bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒11‒22
38 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. Nature. 2020 Nov 18.
    Adelmann CH, Traunbauer AK, Chen B, Condon KJ, Chan SH, Kunchok T, Lewis CA, Sabatini DM.
      Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome-the organelle, related to the lysosome, that synthesizes pigment-but have unclear functions1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3,4. We find that MFSD12 is required to maintain normal levels of cystine-the oxidized dimer of cysteine-in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7-9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes.
    DOI:  https://doi.org/10.1038/s41586-020-2937-x
  2. Autophagy. 2020 Nov 19.
    Fraiberg M, Tamim-Yecheskel BC, Kokabi K, Subic N, Heimer G, Eck F, Nalbach K, Behrends C, Ben-Zeev B, Shatz O, Elazar Z.
      TECPR2 (tectonin beta-propeller repeat containing 2) is a large, multi-domain protein comprised of an amino-terminal WD domain, a middle unstructured region and a carboxy-terminal TEPCR domain comprises of six TECPR repeats followed by a functional LIR motif. Human TECPR2 mutations are linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder. Here we show that basal macroautophagic/autophagic flux is impaired in SPG49 patient fibroblasts in the form of accumulated autophagosomes. Ectopic expression of either full length TECPR2 or the TECPR domain rescued autophagy in patient fibroblasts in a LIR-dependent manner. Moreover, this domain is recruited to the cytosolic leaflet of autophagosomal and lysosomal membranes in a LIR- and VAMP8-dependent manner, respectively. These findings provide evidence for a new role of the TECPR domain in particular, and TECPR2 in general, in lysosomal targeting of autophagosomes via association with Atg8-family proteins on autophagosomes and VAMP8 on lysosomes.
    Keywords:  SPG49; TECPR2; autophagy; lysosome; neurodegeneration
    DOI:  https://doi.org/10.1080/15548627.2020.1852727
  3. FEBS Open Bio. 2020 Nov 18.
    Doray B, Liu L, Lee WS, Jennings BC, Kornfeld S.
      The Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral puncta to the trans-Golgi network. In comparison, GNPTAB-/- HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple knock-out cells is mediated by AP-1.
    Keywords:  AP-1; CI-MPR; GGA1; GGA2; GGA3; cathepsin D sorting
    DOI:  https://doi.org/10.1002/2211-5463.13040
  4. EMBO J. 2020 Nov 20. e103661
    Villari G, Enrico Bena C, Del Giudice M, Gioelli N, Sandri C, Camillo C, Fiorio-Pla A, Bosia C, Serini G.
      Although subcellular positioning of endosomes significantly impacts on their functions, the molecular mechanisms governing the different steady-state distribution of early endosomes (EEs) and late endosomes (LEs)/lysosomes (LYs) in peripheral and perinuclear eukaryotic cell areas, respectively, are still unsolved. We unveil that such differences arise because, while LE retrograde transport depends on the dynein microtubule (MT) motor only, the one of EEs requires the cooperative antagonism of dynein and kinesin-14 KIFC1, a MT minus end-directed motor involved in cancer progression. Mechanistically, the Ser-x-Ile-Pro (SxIP) motif-mediated interaction of the endoplasmic reticulum transmembrane protein stromal interaction molecule 1 (STIM1) with the MT plus end-binding protein 1 (EB1) promotes its association with the p150Glued subunit of the dynein activator complex dynactin and the distinct location of EEs and LEs/LYs. The peripheral distribution of EEs requires their p150Glued-mediated simultaneous engagement with dynein and SxIP motif-containing KIFC1, via HOOK1 and HOOK3 adaptors, respectively. In sum, we provide evidence that distinct minus end-directed MT motor systems drive the differential transport and subcellular distribution of EEs and LEs in mammalian cells.
    Keywords:  endoplasmic reticulum; motor; movement; traffic; vesicles
    DOI:  https://doi.org/10.15252/embj.2019103661
  5. FASEB J. 2020 Nov 15.
    Arora K, Liyanage P, Zhong Q, Naren AP.
      Autophagy, a cellular stress response to starvation and bacterial infection, is executed by double-membrane-bound organelles called autophagosomes. Autophagosomes transfer cytosolic material to acidified lysosomes for degradation following soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-dependent fusion processes. Many of the autophagy-related disorders stem from defective end-step proteolysis inside lysosomes. The role of epithelial cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel has been argued to be critical for efficient lysosomal clearance; however, its context to autophagic clearance and the underlying mechanism is poorly defined. Here, we report that syntaxin17 (Stx17), an autophagic SNARE protein interacts with CFTR under nutritional stress and bacterial infection and incorporates it into mature autophagosomes to mediate an efficient lysosomal clearance. Lack of CFTR function and Stx17 and loss of CFTR-Stx17 interaction impairs bacterial clearance. We discover a specialized role of the Stx17-CFTR protein complex that is critical to prevent defective autophagy as has been the reported scenario in CF airway epithelial cells, infectious diseases, and lysosomal clearance disorders.
    Keywords:  SNARE proteins; autophagy; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; lysosomal clearance
    DOI:  https://doi.org/10.1096/fj.201903210R
  6. Cancers (Basel). 2020 Nov 16. pii: E3388. [Epub ahead of print]12(11):
    Min KJ, Kwon TK.
      FTY720, a sphingosine-1-phosphate (S1P) receptor modulator, is a synthetic compound produced by the modification of a metabolite from I. sinclairii. Here, we found that FTY720 induced non-apoptotic cell death in human glioma cells (U251MG, U87MG, and U118MG). FTY720 (10 µM) dramatically induced cytoplasmic vacuolation in glioma cells. However, FTY720-mediated vacuolation and cell death are not associated with autophagy. Genetic or pharmacological inhibition of autophagy did not inhibit FTY720-induced cell death. Herein, we detected that FTY720-induced cytoplasmic vacuoles were stained with lysotracker red, and FTY720 induced lysosomal membrane permeabilization (LMP). Interestingly, cathepsin inhibitors (E64D and pepstatin A) and ectopic expression of heat shock protein 70 (HSP70), which is an endogenous inhibitor of LMP, markedly inhibited FTY720-induced cell death. Our results demonstrated that FTY720 induced non-apoptotic cell death via the induction of LMP in human glioma cells.
