bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒11‒21
twenty-nine papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. GRASPing the unconventional secretory machinery to bridge cellular stress signaling to the extracellular proteome.
  2. The TSC Complex-mTORC1 Axis: From Lysosomes to Stress Granules and Back.
  3. mTOR-mediated phosphorylation of VAMP8 and SCFD1 regulates autophagosome maturation.
  4. The PI3 kinase complex II-PI3P-Vps27 axis on vacuolar membranes is critical for microautophagy induction and nutrient stress adaptation.
  5. Ca2+ administration prevents α-synuclein proteotoxicity by stimulating calcineurin-dependent lysosomal proteolysis.
  6. CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.
  7. Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2.
  8. Folliculin impairs breast tumor growth by repressing TFE3-dependent induction of the Warburg effect and angiogenesis.
  9. Emerging role of lysosomal calcium store as a hub of neuroprotection.
  10. Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly.
  11. Convergent molecular mechanisms underlying cognitive impairment in mucopolysaccharidosis type II.
  12. Dysregulated endolysosomal trafficking in cells arrested in the G1 phase of the host cell cycle impairs Salmonella vacuolar replication.
  13. Molecular and histological traits of reduced lysosomal acid lipase activity in the fatty liver.
  14. mTORC1 Activation in Chx10-Specific Tsc1 Knockout Mice Accelerates Retina Aging and Degeneration.
  15. Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors.
  16. Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization.
  17. ATG2 and VPS13 proteins: Molecular highways transporting lipids to drive membrane expansion and organelle communication.
  18. mTOR-dependent translation drives tumor infiltrating CD8+ effector and CD4+ Treg cells expansion.
  19. Clathrin adapters AP-1 and GGA2 support expression of epidermal growth factor receptor for cell growth.
  20. The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE-interactions.
  21. Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.
  22. Nde1 is a Rab9 effector for loading late endosomes to cytoplasmic dynein motor complex.
  23. The K63 deubiquitinase CYLD modulates autism-like behaviors and hippocampal plasticity by regulating autophagy and mTOR signaling.
  24. JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila.
  25. Impacts of essential amino acids on energy balance.
  26. Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs).
  27. Editorial: The Role of Different Subcellular Organelles in DNA Damage Response.
  28. Targeting Phosphatases and Kinases: How to Checkmate Cancer.
  29. Bi-directional ribosome scanning controls the stringency of start codon selection.
  1. Cell Stress. 2021 Nov;5(11): 173-175
    GRASPing the unconventional secretory machinery to bridge cellular stress signaling to the extracellular proteome.
    Constantinos Demetriades, Julian Nuüchel, Markus Plomann.
      Cellular adaptation to stress is a crucial homeostatic process for survival, metabolism, physiology, and disease. Cells respond to stress stimuli (e.g., nutrient starvation, growth factor deprivation, hypoxia, low energy, etc.) by changing the activity of signaling pathways, and interact with their environment by qualitatively and quantitatively modifying their intracellular, surface, and extracellular proteomes. How this delicate communication takes place is a hot topic in cell biological research, and has important implications for human disease.
    Keywords:  GRASP55; Golgi; Tuberous Sclerosis Complex (TSC); cellular stress response; mTORC1; rapamycin; unconventional protein secretion (UPS)
    DOI:  https://doi.org/10.15698/cst2021.11.259
  2. Front Cell Dev Biol. 2021 ;9 751892
    The TSC Complex-mTORC1 Axis: From Lysosomes to Stress Granules and Back.
    Ulrike Rehbein, Mirja Tamara Prentzell, Marti Cadena Sandoval, Alexander Martin Heberle, Elizabeth P Henske, Christiane A Opitz, Kathrin Thedieck.
      The tuberous sclerosis protein complex (TSC complex) is a key integrator of metabolic signals and cellular stress. In response to nutrient shortage and stresses, the TSC complex inhibits the mechanistic target of rapamycin complex 1 (mTORC1) at the lysosomes. mTORC1 is also inhibited by stress granules (SGs), RNA-protein assemblies that dissociate mTORC1. The mechanisms of lysosome and SG recruitment of mTORC1 are well studied. In contrast, molecular details on lysosomal recruitment of the TSC complex have emerged only recently. The TSC complex subunit 1 (TSC1) binds lysosomes via phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2]. The SG assembly factors 1 and 2 (G3BP1/2) have an unexpected lysosomal function in recruiting TSC2 when SGs are absent. In addition, high density lipoprotein binding protein (HDLBP, also named Vigilin) recruits TSC2 to SGs under stress. In this mini-review, we integrate the molecular mechanisms of lysosome and SG recruitment of the TSC complex. We discuss their interplay in the context of cell proliferation and migration in cancer and in the clinical manifestations of tuberous sclerosis complex disease (TSC) and lymphangioleiomyomatosis (LAM).
    Keywords:  G3BP1 (G3BP stress granule assembly factor 1); HDLBP; TSC complex; autophagy; lymphangioleiomyomatosis (LAM); lysosomes; mTORC1 (mechanistic target of rapamycin complex 1); stress granules (SG)
    DOI:  https://doi.org/10.3389/fcell.2021.751892
  3. Nat Commun. 2021 Nov 16. 12(1): 6622
    mTOR-mediated phosphorylation of VAMP8 and SCFD1 regulates autophagosome maturation.
    Hong Huang, Qinqin Ouyang, Min Zhu, Haijia Yu, Kunrong Mei, Rong Liu.
