bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒09‒20
forty-five papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. JCI Insight. 2020 Sep 15. pii: 136676. [Epub ahead of print]
    Liu EA, Schultz ML, Mochida C, Chung C, Paulson HL, Lieberman AP.
      A critical response to lysosomal membrane permeabilization (LMP) is the clearance of damaged lysosomes through a selective form of macroautophagy known as lysophagy. Although regulators of this process are emerging, whether organ and cell specific components contribute to the control of lysophagy remains incompletely understood. Here, we examine LMP and lysophagy in Niemann-Pick type C disease (NPC), an autosomal recessive disorder characterized by the accumulation of unesterified cholesterol within late endosomes and lysosomes, leading to neurodegeneration and early death. We demonstrate that NPC patient fibroblasts show enhanced sensitivity to lysosomal damage as a consequence of lipid storage. Moreover, we describe a role for the glycan binding F-box protein Fbxo2 in CNS lysophagy. Fbxo2 functions as a component of the SCF ubiquitin ligase complex. Loss of Fbxo2 in mouse primary cortical cultures delays clearance of damaged lysosomes and decreases viability following lysosomal damage. Moreover, Fbxo2 deficiency in a mouse model of NPC exacerbates deficits in motor function, enhances neurodegeneration, and reduces survival. Collectively, our data identify a role for Fbxo2 in CNS lysophagy and establish its functional importance in NPC.
    Keywords:  Autophagy; Lysosomes; Neurodegeneration; Neuroscience
    DOI:  https://doi.org/10.1172/jci.insight.136676
  2. Mol Genet Metab. 2020 Sep 04. pii: S1096-7192(20)30190-6. [Epub ahead of print]
    Schwarz JM, Pedrazza L, Stenzel W, Rosa JL, Schuelke M, Straussberg R.
      The giant 532 kDa HERC1 protein is a ubiquitin ligase that interacts with tuberous sclerosis complex subunit 2 (TSC2), a negative upstream regulator of the mammalian target of rapamycin complex 1 (mTORC1). TSC2 regulates anabolic cell growth through its influence on protein synthesis, cell growth, proliferation, autophagy, and differentiation. TSC subunit 1 (TSC1) stabilizes TSC2 by inhibiting the interaction between TSC2 and HERC1, forming a TSC1-TSC2 complex that negatively regulates mTORC1. HERC1-TSC2 interaction destabilizes and degrades TSC2. Recessive mutations in HERC1 have been reported in patients with intellectual disability. Some patients exhibit epilepsy, macrocephaly, somatic overgrowth, and dysmorphic facial features as well. Here we describe two sisters from a consanguineous marriage with a novel homozygous missense variant in the C-terminal HECT domain of HERC1 [chr15:g63,907,989C>G GRCh37.p11 | c.14,072G>C NM_003922 | p.(Arg4,691Pro)]. Symptoms compris global developmental delay, macrocephaly, somatic overgrowth, intellectual disability, seizures, schizoaffective disorder, and pyramidal tract signs. We functionally assessed the HERC1 mutation by investigation of patient and control fibroblasts under normal and nutrient starving conditions. During catabolic state, mTORC1 activity remained high in patient fibroblasts, which stands in stark contrast to its downregulation in controls. This was corroborated by an abnormally high phosphorylation of S6K1-kinase, a direct downstream target of mTORC1, in patients. Moreover, autophagy, usually enhanced in catabolic states, was down-regulated in patient fibroblasts. These data confirm that the missense variant found in both patients results in a gain-of-function for the mutant HERC1 protein.
    Keywords:  Autophagy; MDFPMR; Next generation sequencing; Seizures; Tuberous sclerosis complex; Ubiquitination; mTORC1
    DOI:  https://doi.org/10.1016/j.ymgme.2020.08.008
  3. Neurobiol Dis. 2020 Sep 09. pii: S0969-9961(20)30356-9. [Epub ahead of print] 105081
    Kuwahara T, Funakawa K, Komori T, Sakurai M, Yoshii G, Eguchi T, Fukuda M, Iwatsubo T.
      Leucine-rich repeat kinase 2 (LRRK2), the major causative gene product of autosomal-dominant Parkinson's disease, is a protein kinase that phosphorylates a subset of Rab GTPases. Since pathogenic LRRK2 mutations increase its ability to phosphorylate Rab GTPases, elucidating the mechanisms of how Rab phosphorylation is regulated by LRRK2 is of great importance. We have previously reported that chloroquine-induced lysosomal stress facilitates LRRK2 phosphorylation of Rab10 to maintain lysosomal homeostasis. Here we reveal that Rab10 phosphorylation by LRRK2 is potently stimulated by treatment of cells with a set of lysosome stressors and clinically used lysosomotropic drugs. These agents commonly promoted the formation of LRRK2-coated enlarged lysosomes and extracellular release of lysosomal enzyme cathepsin B, the latter being dependent on LRRK2 kinase activity. In contrast to the increase in Rab10 phosphorylation, treatment with lysosomotropic drugs did not increase the enzymatic activity of LRRK2, as monitored by its autophosphorylation at Ser1292 residue, but rather enhanced the molecular proximity between LRRK2 and its substrate Rab GTPases on the cytosolic surface of lysosomes. Lysosomotropic drug-induced upregulation of Rab10 phosphorylation was likely a downstream event of Rab29 (Rab7L1)-mediated enzymatic activation of LRRK2. These results suggest a regulated process of Rab10 phosphorylation by LRRK2 that is associated with lysosomal overload stress, and provide insights into the novel strategies to halt the aberrant upregulation of LRRK2 kinase activity.
    Keywords:  LRRK2; Lysosome; Lysosomotropic agents; Parkinson's disease; Phosphorylation; Rab
    DOI:  https://doi.org/10.1016/j.nbd.2020.105081
  4. Life (Basel). 2020 Sep 11. pii: E194. [Epub ahead of print]10(9):
    Goker-Alpan O, Kasturi VG, Sohi MK, Limgala RP, Austin SL, Jennelle T, Banikazemi M, Kishnani PS.
      There is limited data on pregnancy outcomes in Pompe Disease (PD) resulting from deficiency of the lysosomal enzyme acid alpha-glucosidase. Late-onset PD is characterized by progressive proximal muscle weakness and decline of respiratory function secondary to the involvement of the respiratory muscles. In a cohort of twenty-five females, the effects of both PD on the course of pregnancy and the effects of pregnancy on PD were investigated. Reproductive history, course of pregnancy, use of Enzyme replacement therapy (ERT), PD symptoms, and outcomes of each pregnancy were obtained through a questionnaire. Among 20 subjects that reported one or more pregnancies, one subject conceived while on ERT and continued therapy through two normal pregnancies with worsening of weakness during pregnancy and improvement postpartum. While fertility was not affected, pregnancy may worsen symptoms, or cause initial symptoms to arise. Complications with pregnancy or birth were not higher, except for an increase in the rate of stillbirths (3.8% compared to the national average of 0.2-0.7%). Given small sample size and possible bias of respondents being only women who have been pregnant, further data may be needed to better analyze the effects of pregnancy on PD, and the effects of ERT on pregnancy outcomes.
    Keywords:  LOPD; enzyme replacement therapy; glycogen storage disease type II; lysosomal disorders; pompe disease; pregnancy
    DOI:  https://doi.org/10.3390/life10090194
  5. Trends Mol Med. 2020 Sep 15. pii: S1471-4914(20)30183-0. [Epub ahead of print]
    Tayebi N, Lopez G, Do J, Sidransky E.
