bims-lysosi 2021-09-26 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–09–26
33 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Dissecting the biology of mTORC1 beyond rapamycin.
KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis.
Interleukin-6 mediates PSAT1 expression and serine metabolism in TSC2-deficient cells.
RORα Enhances Lysosomal Acidification and Autophagic Flux in the Hepatocytes.
GM1 Gangliosidosis-A Mini-Review.
Dysregulation of DPYSL2 expression by mTOR signaling in schizophrenia: multi-level study of postmortem brain.
Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells.
Dual mTORC1/2 inhibitor AZD2014 diminishes myeloid-derived suppressor cells accumulation in ovarian cancer and delays tumor growth.
Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.
Translational Aspects of the Mammalian Target of Rapamycin Complexes in Diabetic Nephropathy.
Structural basis of human PDZD8-Rab7 interaction for the ER-late endosome tethering.
Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.
STAT3 contributes to cisplatin resistance, modulating EMT markers, and the mTOR signaling in lung adenocarcinoma.
Germline and sporadic mTOR pathway mutations in low-grade oncocytic tumor of the kidney.
Heterozygous GBA D490V and ATP13a2 mutations do not exacerbate pathological α-synuclein spread in the prodromal preformed fibrils model in young mice.
Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense.
Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.
Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion.
Adipocyte-specific mTORC2 deficiency impairs BAT and iWAT thermogenic capacity without affecting glucose uptake and energy expenditure in cold-acclimated mice.
Proximity Interactome Map of the Vac14-Fig4 Complex Using BioID.
Co-treatment With Everolimus, an mTOR-Specific Antagonist, or Downregulation of ELK1 Enhances the Sensitivity of Pancreatic Cancer Cells to Genistein.
Vtc5 Is Localized to the Vacuole Membrane by the Conserved AP-3 Complex to Regulate Polyphosphate Synthesis in Budding Yeast.
Ablation of Collagen VI leads to the release of platelets with altered function.
Current progress in plant V-ATPase: From biochemical properties to physiological functions.
Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum.
Glucocerebrosidase Activity is not Associated with Parkinson's Disease Risk or Severity.
The enigma of phosphoinositides and their derivatives: Their role in regulation of subcellular compartment morphology.
Molecular mechanisms of endomembrane trafficking in plants.
SnapShot: Cancer metabolism.
Control of the eIF4E activity: structural insights and pharmacological implications.
Supply and demand: Cellular nutrient uptake and exchange in cancer.
AMPK: restoring metabolic homeostasis over space and time.
Longevity lost in translation.

Sci Signal. 2021 Sep 21. 14(701): eabe0161

Dissecting the biology of mTORC1 beyond rapamycin.

Guang Yang, Deanne Francis, James R Krycer, Mark Larance, Ziyang Zhang, Chris J Novotny, Alexis Diaz-Vegas, Kevan M Shokat, David E James.

[Figure: see text].

DOI: https://doi.org/10.1126/scisignal.abe0161
Mol Cell. 2021 Sep 14. pii: S1097-2765(21)00735-8. [Epub ahead of print]

KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis.

Hiroshi Senoo, Daisuke Murata, May Wai, Kenta Arai, Wakiko Iwata, Hiromi Sesaki, Miho Iijima.

  AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.

Keywords:  AKT; KRAS GTPase; PKA; RHOA GTPase; insulin; mTORC2

DOI:  https://doi.org/10.1016/j.molcel.2021.09.001
Proc Natl Acad Sci U S A. 2021 09 28. pii: e2101268118. [Epub ahead of print]118(39):

Interleukin-6 mediates PSAT1 expression and serine metabolism in TSC2-deficient cells.

Ji Wang, Harilaos Filippakis, Thomas Hougard, Heng Du, Chenyang Ye, Heng-Jia Liu, Long Zhang, Khadijah Hindi, Shefali Bagwe, Julie Nijmeh, John M Asara, Wei Shi, Souheil El-Chemaly, Elizabeth P Henske, Hilaire C Lam.

  Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2 Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking up-regulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed up-regulation of IL-6 transcription and secretion compared to wild-type cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wild-type cells. Treatment with anti-IL-6 (αIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2 +/- mice while reducing PSAT1 expression. These data reveal a mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.

Keywords:  interleukin 6; lymphangioleiomyomatosis; mTORC1; phosphoserine aminotransferase 1 (PSAT1); tuberous sclerosis complex

DOI:  https://doi.org/10.1073/pnas.2101268118
Hepatol Commun. 2021 Aug 24.

RORα Enhances Lysosomal Acidification and Autophagic Flux in the Hepatocytes.

Hyeon-Ji Kim, Yong-Hyun Han, Ju-Yeon Kim, Mi-Ock Lee.

Lysosomes are intracellular acidic organelles with catabolic functions that contribute to the activation of autophagy. Although autophagy abnormality is associated with defects in lysosomal acidification during the progression of nonalcoholic fatty liver disease (NAFLD), the mechanisms of control of lysosomal acidification are not well understood at the molecular level. Thus, we aimed to elucidate the role of the orphan nuclear receptor retinoic acid-related orphan receptor α (RORα) in lysosomal acidification and autophagic flux, particularly in nutrition-enriched hepatocytes. First, lysosomal acidity was much lower in the hepatocytes obtained from hepatocyte-specific RORα-deleted (RORα-LKO) mice, whereas the infusion of an adenovirus encoding RORα in wild-type hepatocytes increased lysosomal acidity, as determined by LysoSensor. Second, the lysosomal translocation of the mechanistic target of rapamycin was increased and immature cathepsin D was accumulated in the liver of RORα-LKO mice. Third, the accumulation of LC3-II, p62/sequestosome 1 (SQSTM1), and neighbor of BRCA1 gene 1 (NBR1) was increased in the livers of RORα-LKO mice, indicating an impaired autophagic flux in the livers. Consistently, the number of autolysosomes containing mitochondria and lipid droplets was dramatically reduced in the RORα-deleted hepatocytes. Finally, we found that RORα induced the transcription of genes involved in lysosomal function, such as Atp6v1g1, a vacuolar H+ -ATPase (v-ATPase) subunit, which were largely down-regulated in the livers of mice with high-fat diet-induced NAFLD and patients with hepatitis. Conclusion: Targeting RORα may be a potential therapeutic strategy to restore lysosomal acidification, which inhibits the progression of NAFLD.

DOI: https://doi.org/10.1002/hep4.1785
Front Genet. 2021 ;12 734878

GM1 Gangliosidosis-A Mini-Review.

Elena-Raluca Nicoli, Ida Annunziata, Alessandra d'Azzo, Frances M Platt, Cynthia J Tifft, Karolina M Stepien.