    Keywords:  FTY720; LMP; cathepsins; glioma; non-apoptotic cell death
    DOI:  https://doi.org/10.3390/cancers12113388
  7. Intern Med J. 2020 Nov;50 Suppl 4 5-27
    Peters H, Ellaway C, Nicholls K, Reardon K, Szer J.
      Lysosomal storage diseases (LSD) comprise a rare and heterogeneous group of nearly 50 heritable metabolic disorders caused by mutations in proteins critical for cellular lysosomal function. Defects in the activity of these proteins in multiple organs leads to progressive intra-lysosomal accumulation of specific substrates, resulting in disruption of cellular functions, extracellular inflammatory responses, tissue damage and organ dysfunction. The classification and clinical presentation of different LSD are dependent on the type of accumulated substrate. Some clinical signs and symptoms are common across multiple LSD, while others are more specific to a particular syndrome. Due to the rarity and wide clinical diversity of LSD, identification and diagnosis can be challenging, and in many cases diagnosis is delayed for months or years. Treatments, such as enzyme replacement therapy, haemopoietic stem cell transplantation and substrate reduction therapy, are now available for some of the LSD. For maximum effect, therapy must be initiated prior to the occurrence of irreversible tissue damage, highlighting the importance of prompt diagnosis. Herein, we discuss the clinical presentation, diagnosis and treatment of four of the treatable LSD: Gaucher disease, Fabry disease, Pompe disease, and two of the mucopolysaccharidoses (I and II). For each disease, we present illustrative case studies to help increase awareness of their clinical presentation and possible treatment outcomes.
    Keywords:  Fabry; Gaucher; Pompe; lysosomal storage disease; mucopolysaccharidosis
    DOI:  https://doi.org/10.1111/imj.15100
  8. Am J Hum Genet. 2020 Nov 13. pii: S0002-9297(20)30401-8. [Epub ahead of print]
    Polovitskaya MM, Barbini C, Martinelli D, Harms FL, Cole FS, Calligari P, Bocchinfuso G, Stella L, Ciolfi A, Niceta M, Rizza T, Shinawi M, Sisco K, Johannsen J, Denecke J, Carrozzo R, Wegner DJ, Kutsche K, Tartaglia M, Jentsch TJ.
      Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.
    Keywords:  anion/proton antiport; channelopathy; chloride channel; chloride/proton exchange; copper metabolism; gain of function; gating glutamate; luminal pH; neurogenic bladder; vacuole fusion
    DOI:  https://doi.org/10.1016/j.ajhg.2020.11.004
  9. JIMD Rep. 2020 Nov;56(1): 46-57
    Chen OCW, Colaco A, Davis LC, Kiskin FN, Farhat NY, Speak AO, Smith DA, Morris L, Eden E, Tynan P, Churchill GC, Galione A, Porter FD, Platt FM.
      Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in either NPC1 (95% of cases) or NPC2. Reduced late endosome/lysosome calcium (Ca2+) levels and the accumulation of unesterified cholesterol and sphingolipids within the late endocytic system characterize this disease. We previously reported impaired lysosome-related organelle (LRO) function in Npc1 -/- Natural Killer cells; however, the potential contribution of impaired acid compartment Ca2+ flux and LRO function in other cell types has not been determined. Here, we investigated LRO function in NPC1 disease platelets. We found elevated numbers of circulating platelets, impaired platelet aggregation and prolonged bleeding times in a murine model of NPC1 disease. Electron microscopy revealed abnormal ultrastructure in murine platelets, consistent with that seen in a U18666A (pharmacological inhibitor of NPC1) treated megakaryocyte cell line (MEG-01) exhibiting lipid storage and acidic compartment Ca2+ flux defects. Furthermore, platelets from NPC1 patients across different ages were found to cluster at the lower end of the normal range when platelet numbers were measured and had platelet volumes that were clustered at the top of the normal range. Taken together, these findings highlight the role of acid compartment Ca2+ flux in the function of platelet LROs.
    Keywords:  Niemann‐Pick disease type C; calcium (Ca2+); lysosome; lysosome‐related organelle
    DOI:  https://doi.org/10.1002/jmd2.12148
  10. Front Oncol. 2020 ;10 562196
    Varalda M, Antona A, Bettio V, Roy K, Vachamaram A, Yellenki V, Massarotti A, Baldanzi G, Capello D.
      Background and Purpose: Drug repositioning is a promising strategy for discovering new therapeutic strategies for cancer therapy. We investigated psychotropic drugs for their antitumor activity because of several epidemiological studies reporting lower cancer incidence in individuals receiving long term drug treatment. Experimental Approach: We investigated 27 psychotropic drugs for their cytotoxic activity in colorectal carcinoma, glioblastoma and breast cancer cell lines. Consistent with the cationic amphiphilic structure of the most cytotoxic compounds, we investigated their effect on mitochondrial and lysosomal compartments. Results: Penfluridol, ebastine, pimozide and fluoxetine, fluspirilene and nefazodone showed significant cytotoxicity, in the low micromolar range, in all cell lines tested. In MCF7 cells these drugs caused mitochondrial membrane depolarization, increased the acidic vesicular compartments and induced phospholipidosis. Both penfluridol and spiperone induced AMPK activation and autophagy. Neither caspase nor autophagy inhibitors rescued cells from death induced by ebastine, fluoxetine, fluspirilene and nefazodone. Treatment with 3-methyladenine partially rescued cell death induced by pimozide and spiperone, whereas enhanced the cytotoxic activity of penfluridol. Conversely, inhibition of lysosomal cathepsins significantly reduced cell death induced by ebastin, penfluridol, pimozide, spiperone and mildly in fluoxetine treated cells. Lastly, Spiperone cytotoxicity was restricted to colorectal cancer and breast cancer and caused apoptotic cell death in MCF7 cells. Conclusions: The cytotoxicity of psychotropic drugs with cationic amphiphilic structures relied on simultaneous mitochondrial and lysosomal disruption and induction of cell death that not necessarily requires apoptosis. Since dual targeting of lysosomes and mitochondria constitutes a new promising therapeutic approach for cancer, particularly those in which the apoptotic machinery is defective, these data further support their clinical development for cancer therapy.