      The mammalian target of rapamycin (mTORC1) has been shown to regulate autophagy at different steps. However, how mTORC1 regulates the N-ethylmaleimide-sensitive protein receptor (SNARE) complex remains elusive. Here we show that mTORC1 inhibits formation of the SNARE complex (STX17-SNAP29-VAMP8) by phosphorylating VAMP8, thereby blocking autophagosome-lysosome fusion. A VAMP8 phosphorylation mimic mutant is unable to promote autophagosome-lysosome fusion in vitro. Furthermore, we identify SCFD1, a Sec1/Munc18-like protein, that localizes to the autolysosome and is required for SNARE complex formation and autophagosome-lysosome fusion. VAMP8 promotes SCFD1 recruitment to autolysosomes when dephosphorylated. Consistently, phosphorylated VAMP8 or SCFD1 depletion inhibits autophagosome-lysosome fusion, and expression of phosphomimic VAMP8 leads to increased lipid droplet accumulation when expressed in mouse liver. Thus, our study supports that mTORC1-mediated phosphorylation of VAMP8 blocks SCFD1 recruitment, thereby inhibiting STX17-SNAP29-VAMP8 complex formation and autophagosome-lysosome fusion.
    DOI:  https://doi.org/10.1038/s41467-021-26824-5
  4. J Mol Biol. 2021 Nov 16. pii: S0022-2836(21)00597-0. [Epub ahead of print] 167360
    The PI3 kinase complex II-PI3P-Vps27 axis on vacuolar membranes is critical for microautophagy induction and nutrient stress adaptation.
    Most Naoshia Tasnin, Kisara Ito, Haruko Katsuta, Tsuneyuki Takuma, Tasnuva Sharmin, Takashi Ushimaru.
      Phosphatidylinositol 3-phosphate (PI3P), a scaffold of membrane-associated proteins required for diverse cellular events, is produced by Vps34-containing phosphatidylinositol 3-kinase (PI3K). PI3K complex I (PI3KCI)-generated PI3P is required for macroautophagy, whereas PI3K complex II (PI3KCII)-generated PI3P is required for endosomal sorting complex required for transport (ESCRT)-mediated multi-vesicular body (MVB) formation in late endosomes. ESCRT also promotes vacuolar membrane remodeling in microautophagy after nutrient starvation and inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Whereas PI3KCI and macroautophagy are critical for the nutrient starvation response, the physiological roles of PI3KCII and microautophagy during starvation are largely unknown. Here, we showed that PI3KCII-produced PI3P on vacuolar membranes is required for microautophagy induction and survival in nutrient-stressed conditions. PI3KCII is required for Vps27 (an ESCRT-0 component) recruitment and ESCRT-0 complex formation on vacuolar surfaces after TORC1 inactivation. Forced recruitment of Vps27 onto vacuolar membranes rescued the defect in microautophagy induction in PI3KCII-deficient cells, indicating that a critical role of PI3P on microautophagy induction is Vps27 recruitment onto vacuolar surfaces. Finally, vacuolar membrane-associated Vps27 was able to recover survival during nutrient starvation in cells lacking PI3KCII or Vps27. This study revealed that the PI3KCII-PI3P-Vps27 axis on vacuolar membranes is critical for ESCRT-mediated microautophagy induction and nutrient stress adaptation.
    Keywords:  ESCRT; TORC1; microautophagy; phosphatidylinositol 3-kinase complex II; phosphatidylinositol 3-phosphate
    DOI:  https://doi.org/10.1016/j.jmb.2021.167360
  5. PLoS Genet. 2021 Nov 15. 17(11): e1009911
    Ca2+ administration prevents α-synuclein proteotoxicity by stimulating calcineurin-dependent lysosomal proteolysis.
    Lukas Habernig, Filomena Broeskamp, Andreas Aufschnaiter, Jutta Diessl, Carlotta Peselj, Elisabeth Urbauer, Tobias Eisenberg, Ana de Ory, Sabrina Büttner.
      The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. Here, we report that proteotoxicity in yeast and Drosophila models for Parkinson's disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson's disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, we establish a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.
    DOI:  https://doi.org/10.1371/journal.pgen.1009911
  6. Nature. 2021 Nov 18.
    CRISPR screens unveil signal hubs for nutrient licensing of T cell immunity.
    Lingyun Long, Jun Wei, Seon Ah Lim, Jana L Raynor, Hao Shi, Jon P Connelly, Hong Wang, Cliff Guy, Boer Xie, Nicole M Chapman, Guotong Fu, Yanyan Wang, Hongling Huang, Wei Su, Jordy Saravia, Isabel Risch, Yong-Dong Wang, Yuxin Li, Mingming Niu, Yogesh Dhungana, Anil Kc, Peipei Zhou, Peter Vogel, Jiyang Yu, Shondra M Pruett-Miller, Junmin Peng, Hongbo Chi.
      Nutrients are emerging regulators of adaptive immunity1. Selective nutrients interplay with immunological signals to activate mechanistic target of rapamycin complex 1 (mTORC1), a key driver of cell metabolism2-4, but how these environmental signals are integrated for immune regulation remains unclear. Here we use genome-wide CRISPR screening combined with protein-protein interaction networks to identify regulatory modules that mediate immune receptor- and nutrient-dependent signalling to mTORC1 in mouse regulatory T (Treg) cells. SEC31A is identified to promote mTORC1 activation by interacting with the GATOR2 component SEC13 to protect it from SKP1-dependent proteasomal degradation. Accordingly, loss of SEC31A impairs T cell priming and Treg suppressive function in mice. In addition, the SWI/SNF complex restricts expression of the amino acid sensor CASTOR1, thereby enhancing mTORC1 activation. Moreover, we reveal that the CCDC101-associated SAGA complex is a potent inhibitor of mTORC1, which limits the expression of glucose and amino acid transporters and maintains T cell quiescence in vivo. Specific deletion of Ccdc101 in mouse Treg cells results in uncontrolled inflammation but improved antitumour immunity. Collectively, our results establish epigenetic and post-translational mechanisms that underpin how nutrient transporters, sensors and transducers interplay with immune signals for three-tiered regulation of mTORC1 activity and identify their pivotal roles in licensing T cell immunity and immune tolerance.