      Mutations in GBA1, the gene encoding the lysosomal hydrolase glucocerebrosidase (GCase), are a risk factor for parkinsonism. Pursuing the potential mechanisms underlying this risk in aging neurons, we propose a new network uniting three major lysosomal proteins: (i) cathepsin D (CTSD), which plays a major role in α-synuclein (SNCA) degradation and prosaposin (PSAP) cleavage; (ii) PSAP, essential for GCase activation and progranulin (PGRN) transport; and (iii) PGRN, impacting lysosomal biogenesis, PSAP trafficking, and CTSD maturation. We hypothesize that alterations to this network and associated receptors modify lysosomal function and subsequently impact both SNCA degradation and GCase activity. By exploring the interactions between this protein trio and each of their respective transporters and receptors, we may identify secondary risk factors that provide insight into the relationship between these lysosomal proteins, GCase, and SNCA, and reveal novel therapeutic targets.
    Keywords:  Parkinson disease; alpha-synuclein; cathepsin D; glucocerebrosidase; lysosome; progranulin; prosaposin
    DOI:  https://doi.org/10.1016/j.molmed.2020.07.004
  6. Sci Adv. 2020 Jun;pii: eabb2210. [Epub ahead of print]6(25):
    Fassl A, Brain C, Abu-Remaileh M, Stukan I, Butter D, Stepien P, Feit AS, Bergholz J, Michowski W, Otto T, Sheng Q, Loo A, Michael W, Tiedt R, DeAngelis C, Schiff R, Jiang B, Jovanovic B, Nowak K, Ericsson M, Cameron M, Gray N, Dillon D, Zhao JJ, Sabatini DM, Jeselsohn R, Brown M, Polyak K, Sicinski P.
      Inhibitors of cyclin-dependent kinases CDK4 and CDK6 have been approved for treatment of hormone receptor-positive breast cancers. In contrast, triple-negative breast cancers (TNBCs) are resistant to CDK4/6 inhibition. Here, we demonstrate that a subset of TNBC critically requires CDK4/6 for proliferation, and yet, these TNBC are resistant to CDK4/6 inhibition due to sequestration of CDK4/6 inhibitors into tumor cell lysosomes. This sequestration is caused by enhanced lysosomal biogenesis and increased lysosomal numbers in TNBC cells. We developed new CDK4/6 inhibitor compounds that evade the lysosomal sequestration and are efficacious against resistant TNBC. We also show that coadministration of lysosomotropic or lysosome-destabilizing compounds (an antibiotic azithromycin, an antidepressant siramesine, an antimalaria compound chloroquine) renders resistant tumor cells sensitive to currently used CDK4/6 inhibitors. Lastly, coinhibition of CDK2 arrested proliferation of CDK4/6 inhibitor-resistant cells. These observations may extend the use of CDK4/6 inhibitors to TNBCs that are refractory to current anti-CDK4/6 therapies.
    DOI:  https://doi.org/10.1126/sciadv.abb2210
  7. Sci Adv. 2020 Jul;pii: eabb8725. [Epub ahead of print]6(31):
    Xu F, Li X, Huang X, Pan J, Wang Y, Zhou S.
      Autophagy is involved in the occurrence and development of tumors. Here, a pH-responsive polymersome codelivering hydroxychloroquine (HCQ) and tunicamycin (Tuni) drugs is developed to simultaneously induce endoplasmic reticulum (ER) stress and autophagic flux blockade for achieving an antitumor effect and inhibiting tumor metastasis. The pH response of poly(β-amino ester) and HCQ synergistically deacidifies the lysosomes, thereby blocking the fusion of autophagosomes and lysosomes and lastly blocking autophagic flux. The function mechanism of regulating autophagy was systematically investigated on orthotopic luciferase gene-transfected, 4T1 tumor-bearing BALB/c mice through Western blot and immunohistochemistry analyses. The Tuni triggers ER stress to regulate the PERK/Akt signaling pathway to increase the autophagic level. The "autophagic stress" generated by triggering ER stress-induced autophagy and blocking autophagic flux is effective against tumors. The reduced expression of matrix metalloproteinase-2 due to ER stress and reduced focal adhesions turnover due to the blockade of autophagic flux synergistically inhibit tumor metastasis.
    DOI:  https://doi.org/10.1126/sciadv.abb8725
  8. J Inherit Metab Dis. 2020 Sep 17.
    Yilmaz BS, Davison J, Jones SA, Baruteau J.
      Mucopolysaccharidosis type III (MPS III) or Sanfilippo disease is an orphan inherited lysosomal storage disease and one of the most common MPS subtypes. The classical presentation is an infantile-onset neurodegenerative disease characterised by intellectual regression, behavioural and sleep disturbances, loss of ambulation and early death. Unlike other MPS, no disease-modifying therapy has yet been approved. Here, we review the numerous approaches of curative therapy developed for MPS III from historical ineffective haematopoietic stem cell transplantation and substrate reduction therapy to the promising ongoing clinical trials based on enzyme replacement therapy or adeno-associated (AAV) or lentiviral vectors mediated gene therapy. Preclinical studies are presented alongside the most recent translational first-in-man trials. In addition, we present experimental research with preclinical mRNA and gene editing strategies. Lessons from animal studies and clinical trials have highlighted the importance of an early therapy before extensive neuronal loss. A disease-modifying therapy for MPS III will undoubtedly mandate development of new strategies for early diagnosis. This article is protected by copyright. All rights reserved.
    Keywords:  Adeno-associated virus; Enzyme replacement therapy; Gene therapy; Mucopolysaccharidosis type III; Sanfilippo disease; gene editing; heparan sulphate; lentivirus; lysosomal storage disease; mRNA; substrate reduction therapy
    DOI:  https://doi.org/10.1002/jimd.12316
  9. Sci Adv. 2020 Jul;pii: eaay9131. [Epub ahead of print]6(31):
    Shin SH, Lee JS, Zhang JM, Choi S, Boskovic ZV, Zhao R, Song M, Wang R, Tian J, Lee MH, Kim JH, Jeong M, Lee JH, Petukhov M, Lee SW, Kim SG, Zou L, Byun S.
      Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.
    DOI:  https://doi.org/10.1126/sciadv.aay9131
  10. Cells. 2020 Sep 10. pii: E2072. [Epub ahead of print]9(9):
    Guijarro MV, Danielson LS, Cañamero M, Nawab A, Abrahan C, Hernando E, Palmer GD.
      TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.
    Keywords:  TSC1; mammalian target of rapamycin (mTOR); mesenchymal stem cell; senescence; stem cell proliferation; tuberous sclerosis
    DOI:  https://doi.org/10.3390/cells9092072
  11. Mol Cell Oncol. 2020 ;7(5): 1789418
    Hernández-Cáceres MP, Cereceda K, Hernández S, Li Y, Narro C, Rivera P, Silva P, Ávalos Y, Jara C, Burgos P, Toledo-Valenzuela L, Lagos P, Cifuentes Araneda F, Perez-Leighton C, Bertocchi C, Clegg DJ, Criollo A, Tapia-Rojas C, Burgos PV, Morselli E.