    Keywords:  autophagy; cancer; cationic amphiphilic drugs (CADs); lysosomotropism; psychotropic drug; repositioning
    DOI:  https://doi.org/10.3389/fonc.2020.562196
  11. J Gen Physiol. 2021 Jan 04. pii: e202012583. [Epub ahead of print]153(1):
    Pusch M, Zifarelli G.
      ClC-7 is a lysosomal 2 Cl-/1 H+ antiporter of the CLC protein family, which comprises Cl- channels and other Cl-/H+ antiporters. Mutations in ClC-7 and its associated β subunit Ostm1 lead to osteopetrosis and lysosomal storage disease in humans and mice. Previous studies on other mammalian CLC transporters showed that mutations of a conserved, intracellularly located glutamate residue, the so-called proton glutamate, abolish steady-state transport activity but increase transient capacitive currents associated with partial reactions of the transport cycle. In contrast, we observed large, transient capacitive currents for the wild-type ClC-7, which depend on external pH and internal, but not external, Cl-. Very similar transient currents were observed for the E312A mutant of the proton glutamate. Interestingly, and unlike in other mammalian CLC transporters investigated so far, the E312A mutation strongly reduces, but does not abolish, stationary transport currents, potentially explaining the intermediate phenotype observed in the E312A mouse line.
    DOI:  https://doi.org/10.1085/jgp.202012583
  12. Sci Rep. 2020 Nov 18. 10(1): 20125
    Kunishige R, Mizoguchi M, Tsubouchi A, Hanaoka K, Miura Y, Kurosu H, Urano Y, Kuro-O M, Murata M.
      Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.
    DOI:  https://doi.org/10.1038/s41598-020-77308-3
  13. Cell Metab. 2020 Nov 11. pii: S1550-4131(20)30591-X. [Epub ahead of print]
    Su W, Chapman NM, Wei J, Zeng H, Dhungana Y, Shi H, Saravia J, Zhou P, Long L, Rankin S, Kc A, Vogel P, Chi H.
      Effector regulatory T (eTreg) cells are essential for immune tolerance and depend upon T cell receptor (TCR) signals for generation. The immunometabolic signaling mechanisms that promote the differentiation and maintenance of eTreg cells remain unclear. Here, we show that isoprenoid-dependent posttranslational lipid modifications dictate eTreg cell accumulation and function by intersecting with TCR-induced intracellular signaling. We find that isoprenoids are essential for activated Treg cell suppressive activity, and Treg cell-specific deletion of the respective farnesylation- and geranylgeranylation-promoting enzymes Fntb or Pggt1b leads to the development of fatal autoimmunity, associated with reduced eTreg cell accumulation. Mechanistically, Fntb promotes eTreg cell maintenance by regulating mTORC1 activity and ICOS expression. In contrast, Pggt1b acts as a rheostat of TCR-dependent transcriptional programming and Rac-mediated signaling for establishment of eTreg cell differentiation and immune tolerance. Therefore, our results identify bidirectional metabolic signaling, specifically between immunoreceptor signaling and metabolism-mediated posttranslational lipid modifications, for the differentiation and maintenance of eTreg cells.
    Keywords:  Fntb; Pggt1b; Treg cells; immunometabolism; mTOR; protein prenylation
    DOI:  https://doi.org/10.1016/j.cmet.2020.10.022
  14. Front Cell Dev Biol. 2020 ;8 540726
    Pierzynowska K, Gaffke L, Jankowska E, Rintz E, Witkowska J, Wieczerzak E, Podlacha M, Węgrzyn G.
      In this study, we have asked whether proteasome composition and function are affected in cells derived from patients suffering from all types of mucopolysaccharidosis (MPS), an inherited metabolic disease caused by accumulation of undegraded glycosaminoglycans (GAGs). Moreover, we have tested if genistein, a small molecule proposed previously as a potential therapeutic agent in MPS, can modulate proteasomes, which might shed a new light on the molecular mechanisms of action of this isoflavone as a potential drug for macromolecule storage diseases. Significant changes in expression of various proteasome-linked genes have been detected during transcriptomic (RNA-seq) analyses in vast majority of MPS types. These results were corroborated by demonstration of increased proteasomal activities in MPS cells. However, GAGs were not able to stimulate the 26S proteasome in vitro, suggesting that the observed activation in cells is indirect rather than arising from direct GAG-proteasome interactions. Genistein significantly reduced proteasomal activities in fibroblasts derived from patients suffering from all MPS types, while its effects on in vitro 26S proteasome activity were negligible. Unexpectedly, levels of many proteasomal subunits were increased in genistein-treated MPS cells. On the other hand, this ostensible discrepancy between results of experiments designed for estimation of effects of genistein on proteasome activities and abundance of proteasomal subunits can be explained by demonstration that in the presence of this isoflavone, levels of ubiquitinated proteins were decreased. The genistein-mediated reduction of proteasomal activities might have beneficial effects in cells of MPS patients due to potential increasing of residual activities of defective lysosomal enzymes which would otherwise be subjected to efficient ubiquitination and proteasomal degradation as misfolded proteins. These results indicate another activity of genistein (apart from previously demonstrated reduction of GAG synthesis efficiency, stimulation of lysosomal biogenesis, and activation of the autophagy process) which can be beneficial in the use of this small molecule in treatment of MPS.
