bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒12‒05
twenty-nine papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C.
  2. From mouse genetics to targeting the Rag GTPase pathway.
  3. Targeting neurological abnormalities in lysosomal storage diseases.
  4. Role of mammalian target of rapamycin complex 2 in primary and secondary liver cancer.
  5. RUNX1/EGFR pathway contributes to STAT3 activation and tumor growth caused by hyperactivated mTORC1.
  6. Idiosyncratic Biogenesis of Intracellular Pathogens-Containing Vacuoles.
  7. RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli.
  8. The nutrient sensor mTORC1 regulates insulin secretion by modulating ß-cell autophagy.
  9. Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis.
  10. A novel synaptopathy defective synaptic vesicle protein trafficking in the mutant CHMP2B mouse model of frontotemporal dementia.
  11. Neuropathology of murine Sanfilippo D syndrome.
  12. Can We Use mTOR Inhibitors for COVID-19 Therapy?
  13. Vesicle cholesterol controls exocytotic fusion pore.
  14. A novel live-cell imaging assay reveals regulation of endosome maturation.
  15. Lysosomal Function Impacts the Skeletal Muscle Extracellular Matrix.
  16. Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity.
  17. GRB10 regulates β cell mass by inhibiting β cell proliferation and stimulating β cell dedifferentiation.
  18. Probes for Fluorescent Visualization of Specific Cellular Organelles.
  19. Expression of 4E-BP1 in juvenile mice alleviates mTOR-induced neuronal dysfunction and epilepsy.
  20. Personalized phosphoproteomics identifies functional signaling.
  21. A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells.
  22. Targeting TBK1 Attenuates LPS-Induced NLRP3 Inflammasome Activation by Regulating of mTORC1 Pathways in Trophoblasts.
  23. Oscillatory neural network alterations in young people with tuberous sclerosis complex and associations with co-occurring symptoms of autism spectrum disorder and attention-deficit/hyperactivity disorder.
  24. Cell cycle regulation of ER membrane biogenesis protects against chromosome missegregation.
  25. TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae.
  26. Dyrk1b promotes hepatic lipogenesis by bypassing canonical insulin signaling and directly activating mTORC2 in mice.
  27. Structural basis for substrate specificity of heteromeric transporters of neutral amino acids.
  28. A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.
  29. Smaug1 membrane-less organelles respond to AMPK/mTOR and affect mitochondrial function‡.
  1. Sci Transl Med. 2021 Dec;13(622): eabg2919
    The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C.
    John V Pluvinage, Jerry Sun, Christel Claes, Ryan A Flynn, Michael S Haney, Tal Iram, Xiangling Meng, Rachel Lindemann, Nicholas M Riley, Emma Danhash, Jean Paul Chadarevian, Emma Tapp, David Gate, Sravani Kondapavulur, Inma Cobos, Sundari Chetty, Anca M Pașca, Sergiu P Pașca, Elizabeth Berry-Kravis, Carolyn R Bertozzi, Mathew Blurton-Jones, Tony Wyss-Coray.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/scitranslmed.abg2919
  2. Mol Cell Oncol. 2021 ;8(5): 1979370
    From mouse genetics to targeting the Rag GTPase pathway.
    Ana Ortega-Molina, Alejo Efeyan.
      The identification of the Rag GTPases initiated the deciphering of the molecular puzzle of nutrient signaling to the mechanistic target of rapamycin (mTOR), and spurred interest in targeting this pathway to combat human disease. Recent mouse genetic studies have provided pathophysiological insight and pointed to potential indications for inhibitors of the Rag GTPase pathway.
    Keywords:  B cells; Mtor; Rag GTPases; nutrients; lymphoma; mice; small molecules
    DOI:  https://doi.org/10.1080/23723556.2021.1979370
  3. Trends Pharmacol Sci. 2021 Nov 27. pii: S0165-6147(21)00222-4. [Epub ahead of print]
    Targeting neurological abnormalities in lysosomal storage diseases.
    Raquel van Gool, Anthony Tucker-Bartley, Edward Yang, Nicholas Todd, Frank Guenther, Benjamin Goodlett, Walla Al-Hertani, Olaf A Bodamer, Jaymin Upadhyay.
      Central nervous system (CNS) abnormalities and corresponding neurological and psychiatric symptoms are frequently observed in lysosomal storage disorders (LSDs). The genetic background of individual LSDs is indeed unique to each illness. However, resulting defective lysosomal function within the CNS can transition normal cellular processes (i.e., autophagy) into aberrant mechanisms, facilitating overlapping downstream consequences including neurocircuitry dysfunction, neurodegeneration as well as sensory, motor, cognitive, and psychological symptoms. Here, the neurological and biobehavioral phenotypes of major classes of LSDs are discussed alongside therapeutic strategies in development that aim to tackle neuropathology among other disease elements. Finally, focused ultrasound blood-brain barrier opening is proposed to enhance therapeutic delivery thereby overcoming the key hurdle of central distribution of disease modifying therapies in LSDs.
