bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒12‒06
33 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. Biochem Biophys Res Commun. 2020 Oct 22. pii: S0006-291X(20)31950-1. [Epub ahead of print]
    Kuang F, Liu J, Li C, Kang R, Tang D.
      Ferroptosis is a type of non-apoptotic regulated cell death that involves excessive iron accumulation and subsequent lipid peroxidation. Although the antioxidant mechanisms of ferroptosis have been extensively studied recently, little is known about the interactions between the different organelles that control ferroptosis. Here, we show that the translocation of lysosomal cysteine protease cathepsin B (CTSB) into the nucleus is an important molecular event that mediates organelle-specific initiation of ferroptosis in human pancreatic cancer cells. Iron-dependent lysosomal membrane permeability triggers the release of CTSB from the lysosome to nucleus during ferroptosis. Mechanistically, nuclear CTSB accumulation causes DNA damage and subsequent activation of the stimulator of interferon response CGAMP interactor 1 (STING1/STING)-dependent DNA sensor pathway, which ultimately leads to autophagy-dependent ferroptosis. Consequently, the genetic inhibition of CTSB-dependent STING1 activation by RNAi prevents ferroptosis in cell culture and animal models. These new findings not only enhance our understanding of the mechanism by which organelles specifically trigger ferroptosis, but also may provide a potential way to enhance the anticancer activity of ferroptosis therapy.
    Keywords:  Autophagy; Cathepsin B; DNA damage; Ferroptosis; Lysosome; STING1
    DOI:  https://doi.org/10.1016/j.bbrc.2020.10.035
  2. Mol Cell. 2020 Nov 23. pii: S1097-2765(20)30786-3. [Epub ahead of print]
    Najafov A, Luu HS, Mookhtiar AK, Mifflin L, Xia HG, Amin PP, Ordureau A, Wang H, Yuan J.
      The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.
    Keywords:  AMPK; CASP8; MLKL; RIPK1; RIPK3; TSC2; lysosome; mTORC1; neonatal lethality
    DOI:  https://doi.org/10.1016/j.molcel.2020.11.008
  3. J Neurochem. 2020 Dec 01.
    Pang W, Hu F.
      The hexanucleotide repeat expansion (HRE) in the C9orf72 gene is the main cause of two tightly linked neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). HRE leads to not only a gain of toxicity from RNA repeats and dipeptide repeats but also reduced levels of C9orf72 protein. However, the cellular and physiological functions of C9orf72 were unknown until recently. Through proteomic analysis, Smith-Magenis Chromosome Regions 8 (SMCR8) and WD repeat-containing protein (WDR41) were identified as binding partners of C9orf72. These three proteins have been shown to form a tight complex, but the exact functions of this complex remain to be characterized. Both C9orf72 and SMCR8 contain a DENN domain, which has been shown to regulate the activities of small GTPases. The C9orf72 complex has been implicated in many cellular processes, including vesicle trafficking, lysosome homeostasis, mTORC1 signaling pathway, and autophagy. C9orf72 deficiency in mice results in exaggerated inflammatory responses and human patients with C9orf72 mutations have neuroinflammation phenotype. Recent studies indicate that C9orf72 regulates trafficking and lysosomal degradation of inflammatory mediators, including toll-like receptors (TLRs) and STING, to affect inflammatory outputs. Further exploration of cellular and physiological functions of C9orf72 will help dissect the pathological mechanism of ALS/FTD caused by C9orf72 mutations.
    Keywords:  ALS; C9orf72; FTLD; SMCR8; WDR41; autophagy; inflammation; lysosome; mTORC1
    DOI:  https://doi.org/10.1111/jnc.15255
  4. J Biol Chem. 2020 Dec 03. pii: jbc.RA120.014960. [Epub ahead of print]
    Beauchamp RL, Erdin S, Witt L, Jordan JT, Plotkin SR, Gusella JF, Ramesh V.
      Meningiomas (MN) arise from the arachnoid/meningeal layer and are non-responsive to chemotherapies, with ~50-60% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 signaling, leading to clinical trials for NF2 and meningioma. Recently our 'omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in both NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly NRG1/neuregulin 1, and confirm increased NRG1 secretion and activation of ERBB3 receptor tyrosine kinase to which NRG1 binds. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2 and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated basal mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a PDK1-dependent signaling mechanism independent of NRG1-ERBB3, but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of another receptor tyrosine kinase, IGF1R Drug treatment co-targeting mTORC1/2 and IGF1R/IR in NF2-null cells attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion of NRG1 and activation of ERBB3. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/IR, suggesting that co-targeting these pathways may prove effective for treatment of NF2-deficient meningioma.
    Keywords:  Akt PKB; NF2; NRG1-ERBB3; brain tumor; dual mTORC1/mTORC2 inhibition; insulin-like growth factor (IGF) receptor; mammalian target of rapamycin (mTOR); meningioma; signaling; tumor suppressor gene
    DOI:  https://doi.org/10.1074/jbc.RA120.014960
  5. Nat Commun. 2020 11 30. 11(1): 6088
    Zhou X, Zhong Y, Molinar-Inglis O, Kunkel MT, Chen M, Sun T, Zhang J, Shyy JY, Trejo J, Newton AC, Zhang J.