    Keywords:  genistein; mucopolysaccharidosis; proteasome; transcriptomics; ubiquitinated proteins
    DOI:  https://doi.org/10.3389/fcell.2020.540726
  15. Proc Natl Acad Sci U S A. 2020 Nov 16. pii: 202016959. [Epub ahead of print]
    Griffin CS, Alvarado MG, Yamasaki E, Drumm BT, Krishnan V, Ali S, Nagle EM, Sanders KM, Earley S.
      TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1 -/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1 -/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1 -/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1 -/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.
    Keywords:  calcium signaling; endolysosomes; ion channels; lower urinary tract; superresolution microscopy
    DOI:  https://doi.org/10.1073/pnas.2016959117
  16. Mov Disord. 2020 Nov 21.
    García-Sanz P, M F G Aerts J, Moratalla R.
      Parkinson's disease (PD) is a progressive neurodegenerative disease where dopaminergic neurons in the substantia nigra are lost, resulting in a decrease in striatal dopamine and, consequently, motor control. Dopaminergic degeneration is associated with the appearance of Lewy bodies, which contain membrane structures and proteins, including α-synuclein (α-Syn), in surviving neurons. PD displays a multifactorial pathology and develops from interactions between multiple elements, such as age, environmental conditions, and genetics. Mutations in the GBA1 gene represent one of the major genetic risk factors for PD. This gene encodes an essential lysosomal enzyme called β-glucocerebrosidase (GCase), which is responsible for degrading the glycolipid glucocerebroside into glucose and ceramide. GCase can generate glucosylated cholesterol via transglucosylation and can also degrade the sterol glucoside. Although the molecular mechanisms that predispose an individual to neurodegeneration remain unknown, the role of cholesterol in PD pathology deserves consideration. Disturbed cellular cholesterol metabolism, as reflected by accumulation of lysosomal cholesterol in GBA1-associated PD cellular models, could contribute to changes in lipid rafts, which are necessary for synaptic localization and vesicle cycling and modulation of synaptic integrity. α-Syn has been implicated in the regulation of neuronal cholesterol, and cholesterol facilitates interactions between α-Syn oligomers. In this review, we integrate the results of previous studies and describe the cholesterol landscape in cellular homeostasis and neuronal function. We discuss its implication in α-Syn and Lewy body pathophysiological mechanisms underlying PD, focusing on the role of GCase and cholesterol. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC. on behalf of International Parkinson and Movement Disorder Society.
    Keywords:  autophagy; glycosphingolipid; lipid storage diseaseslysosomesmultilamellar bodiesneurodegeneration
    DOI:  https://doi.org/10.1002/mds.28396
  17. Front Cell Dev Biol. 2020 ;8 597608
    Sneeggen M, Guadagno NA, Progida C.
      Tumor progression is a complex process consisting of several steps characterized by alterations in cellular behavior and morphology. These steps include uncontrolled cell division and proliferation, invasiveness and metastatic ability. Throughout these phases, cancer cells encounter a changing environment and a variety of metabolic stress. To meet their needs for energy while they proliferate and survive in their new environment, tumor cells need to continuously fine-tune their metabolism. The connection between intracellular transport and metabolic reprogramming during cancer progression is emerging as a central process of cellular adaptation to these changes. The trafficking of proteolytic enzymes, surface receptors, but also the regulation of downstream pathways, are all central to cancer progression. In this review, we summarize different hallmarks of cancer with a special focus on the role of intracellular trafficking in cell proliferation, epithelial to mesenchymal transition as well as invasion. We will further emphasize how intracellular trafficking contributes to the regulation of energy consumption and metabolism during these steps of cancer progression.
    Keywords:  cancer cell metabolism; cell proliferation; epithelial to mesenchymal transition; invasion; membrane trafficking
    DOI:  https://doi.org/10.3389/fcell.2020.597608
  18. Cell Chem Biol. 2020 Nov 19. pii: S2451-9456(20)30429-3. [Epub ahead of print]27(11): 1329-1331
    Turocy T, Crawford JM.
      Chemotherapeutic treatments are frequently impeded by the development of multidrug resistance (MDR). In this issue of Cell Chemical Biology, Wang et al. (2020) identify the natural product verucopeptin as having therapeutic potential toward MDR cancer cell types by targeting v-ATPase and mTORC1 signaling.
    DOI:  https://doi.org/10.1016/j.chembiol.2020.10.013
  19. J Biol Chem. 2020 Nov 20. pii: jbc.RA120.014682. [Epub ahead of print]
    Chen G, Zhou G, Lotvola A, Granneman JG, Wang J.
      ABHD5 is an essential coactivator of ATGL, the rate-limiting triglyceride (TG) lipase in many cell types. Importantly, ABHD5 also functions as a tumor suppressor, and ABHD5 mRNA expression levels correlate with patient survival for several cancers. Nevertheless, the mechanisms involved in ABHD5-dependent tumor suppression are not known. We found that overexpression of ABHD5 induces cell-cycle arrest at the G1 phase and causes growth retardation in a panel of prostate cancer cells. Transcriptomic profiling and biochemical analysis revealed that genetic or pharmacological activation of lipolysis by ABHD5 potently inhibits mTORC1 signaling, leading to a significant downregulation of protein synthesis. Mechanistically, we found that ABHD5 elevates intracellular AMP content, which activates AMPK, leading to inhibition of mTORC1. Interestingly, ABHD5-dependent suppression of mTORC1 was abrogated by pharmacological inhibition of DGAT1 or DGAT2, isoenzymes that re-esterify fatty acids in a process that consumes ATP. Collectively, this study maps out a novel molecular pathway crucial for limiting cancer cell proliferation, in which ABHD5-mediated lipolysis creates an energy-consuming futile cycle between TG hydrolysis and resynthesis, leading to inhibition of mTORC1 and cancer cell growth arrest.
    Keywords:  lipid signaling; lipolysis; metabolic regulation; tumor cell biology; tumor metabolism
    DOI:  https://doi.org/10.1074/jbc.RA120.014682
  20. Front Genet. 2020 ;11 582368
    Li S, Ren C, Stone C, Chandra A, Xu J, Li N, Han C, Ding Y, Ji X, Shao G.