    Keywords:  autophagy; central nervous system abnormalities; lysosomal storage disease; neurological symptoms; psychiatric symptoms
    DOI:  https://doi.org/10.1016/j.tips.2021.11.005
  4. World J Gastrointest Oncol. 2021 Nov 15. 13(11): 1632-1647
    Role of mammalian target of rapamycin complex 2 in primary and secondary liver cancer.
    Katharina Joechle, Jessica Guenzle, Claus Hellerbrand, Pavel Strnad, Thorsten Cramer, Ulf Peter Neumann, Sven Arke Lang.
      The mammalian target of rapamycin (mTOR) acts in two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Upon deregulation, activated mTOR signaling is associated with multiple processes involved in tumor growth and metastasis. Compared with mTORC1, much less is known about mTORC2 in cancer, mainly because of the unavailability of a selective inhibitor. However, existing data suggest that mTORC2 with its two distinct subunits Rictor and mSin1 might play a more important role than assumed so far. It is one of the key effectors of the PI3K/AKT/mTOR pathway and stimulates cell growth, cell survival, metabolism, and cytoskeletal organization. It is not only implicated in tumor progression, metastasis, and the tumor microenvironment but also in resistance to therapy. Rictor, the central subunit of mTORC2, was found to be upregulated in different kinds of cancers and is associated with advanced tumor stages and a bad prognosis. Moreover, AKT, the main downstream regulator of mTORC2/Rictor, is one of the most highly activated proteins in cancer. Primary and secondary liver cancer are major problems for current cancer therapy due to the lack of specific medical treatment, emphasizing the need for further therapeutic options. This review, therefore, summarizes the role of mTORC2/Rictor in cancer, with special focus on primary liver cancer but also on liver metastases.
    Keywords:  Cholangiocellular carcinoma; Hepatocellular carcinoma; Liver cancer; Liver metastases; Mammalian target of rapamycin; Mammalian target of rapamycin complex 2; Rictor
    DOI:  https://doi.org/10.4251/wjgo.v13.i11.1632
  5. Mol Ther Oncolytics. 2021 Dec 17. 23 387-401
    RUNX1/EGFR pathway contributes to STAT3 activation and tumor growth caused by hyperactivated mTORC1.
    Wei Lin, Xiaofeng Wan, Anjiang Sun, Meng Zhou, Xu Chen, Yanling Li, Zixi Wang, Hailiang Huang, Hongwu Li, Xianguo Chen, Juan Hua, Xiaojun Zha.
      Loss of function of tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) leads to the activation of mammalian target of rapamycin complex 1 (mTORC1). Hyperactivated mTORC1 plays a critical role in tumor growth, but the underlying mechanism is still not completely elucidated. Here, by analyzing Tsc1- or Tsc2-null mouse embryonic fibroblasts, rat Tsc2-null ELT3 cells, and human cancer cells, we present evidence for the involvement of epidermal growth factor receptor (EGFR) as a downstream target of mTORC1 in tumor growth. We show that mTORC1 leads to increased EGFR expression through upregulation of runt-related transcriptional factor 1 (RUNX1). Knockdown of EGFR impairs proliferation and tumoral growth of Tsc-deficient cells, while overexpression of EGFR promotes the proliferation of the control cells. Moreover, the mTOR signaling pathway has been shown to be positively correlated with EGFR in human cancers. In addition, we demonstrated that EGFR enhances cell growth through activation of signal transducer and activator of transcription 3 (STAT3). We conclude that activation of the RUNX1/EGFR/STAT3 signaling pathway contributes to tumorigenesis caused by hyperactivated mTORC1 and should be targeted for the treatment of mTORC1-related tumors, particularly TSC.
    Keywords:  EGFR; RUNX1; STAT3; mTOR; tuberous sclerosis complex; tumorigenesis
    DOI:  https://doi.org/10.1016/j.omto.2021.10.009
  6. Front Cell Infect Microbiol. 2021 ;11 722433
    Idiosyncratic Biogenesis of Intracellular Pathogens-Containing Vacuoles.
    Bethany Vaughn, Yousef Abu Kwaik.
      While most bacterial species taken up by macrophages are degraded through processing of the bacteria-containing vacuole through the endosomal-lysosomal degradation pathway, intravacuolar pathogens have evolved to evade degradation through the endosomal-lysosomal pathway. All intra-vacuolar pathogens possess specialized secretion systems (T3SS-T7SS) that inject effector proteins into the host cell cytosol to modulate myriad of host cell processes and remodel their vacuoles into proliferative niches. Although intravacuolar pathogens utilize similar secretion systems to interfere with their vacuole biogenesis, each pathogen has evolved a unique toolbox of protein effectors injected into the host cell to interact with, and modulate, distinct host cell targets. Thus, intravacuolar pathogens have evolved clear idiosyncrasies in their interference with their vacuole biogenesis to generate a unique intravacuolar niche suitable for their own proliferation. While there has been a quantum leap in our knowledge of modulation of phagosome biogenesis by intravacuolar pathogens, the detailed biochemical and cellular processes affected remain to be deciphered. Here we discuss how the intravacuolar bacterial pathogens Salmonella, Chlamydia, Mycobacteria, Legionella, Brucella, Coxiella, and Anaplasma utilize their unique set of effectors injected into the host cell to interfere with endocytic, exocytic, and ER-to-Golgi vesicle traffic. However, Coxiella is the main exception for a bacterial pathogen that proliferates within the hydrolytic lysosomal compartment, but its T4SS is essential for adaptation and proliferation within the lysosomal-like vacuole.
