apr 08. pii: S0021-9258(21)00433-6. [Epub ahead of print] 100647
    Major Advances in Targeted Protein Degradation: PROTACs, LYTACs, and MADTACs.
    Shanique Alabi, Craig Crews.
      Of late, targeted protein degradation (TPD) has surfaced as a novel and innovative chemical tool and therapeutic modality. By co-opting protein degradation pathways, TPD facilitates complete removal of the protein molecules from within or outside the cell. While the pioneering Proteolysis Targeting Chimera (PROTAC) technology and molecular glues hijack the ubiquitin-proteasome system, newer modalities co-opt autophagy or the endo-lysosomal pathway. Using this mechanism, TPD is posited to largely expand the druggable space far beyond small molecule inhibitors. In this review, we discuss the major advances in TPD, highlight our current understanding, and explore outstanding questions in the field.
    Keywords:  AUTACs; LYTACs; Molecular Glues; PROTACs; chemical biology; drug action; lysosome; protein degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2021.100647
  18. Neurosci Res. 2021 Apr 09. pii: S0168-0102(21)00074-2. [Epub ahead of print]
    Destroy the Old to Build the New: Activity-Dependent Lysosomal Exocytosis in Neurons.
    Keiji Ibata, Michisuke Yuzaki.
      Lysosomes are organelles that support diverse cellular functions such as terminal degradation of macromolecules and nutrient recycling. Additionally, lysosomes can fuse with the plasma membrane, a phenomenon referred to as lysosomal exocytosis, to release their contents, including hydrolytic enzymes and cargo proteins. Recently, neuronal activity has been shown to induce lysosomal exocytosis in dendrites and axons. Secreted lysosomal enzyme cathepsin B induces and stabilizes synaptic structural changes by degrading the local extracellular matrix. Extracellular matrix reorganization could also enhance the lateral diffusion of the co-released synaptic organizer Cbln1 along the surface of axons to facilitate new synapse formation. Similarly, lateral diffusion of dendritic AMPA-type glutamate receptors could be facilitated to enhance functional synaptic plasticity. Therefore, lysosomal exocytosis is a powerful way of building new cellular structures through the coordinated destruction of the old environment. Understanding the mechanisms by which lysosomal exocytosis is regulated in neurons is expected to lead to the development of new therapeutics for neuronal plasticity following spinal cord injury or neurodegenerative disease.
    Keywords:  Cathepsin; Cbln1; Degradation; Exocytosis; Long-term potentiation; SNARE complex; Synaptic plasticity
    DOI:  https://doi.org/10.1016/j.neures.2021.03.011
  19. Nat Commun. 2021 04 14. 12(1): 2238
    Acetylated tau inhibits chaperone-mediated autophagy and promotes tau pathology propagation in mice.
    Benjamin Caballero, Mathieu Bourdenx, Enrique Luengo, Antonio Diaz, Peter Dongmin Sohn, Xu Chen, Chao Wang, Yves R Juste, Susanne Wegmann, Bindi Patel, Zapporah T Young, Szu Yu Kuo, Jose Antonio Rodriguez-Navarro, Hao Shao, Manuela G Lopez, Celeste M Karch, Alison M Goate, Jason E Gestwicki, Bradley T Hyman, Li Gan, Ana Maria Cuervo.
      Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression.
    DOI:  https://doi.org/10.1038/s41467-021-22501-9
  20. Sci Rep. 2021 Apr 16. 11(1): 8392
    Leucyl-tRNA synthetase deficiency systemically induces excessive autophagy in zebrafish.
    Masanori Inoue, Hiroaki Miyahara, Hiroshi Shiraishi, Nobuyuki Shimizu, Mika Tsumori, Kyoko Kiyota, Miwako Maeda, Ryohei Umeda, Tohru Ishitani, Reiko Hanada, Kenji Ihara, Toshikatsu Hanada.
      Leucyl-tRNA synthetase (LARS) is an enzyme that catalyses the ligation of leucine with leucine tRNA. LARS is also essential to sensitize the intracellular leucine concentration to the mammalian target of rapamycin complex 1 (mTORC1) activation. Biallelic mutation in the LARS gene causes infantile liver failure syndrome type 1 (ILFS1), which is characterized by acute liver failure, anaemia, and neurological disorders, including microcephaly and seizures. However, the molecular mechanism underlying ILFS1 under LARS deficiency has been elusive. Here, we generated Lars deficient (larsb-/-) zebrafish that showed progressive liver failure and anaemia, resulting in early lethality within 12 days post fertilization. The atg5-morpholino knockdown and bafilomycin treatment partially improved the size of the liver and survival rate in larsb-/- zebrafish. These findings indicate the involvement of autophagy in the pathogenesis of larsb-/- zebrafish. Indeed, excessive autophagy activation was observed in larsb-/- zebrafish. Therefore, our data clarify a mechanistic link between LARS and autophagy in vivo. Furthermore, autophagy regulation by LARS could lead to development of new therapeutics for IFLS1.
    DOI:  https://doi.org/10.1038/s41598-021-87879-4
  21. Sci Signal. 2021 Apr 13. pii: eabe4509. [Epub ahead of print]14(678):
    mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.
    Timothy R Baffi, Gema Lordén, Jacob M Wozniak, Andreas Feichtner, Wayland Yeung, Alexandr P Kornev, Charles C King, Jason C Del Rio, Ameya J Limaye, Julius Bogomolovas, Christine M Gould, Ju Chen, Eileen J Kennedy, Natarajan Kannan, David J Gonzalez, Eduard Stefan, Susan S Taylor, Alexandra C Newton.
      The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x3-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.
    DOI:  https://doi.org/10.1126/scisignal.abe4509
  22. Front Neurosci. 2021 ;15 621076
    Manipulation of EGFR-Induced Signaling for the Recruitment of Quiescent Neural Stem Cells in the Adult Mouse Forebrain.
