bims-lysosi 2021-04-25 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒04‒25
forty papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C.
MCOLN1/TRPML1 finely controls oncogenic autophagy in cancer by mediating zinc influx.
Bidirectional interconversion between PtdIns4P and PtdIns(4,5)P2 is required for autophagic lysosome reformation and protection from skeletal muscle disease.
4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold.
HOPS-associated neurological disorders (HOPSANDs): linking endolysosomal dysfunction to the pathogenesis of dystonia.
Biophysical impact of sphingosine and other abnormal lipid accumulation in Niemann-Pick disease type C cell models.
Choreographing endo-lysosomal Ca2+ throughout the life of a phagosome.
Analysis of TORC1-body Formation in Budding Yeast.
DDRGK1, a crucial player of ufmylation system, is indispensable for autophagic degradation by regulating lysosomal function.
A non-immunological role for γ-interferon-inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption.
Glycogen accumulation in smooth muscle of a Pompe disease mouse model.
Distinct signaling by insulin and IGF-1 receptors and their extra- and intracellular domains.
Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity.
Structural basis of DEPTOR to recognize phosphatidic acid using its tandem DEP domains.
Endosomal-lysosomal dysfunctions in Alzheimer's disease: Pathogenesis and therapeutic interventions.
Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis.
Pharmacologic activation of autophagy without direct mTOR inhibition as a therapeutic strategy for treating dry macular degeneration.
Advances in the Role of Leucine-Sensing in the Regulation of Protein Synthesis in Aging Skeletal Muscle.
CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage.
Evaluation of Hsp90 and mTOR inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer.
HIV-1 Vpr protein impairs lysosome clearance causing SNCA/alpha-synuclein accumulation in neurons.
JPT2: The missing link between intracellular Ca2+ release channels and NAADP?
mTOR attenuation with rapamycin reverses neurovascular uncoupling and memory deficits in mice modeling Alzheimer's disease.
eIF4E3 forms an active eIF4F complex during stresses (eIF4FS) targeting mTOR and re-programs the translatome.
Maturing secretory granules: Where secretory and endocytic pathways converge.
TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation.
UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling.
Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.
Selectivity of mRNA degradation by autophagy in yeast.
Regulation of Golgi turnover by CALCOCO1-mediated selective autophagy.
Tissue-specific modulation of gene expression in response to lowered insulin signalling in Drosophila.
Flavonoids increase melanin production and reduce proliferation, migration and invasion of melanoma cells by blocking endolysosomal/melanosomal TPC2.
Single Molecule Assay for Ultrasensitive Detection of Cathepsin B in Human Blood.
The Regulation of Polyamine Pathway Proteins in Models of Skeletal Muscle Hypertrophy and Atrophy - a potential role for mTORC1.
The adverse metabolic effects of branched-chain amino acids are mediated by isoleucine and valine.
Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy.
High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.
Self-organization of apical membrane protein sorting in epithelial cells.
Role of mTORC2 in biphasic regulation of brown fat metabolism in response to mild and severe cold.
VPS39-deficiency observed in type 2 diabetes impairs muscle stem cell differentiation via altered autophagy and epigenetics.

Dev Cell. 2021 Apr 13. pii: S1534-5807(21)00307-5. [Epub ahead of print]

Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C.

Joseph C Roney, Sunan Li, Tamar Farfel-Becker, Ning Huang, Tao Sun, Yuxiang Xie, Xiu-Tang Cheng, Mei-Yao Lin, Frances M Platt, Zu-Hang Sheng.

  Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-β-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.

Keywords:  Niemann-Pick disease type C; autophagosome; axonal dystrophy; axonal transport; cholesterol; kinesin; lipid; lysosomal storage disorder; lysosome; neurodegeneration

DOI:  https://doi.org/10.1016/j.devcel.2021.03.032
Autophagy. 2021 Apr 23. 1-22

MCOLN1/TRPML1 finely controls oncogenic autophagy in cancer by mediating zinc influx.

Jiansong Qi, Yanhong Xing, Yucheng Liu, Meng-Meng Wang, Xiangqing Wei, Zhongheng Sui, Lin Ding, Yang Zhang, Chen Lu, Yuan-Hui Fei, Nan Liu, Rong Chen, Mengmei Wu, Lijuan Wang, Zhenyu Zhong, Ting Wang, Yifan Liu, Yuqing Wang, Jiamei Liu, Haoxing Xu, Feng Guo, Wuyang Wang.

  Macroautophagy/autophagy is elevated to ensure the high demand for nutrients for the growth of cancer cells. Here we demonstrated that MCOLN1/TRPML1 is a pharmaceutical target of oncogenic autophagy in cancers such as pancreatic cancer, breast cancer, gastric cancer, malignant melanoma, and glioma. First, we showed that activating MCOLN1, by increasing expression of the channel or using the MCOLN1 agonists, ML-SA5 or MK6-83, arrests autophagic flux by perturbing fusion between autophagosomes and lysosomes. Second, we demonstrated that MCOLN1 regulates autophagy by mediating the release of zinc from the lysosome to the cytosol. Third, we uncovered that zinc influx through MCOLN1 blocks the interaction between STX17 (syntaxin 17) in the autophagosome and VAMP8 in the lysosome and thereby disrupting the fusion process that is determined by the two SNARE proteins. Furthermore, we demonstrated that zinc influx originating from the extracellular fluid arrests autophagy by the same mechanism as lysosomal zinc, confirming the fundamental function of zinc as a participant in membrane trafficking. Last, we revealed that activating MCOLN1 with the agonists, ML-SA5 or MK6-83, triggers cell death of a number of cancer cells by evoking autophagic arrest and subsequent apoptotic response and cell cycle arrest, with little or no effect observed on normal cells. Consistent with the in vitro results, administration of ML-SA5 in Patu 8988 t xenograft mice profoundly suppresses tumor growth and improves survival. These results establish that a lysosomal cation channel, MCOLN1, finely controls oncogenic autophagy in cancer by mediating zinc influx into the cytosol.

Keywords:  Autophagic arrest; MCOLN1; autophagosome-lysosome fusion; cancer; zinc influx

DOI:  https://doi.org/10.1080/15548627.2021.1917132
Autophagy. 2021 Apr 20. 1-3

Bidirectional interconversion between PtdIns4P and PtdIns(4,5)P2 is required for autophagic lysosome reformation and protection from skeletal muscle disease.

Matthew J Eramo, Rajendra Gurung, Christina A Mitchell, Meagan J McGrath.

  Autophagic lysosome reformation (ALR) recycles autolysosome membranes formed during autophagy, to make lysosomes and is essential for continued autophagy function. Localized membrane remodeling on autolysosomes leads to the extension of reformation tubules, which undergo scission to form new lysosomes. The phosphoinositides phosphatidylinositol-4-phosphate (PtdIns4P) and phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) induce this remodeling by recruiting protein effectors to membranes. We identified the inositol polyphosphate 5-phosphatase INPP5K, which converts PtdIns(4,5)P2 to PtdIns4P is essential for ALR in skeletal muscle. INPP5K mutations that reduce its 5-phosphatase activity are known to cause muscular dystrophy, via an undefined mechanism. We generated skeletal muscle-specific inpp5k knockout mice which exhibited severe muscle disease, with lysosome depletion and marked autophagy inhibition. This was due to decreased PtdIns4P and increased PtdIns(4,5)P2 on autolysosomes, causing reduced scission of reformation tubules. ALR was restored in cells with loss of INPP5K by expression of wild-type INPP5K, but not muscle-disease causing mutants. Therefore on autolysosomes, both PtdIns(4,5)P2 generation and its removal by INPP5K is required for completion of ALR. Furthermore, skeletal muscle shows a dependence on the membrane recycling ALR pathway to maintain lysosome homeostasis and ensure the protective role of autophagy against disease.