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth, nutrient and energy status cues to control cell growth and metabolism. While mTORC1 activation at the lysosome is well characterized, it is not clear how this complex is regulated at other subcellular locations. Here, we combine location-selective kinase inhibition, live-cell imaging and biochemical assays to probe the regulation of growth factor-induced mTORC1 activity in the nucleus. Using a nuclear targeted Akt Substrate-based Tandem Occupancy Peptide Sponge (Akt-STOPS) that we developed for specific inhibition of Akt, a critical upstream kinase, we show that growth factor-stimulated nuclear mTORC1 activity requires nuclear Akt activity. Further mechanistic dissection suggests that nuclear Akt activity mediates growth factor-induced nuclear translocation of Raptor, a regulatory scaffolding component in mTORC1, and localization of Raptor to the nucleus results in nuclear mTORC1 activity in the absence of growth factor stimulation. Taken together, these results reveal a mode of regulation of mTORC1 that is distinct from its lysosomal activation, which controls mTORC1 activity in the nuclear compartment.
    DOI:  https://doi.org/10.1038/s41467-020-19937-w
  6. Biol Pharm Bull. 2020 ;43(12): 1983-1986
    Iwahashi S, Tokumura K, Park G, Ochiai S, Okayama Y, Fusawa H, Ohta K, Fukasawa K, Iezaki T, Hinoi E.
      The mechanistic/mammalian target of rapamycin complex-1 (mTORC1) integrates multiple signaling pathways and regulates various cellular processes. Tuberous sclerosis complex 1 (Tsc1) and complex 2 (Tsc2) are critical negative regulators of mTORC1. Mouse genetic studies, including ours, have revealed that inactivation of mTORC1 in undifferentiated mesenchymal cells and chondrocytes leads to severe skeletal abnormalities, indicating a pivotal role for mTORC1 in skeletogenesis. Here, we show that hyperactivation of mTORC1 influences skeletal development through its expression in undifferentiated mesenchymal cells at the embryonic stage. Inactivation of Tsc1 in undifferentiated mesenchymal cells by paired-related homeobox 1 (Prx1)-Cre-mediated recombination led to skeletal abnormalities in appendicular skeletons. In contrast, Tsc1 deletion in chondrocytes using collagen type II α1 (Col2a1)-Cre or in osteoprogenitors using Osterix (Osx)-Cre did not result in skeletal defects in either appendicular or axial skeletons. These findings indicate that Tsc complex-mediated chronic overactivation of mTORC1 influences skeletal development at the embryonic stage through its expression in undifferentiated mesenchymal cells but not in chondrocytes or osteoprogenitors.
    Keywords:  mechanistic/mammalian target of rapamycin complex-1 (mTORC1); skeletogenesis; tuberous sclerosis complex 1; undifferentiated mesenchymal cell
    DOI:  https://doi.org/10.1248/bpb.b20-00619
  7. Diabetes. 2020 Dec 04. pii: db200474. [Epub ahead of print]
    Brouwers B, Coppola I, Vints K, Dislich B, Jouvet N, Van Lommel L, Segers C, Gounko NV, Thorrez L, Schuit F, Lichtenthaler SF, Estall JL, Declercq J, Ramos-Molina B, Creemers JWM.
      FURIN is a proprotein convertase (PC) responsible for proteolytic activation of a wide array of precursor proteins within the secretory pathway. It maps to the PRC1 locus, a type 2 diabetes susceptibility locus, yet its specific role in pancreatic β cells is largely unknown. The aim of this study was to determine the role of FURIN in glucose homeostasis. We show that FURIN is highly expressed in human islets, while PCs that potentially could provide redundancy are expressed at considerably lower levels. β cell-specific Furin knockout (βFurKO) mice are glucose intolerant, due to smaller islets with lower insulin content and abnormal dense core secretory granule morphology. mRNA expression analysis and differential proteomics on βFurKO islets revealed activation of Activating Transcription Factor 4 (ATF4), which was mediated by mammalian target of rapamycin C1 (mTORC1). βFurKO cells show impaired cleavage or shedding of the V-ATPase subunits Ac45 and prorenin receptor (PRR), respectively, and impaired lysosomal acidification. Blocking the V-ATPase pharmacologically in β cells increases mTORC1 activity, suggesting the involvement of the V-ATPase proton pump in the phenotype. Taken together, these results suggest a model of mTORC1-ATF4 hyperactivation and impaired lysosomal acidification in β cells lacking Furin, which causes β cell dysfunction.
    DOI:  https://doi.org/10.2337/db20-0474
  8. Trends Cell Biol. 2020 Nov 30. pii: S0962-8924(20)30226-9. [Epub ahead of print]
    Odle RI, Florey O, Ktistakis NT, Cook SJ.
      Autophagy and cap-dependent mRNA translation are tightly regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signalling complex in response to nutrient availability. However, the regulation of these processes, and mTORC1 itself, is different during mitosis, and this has remained an area of significant controversy; for example, studies have argued that autophagy is either repressed or highly active during mitosis. Recent studies have shown that autophagy initiation is repressed, and cap-dependent mRNA translation is maintained during mitosis despite mTORC1 activity being repressed. This is achieved in large part by a switch from mTORC1- to cyclin-dependent kinase 1 (CDK1)-mediated regulation. Here, we review the history and recent advances and seek to present a unifying model to inform the future study of autophagy and mTORC1 during mitosis.
    Keywords:  CDK1; autophagy; mTORC1; mitosis; translation
    DOI:  https://doi.org/10.1016/j.tcb.2020.11.001
  9. Fundam Clin Pharmacol. 2020 Dec 01.
    da Costa A, Metais T, Mouthon F, Kerkovich D, Charvériat M.
      TFEB is a mammalian transcription factor that binds directly to the CLEAR consensus sequence (5'-GTCACGTGAC-3') present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, lysosomal exocytosis and lysosomal housekeeping functions. By modulating these activities, TFEB coordinates on-demand control over each cell's degradation pathway. Thus, a nuclear signaling pathway regulates cellular energy metabolism through TFEB. Our growing understanding of the role of TFEB and CLEAR in the promotion of healthy clearance and subsequent in vitro and in vivo preclinical findings in various animal models of disease supports the conclusion that the pharmacological activation of TFEB could clear toxic proteins in multiple rare and common illnesses including pediatric and adult neurodegenerative disease.