      Hypoxic/ischemic preconditioning (HPC/IPC) is an innate neuroprotective mechanism in which a number of endogenous molecules are known to be involved. Tuberous sclerosis complex 1 (TSC1), also known as hamartin, is thought to be one such molecule. It is also known that hamartin is involved as a target in the rapamycin (mTOR) signaling pathway, which functions to integrate a variety of environmental triggers in order to exert control over cellular metabolism and homeostasis. Understanding the role of hamartin in ischemic/hypoxic neuroprotection will provide a novel target for the treatment of hypoxic-ischemic disease. Therefore, the proposed molecular mechanisms of this neuroprotective role and its preconditions are reviewed in this paper, with emphases on the mTOR pathway and the relationship between the expression of hamartin and DNA methylation.
    Keywords:  TSC1; hamartin; hypoxia; ischemia; neuroprotection
    DOI:  https://doi.org/10.3389/fgene.2020.582368
  21. Front Med (Lausanne). 2020 ;7 576221
    Shaimardanova AA, Chulpanova DS, Solovyeva VV, Mullagulova AI, Kitaeva KV, Allegrucci C, Rizvanov AA.
      Metachromatic leukodystrophy is a lysosomal storage disease, which is characterized by damage of the myelin sheath that covers most of nerve fibers of the central and peripheral nervous systems. The disease occurs due to a deficiency of the lysosomal enzyme arylsulfatase A (ARSA) or its sphingolipid activator protein B (SapB) and it clinically manifests as progressive motor and cognitive deficiency. ARSA and SapB protein deficiency are caused by mutations in the ARSA and PSAP genes, respectively. The severity of clinical course in metachromatic leukodystrophy is determined by the residual ARSA activity, depending on the type of mutation. Currently, there is no effective treatment for this disease. Clinical cases of bone marrow or cord blood transplantation have been reported, however the therapeutic effectiveness of these methods remains insufficient to prevent aggravation of neurological disorders. Encouraging results have been obtained using gene therapy for delivering the wild-type ARSA gene using vectors based on various serotypes of adeno-associated viruses, as well as using mesenchymal stem cells and combined gene-cell therapy. This review discusses therapeutic strategies for the treatment of metachromatic leukodystrophy, as well as diagnostic methods and modeling of this pathology in animals to evaluate the effectiveness of new therapies.
    Keywords:  arylsulfatase A; bone marrow transplantation; gene therapy; lysosomal storage diseases; mesenchymal stem cells; metachromatic leukodystrophy; replacement therapy; sulfatide
    DOI:  https://doi.org/10.3389/fmed.2020.576221
  22. Front Cell Dev Biol. 2020 ;8 594464
    Nałęcz KA.
      Cancer cells are characterized by quick growth and proliferation, demanding constant supply of various nutrients. Several plasma membrane transporters delivering such compounds are upregulated in cancer. Solute carrier family 6 member 14 (SLC6A14), known as amino acid transporter B0,+ (ATB0,+) transports all amino acids with exception of the acidic ones: aspartate and glutamate. Its malfunctioning is correlated with several pathological states and it is upregulated in solid tumors. The high expression of SLC6A14 is prognostic and unfavorable in pancreatic cancer, while in breast cancer it is expressed in estrogen receptor positive cells. As many plasma membrane transporters it resides in endoplasmic reticulum (ER) membrane after translation before further trafficking through Golgi to the cell surface. Transporter exit from ER is strictly controlled. The proper folding of SLC6A14 was shown to be controlled from the cytoplasmic side by heat shock proteins, further exit from ER and formation of coatomer II (COPII) coated vesicles depends on specific interaction with COPII cargo-recognizing subunit SEC24C, phosphorylated by kinase AKT. Inhibition of heat shock proteins, known to be upregulated in cancer, directs SLC6A14 to degradation. Targeting proteins regulating SLC6A14 trafficking is proposed as an additional pharmacological treatment of cancer.
    Keywords:  AKT 3; SLC6A14; amino acid transporter; cancer; estrogen receptor; heat shock proteins; trafficking
    DOI:  https://doi.org/10.3389/fcell.2020.594464
  23. Biomolecules. 2020 Nov 14. pii: E1553. [Epub ahead of print]10(11):
    Boutouja F, Platta HW.
      The changing accessibility of nutrient resources induces the reprogramming of cellular metabolism in order to adapt the cell to the altered growth conditions. The nutrient-depending signaling depends on the kinases mTOR (mechanistic target of rapamycin), which is mainly activated by nitrogen-resources, and PKA (protein kinase A), which is mainly activated by glucose, as well as both of their associated factors. These systems promote protein synthesis and cell proliferation, while they inhibit degradation of cellular content by unselective bulk autophagy. Much less is known about their role in selective autophagy pathways, which have a more regulated cellular function. Especially, we were interested to analyse the central Ras2-module of the PKA-pathway in the context of peroxisome degradation. Yeast Ras2 is homologous to the mammalian Ras proteins, whose mutant forms are responsible for 33% of human cancers. In the present study, we were able to demonstrate a context-dependent role of Ras2 activity depending on the type of mTOR-inhibition and glucose-sensing situation. When mTOR was inhibited directly via the macrolide rapamycin, peroxisome degradation was still partially suppressed by Ras2, while inactivation of Ras2 resulted in an enhanced degradation of peroxisomes, suggesting a role of Ras2 in the inhibition of peroxisome degradation in glucose-grown cells. In contrast, the inhibition of mTOR by shifting cells from oleate-medium, which lacks glucose, to pexophagy-medium, which contains glucose and is limited in nitrogen, required Ras2-activity for efficient pexophagy, strongly suggesting that the role of Ras2 in glucose sensing-associated signaling is more important in this context than its co-function in mTOR-related autophagy-inhibition.