Keywords:  Autophagic lysosome reformation; INPP5K; PtdIns(4, 5)P2; PtdIns4P; autophagy; inositol polyphosphate 5-phosphatase; lysosome; muscular dystrophy; phosphoinositide; skeletal muscle

DOI:  https://doi.org/10.1080/15548627.2021.1916195
Proc Natl Acad Sci U S A. 2021 Apr 13. pii: e2025522118. [Epub ahead of print]118(15):

4E-BP2-dependent translation in parvalbumin neurons controls epileptic seizure threshold.

Vijendra Sharma, Rapita Sood, Danning Lou, Tzu-Yu Hung, Maxime Lévesque, Yelin Han, Jeremy Y Levett, Peng Wang, Shravan Murthy, Shannon Tansley, Siyan Wang, Nadeem Siddiqui, Soroush Tahmasebi, Kobi Rosenblum, Massimo Avoli, Jean-Claude Lacaille, Nahum Sonenberg, Arkady Khoutorsky.

  The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)-induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3ca H1047R-Pvalb ) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

Keywords:  epilepsy; mRNA translation; mTORC1

DOI:  https://doi.org/10.1073/pnas.2025522118
Brain. 2021 Apr 19. pii: awab161. [Epub ahead of print]

HOPS-associated neurological disorders (HOPSANDs): linking endolysosomal dysfunction to the pathogenesis of dystonia.

Edoardo Monfrini, Michael Zech, Dora Steel, Manju A Kurian, Juliane Winkelmann, Alessio Di Fonzo.

  The "homotypic fusion and protein sorting" (HOPS) complex is the structural bridge necessary for the fusion of late endosomes and autophagosomes with lysosomes. Recent publications linked mutations in genes encoding HOPS complex proteins with the etiopathogenesis of inherited dystonias (i.e., VPS16, VPS41, and VPS11). Functional and microstructural studies conducted on patient-derived fibroblasts carrying mutations of HOPS complex subunits displayed clear abnormalities of the lysosomal and autophagic compartments. We propose to name HOPS-associated Neurological Disorders (HOPSANDs) this group of diseases, which are mainly characterized by dystonic presentations. The delineation of HOPSANDs further confirms the connection of lysosomal and autophagic dysfunction with the pathogenesis of dystonia, prompting researchers to find innovative therapies targeting this pathway.

Keywords:  HOPS; HOPSANDs; autophagy; dystonia genetics; lysosome

DOI:  https://doi.org/10.1093/brain/awab161
Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Apr 20. pii: S1388-1981(21)00070-6. [Epub ahead of print] 158944

Biophysical impact of sphingosine and other abnormal lipid accumulation in Niemann-Pick disease type C cell models.

Ana C Carreira, Sarka Pokorna, Ana E Ventura, Mathew W Walker, Anthony H Futerman, Emyr Lloyd-Evans, Rodrigo F M de Almeida, Liana C Silva.

  Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC disease, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC disease and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC disease.

Keywords:  Cholesterol; Lipid domains; Lysosomal storage diseases; Membrane fluidity; Niemann-Pick disease type C; Sphingosine

DOI:  https://doi.org/10.1016/j.bbalip.2021.158944
Biochim Biophys Acta Mol Cell Res. 2021 Apr 16. pii: S0167-4889(21)00094-X. [Epub ahead of print] 119040

Choreographing endo-lysosomal Ca2+ throughout the life of a phagosome.

Anthony J Morgan, Lianne C Davis, Antony Galione.

  The emergence of endo-lysosomes as ubiquitous Ca2+ stores with their unique cohort of channels has resulted in their being implicated in a growing number of processes in an ever-increasing number of cell types. The architectural and regulatory constraints of these acidic Ca2+ stores distinguishes them from other larger Ca2+ sources such as the ER and influx across the plasma membrane. In view of recent advances in the understanding of the modes of operation, we discuss phagocytosis as a template for how endo-lysosomal Ca2+ signals (generated via TPC and TRPML channels) can be integrated in multiple sophisticated ways into biological processes. Phagocytosis illustrates how different endo-lysosomal Ca2+ signals drive different phases of a process, and how these can be altered by disease or infection.

Keywords:  Calcium; Lysosome; Macrophage; NAADP; Phagocytosis; TPC

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119040
Bio Protoc. 2021 Apr 05. 11(7): e3975

Analysis of TORC1-body Formation in Budding Yeast.

Ryan L Wallace, Eric Lu, Arron Sullivan, James E Hughes Hallett, Andrew P Capaldi.

  The Target of Rapamycin kinase Complex I (TORC1) is the master regulator of cell growth and metabolism in eukaryotes. In the presence of pro-growth hormones and abundant nutrients, TORC1 is active and drives protein, lipid, and nucleotide synthesis by phosphorylating a wide range of proteins. In contrast, when nitrogen and/or glucose levels fall, TORC1 is inhibited, causing the cell to switch from anabolic to catabolic metabolism, and eventually enter a quiescent state. In the budding yeast Saccharomyces cerevisiae, TORC1 inhibition triggers the movement of TORC1 from its position around the vacuole to a single focus/body on the edge of the vacuolar membrane. This relocalization depends on the activity of numerous key TORC1 regulators and thus analysis of TORC1 localization can be used to follow signaling through the TORC1 pathway. Here we provide a detailed protocol for measuring TORC1 (specifically, Kog1-YFP) relocalization/signaling using fluorescence microscopy. Emphasis is placed on procedures that ensure: (1) TORC1-bodies are identified (and counted) correctly despite their relatively low fluorescence and the accumulation of autofluorescent foci during glucose and nitrogen starvation; (2) Cells are kept in log-phase growth at the start of each experiment so that the dynamics of TORC1-body formation are monitored correctly; (3) The appropriate fluorescent tags are used to avoid examining mislocalized TORC1.

Keywords:  Kog1; Kog1-body; TORC1; TORC1-body; Tor1

DOI:  https://doi.org/10.21769/BioProtoc.3975
Cell Death Dis. 2021 Apr 20. 12(5): 416

DDRGK1, a crucial player of ufmylation system, is indispensable for autophagic degradation by regulating lysosomal function.

Yan Cao, Rongyang Li, Ming Shen, Chengyu Li, Yan Zou, Qiang Jiang, Shuo Liu, Chunwan Lu, Honglin Li, Honglin Liu, Yafei Cai.