      High-fat diet (HFD)-induced obesity is associated with increased cancer risk. Long-term feeding with HFD increases the concentration of the saturated fatty acid palmitic acid (PA) in the hypothalamus. We previously showed that, in hypothalamic neuronal cells, exposure to PA inhibits the autophagic flux, which is the whole autophagic process from the synthesis of the autophagosomes, up to their lysosomal fusion and degradation. However, the mechanism by which PA impairs autophagy in hypothalamic neurons remains unknown. Here, we show that PA-mediated reduction of the autophagic flux is not caused by lysosomal dysfunction, as PA treatment does not impair lysosomal pH or the activity of cathepsin B.Instead, PA dysregulates autophagy by reducing autophagosome-lysosome fusion, which correlates with the swelling of endolysosomal compartments that show areduction in their dynamics. Finally, because lysosomes undergo constant dynamic regulation by the small Rab7 GTPase, we investigated the effect of PA treatment on its activity. Interestingly, we found PA treatment altered the activity of Rab7. Altogether, these results unveil the cellular process by which PA exposure impairs the autophagic flux. As impaired autophagy in hypothalamic neurons promotes obesity, and balanced autophagy is required to inhibit malignant transformation, this could affect tumor initiation, progression, and/or response to therapy of obesity-related cancers.
    Keywords:  Palmitic acid; Rab7; autophagy; central nervous system; electron microscopy; high-fat diet; hypothalamic neuronal cells; lysosomes; obesity-associated cancers
    DOI:  https://doi.org/10.1080/23723556.2020.1789418
  12. Nat Commun. 2020 Sep 17. 11(1): 4684
    Gremke N, Polo P, Dort A, Schneikert J, Elmshäuser S, Brehm C, Klingmüller U, Schmitt A, Reinhardt HC, Timofeev O, Wanzel M, Stiewe T.
      Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.
    DOI:  https://doi.org/10.1038/s41467-020-18504-7
  13. Front Oncol. 2020 ;10 1615
    Cong Y, Li Q, Zhang X, Chen Y, Yu K.
      Mechanistic target of rapamycin (mTOR) signaling pathway mediates the function of oncogenic receptor tyrosine kinases (RTKs). We aimed to elucidate new role of mTOR in EGFR-mutant (EGFR-mut) non-small cell lung cancer (NSCLC) and glioblastoma (GBM) with a focus on tumor microenvironments. Here, we report a novel regulatory link between mTOR complexes (mTORCs) and tissue factor (TF), an initiator of tumor-derived thrombosis. TF is elevated in EGFR-mut NSCLC/GBM cell lines and tumors from patients with poor prognosis. Application of mTORC1/2 inhibitors (AZD8055, WYE-125132, MTI-31, and rapamycin) or genetic mTORC-depletion all reduced TF expression, which appeared to be differentially mediated depending on cellular context. In U87MG and HCC827 cells, mTORC1 exerted a dominant role via promoting TF mRNA transcription. In EGFR-TKI-resistant H1975 and PC9 cells, it was mTORC2 that played a major role in specific repression of lysosomal-targeted TF protein degradation. Successful inhibition of TF expression was demonstrated in AZD8055- or MTI-31-treated H1975 and U87MG tumors in mice, while a TF-targeted antibody antagonized TF activity without reducing TF protein. Both the mTOR- and TF-targeted therapy induced a multifaceted remodeling of tumor microenvironment reflecting not only a diminished hypercoagulopathy state (fibrin level) but also a reduced stromal fibrosis (collagen distribution), compromised vessel density and/or maturity (CD31 and/or α-SMA) as well as a substantially decreased infiltration of immune-suppressive M2-type tumor-associated macrophages (CD206/F4/80 ratio). Thus, our results have identified TF as a functional biomarker of mTOR. Downregulation of mTOR-TF axis activity likely contributes to the therapeutic mechanism of mTORC1/2- and TF-targeted agents in EGFR-mut advanced NSCLC and GBM.
    Keywords:  EGFR mutant cancer; mTOR inhibitor; mTOR-TF axis; tissue factor; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2020.01615
  14. Autophagy. 2020 Sep 14.
    Tian X, Teng J, Chen J.
      Macroautophagy/autophagy refers to the engulfment of cellular contents selected for lysosomal degradation. The final step in autophagy is the fusion of autophagosome with the lysosome, which is mediated by SNARE proteins. Of the SNAREs, autophagosome-localized Q-SNAREs, such as STX17 and SNAP29, and lysosome-localized R-SNAREs, such as VAMP8 or VAMP7, have been reported to be involved. Recent studies also reveal participation of the R-SNARE, YKT6, in autophagosome-lysosome fusion. These SNAREs, with the help of other regulatory factors, act coordinately to spatiotemporally control the fusion process. Besides regulating autophagosome-lysosome fusion, some SNAREs, such as STX17, also function in other autophagic processes, including autophagosome formation and mitophagy. A better understanding of the functions of SNAREs will shed light on the molecular mechanisms of autophagosome-lysosome fusion as well as on the mechanisms by which autophagy is globally regulated.
    Keywords:  Autophagosome-lysosome fusion; SNARE; STX17; autophagy; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2020.1823124
  15. Anal Methods. 2020 Jun 18. 12(23): 2978-2984
    Li L, Xiong Z, Dang Y, Li Y, Zhang A, Ding C, Xu Z, Zhang W.
      Visual detection of pH changes in lysosomes is critical because lysosomes not only play an important role in diverse cellular functions but also are closely related to various physiological and pathological processes. Herein, we disclose a donor-acceptor-donor (D-A-D) type fluorescent probe DBTD for detecting pH fluctuation in lysosomes. DBTD was rationally designed by using benzothiadiazole as the electron acceptor and N,N-diethylamino groups as the electron donor. Owing to its unique D-A-D structure, DBTD displayed a red-emission centered at 614 nm. It showed a sensitive and a linear response to pH from 4.5 to 5.2 with a pKa of 5.0, which is very suitable for lysosomal pH imaging. The response was based on the intramolecular charge transfer (ICT) effect owing to the protonation of the diethylamino group. Furthermore, DBTD could accurately monitor lysosomal pH variations in SGC-7901 cells. More importantly, it was able to image the pH change in lysosomes during the autophagy process successfully, suggesting the great potential of DBTD acting as a powerful tool for monitoring lysosomal pH-related biological processes.
    DOI:  https://doi.org/10.1039/d0ay00418a
  16. Oncoimmunology. 2020 Jun 03. 9(1): 1774281
    He Y, Du J, Dong Z.
      A large number of heterogeneous macrophages can be observed in solid tumor lesions. Classically activated M1 macrophages are a powerful killer of cancer cells. In contrast, tumor-associated macrophages (TAMs) are often referred to as M2 phenotype and usually impair tumor immunity mediated by cytotoxic lymphocytes, natural killer (NK) cells and CD8+ T cells. Therefore, orchestrating M2 to M1 reprogramming will provide a promising approach to tumor immunotherapy. Here we used a PyMT-induced spontaneous breast cancer model in which M2-polarized macrophages were abundant. This M2 phenotype was closely related to tumor progression and immune dysfunction of NK cells and CD8+ T cells. We then found that these TAMs showed increased energy expenditure and over-activation of two kinases, Akt and mammalian target of rapamycin (mTOR). Myeloid inactivation of phosphoinositide-dependent kinase-1 (PDK1), the upstream regulator for Akt and mTOR signaling, significantly reduced excessive metabolic activation of macrophages. Notably, the loss of PDK1 significantly led to regression of breast cancer and prevented lung metastasis. Mechanistically, PDK1 deficiency mainly inhibited the activation of mTOR complex 1 (mTORC1), transforming TAMs into M1 phenotype, thereby reversing tumor-related dysfunction of T cells and NK cells. Therefore, targeting PDK1 may be a new approach for M2 macrophage-enriched solid tumor immunotherapy.