    Keywords:  Ras2; autophagy; mTOR; peroxisomes; pexophagy; rapamycin
    DOI:  https://doi.org/10.3390/biom10111553
  24. Mol Cell. 2020 Nov 05. pii: S1097-2765(20)30737-1. [Epub ahead of print]
    Wilfling F, Lee CW, Erdmann PS, Zheng Y, Sherpa D, Jentsch S, Pfander B, Schulman BA, Baumeister W.
      Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.
    Keywords:  Atg11; Atg8; Ede1; clathrin-mediated endocytosis; intrinsic autophagy receptor; liquid-liquid phase separation; selective autophagy
    DOI:  https://doi.org/10.1016/j.molcel.2020.10.030
  25. J Biol Chem. 2020 11 16. pii: jbc.REV120.011985. [Epub ahead of print]
    Shetty S, Varshney U.
      Protein synthesis is an energetically costly cellular activity. It is therefore important that the process of mRNA translation remains in excellent synchrony with cellular metabolism and its energy reserves. Unregulated translation could lead to the production of incomplete, mistranslated, or misfolded proteins, squandering the energy needed for cellular sustenance, and causing cytotoxicity. One-carbon metabolism (OCM), an integral part of cellular intermediary metabolism, produces a number of one-carbon unit intermediates (formyl, methylene, methenyl, methyl). These OCM intermediates are required for the production of amino acids like methionine, and biomolecules such as purines, thymidylate, and redox regulators. In this review, we discuss how OCM impacts the translation apparatus (composed of ribosome, tRNA, mRNA, and translation factors) and regulates crucial steps in protein synthesis. More specifically, we address how the OCM metabolites regulate the fidelity and rate of translation initiation in bacteria and eukaryotic organelles such as mitochondria. Modulation of the fidelity of translation initiation by OCM opens new avenues to understand alternative translation mechanisms involved in stress tolerance and drug resistance.
    Keywords:  3GC base pairs; RNA; RNA methylation; RNA methyltransferase; RNA modification; S-adenosylmethionine (SAM); folate; formylation; initiator tRNA; methylations; mitochondria; one-carbon metabolism; ribosome; ribosome heterogeneity
    DOI:  https://doi.org/10.1074/jbc.REV120.011985
  26. Mol Neurobiol. 2020 Nov 20.
    Chen YP, Gu XJ, Ou RW, Zhang LY, Hou YB, Liu KC, Cao B, Wei QQ, Song W, Zhao B, Wu Y, Cheng JQ, Shang HF.
      Recent genetic studies clearly indicate that variants in several lysosomal genes act as risk factors for idiopathic Parkinson's disease (PD). Variants in the co-activator of glucocerebrosidase gene (GBA) and the four active saposins (Sap A-D) which are encoded by the prosaposin gene (PSAP) are of particular interest; however, their genetic roles in PD are unknown. Whole-exome sequencing and Sanger sequencing were used to assess the genetic etiology of 400 autosomal dominant inherited PD (ADPD) and 300 sporadic PD (SPD) patients. Variants from public databases, including Genome Aggregation Database-East Asian (GnomAD_EAS) and Chinese Millionome Database (CMDB), were used as control groups. Burden analysis based on gene and domains level were performed to investigate the role of rare PSAP variants in PD. Six rare and likely pathogenic variants, located in the Sap A-D domains, were identified and accounted for 0.75% (3/400) of ADPD and 1.33% (4/300) of SPD in the Chinese population. Based on the gene or domain, burden analysis showed that damaging missense variants in SapC had statistical significance on the risk of developing PD. Interestingly, rs4747203, an intronic variant potentially linked to PSAP expression, was associated with reduced risk for PD (p = 8.6e-7 in GnomAD EAS and p = 0.002 in Chinese). Clinically, patients carrying the likely pathogenic variants presented typical PD motor symptoms and responded well to levodopa treatment. Six out of seven patients carrying the likely pathogenic variants of PSAP presented slow disease progression, and none of the patients developed cognitive impairment. Our study expands the spectrum of mutations associated with the risk of developing PD and enhances the understanding of the relationship of the clinical phenotype of PD with PSAP variants.
    Keywords:  Association analysis; Burden analysis; Parkinson’s disease; Prosaposin; Rare variants
    DOI:  https://doi.org/10.1007/s12035-020-02218-4
  27. Anal Chem. 2020 Nov 17.
    Frankenfield AM, Fernandopulle MS, Hasan S, Ward ME, Hao L.
      Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batches of APEX labeling and different APEX probes. We conducted a comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.
    DOI:  https://doi.org/10.1021/acs.analchem.0c03107
  28. EMBO J. 2020 Nov 20. e104532
    Ko CJ, Zhang L, Jie Z, Zhu L, Zhou X, Xie X, Gao T, Yang JY, Cheng X, Sun SC.
      Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.
    Keywords:  Peli1; T cell metabolism; antitumor immunity; mTORC1; ubiquitination
    DOI:  https://doi.org/10.15252/embj.2020104532
  29. Sci Transl Med. 2020 Nov 18. pii: eabc1492. [Epub ahead of print]12(570):
    Hsieh LS, Wen JH, Nguyen LH, Zhang L, Getz SA, Torres-Reveron J, Wang Y, Spencer DD, Bordey A.
      The causative link between focal cortical malformations (FCMs) and epilepsy is well accepted, especially among patients with focal cortical dysplasia type II (FCDII) and tuberous sclerosis complex (TSC). However, the mechanisms underlying seizures remain unclear. Using a mouse model of TSC- and FCDII-associated FCM, we showed that FCM neurons were responsible for seizure activity via their unexpected abnormal expression of the hyperpolarization-activated cyclic nucleotide-gated potassium channel isoform 4 (HCN4), which is normally not present in cortical pyramidal neurons after birth. Increasing intracellular cAMP concentrations, which preferentially affects HCN4 gating relative to the other isoforms, drove repetitive firing of FCM neurons but not control pyramidal neurons. Ectopic HCN4 expression was dependent on the mechanistic target of rapamycin (mTOR), preceded the onset of seizures, and was also found in diseased neurons in tissue resected from patients with TSC and FCDII. Last, blocking HCN4 channel activity in FCM neurons prevented epilepsy in the mouse model. These findings suggest that HCN4 play a main role in seizure and identify a cAMP-dependent seizure mechanism in TSC and FCDII. Furthermore, the unique expression of HCN4 exclusively in FCM neurons suggests that gene therapy targeting HCN4 might be effective in reducing seizures in FCDII or TSC.