DDRGK domain-containing protein 1 (DDRGK1) is an important component of the newly discovered ufmylation system and its absence has been reported to induce extensive endoplasmic reticulum (ER) stress. Recently, emerging evidence indicates that the ufmylation system is correlated with autophagy, although the exact mechanism remains largely unknown. To explore the regulation mechanism of DDRGK1 on autophagy, in this study, we established an immortalized mouse embryonic fibroblast (MEF) cell lines harvested from the DDRGK1F/F:ROSA26-CreERT2 mice, in which DDRGK1 depletion can be induced by 4-hydroxytamoxifen (4-OHT) treatment. Here, we show that DDRGK1 deficiency in MEFs has a dual effect on autophagy, which leads to a significant accumulation of autophagosomes. On one hand, it promotes autophagy induction by impairing mTOR signaling; on the other hand, it blocks autophagy degradation by inhibiting autophagosome-lysosome fusion. This dual effect of DDRGK1 depletion on autophagy ultimately aggravates apoptosis in MEFs. Further studies reveal that DDRGK1 loss is correlated with suppressed lysosomal function, including impaired Cathepsin D (CTSD) expression, aberrant lysosomal pH, and v-ATPase accumulation, which might be a potential trigger for impairment in autophagy process. Hence, this study confirms a crucial role of DDRGK1 as an autophagy regulator by controlling lysosomal function. It may provide a theoretical basis for the treatment strategies of various physiological diseases caused by DDRGK1 deficiency.

DOI: https://doi.org/10.1038/s41419-021-03694-9
Sci Adv. 2021 Apr;pii: eabd3684. [Epub ahead of print]7(17):

A non-immunological role for γ-interferon-inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption.

Benjamin W Ewanchuk, Corey R Arnold, Dale R Balce, Priyatha Premnath, Tanis L Orsetti, Amy L Warren, Alexandra Olsen, Roman J Krawetz, Robin M Yates.

The extracellular bone resorbing lacuna of the osteoclast shares many characteristics with the degradative lysosome of antigen-presenting cells. γ-Interferon-inducible lysosomal thiol reductase (GILT) enhances antigen processing within lysosomes through direct reduction of antigen disulfides and maintenance of cysteine protease activity. In this study, we found the osteoclastogenic cytokine RANKL drove expression of GILT in osteoclast precursors in a STAT1-dependent manner, resulting in high levels of GILT in mature osteoclasts, which could be further augmented by γ-interferon. GILT colocalized with the collagen-degrading cysteine protease, cathepsin K, suggesting a role for GILT inside the osteoclastic resorption lacuna. GILT-deficient osteoclasts had reduced bone-resorbing capacity, resulting in impaired bone turnover and an osteopetrotic phenotype in GILT-deficient mice. We demonstrated that GILT could directly reduce the noncollagenous bone matrix protein SPARC, and additionally, enhance collagen degradation by cathepsin K. Together, this work describes a previously unidentified, non-immunological role for GILT in osteoclast-mediated bone resorption.

DOI: https://doi.org/10.1126/sciadv.abd3684
J Smooth Muscle Res. 2021 ;57(0): 8-18

Glycogen accumulation in smooth muscle of a Pompe disease mouse model.

Angela L McCall, Justin S Dhindsa, Aidan M Bailey, Logan A Pucci, Laura M Strickland, Mai K ElMallah.

  Pompe disease is a lysosomal storage disease caused by mutations within the GAA gene, which encodes acid α-glucosidase (GAA)-an enzyme necessary for lysosomal glycogen degradation. A lack of GAA results in an accumulation of glycogen in cardiac and skeletal muscle, as well as in motor neurons. The only FDA approved treatment for Pompe disease-an enzyme replacement therapy (ERT)-increases survival of patients, but has unmasked previously unrecognized clinical manifestations of Pompe disease. These clinical signs and symptoms include tracheo-bronchomalacia, vascular aneurysms, and gastro-intestinal discomfort. Together, these previously unrecognized pathologies indicate that GAA-deficiency impacts smooth muscle in addition to skeletal and cardiac muscle. Thus, we sought to characterize smooth muscle pathology in the airway, vascular, gastrointestinal, and genitourinary in the Gaa-/- mouse model. Increased levels of glycogen were present in smooth muscle cells of the aorta, trachea, esophagus, stomach, and bladder of Gaa-/- mice, compared to wild type mice. In addition, there was an increased abundance of both lysosome membrane protein (LAMP1) and autophagosome membrane protein (LC3) indicating vacuolar accumulation in several tissues. Taken together, we show that GAA deficiency results in subsequent pathology in smooth muscle cells, which may lead to life-threatening complications if not properly treated.

Keywords:  Pompe disease; autophagy; smooth muscle

DOI:  https://doi.org/10.1540/jsmr.57.8
Proc Natl Acad Sci U S A. 2021 Apr 27. pii: e2019474118. [Epub ahead of print]118(17):

Distinct signaling by insulin and IGF-1 receptors and their extra- and intracellular domains.

Hirofumi Nagao, Weikang Cai, Nicolai J Wewer Albrechtsen, Martin Steger, Thiago M Batista, Hui Pan, Jonathan M Dreyfuss, Matthias Mann, C Ronald Kahn.

  Insulin and insulin-like growth factor 1 (IGF-1) receptors share many downstream signaling pathways but have unique biological effects. To define the molecular signals contributing to these distinct activities, we performed global phosphoproteomics on cells expressing either insulin receptor (IR), IGF-1 receptor (IGF1R), or chimeric IR-IGF1R receptors. We show that IR preferentially stimulates phosphorylations associated with mammalian target of rapamycin complex 1 (mTORC1) and Akt pathways, whereas IGF1R preferentially stimulates phosphorylations on proteins associated with the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases), and cell cycle progression. There were also major differences in the phosphoproteome between cells expressing IR versus IGF1R in the unstimulated state, including phosphorylation of proteins involved in membrane trafficking, chromatin remodeling, and cell cycle. In cells expressing chimeric IR-IGF1R receptors, these differences in signaling could be mapped to contributions of both the extra- and intracellular domains of these receptors. Thus, despite their high homology, IR and IGF1R preferentially regulate distinct networks of phosphorylation in both the basal and stimulated states, allowing for the unique effects of these hormones on organismal function.

Keywords:  IGF-1 signaling; cellular signaling; insulin signaling; kinases; protein phosphorylation

DOI:  https://doi.org/10.1073/pnas.2019474118
J Immunother Cancer. 2021 Apr;pii: e002137. [Epub ahead of print]9(4):

Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity.

Yiwen Zhang, Hongrong Hu, Weiwei Liu, Shu-Mei Yan, Yuzhuang Li, Likai Tan, Yingshi Chen, Jun Liu, Zhilin Peng, Yaochang Yuan, Wenjing Huang, Fei Yu, Xin He, Bo Li, Hui Zhang.

  BACKGROUND: In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+ tumor-infiltrating lymphocytes (TILs) is unclear.METHOD: In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8+ TILs.
RESULTS: We discovered that the Rag complex, particularly RagD, was crucial for CD8+ T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8+ TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8+ T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8+ T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo.
CONCLUSION: Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.