  GM1 gangliosidosis is a progressive, neurosomatic, lysosomal storage disorder caused by mutations in the GLB1 gene encoding the enzyme β-galactosidase. Absent or reduced β-galactosidase activity leads to the accumulation of β-linked galactose-containing glycoconjugates including the glycosphingolipid (GSL) GM1-ganglioside in neuronal tissue. GM1-gangliosidosis is classified into three forms [Type I (infantile), Type II (late-infantile and juvenile), and Type III (adult)], based on the age of onset of clinical symptoms, although the disorder is really a continuum that correlates only partially with the levels of residual enzyme activity. Severe neurocognitive decline is a feature of Type I and II disease and is associated with premature mortality. Most of the disease-causing β-galactosidase mutations reported in the literature are clustered in exons 2, 6, 15, and 16 of the GLB1 gene. So far 261 pathogenic variants have been described, missense/nonsense mutations being the most prevalent. There are five mouse models of GM1-gangliosidosis reported in the literature generated using different targeting strategies of the Glb1 murine locus. Individual models differ in terms of age of onset of the clinical, biochemical, and pathological signs and symptoms, and overall lifespan. However, they do share the major abnormalities and neurological symptoms that are characteristic of the most severe forms of GM1-gangliosidosis. These mouse models have been used to study pathogenic mechanisms, to identify biomarkers, and to evaluate therapeutic strategies. Three GLB1 gene therapy trials are currently recruiting Type I and Type II patients (NCT04273269, NCT03952637, and NCT04713475) and Type II and Type III patients are being recruited for a trial utilizing the glucosylceramide synthase inhibitor, venglustat (NCT04221451).

Keywords:  GM1 gangliosidosis; beta galactosidase; gene therapy; glycoconjugates metabolism; mouse model

DOI:  https://doi.org/10.3389/fgene.2021.734878
Neurosci Res. 2021 Sep 17. pii: S0168-0102(21)00206-6. [Epub ahead of print]

Dysregulation of DPYSL2 expression by mTOR signaling in schizophrenia: multi-level study of postmortem brain.

Ryuta Izumi, Mizuki Hino, Atsuko Nagaoka, Risa Shishido, Akiyoshi Kakita, Mikio Hoshino, Yasuto Kunii, Hirooki Yabe.

  The mechanistic target of rapamycin (mTOR)-signaling and dihydropyrimidinase-like 2 (DPYSL2), which are increasingly gaining attention as potential therapeutic targets for schizophrenia, are connected via Cap-dependent translation of the 5'TOP motif. We quantified the expression of molecules constituting the mTOR-signaling and DPYSL2 in the prefrontal cortex (PFC) and superior temporal gyrus (STG) of postmortem brain tissue samples from 24 patients with schizophrenia and 32 control individuals and conducted association analysis to examine abnormal regulation of DPYSL2 expression by the mTOR-signaling in schizophrenia. The average ribosomal protein S6 (S6) levels in the PFC and STG were lower in patients with schizophrenia (p < 0.01). DPYSL2 expression showed a significant positive correlation with phospho-S6 expression levels, which were effectors of mTOR translational regulation, and the correlation slope between phospho-S6 and DPYSL2 expressions differed between cases and controls. Association analyses of these mTOR-signaling and DPYSL2 alterations with genetic polymorphisms and the clinical profile suggested that certain genetic variants of DPYSL2 require high mTOR-signaling activity. Thus, the findings confirmed decreased S6 expression levels in schizophrenia and supported the relationship between the mTOR-signaling and DPYSL2 via 5'TOP Cap-dependent translation, thus providing insights connecting the two major schizophrenia treatment strategies associated with the mTOR-signaling and DPYSL2.

Keywords:  DPYSL2; RPS6; mTOR; postmortem-brain; schizophrenia

DOI:  https://doi.org/10.1016/j.neures.2021.09.004
FASEB J. 2021 Oct;35(10): e21909

Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells.

Sung Eun Kim, Justin Zhang, Enoch Jiang, Michael Overholtzer.

  Metabolic stress contributes to the regulation of cell death in normal and diseased tissues. While different forms of cell death are known to be regulated by metabolic stress, how the cell engulfment and killing mechanism entosis is regulated is not well understood. Here we find that the death of entotic cells is regulated by the presence of amino acids and activity of the mechanistic target of rapamycin (mTOR). Amino acid withdrawal or mTOR inhibition induces apoptosis of engulfed cells and blocks entotic cell death that is associated with the lipidation of the autophagy protein microtubule-associated protein light chain 3 (LC3) to entotic vacuoles. Two other live cell engulfment programs, homotypic cell cannibalism (HoCC) and anti-CD47 antibody-mediated phagocytosis, known as phagoptosis, also undergo a similar vacuole maturation sequence involving LC3 lipidation and lysosome fusion, but only HoCC involves mTOR-dependent regulation of vacuole maturation and engulfed cell death similar to entosis. We further find that the regulation of cell death by mTOR is independent of autophagy activation and instead involves the 4E-BP1/2 proteins that are known regulators of mRNA translation. Depletion of 4E-BP1/2 proteins can restore the mTOR-regulated changes of entotic death and apoptosis rates of engulfed cells. These results identify amino acid signaling and the mTOR-4E-BP1/2 pathway as an upstream regulation mechanism for the fate of live engulfed cells formed by entosis and HoCC.

Keywords:  amino acids; cell death; entosis; mTOR; metabolism

DOI:  https://doi.org/10.1096/fj.202100870R
Cancer Lett. 2021 Sep 21. pii: S0304-3835(21)00464-X. [Epub ahead of print]

Dual mTORC1/2 inhibitor AZD2014 diminishes myeloid-derived suppressor cells accumulation in ovarian cancer and delays tumor growth.

Ruyu Pi, Yang Yang, Xiaoyi Hu, Hongyi Li, Houhui Shi, Yu Liu, Xi Wang, An Tong, Tianqi Lu, Yuquan Wei, Xia Zhao, Xiawei Wei.

  Mechanistic target of rapamycin (mTOR) forms two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. Here we investigated the antitumor effect of dual mTORC1/2 inhibitor AZD2014 on epithelial ovarian cancer (EOC) and its potential effect on immunosuppressive myeloid-derived suppressor cells (MDSCs). Immunohistochemical analysis of mTORC1 and mTORC2 was performed on a human ovarian cancer tissue microarray. High mTORC2 expression level was associated with shorter survival in EOC, whereas mTORC1 was not correlate with patients' prognosis. AZD2014 suppressed mTOR signaling pathway in ovarian cancer cells, inhibited proliferation and induced G1-phase cell cycle arrest and apoptosis. In tumor-bearing mice, AZD2014 treatment limited tumor growth, reduced peritoneal ascites, and prolonged survival. AZD2014 specifically reduced MDSCs migration and accumulation in EOC peritoneal fluid but not in the spleen. Moreover, subsequent AZD2014 treatment after cisplatin chemotherapy delayed EOC recurrence. Collectively, we observed that high mTORC2 expression level in EOC indicated a poor prognosis. Remarkably, in tumor-bearing mice, AZD2014 diminished MDSC accumulation and delayed tumor growth and recurrence.