    Keywords:  CNS disorders; TFEB; autophagy; lysosomal functions
    DOI:  https://doi.org/10.1111/fcp.12634
  10. Mol Oncol. 2020 Nov 30.
    Astanina E, Bussolino F, Doronzo G.
      Transcription factor EB (TFEB) represents an emerging player in cancer biology. Together with microphthalmia-associated transcription factor, transcription factor E3 and transcription factor EC, TFEB belongs to the microphthalmia family of bHLH-leucine zipper transcription factors that may be implicated in human melanomas, renal and pancreatic cancers. In particular, TFEB was originally described to be translocated in a juvenile subset of paediatric renal cell carcinoma, however, whole genome sequencing reported somatic mutations sporadically found in many different cancers. Besides its oncogenic activity, TFEB controls the autophagy-lysosomal pathway by recognizing a recurrent motif present in the promoter regions of a set of genes that participate in lysosome biogenesis, furthermore its dysregulation was found to have a crucial pathogenic role in different tumors by modulating the autophagy process. Other than to regulate cancer cell-autonomous responses, recent findings indicate that TFEB participates in the regulation of cellular functions of the tumor microenvironment. Here, we review the emerging role of TFEB in regulating cancer cell behaviour and choreographing tumor-microenvironment interaction. Recognizing TFEB as a hub of network of signals exchanged within the tumor between cancer and stroma cells provides a fresh perspective on the molecular principles of tumor self-organization, promising to unveil numerous new and potentially druggable vulnerabilities.
    Keywords:  angiogenesis; autophagy; cell-cycle; lysosome; metabolism; tumor microenvironment
    DOI:  https://doi.org/10.1002/1878-0261.12867
  11. FEBS Open Bio. 2020 Dec 04.
    Kobayashi M, Yasukawa H, Arikawa T, Deguchi Y, Mizushima N, Sakurai M, Onishi S, Tagawa R, Sudo Y, Okita N, Higashi K, Higami Y.
      Adipocytes, which comprise the majority of white adipose tissue (WAT), are involved in obesity-related pathology via various mechanisms, including disturbed lysosomal enzymatic activity and accumulation of oxidative stress. Sequestosome 1 (SQSTM1/p62) is an autophagy marker that participates in antioxidative responses via the activation of nuclear factor erythroid-derived 2-like 2 (NRF2). Trehalose is a non-reducing disaccharide reported to suppress adipocyte hypertrophy in obese mice and improve glucose tolerance in humans. We recently revealed that trehalose increases SQSTM1 levels and enhances antioxidative capacity in hepatocytes. Here, to further evaluate the mechanism behind the beneficial effects of trehalose on metabolism, we examined SQSTM1 levels, autophagy, and oxidative stress in trehalose-treated adipocytes. We initially confirmed that trehalose increases SQSTM1 transcription and protein levels without affecting autophagy in adipocytes. Trehalose also elevated transcription of several lysosomal genes and the activity of cathepsin L, a lysosomal enzyme, independently of the transcription factor EB. In agreement with our data from hepatocytes, trehalose induced the nuclear translocation of NRF2 and the transcription of its downstream antioxidative genes, resulting in reduced cellular reactive oxygen species levels. Moreover, some cellular trehalose was detected in trehalose-treated adipocytes, implying that extracellular trehalose is taken into cells. These observations reveal the mechanism behind the beneficial effects of trehalose on metabolism and suggest its potential for preventing or treating obesity-related pathology.
    Keywords:  Adipocyte; Lysosome; Oxidative Stress; SQSTM1; Trehalose
    DOI:  https://doi.org/10.1002/2211-5463.13055
  12. J Exp Clin Cancer Res. 2020 Nov 30. 39(1): 266
    Quan L, Ohgaki R, Hara S, Okuda S, Wei L, Okanishi H, Nagamori S, Endou H, Kanai Y.
      BACKGROUND: Tumor angiogenesis is regarded as a rational anti-cancer target. The efficacy and indications of anti-angiogenic therapies in clinical practice, however, are relatively limited. Therefore, there still exists a demand for revealing the distinct characteristics of tumor endothelium that is crucial for the pathological angiogenesis. L-type amino acid transporter 1 (LAT1) is well known to be highly and broadly upregulated in tumor cells to support their growth and proliferation. In this study, we aimed to establish the upregulation of LAT1 as a novel general characteristic of tumor-associated endothelial cells as well, and to explore the functional relevance in tumor angiogenesis.METHODS: Expression of LAT1 in tumor-associated endothelial cells was immunohistologically investigated in human pancreatic ductal adenocarcinoma (PDA) and xenograft- and syngeneic mouse tumor models. The effects of pharmacological and genetic ablation of endothelial LAT1 were examined in aortic ring assay, Matrigel plug assay, and mouse tumor models. The effects of LAT1 inhibitors and gene knockdown on cell proliferation, regulation of translation, as well as on the VEGF-A-dependent angiogenic processes and intracellular signaling were investigated in in vitro by using human umbilical vein endothelial cells.
    RESULTS: LAT1 was highly expressed in vascular endothelial cells of human PDA but not in normal pancreas. Similarly, high endothelial LAT1 expression was observed in mouse tumor models. The angiogenesis in ex/in vivo assays was suppressed by abrogating the function or expression of LAT1. Tumor growth in mice was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. LAT1-mediated amino acid transport was fundamental to support endothelial cell proliferation and translation initiation in vitro. Furthermore, LAT1 was required for the VEGF-A-dependent migration, invasion, tube formation, and activation of mTORC1, suggesting a novel cross-talk between pro-angiogenic signaling and nutrient-sensing in endothelial cells.