Keywords:  CD8-positive T-lymphocytes; lymphocyte activation; lymphocytes; metabolic networks and pathways; tumor microenvironment; tumor-infiltrating

DOI:  https://doi.org/10.1136/jitc-2020-002137
J Mol Biol. 2021 Apr 15. pii: S0022-2836(21)00190-X. [Epub ahead of print] 166989

Structural basis of DEPTOR to recognize phosphatidic acid using its tandem DEP domains.

Zhuangfeng Weng, Xinxin Shen, Jiefu Zheng, Huanhuan Liang, Yingfang Liu.

  DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.

Keywords:  Anionic lipid; Crystal structure; DDEX motif; DEPDC6; mTOR

DOI:  https://doi.org/10.1016/j.jmb.2021.166989
Metab Brain Dis. 2021 Apr 21.

Endosomal-lysosomal dysfunctions in Alzheimer's disease: Pathogenesis and therapeutic interventions.

Shereen Shi Min Lai, Khuen Yen Ng, Rhun Yian Koh, Kian Chung Chok, Soi Moi Chye.

  The endosomal-lysosomal system mediates the process of protein degradation through endocytic pathway. This system consists of early endosomes, late endosomes, recycling endosomes and lysosomes. Each component in the endosomal-lysosomal system plays individual crucial role and they work concordantly to ensure protein degradation can be carried out functionally. Dysregulation in the endosomal-lysosomal system can contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). In AD endosomal-lysosomal abnormalities are the earliest pathological features to note and hence it is important to understand the involvement of endosomal-lysosomal dysfunction in the pathogenesis of AD. In-depth understanding of this dysfunction can allow development of new therapeutic intervention to prevent and treat AD.

Keywords:  Alzheimer's disease; Endosomal‐lysosomal system; Endosome trafficking; Lysosomal defects; Neurodegeneration; Pathogenesis; Therapeutic interventions

DOI:  https://doi.org/10.1007/s11011-021-00737-0
Cell Signal. 2021 Apr 16. pii: S0898-6568(21)00098-X. [Epub ahead of print] 110010

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis.

Siddharth Sunilkumar, Scot R Kimball, Michael D Dennis.

  Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.

Keywords:  Cyclic AMP; Diabetes; Glucagon; Insulin; Liver; Protein synthesis

DOI:  https://doi.org/10.1016/j.cellsig.2021.110010
Aging (Albany NY). 2021 Apr 19. 13

Pharmacologic activation of autophagy without direct mTOR inhibition as a therapeutic strategy for treating dry macular degeneration.

Qitao Zhang, Feriel Presswalla, Robin R Ali, David N Zacks, Debra A Thompson, Jason M L Miller.

  Dry age-related macular degeneration (AMD) is marked by the accumulation of extracellular and intracellular lipid-rich deposits within and around the retinal pigment epithelium (RPE). Inducing autophagy, a conserved, intracellular degradative pathway, is a potential treatment strategy to prevent disease by clearing these deposits. However, mTOR inhibition, the major mechanism for inducing autophagy, disrupts core RPE functions. Here, we screened autophagy inducers that do not directly inhibit mTOR for their potential as an AMD therapeutic in primary human RPE culture. Only two out of more than thirty autophagy inducers tested reliably increased autophagy flux in RPE, emphasizing that autophagy induction mechanistically differs across distinct tissues. In contrast to mTOR inhibitors, these compounds preserved RPE health, and one inducer, the FDA-approved compound flubendazole (FLBZ), reduced the secretion of apolipoprotein that contributes to extracellular deposits termed drusen. Simultaneously, FLBZ increased production of the lipid-degradation product β-hydroxybutyrate, which is used by photoreceptor cells as an energy source. FLBZ also reduced the accumulation of intracellular deposits, termed lipofuscin, and alleviated lipofuscin-induced cellular senescence and tight-junction disruption. FLBZ triggered compaction of lipofuscin-like granules into a potentially less toxic form. Thus, induction of RPE autophagy without direct mTOR inhibition is a promising therapeutic approach for dry AMD.

Keywords:  age-related macular degeneration (AMD); autophagy; drusen; lipofuscin; retinal pigment epithelium (RPE)

DOI:  https://doi.org/10.18632/aging.202974
Front Cell Dev Biol. 2021 ;9 646482

Advances in the Role of Leucine-Sensing in the Regulation of Protein Synthesis in Aging Skeletal Muscle.

Yan Zhao, Jason Cholewa, Huayu Shang, Yueqin Yang, Xiaomin Ding, Qianjin Wang, Quansheng Su, Nelo Eidy Zanchi, Zhi Xia.

  Skeletal muscle anabolic resistance (i.e., the decrease in muscle protein synthesis (MPS) in response to anabolic stimuli such as amino acids and exercise) has been identified as a major cause of age-related sarcopenia, to which blunted nutrition-sensing contributes. In recent years, it has been suggested that a leucine sensor may function as a rate-limiting factor in skeletal MPS via small-molecule GTPase. Leucine-sensing and response may therefore have important therapeutic potential in the steady regulation of protein metabolism in aging skeletal muscle. This paper systematically summarizes the three critical processes involved in the leucine-sensing and response process: (1) How the coincidence detector mammalian target of rapamycin complex 1 localizes on the surface of lysosome and how its crucial upstream regulators Rheb and RagB/RagD interact to modulate the leucine response; (2) how complexes such as Ragulator, GATOR, FLCN, and TSC control the nucleotide loading state of Rheb and RagB/RagD to modulate their functional activity; and (3) how the identified leucine sensor leucyl-tRNA synthetase (LARS) and stress response protein 2 (Sestrin2) participate in the leucine-sensing process and the activation of RagB/RagD. Finally, we discuss the potential mechanistic role of exercise and its interactions with leucine-sensing and anabolic responses.

Keywords:  age-related sarcopenia; anabolic resistance; leucine response; leucine-sensing; protein synthesis

DOI:  https://doi.org/10.3389/fcell.2021.646482
Stroke. 2021 Apr 20. STROKEAHA120032749

CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage.

Yueyang Liu, Xiaohang Che, Haotian Zhang, Xiaoxiao Fu, Yang Yao, Jun Luo, Yu Yang, Ruiping Cai, Xiangnan Yu, Jingyu Yang, Ming-Sheng Zhou.

  BACKGROUND AND PURPOSE: CAPN1 (calpain1)-an intracellular Ca2+-regulated cysteine protease-can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined.METHODS: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose-deprived primary neuron in vitro model.
RESULTS: CAPN1 was activated in the cortex of permanent middle cerebral occlusion-operated rats and oxygen-glucose deprivation-exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage.
CONCLUSIONS: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia-mediated autophagy-lysosomal pathway defects and neuronal damage.

Keywords:  autophagy; glucose; ischemia; lysosomes; neurons

DOI:  https://doi.org/10.1161/STROKEAHA.120.032749
PLoS One. 2021 ;16(4): e0248380

Evaluation of Hsp90 and mTOR inhibitors as potential drugs for the treatment of TSC1/TSC2 deficient cancer.

Evelyn M Mrozek, Vineeta Bajaj, Yanan Guo, Izabela A Malinowska, Jianming Zhang, David J Kwiatkowski.