    Keywords:  biogenesis; intracellular pathogens; lysosomes; trafficking ; vacuoles
    DOI:  https://doi.org/10.3389/fcimb.2021.722433
  7. Am J Physiol Cell Physiol. 2021 Dec 01.
    RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli.
    Nathan Hodson, Michael Mazzulla, Maksym N H Holowaty, Dinesh Kumbhare, Daniel R Moore.
      Following anabolic stimuli (mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or prior to translocation (i.e. in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7;23±5yrs;76.8±3.6kg;13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7;22±2yrs;78.1±3.6kg;12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.
    Keywords:  Focal Adhesions; RPS6; Resistance Exercise; Skeletal muscle; mTOR
    DOI:  https://doi.org/10.1152/ajpcell.00357.2021
  8. Diabetes. 2021 Dec 03. pii: db210281. [Epub ahead of print]
    The nutrient sensor mTORC1 regulates insulin secretion by modulating ß-cell autophagy.
    Tal Israeli, Yael Riahi, Perla Garzon, Ruy Andrade Louzada, Joao Pedro Werneck-de-Castro, Manuel Blandino-Rosano, Roni Yeroslaviz-Stolper, Liat Kadosh, Sharona Tornovsky-Babeay, Gilad Hacker, Nitzan Israeli, Orly Agmon, Boaz Tirosh, Erol Cerasi, Ernesto Bernal-Mizrachi, Gil Leibowitz.
      The dynamic regulation of autophagy in β-cells by cycles of fasting-feeding and its effects on insulin secretion are unknown. In β-cells mTORC1 is inhibited while fasting, and is rapidly stimulated during refeeding by a single amino acid, leucine, and glucose. Stimulation of mTORC1 by nutrients inhibited the autophagy initiator ULK1 and the transcription factor TFEB, thereby preventing autophagy when β-cells are continuously exposed to nutrients. Inhibition of mTORC1 by Raptor knockout mimicked the effects of fasting and stimulated autophagy while inhibiting insulin secretion, whereas moderate inhibition of autophagy under these conditions rescued insulin secretion. These results show that mTORC1 regulates insulin secretion through modulation of autophagy under different nutritional situations. In the fasting state, autophagy is regulated in an mTORC1-dependent manner and its stimulation is required to keep insulin levels low, thereby preventing hypoglycemia. Reciprocally, stimulation of mTORC1 by elevated leucine and glucose, which is common in obesity, may promote hyperinsulinemia by inhibiting autophagy.
    DOI:  https://doi.org/10.2337/db21-0281
  9. Cell Death Dis. 2021 Dec 03. 12(12): 1127
    Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis.
    Hyeon-Ok Jin, Sung-Eun Hong, Ji-Young Kim, Se-Kyeong Jang, In-Chul Park.
      Amino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.
    DOI:  https://doi.org/10.1038/s41419-021-04417-w
  10. J Neurochem. 2021 Dec 02.
    A novel synaptopathy defective synaptic vesicle protein trafficking in the mutant CHMP2B mouse model of frontotemporal dementia.
    Emma L Clayton, Katherine Bonnycastle, Adrian M Isaacs, Michael A Cousin, Stephanie Schorge.
      Mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C-terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse.
    Keywords:  CHMP2B; ESCRT; endosome; frontotemporal dementia; lysosome; synaptic vesicle
    DOI:  https://doi.org/10.1111/jnc.15551
  11. Mol Genet Metab. 2021 Nov 24. pii: S1096-7192(21)00828-3. [Epub ahead of print]
    Neuropathology of murine Sanfilippo D syndrome.
    Keigo Takahashi, Steven Q Le, Shih-Hsin Kan, Matthew J Jansen, Patricia I Dickson, Jonathan D Cooper.
      Sanfilippo D syndrome (mucopolysaccharidosis type IIID) is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulfatase (GNS). A mouse model was generated by constitutive knockout of the Gns gene. We studied affected mice and controls at 12, 24, 36, and 48 weeks of age for neuropathological markers of disease in the somatosensory cortex, primary motor cortex, ventral posterior nuclei of the thalamus, striatum, hippocampus, and lateral and medial entorhinal cortex. We found significantly increased immunostaining for glial fibrillary associated protein (GFAP), CD68 (a marker of activated microglia), and lysosomal-associated membrane protein-1 (LAMP-1) in Sanfilippo D mice compared to controls at 12 weeks of age in all brain regions. Intergroup differences were marked for GFAP and CD68 staining, with levels in Sanfilippo D mice consistently above controls at all age groups. Intergroup differences in LAMP-1 staining were more pronounced in 12- and 24-week age groups compared to 36- and 48-week groups, as control animals showed some LAMP-1 staining at later timepoints in some brain regions. We also evaluated the somatosensory cortex, medial entorhinal cortex, reticular nucleus of the thalamus, medial amygdala, and hippocampal hilus for subunit c of mitochondrial ATP synthase (SCMAS). We found a progressive accumulation of SCMAS in most brain regions of Sanfilippo D mice compared to controls by 24 weeks of age. Cataloging the regional neuropathology of Sanfilippo D mice may aid in understanding the disease pathogenesis and designing preclinical studies to test brain-directed treatments.