    Loïc M Cochard, Louis-Charles Levros, Sandra E Joppé, Federico Pratesi, Anne Aumont, Karl J L Fernandes.
      The ventricular-subventricular zone (V-SVZ) is the principal neurogenic niche in the adult mammalian forebrain. Neural stem/progenitor cell (NSPC) activity within the V-SVZ is controlled by numerous of extrinsic factors, whose downstream effects on NSPC proliferation, survival and differentiation are transduced via a limited number of intracellular signaling pathways. Here, we investigated the relationship between age-related changes in NSPC output and activity of signaling pathways downstream of the epidermal growth factor receptor (EGFR), a major regulator of NSPC activity. Biochemical experiments indicated that age-related decline of NSPC activity in vivo is accompanied by selective deficits amongst various EGFR-induced signal pathways within the V-SVZ niche. Pharmacological loss-of-function signaling experiments with cultured NSPCs revealed both overlap and selectivity in the biological functions modulated by the EGFR-induced PI3K/AKT, MEK/ERK and mTOR signaling modules. Specifically, while all three modules promoted EGFR-mediated NSPC proliferation, only mTOR contributed to NSPC survival and only MEK/ERK repressed NSPC differentiation. Using a gain-of-function in vivo genetic approach, we electroporated a constitutively active EGFR construct into a subpopulation of quiescent, EGFR-negative neural stem cells (qNSCs); this ectopic activation of EGFR signaling enabled qNSCs to divide in 3-month-old early adult mice, but not in mice at middle-age or carrying familial Alzheimer disease mutations. Thus, (i) individual EGFR-induced signaling pathways have dissociable effects on NSPC proliferation, survival, and differentiation, (ii) activation of EGFR signaling is sufficient to stimulate qNSC cell cycle entry during early adulthood, and (iii) the proliferative effects of EGFR-induced signaling are dominantly overridden by anti-proliferative signals associated with aging and Alzheimer's disease.
    Keywords:  Alzheimer’s disease; aging; electroporation; epidermal growth factor; neural stem cell; neurogenesis; quiescence; ventricular-subventricular zone
    DOI:  https://doi.org/10.3389/fnins.2021.621076
  23. Mol Genet Metab Rep. 2021 Jun;27 100748
    Examination of a blood-brain barrier targeting β-galactosidase-monoclonal antibody fusion protein in a murine model of GM1-gangliosidosis.
    Michael J Przybilla, Christine Stewart, Timothy W Carlson, Li Ou, Brenda L Koniar, Rohini Sidhu, Pamela J Kell, Xuntian Jiang, Jeanine R Jarnes, M Gerard O'Sullivan, Chester B Whitley.
      GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase β-galactosidase (β-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing β-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (β-gal-/-). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult β-gal-/- mice showed clearance of enzyme activity in the plasma and an increase in β-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating β-gal-/- mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated β-gal-/- mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed β-gal-/- mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in β-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active β-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to β-gal-/- mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in β-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).
    Keywords:  Enzyme replacement therapy; Fusion enzyme; GM1-gangliosidosis
    DOI:  https://doi.org/10.1016/j.ymgmr.2021.100748
  24. Sci Rep. 2021 Apr 12. 11(1): 7845
    Fusion and fission events regulate endosome maturation and viral escape.
    Mario Castro, Grant Lythe, Jolanda Smit, Carmen Molina-París.
      Endosomes are intracellular vesicles that mediate the communication of the cell with its extracellular environment. They are an essential part of the cell's machinery regulating intracellular trafficking via the endocytic pathway. Many viruses, which in order to replicate require a host cell, attach themselves to the cellular membrane; an event which usually initiates uptake of a viral particle through the endocytic pathway. In this way viruses hijack endosomes for their journey towards intracellular sites of replication and avoid degradation without host detection by escaping the endosomal compartment. Recent experimental techniques have defined the role of endosomal maturation in the ability of enveloped viruses to release their genetic material into the cytoplasm. Endosome maturation depends on a family of small hydrolase enzymes (or GTPases) called Rab proteins, arranged on the cytoplasmic surface of its membrane. Here, we model endosomes as intracellular compartments described by two variables (its levels of active Rab5 and Rab7 proteins) and which can undergo coagulation (or fusion) and fragmentation (or fission). The key element in our approach is the "per-cell endosomal distribution" and its dynamical (Boltzmann) equation. The Boltzmann equation allows us to derive the dynamics of the total number of endosomes in a cell, as well as the mean and the standard deviation of its active Rab5 and Rab7 levels. We compare our mathematical results with experiments of Dengue viral escape from endosomes. The relationship between endosomal active Rab levels and pH suggests a mechanism that can account for the observed variability in viral escape times, which in turn regulate the viability of a viral intracellular infection.
    DOI:  https://doi.org/10.1038/s41598-021-86877-w
  25. Nature. 2021 Apr 14.
    CRL4AMBRA1 is a master regulator of D-type cyclins.
    Daniele Simoneschi, Gergely Rona, Nan Zhou, Yeon-Tae Jeong, Shaowen Jiang, Giacomo Milletti, Arnaldo A Arbini, Alfie O'Sullivan, Andrew A Wang, Sorasicha Nithikasem, Sarah Keegan, Yik Siu, Valentina Cianfanelli, Emiliano Maiani, Francesca Nazio, Francesco Cecconi, Francesco Boccalatte, David Fenyö, Drew R Jones, Luca Busino, Michele Pagano.
      D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.
    DOI:  https://doi.org/10.1038/s41586-021-03445-y
  26. J Mol Biol. 2021 Apr 09. pii: S0022-2836(21)00188-1. [Epub ahead of print] 166987
    Phosphorylation of the LIR domain of SCOC modulates ATG8 binding affinity and specificity.