    CONCLUSIONS: These results demonstrate that the endothelial LAT1 is a novel key player in tumor angiogenesis, which regulates proliferation, translation, and pro-angiogenic VEGF-A signaling. This study furthermore indicates a new insight into the dual functioning of LAT1 in tumor progression both in tumor cells and stromal endothelium. Therapeutic inhibition of LAT1 may offer an ideal option to potentiate anti-angiogenic therapies.
    Keywords:  Amino acid transporter; Endothelial cell; Tumor angiogenesis; VEGF-A; mTORC1
    DOI:  https://doi.org/10.1186/s13046-020-01762-0
  13. Cancers (Basel). 2020 Nov 22. pii: E3476. [Epub ahead of print]12(11):
    Soond SM, Savvateeva LV, Makarov VA, Gorokhovets NV, Townsend PA, Zamyatnin AA.
      While viewed as the "guardian of the genome", the importance of the tumor suppressor p53 protein has increasingly gained ever more recognition in modulating additional modes of action related to cell death. Slowly but surely, its importance has evolved from a mutated genetic locus heavily implicated in a wide array of cancer types to modulating lysosomal-mediated cell death either directly or indirectly through the transcriptional regulation of the key signal transduction pathway intermediates involved in this. As an important step in determining the fate of cells in response to cytotoxicity or during stress response, lysosomal-mediated cell death has also become strongly interwoven with the key components that give the lysosome functionality in the form of the cathepsin proteases. While a number of articles have been published highlighting the independent input of p53 or cathepsins to cellular homeostasis and disease progression, one key area that warrants further focus is the regulatory relationship that p53 and its isoforms share with such proteases in regulating lysosomal-mediated cell death. Herein, we review recent developments that have shaped this relationship and highlight key areas that need further exploration to aid novel therapeutic design and intervention strategies.
    Keywords:  MOMP; apoptosis; cancer; cathepsin; lysosomal membrane permeabilization; p53
    DOI:  https://doi.org/10.3390/cancers12113476
  14. PLoS One. 2020 ;15(12): e0243006
    Cavender C, Mangini L, Van Vleet JL, Corado C, McCullagh E, Gray-Edwards HL, Martin DR, Crawford BE, Lawrence R.
      β-hexosaminidase is an enzyme responsible for the degradation of gangliosides, glycans, and other glycoconjugates containing β-linked hexosamines that enter the lysosome. GM2 gangliosidoses, such as Tay-Sachs and Sandhoff, are lysosomal storage disorders characterized by β-hexosaminidase deficiency and subsequent lysosomal accumulation of its substrate metabolites. These two diseases result in neurodegeneration and early mortality in children. A significant difference between these two disorders is the accumulation in Sandhoff disease of soluble oligosaccharide metabolites that derive from N- and O-linked glycans. In this paper we describe our results from a longitudinal biochemical study of a feline model of Sandhoff disease and an ovine model of Tay-Sachs disease to investigate the accumulation of GM2/GA2 gangliosides, a secondary biomarker for phospholipidosis, bis-(monoacylglycero)-phosphate, and soluble glycan metabolites in both tissue and fluid samples from both animal models. While both Sandhoff cats and Tay-Sachs sheep accumulated significant amounts of GM2 and GA2 gangliosides compared to age-matched unaffected controls, the Sandhoff cats having the more severe disease, accumulated larger amounts of gangliosides compared to Tay-Sachs sheep in their occipital lobes. For monitoring glycan metabolites, we developed a quantitative LC/MS assay for one of these free glycans in order to perform longitudinal analysis. The Sandhoff cats showed significant disease-related increases in this glycan in brain and in other matrices including urine which may provide a useful clinical tool for measuring disease severity and therapeutic efficacy. Finally, we observed age-dependent increasing accumulation for a number of analytes, especially in Sandhoff cats where glycosphingolipid, phospholipid, and glycan levels showed incremental increases at later time points without signs of peaking. This large animal natural history study for Sandhoff and Tay-Sachs is the first of its kind, providing insight into disease progression at the biochemical level. This report may help in the development and testing of new therapies to treat these disorders.
    DOI:  https://doi.org/10.1371/journal.pone.0243006
  15. Sci Rep. 2020 Dec 03. 10(1): 21160
    Gen S, Matsumoto Y, Kobayashi KI, Suzuki T, Inoue J, Yamamoto Y.
      Mutations in genes that encode components of tuberous sclerosis complex 2 (TSC2) are associated with tuberous sclerosis complex disease. TSC2 interacts with tuberous sclerosis complex 1 to form a complex that negatively regulates cell growth and proliferation via the inactivation of mechanistic target of rapamycin complex 1. The activity of TSC2 is mainly regulated via posttranslational modifications such as phosphorylation. However, the control of TSC2 activity is not entirely achieved by phosphorylation. In this study, we show that TSC2 is methylated at R1457 and R1459 by protein arginine methyltransferase 1 (PRMT1). Methylation of these two residues can affect the phosphorylation status through protein kinase B (Akt) of TSC2 at T1462 and is essential for TSC2 stability. Taken together, these findings indicate that novel posttranslational modifications are important for the regulation of TSC2 stability through PRMT1-mediated methylation.
    DOI:  https://doi.org/10.1038/s41598-020-78274-6
  16. J Cell Sci. 2020 Dec 01. pii: jcs.250241. [Epub ahead of print]
    Chatterjee S, Chakrabarty Y, Banerjee S, Ghosh S, Bhattacharyya SN.