Keywords:  MDSC; Ovarian cancer; mTOR inhibitor

DOI:  https://doi.org/10.1016/j.canlet.2021.09.017
mBio. 2021 Sep 21. e0229921

Anaplasma phagocytophilum Hijacks Flotillin and NPC1 Complex To Acquire Intracellular Cholesterol for Proliferation, Which Can Be Inhibited with Ezetimibe.

Weiyan Huang, Qingming Xiong, Mingqun Lin, Yasuko Rikihisa.

  The intracellular cholesterol transport protein Niemann-Pick type C1 (NPC1) and lipid-raft protein flotillin (FLOT) are required for cholesterol uptake by the obligatory intracellular bacterium Anaplasma phagocytophilum and for infection, and each protein localizes to membrane-bound inclusions containing replicating bacteria. Here, we found striking localization of FLOT2 in NPC1-lined vesicles and a physical interaction between FLOT2 and NPC1. This interaction was cholesterol dependent, as a CRAC (cholesterol recognition/interaction amino acid cholesterol-binding) domain mutant of FLOT2 did not interact with NPC1, and the cholesterol-sequestering agent methyl-β-cyclodextrin reduced the interaction. The stomatin-prohibitin-flotillin-HflC/K domain of FLOT2, FLOT21-183, was sufficient for the unique FLOT2 localization and interaction with NPC1. NPC1, FLOT2, and FLOT21-183 trafficked to the lumen of Anaplasma inclusions. A loss-of-function mutant, NPC1P691S (mutation in the sterol-sensing domain), did not colocalize or interact with FLOT2 or with Anaplasma inclusions and inhibited infection. Ezetimibe is a drug that blocks cholesterol absorption in the small intestine by inhibiting plasma membrane Niemann-Pick C1-like 1 interaction with FLOTs. Ezetimibe blocked the interaction between NPC1 and FLOT2 and inhibited Anaplasma infection. Ezetimibe did not directly inhibit Anaplasma proliferation but inhibited host membrane lipid and cholesterol traffic to the bacteria in the inclusion. These data suggest that Anaplasma hijacks NPC1 vesicles containing cholesterol bound to FLOT2 to deliver cholesterol into Anaplasma inclusions to assimilate cholesterol for its proliferation. These results provide insights into mechanisms of intracellular cholesterol transport and a potential approach to inhibit Anaplasma infection by blocking cholesterol delivery into the lumen of bacterial inclusions. IMPORTANCE Cholesterol influences membrane fluidity and forms membrane microdomains called lipid rafts that serve as organizing centers for the assembly of signaling molecules. Flotillin (FLOT) is a cholesterol-binding lipid-raft protein. The cholesterol-binding membrane glycoprotein Niemann-Pick type C1 (NPC1) is critical for managing cellular cholesterol level and its intracellular transport, and mutation of the gene encoding NPC1 causes the fatal cholesterol storage disease, Niemann-Pick disease, type C. Both FLOT and NPC1 are trafficked to inclusions created by the cholesterol-dependent bacterium Anaplasma phagocytophilum and required for cholesterol uptake by this bacterium for replication. Our novel findings that FLOT2 interacts physically with NPC1 and resides inside both bacterial inclusions and NPC1-containing vesicles underscore the important role for FLOT2 in infection, the intracellular transport of cholesterol in NPC1 vesicles, and cholesterol homeostasis. Both NPC1-FLOT2 interaction and A. phagocytophilum infection can be inhibited by ezetimibe, suggesting possible pharmacological intervention of intracellular cholesterol hijacking by Anaplasma.

Keywords:  Anaplasma; NPC1; cholesterol; ezetimibe; flotillin; intracellular bacteria; intracellular cholesterol transport

DOI:  https://doi.org/10.1128/mBio.02299-21
Antioxid Redox Signal. 2021 Sep 21.

Translational Aspects of the Mammalian Target of Rapamycin Complexes in Diabetic Nephropathy.

Alaa Abou Daher, Sahar Alkhansa, William S Azar, Rim Rafeh, Hilda E Ghadieh, Assaad Antoine Eid.

SIGNIFICANCE: Despite the many efforts put into understanding diabetic nephropathy (DN), direct treatments for DN have yet to be discovered. Understanding the mechanisms behind DN is an essential step in the development of novel therapeutic regimens. The mTOR pathway has emerged as an important candidate in the quest for drug discovery because of its role in regulating growth, proliferation, as well as protein and lipid metabolism. Recent Advances: Kidney cells have been found to rely on basal autophagy for survival and for conserving kidney integrity. Recent studies have shown that diabetes induces renal autophagy deregulation, leading to kidney injury. Hyper-activation of the mTOR pathway and oxidative stress have been suggested to play a role in diabetes-induced autophagy imbalance.
CRITICAL ISSUES: A detailed understanding of the role of mTOR signaling in diabetes-associated complications is of major importance in the search for a cure. In this review, we provide evidence that mTOR is heavily implicated in diabetes-induced kidney injury. We suggest possible mechanisms through which mTOR exerts its negative effects by increasing insulin resistance, upregulating oxidative stress, and inhibiting autophagy.
FUTURE DIRECTIONS: Increased oxidative stress and autophagy deregulation are both deeply embedded in DN. However, the mechanisms controlling oxidative stress and autophagy are not well understood. While Akt/mTOR signaling seems to play an important role in oxidative stress and autophagy, further investigation is required to uncover the details of this signaling pathway.

DOI: https://doi.org/10.1089/ars.2021.0217
Sci Rep. 2021 Sep 22. 11(1): 18859

Structural basis of human PDZD8-Rab7 interaction for the ER-late endosome tethering.

Haider Khan, Lin Chen, Lingchen Tan, Young Jun Im.

The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8-Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC-Rab7 complex correlates with the recruitment of PDZD8 at the LE-ER interface and its role in lipid transport and regulation.

DOI: https://doi.org/10.1038/s41598-021-98419-5
Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00688-2. [Epub ahead of print]81(18): 3786-3802.e13

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Max-Hinderk Schuler, Alyssa M English, Tianyao Xiao, Thane J Campbell, Janet M Shaw, Adam L Hughes.

  Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Keywords:  MDC; Tom70; amino acid; lysosome; mitochondria; nutrient carrier; vacuole

DOI:  https://doi.org/10.1016/j.molcel.2021.08.021
Neoplasia. 2021 Sep 17. pii: S1476-5586(21)00068-3. [Epub ahead of print]23(10): 1048-1058

STAT3 contributes to cisplatin resistance, modulating EMT markers, and the mTOR signaling in lung adenocarcinoma.