Inactivating mutations in either TSC1 or TSC2 cause Tuberous Sclerosis Complex, an autosomal dominant disorder, characterized by multi-system tumor and hamartoma development. Mutation and loss of function of TSC1 and/or TSC2 also occur in a variety of sporadic cancers, and rapamycin and related drugs show highly variable treatment benefit in patients with such cancers. The TSC1 and TSC2 proteins function in a complex that inhibits mTORC1, a key regulator of cell growth, which acts to enhance anabolic biosynthetic pathways. In this study, we identified and validated five cancer cell lines with TSC1 or TSC2 mutations and performed a kinase inhibitor drug screen with 197 compounds. The five cell lines were sensitive to several mTOR inhibitors, and cell cycle kinase and HSP90 kinase inhibitors. The IC50 for Torin1 and INK128, both mTOR kinase inhibitors, was significantly increased in three TSC2 null cell lines in which TSC2 expression was restored. Rapamycin was significantly more effective than either INK128 or ganetespib (an HSP90 inhibitor) in reducing the growth of TSC2 null SNU-398 cells in a xenograft model. Combination ganetespib-rapamycin showed no significant enhancement of growth suppression over rapamycin. Hence, although HSP90 inhibitors show strong inhibition of TSC1/TSC2 null cell line growth in vitro, ganetespib showed little benefit at standard dosage in vivo. In contrast, rapamycin which showed very modest growth inhibition in vitro was the best agent for in vivo treatment, but did not cause tumor regression, only growth delay.

DOI: https://doi.org/10.1371/journal.pone.0248380
Autophagy. 2021 Apr 23. 1-15

HIV-1 Vpr protein impairs lysosome clearance causing SNCA/alpha-synuclein accumulation in neurons.

Maryline Santerre, Sterling P Arjona, Charles Ns Allen, Shannon Callen, Shilpa Buch, Bassel E Sawaya.

  Despite the promising therapeutic effects of combinatory antiretroviral therapy (cART), 20% to 30% of HIV/AIDS patients living with long term infection still exhibit related cognitive and motor disorders. Clinical studies in HIV-infected patients revealed evidence of basal ganglia dysfunction, tremors, fine motor movement deficits, gait, balance, and increased risk of falls. Among older HIV+ adults, the frequency of cases with SNCA/α-synuclein staining is higher than in older healthy persons and may predict an increased risk of developing a neurodegenerative disease. The accumulation of SNCA aggregates known as Lewy Bodies is widely described to be directly linked to motor dysfunction. These aggregates are naturally removed by Macroautophagy/autophagy, a cellular housekeeping mechanism, that can be disturbed by HIV-1. The molecular mechanisms involved in linking HIV-1 proteins and autophagy remain mostly unclear and necessitates further exploration. We showed that HIV-1 Vpr protein triggers the accumulation of SNCA in neurons after decreasing lysosomal acidification, deregulating lysosome positioning, and the expression levels of several proteins involved in lysosomal maturation. Viruses and retroviruses such as HIV-1 are known to manipulate autophagy in order to use it for their replication while blocking the degradative final step, which could destroy the virus itself. Our study highlights how the suppression of neuronal autophagy by HIV-1 Vpr is a mechanism leading to toxic protein aggregation and neurodegeneration.

Keywords:  Alpha-synuclein; HIV-1; Snapin; autophagy; lysosomes; motor dysfunction; neurons

DOI:  https://doi.org/10.1080/15548627.2021.1915641
Cell Calcium. 2021 Apr 14. pii: S0143-4160(21)00059-2. [Epub ahead of print]97 102405

JPT2: The missing link between intracellular Ca2+ release channels and NAADP?

Einar Krogsaeter, Rachel Tang, Christian Grimm.

  NAADP (nicotinic acid adenine dinucleotide phosphate) is a potent second messenger releasing Ca2+ from endolysosomes through two-pore channels (TPCs) and from the endoplasmic reticulum (ER) through type 1 ryanodine receptors (RyR1). How NAADP activates these channels, whether directly or indirectly, has been a matter of debate for more than a decade. Now a protein has emerged possibly providing the missing link between TPCs/RyR1 and NAADP.

Keywords:  Jpt2; Lysosome; TPC; TPC1; TPC2; Two-pore channel

DOI:  https://doi.org/10.1016/j.ceca.2021.102405
J Neurosci. 2021 Apr 20. pii: JN-RM-2144-20. [Epub ahead of print]

mTOR attenuation with rapamycin reverses neurovascular uncoupling and memory deficits in mice modeling Alzheimer's disease.

Candice E Van Skike, Stacy A Hussong, Stephen F Hernandez, Andy Q Banh, Nicholas DeRosa, Veronica Galvan.

Vascular dysfunction is a universal feature of aging and decreased cerebral blood flow has been identified as an early event in the pathogenesis of Alzheimer's disease (AD). Cerebrovascular dysfunction in AD includes deficits in neurovascular coupling, a mechanism that ensures rapid delivery of energy substrates to active neurons through the blood supply. The mechanisms underlying NVC impairment in AD, however, are not well understood. We have previously shown that mechanistic/mammalian target of rapamycin (mTOR) drives cerebrovascular dysfunction in models of AD by reducing the activity of endothelial nitric oxide synthase (eNOS), and that attenuation of mTOR activity with rapamycin is sufficient to restore eNOS-dependent cerebrovascular function. Here we show mTOR drives NVC impairments in an AD model through the inhibition of neuronal NOS and non-NOS dependent components of NVC, and that mTOR attenuation with rapamycin is sufficient to restore NVC and even enhance it above WT responses. Restoration of NVC and concomitant reduction cortical amyloid-beta levels effectively treated memory deficits in 12-month-old hAPP(J20) mice. These data indicate mTOR is a critical driver of NVC dysfunction and underlies cognitive impairment in an AD model. Together with our previous findings, the present studies suggest that mTOR promotes cerebrovascular dysfunction in AD, which is associated with early disruption of nNOS activation, through its broad negative impact on nNOS as well as on non-NOS components of NVC. Our studies highlight the potential of mTOR attenuation as an efficacious treatment for AD and potentially other neurological diseases of aging.SIGNIFICANCE STATEMENT:Failure of the blood flow response to neuronal activation (neurovascular coupling, NVC) in a model of AD precedes the onset of AD-like cognitive symptoms and is driven, to a large extent, by mTOR-dependent inhibition of nitric oxide synthase activity. Our studies show that mTOR also drives AD-like failure of non-NO-mediated components of NVC, and importantly that it suppresses NVC in non-diseased animals. Thus, mTOR attenuation may serve to treat AD, where we find that nNOS is profoundly reduced early in disease progression, and potentially other neurological diseases of aging with cerebrovascular dysfunction as part of their etiology. Our studies also provide, to our knowledge, the first evidence for a role of mTOR in regulating NVC in non-diseased conditions in mice.

DOI: https://doi.org/10.1523/JNEUROSCI.2144-20.2021
Nucleic Acids Res. 2021 Apr 24. pii: gkab267. [Epub ahead of print]

eIF4E3 forms an active eIF4F complex during stresses (eIF4FS) targeting mTOR and re-programs the translatome.

Benjamin Weiss, George Edward Allen, Joachim Kloehn, Karim Abid, Pascale Jaquier-Gubler, Joseph Alphonsus Curran.