    Keywords:  Glycosaminoglycan; Lysosomal storage disease; Mucopolysaccharidosis
    DOI:  https://doi.org/10.1016/j.ymgme.2021.11.010
  12. Comb Chem High Throughput Screen. 2021 Nov 30.
    Can We Use mTOR Inhibitors for COVID-19 Therapy?
    Ina Y Aneva, Solomon Habtemariam, Maciej Banach, Parames C Sil, Kasturi Sarkar, Adeleh Sahebnasagh, Mohammad Amjad Kamal, Maryam Khayatkashani, Hamid Reza Khayat Kashani.
      Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes acute inflammation due to extensive replication of the virus in the epithelial cells of the upper and lower respiratory system. The mammalian target of rapamycin (mTOR) is a l signalling protein with critical functions in cell growth, metabolism, and proliferation. It is known for its regulatory functions in protein synthesis and angiogenesis cascades. The structure of mTOR consists of two distinct complexes (mTORC1 and mTORC2) with diverse functions at different levels of the signaling pathway. By activating mRNA translation, the mTORC1 plays a key role in regulating protein synthesis and cellular growth. On the other hand, the functions of mTORC2 are mainly associated with cell proliferation and survival. By using an appropriate inhibitor at the right time, mTOR modulation could provide immunosuppressive opportunities as antirejection regimens in organ transplantation as well as in the treatment of autoimmune diseases and solid tumours. The mTOR has an important role in the inflammatory process, too. Inhibitors of mTOR might indeed be promising agents in the treatment of viral infections. They have further been successfully used in patients with severe influenza A/H1N1 pneumonia and acute respiratory failure. The officially accepted mTOR inhibitors that have undergone clinical testing are sirolimus, everolimus, temsirolimus, and tacrolimus. Thus, further studies on mTOR inhibitors for SARS-CoV-2 infection or COVID-19 therapy are well merited.
    Keywords:  COVID-19; SARS-CoV-2; everolimus; mTOR inhibitors; rapamycin; sirolimus; temsirolimus
    DOI:  https://doi.org/10.2174/1386207325666211130140923
  13. Cell Calcium. 2021 Nov 20. pii: S0143-4160(21)00157-3. [Epub ahead of print]101 102503
    Vesicle cholesterol controls exocytotic fusion pore.
    Boštjan Rituper, Alenka Guček, Marjeta Lisjak, Urszula Gorska, Aleksandra Šakanović, Saša Trkov Bobnar, Eva Lasič, Mićo Božić, Prabhodh S Abbineni, Jernej Jorgačevski, Marko Kreft, Alexei Verkhratsky, Frances M Platt, Gregor Anderluh, Matjaž Stenovec, Bojan Božič, Jens R Coorssen, Robert Zorec.
      In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.
    Keywords:  Cholesterol; Constriction force; Exocytosis; Fusion pore conductance; Fusion pore widening and constriction; Lysosomal storage disease
    DOI:  https://doi.org/10.1016/j.ceca.2021.102503
  14. Elife. 2021 11 30. pii: e70982. [Epub ahead of print]10
    A novel live-cell imaging assay reveals regulation of endosome maturation.
    Maria Podinovskaia, Cristina Prescianotto-Baschong, Dominik P Buser, Anne Spang.
      Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.
    Keywords:  Rab GTPases; cell biology; endocytosis; endosome maturation; human; live cell imaging assay; membrane transport; organelle acidification; pH; recycling; sorting; sorting nexin
    DOI:  https://doi.org/10.7554/eLife.70982
  15. J Dev Biol. 2021 Nov 23. pii: 52. [Epub ahead of print]9(4):
    Lysosomal Function Impacts the Skeletal Muscle Extracellular Matrix.
    Elizabeth C Coffey, Mary Astumian, Sarah S Alrowaished, Claire Schaffer, Clarissa A Henry.