Ana Paula Morelli, Tharcísio Citrângulo Tortelli, Mariana Camargo Silva Mancini, Isadora Carolina Betim Pavan, Luiz Guilherme Salvino Silva, Matheus Brandemarte Severino, Daniela Campos Granato, Nathalie Fortes Pestana, Luis Gustavo Saboia Ponte, Guilherme Francisco Peruca, Bianca Alves Pauletti, Daniel Francisco Guimarães Dos Santos, Leandro Pereira de Moura, Rosângela Maria Neves Bezerra, Adriana Franco Paes Leme, Roger Chammas, Fernando Moreira Simabuco.

  Lung cancer is the second leading cause of cancer death worldwide and is strongly associated with cisplatin resistance. The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells and coordinates critical cellular processes as survival, self-renewal, and inflammation. In several types of cancer, STAT3 controls the development, immunogenicity, and malignant behavior of tumor cells while it dictates the responsiveness to radio- and chemotherapy. It is known that STAT3 phosphorylation at Ser727 by mechanistic target of rapamycin (mTOR) is necessary for its maximal activation, but the crosstalk between STAT3 and mTOR signaling in cisplatin resistance remains elusive. In this study, using a proteomic approach, we revealed important targets and signaling pathways altered in cisplatin-resistant A549 lung adenocarcinoma cells. STAT3 had increased expression in a resistance context, which can be associated with a poor prognosis. STAT3 knockout (SKO) resulted in a decreased mesenchymal phenotype in A549 cells, observed by clonogenic potential and by the expression of epithelial-mesenchymal transition markers. Importantly, SKO cells did not acquire the mTOR pathway overactivation induced by cisplatin resistance. Consistently, SKO cells were more responsive to mTOR inhibition by rapamycin and presented impairment of the feedback activation loop in Akt. Therefore, rapamycin was even more potent in inhibiting the clonogenic potential in SKO cells and sensitized to cisplatin treatment. Mechanistically, STAT3 partially coordinated the cisplatin resistance phenotype via the mTOR pathway in non-small cell lung cancer. Thus, our findings reveal important targets and highlight the significance of the crosstalk between STAT3 and mTOR signaling in cisplatin resistance. The synergic inhibition of STAT3 and mTOR potentially unveil a potential mechanism of synthetic lethality to be explored for human lung cancer treatment.

Keywords:  Cisplatin; Lung cancer; Proteomics; Rapamycin; STAT3; mTOR

DOI:  https://doi.org/10.1016/j.neo.2021.08.003
Mod Pathol. 2021 Sep 20.

Germline and sporadic mTOR pathway mutations in low-grade oncocytic tumor of the kidney.

Payal Kapur, Ming Gao, Hua Zhong, Suneetha Chintalapati, Midori Mitui, Spencer D Barnes, Qinbo Zhou, Jeffrey Miyata, Deyssy Carrillo, Venkat S Malladi, Dinesh Rakheja, Ivan Pedrosa, Lin Xu, Lisa Kinch, James Brugarolas.

Low-grade oncocytic tumor (LOT) of the kidney is a recently described entity with poorly understood pathogenesis. Using next-generation sequencing (NGS) and complementary approaches, we provide insight into its biology. We describe 22 LOT corresponding to 7 patients presenting with a median age of 75 years (range 63-86 years) and male to female ratio 2:5. All 22 tumors demonstrated prototypical microscopic features. Tumors were well-circumscribed and solid. They were composed of sheets of tumor cells in compact nests. Tumor cells had eosinophilic cytoplasm, round to oval nuclei (without nuclear membrane irregularities), focal subtle perinuclear halos, and occasional binucleation. Sharply delineated edematous stromal islands were often observed. Tumor cells were positive for PAX8, negative for CD117, and exhibited diffuse and strong cytokeratin-7 expression. Six patients presented with pT1 tumors. At a median follow-up of 29 months, four patients were alive without recurrence (three patients had died from unrelated causes). All tumors were originally classified as chromophobe renal cell carcinoma, eosinophilic variant (chRCC-eo). While none of the patients presented with known syndromic features, one patient with multiple bilateral LOTs was subsequently found to have a likely pathogenic germline TSC1 mutation. Somatic, likely activating, mutations in MTOR and RHEB were identified in all other evaluable LOTs. As assessed by phospho-S6 and phospho-4E-BP1, mTOR complex 1 (mTORC1) was activated across all cases but to different extent. MTOR mutant LOT exhibited lower levels of mTORC1 activation, possibly related to mTORC1 dimerization and the preservation of a wild-type MTOR copy (retained chromosome 1). Supporting its distinction from related entities, gene expression analyses showed that LOT clustered separately from classic chRCC, chRCC-eo, and RO. In summary, converging mTORC1 pathway mutations, mTORC1 complex activation, and a distinctive gene expression signature along with characteristic phenotypic features support LOT designation as a distinct entity with both syndromic and non-syndromic cases associated with an indolent course.

DOI: https://doi.org/10.1038/s41379-021-00896-6
Neurobiol Dis. 2021 Sep 16. pii: S0969-9961(21)00262-X. [Epub ahead of print]159 105513

Heterozygous GBA D490V and ATP13a2 mutations do not exacerbate pathological α-synuclein spread in the prodromal preformed fibrils model in young mice.

Michaela E Johnson, Liza Bergkvist, Lucas Stetzik, Jennifer A Steiner, Lindsay Meyerdirk, Emily Schulz, Emily Wolfrum, Kelvin C Luk, Daniel W Wesson, Dimitri Krainc, Patrik Brundin.

  Autophagic dysregulation and lysosomal impairment have been implicated in the pathogenesis of Parkinson's disease, partly due to the identification of mutations in multiple genes involved in these pathways such as GBA, SNCA, ATP13a2 (also known as PARK9), TMEM175 and LRRK2. Mutations resulting in lysosomal dysfunction are proposed to contribute to Parkinson's disease by increasing α-synuclein levels, that in turn may promote aggregation of this protein. Here, we used two different genetic models-one heterozygous for a mutated form of the GBA protein (D409V), and the other heterozygous for an ATP13a2 loss-of-function mutation, to test whether these mutations exacerbate the spread of α-synuclein pathology following injection of α-synuclein preformed fibrils in the olfactory bulb of 12-week-old mice. Contrary to our hypothesis, we found that mice harboring GBA D409V+/- and ATP13a2+/- mutations did not have exacerbated behavioral impairments or histopathology (α-synuclein, LAMP2, and Iba1) when compared to their wildtype littermates. This indicates that in the young mouse brain, neither the GBA D409V mutation or ATP13a2 loss-of-function mutation accelerate the spread of α-synuclein pathology. As a consequence, we postulate that these mutations increase Parkinson's disease risk only by acting in one of the initial, upstream events in the Parkinson's disease pathogenic process. Further, the mutations, and the molecular pathways they impact, appear to play a less important role once the pathogenic process has been triggered and therefore do not specifically influence α-synuclein pathology spread.