    Martina Wirth, Stephane Mouilleron, Wenxin Zhang, Eva Sjottem, Yakubu Princely Abudu, Ashish Jain, Hallvard Lauritz Olsvik, Jack-Ansgar Bruun, Minoo Razi, Harold B J Jefferies, Rebecca Lee, Dhira Joshi, Nicola O'Reilly, Terje Johansen, Sharon A Tooze.
      Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.
    Keywords:  Autophagy; Bio-layer interferometry; LIR motif; crystal structure; phosphorylation
    DOI:  https://doi.org/10.1016/j.jmb.2021.166987
  27. Glia. 2021 Apr 14.
    Mechanisms of demyelination and neurodegeneration in globoid cell leukodystrophy.
    M Laura Feltri, Nadav I Weinstock, Jacob Favret, Narayan Dhimal, Lawrence Wrabetz, Daesung Shin.
      Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a lysosomal storage disorder causing extensive demyelination in the central and peripheral nervous systems. GLD is caused by loss-of-function mutations in the lysosomal hydrolase, galactosylceramidase (GALC), which catabolizes the myelin sphingolipid galactosylceramide. The pathophysiology of GLD is complex and reflects the expression of GALC in a number of glial and neural cell types in both the central and peripheral nervous systems (CNS and PNS), as well as leukocytes and kidney in the periphery. Over the years, GLD has garnered a wide range of scientific and medical interests, especially as a model system to study gene therapy and novel preclinical therapeutic approaches to treat the spontaneous murine model for GLD. Here, we review recent findings in the field of Krabbe disease, with particular emphasis on novel aspects of GALC physiology, GLD pathophysiology, and therapeutic strategies.
    Keywords:  Krabbe disease; demyelination; leukodystrophy; neurodegeneration
    DOI:  https://doi.org/10.1002/glia.24008
  28. Cell Discov. 2021 Apr 13. 7(1): 24
    Distinct uptake, amplification, and release of SARS-CoV-2 by M1 and M2 alveolar macrophages.
    Jiadi Lv, Zhenfeng Wang, Yajin Qu, Hua Zhu, Qiangqiang Zhu, Wei Tong, Linlin Bao, Qi Lv, Ji Cong, Dan Li, Wei Deng, Pin Yu, Jiangping Song, Wei-Min Tong, Jiangning Liu, Yuying Liu, Chuan Qin, Bo Huang.
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the alveoli, where abundant alveolar macrophages (AMs) reside. How AMs respond to SARS-CoV-2 invasion remains elusive. Here, we show that classically activated M1 AMs facilitate viral spread; however, alternatively activated M2 AMs limit the spread. M1 AMs utilize cellular softness to efficiently take up SARS-CoV-2. Subsequently, the invaded viruses take over the endo-lysosomal system to escape. M1 AMs have a lower endosomal pH, favoring membrane fusion and allowing the entry of viral RNA from the endosomes into the cytoplasm, where the virus achieves replication and is packaged to be released. In contrast, M2 AMs have a higher endosomal pH but a lower lysosomal pH, thus delivering the virus to lysosomes for degradation. In hACE2 transgenic mouse model, M1 AMs are found to facilitate SARS-CoV-2 infection of the lungs. These findings provide insights into the complex roles of AMs during SARS-CoV-2 infection, along with potential therapeutic targets.
    DOI:  https://doi.org/10.1038/s41421-021-00258-1
  29. Nat Commun. 2021 04 12. 12(1): 2176
    Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.
    Sabine Ruegenberg, Felix A M C Mayr, Ilian Atanassov, Ulrich Baumann, Martin S Denzel.
      The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.
    DOI:  https://doi.org/10.1038/s41467-021-22320-y
  30. Oncogene. 2021 Apr 12.
    Nuclear translocation of ASPL-TFE3 fusion protein creates favorable metabolism by mediating autophagy in translocation renal cell carcinoma.
    Ru Fang, Xiaotong Wang, Qiuyuan Xia, Ming Zhao, Hao Zhang, Xuan Wang, Shengbing Ye, Kai Cheng, Yan Liang, Yang Cheng, Yayun Gu, Qiu Rao.
      The ASPL-TFE3 fusion gene, resulting from t(X;17)(p11.2;q25.3), is one of the most commonly identified fusion genes in Xp11 translocation renal cell carcinoma (tRCC). However, its roles and underlying mechanism in RCC development are not yet clear. Here, we identified ASPL-TFE3 fusion as the most common tRCC subtype in a Chinese population (29/126, 23.03%). This fusion protein translocated into the nucleus and promoted RCC cell proliferation both in vitro and in vivo. Mechanistically, the fusion protein transcriptionally activated the lysosome-autophagy pathway by binding to the promoters of lysosome-related genes. Autophagy, activated by ASPL-TFE3, enabled RCC cells to escape energy stress by promoting the utilization of proteins and lipids. Moreover, we found that the ASPL-TFE3 fusion escaped regulation by the classic mTOR-TFE3 signal and instead activated phospho-mTOR and its downstream targets. Finally, targeting both autophagy and the mTOR axis resulted in a greater antiproliferative effect than single pathway inhibition. In summary, these results confirmed the ASPL-TFE3 fusion as a master regulator of metabolic adaptation mediated by autophagy in tRCC. The simultaneous manipulation of autophagy and the mTOR axis may represent a novel treatment strategy for ASPL-TFE3 fusion RCC.
    DOI:  https://doi.org/10.1038/s41388-021-01776-8
  31. Front Physiol. 2021 ;12 638983
    The Role of Autophagy in Skeletal Muscle Diseases.
    Qianghua Xia, Xubo Huang, Jieru Huang, Yongfeng Zheng, Michael E March, Jin Li, Yongjie Wei.