      Muscle development and homeostasis are critical for normal muscle function. A key aspect of muscle physiology during development, growth, and homeostasis is modulation of protein turnover, the balance between synthesis and degradation of muscle proteins. Protein degradation depends upon lysosomal pH, generated and maintained by proton pumps. Sphingolipid transporter 1 (spns1), a highly conserved gene encoding a putative late endosome/lysosome carbohydrate/H+ symporter, plays a pivotal role in maintaining optimal lysosomal pH and spns1-/- mutants undergo premature senescence. However, the impact of dysregulated lysosomal pH on muscle development and homeostasis is not well understood. We found that muscle development proceeds normally in spns1-/- mutants prior to the onset of muscle degeneration. Dysregulation of the extracellular matrix (ECM) at the myotendinous junction (MTJ) coincided with the onset of muscle degeneration in spns1-/- mutants. Expression of the ECM proteins laminin 111 and MMP-9 was upregulated. Upregulation of laminin 111 mitigated the severity of muscle degeneration, as inhibition of adhesion to laminin 111 exacerbated muscle degeneration in spns1-/- mutants. MMP-9 upregulation was induced by tnfsf12 signaling, but abrogation of MMP-9 did not impact muscle degeneration in spns1-/- mutants. Taken together, these data indicate that dysregulated lysosomal pH impacts expression of ECM proteins at the myotendinous junction.
    Keywords:  basement membrane; lysosomal myopathy; myotendinous junction; skeletal muscle; spinster; zebrafish
    DOI:  https://doi.org/10.3390/jdb9040052
  16. EMBO Mol Med. 2021 Dec 01. e13968
    Muscle-directed gene therapy corrects Pompe disease and uncovers species-specific GAA immunogenicity.
    Michelle Eggers, Charles H Vannoy, Jianyong Huang, Pravinkumar Purushothaman, Jacqueline Brassard, Carlos Fonck, Hui Meng, Mariah J Prom, Michael W Lawlor, Justine Cunningham, Chanchal Sadhu, Fulvio Mavilio.
      Pompe disease is a severe disorder caused by loss of acid α-glucosidase (GAA), leading to glycogen accumulation in tissues and neuromuscular and cardiac dysfunction. Enzyme replacement therapy is the only available treatment. AT845 is an adeno-associated viral vector designed to express human GAA specifically in skeletal muscle and heart. Systemic administration of AT845 in Gaa-/- mice led to a dose-dependent increase in GAA activity, glycogen clearance in muscles and heart, and functional improvement. AT845 was tolerated in cynomolgus macaques at low doses, while high doses caused anti-GAA immune response, inflammation, and cardiac abnormalities resulting in unscheduled euthanasia of two animals. Conversely, a vector expressing the macaque GAA caused no detectable pathology, indicating that the toxicity observed with AT845 was an anti-GAA xenogeneic immune response. Western blot analysis showed abnormal processing of human GAA in cynomolgus muscle, adding to the species-specific effects of enzyme expression. Overall, these studies show that AAV-mediated GAA delivery to muscle is efficacious in Gaa-/- mice and highlight limitations in predicting the toxicity of AAV vectors encoding human proteins in non-human species.
    Keywords:  adeno-associated virus; glycogen accumulation; heart inflammation; lysosomal storage disease; xenogeneic immune response
    DOI:  https://doi.org/10.15252/emmm.202113968
  17. J Genet Genomics. 2021 Nov 30. pii: S1673-8527(21)00359-3. [Epub ahead of print]
    GRB10 regulates β cell mass by inhibiting β cell proliferation and stimulating β cell dedifferentiation.
    Zixin Cai, Fen Liu, Yan Yang, Dandan Li, Shanbiao Hu, Lei Song, Shaojie Yu, Ting Li, Bilian Liu, Hairong Luo, Weiping Zhang, Zhiguang Zhou, Jingjing Zhang.
      Decreased functional β-cell mass is the hallmark of diabetes, but the cause of this metabolic defect remains elusive. Here, we show that the expression levels of the growth factor receptor-bound protein 10 (GRB10), a negative regulator of insulin and mTORC1 signaling, are markedly induced in islets of diabetic mice and high glucose-treated insulinoma cell line INS-1cells. β-cell-specific knockout of Grb10 in mice increased β-cell mass and improved β-cell function. Grb10-deficient β-cells exhibit enhanced mTORC1 signaling and reduced β-cell dedifferentiation, which could be blocked by rapamycin. On the contrary, Grb10 overexpression induced β-cell dedifferentiation in MIN6 cells. Our study identifies GRB10 as a critical regulator of β-cell dedifferentiation and β-cell mass, which exerts its effect by inhibiting mTORC1 signaling.
    Keywords:  Grb10; dedifferentiation; mTORC1; β cell mass
    DOI:  https://doi.org/10.1016/j.jgg.2021.11.006
  18. Methods Mol Biol. 2022 ;2422 85-124
    Probes for Fluorescent Visualization of Specific Cellular Organelles.
    Timothy Paul Foster.
      The defining characteristic of eukaryotic cells is the segregation of critical cellular functions within various membrane bound cellular organelles, including the nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria. Cell biologists therefore have extensively utilized organelle specific counterstains to help identify the localization of specific proteins or other targets of interest in order to garner an understanding of either their potential functions or their effects on the cell. There currently is a wide array of fluorescent dyes and reagents that can be utilized in live and fixed cells to identify organelles, thereby creating challenges in both choosing between the plethora of options and optimizing their use. Here we present a discussion of commonly utilized commercially available organelle dyes and summarize the factors that influence selection of the various dyes for: a given organelle; live versus fixed cellular conditions; adaptation to a specific protocol; spectral multiplexing; or matching excitation/emission spectra to available imaging equipment. Also presented are recommended protocols for a typical example reagent that can be reliably utilized to visualize its target cellular organelle.