Keywords:  ATP13a2; GBA; Olfactory bulb; Parkinson's disease; α-Synuclein preformed fibrils

DOI:  https://doi.org/10.1016/j.nbd.2021.105513
PLoS Pathog. 2021 Sep 23. 17(9): e1009943

Salmonella Typhimurium impairs glycolysis-mediated acidification of phagosomes to evade macrophage defense.

Saray Gutiérrez, Julia Fischer, Raja Ganesan, Nina Judith Hos, Gökhan Cildir, Martina Wolke, Alberto Pessia, Peter Frommolt, Vincenzo Desiderio, Vidya Velagapudi, Nirmal Robinson.

Regulation of cellular metabolism is now recognized as a crucial mechanism for the activation of innate and adaptive immune cells upon diverse extracellular stimuli. Macrophages, for instance, increase glycolysis upon stimulation with pathogen-associated molecular patterns (PAMPs). Conceivably, pathogens also counteract these metabolic changes for their own survival in the host. Despite this dynamic interplay in host-pathogen interactions, the role of immunometabolism in the context of intracellular bacterial infections is still unclear. Here, employing unbiased metabolomic and transcriptomic approaches, we investigated the role of metabolic adaptations of macrophages upon Salmonella enterica serovar Typhimurium (S. Typhimurium) infections. Importantly, our results suggest that S. Typhimurium abrogates glycolysis and its modulators such as insulin-signaling to impair macrophage defense. Mechanistically, glycolysis facilitates glycolytic enzyme aldolase A mediated v-ATPase assembly and the acidification of phagosomes which is critical for lysosomal degradation. Thus, impairment in the glycolytic machinery eventually leads to decreased bacterial clearance and antigen presentation in murine macrophages (BMDM). Collectively, our results highlight a vital molecular link between metabolic adaptation and phagosome maturation in macrophages, which is targeted by S. Typhimurium to evade cell-autonomous defense.

DOI: https://doi.org/10.1371/journal.ppat.1009943
Nat Microbiol. 2021 Sep 23.

Restriction factor compendium for influenza A virus reveals a mechanism for evasion of autophagy.

Laura Martin-Sancho, Shashank Tripathi, Ariel Rodriguez-Frandsen, Lars Pache, Maite Sanchez-Aparicio, Michael J McGregor, Kelsey M Haas, Danielle L Swaney, Thong T Nguyen, João I Mamede, Christopher Churas, Dexter Pratt, Sara B Rosenthal, Laura Riva, Courtney Nguyen, Nish Beltran-Raygoza, Stephen Soonthornvacharin, Guojun Wang, David Jimenez-Morales, Paul D De Jesus, Hong M Moulton, David A Stein, Max W Chang, Chris Benner, Trey Ideker, Randy A Albrecht, Judd F Hultquist, Nevan J Krogan, Adolfo García-Sastre, Sumit K Chanda.

The fate of influenza A virus (IAV) infection in the host cell depends on the balance between cellular defence mechanisms and viral evasion strategies. To illuminate the landscape of IAV cellular restriction, we generated and integrated global genetic loss-of-function screens with transcriptomics and proteomics data. Our multi-omics analysis revealed a subset of both IFN-dependent and independent cellular defence mechanisms that inhibit IAV replication. Amongst these, the autophagy regulator TBC1 domain family member 5 (TBC1D5), which binds Rab7 to enable fusion of autophagosomes and lysosomes, was found to control IAV replication in vitro and in vivo and to promote lysosomal targeting of IAV M2 protein. Notably, IAV M2 was observed to abrogate TBC1D5-Rab7 binding through a physical interaction with TBC1D5 via its cytoplasmic tail. Our results provide evidence for the molecular mechanism utilised by IAV M2 protein to escape lysosomal degradation and traffic to the cell membrane, where it supports IAV budding and growth.

DOI: https://doi.org/10.1038/s41564-021-00964-2
J Extracell Vesicles. 2021 Sep;10(11): e12146

Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion.

Lei Xi, Meixi Peng, Shuiqing Liu, Yongcan Liu, Xueying Wan, Yixuan Hou, Yilu Qin, Liping Yang, Shanchun Chen, Huan Zeng, Yong Teng, Xiaojiang Cui, Manran Liu.

  Cancer-associated fibroblasts (CAFs) as a predominant cell component in the tumour microenvironment (TME) play an essential role in tumour progression. Our earlier studies revealed oxidized ATM activation in breast CAFs, which is independent of DNA double-strand breaks (DSBs). Oxidized ATM has been found to serve as a redox sensor to maintain cellular redox homeostasis. However, whether and how oxidized ATM in breast CAFs regulates breast cancer progression remains poorly understood. In this study, we found that oxidized ATM phosphorylates BNIP3 to induce autophagosome accumulation and exosome release from hypoxic breast CAFs. Inhibition of oxidized ATM kinase by KU60019 (a small-molecule inhibitor of activated ATM) or shRNA-mediated knockdown of endogenous ATM or BNIP3 blocks autophagy and exosome release from hypoxic CAFs. We also show that oxidized ATM phosphorylates ATP6V1G1, a core proton pump in maintaining lysosomal acidification, leading to lysosomal dysfunction and autophagosome fusion with multi-vesicular bodies (MVB) but not lysosomes to facilitate exosome release. Furthermore, autophagy-associated GPR64 is enriched in hypoxic CAFs-derived exosomes, which stimulates the non-canonical NF-κB signalling to upregulate MMP9 and IL-8 in recipient breast cancer cells, enabling cancer cells to acquire enhanced invasive abilities. Collectively, these results provide novel insights into the role of stromal CAFs in promoting tumour progression and reveal a new function of oxidized ATM in regulating autophagy and exosome release.

Keywords:  autophagy; cancer-associated fibroblasts; exosomes; invasion; oxidized ATM

DOI:  https://doi.org/10.1002/jev2.12146
Am J Physiol Endocrinol Metab. 2021 Sep 20.

Adipocyte-specific mTORC2 deficiency impairs BAT and iWAT thermogenic capacity without affecting glucose uptake and energy expenditure in cold-acclimated mice.

Erique Castro, Thayna S Vieira, Tiago E Oliveira, Milene Ortiz-Silva, Maynara L Andrade, Caroline A Tomazelli, Albert S Peixoto, Cleyton R Sobrinho, Mayara F Moreno, Gustavo R Gilio, Rafael Junges Moreira, Raphael C Guimarães, Luiz Augusto Perandini, Patricia Chimin, Patricia Reckziegel, Eduardo H Moretti, Alexandre A Steiner, Mathieu Laplante, William T Festuccia.