      Defective intracellular trafficking and export of microRNAs have been observed in growth retarded mammalian cells having impaired mitochondrial potential and dynamics. Uncoupling Protein 2 mediated depolarization of mitochondrial membrane also results in progressive sequestration of microRNAs with polysomes and lowered their release via extracellular vesicles. Interestingly, impaired miRNA-trafficking process in growth retarded human cells could be reversed in presence of Genipin an inhibitor of Uncoupling Protein 2. Mitochondrial detethering of endoplasmic reticulum, observed in mitochondria depolarized cells, found to be responsible for defective compartmentalization of translation initiation factor eIF4E to endoplasmic reticulum attached polysomes. It causes retarded translation process accompanied by enhanced retention of miRNAs and target mRNAs with endoplasmic reticulum attached polysomes to restrict extracellular export of miRNAs. Reduced compartment specific activity of mTORC1 complex, the master regulator of protein synthesis, in mitochondria defective or ER- detethered cells, causes reduced phosphorylation of eIF4E-BP1 to prevent eIF-4E targeting to ER attached polysome and microRNA export. These data suggest how mitochondrial membrane potential and dynamics, by affecting mTORC1 activity and compartmentalization, determine sub-cellular localization and export of microRNAs.
    Keywords:  EIF4E and mTORC1; Exosomes; Extracellular vesicles; MiRNA; Mitochondria; P-body; Polysome; Processing bodies
    DOI:  https://doi.org/10.1242/jcs.250241
  17. J Cell Physiol. 2020 Dec 02.
    Liang Z, Zhang Q, Dong X, Zhang Z, Wang H, Zhang J, Zhao Y.
      The differentiation of mature medullary thymic epithelial cells (mTECs) is critical for the induction of central immune tolerance. Although the critical effect of mechanistic target of rapamycin complex 1 (mTORC1) in shaping mTEC differentiation has been studied, the regulatory role of mTORC2 in the differentiation and maturation of mTECs is poorly understood. We herein reported that TEC-specific ablation of a rapamycin-insensitive companion of mTOR (RICTOR), a key component of mTORC2, significantly decreased the thymus size and weight, the total cell number of TECs, and the cell number of mTECs with a smaller degree of reduced cortical thymic epithelial cells. Interestingly, RICTOR deficiency significantly accelerated the mTEC maturation process, as indicated by the increased ratios of mature mTECs (MHCIIhi , CD80+ , and Aire+ ) to immature mTECs (MHCIIlo , CD80- , and Aire- ) in Rictor-deficient mice. The RNA-sequencing assays showed that the upregulated nuclear factor-κB (NF-κB) signaling pathway in Rictor-deficient mTECs was one of the obviously altered pathways compared with wild-type mTECs. Our studies further showed that Rictor-deficient mTECs exhibited upregulated expression of receptor activator of NF-κB (RANK) and lymphotoxin β receptor (LTβR), as well as increased activity of canonical and noncanonical NF-κB signaling pathways as determined by ImageStream and Simple Western. Finally, our results showed that inhibition of NF-κB signaling pathways could partially reverse the accelerated maturation of mTECs in Rictor conditional KO mice. Thus, mTORC2 negatively controls the kinetics of the mTEC maturation process by inhibiting the LTβR/RANK-NF-κB signal axis.
    Keywords:  T cell development; mTORC2; thymic epithelial cells; thymus
    DOI:  https://doi.org/10.1002/jcp.30192
  18. Int J Mol Sci. 2020 Nov 25. pii: E8933. [Epub ahead of print]21(23):
    Neutel CHG, Hendrickx JO, Martinet W, De Meyer GRY, Guns PJ.
      BACKGROUND: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN).OBJECTIVE: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy.
    DATA SOURCES: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015-2020 (92%).
    CONCLUSION: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.
    Keywords:  aortic valve calcification; autophagy; lysosomes; valvular interstitial cell (VIC); vascular calcification; vascular smooth muscle cell (VSMC)
    DOI:  https://doi.org/10.3390/ijms21238933
  19. Int J Mol Sci. 2020 Nov 26. pii: E8979. [Epub ahead of print]21(23):
    Pallottini V, Pfrieger FW.
      Biomedical research aims to understand the molecular mechanisms causing human diseases and to develop curative therapies. So far, these goals have been achieved for a small fraction of diseases, limiting factors being the availability, validity, and use of experimental models. Niemann-Pick type C (NPC) is a prime example for a disease that lacks a curative therapy despite substantial breakthroughs. This rare, fatal, and autosomal-recessive disorder is caused by defects in NPC1 or NPC2. These ubiquitously expressed proteins help cholesterol exit from the endosomal-lysosomal system. The dysfunction of either causes an aberrant accumulation of lipids with patients presenting a large range of disease onset, neurovisceral symptoms, and life span. Here, we note general aspects of experimental models, we describe the line-up used for NPC-related research and therapy development, and we provide an outlook on future topics.
    Keywords:  C. elegans; Drosophila; cell culture; cholesterol; feline; induced pluripotent stem cells; lysosomal disorder; neurodegeneration; transgenic; zebrafish
    DOI:  https://doi.org/10.3390/ijms21238979
  20. FEBS Lett. 2020 Nov 29.
    Cheng X, Ge M, Zhu S, Li D, Wang R, Xu Q, Chen Z, Xie S, Liu H.
      Transplantation of in vitro-manipulated cells is widely used in hematology. While transplantation is well recognized to impose severe stress on transplanted cells, the nature of transplant-induced stress remains elusive. Here we propose that the lack of amino acids in serum is the major cause of transplant-induced stress. Mechanistically, amino acid deficiency decreases protein synthesis and nutrient consummation. However, in cells with overactive AKT and ERK, mTORC1 is not inhibited and protein synthesis remains relatively high. This impaired signaling causes nutrient depletion, cell cycle block, and eventually autophagy and cell death, which can be inhibited by cycloheximide or mTORC1 inhibitors. Thus, mTORC1-mediated amino acid signaling is critical in cell fate determination under transplant-induced stress, and protein synthesis inhibition can improve transplantation efficiency.