    Keywords:  Macrophage; NK cells; PDK1; mTOR; macrophage polarization
    DOI:  https://doi.org/10.1080/2162402X.2020.1774281
  17. Eur J Hum Genet. 2020 Sep 18.
    Stamerra CA, De Feo M, Castelli V, d'Angelo M, Cimini A, Grassi D, Ferri C.
      Fabry is an X-linked disorder of glycosphingolipid metabolism that is caused by variants of the GLA gene that codes for α-galactosidase A, leading to lysosomal accumulation of globotriaosylceramide in many cell types. As a result, affected patients manifest with an increased risk of developing ischemic stroke, peripheral neuropathy, cardiac dysfunction, and chronic kidney disease. The protective effects of enzyme replacement therapy (ERT), the milestone in Fabry disease treatment, against globotriaosylceramide (GL-3) accumulation and Fabry disease progression are well known. However, the mechanism of action of ERT is not well understood. Since GL-3 also accumulates in the vascular endothelium, we investigated the effects of agalsidase-β, a recombinant human α-Gal enzyme approved for the treatment of Fabry disease. In this study, vascular function and blood pressure in four adult siblings affected by Fabry disease were evaluated upon agalsidase-β. In all patients, agalsidase-β infusion improves flow-mediated dilation and augmentation index. These changes occurred after the first infusion and were then maintained for the whole period of observation, i.e., 1 year, with more pronounced additional increments in flow-mediated dilation after the second agalsidase-β infusion. Blood pressure was also maintained at optimal levels in all of the patients for the whole period of observation. Our findings show that agalsidase-β administration can improve vascular function in patients suffering from Fabry disease. Changes in flow-mediated dilation and augmentation index persisted for the whole period of observation (1 year), thus suggesting that early substitutive therapy should be promoted in order to protect the cardiovascular system.
    DOI:  https://doi.org/10.1038/s41431-020-00721-9
  18. EMBO Rep. 2020 Sep 14. e50241
    Werner G, Damme M, Schludi M, Gnörich J, Wind K, Fellerer K, Wefers B, Wurst W, Edbauer D, Brendel M, Haass C, Capell A.
      Single nucleotide polymorphisms (SNPs) in TMEM106B encoding the lysosomal type II transmembrane protein 106B increase the risk for frontotemporal lobar degeneration (FTLD) of GRN (progranulin gene) mutation carriers. Currently, it is unclear if progranulin (PGRN) and TMEM106B are synergistically linked and if a gain or a loss of function of TMEM106B is responsible for the increased disease risk of patients with GRN haploinsufficiency. We therefore compare behavioral abnormalities, gene expression patterns, lysosomal activity, and TDP-43 pathology in single and double knockout animals. Grn-/- /Tmem106b-/- mice show a strongly reduced life span and massive motor deficits. Gene expression analysis reveals an upregulation of molecular signature characteristic for disease-associated microglia and autophagy. Dysregulation of maturation of lysosomal proteins as well as an accumulation of ubiquitinated proteins and widespread p62 deposition suggest that proteostasis is impaired. Moreover, while single Grn-/- knockouts only occasionally show TDP-43 pathology, the double knockout mice exhibit deposition of phosphorylated TDP-43. Thus, a loss of function of TMEM106B may enhance the risk for GRN-associated FTLD by reduced protein turnover in the lysosomal/autophagic system.
    Keywords:   FTD ; TDP-43; TMEM106B; neurodegeneration; progranulin
    DOI:  https://doi.org/10.15252/embr.202050241
  19. Orphanet J Rare Dis. 2020 Sep 14. 15(1): 247
    van den Dorpel JJA, Poelman E, Harlaar L, van Kooten HA, van der Giessen LJ, van Doorn PA, van der Ploeg AT, van den Hout JMP, van der Beek NAME.
      BACKGROUND: Enzyme replacement therapy (ERT; alglucosidase alfa) has improved the prospects for patients with classic infantile Pompe disease considerably. However, over time we noticed that many of these children exhibit distal muscle weakness at an early age, which is in contrast to the primarily proximal and axial muscle weakness in patients with late-onset Pompe disease. This was reason to study the prevalence and severity of distal muscle weakness, and the sequence of muscle involvement over time in patients that had learned to walk under ERT.METHODS: In this prospective, single-center cohort study, we studied 16 classic infantile patients. We used video recordings that were made during regular standardized assessments to investigate distal muscle function (active dorsiflexion of the feet during walking; ability to use a pincer grasp/actively extend the fingers) and proximal muscle function (standing up from a supine position; raising the arms above the head).
    RESULTS: Median age at start of ERT was 3.2 months (0.1-5.8 months), median age at study end was 5.6 years (2.9-18.2 years). Six patients (6/16, 38%) initially had no evident signs of distal muscle weakness and developed a gait with active dorsiflexion of the feet. The other 10 patients never exhibited active dorsiflexion of the feet during walking. At study-end two patients showed no loss of distal muscle function. A subset of five patients (5/16, 31%) developed also weakness of the hands, particularly of the extensors of the 3rd and 4th digit.
    CONCLUSIONS: We found that the majority (14/16, 88%) of patients who had learned to walk exhibited distal muscle weakness of the lower extremities, while a subset (5/16, 31%) also developed weakness of the hands. The distal muscle weakness was often more serious than, and preceded the development of, the proximal muscle weakness.
    Keywords:  Distal muscle weakness; Enzyme replacement therapy (ERT); Glycogen storage disease type II; Muscle function; Pompe disease
    DOI:  https://doi.org/10.1186/s13023-020-01482-w
  20. Cell Death Dis. 2020 Sep 12. 11(9): 747
    Ma X, Du W, Wang W, Luo L, Huang M, Wang H, Lin R, Li Z, Shi H, Yuan T, Jiang W, Worley PF, Xu T.
      The small GTPase Ras homolog enriched in the brain (Rheb) can activate mammalian target of rapamycin (mTOR) and regulate the growth and cell cycle progression. We investigated the role of Rheb-mediated mTORC1 signaling in neuropathic pain. A chronic constriction injury (CCI) model was dopted. CCI induced obvious spinal Rheb expression and phosphorylation of mTOR, S6, and 4-E-BP1. Blocking mTORC1 signal with rapamycin alleviated the neuropathic pain and restored morphine efficacy in CCI model. Immunofluoresence showed a neuronal co-localization of CCI-induced Rheb and pS6. Rheb knockin mouse showed a similar behavioral phenotype as CCI. In spinal slice recording, CCI increased the firing frequency of neurons expressing HCN channels; inhibition of mTORC1 with rapamycin could reverse the increased spinal neuronal activity in neuropathic pain. Spinal Rheb is induced in neuropathic pain, which in turn active the mTORC1 signaling in CCI. Spinal Rheb-mTOR signal plays an important role in regulation of spinal sensitization in neuropathic pain, and targeting mTOR may give a new strategy for pain management.
    DOI:  https://doi.org/10.1038/s41419-020-02966-0
  21. Dev Cell. 2020 Sep 08. pii: S1534-5807(20)30667-5. [Epub ahead of print]
    Funato Y, Yoshida A, Hirata Y, Hashizume O, Yamazaki D, Miki H.