    Keywords:  Actin; Cell biology; Endoplasmic reticulum; Fluorescent dyes; Fluorescent labeling; Golgi; Lysosome; Mitochondria; Nucleus; Organelles; Plasma membrane
    DOI:  https://doi.org/10.1007/978-1-0716-1948-3_7
  19. Brain. 2021 Nov 25. pii: awab390. [Epub ahead of print]
    Expression of 4E-BP1 in juvenile mice alleviates mTOR-induced neuronal dysfunction and epilepsy.
    Lena H Nguyen, Youfen Xu, Travorn Mahadeo, Longbo Zhang, Tiffany V Lin, Heather A Born, Anne E Anderson, Angélique Bordey.
      Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway during fetal neurodevelopment alters neuron structure and function, leading to focal malformation of cortical development (FMCD) and intractable epilepsy. Recent evidence suggests a role for dysregulated cap-dependent translation downstream of mTOR in the formation of FMCD and seizures. However, it is unknown whether modifying translation once the developmental pathologies are established can reverse neuronal abnormalities and seizures. Addressing these issues is crucial with regards to therapeutics since these neurodevelopmental disorders are predominantly diagnosed during childhood, when patients present with symptoms. Here, we report increased phosphorylation of the mTOR effector and translational repressor, 4E-BP1, in patient FMCD tissue and in a mouse model of FMCD. Using temporally regulated conditional gene expression systems, we found that expression of a constitutively active form of 4E-BP1 that resists phosphorylation by mTOR in juvenile mice reduced neuronal cytomegaly and corrected several neuronal electrophysiological alterations, including depolarized resting membrane potential, irregular firing pattern, and aberrant expression of HCN4 channels. Further, 4E-BP1 expression in juvenile FMCD mice after epilepsy onset resulted in improved cortical spectral activity and decreased spontaneous seizure frequency in adults. Overall, our study uncovered a remarkable plasticity of the juvenile brain that facilitates novel therapeutic opportunities to treat FMCD-related epilepsy during childhood with potentially long-lasting effects in adults.
    Keywords:  cap-dependent translation; hyperpolarization-activated cyclic nucleotide-gated (HCN) channels; in utero electroporation; malformation of cortical development; seizures
    DOI:  https://doi.org/10.1093/brain/awab390
  20. Nat Biotechnol. 2021 Dec 02.
    Personalized phosphoproteomics identifies functional signaling.
    Elise J Needham, Janne R Hingst, Benjamin L Parker, Kaitlin R Morrison, Guang Yang, Johan Onslev, Jonas M Kristensen, Kurt Højlund, Naomi X Y Ling, Jonathan S Oakhill, Erik A Richter, Bente Kiens, Janni Petersen, Christian Pehmøller, David E James, Jørgen F P Wojtaszewski, Sean J Humphrey.
      Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.
    DOI:  https://doi.org/10.1038/s41587-021-01099-9
  21. Nat Commun. 2021 Nov 30. 12(1): 6979
    A DAP5/eIF3d alternate mRNA translation mechanism promotes differentiation and immune suppression by human regulatory T cells.
    Viviana Volta, Sandra Pérez-Baos, Columba de la Parra, Olga Katsara, Amanda Ernlund, Sophie Dornbaum, Robert J Schneider.
      Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4+ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5' noncoding regions. Silencing DAP5 in isolated human naive CD4+ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation.
    DOI:  https://doi.org/10.1038/s41467-021-27087-w
  22. Front Immunol. 2021 ;12 743700
    Targeting TBK1 Attenuates LPS-Induced NLRP3 Inflammasome Activation by Regulating of mTORC1 Pathways in Trophoblasts.
    Sohee Lee, Jiha Shin, Jong-Seok Kim, Jongdae Shin, Sung Ki Lee, Hwan-Woo Park.
      Pathological maternal inflammation and abnormal placentation contribute to several pregnancy-related disorders, including preterm birth, intrauterine growth restriction, and preeclampsia. TANK-binding kinase 1 (TBK1), a serine/threonine kinase, has been implicated in the regulation of various physiological processes, including innate immune response, autophagy, and cell growth. However, the relevance of TBK1 in the placental pro-inflammatory environment has not been investigated. In this study, we assessed the effect of TBK1 inhibition on lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation and its underlying mechanisms in human trophoblast cell lines and mouse placenta. TBK1 phosphorylation was upregulated in the trophoblasts and placenta in response to LPS. Pharmacological and genetic inhibition of TBK1 in trophoblasts ameliorated LPS-induced NLRP3 inflammasome activation, placental inflammation, and subsequent interleukin (IL)-1 production. Moreover, maternal administration of amlexanox, a TBK1 inhibitor, reversed LPS-induced adverse pregnancy outcomes. Notably, TBK1 inhibition prevented LPS-induced NLRP3 inflammasome activation by targeting the mammalian target of rapamycin complex 1 (mTORC1). Thus, this study provides evidence for the biological significance of TBK1 in placental inflammation, suggesting that amlexanox may be a potential therapeutic candidate for treating inflammation-associated pregnancy-related complications.