  Deletion of mTORC2 essential component Rictor by a Cre recombinase under control of the broad, non-adipocyte specific aP2/FABP4 promoter impairs thermoregulation and brown adipose tissue (BAT) glucose uptake upon acute cold exposure. We investigated herein whether adipocyte-specific mTORC2 deficiency affects BAT and inguinal white adipose tissue (iWAT) signaling, metabolism and thermogenesis in cold-acclimated mice. For this, 8-weeks old male mice bearing Rictor deletion and therefore mTORC2 deficiency in adipocytes (adiponectin-Cre) and littermates controls were either kept at thermoneutrality (30 ± 1ºC) or cold-acclimated (10 ± 1ºC) for 14 days and evaluated for BAT and iWAT signaling, metabolism and thermogenesis. Cold acclimation inhibited mTORC2 in BAT and iWAT, but its residual activity is still required for the cold-induced increases in BAT adipocyte number, total UCP-1 content and mRNA levels of proliferation markers Ki67 and cyclin 1D and de novo lipogenesis enzymes ATP-citrate lyase and acetyl-CoA carboxylase. In iWAT, mTORC2 residual activity is partially required for the cold-induced increases in multilocular adipocytes, mitochondrial mass and UCP-1 content. Conversely, BAT mTORC1 activity and BAT and iWAT glucose uptake were upregulated by cold independently of mTORC2. Noteworthy, the impairment in BAT and iWAT total UCP-1 content and thermogenic capacity induced by adipocyte mTORC2 deficiency had no major impact on whole-body energy expenditure in cold-acclimated mice due to a compensatory activation of muscle shivering. In conclusion, adipocyte mTORC2 deficiency impairs, through different mechanisms, BAT and iWAT total UCP-1 content and thermogenic capacity in cold-acclimated mice, without affecting glucose uptake and whole-body energy expenditure.

Keywords:  brown adipose tissue (BAT); glucose uptake; mTORC2; subcutaneous inguinal white adipose tissue (iWAT); thermogenesis

DOI:  https://doi.org/10.1152/ajpendo.00587.2020
J Proteome Res. 2021 Sep 23.

Proximity Interactome Map of the Vac14-Fig4 Complex Using BioID.

Shirley Qiu, Mathieu Lavallée-Adam, Marceline Côté.

  Conversion between phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate on endosomal membranes is critical for the maturation of early endosomes to late endosomes/lysosomes and is regulated by the PIKfyve-Vac14-Fig4 complex. Despite the importance of this complex for endosomal homeostasis and vesicular trafficking, there is little known about how its activity is regulated or how it interacts with other cellular proteins. Here, we screened for the cellular interactome of Vac14 and Fig4 using proximity-dependent biotin labeling (BioID). After independently screening the interactomes of Vac14 and Fig4, we identified 89 high-confidence protein hits shared by both proteins. Network analysis of these hits revealed pathways with known involvement of the PIKfyve-Vac14-Fig4 complex, including vesicular organization and PI3K/Akt signaling, as well as novel pathways including cell cycle and mitochondrial regulation. We also identified subunits of coatomer complex I (COPI), a Golgi-associated complex with an emerging role in endosomal dynamics. Using proximity ligation assays, we validated the interaction between Vac14 and COPI subunit COPB1 and between Vac14 and Arf1, a GTPase required for COPI assembly. In summary, this study used BioID to comprehensively map the Vac14-Fig4 interactome, revealing potential roles for these proteins in diverse cellular processes and pathways, including preliminary evidence of an interaction between Vac14 and COPI. Data are available via ProteomeXchange with the identifier PXD027917.

Keywords:  BioID; COPI; Fig4; PCSF; PIKfyve; Vac14; endosomal trafficking; protein−protein interactions; proximity ligation assay

DOI:  https://doi.org/10.1021/acs.jproteome.1c00408
Front Cell Dev Biol. 2021 ;9 633035

Co-treatment With Everolimus, an mTOR-Specific Antagonist, or Downregulation of ELK1 Enhances the Sensitivity of Pancreatic Cancer Cells to Genistein.

Tianyu Li, Tiantao Kuang, Zhaoshuo Yang, Qiqi Zhang, Wen Zhang, Yue Fan.

  Genistein is a natural isoflavone with pharmacological or potentially anti-tumor properties. However, the resistance of cancer cells to genistein remains a major obstacle. This study focused on the mechanism implicated in the resistance of pancreatic cancer (PC) cells to genistein and the mechanism of action. First, key molecules and signaling pathways related to genistein resistance in PC cells were explored using bioinformatics tools. DEP domain containing MTOR interacting protein (DEPTOR), a typical inhibitor of the mammalian target of rapamycin (mTOR) signaling, was predicted to be poorly expressed in the genistein-resistant PC cells. Thereafter, genistein-resistant PC cells (Panc-1 and PaCa) were constructed. Altered expression of DEPTOR was introduced in cells, and everolimus (ELM), an mTOR-specific antagonist, was administrated in cells as well to examine their roles in genistein resistance. The cell apoptosis was examined in vitro and in vivo in mouse xenograft tumors. The upstream regulator of DEPTOR was predicted via bioinformatic tools. The bioinformatic analyses showed that the PI3K/AKT/mTOR signaling pathway was activated in the setting of DEPTOR downregulation in genistein-resistant PC cells. DEPTOR overexpression reduced the 50% inhibiting concentration (IC50) of genistein in PC cells and suppressed mTOR phosphorylation, and it increased caspase-3 activity, LDH release and apoptosis in PC cells. ELM treatment enhanced the sensitivity of PC cells to genistein in vitro and it strengthened the tumor-eliminating role of genistein in mice. ETS transcription factor ELK1 (ELK1), a transcription factor that negatively regulated DEPTOR transcription, was suppressed by genistein. Upregulation of ELK1 suppressed DEPTOR transcription and reduced the genistein sensitivity of cells, and it also blocked the genistein-sensitizing roles of ELM in PC cells. In conclusion, this study demonstrated that ELK1 reduces DEPTOR transcription, leading to mTOR phosphorylation and the drug resistance of PC cells.

Keywords:  DEPTOR; Elk1; everolimus; genistein; mTOR; pancreatic cancer

DOI:  https://doi.org/10.3389/fcell.2021.633035
mBio. 2021 Sep 21. e0099421

Vtc5 Is Localized to the Vacuole Membrane by the Conserved AP-3 Complex to Regulate Polyphosphate Synthesis in Budding Yeast.

Amanda Bentley-DeSousa, Michael Downey.

  Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3 residues to thousands of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound vacuolar transporter chaperone (VTC) complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen, where it is stored at high concentrations. VTC's activity is promoted by an accessory subunit called Vtc5. In this work, we found that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we found that Vtc5 is rerouted to the vacuole lumen by the endosomal sorting complex required for transport (ESCRT), where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes. IMPORTANCE Long polymers of inorganic phosphates called polyphosphates are ubiquitous across biological kingdoms. From bacteria to humans, they have diverse functions related to protein homeostasis, energy metabolism, and cell signaling. In this study, we provide new insights into the intracellular trafficking of the polyphosphate biosynthetic machinery in the budding yeast S. cerevisiae. The critical advances of the work are 2-fold. First, it provides an explanation for decreased polyphosphate levels observed in cells mutated for a conserved intracellular trafficking machine. Second, it defines critical pathways that are highly likely to serve as hubs for polyphosphate regulation in yeast and other species.

Keywords:  AP-3 complex; Apl5; ESCRT; Pep4; S. cerevisiae; VTC complex; Vtc5; budding yeast; polyP; polyphosphate; vacuole

DOI:  https://doi.org/10.1128/mBio.00994-21
Blood Adv. 2021 Sep 21. pii: bloodadvances.2020002671. [Epub ahead of print]

Ablation of Collagen VI leads to the release of platelets with altered function.

Vittorio Abbonante, Cristian Gruppi, Monica Battiston, Alessandra Zulian, Christian Andrea Di Buduo, Martina Chrisam, Lucia Sereni, Pierre-Alexandre Laurent, Claudio Semplicini, Elisabetta Lombardi, Mario Mazzucato, Francesco Moccia, Valeria Petronili, Anna Villa, Luca Bello, Elena Pegoraro, Paolo Bernardi, Paola Braghetta, Luigi De Marco, Paolo Bonaldo, Alessandra Balduini.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by Collagen VI-related disorders and investigated the defects in platelet functionality whose mechanisms are unknown. We demonstrated that megakaryocytes express Collagen VI that is involved in the regulation of functional platelet production. By exploiting a Collagen VI null mouse model (Col6a1-/-), we found that Collagen VI null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of STIM1 and ORAI1, the components of Store-Operated Calcium Entry (SOCE), and activation of the mTOR signaling pathway. In vivo mTOR inhibition by rapamycin reduced both STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell-autonomous, as transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation as compared to Col6a1-/- mice. Peripheral blood platelets of patients affected by Collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of Collagen VI in the production of functional platelets by megakaryocytes in mouse models and in Collagen VI-related diseases.

DOI: https://doi.org/10.1182/bloodadvances.2020002671
J Plant Physiol. 2021 Sep 15. pii: S0176-1617(21)00164-4. [Epub ahead of print]266 153525

Current progress in plant V-ATPase: From biochemical properties to physiological functions.

Chao Wang, Yun Xiang, Dong Qian.

  Vacuolar-type adenosine triphosphatase (V-ATPase, VHA) is a highly conserved, ATP-driven multisubunit proton pump that is widely distributed in all eukaryotic cells. V-ATPase consists of two domains formed by at least 13 different subunits, the membrane peripheral V1 domain responsible for ATP hydrolysis, and the membrane-integral V0 domain responsible for proton translocation. V-ATPase plays an essential role in energizing secondary active transport and is indispensable to plants. In addition to multiple stress responses, plant V-ATPase is also implicated in physiological processes such as growth, development, and morphogenesis. Based on the identification of distinct V-ATPase mutants and advances in luminal pH measurements in vivo, it has been revealed that this holoenzyme complex plays a pivotal role in pH homeostasis of the plant endomembrane system and endocytic and secretory trafficking. Here, we review recent progress in comprehending the biochemical properties and physiological functions of plant V-ATPase and explore the topics that require further elucidation.

Keywords:  Stress response; Structure; TGN/EE; V-ATPase; Vacuole; pH homeostasis

DOI:  https://doi.org/10.1016/j.jplph.2021.153525
ACS Sens. 2021 Sep 21.

Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum.

Yang Song, Jestin Gage Wright, Morgan J Anderson, Sabari Rajendran, Zhaoyang Ren, Duy H Hua, Jessica E Koehne, M Meyyappan, Jun Li.

  Proteases are critical signaling molecules and prognostic biomarkers for many diseases including cancer. There is a strong demand for multiplex bioanalytical techniques that can rapidly detect the activity of extracellular proteases with high sensitivity and specificity. This study demonstrates an activity-based electrochemical biosensor of a 3 × 3 gold microelectrode array for the detection of cathepsin B activity in human serum diluted in a neutral buffer. Proteolysis of ferrocene-labeled peptide substrates functionalized on 200 × 200 μm microelectrodes is measured simultaneously over the nine channels by AC voltammetry. The protease activity is represented by the inverse of the exponential decay time constant (1/τ), which equals to (kcat/KM)[CB] based on the Michaelis-Menten model. An enhanced activity of the recombinant human cathepsin B (rhCB) is observed in a low-ionic-strength phosphate buffer at pH = 7.4, giving a very low limit of detection of 8.49 × 10-4 s-1 for activity and 57.1 pM for the active rhCB concentration that is comparable to affinity-based enzyme-linked immunosorbent assay (ELISA). The cathepsin B presented in the human serum sample is validated by ELISA, which mainly detects the inactive proenzyme, while the electrochemical biosensor specifically measures the active cathepsin B and shows significantly higher decay rates when rhCB and human serum are activated. Analyses of the kinetic electrochemical measurements with spiked active cathepsin B in human serum provide further assessment of the protease activity in the complex sample. This study lays the foundation to develop the gold microelectrode array into a multiplex biosensor for rapid detection of the activity of extracellular proteases toward cancer diagnosis and treatment assessment.

Keywords:  cathepsin B; enzyme-linked immunosorbent assay; extracellular protease analysis; human serum; microelectrode array; multiplex detection; protease activity profiling

DOI:  https://doi.org/10.1021/acssensors.1c01175
Mov Disord. 2021 Sep 22.

Glucocerebrosidase Activity is not Associated with Parkinson's Disease Risk or Severity.

Nurit Omer, Nir Giladi, Tanya Gurevich, Anat Bar-Shira, Mali Gana-Weisz, Tal Glinka, Orly Goldstein, Meir Kestenbaum, Jesse M Cedarbaum, Omar S Mabrouk, Kyle B Fraser, Julia C Shirvan, Avi Orr-Urtreger, Anat Mirelman, Avner Thaler.