    Keywords:  amino acid; leukemia; mTORC1; metabolism; transplant-induced stress
    DOI:  https://doi.org/10.1002/1873-3468.14008
  21. Mol Genet Metab. 2020 Nov 18. pii: S1096-7192(20)30248-1. [Epub ahead of print]
    Sidhu R, Kell P, Dietzen DJ, Farhat NY, Do AND, Porter FD, Berry-Kravis E, Reunert J, Marquardt T, Giugliani R, Lourenço CM, Wang RY, Movsesyan N, Plummer E, Schaffer JE, Ory DS, Jiang X.
      Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3β,5α,6β-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-β-cyclodextrin (HPβCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPβCD treatment.
    Keywords:  2-hydroxypropyl-β-cyclodextrin; Bile acid; N-(3β,5α,6β-trihydroxy-cholan-24-oyl)glycine; Niemann-Pick disease type C; diagnosis; treatment assessment
    DOI:  https://doi.org/10.1016/j.ymgme.2020.11.005
  22. EMBO Rep. 2020 Dec 03. e51869
    Barz S, Kriegenburg F, Henning A, Bhattacharya A, Mancilla H, Sánchez-Martín P, Kraft C.
      Autophagy mediates the degradation of cytoplasmic material. Upon autophagy induction, autophagosomes form a sealed membrane around the cargo and fuse with the lytic compartment to release the cargo for degradation. In order to avoid premature fusion of immature autophagosomal membranes with the lytic compartment, this process needs to be tightly regulated. Several factors mediating autophagosome-vacuole fusion have recently been identified. In budding yeast, autophagosome-vacuole fusion requires the R-SNARE Ykt6 on the autophagosome, together with the three Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. However, how these SNAREs are regulated during the fusion process is poorly understood. In this study, we investigate the regulation of Ykt6. We found that Ykt6 is directly phosphorylated by Atg1 kinase, which keeps this SNARE in an inactive state. Ykt6 phosphorylation prevents SNARE bundling by disrupting its interaction with the vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion. These findings shed new light on the regulation of autophagosome-vacuole fusion and reveal a further step in autophagy controlled by the Atg1 kinase.
    Keywords:  Atg1; SNARE; Ykt6; autophagosome; autophagy
    DOI:  https://doi.org/10.15252/embr.202051869
  23. Autophagy. 2020 Nov 29.
    Singh SR, Meyer-Jens M, Alizoti E, Bacon WC, Davis G, Osinska H, Gulick J, Reischmann-Düsener S, Orthey E, McLendon PM, Molkentin JD, Schlossarek S, Robbins J, Carrier L.
      The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (UbG76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both UbG76V-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of Dapk2 and Fyco1, which are involved in autophagy. RNA-seq from Zfp418 knockdown also revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function.
    Keywords:  ALP; UPS; ZFP418; ZNF418; autophagy; cardiomyocyte proteasome; protein degradation; screen; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2020.1856493
  24. Front Med. 2020 Dec 01.
    Zhang J, Yin Y, Wang J, Zhang J, Liu H, Feng W, Yang W, Zetter B, Xu Y.
      The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer. The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells. The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth. Unsurprisingly, it is also one of the most commonly attempted targets for cancer therapy. FK506 binding protein 8 (FKBP8) is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function. We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a "switch" type regulator. We identified through immunoprecipitation-mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1 (PHB1) specifically interacts with FKBP8. Furthermore, the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway, whereas the FKBP8 level in the mitochondria was substantially reduced. Moreover, concomitant with these changes, the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1. Collectively, our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.
    Keywords:  FKBP8; cancer; cell proliferation; mTOR; prohibitin 1
    DOI:  https://doi.org/10.1007/s11684-020-0805-6
  25. J Pharmacol Exp Ther. 2020 Dec 04. pii: JPET-AR-2020-000385. [Epub ahead of print]
    Liu X, Jusko WJ.
      A semi-mechanistic physiologically-based pharmacokinetic (PBPK) model for chloroquine (CQ), a highly lysosomotropic weak base, was applied to digitized rat (Adelusi and Salako, 1982a) and human (Frisk-Holmberg et al, 1984) concentration versus time data. The PBPK model in rat featured plasma and RBC concentrations, extensive and apparent nonlinear tissue distribution, fitted hepatic and renal intrinsic clearances, and a plasma half-life of about 1 day. Tissue to plasma CQ ratios at 50 h after dosing were highest in lung, kidney, liver, and spleen (182-318) and lower in heart, muscle, brain, eye, and skin (11-66). The RBC to plasma ratio of 11.6 was assumed to reflect cell lipid partitioning. A lysosome-based extended model was used to calculate subcellular CQ concentrations based on tissue mass balances, fitted plasma, interstitial, and free cytosol concentrations, and literature-based pH and pKa values. The CQ tissue component concentrations ranked: lysosome >> acidic phospholipid > plasma = interstitial = cytosol {greater than or equal to} neutral lipids. The extensive lysosome sequestration appeared to change over time and was attributed to lowering pH values caused by proton pump influx of hydrogen ions. The man-to-rat volume of distribution (Vss) ratio of 7 used to scale rat tissue partitioning to man along with estimation of hepatic clearance allowed excellent fitting of oral dose PK (150-600 mg) of CQ with a 50-day half-life in man. The prolonged PK of chloroquine was well-characterized for rat and man with this PBPK model. The calculated intra-tissue concentrations and lysosomal effects have therapeutic relevance for CQ and other cationic drugs. Significance Statement Sequestration in lysosomes is a major factor controlling the pharmacokinetics and pharmacology of chloroquine and other cationic drugs. This report provides comprehensive physiologic modeling of chloroquine distribution in tissues and overall disposition in rat and man revealing expected complexities and inferences related to its subcellular association with various tissue components.