      Skeletal muscle is the most abundant type of tissue in human body, being involved in diverse activities and maintaining a finely tuned metabolic balance. Autophagy, characterized by the autophagosome-lysosome system with the involvement of evolutionarily conserved autophagy-related genes, is an important catabolic process and plays an essential role in energy generation and consumption, as well as substance turnover processes in skeletal muscles. Autophagy in skeletal muscles is finely tuned under the tight regulation of diverse signaling pathways, and the autophagy pathway has cross-talk with other pathways to form feedback loops under physiological conditions and metabolic stress. Altered autophagy activity characterized by either increased formation of autophagosomes or inhibition of lysosome-autophagosome fusion can lead to pathological cascades, and mutations in autophagy genes and deregulation of autophagy pathways have been identified as one of the major causes for a variety of skeleton muscle disorders. The advancement of multi-omics techniques enables further understanding of the molecular and biochemical mechanisms underlying the role of autophagy in skeletal muscle disorders, which may yield novel therapeutic targets for these disorders.
    Keywords:  AMPK; autophagy; mTOR; muscle cell homeostasis; skeletal muscle diseases; transcriptional regulation
    DOI:  https://doi.org/10.3389/fphys.2021.638983
  32. Proc Natl Acad Sci U S A. 2021 Apr 20. pii: e2101562118. [Epub ahead of print]118(16):
    A model for a partnership of lipid transfer proteins and scramblases in membrane expansion and organelle biogenesis.
    Alireza Ghanbarpour, Diana P Valverde, Thomas J Melia, Karin M Reinisch.
      The autophagy protein ATG2, proposed to transfer bulk lipid from the endoplasmic reticulum (ER) during autophagosome biogenesis, interacts with ER residents TMEM41B and VMP1 and with ATG9, in Golgi-derived vesicles that initiate autophagosome formation. In vitro assays reveal TMEM41B, VMP1, and ATG9 as scramblases. We propose a model wherein membrane expansion results from the partnership of a lipid transfer protein, moving lipids between the cytosolic leaflets of apposed organelles, and scramblases that reequilibrate the leaflets of donor and acceptor organelle membranes as lipids are depleted or augmented. TMEM41B and VMP1 are implicated broadly in lipid homeostasis and membrane dynamics processes in which their scrambling activities likely are key.
    Keywords:  ATG9A; TMEM41B; VMP1; scramblase
    DOI:  https://doi.org/10.1073/pnas.2101562118
  33. Adv Cancer Res. 2021 ;pii: S0065-230X(21)00018-X. [Epub ahead of print]150 335-363
    mTOR as a senescence manipulation target: A forked road.
    Sarah Saoudaoui, Monique Bernard, Guillaume B Cardin, Nicolas Malaquin, Apostolos Christopoulos, Francis Rodier.
      Cellular senescence, cancer and aging are highly interconnected. Among many important molecular machines that lie at the intersection of this triad, the mechanistic (formerly mammalian) target of rapamycin (mTOR) is a central regulator of cell metabolism, proliferation, and survival. The mTOR signaling cascade is essential to maintain cellular homeostasis in normal biological processes or in response to stress, and its dysregulation is implicated in the progression of many disorders, including age-associated diseases. Accordingly, the pharmacological implications of mTOR inhibition using rapamycin or others rapalogs span the treatment of various human diseases from immune disorders to cancer. Importantly, rapamycin is one of the only known pan-species drugs that can extend lifespan. The molecular and cellular mechanisms explaining the phenotypic consequences of mTOR are vast and heavily studied. In this review, we will focus on the potential role of mTOR in the context of cellular senescence, a tumor suppressor mechanism and a pillar of aging. We will explore the link between senescence, autophagy and mTOR and discuss the opportunities to exploit senescence-associated mTOR functions to manipulate senescence phenotypes in age-associated diseases and cancer treatment.
    Keywords:  Aging; Apoptosis; Autophagy; Lifespan; Metabolism; Senescence; Senolytics; mTOR
    DOI:  https://doi.org/10.1016/bs.acr.2021.02.002
  34. Sci Adv. 2021 Apr;pii: eabe5544. [Epub ahead of print]7(16):
    Kog1/Raptor mediates metabolic rewiring during nutrient limitation by controlling SNF1/AMPK activity.
    Zeenat Rashida, Rajalakshmi Srinivasan, Meghana Cyanam, Sunil Laxman.
      In changing environments, cells modulate resource budgeting through distinct metabolic routes to control growth. Accordingly, the TORC1 and SNF1/AMPK pathways operate contrastingly in nutrient replete or limited environments to maintain homeostasis. The functions of TORC1 under glucose and amino acid limitation are relatively unknown. We identified a modified form of the yeast TORC1 component Kog1/Raptor, which exhibits delayed growth exclusively during glucose and amino acid limitations. Using this, we found a necessary function for Kog1 in these conditions where TORC1 kinase activity is undetectable. Metabolic flux and transcriptome analysis revealed that Kog1 controls SNF1-dependent carbon flux apportioning between glutamate/amino acid biosynthesis and gluconeogenesis. Kog1 regulates SNF1/AMPK activity and outputs and mediates a rapamycin-independent activation of the SNF1 targets Mig1 and Cat8. This enables effective glucose derepression, gluconeogenesis activation, and carbon allocation through different pathways. Therefore, Kog1 centrally regulates metabolic homeostasis and carbon utilization during nutrient limitation by managing SNF1 activity.
    DOI:  https://doi.org/10.1126/sciadv.abe5544
  35. Cell. 2021 Apr 13. pii: S0092-8674(21)00366-4. [Epub ahead of print]
    ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.