    DOI:  https://doi.org/10.1038/s41586-021-04109-7
  7. Mol Cell. 2021 Nov 15. pii: S1097-2765(21)00949-7. [Epub ahead of print]
    Cryo-EM reveals mechanistic insights into lipid-facilitated polyamine export by human ATP13A2.
    Atsuhiro Tomita, Takashi Daiho, Tsukasa Kusakizako, Keitaro Yamashita, Satoshi Ogasawara, Takeshi Murata, Tomohiro Nishizawa, Osamu Nureki.
      The cytoplasmic polyamine maintains cellular homeostasis by chelating toxic metal cations, regulating transcriptional activity, and protecting DNA. ATP13A2 was identified as a lysosomal polyamine exporter responsible for polyamine release into the cytosol, and its dysfunction is associated with Alzheimer's disease and other neural degradation diseases. ATP13A2 belongs to the P5 subfamily of the P-type ATPase family, but its mechanisms remain unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human ATP13A2 under four different conditions, revealing the structural coupling between the polyamine binding and the dephosphorylation. Polyamine is bound at the luminal tunnel and recognized through numerous electrostatic and π-cation interactions, explaining its broad specificity. The unique N-terminal domain is anchored to the lipid membrane to stabilize the E2P conformation, thereby accelerating the E1P-to-E2P transition. These findings reveal the distinct mechanism of P5B ATPases, thereby paving the way for neuroprotective therapy by activating ATP13A2.
    Keywords:  ATP13A2; MD simulation; P-type ATPase; P5B-ATPase; PARK9; Parkinson's disease; cryo-EM; lysosome; membrane protein; polyamine
    DOI:  https://doi.org/10.1016/j.molcel.2021.11.001
  8. J Clin Invest. 2021 Nov 15. pii: e144871. [Epub ahead of print]131(22):
    Folliculin impairs breast tumor growth by repressing TFE3-dependent induction of the Warburg effect and angiogenesis.
    Leeanna El-Houjeiri, Marco Biondini, Mathieu Paquette, Helen Kuasne, Alain Pacis, Morag Park, Peter M Siegel, Arnim Pause.
      Growing tumors exist in metabolically compromised environments that require activation of multiple pathways to scavenge nutrients to support accelerated rates of growth. The folliculin (FLCN) tumor suppressor complex (FLCN, FNIP1, FNIP2) is implicated in the regulation of energy homeostasis via 2 metabolic master kinases: AMPK and mTORC1. Loss-of-function mutations of the FLCN tumor suppressor complex have only been reported in renal tumors in patients with the rare Birt-Hogg-Dube syndrome. Here, we revealed that FLCN, FNIP1, and FNIP2 are downregulated in many human cancers, including poor-prognosis invasive basal-like breast carcinomas where AMPK and TFE3 targets are activated compared with the luminal, less aggressive subtypes. FLCN loss in luminal breast cancer promoted tumor growth through TFE3 activation and subsequent induction of several pathways, including autophagy, lysosomal biogenesis, aerobic glycolysis, and angiogenesis. Strikingly, induction of aerobic glycolysis and angiogenesis in FLCN-deficient cells was dictated by the activation of the PGC-1α/HIF-1α pathway, which we showed to be TFE3 dependent, directly linking TFE3 to Warburg metabolic reprogramming and angiogenesis. Conversely, FLCN overexpression in invasive basal-like breast cancer models attenuated TFE3 nuclear localization, TFE3-dependent transcriptional activity, and tumor growth. These findings support a general role of a deregulated FLCN/TFE3 tumor suppressor pathway in human cancers.
    Keywords:  Angiogenesis; Breast cancer; Cancer; Metabolism
    DOI:  https://doi.org/10.1172/JCI144871
  9. Neural Regen Res. 2022 Jun;17(6): 1259-1260
    Emerging role of lysosomal calcium store as a hub of neuroprotection.
    Valentina Tedeschi, Agnese Secondo.
      
    DOI:  https://doi.org/10.4103/1673-5374.327340
  10. Elife. 2021 11 17. pii: e71575. [Epub ahead of print]10
    Control of protein synthesis and memory by GluN3A-NMDA receptors through inhibition of GIT1/mTORC1 assembly.
    María J Conde-Dusman, Partha N Dey, Óscar Elía-Zudaire, Luis G Rabaneda, Carmen García-Lira, Teddy Grand, Victor Briz, Eric R Velasco, Raül Andero, Sergio Niñerola, Angel Barco, Pierre Paoletti, John F Wesseling, Fabrizio Gardoni, Steven J Tavalin, Isabel Perez-Otaño.
      De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement.
    Keywords:  BDNF; GIT1; GluN3A; NMDA receptor; cell biology; mTOR; memory; mouse; protein synthesis; rat; synapse
    DOI:  https://doi.org/10.7554/eLife.71575
  11. Metab Brain Dis. 2021 Nov 19.
    Convergent molecular mechanisms underlying cognitive impairment in mucopolysaccharidosis type II.
    Thiago Corrêa, Fabiano Poswar, Cíntia B Santos-Rebouças.
      Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by pathogenic variants in the iduronate-2-sulfatase gene (IDS), responsible for the degradation of glycosaminoglycans (GAGs) heparan and dermatan sulfate. IDS enzyme deficiency results in the accumulation of GAGs within cells and tissues, including the central nervous system (CNS). The progressive neurological outcome in a representative number of MPSII patients (neuronopathic form) involves cognitive impairment, behavioral difficulties, and regression in developmental milestones. In an attempt to dissect part of the influence of axon guidance instability over the cognitive impairment presentation in MPS II, we used brain expression data, network propagation, and clustering algorithm to prioritize in the human interactome a disease module associated with the MPS II context. We identified new candidate genes and pathways that act in focal adhesion, integrin cell surface, laminin interactions, ECM proteoglycans, cytoskeleton, and phagosome that converge into functional mechanisms involved in early neural circuit formation defects and could indicate clues about cognitive impairment in patients with MPSII. Such molecular changes during neurodevelopment may precede the morphological and clinical evidence, emphasizing the importance of an early diagnosis and directing the development of potential drug leads. Furthermore, our data also support previous hypotheses pointing to shared pathogenic mechanisms in some neurodegenerative diseases.
    Keywords:  Axon guidance; Cognitive impairment; Extracellular matrix; MPS II; Signaling pathways
    DOI:  https://doi.org/10.1007/s11011-021-00872-8
  12. Autophagy. 2021 Nov 15. 1-16
    Dysregulated endolysosomal trafficking in cells arrested in the G1 phase of the host cell cycle impairs Salmonella vacuolar replication.
    Clivia Lisowski, Jane Dias, Susana Costa, Ricardo Jorge Silva, Miguel Mano, Ana Eulalio.
      Modulation of the host cell cycle has emerged as a common theme among the pathways regulated by bacterial pathogens, arguably to promote host cell colonization. However, in most cases the exact benefit ensuing from such interference to the infection process remains unclear. Previously, we have shown that Salmonella actively induces G2/M arrest of host cells, and that infection is severely inhibited in cells arrested in G1. In this study, we demonstrate that Salmonella vacuolar replication is inhibited in host cells blocked in G1, whereas the cytosolic replication of the closely related pathogen Shigella is not affected. Mechanistically, we show that cells arrested in G1, but not cells arrested in G2, present dysregulated endolysosomal trafficking, displaying an abnormal accumulation of vesicles positive for late endosomal and lysosomal markers. In addition, the macroautophagic/autophagic flux and degradative lysosomal function are strongly impaired. This endolysosomal trafficking dysregulation results in sustained activation of the SPI-1 type III secretion system and lack of vacuole repair by the autophagy pathway, ultimately compromising the maturation and integrity of the Salmonella-containing vacuole. As such, Salmonella is released in the host cytosol. Collectively, our findings demonstrate that the modulation of the host cell cycle occurring during Salmonella infection is related to a disparity in the permissivity of cells arrested in G1 and G2/M, due to their intrinsic characteristics.
    Keywords:  Autophagy; G1 arrest; Salmonella; Salmonella cytosolic replication; Salmonella-containing vacuole; cell cycle; endolysosomal trafficking; type III secretion system
    DOI:  https://doi.org/10.1080/15548627.2021.1999561
  13. Cell Death Dis. 2021 Nov 18. 12(12): 1092
    Molecular and histological traits of reduced lysosomal acid lipase activity in the fatty liver.
    Simone Carotti, Daniele Lettieri-Barbato, Fiorella Piemonte, Sergio Ruggiero, Marco Rosina, Francesca Zalfa, Maria Zingariello, Francesca Arciprete, Francesco Valentini, Maria Francesconi, Jessica D'Amico, Antonio De Vincentis, Andrea Baiocchini, Giuseppe Perrone, Raffaele Antonelli-Incalzi, Sergio Morini, Antonio Picardi, Katia Aquilano, Umberto Vespasiani-Gentilucci.
      Recent studies demonstrated reduced blood lysosomal acid lipase (LAL) activity in patients with nonalcoholic fatty liver disease (NAFLD). We aimed to verify hepatic LAL protein content and activity in in vitro and in vivo models of fat overload and in NAFLD patients. LAL protein content and activity were firstly evaluated in Huh7 cells exposed to high-glucose/high-lipid (HGHL) medium and in the liver of C57BL/6 mice fed with high-fat diet (HFD) for 4 and 8 months. LAL protein was also evaluated by immunohistochemistry in liver biopsies from 87 NAFLD patients and 10 controls, and correlated with hepatic histology. Huh7 cells treated with HGHL medium showed a significant reduction of LAL activity, which was consistent with reduced LAL protein levels by western blotting using an antibody towards the N-term of the enzyme. Conversely, antibodies towards the C-term of the enzyme evidenced LAL accumulation, suggesting a post-translational modification that masks the LAL N-term epitope and affects enzymatic activity. Indeed, we found a high rate of ubiquitination and extra-lysosomal localization of LAL protein in cells treated with HGHL medium. Consistent with these findings, inhibition of proteasome triggered dysfunctional LAL accumulation and affected LAL activity. Accumulation of ubiquitinated/dysfunctional LAL was also found in the liver of HFD fed mice. In NAFLD patients, hepatic levels of non-ubiquitinated/functional LAL were lower than in controls and inversely correlated with disease activity and some of the hallmarks of reduced LAL. Fat overload leads to LAL ubiquitination and impairs its function, possibly reducing hepatic fat disposal and promoting NAFLD activity.
    DOI:  https://doi.org/10.1038/s41419-021-04382-4
  14. Oxid Med Cell Longev. 2021 ;2021 6715758
    mTORC1 Activation in Chx10-Specific Tsc1 Knockout Mice Accelerates Retina Aging and Degeneration.
    Yu-Qing Rao, Yu-Tong Zhou, Wenchuan Zhou, Jia-Kai Li, Baojie Li, Jing Li.