      Extracellular pH is usually maintained around 7.4 in multicellular organisms, and cells are optimized to proliferate under this condition. Here, we find cells can adapt to a more acidic pH of 6.5 and become addicted to this acidic microenvironment by expressing phosphatase of regenerating liver (PRL), a driver of cancer malignancy. Genome-scale CRISPR-Cas9 knockout screening and subsequent analyses revealed that PRL promotes H+ extrusion and acid addiction by stimulating lysosomal exocytosis. Further experiments using cultured cells and Caenorhabditis elegans clarified the molecular link between PRL and lysosomal exocytosis across species, involving activation of lysosomal Ca2+ channel TRPML by ROS. Indeed, disruption of TRPML in cancer cells abolished PRL-stimulated lysosomal exocytosis, acid addiction, and metastasis. Thus, PRL is the molecular switch turning cells addicted to an acidic condition, which should benefit cancer cells to thrive in an acidic tumor microenvironment.
    Keywords:  PRL; TRPML; acid addiction; lysosomal exocytosis; phosphatase of regenerating liver; transient receptor potential mucolipin; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.devcel.2020.08.009
  22. Sci Signal. 2020 Sep 15. pii: eaba5665. [Epub ahead of print]13(649):
    De Zan E, van Stiphout R, Gapp BV, Blomen VA, Brummelkamp TR, Nijman SMB.
      Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown "synthetic-sick" interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative "perturb and observe" genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.
    DOI:  https://doi.org/10.1126/scisignal.aba5665
  23. Free Radic Res. 2020 Sep 16. 1-11
    Alboaklah HKM, Leake DS.
      Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. α-Tocopherol has been shown to inhibit LDL oxidation in vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of α-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with α-tocopherol by incubating human plasma with α-tocopherol followed by LDL isolation by ultracentrifugation. The α-tocopherol content of LDL was increased from 14.4 ± 0.2 to 24.3 ± 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with α-tocopherol was oxidised more slowly than control LDL by Cu2+ at pH 7.4, but was not protected against oxidation by Cu2+ or Fe3+ or a low concentration of Fe2+ at pH 4.5 (it was sometimes oxidised faster by α-tocopherol with Cu2+ or Fe3+ at pH 4.5). α-Tocopherol-enriched LDL reduced Cu2+ and Fe3+ into the more pro-oxidant Cu+ and Fe2+ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of α-tocopherol did not protect against cardiovascular disease.
    Keywords:  Atherosclerosis; LDL oxidation; iron; lysosome; vitamin E; α-Tocopherol
    DOI:  https://doi.org/10.1080/10715762.2020.1817912
  24. Pediatr Int. 2020 Sep 15.
    Iijima M, Hirano D, Yokoi K, Kobayashi H, Fujiwara M, Ida H, Oishi K.
      BACKGROUND: Hunter syndrome (HS) is an X-linked recessive lysosomal storage disease caused by a deficiency of lysosomal enzyme, iduronate sulfatase (IDS). It is characterized by multisystem accumulations of glycosaminoglycans and upper airway obstruction is one of the major causes of death. While the current disease severity classifications for HS are mainly based on the degree of neurocognitive impairment, its association with the level of upper airway obstruction has not been assessed.METHODS: Retrospective chart review of HS patients who were followed at the Jikei University School of Medicine was performed. Association between the degree of airway obstruction and the currently used disease severity scores was evaluated.
    RESULTS: We identified eight HS patients and they were enrolled in the study. Modified Mallampati classification (MMC) score to predict difficulties for oropharyngeal procedures was significantly correlated with the disease severity of HS. It was also correlated with Apnea-Hypopnea Index (AHI). No significant correlation between IDS enzymatic activity and the disease severity of HS was identified.
    CONCLUSIONS: Variable clinical expressivities exist in HS, but the risk of respiratory complications can likely be associated with the disease severity assessed by the previously recognized neurocognitive function-based severity scoring systems. MMC can be a simple supplementary tool to evaluate the disease severity and also to predict difficulties for oropharyngeal procedures and respiratory functional complications such as sleep apnea in HS.
    Keywords:  AHI; Hunter syndrome; Modified Mallampati classification; airway obstruction; difficult intubation
    DOI:  https://doi.org/10.1111/ped.14467
  25. Nat Commun. 2020 09 16. 11(1): 4677
    Vasudevan D, Neuman SD, Yang A, Lough L, Brown B, Bashirullah A, Cardozo T, Ryoo HD.
      The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.
    DOI:  https://doi.org/10.1038/s41467-020-18453-1
  26. Cell Death Dis. 2020 Sep 17. 11(9): 768
    Geng J, Zhang R, Yuan X, Xu H, Zhu Z, Wang X, Wang Y, Xu G, Guo W, Wu J, Qin ZH.
      Lung cancer is the leading cause of cancer-associated mortality worldwide. DNA damage-regulated autophagy modulator 1 (DRAM1) plays an important roles in autophagy and tumor progression. However, the mechanisms by which DRAM1 inhibits tumor growth are not fully understood. Here, we report that DRAM1 was decreased in nonsmall-cell lung carcinoma (NSCLC) and was associated with poor prognosis. We confirmed that DRAM1 inhibited the growth, migration, and invasion of NSCLC cells in vitro. Furthermore, overexpression of DRAM1 suppressed xenografted NSCLC tumors in vivo. DRAM1 increased EGFR endocytosis and lysosomal degradation, downregulating EGFR signaling pathway. On one side, DRAM1 interacted with EPS15 to promote EGFR endocytosis, as evidence by the results of proximity labeling followed by proteomics; on the other, DRAM1 recruited V-ATP6V1 subunit to lysosomes, thereby increasing the assemble of the V-ATPase complex, resulting in decreased lysosomal pH and increased activation of lysosomal proteases. These two actions of DRAM1 results in acceleration of EGFR degradation. In summary, these in vitro and in vivo studies uncover a novel mechanism through which DRAM1 suppresses oncogenic properties of NSCLC by regulating EGFR trafficking and degradation and highlights the potential value of DRAM1 as a prognostic biomarker in lung cancers.
    DOI:  https://doi.org/10.1038/s41419-020-02979-9
  27. Mol Cell Oncol. 2020 ;7(4): 1763150
    Klann K, Münch C.
      The mammalian target of rapamycin and the integrated stress response are central cellular hubs regulating translation upon stress. The precise proteins and pathway specificity of translation targets of these pathways remained largely unclear. We recently described a new method for quantitative translation proteomics and found that both pathways control translation of the same sets of proteins.
    Keywords:  ISR; Translation proteomics; integrated stress response; mTOR; proteostasis; stress; translatome
    DOI:  https://doi.org/10.1080/23723556.2020.1763150
  28. Sci Rep. 2020 Sep 16. 10(1): 15157
    Nelvagal HR, Hurtado ML, Eaton SL, Kline RA, Lamont DJ, Sands MS, Wishart TM, Cooper JD.