    Aubrey V Weigel, Chi-Lun Chang, Gleb Shtengel, C Shan Xu, David P Hoffman, Melanie Freeman, Nirmala Iyer, Jesse Aaron, Satya Khuon, John Bogovic, Wei Qiu, Harald F Hess, Jennifer Lippincott-Schwartz.
      Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.
    Keywords:  COPI; COPII; cholesterol; correlative light and electron microscopy; endoplasmic reticulum exit sites; endoplasmic reticulum to Golgi transport intermediate; focused ion beam-scanning electron microscopy; membrane trafficking; retention using selective hook system; secretory pathway
    DOI:  https://doi.org/10.1016/j.cell.2021.03.035
  36. Nature. 2021 Apr 14.
    AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.
    Emiliano Maiani, Giacomo Milletti, Francesca Nazio, Søs Grønbæk Holdgaard, Jirina Bartkova, Salvatore Rizza, Valentina Cianfanelli, Mar Lorente, Daniele Simoneschi, Miriam Di Marco, Pasquale D'Acunzo, Luca Di Leo, Rikke Rasmussen, Costanza Montagna, Marilena Raciti, Cristiano De Stefanis, Estibaliz Gabicagogeascoa, Gergely Rona, Nélida Salvador, Emanuela Pupo, Joanna Maria Merchut-Maya, Colin J Daniel, Marianna Carinci, Valeriana Cesarini, Alfie O'sullivan, Yeon-Tae Jeong, Matteo Bordi, Francesco Russo, Silvia Campello, Angela Gallo, Giuseppe Filomeni, Letizia Lanzetti, Rosalie C Sears, Petra Hamerlik, Armando Bartolazzi, Robert E Hynds, David R Pearce, Charles Swanton, Michele Pagano, Guillermo Velasco, Elena Papaleo, Daniela De Zio, Apolinar Maya-Mendoza, Franco Locatelli, Jiri Bartek, Francesco Cecconi.
      Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.
    DOI:  https://doi.org/10.1038/s41586-021-03422-5
  37. Nature. 2021 Apr 14.
    The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.
    Andrea C Chaikovsky, Chuan Li, Edwin E Jeng, Samuel Loebell, Myung Chang Lee, Christopher W Murray, Ran Cheng, Janos Demeter, Danielle L Swaney, Si-Han Chen, Billy W Newton, Jeffrey R Johnson, Alexandros P Drainas, Yan Ting Shue, Jose A Seoane, Preethi Srinivasan, Andy He, Akihiro Yoshida, Susan Q Hipkins, Edel McCrea, Carson D Poltorack, Nevan J Krogan, J Alan Diehl, Christina Kong, Peter K Jackson, Christina Curtis, Dmitri A Petrov, Michael C Bassik, Monte M Winslow, Julien Sage.
      The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.
    DOI:  https://doi.org/10.1038/s41586-021-03474-7
  38. J Cell Biol. 2021 May 03. pii: e202103128. [Epub ahead of print]220(5):
    Food for thought: How cell adhesion coordinates nutrient sensing.
    Hellyeh Hamidi, Johanna Ivaska.
      Cell adhesion controls cell survival and proliferation via multiple mechanisms. Rabanal-Ruiz et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202004010) demonstrate that focal adhesions are key signaling hubs for cellular nutrient sensing and signaling.
    DOI:  https://doi.org/10.1083/jcb.202103128
  39. Blood Cancer Discov. 2021 Mar;2(2): 162-185
    TFEB links MYC signaling to epigenetic control of myeloid differentiation and acute myeloid leukemia.
    Seongseok Yun, Nicole D Vincelette, Xiaoqing Yu, Gregory W Watson, Mario R Fernandez, Chunying Yang, Taro Hitosugi, Chia-Ho Cheng, Audrey R Freischel, Ling Zhang, Weimin Li, Hsinan Hou, Franz X Schaub, Alexis R Vedder, Ling Cen, Kathy L McGraw, Jungwon Moon, Daniel J Murphy, Andrea Ballabio, Scott H Kaufmann, Anders E Berglund, John L Cleveland.
      MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.
    DOI:  https://doi.org/10.1158/2643-3230.bcd-20-0029
  40. Cell Rep. 2021 Apr 13. pii: S2211-1247(21)00305-3. [Epub ahead of print]35(2): 108991
    Cell-type-specific profiling of human cellular models of fragile X syndrome reveal PI3K-dependent defects in translation and neurogenesis.
    Nisha Raj, Zachary T McEachin, William Harousseau, Ying Zhou, Feiran Zhang, Megan E Merritt-Garza, J Matthew Taliaferro, Magdalena Kalinowska, Samuele G Marro, Chadwick M Hales, Elizabeth Berry-Kravis, Marisol W Wolf-Ochoa, Veronica Martinez-Cerdeño, Marius Wernig, Lu Chen, Eric Klann, Stephen T Warren, Peng Jin, Zhexing Wen, Gary J Bassell.
      Transcriptional silencing of the FMR1 gene in fragile X syndrome (FXS) leads to the loss of the RNA-binding protein FMRP. In addition to regulating mRNA translation and protein synthesis, emerging evidence suggests that FMRP acts to coordinate proliferation and differentiation during early neural development. However, whether loss of FMRP-mediated translational control is related to impaired cell fate specification in the developing human brain remains unknown. Here, we use human patient induced pluripotent stem cell (iPSC)-derived neural progenitor cells and organoids to model neurogenesis in FXS. We developed a high-throughput, in vitro assay that allows for the simultaneous quantification of protein synthesis and proliferation within defined neural subpopulations. We demonstrate that abnormal protein synthesis in FXS is coupled to altered cellular decisions to favor proliferative over neurogenic cell fates during early development. Furthermore, pharmacologic inhibition of elevated phosphoinositide 3-kinase (PI3K) signaling corrects both excess protein synthesis and cell proliferation in a subset of patient neural cells.