    Keywords:  kinetic modeling; pharmacokinetic; physiologically-based pharmacokinetics
    DOI:  https://doi.org/10.1124/jpet.120.000385
  26. FEBS Lett. 2020 Nov 28.
    Yuan Q, Chen M, Yang W, Xiao B.
      The morphological structure and metabolic activity of mitochondria are coordinately regulated by circadian mechanisms. However, the mechanistic interplay between circadian mechanisms and mitochondrial architecture remains poorly understood. Here, we demonstrate circadian rhythmicity of Rheb protein in liver, in line with that of Per2. Using genetic mouse models, we show that Rheb, a small GTPase that binds mTOR, is critical for circadian oscillation of mTORC1 activity in liver. Disruption of Rheb oscillation in hepatocytes by persistent expression of Rheb transgene interrupted mTORC1 oscillation. We further show that Rheb-regulated mTORC1 altered mitochondrial fission factor DRP1 in liver, leading to altered mitochondrial dynamics. Our results suggest that Rheb/mTORC1 regulated DRP1 oscillation involves ubiquitin-mediated proteolysis. This study identifies Rheb as a nodal point that couples circadian clock and mitochondrial architecture for optimal mitochondrial metabolism.
    Keywords:  Circadian clock; DRP1; Rheb/mTORC1; mitochondrial dynamics
    DOI:  https://doi.org/10.1002/1873-3468.14009
  27. Sci Transl Med. 2020 Dec 02. pii: eabb5413. [Epub ahead of print]12(572):
    Sondhi D, Kaminsky SM, Hackett NR, Pagovich OE, Rosenberg JB, De BP, Chen A, Van de Graaf B, Mezey JG, Mammen GW, Mancenido D, Xu F, Kosofsky B, Yohay K, Worgall S, Kaner RJ, Souwedaine M, Greenwald BM, Kaplitt M, Dyke JP, Ballon DJ, Heier LA, Kiss S, Crystal RG.
      Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.
    DOI:  https://doi.org/10.1126/scitranslmed.abb5413
  28. Biomolecules. 2020 Nov 25. pii: E1598. [Epub ahead of print]10(12):
    Topolska M, Roelants FM, Si EP, Thorner J.
      Membrane-tethered sterol-binding Lam/Ltc proteins localize at junctions between the endoplasmic reticulum (ER) membrane and other organelles. Two of the six family members-Lam2/Ltc4 (initially Ysp2) and paralog Lam4/Ltc3-localize to ER-plasma membrane (PM) contact sites (CSs) and mediate retrograde ergosterol transport from the PM to the ER. Our prior work demonstrated that Lam2 and Lam4 are substrates of TORC2-regulated protein kinase Ypk1, that Ypk1-mediated phosphorylation inhibits their function in retrograde sterol transport, and that PM sterol retention bolsters cell survival under stressful conditions. At ER-PM CSs, Lam2 and Lam4 associate with Laf1/Ymr102c and Dgr2/Ykl121w (paralogous WD40 repeat-containing proteins) that reportedly bind sterol. Using fluorescent tags, we found that Lam2 and Lam4 remain at ER-PM CSs when Laf1 and Dgr2 are absent, whereas neither Laf1 nor Dgr2 remain at ER-PM CSs when Lam2 and Lam4 are absent. Loss of Laf1 (but not Dgr2) impedes retrograde ergosterol transport, and a laf1∆ mutation does not exacerbate the transport defect of lam2∆ lam4∆ cells, indicating a shared function. Lam2 and Lam4 bind Laf1 and Dgr2 in vitro in a pull-down assay, and the PH domain in Lam2 hinders its interaction with Laf1. Lam2 phosphorylated by Ypk1, and Lam2 with phosphomimetic (Glu) replacements at its Ypk1 sites, exhibited a marked reduction in Laf1 binding. Thus, phosphorylation prevents Lam2 interaction with Laf1 at ER-PM CSs, providing a mechanism by which Ypk1 action inhibits retrograde sterol transport.
    Keywords:  ergosterol; homeostasis; membrane contact sites; protein kinases; regulation
    DOI:  https://doi.org/10.3390/biom10121598
  29. Curr Mol Med. 2020 Dec 03.
    Deng CY, Lv M, Luo BH, Zhao SZ, Mo ZC, Xie YJ.
      Male fertility is closely related to the normal function of the hypothalamic-pituitary-testicular axis. The testis is an important male reproductive organ that secretes androgen and produces sperm through spermatogenesis. Spermatogenesis refers to the process by which spermatogonial stem cells (SSCs) produce highly differentiated spermatozoa and is divided into three stages: mitosis, meiosis and spermiogenesis. Spermatogenesis requires SSCs to strike a proper balance between self-renewal and differentiation and the commitment of spermatocytes to meiosis, which involves many molecules and signalling pathways. Abnormal gene expression or signal transduction in the hypothalamus and pituitary, but particularly in the testis, may lead to spermatogenic disorders and male infertility. The phosphoinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathway is involved in many stages of male reproduction, including the regulation of the hypothalamus-pituitary-gonad (HPG) axis during spermatogenesis, the proliferation and differentiation of spermatogonia and somatic cells, and the regulation of sperm autophagy and testicular endocrine function in the presence of environmental pollutants, particularly endocrine-disrupting chemicals (EDCs). In the PI3K/AKT/mTOR signalling pathway, mTOR is considered the central integrator of several signals, regulating metabolism, cell growth and proliferation. In particular, mTOR plays an important role in the maintenance and differentiation of SSCs, as well as in regulating the redox balance and metabolic activity of Sertoli cells, which play an important role in nutritional support during spermatogenesis.