The eIF4E are a family of initiation factors that bind the mRNA 5' cap, regulating the proteome and the cellular phenotype. eIF4E1 mediates global translation and its activity is controlled via the PI3K/AKT/mTOR pathway. mTOR down-regulation results in eIF4E1 sequestration into an inactive complex with the 4E binding proteins (4EBPs). The second member, eIF4E2, regulates the translatome during hypoxia. However, the exact function of the third member, eIF4E3, has remained elusive. We have dissected its function using a range of techniques. Starting from the observation that it does not interact with 4EBP1, we demonstrate that eIF4E3 recruitment into an eIF4F complex occurs when Torin1 inhibits the mTOR pathway. Ribo-seq studies demonstrate that this complex (eIF4FS) is translationally active during stress and that it selects specific mRNA populations based on 5' TL (UTR) length. The interactome reveals that it associates with cellular proteins beyond the cognate initiation factors, suggesting that it may have 'moon-lighting' functions. Finally, we provide evidence that cellular metabolism is altered in an eIF4E3 KO background but only upon Torin1 treatment. We propose that eIF4E3 acts as a second branch of the integrated stress response, re-programming the translatome to promote 'stress resistance' and adaptation.

DOI: https://doi.org/10.1093/nar/gkab267
Adv Biol Regul. 2021 Mar 25. pii: S2212-4926(21)00023-3. [Epub ahead of print]80 100807

Maturing secretory granules: Where secretory and endocytic pathways converge.

Cheng-I Jonathan Ma, Jason Burgess, Julie A Brill.

  Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.

Keywords:  Endocytic trafficking; Organelle biogenesis; Post-Golgi trafficking; Regulated secretion; Secretory granule

DOI:  https://doi.org/10.1016/j.jbior.2021.100807
Mol Cancer Res. 2021 Apr 22. pii: molcanres.MCR-20-1046-A.2020. [Epub ahead of print]

TSC2 Interacts with HDLBP/Vigilin and Regulates Stress Granule Formation.

Kosmas Kosmas, Harilaos Filippakis, Damir Khabibullin, Michal Turkiewicz, Hilaire C Lam, Jane Yu, Nancy L Kedersha, Paul Joseph Anderson, Elizabeth P Henske.

Tuberous sclerosis complex (TSC) is caused by mutations of either the TSC1 or TSC2 tumor suppressor gene. TSC causes tumors of the brain, heart, kidney, lung and skin. Here we report that the TSC2 protein physically binds to high-density lipoprotein binding protein (HDLBP), also called vigilin, a core stress granule (SG) protein, and that TSC2 localizes to SGs. SGs contain mRNAs and translation initiation complexes, and regulate gene expression by sequestering specific transcripts, thereby serving a cytoprotective role. TSC2 has never before been shown to localize to SGs and knocking down vigilin impacts SG translocation of TSC2. TSC2-deficient cells showed a striking increase in the number of SGs after thermal shock and arsenite treatment relative to Tsc2-expressing cells. Our findings also show that murine kidney lysates from a model of TSC have increased levels of SG components including G3BP1 and Caprin1. G3BP1 and Caprin are elevated in renal angiomyolipomas (a renal tumor common in TSC patients) compared to control normal kidney. G3BP1 is also elevated in TSC-associated Subependymal Giant Cell Astrocytomas. We found that genetic inhibition of G3BP1 inhibits the proliferation of TSC2- deficient cells in vitro. Finally, in a mouse model of TSC, genetic inhibition of SGs suppresses cell growth, suggesting that targeting SGs may have efficacy in the therapy of TSC. Implications: This study demonstrates that TSC2 physically interacts with HDLBP/vigilin, a component of stress granules, that TSC2 localizes to SG and that TSC2-deficient cells have more SGs, suggesting that SGs represent a novel therapeutic target in TSC.

DOI: https://doi.org/10.1158/1541-7786.MCR-20-1046
Elife. 2021 Apr 22. pii: e68348. [Epub ahead of print]10

UBTD1 regulates ceramide balance and endolysosomal positioning to coordinate EGFR signaling.

Stéphanie Torrino, Victor Tiroille, Bastien Dolfi, Maeva Dufies, Charlotte Hinault, Laurent Bonesso, Sonia Dagnino, Jennifer Uhler, Marie Irondelle, Anne-Sophie Gay, Lucile Fleuriot, Delphine Debayle, Sandra Lacas-Gervais, Mireille Cormont, Thomas Bertero, Frederic Bost, Jerome Gilleron, Stephan Clavel.

  To adapt in an ever-changing environment, cells must integrate physical and chemical signals and translate them into biological meaningful information through complex signaling pathways. By combining lipidomic and proteomic approaches with functional analysis, we have shown that UBTD1 (Ubiquitin domain-containing protein 1) plays a crucial role in both the EGFR (Epidermal Growth Factor Receptor) self-phosphorylation and its lysosomal degradation. On the one hand, by modulating the cellular level of ceramides through ASAH1 (N-Acylsphingosine Amidohydrolase 1) ubiquitination, UBTD1 controls the ligand-independent phosphorylation of EGFR. On the other hand, UBTD1, via the ubiquitination of SQSTM1/p62 (Sequestosome 1) by RNF26 and endolysosome positioning, participates in the lysosomal degradation of EGFR. The coordination of these two ubiquitin-dependent processes contributes to the control of the duration of the EGFR signal. Moreover, we showed that UBTD1 depletion exacerbates EGFR signaling and induces cell proliferation emphasizing a hitherto unknown function of UBTD1 in EGFR-driven human cell proliferation.

Keywords:  cell biology; human

DOI:  https://doi.org/10.7554/eLife.68348
Cell. 2021 Apr 14. pii: S0092-8674(21)00379-2. [Epub ahead of print]

Chaperone-mediated autophagy prevents collapse of the neuronal metastable proteome.

Mathieu Bourdenx, Adrián Martín-Segura, Aurora Scrivo, Jose A Rodriguez-Navarro, Susmita Kaushik, Inmaculada Tasset, Antonio Diaz, Nadia J Storm, Qisheng Xin, Yves R Juste, Erica Stevenson, Enrique Luengo, Cristina C Clement, Se Joon Choi, Nevan J Krogan, Eugene V Mosharov, Laura Santambrogio, Fiona Grueninger, Ludovic Collin, Danielle L Swaney, David Sulzer, Evripidis Gavathiotis, Ana Maria Cuervo.

  Components of the proteostasis network malfunction in aging, and reduced protein quality control in neurons has been proposed to promote neurodegeneration. Here, we investigate the role of chaperone-mediated autophagy (CMA), a selective autophagy shown to degrade neurodegeneration-related proteins, in neuronal proteostasis. Using mouse models with systemic and neuronal-specific CMA blockage, we demonstrate that loss of neuronal CMA leads to altered neuronal function, selective changes in the neuronal metastable proteome, and proteotoxicity, all reminiscent of brain aging. Imposing CMA loss on a mouse model of Alzheimer's disease (AD) has synergistic negative effects on the proteome at risk of aggregation, thus increasing neuronal disease vulnerability and accelerating disease progression. Conversely, chemical enhancement of CMA ameliorates pathology in two different AD experimental mouse models. We conclude that functional CMA is essential for neuronal proteostasis through the maintenance of a subset of the proteome with a higher risk of misfolding than the general proteome.