    DOI:  https://doi.org/10.1126/scitranslmed.abc1492
  30. Biochemistry (Mosc). 2020 Oct;85(10): 1169-1177
    Ratushnyy AY, Rudimova YV, Buravkova LB.
      Cell senescence leads to a number of changes in the properties of mesenchymal stromal cells (MSCs). In particular, the number of damaged structures is increased producing negative effect on intracellular processes. Elimination of the damaged molecules and organelles occurs via autophagy that can be important in the context of aging. Cultivation under low oxygen level can be used as an approach for enhancement of MSC therapeutic properties and "slowing down" cell senescence. The goal of this work was to study some morphological and functional characteristics and expression of autophagy-associated genes during replicative senescence of MSCs under different oxygen concentration. The study revealed changes in the regulation of autophagy at the transcriptional level. Upregulation of the expression of autophagosome membrane growth genes ATG9A and ULK1, of the autophagosome maturation genes CTSD, CLN3, GAA, and GABARAPL1, of the autophagy regulation genes TP53, TGFB1, BCL2L1, FADD, and HTT was shown. These changes were accompanied by downregulation of IGF1 and TGM2 expression. Increase of the lysosomal compartment volume was observed in the senescent MSCs that also indicated increase of their degradation activity. The number of lysosomes was decreased following prolonged cultivation under low oxygen concentration (5%). The replicative senescence of MSCs under conditions of different oxygen levels led to the similar modifications in the expression of the autophagy-associated genes.
    DOI:  https://doi.org/10.1134/S0006297920100053
  31. Autophagy. 2020 Nov 19.
    Dolai S, Takahashi T, Qin T, Liang T, Xie L, Kang F, Miao YF, Xie H, Kang Y, Manuel J, Winter E, Roche PA, Cattral MS, Gaisano HY.
      Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKβ-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKβ inhibitors, and confirmed by the inability of IKBKB/IKKβ phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKβ's involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.
    Keywords:  Autophagy; IKKβ; SNAREs; caerulein; experimental pancreatitis; pancreatic acinar cell
    DOI:  https://doi.org/10.1080/15548627.2020.1852725
  32. Oncogene. 2020 Nov 15.
    Goss KL, Koppenhafer SL, Waters T, Terry WW, Wen KK, Wu M, Ostergaard J, Gordon PM, Gordon DJ.
      Ribonucleotide reductase (RNR), which is a heterodimeric tetramer composed of RRM1 and RRM2 subunits, is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and essential for both DNA replication and the repair of DNA damage. The activity of RNR is coordinated with the cell cycle and regulated by fluctuations in the level of the RRM2 subunit. Multiple cancer types, including Ewing sarcoma tumors, are sensitive to inhibitors of RNR or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. Here, we show that the expression of the RRM2 protein is dependent on active protein synthesis and that 4E-BP1, a repressor of cap-dependent protein translation, specifically regulates the level of the RRM2 protein. Furthermore, inhibition of mTORC1/2, but not mTORC1, activates 4E-BP1, inhibits protein synthesis, and reduces the level of the RRM2 protein in multiple sarcoma cell lines. This effect of mTORC1/2 inhibitors on protein synthesis and RRM2 levels was rescued in cell lines with the CRISPR/Cas9-mediated knockout of 4E-BP1. In addition, the inducible expression of a mutant 4E-BP1 protein that cannot be phosphorylated by mTOR blocked protein synthesis and inhibited the growth of Ewing sarcoma cells in vitro and in vivo in a xenograft. Overall, these results provide insight into the multifaceted regulation of RRM2 protein levels and identify a regulatory link between protein translation and DNA replication.
    DOI:  https://doi.org/10.1038/s41388-020-01552-0
  33. J Biol Chem. 2020 Nov 15. pii: jbc.RA120.016193. [Epub ahead of print]
    Hedman AC, Li Z, Gorisse L, Parvathaneni S, Morgan CJ, Sacks DB.
      AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+ /calmodulin-dependent protein kinase kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.
    Keywords:  AMP-activated kinase (AMPK); IQGAP1; calcium; calmodulin (CaM); cell signaling; homeostasis; metabolic regulation; metformin; protein-protein interaction; scaffold protein; signaling
    DOI:  https://doi.org/10.1074/jbc.RA120.016193
  34. Autophagy. 2020 Nov 19.
    Deng R, Zhang HL, Huang JH, Cai RZ, Wang Y, Chen YH, Hu BX, Ye ZP, Li ZL, Mai J, Huang Y, Li X, Peng XD, Feng GK, Li JD, Tang J, Zhu XF.
      The function of mitophagy in cancer is controversial. ULK1 is critical for induction of macroautophagy/autophagy and has a more specific role in mitophagy in response to hypoxia. Here, we show that ULK1 deficiency induces an invasive phenotype of breast cancer cells under hypoxia and increases osteolytic bone metastasis. Mechanistically, ULK1 depletion attenuates mitophagy ability during hypoxia. As a result, the accumulation of damaged, ROS-generating mitochondria leads to activation of the NLRP3 inflammasome, which induces abnormal soluble cytokines secretion, then promotes the differentiation and maturation of osteoclasts, and ultimately results in bone metastasis. Notably, phosphorylation of ULK1 by MAPK1/ERK2-MAPK3/ERK1 kinase triggers its interaction with BTRC and subsequent K48-linked ubiquitination and proteasome degradation. Also, a clearly negative correlation between the expression levels of ULK1 and p-MAPK1/3 was observed in human breast cancer tissues. The MAP2K/MEK inhibitor trametinib is sufficient to restore mitophagy function via upregulation of ULK1, leading to inhibition of NLRP3 inflammasome activation, thereby reduces bone metastasis. These results indicate that ULK1 knockout-mediated mitophagy defect promotes breast cancer bone metastasis and provide evidence to explore MAP2K/MEK- MAPK1/3 pathway inhibitors for therapy, especially in cancers displaying low levels of ULK1.