    Keywords:  FMRP; Fragile X syndrome; autism; flow cytometry; iPSCs; neural stem cells; neurogenesis; protein synthesis; translation
    DOI:  https://doi.org/10.1016/j.celrep.2021.108991
  41. EMBO Rep. 2021 Apr 14. e50684
    The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.
    Hyunju Ryu, Xiao-Xin Sun, Yingxiao Chen, Yanping Li, Xiaoyan Wang, Roselyn S Dai, Hong-Ming Zhu, John Klimek, Larry David, Lev M Fedorov, Yoshiaki Azuma, Rosalie C Sears, Mu-Shui Dai.
      SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.
    Keywords:  SUMOylation; USP36; deubiquitinating enzyme; ribosome biogenesis; snoRNP
    DOI:  https://doi.org/10.15252/embr.202050684
  42. Genes Dev. 2021 Apr 01. 35(7-8): 449-469
    Interorganelle communication, aging, and neurodegeneration.
    Maja Petkovic, Caitlin E O'Brien, Yuh Nung Jan.
      Our cells are comprised of billions of proteins, lipids, and other small molecules packed into their respective subcellular organelles, with the daunting task of maintaining cellular homeostasis over a lifetime. However, it is becoming increasingly evident that organelles do not act as autonomous discrete units but rather as interconnected hubs that engage in extensive communication through membrane contacts. In the last few years, our understanding of how these contacts coordinate organelle function has redefined our view of the cell. This review aims to present novel findings on the cellular interorganelle communication network and how its dysfunction may contribute to aging and neurodegeneration. The consequences of disturbed interorganellar communication are intimately linked with age-related pathologies. Given that both aging and neurodegenerative diseases are characterized by the concomitant failure of multiple cellular pathways, coordination of organelle communication and function could represent an emerging regulatory mechanism critical for long-term cellular homeostasis. We anticipate that defining the relationships between interorganelle communication, aging, and neurodegeneration will open new avenues for therapeutics.
    Keywords:  aging; cellular homeostasis; communication; contact sites; endolysosomal pathway; interorganelle communication; lipid metabolism; mitochondria; neurodegeneration
    DOI:  https://doi.org/10.1101/gad.346759.120
  43. Bone. 2021 Apr 07. pii: S8756-3282(21)00108-3. [Epub ahead of print]148 115946
    Loss of function of lysosomal acid lipase (LAL) profoundly impacts osteoblastogenesis and increases fracture risk in humans.
    Ron C Helderman, Daniel G Whitney, Madalina Duta-Mare, Alena Akhmetshina, Nemanja Vujic, Shobana Jayapalan, Jeffry S Nyman, Biswapriya B Misra, Clifford J Rosen, Michael P Czech, Dagmar Kratky, Elizabeth Rendina-Ruedy.
      Lysosomal acid lipase (LAL) is essential for cholesteryl ester (CE) and triacylglycerol (TAG) hydrolysis in the lysosome. Clinically, an autosomal recessive LIPA mutation causes LAL deficiency (LALD), previously described as Wolman Disease or Cholesteryl Ester Storage Disease (CESD). LAL-D is associated with ectopic lipid accumulation in the liver, small intestine, spleen, adrenal glands, and blood. Considering the importance of unesterified cholesterol and fatty acids in bone metabolism, we hypothesized that LAL is essential for bone formation, and ultimately, skeletal health. To investigate the role of LAL in skeletal homeostasis, we used LAL-deficient (-/-) mice, in vitro osteoblast cultures, and novel clinical data from LAL-D patients. Both male and female LAL-/- mice demonstarted lower trabecular and cortical bone parameters , which translated to reduced biomechanical properties. Further histological analyses revealed that LAL-/- mice had fewer osteoblasts, with no change in osteoclast or marrow adipocyte numbers. In studying the cell-autonomous role of LAL, we observed impaired differentiation of LAL-/- calvarial osteoblasts and in bone marrow stromal cells treated with the LAL inhibitor lalistat. Consistent with LAL's role in other tissues, lalistat resulted in profound lipid puncta accumulation and an altered intracellular lipid profile. Finally, we analyzed a large de-identified national insurance database (i.e. 2016/2017 Optum Clinformatics®) which revealed that adults (≥18 years) with CESD (n = 3076) had a higher odds ratio (OR = 1.21; 95% CI = 1.03-1.41) of all-cause fracture at any location compared to adults without CESD (n = 13.7 M) after adjusting for demographic variables and osteoporosis. These data demonstrate that alterations in LAL have significant clinical implications related to fracture risk and that LAL's modulation of lipid metabolism is a critical for osteoblast function.
    Keywords:  Bone; Cholesterol; Lipid; Lipophagy; Metabolism; Osteoblast; Skeleton
    DOI:  https://doi.org/10.1016/j.bone.2021.115946
  44. Autophagy. 2021 Apr 13. 1-18
    A NOVEL NOX/PHOX-CD38-NAADP-TFEB AXIS IMPORTANT FOR MACROPHAGE ACTIVATION DURING BACTERIAL PHAGOCYTOSIS.
    Mehran Najibi, Havisha H Honwad, Joseph A Moreau, Stephanie M Becker, Javier E Irazoqui.