Keywords:  Alzheimer’s disease; aging; chaperones; chemical activators of autophagy; lysosomes; neurodegeneration; protein aggregation; proteotoxicity; supersaturated proteome; tau

DOI:  https://doi.org/10.1016/j.cell.2021.03.048
Nat Commun. 2021 Apr 19. 12(1): 2316

Selectivity of mRNA degradation by autophagy in yeast.

Shiho Makino, Tomoko Kawamata, Shintaro Iwasaki, Yoshinori Ohsumi.

Synthesis and degradation of cellular constituents must be balanced to maintain cellular homeostasis, especially during adaptation to environmental stress. The role of autophagy in the degradation of proteins and organelles is well-characterized. However, autophagy-mediated RNA degradation in response to stress and the potential preference of specific RNAs to undergo autophagy-mediated degradation have not been examined. In this study, we demonstrate selective mRNA degradation by rapamycin-induced autophagy in yeast. Profiling of mRNAs from the vacuole reveals that subsets of mRNAs, such as those encoding amino acid biosynthesis and ribosomal proteins, are preferentially delivered to the vacuole by autophagy for degradation. We also reveal that autophagy-mediated mRNA degradation is tightly coupled with translation by ribosomes. Genome-wide ribosome profiling suggested a high correspondence between ribosome association and targeting to the vacuole. We propose that autophagy-mediated mRNA degradation is a unique and previously-unappreciated function of autophagy that affords post-transcriptional gene regulation.

DOI: https://doi.org/10.1038/s41467-021-22574-6
J Cell Biol. 2021 Jun 07. pii: e202006128. [Epub ahead of print]220(6):

Regulation of Golgi turnover by CALCOCO1-mediated selective autophagy.

Thaddaeus Mutugi Nthiga, Birendra Kumar Shrestha, Jack-Ansgar Bruun, Kenneth Bowitz Larsen, Trond Lamark, Terje Johansen.

The Golgi complex is essential for the processing, sorting, and trafficking of newly synthesized proteins and lipids. Golgi turnover is regulated to meet different cellular physiological demands. The role of autophagy in the turnover of Golgi, however, has not been clarified. Here we show that CALCOCO1 binds the Golgi-resident palmitoyltransferase ZDHHC17 to facilitate Golgi degradation by autophagy during starvation. Depletion of CALCOCO1 in cells causes expansion of the Golgi and accumulation of its structural and membrane proteins. ZDHHC17 itself is degraded by autophagy together with other Golgi membrane proteins such as TMEM165. Taken together, our data suggest a model in which CALCOCO1 mediates selective Golgiphagy to control Golgi size and morphology in eukaryotic cells via its interaction with ZDHHC17.

DOI: https://doi.org/10.1083/jcb.202006128
Elife. 2021 Apr 21. pii: e67275. [Epub ahead of print]10

Tissue-specific modulation of gene expression in response to lowered insulin signalling in Drosophila.

Luke Stephen Tain, Robert Sehlke, Ralf Leslie Meilenbrock, Thomas Leech, Jonathan Paulitz, Manopriya Chokkalingam, Nagarjuna Nagaraj, Sebastian Grönke, Jenny Fröhlich, Ilian Atanassov, Matthias Mann, Andreas Beyer, Linda Partridge.

  Reduced activity of the insulin/IGF signalling network increases health during ageing in multiple species. Diverse and tissue-specific mechanisms drive the health improvement. Here, we performed tissue-specific transcriptional and proteomic profiling of long-lived Drosophila dilp2-3,5 mutants, and identified tissue-specific regulation of >3600 transcripts and >3700 proteins. Most expression changes were regulated post-transcriptionally in the fat body, and only in mutants infected with the endosymbiotic bacteria, Wolbachia pipientis, which increases their lifespan. Bioinformatic analysis identified reduced co-translational ER targeting of secreted and membrane-associated proteins and increased DNA damage/repair response proteins. Accordingly, age-related DNA damage and genome instability were lower in fat body of the mutant, and overexpression of a minichromosome maintenance protein subunit extended lifespan. Proteins involved in carbohydrate metabolism showed altered expression in the mutant intestine, and gut-specific overexpression of a lysosomal mannosidase increased autophagy, gut homeostasis, and lifespan. These processes are candidates for combatting ageing-related decline in other organisms.

Keywords:  D. melanogaster; DNA damage; chromosomes; computational biology; gene expression; lifespan; mannosidase; proteomics; systems biology; transcriptomics

DOI:  https://doi.org/10.7554/eLife.67275
Sci Rep. 2021 Apr 19. 11(1): 8515

Flavonoids increase melanin production and reduce proliferation, migration and invasion of melanoma cells by blocking endolysosomal/melanosomal TPC2.

Ponsawan Netcharoensirisuk, Carla Abrahamian, Rachel Tang, Cheng-Chang Chen, Anna Scotto Rosato, Wyatt Beyers, Yu-Kai Chao, Antonio Filippini, Santiago Di Pietro, Karin Bartel, Martin Biel, Angelika M Vollmar, Kaoru Umehara, Wanchai De-Eknamkul, Christian Grimm.

Two-pore channel 2 (TPC2) resides in endolysosomal membranes but also in lysosome-related organelles such as the melanin producing melanosomes. Gain-of-function polymorphisms in hTPC2 are associated with decreased melanin production and blond hair color. Vice versa genetic ablation of TPC2 increases melanin production. We show here an inverse correlation between melanin production and melanoma proliferation, migration, and invasion due to the dual activity of TPC2 in endolysosomes and melanosomes. Our results are supported by both genetic ablation and pharmacological inhibition of TPC2. Mechanistically, our data show that loss/block of TPC2 results in reduced protein levels of MITF, a major regulator of melanoma progression, but an increased activity of the melanin-generating enzyme tyrosinase. TPC2 inhibition thus provides a twofold benefit in melanoma prevention and treatment by increasing, through interference with tyrosinase activity, the synthesis of UV blocking melanin in melanosomes and by decreasing MITF-driven melanoma progression by increased GSK3β-mediated MITF degradation.

DOI: https://doi.org/10.1038/s41598-021-88196-6
ACS Omega. 2021 Apr 13. 6(14): 9609-9616

Single Molecule Assay for Ultrasensitive Detection of Cathepsin B in Human Blood.

Bharani Thangavelu, Angela M Boutté.