      Age-associated decline in retina function is largely responsible for the irreversible vision deterioration in the elderly population. It is also an important risk factor for the development of degenerative and angiogenic diseases. However, the molecular mechanisms involved in the process of aging in the retina remain largely elusive. This study investigated the role of mTORC1 signaling in aging of the retina. We showed that mTORC1 was activated in old-aged retina, particularly in the ganglion cells. The role of mTORC1 activation was further investigated in Chx10-Cre;Tsc1fx/fx mouse (Tsc1-cKO). Activation of mTORC1 was found in bipolar and some of the ganglion and amacrine cells in the adult Tsc1-cKO retina. Bipolar cell hypertrophy and Müller gliosis were observed in Tsc1-cKO since 6 weeks of age. The abnormal endings of bipolar cell dendritic tips at the outer nuclear layer resembled that of the old-aged mice. Microglial cell activation became evident in 6-week-old Tsc1-cKO. At 5 months, the Tsc1-cKO mice exhibited advanced features of old-aged retina, including the expression of p16Ink4a and p21, expression of SA-β-gal in ganglion cells, decreased photoreceptor cell numbers, decreased electroretinogram responses, increased oxidative stress, microglial cell activation, and increased expression of immune and inflammatory genes. Inhibition of microglial cells by minocycline partially prevented photoreceptor cell loss and restored the electroretinogram responses. Collectively, our study showed that the activation of mTORC1 signaling accelerated aging of the retina by both cell autonomous and nonautonomous mechanisms. Our study also highlighted the role of microglia cells in driving the decline in retina function.
    DOI:  https://doi.org/10.1155/2021/6715758
  15. Elife. 2021 Nov 19. pii: e65910. [Epub ahead of print]10
    Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors.
    Dawei Wang, Zuodong Ye, Wenjie Wei, Jingting Yu, Lihong Huang, Hongmin Zhang, Jianbo Yue.
      Actin filaments (F-actin) have been implicated in various steps of endosomal trafficking, and the length of F-actin is controlled by actin capping proteins, such as CapZ, which is a stable heterodimeric protein complex consisting of a and β subunits. However, the role of these capping proteins in endosomal trafficking remains elusive. Here, we found that CapZ docks to endocytic vesicles via its C-terminal actin-binding motif. CapZ knockout significantly increases the F-actin density around immature early endosomes, and this impedes fusion between these vesicles, manifested by the accumulation of small endocytic vesicles in CapZ-knockout cells. CapZ also recruits several RAB5 effectors, such as Rabaptin-5, to RAB5-positive early endosomes via its N-terminal domain, and this further activates RAB5. Collectively, our results indicate that CapZ regulates endosomal trafficking by controlling actin density around early endosomes and recruiting RAB5 effectors.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.65910
  16. Proc Natl Acad Sci U S A. 2021 Nov 23. pii: e2100122118. [Epub ahead of print]118(47):
    Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization.
    Chendong Pan, Kalpita Banerjee, Guillermo L Lehmann, Dena Almeida, Katherine A Hajjar, Ignacio Benedicto, Zhichun Jiang, Roxana A Radu, David H Thompson, Enrique Rodriguez-Boulan, Marcelo M Nociari.
      Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.
    Keywords:  LMP; Lipofuscin; aging; lipid-bisretinoids; necroptosis
    DOI:  https://doi.org/10.1073/pnas.2100122118
  17. FEBS J. 2021 Nov 16.
    ATG2 and VPS13 proteins: Molecular highways transporting lipids to drive membrane expansion and organelle communication.
    David G McEwan, Kevin Ryan.
      Communication between organelles is an essential process that helps maintain cellular homeostasis and organelle contact sites have emerged recently as crucial mediators of this communication. The emergence of a class of molecular bridges that span the inter-organelle gaps has now been shown to direct the flow of lipid traffic from one lipid bilayer to another. One of the keys components of these molecular bridges is the presence of an N-terminal Chorein/VPS13 domain. This is an evolutionarily conserved domain present in multiple proteins within the endocytic and autophagy trafficking pathways. Herein, we discuss the current state-of-the-art of this class of proteins, focusing on the role of these lipid transporters in the autophagy and endocytic pathways. We discuss the recent biochemical and structural advances that have highlighted the essential role Chorein-N domain containing ATG2 proteins play in driving the formation of the autophagosome and how lipids are transported from the endoplasmic reticulum to the growing phagophore. We also consider the VPS13 proteins, their role in organelle contacts and the endocytic pathway and highlight how disease-causing mutations disrupt these contact sites. Finally, we open the door to discuss other Chorein_N domain containing proteins, for instance UHRF1BP1/1L, their role in disease and look towards prokaryote examples of Chorein_N-like domains. Taken together, recent advances have highlighted an exciting opportunity to delve deeper into inter-organelle communication and understand how lipids are transported between membrane bi-layers and how this process is disrupted in multiple diseases.
    Keywords:  ATG2; Autophagy; Chorein; Lipid transfer; UHRF1BP1; VPS13; endosome; organelle contacts
    DOI:  https://doi.org/10.1111/febs.16280
  18. Elife. 2021 Nov 17. pii: e69015. [Epub ahead of print]10
    mTOR-dependent translation drives tumor infiltrating CD8+ effector and CD4+ Treg cells expansion.
    Benedetta De Ponte Conti, Annarita Miluzio, Fabio Grassi, Sergio Abrignani, Stefano Biffo, Sara Ricciardi.