    Keywords:  NLRP3 inflammasome; TBK1; mTORC1; maternal inflammation; placenta; trophoblast
    DOI:  https://doi.org/10.3389/fimmu.2021.743700
  23. Cortex. 2021 Nov 04. pii: S0010-9452(21)00343-9. [Epub ahead of print]146 50-65
    Oscillatory neural network alterations in young people with tuberous sclerosis complex and associations with co-occurring symptoms of autism spectrum disorder and attention-deficit/hyperactivity disorder.
    TS2000 Study Team
      Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations on the TSC1/TSC2 genes, which result in alterations in molecular signalling pathways involved in neurogenesis and hamartomas in the brain and other organs. TSC carries a high risk for autism spectrum disorder (ASD) and attention-deficit/hyperactivity disorder (ADHD), although the reasons for this are unclear. One proposal is that TSC-related alterations in molecular signalling during neurogenesis lead to atypical development of neural networks, which are involved in the occurrence of ASD and ADHD in TSC. We investigated this proposal in young people with TSC who have been studied longitudinally since their diagnosis in childhood. Electroencephalography (EEG) was used to examine oscillatory connectivity in functional neural networks and local and global network organisation during three tasks (resting-state, attentional and inhibitory control Go/Nogo task, upright and inverted face processing task) in participants with TSC (n = 48) compared to an age- and sex-matched group of typically developing Controls (n = 20). Compared to Controls, the TSC group showed hypoconnected neural networks in the alpha frequency during the resting-state and in the theta and alpha frequencies during the Go/Nogo task (P ≤ .008), as well as reduced local network organisation in the theta and alpha frequencies during the Go/Nogo task (F = 3.95, P = .010). There were no significant group differences in network metrics during the face processing task. Increased connectivity in the hypoconnected alpha-range resting-state network was associated with greater ASD and inattentive ADHD symptoms (rho≥.40, P ≤ .036). Reduced local network organisation in the theta-range during the Go/Nogo task was significantly associated with higher hyperactive/impulsive ADHD symptoms (rho = -.43, P = .041). These findings suggest that TSC is associated with widespread hypoconnectivity in neural networks and support the proposal that altered network function may be involved in the co-occurrence of ASD and ADHD in TSC.
    Keywords:  Attention-deficit/hyperactivity disorder (ADHD); Autism spectrum disorder (ASD); Electrophysiology (EEG); Oscillatory neural networks; Tuberous sclerosis complex (TSC)
    DOI:  https://doi.org/10.1016/j.cortex.2021.10.007
  24. Dev Cell. 2021 Nov 24. pii: S1534-5807(21)00894-7. [Epub ahead of print]
    Cell cycle regulation of ER membrane biogenesis protects against chromosome missegregation.
    Holly Merta, Jake W Carrasquillo Rodríguez, Maya I Anjur-Dietrich, Tevis Vitale, Mitchell E Granade, Thurl E Harris, Daniel J Needleman, Shirin Bahmanyar.
      Failure to reorganize the endoplasmic reticulum (ER) in mitosis results in chromosome missegregation. Here, we show that accurate chromosome segregation in human cells requires cell cycle-regulated ER membrane production. Excess ER membranes increase the viscosity of the mitotic cytoplasm to physically restrict chromosome movements, which impedes the correction of mitotic errors leading to the formation of micronuclei. Mechanistically, we demonstrate that the protein phosphatase CTDNEP1 counteracts mTOR kinase to establish a dephosphorylated pool of the phosphatidic acid phosphatase lipin 1 in interphase. CTDNEP1 control of lipin 1 limits the synthesis of fatty acids for ER membrane biogenesis in interphase that then protects against chromosome missegregation in mitosis. Thus, regulation of ER size can dictate the biophysical properties of mitotic cells, providing an explanation for why ER reorganization is necessary for mitotic fidelity. Our data further suggest that dysregulated lipid metabolism is a potential source of aneuploidy in cancer cells.
    Keywords:  CTDNEP1; aneuploidy; lipid homeostasis; lipin; mTOR; medulloblastoma; micronuclei; mitosis; nuclear assembly
    DOI:  https://doi.org/10.1016/j.devcel.2021.11.009
  25. Genetics. 2021 Oct 05. pii: iyab168. [Epub ahead of print]
    TORC1 signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae.
    Riley Horvath, Nicole Hawe, Cindy Lam, Konstantin Mestnikov, Mariam Eji-Lasisi, John Rohde, Ivan Sadowski.
      Cdk8 of the RNA polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55 and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.
    Keywords:   GAL genes; Cdc55; Cdk8; Gal4; PP2A; Tor; phosphorylation; transcription; yeast
    DOI:  https://doi.org/10.1093/genetics/iyab168
  26. J Clin Invest. 2021 Dec 02. pii: e153724. [Epub ahead of print]
    Dyrk1b promotes hepatic lipogenesis by bypassing canonical insulin signaling and directly activating mTORC2 in mice.