      CLN1 disease is a fatal inherited neurodegenerative lysosomal storage disease of early childhood, caused by mutations in the CLN1 gene, which encodes the enzyme Palmitoyl protein thioesterase-1 (PPT-1). We recently found significant spinal pathology in Ppt1-deficient (Ppt1-/-) mice and human CLN1 disease that contributes to clinical outcome and precedes the onset of brain pathology. Here, we quantified this spinal pathology at 3 and 7 months of age revealing significant and progressive glial activation and vulnerability of spinal interneurons. Tandem mass tagged proteomic analysis of the spinal cord of Ppt1-/-and control mice at these timepoints revealed a significant neuroimmune response and changes in mitochondrial function, cell-signalling pathways and developmental processes. Comparing proteomic changes in the spinal cord and cortex at 3 months revealed many similarly affected processes, except the inflammatory response. These proteomic and pathological data from this largely unexplored region of the CNS may help explain the limited success of previous brain-directed therapies. These data also fundamentally change our understanding of the progressive, site-specific nature of CLN1 disease pathogenesis, and highlight the importance of the neuroimmune response. This should greatly impact our approach to the timing and targeting of future therapeutic trials for this and similar disorders.
    DOI:  https://doi.org/10.1038/s41598-020-72075-7
  29. Nat Commun. 2020 Sep 17. 11(1): 4706
    O' Neill JS, Hoyle NP, Robertson JB, Edgar RS, Beale AD, Peak-Chew SY, Day J, Costa ASH, Frezza C, Causton HC.
      Yeast physiology is temporally regulated, this becomes apparent under nutrient-limited conditions and results in respiratory oscillations (YROs). YROs share features with circadian rhythms and interact with, but are independent of, the cell division cycle. Here, we show that YROs minimise energy expenditure by restricting protein synthesis until sufficient resources are stored, while maintaining osmotic homeostasis and protein quality control. Although nutrient supply is constant, cells sequester and store metabolic resources via increased transport, autophagy and biomolecular condensation. Replete stores trigger increased H+ export which stimulates TORC1 and liberates proteasomes, ribosomes, chaperones and metabolic enzymes from non-membrane bound compartments. This facilitates translational bursting, liquidation of storage carbohydrates, increased ATP turnover, and the export of osmolytes. We propose that dynamic regulation of ion transport and metabolic plasticity are required to maintain osmotic and protein homeostasis during remodelling of eukaryotic proteomes, and that bioenergetic constraints selected for temporal organisation that promotes oscillatory behaviour.
    DOI:  https://doi.org/10.1038/s41467-020-18330-x
  30. Acta Biomed. 2020 Sep 07. 91(3): e2020075
    Soliani L, Salerno GG, Pisani F, Barigazzi I, Rizzi S, Spagnoli C, Frattini D, Zangrandi A, Fusco C.
      BACKGROUND: Niemann-Pick disease type C (NPC) is a lysosomal storage disease caused by mutations in NPC1 or NPC2 genes.CASE PRESENTATION: We present two brothers with the same compound heterozygous variants in exon 13 of the NPC1 gene (18q11.2), the first one (c.1955C> G, p. Ser652Trp), inherited from the mother, the second (c.2107T>A p.Phe703Ile) inherited from the father, associated to the classical biochemical phenotype of NPC. The two brothers presented unspecific neurologic symptoms with difference in age of onset: one presented dyspraxia and motor clumsiness at age 7 years, the other showed a systemic presentation with hepatosplenomegaly noted at the age of two months and neurological symptoms onset at age 4 with speech disturbance. Clinical evolution and neuroimaging data led to the final diagnosis. Systemic signs did not correlate with the onset of neurological symptoms. Miglustat therapy was started in both patients.
    CONCLUSIONS: We highlight the extreme phenotypic heterogeneity of NP-C in the presence of the same genetic variant and the unspecificity of neurologic signs at onset as previously reported. We report some positive effects of miglustat on disease progression assessed also with neuropsychological follow-up, with an age-dependent response.
    DOI:  https://doi.org/10.23750/abm.v91i3.9272
  31. Cancer Biol Med. 2020 Aug 15. 17(3): 583-598
    Wang Y, Deng S, Xu J.
      Cancer immunotherapy harness the body's immune system to eliminate cancer, by using a broad panel of soluble and membrane proteins as therapeutic targets. Immunosuppression signaling mediated by ligand-receptor interaction may be blocked by monoclonal antibodies, but because of repopulation of the membrane via intracellular organelles, targets must be eliminated in whole cells. Targeted protein degradation, as exemplified in proteolysis targeting chimera (PROTAC) studies, is a promising strategy for selective inhibition of target proteins. The recently reported use of lysosomal targeting molecules to eliminate immune checkpoint proteins has paved the way for targeted degradation of membrane proteins as crucial anti-cancer targets. Further studies on these molecules' modes of action, target-binding "warheads", lysosomal sorting signals, and linker design should facilitate their rational design. Modifications and derivatives may improve their cell-penetrating ability and the in vivo stability of these pro-drugs. These studies suggest the promise of alternative strategies for cancer immunotherapy, with the aim of achieving more potent and durable suppression of tumor growth. Here, the successes and limitations of antibody inhibitors in cancer immunotherapy, as well as research progress on PROTAC- and lysosomal-dependent degradation of target proteins, are reviewed.
    Keywords:  Cancer immunotherapy; PROTAC; membrane protein; targeted degradation
    DOI:  https://doi.org/10.20892/j.issn.2095-3941.2020.0066
  32. Int J Mol Sci. 2020 Sep 11. pii: E6647. [Epub ahead of print]21(18):
    Keeling E, Culling AJ, Johnston DA, Chatelet DS, Page A, Tumbarello DA, Lotery AJ, Ratnayaka JA.
      Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.
    Keywords:  AMD; RPE; aging; autofluorescence; autophagy; diet; lysosomes; oxidized POS; proteolysis; retina
    DOI:  https://doi.org/10.3390/ijms21186647
  33. J Mol Biol. 2020 Sep 09. pii: S0022-2836(20)30532-5. [Epub ahead of print]
    Zhang M, Jang H, Nussinov R.
      PI3K lipid kinases signal through the PI3K/Akt pathway, regulating cell growth and proliferation. While the structural features that distinguish between the active and inactive states of protein kinases are well established, that has not been the case for lipid kinases, and neither was the structural mechanism controlling the switch between the two states. Class I PI3Ks are obligate heterodimers with catalytic and regulatory subunits. Here, we analyze PI3K crystal structures. Structures with the nSH2 (inactive state) are featured by collapsed activation loop (a-loop) and an IN kinase domain helix 11 (kα11). In the active state, the a-loop is extended and kα11 in the OUT conformation. Our analysis suggests that the nSH2 domain in the regulatory subunit regulates activation, catalysis and autoinhibition through the a-loop. Inhibition, activation and catalytic scenarios are shared by class IA PI3Ks; the activation is mimicked by oncogenic mutations and the inhibition offers an allosteric inhibitor strategy.
    Keywords:  Catalytic and regulatory subunits; PI3K mutations; PI3Kα; Ras; nSH2
    DOI:  https://doi.org/10.1016/j.jmb.2020.09.002
  34. Vasc Biol. 2019 ;1(1): H125-H134
    Kobialka P, Graupera M.
      PI3Ks belong to a family of lipid kinases that comprises eight isoforms. They phosphorylate the third position of the inositol ring present in phosphatidylinositol lipids and, in turn, activate a broad range of proteins. The PI3K pathway regulates primal cellular responses, including proliferation, migration, metabolism and vesicular traffic. These processes are fundamental for endothelial cell function during sprouting angiogenesis, the most common type of blood vessel formation. Research in animal models has revealed key functions of PI3K family members and downstream effectors in angiogenesis. In addition, perturbations in PI3K signalling have been associated with aberrant vascular growth including tumour angiogenesis and vascular malformations. Together, this highlights that endothelial cells are uniquely sensitive to fluctuations in PI3K signalling. Here, we aim to update the current view on this important signalling cue in physiological and pathological blood vessel growth.