   BACKGROUND: Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are risk factors for Parkinson's disease (PD).
OBJECTIVE: To explore the association between GCase activity, PD phenotype, and probability for prodromal PD among carriers of mutations in the GBA and LRRK2 genes.
METHODS: Participants were genotyped for the G2019S-LRRK2 and nine GBA mutations common in Ashkenazi Jews. Performance-based measures enabling the calculation of the Movement Disorder Society (MDS) prodromal probability score were collected.
RESULTS: One hundred and seventy PD patients (102 GBA-PD, 38 LRRK2-PD, and 30 idiopathic PD) and 221 non-manifesting carriers (NMC) (129 GBA-NMC, 45 LRRK2-NMC, 15 GBA-LRRK2-NMC, and 32 healthy controls) participated in this study. GCase activity was lower among GBA-PD (3.15 ± 0.85 μmol/L/h), GBA-NMC (3.23 ± 0.91 μmol/L/h), and GBA-LRRK2-NMC (3.20 ± 0.93 μmol/L/h) compared to the other groups of participants, with no correlation to clinical phenotype.
CONCLUSIONS: Low GCase activity does not explain the clinical phenotype or risk for prodromal PD in this cohort. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords:  GBA; GCase; LRRK2; Parkinson's disease

DOI:  https://doi.org/10.1002/mds.28792
Biochim Biophys Acta Biomembr. 2021 Sep 18. pii: S0005-2736(21)00228-5. [Epub ahead of print] 183780

The enigma of phosphoinositides and their derivatives: Their role in regulation of subcellular compartment morphology.

Banafshé Larijani, Lior Pytowski, David J Vaux.

  The general segregation of a molecular class, lipids, from the pathways of cellular communication, via endo-membranes, has resulted in the over-simplification and misconceptions in deciphering cell signalling mechanisms. Mechanisms in signal transduction and protein activation require targeting of proteins to membranous compartments with a specific localised morphology and dynamics that are dependent on their lipid composition. Many posttranslational events define cellular behaviours and without the active role of membranous compartments these events lead to various dysregulations of the signalling pathways. We summarise the key findings, using tools such as the rapalogue dimerisation, in the structural roles and signalling of the inter-related phosphoinositide lipids and their derivative, diacylglycerol, in the regulation of nuclear envelope biogenesis and other subcellular compartments such as the nucleoplasmic reticulum.

Keywords:  CLEM; Membrane fusion; Nuclear envelope; Nucleoplasmic reticula; Phosphoinositides; Rapalogue dimerisation tool

DOI:  https://doi.org/10.1016/j.bbamem.2021.183780
Plant Cell. 2021 Sep 22. pii: koab235. [Epub ahead of print]

Molecular mechanisms of endomembrane trafficking in plants.

Fernando Aniento, Víctor Sánchez de Medina Hernández, Yasin Dagdas, Marcela Rojas-Pierce, Eugenia Russinova.

Endomembrane trafficking is essential for all eukaryotic cells. The best-characterized membrane trafficking organelles include the endoplasmic reticulum (ER), Golgi apparatus, early and recycling endosomes, multivesicular body (MVB) or late endosome (LE), lysosome/vacuole, and plasma membrane (PM). Even although historically plants have given rise to cell biology, our understanding of membrane trafficking has mainly been shaped by the much more studied mammalian and yeast models. Whereas organelles and major protein families that regulate endomembrane trafficking are largely conserved across all eukaryotes, exciting variations are emerging from advances in plant cell biology research. In this review, we summarize the current statet of knowledge on plant endomembrane trafficking, with a focus on four distinct trafficking pathways: ER-to-Golgi transport, endocytosis, trans-Golgi network-to-vacuole transport, and autophagy. We acknowledge the conservation and commonalities in the trafficking machinery across species, with emphasis on diversity and plant-specific features. Understanding the function of organelles and the trafficking machinery currently non-existent in well-known model organisms will provide great opportunities to acquire new insights into the fundamental cellular process of membrane trafficking.

DOI: https://doi.org/10.1093/plcell/koab235
Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00501-3. [Epub ahead of print]81(18): 3878-3878.e1

SnapShot: Cancer metabolism.

Julia S Brunner, Lydia W S Finley.

Metabolic networks support cancer cell survival, proliferation, and malignant progression. Cancer cells take up large amounts of nutrients such as glucose and glutamine whose metabolism provides the energy, reducing equivalents, and biosynthetic precursors required to meet the biosynthetic demands of proliferation. Intermediates of glycolysis and the tricarboxylic acid (TCA) cycle provide critical building blocks for synthesis of non-essential amino acids, nucleotides, and fatty acids. To view this SnapShot, open or download the PDF.

DOI: https://doi.org/10.1016/j.molcel.2021.06.021
Cell Mol Life Sci. 2021 Sep 19.

Control of the eIF4E activity: structural insights and pharmacological implications.

Alice Romagnoli, Mattia D'Agostino, Chiara Ardiccioni, Cristina Maracci, Stefano Motta, Anna La Teana, Daniele Di Marino.

  The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E-eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

Keywords:  4E-binding proteins (4E-BPs); Canonical eIF4E-binding motif; Non-canonical eIF4E-binding motif; Therapeutic target; Translation initiation; eIF4E

DOI:  https://doi.org/10.1007/s00018-021-03938-z
Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00693-6. [Epub ahead of print]81(18): 3731-3748

Supply and demand: Cellular nutrient uptake and exchange in cancer.

Vasileios Papalazarou, Oliver D K Maddocks.

  Nutrient supply and demand delineate cell behavior in health and disease. Mammalian cells have developed multiple strategies to secure the necessary nutrients that fuel their metabolic needs. This is more evident upon disruption of homeostasis in conditions such as cancer, when cells display high proliferation rates in energetically challenging conditions where nutritional sources may be scarce. Here, we summarize the main routes of nutrient acquisition that fuel mammalian cells and their implications in tumorigenesis. We argue that the molecular mechanisms of nutrient acquisition not only tip the balance between nutrient supply and demand but also determine cell behavior upon nutrient limitation and energetic stress and contribute to nutrient partitioning and metabolic coordination between different cell types in inflamed or tumorigenic environments.

Keywords:  SLC proteins; amino acid; cancer; nutrient scavenging; nutrient transport; nutrient transporters

DOI:  https://doi.org/10.1016/j.molcel.2021.08.026
Mol Cell. 2021 Sep 16. pii: S1097-2765(21)00682-1. [Epub ahead of print]81(18): 3677-3690

AMPK: restoring metabolic homeostasis over space and time.

Elijah Trefts, Reuben J Shaw.

The evolution of AMPK and its homologs enabled exquisite responsivity and control of cellular energetic homeostasis. Recent work has been critical in establishing the mechanisms that determine AMPK activity, novel targets of AMPK action, and the distribution of AMPK-mediated control networks across the cellular landscape. The role of AMPK as a hub of metabolic control has led to intense interest in pharmacologic activation as a therapeutic avenue for a number of disease states, including obesity, diabetes, and cancer. As such, critical work on the compartmentalization of AMPK, its downstream targets, and the systems it influences has progressed in recent years. The variegated distribution of AMPK-mediated control of metabolic homeostasis has revealed key insights into AMPK in normal biology and future directions for AMPK-based therapeutic strategies.

DOI: https://doi.org/10.1016/j.molcel.2021.08.015
Nat Rev Mol Cell Biol. 2021 Sep 20.

Longevity lost in translation.

Eytan Zlotorynski.

DOI: https://doi.org/10.1038/s41580-021-00422-1