    Neha Bhat, Anand Narayanan, Mohsen Fathzadeh, Mario Kahn, Dongyan Zhang, Leigh Goedeke, Arpita Neogi, Rebecca L Cardone, Richard G Kibbey, Carlos Fernandez-Hernando, Henry N Ginsberg, Dhanpat Jain, Gerald Shulman, Arya Mani.
      Mutations in Dyrk1b are associated with metabolic syndrome and non-alcoholic fatty liver disease in humans. Our investigations showed that DYRK1B levels are increased in the liver of patients with non-alcoholic liver steatohepatitis (NASH) and in mice fed with a high fat/sucrose diet. Increasing Dyrk1b levels in the mouse liver enhanced de novo lipogenesis (DNL), fatty-acid uptake, and TAG secretion and caused NASH and hyperlipidemia. Conversely, knockdown of Dyrk1b was protective against high-calorie induced hepatic steatosis and fibrosis and hyperlipidemia. Mechanistically, Dyrk1b increased DNL by activating mTORC2 in a kinase independent fashion. Accordingly, the Dyrk1b-induced NASH was fully rescued when mTORC2 was genetically disrupted. The elevated DNL was associated with increased plasma membrane sn-1,2-diacylglyerol levels and increased PKCε-mediated IRKT1150 phosphorylation, which resulted in impaired activation of hepatic insulin signaling and reduced hepatic glycogen storage. These findings provide new insights into the mechanisms that underlie Dyrk1b-induced hepatic lipogenesis and hepatic insulin resistance and identify Dyrk1b as a therapeutic target for NASH and insulin resistance in the liver.
    Keywords:  Hepatology; Insulin signaling; Metabolism; Obesity; Protein kinases
    DOI:  https://doi.org/10.1172/JCI153724
  27. Proc Natl Acad Sci U S A. 2021 Dec 07. pii: e2113573118. [Epub ahead of print]118(49):
    Structural basis for substrate specificity of heteromeric transporters of neutral amino acids.
    Carlos F Rodriguez, Paloma Escudero-Bravo, Lucía Díaz, Paola Bartoccioni, Carmen García-Martín, Joan G Gilabert, Jasminka Boskovic, Víctor Guallar, Ekaitz Errasti-Murugarren, Oscar Llorca, Manuel Palacín.
      Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.
    Keywords:  HATs; amino acid transporters; hLAT2; structure; substrate selectivity
    DOI:  https://doi.org/10.1073/pnas.2113573118
  28. PLoS Pathog. 2021 Nov 29. 17(11): e1009409
    A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.
    Charlotte A Stoneham, Simon Langer, Paul D De Jesus, Jacob M Wozniak, John Lapek, Thomas Deerinck, Andrea Thor, Lars Pache, Sumit K Chanda, David J Gonzalez, Mark Ellisman, John Guatelli.
      The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.
    DOI:  https://doi.org/10.1371/journal.ppat.1009409
  29. J Cell Sci. 2021 Dec 03. pii: jcs.253591. [Epub ahead of print]
    Smaug1 membrane-less organelles respond to AMPK/mTOR and affect mitochondrial function‡.
    Ana J Fernández-Alvarez, María Gabriela Thomas, Malena L Pascual, Martín Habif, Jerónimo Pimentel, Agustín A Corbat, João P Pessoa, Pablo E La Spina, Lara Boscaglia, Anne Plessis, Maria Carmo-Fonseca, Hernán E Grecco, Marta Casado, Graciela L Boccaccio.
      Smaug is a conserved translational regulator that binds numerous mRNAs, including nuclear transcripts that encode mitochondrial enzymes. Smaug orthologs form cytosolic membrane-less organelles (MLOs) in several organisms and cell types. We have performed single-molecule FISH assays that revealed that SDHB and UQCRC1 mRNAs associate with Smaug1 bodies in U2OS cells. Loss of function of Smaug1 and Smaug2 affected both mitochondrial respiration and morphology of the mitochondrial network. Phenotype rescue by Smaug1 transfection depends on the presence of its RNA binding domain. Moreover, we identified specific Smaug1 domains involved in MLO formation, and found that impaired Smaug1 MLO condensation correlates with mitochondrial defects. Mitochondrial Complex I inhibition by rotenone -but not strong mitochondrial uncoupling by CCCP- rapidly induced Smaug1 MLOs dissolution. Metformin and rapamycin elicited similar effects, which were blocked by pharmacological inhibition of AMPK. Finally, we found that Smaug1 MLO dissolution weakens the interaction with target mRNAs, thus enabling their release. We propose that mitochondrial respiration and the AMPK/mTOR balance controls the condensation and dissolution of Smaug1 MLOs, thus regulating nuclear mRNAs that encode key mitochondrial proteins.
    Keywords:  AMPK; Membrane-less organelles; Metformin; Mitochondria; Processing bodies; Smaug; Uqcrc1
    DOI:  https://doi.org/10.1242/jcs.253591
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