      We performed a systematic analysis of the translation rate of tumor-infiltrating lymphocytes (TILs) and the microenvironment inputs affecting it, both in humans and in mice. Measurement of puromycin incorporation, a proxy of protein synthesis, revealed an increase of translating CD4+ and CD8+ cells in tumors, compared to normal tissues. High translation levels are associated with phospho-S6 labeling downstream of mTORC1 activation, whereas low levels correlate with hypoxic areas, in agreement with data showing that T cell receptor stimulation and hypoxia act as translation stimulators and inhibitors, respectively. Additional analyses revealed the specific phenotype of translating TILs. CD8+ translating cells have enriched expression of IFN-γ and CD-39, and reduced SLAMF6, pointing to a cytotoxic phenotype. CD4+ translating cells are mostly regulatory T cells (Tregs) with enriched levels of CTLA-4 and Ki67, suggesting an expanding immunosuppressive phenotype. In conclusion, the majority of translationally active TILs is represented by cytotoxic CD8+ and suppressive CD4+ Tregs, implying that other subsets may be largely composed by inactive bystanders.
    Keywords:  CD4+; CD8+; Treg; cell biology; human; immunology; inflammation; mouse; translation
    DOI:  https://doi.org/10.7554/eLife.69015
  19. Oncogenesis. 2021 Nov 19. 10(11): 80
    Clathrin adapters AP-1 and GGA2 support expression of epidermal growth factor receptor for cell growth.
    Takefumi Uemura, Takehiro Suzuki, Naoshi Dohmae, Satoshi Waguri.
      The role of Golgi/endosome-localized clathrin adapters in the maintenance of steady-state cell surface epidermal growth factor receptor (EGFR) is not well known. Here, we show that EGFR associates preferentially with both AP-1 and GGA2 in vitro. AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation. Triple immunofluorescence microscopy and proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurred more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes. Biochemical recycling assay revealed that the depletion of AP-1 or GGA2 significantly suppressed EGFR recycling to the plasma membrane regardless of the EGF stimulation. Depletion of AP-1 or GGA2 also reduced cell contents of other tyrosine kinases, MET and ErbB4, and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model. Moreover, AP-1 was expressed in endosomes at higher levels in some cancer tissues. Collectively, these results suggest that AP-1 and GGA2 function in recycling endosomes to retrieve endocytosed EGFR, thereby sustaining its cell surface expression and, consequently, cancer cell growth.
    DOI:  https://doi.org/10.1038/s41389-021-00367-2
  20. Elife. 2021 Nov 15. pii: e69236. [Epub ahead of print]10
    The mesoscale organization of syntaxin 1A and SNAP25 is determined by SNARE-SNARE-interactions.
    Jasmin Mertins, Jérôme Finke, Ricarda Antonia Sies, Kerstin Rink, Jan Hasenauer, Thorsten Lang.
      SNARE proteins have been described as the effectors of fusion events in the secretory pathway more than two decades ago. The strong interactions between SNARE-domains are clearly important in membrane fusion, but it is unclear whether they are involved in any other cellular processes. Here, we analyzed two classical SNARE proteins, syntaxin 1A and SNAP25. Although they are supposed to be engaged in tight complexes, we surprisingly find them largely segregated in the plasma membrane. Syntaxin 1A only occupies a small fraction of the plasma membrane area. Yet, we find it is able to redistribute the far more abundant SNAP25 on the mesoscale by gathering crowds of SNAP25 molecules onto syntaxin-clusters in a SNARE-domain dependent manner. Our data suggests that SNARE-domain interactions are not only involved in driving membrane fusion on the nanoscale, but also play an important role in controlling the general organization of proteins on the mesoscale. Further, we propose this mechanisms preserves active syntaxin 1A-SNAP25 complexes at the plasma membrane.
    Keywords:  biochemistry; cell biology; chemical biology; rat
    DOI:  https://doi.org/10.7554/eLife.69236
  21. Cell Rep. 2021 Nov 16. pii: S2211-1247(21)01486-8. [Epub ahead of print]37(7): 110008
    Sterols lower energetic barriers of membrane bending and fission necessary for efficient clathrin-mediated endocytosis.
    Ruthellen H Anderson, Kem A Sochacki, Harika Vuppula, Brandon L Scott, Elizabeth M Bailey, Maycie M Schultz, Jason G Kerkvliet, Justin W Taraska, Adam D Hoppe, Kevin R Francis.
      Clathrin-mediated endocytosis (CME) is critical for cellular signal transduction, receptor recycling, and membrane homeostasis in mammalian cells. Acute depletion of cholesterol disrupts CME, motivating analysis of CME dynamics in the context of human disorders of cholesterol metabolism. We report that inhibition of post-squalene cholesterol biosynthesis impairs CME. Imaging of membrane bending dynamics and the CME pit ultrastructure reveals prolonged clathrin pit lifetimes and shallow clathrin-coated structures, suggesting progressive impairment of curvature generation correlates with diminishing sterol abundance. Sterol structural requirements for efficient CME include 3' polar head group and B-ring conformation, resembling the sterol structural prerequisites for tight lipid packing and polarity. Furthermore, Smith-Lemli-Opitz fibroblasts with low cholesterol abundance exhibit deficits in CME-mediated transferrin internalization. We conclude that sterols lower the energetic costs of membrane bending during pit formation and vesicular scission during CME and suggest that reduced CME activity may contribute to cellular phenotypes observed within disorders of cholesterol metabolism.
    Keywords:  7-dehydrocholesterol; Smith-Lemli-Opitz syndrome; cholesterol; clathrin-mediated endocytosis; lipid metabolism; membrane curvature; sterols
    DOI:  https://doi.org/10.1016/j.celrep.2021.110008
  22. Structure. 2021 Nov 15. pii: S0969-2126(21)00412-3. [Epub ahead of print]
    Nde1 is a Rab9 effector for loading late endosomes to cytoplasmic dynein motor complex.