      Macrophage activation in the presence of bacterial cells and molecules entails complex programs of gene expression. How such triggers elicit specific gene expression programs is incompletely understood. We previously discovered that TFEB (transcription factor EB) is a key contributor to macrophage activation during bacterial phagocytosis. However, the mechanism linking phagocytosis of bacterial cells to TFEB activation and downstream pro-inflammatory cytokine induction remained unknown. We found that macrophages lacking both TFEB and TFE3 (transcription factor E3) were unable to mount a pro-inflammatory phenotype in response to bacterial infection. The NOX/PHOX (NADPH oxidase)-dependent oxidative burst was required for nuclear translocation of TFEB during phagocytosis of Gram-positive or -negative bacteria, and reactive oxygen species (ROS) were sufficient to trigger TFEB activation in a CD38- and NAADP (nicotinic acid adenine dinucleotide phosphate)-dependent manner. Consistent with the Ca2+-releasing activity of NAADP, intracellular Ca2+ chelation and PPP3/calcineurin inhibition prevented TFEB activation by phagocytosis and ROS (reactive oxygen species), impairing the induction of pro-inflammatory cytokines such as IL6 and TNF/TNFα. Therefore, here we describe a previously unknown pathway that links phagocytosis with macrophage pro-inflammatory polarization via TFEB and related transcription factor TFE3. These findings reveal that activation of TFEB and TFE3 is a key regulatory event for the activation of macrophages, and have important implications for infections, inflammation, cancer, obesity, and atherosclerosis.
    Keywords:  CD38; il1α; il1β; il6; innate immunity; phagocyte; reactive oxygen species; tfe3; tfeb; tnfα
    DOI:  https://doi.org/10.1080/15548627.2021.1911548
  45. Nat Commun. 2021 04 15. 12(1): 2259
    SOD1 regulates ribosome biogenesis in KRAS mutant non-small cell lung cancer.
    Xiaowen Wang, Hong Zhang, Russell Sapio, Jun Yang, Justin Wong, Xin Zhang, Jessie Y Guo, Sharon Pine, Holly Van Remmen, Hong Li, Eileen White, Chen Liu, Megerditch Kiledjian, Dimitri G Pestov, X F Steven Zheng.
      SOD1 is known as the major cytoplasmic superoxide dismutase and an anticancer target. However, the role of SOD1 in cancer is not fully understood. Herein we describe the generation of an inducible Sod1 knockout in KRAS-driven NSCLC mouse model. Sod1 knockout markedly reduces tumor burden in vivo and blocks growth of KRAS mutant NSCLC cells in vitro. Intriguingly, SOD1 is enriched in the nucleus and notably in the nucleolus of NSCLC cells. The nuclear and nucleolar, not cytoplasmic, form of SOD1 is essential for lung cancer cell proliferation. Moreover, SOD1 interacts with PeBoW complex and controls its assembly necessary for pre-60S ribosomal subunit maturation. Mechanistically, SOD1 regulates co-localization of PeBoW with and processing of pre-rRNA, and maturation of cytoplasmic 60S ribosomal subunits in KRAS mutant lung cancer cells. Collectively, our study unravels a nuclear SOD1 function essential for ribosome biogenesis and proliferation in KRAS-driven lung cancer.
    DOI:  https://doi.org/10.1038/s41467-021-22480-x
  46. Elife. 2021 Apr 16. pii: e61069. [Epub ahead of print]10
    The Nesprin-1/-2 ortholog ANC-1 regulates organelle positioning in C. elegans independently from its KASH or actin-binding domains.
    Hongyan Hao, Shilpi Kalra, Laura E Jameson, Leslie A Guerrero, Natalie E Cain, Jessica Bolivar, Daniel A Starr.
      KASH proteins in the outer nuclear membrane comprise the cytoplasmic half of LINC complexes that connect nuclei to the cytoskeleton. Caenorhabditis elegans ANC-1, an ortholog of Nesprin-1/2, contains actin-binding and KASH domains at opposite ends of a long spectrin-like region. Deletion of either the KASH or calponin homology (CH) domains does not completely disrupt nuclear positioning, suggesting neither KASH nor CH domains are essential. Deletions in the spectrin-like region of ANC-1 led to significant defects, but only recapitulated the null phenotype in combination with mutations in the trans-membrane span. In anc-1 mutants, the ER, mitochondria, and lipid droplets were unanchored, moving throughout the cytoplasm. The data presented here support a cytoplasmic integrity model where ANC-1 localizes to the ER membrane and extends into the cytoplasm to position nuclei, ER, mitochondria, and likely other organelles in place.
    Keywords:  C. elegans; cell biology
    DOI:  https://doi.org/10.7554/eLife.61069
  47. Curr Opin Cell Biol. 2021 Apr 13. pii: S0955-0674(21)00035-1. [Epub ahead of print]71 120-129
    Unconventional endocytic mechanisms.
    Henri-François Renard, Emmanuel Boucrot.
      Endocytosis mediates the uptake of extracellular proteins, micronutrients and transmembrane cell surface proteins. Importantly, many viruses, toxins and bacteria hijack endocytosis to infect cells. The canonical pathway is clathrin-mediated endocytosis (CME) and is active in all eukaryotic cells to support critical house-keeping functions. Unconventional mechanisms of endocytosis exit in parallel of CME, to internalize specific cargoes and support various cellular functions. These clathrin-independent endocytic (CIE) routes use three distinct mechanisms: acute signaling-induced membrane remodeling drives macropinocytosis, activity-dependent bulk endocytosis (ADBE), massive endocytosis (MEND) and EGFR non-clathrin endocytosis (EGFR-NCE). Cargo capture and local membrane deformation by cytosolic proteins is used by fast endophilin-mediated endocytosis (FEME), IL-2Rβ endocytosis and ultrafast endocytosis at synapses. Finally, the formation of endocytic pits by clustering of extracellular lipids or cargoes according to the Glycolipid-Lectin (GL-Lect) hypothesis mediates the uptake of SV40 virus, Shiga and cholera toxins, and galectin-clustered receptors by the CLIC/GEEC and the endophilin-A3-mediated CIE.