    Keywords:  MAPK1/3 kinase; NLRP3 inflammasome; ULK1; bone metastasis; breast cancer; mitophagy deficiency
    DOI:  https://doi.org/10.1080/15548627.2020.1850609
  35. Cell Death Dis. 2020 Nov 17. 11(11): 991
    Ma WQ, Sun XJ, Zhu Y, Liu NF.
      Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular energy regulation. Previous studies suggested that PDK4 is increased in the calcified vessels of patients with atherosclerosis and is closely associated with mitochondrial function, but the precise regulatory mechanisms remain largely unknown. This study aims to investigate the role of PDK4 in vascular calcification and the molecular mechanisms involved. Using a variety of complementary techniques, we found impaired autophagic activity in the process of vascular smooth muscle cells (VSMCs) calcification, whereas knocking down PDK4 had the opposite effect. PDK4 drives the metabolic reprogramming of VSMCs towards a Warburg effect, and the inhibition of PDK4 abrogates VSMCs calcification. Mechanistically, PDK4 disturbs the integrity of the mitochondria-associated endoplasmic reticulum membrane, concomitantly impairing mitochondrial respiratory capacity, which contributes to a decrease in lysosomal degradation by inhibiting the V-ATPase and lactate dehydrogenase B interaction. PDK4 also inhibits the nuclear translocation of the transcription factor EB, thus inhibiting lysosomal function. These changes result in the interruption of autophagic flux, which accelerates calcium deposition in VSMCs. In addition, glycolysis serves as a metabolic adaptation to improve VSMCs oxidative stress resistance, whereas inhibition of glycolysis by 2-deoxy-D-glucose induces the apoptosis of VSMCs and increases the calcium deposition in VSMCs. Our results suggest that PDK4 plays a key role in vascular calcification through autophagy inhibition and metabolic reprogramming.
    DOI:  https://doi.org/10.1038/s41419-020-03162-w
  36. J Cell Biol. 2020 Dec 07. pii: e202009128. [Epub ahead of print]219(12):
    Fischer TD, Wang C, Padman BS, Lazarou M, Youle RJ.
      Following the detection of cytosolic double-stranded DNA from viral or bacterial infection in mammalian cells, cyclic dinucleotide activation of STING induces interferon β expression to initiate innate immune defenses. STING activation also induces LC3B lipidation, a classical but equivocal marker of autophagy, that promotes a cell-autonomous antiviral response that arose before evolution of the interferon pathway. We report that STING activation induces LC3B lipidation onto single-membrane perinuclear vesicles mediated by ATG16L1 via its WD40 domain, bypassing the requirement of canonical upstream autophagy machinery. This process is blocked by bafilomycin A1 that binds and inhibits the vacuolar ATPase (V-ATPase) and by SopF, a bacterial effector that catalytically modifies the V-ATPase to inhibit LC3B lipidation via ATG16L1. These results indicate that activation of the cGAS-STING pathway induces V-ATPase-dependent LC3B lipidation that may mediate cell-autonomous host defense, an unanticipated mechanism that is distinct from LC3B lipidation onto double-membrane autophagosomes.
    DOI:  https://doi.org/10.1083/jcb.202009128
  37. Clin Transl Sci. 2020 Nov 17.
    Kang J, Kim JW, Heo H, Lee J, Park KY, Yoon JH, Chang J.
      The current diagnosis of Parkinson's disease (PD) mostly relies on clinical rating scales related to motor dysfunction. Given that clinical symptoms of PD appear after significant neuronal cell death in the brain, it is required to identify accessible, objective and quantifiable biomarkers for early diagnosis of PD. In this study, a total of 20 patients with idiopathic PD and 20 age-matched patients with essential tremor according to the UK Brain Bank Criteria were consecutively enrolled to identify peripheral blood biomarkers for PD. Clinical data were obtained by clinical survey and assessment. Using albumin- and IgG-depleted plasma samples, we performed immunoblot analysis of seven autophagy-related proteins and compared the levels of proteins to those of the control group. We also analyzed the correlation between the levels of candidate proteins and clinical characteristics. Finally, we validated our biomarker models using receiver operating characteristic (ROC) curve analysis. We found that the levels of BCL2-associated athanogene 2 (BAG2) and cathepsin D were significantly decreased in plasma of PD patients (p=0.009 and p=0.0077, respectively). The level of BAG2 in PD patients was significantly correlated with Cross-Culture Smell Identification Test (CC-SIT) score, which indicates olfactory dysfunction. We found that our biomarker model distinguishes PD with 87.5% diagnostic accuracy (AUC=0.875, p<0.0001). Our result suggests BAG2 and cathepsin D as candidates for early-diagnosis plasma biomarkers for PD. We provide the possibility of plasma biomarkers related to the autophagy pathway, by which decreased levels of BAG2 and cathepsin D might lead to dysfunction of autophagy.
    Keywords:  BAG2; Parkinson’s disease; ROC curve; autophagy; cathepsin D; plasma biomarkers
    DOI:  https://doi.org/10.1111/cts.12920
  38. FEBS J. 2020 Nov 17.
    Cavinato M, Madreiter-Sokolowski CT, Büttner S, Schosserer M, Zwerschke W, Wedel S, Grillari J, Graier WF, Jansen-Dürr P.
      Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules which would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e. "senolytics") or inactivating/switching damage-inducing properties of senescent cells (i.e. "senostatics/senomorphics"), such as the senescence-associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their inter-organelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.
    Keywords:  Autophagy; RNA modification; calcium signaling homeostasis; caloric restriction(CR) mimetic; interorganellar connectivity; lysosome; mitochondria; mitophagy; proteostasis; senescence; translational control
    DOI:  https://doi.org/10.1111/febs.15631