    Yifan Zhang, Ziyue Chen, Fang Wang, Honghua Sun, Xueliang Zhu, Jianping Ding, Tianlong Zhang.
      Rab9 is mainly located on late endosomes and required for their intracellular transport to trans-Golgi network (TGN). The cytoplasmic dynein motor, together with its regulatory proteins Nde1/Ndel1 and Lis1, controls intracellular retrograde transport of membranous organelles along the microtubule network. How late endosomes are tethered to the microtubule-based motor dynein for their retrograde transport remains unclear. Here, we demonstrate that the guanosine triphosphate (GTP)-bound Rab9A/B specifically uses Nde1/Ndel1 as an effector to interact with the dynein motor complex. We determined the crystal structure of Rab9A-GTP in complex with the Rab9-binding region of Nde1. The functional roles of key residues involved in the Rab9A-Nde1 interaction are verified using biochemical and cell biology assays. Rab9A mutants unable to bind to Nde1 also failed to associate with dynein, Lis1, and dynactin. Therefore, Nde1 is a Rab9 effector that tethers Rab9-associated late endosomes to the dynein motor for their retrograde transport to the TGN.
    Keywords:  Nde1/Ndel1; Rab GTPase; Rab9A/B; crystal structure; effector; late endosome; microtube; motor dynein; small GTPase; vesicle trafficking
    DOI:  https://doi.org/10.1016/j.str.2021.10.013
  23. Proc Natl Acad Sci U S A. 2021 Nov 23. pii: e2110755118. [Epub ahead of print]118(47):
    The K63 deubiquitinase CYLD modulates autism-like behaviors and hippocampal plasticity by regulating autophagy and mTOR signaling.
    Elisa Colombo, Guilherme Horta, Mona K Roesler, Natascha Ihbe, Stuti Chhabra, Konstantin Radyushkin, Giovanni Di Liberto, Mario Kreutzfeldt, Sven Schumann, Jakob von Engelhardt, Doron Merkler, Christian Behl, Thomas Mittmann, Albrecht M Clement, Ari Waisman, Michael J Schmeisser.
      Nondegradative ubiquitin chains attached to specific targets via Lysine 63 (K63) residues have emerged to play a fundamental role in synaptic function. The K63-specific deubiquitinase CYLD has been widely studied in immune cells and lately also in neurons. To better understand if CYLD plays a role in brain and synapse homeostasis, we analyzed the behavioral profile of CYLD-deficient mice. We found that the loss of CYLD results in major autism-like phenotypes including impaired social communication, increased repetitive behavior, and cognitive dysfunction. Furthermore, the absence of CYLD leads to a reduction in hippocampal network excitability, long-term potentiation, and pyramidal neuron spine numbers. By providing evidence that CYLD can modulate mechanistic target of rapamycin (mTOR) signaling and autophagy at the synapse, we propose that synaptic K63-linked ubiquitination processes could be fundamental in understanding the pathomechanisms underlying autism spectrum disorder.
    Keywords:  CYLD; autism spectrum disorder; autophagy; mTOR signaling; synapse
    DOI:  https://doi.org/10.1073/pnas.2110755118
  24. PLoS Genet. 2021 Nov 15. 17(11): e1009893
    JNK and Yorkie drive tumor malignancy by inducing L-amino acid transporter 1 in Drosophila.
    Bojie Cong, Mai Nakamura, Yukari Sando, Takefumi Kondo, Shizue Ohsawa, Tatsushi Igaki.
      Identifying a common oncogenesis pathway among tumors with different oncogenic mutations is critical for developing anti-cancer strategies. Here, we performed transcriptome analyses on two different models of Drosophila malignant tumors caused by Ras activation with cell polarity defects (RasV12/scrib-/-) or by microRNA bantam overexpression with endocytic defects (bantam/rab5-/-), followed by an RNAi screen for genes commonly essential for tumor growth and malignancy. We identified that Juvenile hormone Inducible-21 (JhI-21), a Drosophila homolog of the L-amino acid transporter 1 (LAT1), is upregulated in these malignant tumors with different oncogenic mutations and knocking down of JhI-21 strongly blocked their growth and invasion. JhI-21 expression was induced by simultaneous activation of c-Jun N-terminal kinase (JNK) and Yorkie (Yki) in these tumors and thereby contributed to tumor growth and progression by activating the mTOR-S6 pathway. Pharmacological inhibition of LAT1 activity in Drosophila larvae significantly suppressed growth of RasV12/scrib-/- tumors. Intriguingly, LAT1 inhibitory drugs did not suppress growth of bantam/rab5-/- tumors and overexpression of bantam rendered RasV12/scrib-/- tumors unresponsive to LAT1 inhibitors. Further analyses with RNA sequencing of bantam-expressing clones followed by an RNAi screen suggested that bantam induces drug resistance against LAT1 inhibitors via downregulation of the TMEM135-like gene CG31157. Our observations unveil an evolutionarily conserved role of LAT1 induction in driving Drosophila tumor malignancy and provide a powerful genetic model for studying cancer progression and drug resistance.
    DOI:  https://doi.org/10.1371/journal.pgen.1009893
  25. Mol Metab. 2021 Nov 13. pii: S2212-8778(21)00248-9. [Epub ahead of print] 101393
    Impacts of essential amino acids on energy balance.
    Fei Xiao, Feifan Guo.