    Keywords:  Activity-dependent bulk endocytosis; Cargoes; Clathrin; Clathrin-independent carriers/GPI-AP-enriched early endosomal compartments (CLIC/GEEC); Clathrin-independent endocytosis; Clathrin-mediated endocytosis; EGFR Non-Clathrin endocytosis; Endocytosis; Endophilin-A3/Galectin-8-mediated endocytosis; Fast endophilin-mediated endocytosis (FEME); Glycolipid-Lectin hypothesis; Glycosylphosphatidylinositol-anchored proteins; Interleukin-2 receptor endocytosis; Lipids; Macropinocytosis; Massive Endocytosis; Receptors; Ultrafast endocytosis
    DOI:  https://doi.org/10.1016/j.ceb.2021.03.001
  48. Mol Neurobiol. 2021 Apr 15.
    The mTOR/NF-κB Pathway Mediates Neuroinflammation and Synaptic Plasticity in Diabetic Encephalopathy.
    Ting Xu, Jiao Liu, Xin-Rui Li, Yinghua Yu, Xuan Luo, Xian Zheng, Yuan Cheng, Pei-Quan Yu, Yi Liu.
      Diabetic encephalopathy, a severe complication of diabetes mellitus, is characterized by neuroinflammation and aberrant synaptogenesis in the hippocampus leading to cognitive decline. Mammalian target of rapamycin (mTOR) is associated with cognition impairment. Nuclear factor-κB (NF-κB) is a transcription factor of proinflammatory cytokines. Although mTOR has been ever implicated in processes occurring in neuroinflammation, the role of this enzyme on NF-κB signaling pathway remains unclear in diabetic encephalopathy. In the present study, we investigated whether mTOR regulates the NF-κB signaling pathway to modulate inflammatory cytokines and synaptic plasticity in hippocampal neurons. In vitro model was constructed in mouse HT-22 hippocampal neuronal cells exposed to high glucose. With the inhibition of mTOR or NF-κB by either chemical inhibitor or short-hairpin RNA (shRNA)-expressing lentivirus-vector, we examined the effects of mTOR/NF-κB signaling on proinflammatory cytokines and synaptic proteins. The diabetic mouse model induced by a high-fat diet combined with streptozotocin injection was administrated with rapamycin (mTOR inhibitor) and PDTC (NF-κB inhibitor), respectively. High glucose significantly increased mTOR phosphorylation in HT-22 cells. While inhibiting mTOR by rapamycin or shmTOR significantly suppressed high glucose-induced activation of NF-κB and its regulators IKKβ and IκBα, suggesting mTOR is the upstream regulator of NF-κB. Furthermore, inhibiting NF-κB by PDTC and shNF-κB decreased proinflammatory cytokines expression (IL-6, IL-1β, and TNF-α) and increased brain-derived neurotrophic factor (BDNF) and synaptic proteins (synaptophysin and PSD-95) in HT-22 cells under high glucose conditions. Besides, the mTOR and NF-κB inhibitors improved cognitive decline in diabetic mice. The inhibition of mTOR and NF-κB suppressed mTOR/NF-κB signaling pathway, increased synaptic proteins, and improved ultrastructural synaptic plasticity in the hippocampus of diabetic mice. Activating mTOR/NF-κB signaling pathway regulates the pathogenesis of diabetic encephalopathy, such as neuroinflammation, synaptic proteins loss, and synaptic ultrastructure impairment. The findings provide the implication that mTOR/NF-κB is potential new drug targets to treat diabetic encephalopathy.
    Keywords:  Diabetic encephalopathy; NF-κB; Neuroinflammation; Synaptic plasticity; mTOR
    DOI:  https://doi.org/10.1007/s12035-021-02390-1
  49. Genetics. 2021 Apr 15. pii: iyab017. [Epub ahead of print]217(4):
    N- and C-terminal Gln3-Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization.
    Jennifer J Tate, Rajendra Rai, Claudio De Virgilio, Terrance G Cooper.
      Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656-666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3-Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3-Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3-Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3-Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3-15-fold with three of them by 10-15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3-Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.
    Keywords:  Gln3; Nitrogen metabolism; Signal transduction; Sit4; TOR complex (TorC1); Transcription factors; URS
    DOI:  https://doi.org/10.1093/genetics/iyab017
  50. Nature. 2021 Apr 14.
    Organelle degradation in the lens by PLAAT phospholipases.
    Hideaki Morishita, Tomoya Eguchi, Satoshi Tsukamoto, Yuriko Sakamaki, Satoru Takahashi, Chieko Saito, Ikuko Koyama-Honda, Noboru Mizushima.
      The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.
    DOI:  https://doi.org/10.1038/s41586-021-03439-w
  51. Cell Death Discov. 2021 Apr 12. 7(1): 74
    mTORC1 mediates fiber type-specific regulation of protein synthesis and muscle size during denervation.
    Jae-Sung You, Kookjoo Kim, Nathaniel D Steinert, Jie Chen, Troy A Hornberger.
      Skeletal muscle denervation occurs in diverse conditions and causes severe muscle atrophy. Signaling by mammalian target of rapamycin complex 1 (mTORC1) plays a central role in the maintenance of skeletal muscle mass by regulating net protein balance; yet, its role in denervation-induced atrophy is unclear. In this study, by using skeletal muscle-specific and inducible raptor knockout mice, we demonstrate that signaling through mTORC1 is activated during denervation and plays an essential role in mitigating the atrophy of non-type IIB muscle fibers. Measurements of protein synthesis rates of individual fibers suggest that denervation increases protein synthesis specifically in non-type IIB muscle fibers and that mTORC1 is required for this event. Furthermore, denervation induced a more pronounced increase in the level of phosphorylated ribosomal S6 protein in non-type IIB muscle fibers than in type IIB muscle fibers. Collectively, our results unveil a novel role for mTORC1 in mediating a fiber type-specific regulation of muscle size and protein synthesis during denervation.
    DOI:  https://doi.org/10.1038/s41420-021-00460-w
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