    Keywords:  Autophagy; HPG axis; PI3K/AKT/mTOR signalling; Spermatogenesis
    DOI:  https://doi.org/10.2174/1566524020666201203164910
  30. Int J Mol Sci. 2020 Dec 02. pii: E9189. [Epub ahead of print]21(23):
    Csolle MP, Ooms LM, Papa A, Mitchell CA.
      The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PtdIns(3,4,5)P3 can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P2. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.
    Keywords:  AKT; Src homology 2-containing inositol phosphatase 2 (SHIP2); breast cancer; inositol polyphosphate phosphatases; phosphatase tensin homolog deleted on chromosome 10 (PTEN); phosphoinositide 3-kinase (PI3K); proline rich inositol polyphosphate 5-phosphatase (PIPP); synaptojanin 2 (SYNJ2)
    DOI:  https://doi.org/10.3390/ijms21239189
  31. J Cell Sci. 2020 Dec 01. pii: jcs.250688. [Epub ahead of print]
    Capella M, Caballero LM, Pfander B, Braun S, Jentsch S.
      Misassembled nuclear pore complexes (NPCs) are removed by sealing off the surrounding nuclear envelope (NE), which is conducted by the ESCRT (endosomal sorting complexes required for transport) machinery. Recruitment of ESCRT proteins to the NE is mediated by the interaction between the ESCRT member Chm7 and the inner nuclear membrane protein Heh1, which belongs to the conserved LEM family. Increased ESCRT recruitment results in excessive membrane scission at damage sites but its regulation remains poorly understood. Here, we show that Hub1-mediated alternative splicing of HEH1 pre-mRNA, resulting into its shorter form Heh1-S, is critical for the integrity of the NE in Saccharomyces cerevisiae ESCRT-III mutants lacking Hub1 or Heh1-S display severe growth defects and accumulate improperly assembled NPCs. This depends on the interaction of Chm7 with the conserved MSC domain only present in the longer variant Heh1-L. Heh1 variants assemble into heterodimers and we demonstrate that a unique splice segment in Heh1-S suppresses growth defects associated with uncontrolled interaction between Heh1-L and Chm7. Together, our findings reveal that Hub1-mediated splicing generates Heh1-S to regulate ESCRT recruitment to the nuclear envelope.
    Keywords:  ESCRT; LEM domain; Nuclear envelope surveillance; Nuclear pore complex; Splicing
    DOI:  https://doi.org/10.1242/jcs.250688
  32. J Cell Sci. 2020 Dec 02. pii: jcs.246819. [Epub ahead of print]
    Das S, Maji S, Raj R, Bhattacharya I, Saha T, Naskar N, Gupta A.
      Wilson disease protein, ATP7B maintains copper homeostasis in the liver. ATP7B traffics from trans-Golgi network to endolysosomes to export excess copper. Regulation of ATP7B trafficking to and fro endolysosomes is not well understood. We investigated the fate of ATP7B, post-copper export. At high copper ATP7B traffics primarily to acidic, active hydrolase (Cathepsin-B) positive endolysosomes and upon subsequent copper chelation, returns to trans-Golgi network. At high copper, ATP7B co-localizes with endolysosomal markers and with core member of retromer complex, VPS35. Knocking down VPS35 did not abrogate copper export function of ATP7B or its copper-responsive anterograde trafficking to vesicles; rather upon subsequent copper chelation, ATP7B failed to relocalize to TGN that was rescued by overexpressing wtVPS35. Overexpressing mutants of retromer complex associated proteins, Rab7 and COMMD1 yielded similar non-recycling phenotype of ATP7B. At high copper, VPS35 and ATP7B are juxtaposed on the same endolysosome and form a large complex that is stabilized by in-vivo photoamino acid labeling and UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of copper transporter ATP7B and it is dependent upon intracellular copper.
    Keywords:  ATP7B; Copper metabolism; Endolysosome; Retromer; VPS35; Wilson disease
    DOI:  https://doi.org/10.1242/jcs.246819
  33. J Cell Sci. 2020 Dec 04. pii: jcs.251835. [Epub ahead of print]
    Evans AS, Lennemann NJ, Coyne CB.
      Autophagy is a degradative cellular pathway that targets cytoplasmic contents and organelles for turnover by the lysosome. Various autophagy pathways play key roles in the clearance of viral infections, and many families of viruses have developed unique methods for avoiding degradation. Some positive stranded RNA viruses, such as enteroviruses and flaviviruses, usurp the autophagic pathway to promote their own replication. We previously identified the endoplasmic reticulum-localized protein BPIFB3 as an important negative regulator of non-canonical autophagy that uniquely impacts the replication of enteroviruses and flaviviruses. Here, we find that many components of the canonical autophagy machinery are not required for BPIFB3 depletion induced autophagy and identify the host factors that facilitate its role in the replication of enteroviruses and flaviviruses. Using proximity-dependent biotinylation (BioID) followed by mass spectrometry, we identify ARFGAP1 and TMED9 as two cellular components that interact with BPIFB3 to regulate autophagy and viral replication. Importantly, our data demonstrate that non-canonical autophagy in mammalian cells can be controlled outside of the traditional pathway regulators and define the role of two proteins in BPIFB3 depletion mediated non-canonical autophagy.
    Keywords:  Autophagy; BPI-like proteins; BPIFB3; Enterovirus; Flavivirus
    DOI:  https://doi.org/10.1242/jcs.251835