      BACKGROUND: Obesity develops due to an imbalance in energy homeostasis, wherein energy intake exceeds energy expenditure. Increasing evidence shows that manipulations of dietary protein and their component amino acids affect the energy balance, resulting in changes in fat mass and body weight. Amino acids are not only the building blocks of proteins but also serve as signals regulating multiple biological pathways.SCOPE OF REVIEW: We present the current available knowledge regarding the effects of dietary alterations of a single essential amino acid (EAA) on energy balance and relevant signaling mechanisms at both the central and peripheral levels. We also summarize the association between EAAs and obesity in humans and the clinical use of modifying the dietary EAA composition for therapeutic intervention in obesity. Finally, we describe similar mechanisms underlying diets varying in protein levels and diets altered of a single EAA. This review will expand the understanding of the contribution of protein and amino acids to energy balance control, which could be helpful in the discovery of new therapeutic approaches for obesity and related diseases.
    MAJOR CONCLUSIONS: Changes in circulating EAA levels, particularly increased branched-chain amino acids (BCAAs), have been observed in obese human and animal models. Alterations in dietary EAA intake can lead to improvements in fat and weight loss in rodents, and each has its distinct mechanism. For example, leucine deprivation increases energy expenditure and reduces food intake and fat mass, primarily through regulation of the general control nonderepressible 2 (GCN2) and mammalian target of rapamycin (mTOR) signaling. Methionine restriction by 80 % decreases fat mass and body weight while developing hyperphagia, mainly via fibroblast growth factor (FGF) 21 signaling. Some effects of diets with different protein levels on energy homeostasis are mediated by similar mechanisms. However, reports on the effects and underlying mechanisms of dietary EAA imbalances on human body weight are few, and more investigations are needed.
    Keywords:  FGF21; GCN2; body weight; energy balance; essential amino acid; mTOR; protein
    DOI:  https://doi.org/10.1016/j.molmet.2021.101393
  26. ACS Chem Biol. 2021 Nov 15.
    Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs).
    Po-Han Chen, Zhenyi Hu, Elvira An, Ifunanya Okeke, Sijin Zheng, Xuanmeng Luo, Angela Gong, Saul Jaime-Figueroa, Craig M Crews.
      Protein phosphorylation, which regulates many critical aspects of cell biology, is dynamically governed by kinases and phosphatases. Many diseases are associated with dysregulated hyperphosphorylation of critical proteins, such as retinoblastoma protein in cancer. Although kinase inhibitors have been widely applied in the clinic, growing evidence of off-target effects and increasing drug resistance prompts the need to develop a new generation of drugs. Here, we propose a proof-of-concept study of phosphorylation targeting chimeras (PhosTACs). Similar to PROTACs in their ability to induce ternary complexes, PhosTACs focus on recruiting a Ser/Thr phosphatase to a phosphosubstrate to mediate its dephosphorylation. However, distinct from PROTACs, PhosTACs can uniquely provide target gain-of-function opportunities to manipulate protein activity. In this study, we applied a chemical biology approach to evaluate the feasibility of PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to protein substrates such as PDCD4 and FOXO3a for targeted protein dephosphorylation. For FOXO3a, this dephosphorylation resulted in the transcriptional activation of a FOXO3a-responsive reporter gene.
    DOI:  https://doi.org/10.1021/acschembio.1c00693
  27. Front Cell Dev Biol. 2021 ;9 760023
    Editorial: The Role of Different Subcellular Organelles in DNA Damage Response.
    Guoping Zhao.
      
    Keywords:  DNA damage repair; crosstalk; lysosomes; mitochondria; subcellular organelles
    DOI:  https://doi.org/10.3389/fcell.2021.760023
  28. Front Cell Dev Biol. 2021 ;9 690306
    Targeting Phosphatases and Kinases: How to Checkmate Cancer.
    Alice Turdo, Caterina D'Accardo, Antonino Glaviano, Gaetana Porcelli, Cristina Colarossi, Lorenzo Colarossi, Marzia Mare, Naida Faldetta, Chiara Modica, Giuseppe Pistone, Maria Rita Bongiorno, Matilde Todaro, Giorgio Stassi.
      Metastatic disease represents the major cause of death in oncologic patients worldwide. Accumulating evidence have highlighted the relevance of a small population of cancer cells, named cancer stem cells (CSCs), in the resistance to therapies, as well as cancer recurrence and metastasis. Standard anti-cancer treatments are not always conclusively curative, posing an urgent need to discover new targets for an effective therapy. Kinases and phosphatases are implicated in many cellular processes, such as proliferation, differentiation and oncogenic transformation. These proteins are crucial regulators of intracellular signaling pathways mediating multiple cellular activities. Therefore, alterations in kinases and phosphatases functionality is a hallmark of cancer. Notwithstanding the role of kinases and phosphatases in cancer has been widely investigated, their aberrant activation in the compartment of CSCs is nowadays being explored as new potential Achille's heel to strike. Here, we provide a comprehensive overview of the major protein kinases and phosphatases pathways by which CSCs can evade normal physiological constraints on survival, growth, and invasion. Moreover, we discuss the potential of inhibitors of these proteins in counteracting CSCs expansion during cancer development and progression.
    Keywords:  cancer stem cell; kinase; phosphatase; phosphatase and kinase inhibitors; targeted therapies
    DOI:  https://doi.org/10.3389/fcell.2021.690306
  29. Nat Commun. 2021 Nov 15. 12(1): 6604
    Bi-directional ribosome scanning controls the stringency of start codon selection.
    Yifei Gu, Yuanhui Mao, Longfei Jia, Leiming Dong, Shu-Bing Qian.
      The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5'→3' migration of the pre-initiation complex (PIC) along the 5' UTR. In probing translation initiation from ultra-short 5' UTR, we report that an AUG triplet near the 5' end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5' end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5' end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.
    DOI:  https://doi.org/10.1038/s41467-021-26923-3
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