    Keywords:  PI3K; angiogenesis; vascular malformations
    DOI:  https://doi.org/10.1530/VB-19-0025
  35. Kidney Int. 2020 Sep 05. pii: S0085-2538(20)30687-6. [Epub ahead of print]
    Berquez M, Gadsby JR, Festa BP, Butler R, Jackson SP, Berno V, Luciani A, Devuyst O, Gallop JL.
      Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2.
    Keywords:  cytoskeleton; endocytosis; lipids; proximal tubule; renal Fanconi syndrome
    DOI:  https://doi.org/10.1016/j.kint.2020.05.040
  36. Mol Cell Oncol. 2020 ;7(5): 1776085
    Wu W, Stork B.
      Receptor interacting serine/threonine kinase 1 (RIPK1) is the central mediator of tumor necrosis factor (TNF) signaling. It regulates both pro-survival/pro-inflammatory and cell death pathways. In order to fulfill this complex regulation, RIPK1 is regulated by several post-translational modifications, including ubiquitination, acetylation, and phosphorylation. In our recent work, we show that the unc-51-like autophagy activating kinase 1 (ULK1) phosphorylates RIPK1 at Ser357 and thus blocks TNF-induced cell death.
    Keywords:  Autophagy; RIPK1; TNF; Ulk1; necroptosis
    DOI:  https://doi.org/10.1080/23723556.2020.1776085
  37. Clin Cancer Res. 2020 Sep 15. pii: clincanres.1675.2020. [Epub ahead of print]
    Truong A, Yoo JH, Scherzer M, Sanchez JMS, Dale KJ, Kinsey CG, Richards JR, Shin D, Ghazi P, Onken MD, Blumer KJ, Odelberg SJ, McMahon M.
      PURPOSE: Mutational activation of GNAQ or GNA11 (GNAQ/11), detected in >90% of uveal melanomas, leads to constitutive activation of oncogenic pathways, including MAP kinase and YAP. To date, chemo- or pathway-targeted therapies, either alone or in combination, have proven ineffective in the treatment of metastatic uveal melanoma patients.EXPERIMENTAL DESIGN: We tested the efficacy of chloroquine or hydroxychloroquine, in combination with MAP kinase pathway inhibition in GNAQ/11-mutated cells in vitro and in vivo and identified mechanisms of MEK1/2 inhibitor plus chloroquine-induced cytotoxicity.
    RESULTS: Inhibition of GNAQ/11-mediated activation of MAP kinase signaling resulted in the induction of autophagy. Combined inhibition of Gα and autophagy or lysosome function resulted in enhanced cell death. Moreover, the combination of MEK1/2 inhibition, using trametinib, with the lysosome inhibitor, chloroquine, also increased cytotoxicity. Treatment of mice bearing GNAQ/11-driven melanomas with trametinib plus hydroxychloroquine resulted in inhibition of tumor growth and significantly prolonged survival. Interestingly, lysosomal- and autophagy-specific inhibition with bafilomycin A1 was not sufficient to promote cytotoxicity in combination with trametinib. However, the addition of YAP inhibition with trametinib plus bafilomycin A1 resulted in cell death at comparable levels to trametinib plus chloroquine (T/CQ) treatment. Furthermore, T/CQ-treated cells displayed decreased YAP nuclear localization and decreased YAP transcriptional activity. Expression of a constitutively active YAP5SA mutant conferred resistance to T/CQ-induced cell death.
    CONCLUSIONS: These results suggest that YAP, MEK1/2, and lysosome function are necessary and critical targets for the therapy of GNAQ/11-driven melanoma, and identify trametinib plus hydroxychloroquine as a potential treatment strategy for metastatic uveal melanoma.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-20-1675
  38. ESMO Open. 2020 Sep;pii: e000728. [Epub ahead of print]5(5):
    Kang BW, Chau I.
      The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway is involved in multiple cellular processes, including cell survival, proliferation, differentiation, metabolism and cytoskeletal reorganisation. The downstream effectors of this PI3K pathway are also essential for maintaining physiologic homeostasis, commonly dysregulated in most solid tumours. AKT is the key regulator in PI3K/AKT/mTOR signalling, interacting with multiple intracellular molecules. AKT activation subsequently leads to a number of potential downstream effects, and its aberrant activation results in the pathogenesis of cancer. Accordingly, as an attractive therapeutic target for cancer treatment, several AKT inhibitors are currently under development and in multiple stages of clinical trials for various types of malignancy, including gastric cancer (GC). Therefore, the authors review the significance of AKT and recent studies on AKT inhibitors in GC, focusing on the scientific background with the potential to improve treatment outcomes.
    Keywords:  AKT; gastric cancer
    DOI:  https://doi.org/10.1136/esmoopen-2020-000728
  39. Cancers (Basel). 2020 Sep 10. pii: E2579. [Epub ahead of print]12(9):
    Oelschlaegel D, Weiss Sadan T, Salpeter S, Krug S, Blum G, Schmitz W, Schulze A, Michl P.
      Stroma-infiltrating immune cells, such as tumor-associated macrophages (TAM), play an important role in regulating tumor progression and chemoresistance. These effects are mostly conveyed by secreted mediators, among them several cathepsin proteases. In addition, increasing evidence suggests that stroma-infiltrating immune cells are able to induce profound metabolic changes within the tumor microenvironment. In this study, we aimed to characterize the impact of cathepsins in maintaining the TAM phenotype in more detail. For this purpose, we investigated the molecular effects of pharmacological cathepsin inhibition on the viability and polarization of human primary macrophages as well as its metabolic consequences. Pharmacological inhibition of cathepsins B, L, and S using a novel inhibitor, GB111-NH2, led to changes in cellular recycling processes characterized by an increased expression of autophagy- and lysosome-associated marker genes and reduced adenosine triphosphate (ATP) content. Decreased cathepsin activity in primary macrophages further led to distinct changes in fatty acid metabolites associated with increased expression of key modulators of fatty acid metabolism, such as fatty acid synthase (FASN) and acid ceramidase (ASAH1). The altered fatty acid profile was associated with an increased synthesis of the pro-inflammatory prostaglandin PGE2, which correlated with the upregulation of numerous NFkB-dependent pro-inflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNFα). Our data indicate a novel link between cathepsin activity and metabolic reprogramming in macrophages, demonstrated by a profound impact on autophagy and fatty acid metabolism, which facilitates a pro-inflammatory micromilieu generally associated with enhanced tumor elimination. These results provide a strong rationale for therapeutic cathepsin inhibition to overcome the tumor-promoting effects of the immune-evasive tumor micromilieu.
    Keywords:  activity-based probes; autophagy; cathepsin; inflammation; lipid metabolism; lysosome; tumor-associated macrophage
    DOI:  https://doi.org/10.3390/cancers12092579
  40. J Cell Mol Med. 2020 Sep 15.
    Al-Bari MAA.
      The catabolic autophagy eliminates cytoplasmic components and organelles via lysosomes. Non-selective bulk autophagy and selective autophagy (mitophagy) are linked in intracellular homeostasis both normal and cancer cells. Autophagy has complex and paradoxical dual role in cancers; it can play either tumour suppressor or tumour promoter depending on the tumour type, stage, microenvironment and genetic context. Cancer stem cells (CSCs) cause tumour recurrence and promote resistant to therapy for driving poor clinical consequences. Thus, new healing strategies are urgently needed to annihilate and eradicate CSCs. As chloroquine (CQ) analogues show positive clinical outcome in several clinical trials either standalone or combination with several chemotherapies. Moreover, CQ analogues are known to eliminate CSCs via altering DNA methylation. However, several obstacles such as higher concentrations and dose-dependent toxicity are noticeable in the treatment of cancers. As tumour cells predominantly rely on mitochondrial actions, mitochondrial targeting FDA-approved antibiotics are reported to effectively eradicate CSCs alone or combination with chemotherapy. However, antibiotics cause metabolic glycolytic shift in cancer cells for survival and repopulation. This review will provide a sketch of the inhibiting roles of current chloroquine analogues and antibiotic combination in CSC autophagy process and discuss the possibility that pre-clinical and clinical potential therapeutic strategy for anticancer therapy.
    Keywords:  CSCs therapy; antibiotics; autophagy; chloroquine analogues; drug repurposing; mitochondrial target
    DOI:  https://doi.org/10.1111/jcmm.15879
  41. Elife. 2020 Sep 17. pii: e60541. [Epub ahead of print]9
    Aikin TJ, Peterson AF, Pokrass MJ, Clark HR, Regot S.
      A large fraction of human cancers contain genetic alterations within the Mitogen Activated Protein Kinase (MAPK) signaling network that promote unpredictable phenotypes. Previous studies have shown that the temporal patterns of MAPK activity (i.e. signaling dynamics) differentially regulate cell behavior. However, the role of signaling dynamics in mediating the effects of cancer driving mutations has not been systematically explored. Here, we show that oncogene expression leads to either pulsatile or sustained ERK activity that correlate with opposing cellular behaviors (i.e. proliferation vs. cell cycle arrest, respectively). Moreover, sustained-but not pulsatile-ERK activity triggers ERK activity waves in unperturbed neighboring cells that depend on the membrane metalloprotease ADAM17 and EGFR activity. Interestingly, the ADAM17-EGFR signaling axis coordinates neighboring cell migration toward oncogenic cells and is required for oncogenic cell extrusion. Overall, our data suggests that the temporal patterns of MAPK activity differentially regulate cell autonomous and non-cell autonomous effects of oncogene expression.
    Keywords:  biosensors; cancer biology; cell biology; human; live-cell imaging; signaling dynamics
    DOI:  https://doi.org/10.7554/eLife.60541
  42. Molecules. 2020 Sep 10. pii: E4136. [Epub ahead of print]25(18):
    Yang H, Heyer J, Zhao H, Liang S, Guo R, Zhong L.
      (1) Background: Cathepsin K has been found overexpressed in several malignant tumors. However, there is little information regarding the involvement of Cathepsin K in non-small cell lung cancer (NSCLC). (2) Methods: Cathepsin K expression was tested in human NSCLC cell lines A549 and human embryo lung fibroblast MRC-5 cells using Western blot and immunofluorescence assay. Cathepsin K was transiently overexpressed or knocked down using transfection with a recombinant plasmid and siRNA, respectively, to test the effects on cell proliferation, migration, invasion, and on the mammalian target of rapamycin (mTOR) signaling pathway. (3) Results: Expression of Cathepsin K was increased significantly in A549 cells and diffused within the cytoplasm compared to the MRC-5 cells used as control. Cathepsin K overexpression promoted the proliferation, migration, and invasion of A549 cells, accompanied by mTOR activation. Cathepsin K knockdown reversed the above malignant behavior and inhibited the mTOR signaling activation, suggesting that Cathepsin K may promote the progression of NSCLC by activating the mTOR signaling pathway. (4) Conclusion: Cathepsin K may potentially represent a viable drug target for NSCLC treatment.
    Keywords:  Cathepsin K; NSCLC; cell invasion; cell migration; cell proliferation; mTOR
    DOI:  https://doi.org/10.3390/molecules25184136
  43. Sci Rep. 2020 Sep 16. 10(1): 15169
    Tanaka T, Warner BM, Odani T, Ji Y, Mo YQ, Nakamura H, Jang SI, Yin H, Michael DG, Hirata N, Suizu F, Ishigaki S, Oliveira FR, Motta ACF, Ribeiro-Silva A, Rocha EM, Atsumi T, Noguchi M, Chiorini JA.
      Primary Sjögren's syndrome (pSS) is a complex autoimmune disease characterized by dysfunction of secretory epithelia with only palliative therapy. Patients present with a constellation of symptoms, and the diversity of symptomatic presentation has made it difficult to understand the underlying disease mechanisms. In this study, aggregation of unbiased transcriptome profiling data sets of minor salivary gland biopsies from controls and Sjögren's syndrome patients identified increased expression of lysosome-associated membrane protein 3 (LAMP3/CD208/DC-LAMP) in a subset of Sjögren's syndrome cases. Stratification of patients based on their clinical characteristics suggested an association between increased LAMP3 expression and the presence of serum autoantibodies including anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies. In vitro studies demonstrated that LAMP3 expression induces epithelial cell dysfunction leading to apoptosis. Interestingly, LAMP3 expression resulted in the accumulation and release of intracellular TRIM21 (one component of SSA), La (SSB), and α-fodrin protein, common autoantigens in Sjögren's syndrome, via extracellular vesicles in an apoptosis-independent mechanism. This study defines a clear role for LAMP3 in the initiation of apoptosis and an independent pathway for the extracellular release of known autoantigens leading to the formation of autoantibodies associated with this disease.ClinicalTrials.gov Identifier: NCT00001196, NCT00001390, NCT02327884.
    DOI:  https://doi.org/10.1038/s41598-020-71669-5
  44. Mol Cell. 2020 Sep 09. pii: S1097-2765(20)30580-3. [Epub ahead of print]
    Chen X, Ariss MM, Ramakrishnan G, Nogueira V, Blaha C, Putzbach W, Islam ABMMK, Frolov MV, Hay N.
      Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.
    Keywords:  Akt1; Akt2; breast cancer; insulin; metastasis; neutrophils; therapy
    DOI:  https://doi.org/10.1016/j.molcel.2020.08.017
  45. Cell Rep. 2020 Sep 15. pii: S2211-1247(20)31140-2. [Epub ahead of print]32(11): 108151
    Chen K, Jiao X, Di Rocco A, Shen D, Xu S, Ertel A, Yu Z, Di Sante G, Wang M, Li Z, Pestell TG, Casimiro MC, Skordalakes E, Achilefu S, Pestell RG.
      Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates RB and functions as a collaborative nuclear oncogene. The serine threonine kinase Akt plays a pivotal role in the control of cellular metabolism, survival, and mitogenic signaling. Herein, Akt1-mediated phosphorylation of downstream substrates in the mammary gland is reduced by cyclin D1 genetic deletion and is induced by mammary-gland-targeted cyclin D1 overexpression. Cyclin D1 is associated with Akt1 and augments the rate of onset and maximal cellular Akt1 activity induced by mitogens. Cyclin D1 is identified in a cytoplasmic-membrane-associated pool, and cytoplasmic-membrane-localized cyclin D1-but not nuclear-localized cyclin D1-recapitulates Akt1 transcriptional function. These studies identify a novel extranuclear function of cyclin D1 to enhance proliferative functions via augmenting Akt1 phosphorylation at Ser473.
    Keywords:  Akt1; breast cancer; cyclin D1; phosphorylation
    DOI:  https://doi.org/10.1016/j.celrep.2020.108151