Cathepsin B (catB) is a lysosomal cysteine protease expressed in several cells and organs, where it plays a role in protein degradation and turnover. Extracellular, secreted catB has utility as a biomarker for a host of pathological or physiological states, including a myriad of cancers or neurological diseases and injuries. Analytical or diagnostic assessment may be limited by biological sample volume availability. Pathologically relevant changes in catB levels may occur at low-moderate concentrations that require accurate measurement to differentiate from basal levels. Furthermore, biological samples like plasma and serum, often occlude accurate catB measurements because of background and high variance, vastly limiting the ability to detect catB as a peripheral biomarker. Techniques for ultrasensitive protein detection that require low volumes of sample are necessary. To overcome these challenges, a digital enzyme-linked immunosorbent assay (ELISA) was developed for differential detection of catB within less than 5 μL of serum and plasma using the single molecule array (SiMoA) platform, which offers 1000-times more sensitivity and vastly reduced variance compared to colorimetric tests. In buffer, curve-fitting estimated the limit of detection (LoD) to be ∼1.56 and ∼8.47 pg/mL using two-step or three-step assay configurations, respectively. After correcting for endogenous levels, the estimated LoD was ∼4.7 pg/mL in the serum or plasma with the two-step assay. The lower limit of quantitation was ∼2.3 pg/mL in the buffer and ∼9.4 pg/mL in the serum or plasma, indicting the ability to measure small changes above endogenous levels within blood samples.

DOI: https://doi.org/10.1021/acsomega.1c00180
Am J Physiol Cell Physiol. 2021 Apr 21.

The Regulation of Polyamine Pathway Proteins in Models of Skeletal Muscle Hypertrophy and Atrophy - a potential role for mTORC1.

Michael Tabbaa, Tania Ruz Gomez, Dean G Campelj, Paul Gregorevic, Alan Hayes, Craig A Goodman.

  Polyamines have been shown to be absolutely required for protein synthesis and cell growth. The serine/threonine kinase, the mechanistic target of rapamycin complex 1 (mTORC1), also plays a fundamental role in the regulation of protein turnover and cell size, including in skeletal muscle, where mTORC1 is sufficient to increase protein synthesis and muscle fiber size, and is necessary for mechanical overload-induced muscle hypertrophy. Recent evidence suggests that mTORC1 may regulate the polyamine metabolic pathway; however, there is currently no evidence in skeletal muscle. This study examined changes in polyamine pathway proteins during muscle hypertrophy induced by mechanical overload (7 d), with and without the mTORC1 inhibitor, rapamycin, and during muscle atrophy induced by food deprivation (48 h) and denervation (7 d) in mice. Mechanical overload induced an increase in mTORC1 signalling, protein synthesis and muscle mass, and these were associated with rapamycin-sensitive increases in adenosylmethione decarboxylase 1 (Amd1), spermidine synthase (SpdSyn) and c-Myc. Food deprivation decreased mTORC1 signalling, protein synthesis and muscle mass, accompanied by a decrease in spermidine/spermine acetyltransferase 1 (Sat1). Denervation, resulted increased mTORC1 signalling and protein synthesis, and decreased muscle mass, which was associated with an increase in SpdSyn, spermine synthase (SpmSyn) and c-Myc. Combined, these data show that polyamine pathway enzymes are differentially regulated in models of altered mechanical and metabolic stress, and that Amd1 and SpdSyn are, in part, regulated in a mTORC1-dependent manner. Furthermore, these data suggest that polyamines may play a role in the adaptive response to stressors in skeletal muscle.

Keywords:  mTORC1; muscle atrophy; muscle hypertrophy; polyamine; protein synthesis

DOI:  https://doi.org/10.1152/ajpcell.00078.2021
Cell Metab. 2021 Apr 16. pii: S1550-4131(21)00166-2. [Epub ahead of print]

The adverse metabolic effects of branched-chain amino acids are mediated by isoleucine and valine.

Deyang Yu, Nicole E Richardson, Cara L Green, Alexandra B Spicer, Michaela E Murphy, Victoria Flores, Cholsoon Jang, Ildiko Kasza, Maria Nikodemova, Matthew H Wakai, Jay L Tomasiewicz, Shany E Yang, Blake R Miller, Heidi H Pak, Jacqueline A Brinkman, Jennifer M Rojas, William J Quinn, Eunhae P Cheng, Elizabeth N Konon, Lexington R Haider, Megan Finke, Michelle Sonsalla, Caroline M Alexander, Joshua D Rabinowitz, Joseph A Baur, Kristen C Malecki, Dudley W Lamming.

  Low-protein diets promote metabolic health in rodents and humans, and the benefits of low-protein diets are recapitulated by specifically reducing dietary levels of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we demonstrate that each BCAA has distinct metabolic effects. A low isoleucine diet reprograms liver and adipose metabolism, increasing hepatic insulin sensitivity and ketogenesis and increasing energy expenditure, activating the FGF21-UCP1 axis. Reducing valine induces similar but more modest metabolic effects, whereas these effects are absent with low leucine. Reducing isoleucine or valine rapidly restores metabolic health to diet-induced obese mice. Finally, we demonstrate that variation in dietary isoleucine levels helps explain body mass index differences in humans. Our results reveal isoleucine as a key regulator of metabolic health and the adverse metabolic response to dietary BCAAs and suggest reducing dietary isoleucine as a new approach to treating and preventing obesity and diabetes.

Keywords:  FGF21; GCN2; body mass index; branched-chain amino acids; diabetes; insulin resistance; isoleucine; mTORC1; obesity; valine

DOI:  https://doi.org/10.1016/j.cmet.2021.03.025
Sci Adv. 2021 Apr;pii: eabg4922. [Epub ahead of print]7(17):

Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy.

Chunmei Chang, Xiaoshan Shi, Liv E Jensen, Adam L Yokom, Dorotea Fracchiolla, Sascha Martens, James H Hurley.

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

DOI: https://doi.org/10.1126/sciadv.abg4922
Oncotarget. 2021 Apr 13. 12(8): 859-872

High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.

Elodie Montaudon, Rania El Botty, Sophie Vacher, Olivier Déas, Adnan Naguez, Sophie Chateau-Joubert, Damien Treguer, Ludmilla de Plater, Leïla Zemoura, Fariba Némati, André Nicolas, Alain Chapelier, Alain Livartowski, Stefano Cairo, Catherine Daniel, Marie Brevet, Elisabetta Marangoni, Didier Meseure, Sergio Roman-Roman, Ivan Bieche, Nicolas Girard, Didier Decaudin.

  Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient's outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

Keywords:  NSCLC; PLK1; Pi3K signalling pathway; RAD001 (everolimus); mTORC1

DOI:  https://doi.org/10.18632/oncotarget.27930
FEBS J. 2021 Apr 17.

Self-organization of apical membrane protein sorting in epithelial cells.

Daniel S Levic, Michel Bagnat.

  Polarized epithelial cells are characterized by the asymmetric distribution of proteins between apical and basolateral domains of the plasma membrane. This asymmetry is highly conserved and is fundamental to epithelial cell physiology, development, and homeostasis. How proteins are segregated for apical or basolateral delivery, a process known as sorting, has been the subject of considerable investigation for decades. Despite these efforts, the rules guiding apical sorting are poorly understood and remain controversial. Here, we consider mechanisms of apical membrane protein sorting and argue that they are largely driven by self-organization and biophysical principles. The preponderance of data to date are consistent with the idea that apical sorting is not ruled by a dedicated protein-based sorting machinery and relies instead on the concerted effects of oligomerization, phase separation of lipids and proteins in membranes, and pH-dependent glycan interactions.

Keywords:  V-H+ATPase; acidification; apical membrane; apical sorting; clustering; epithelial cell; glycans; lipid raft; oligomerization; polarization; segregation; zebrafish

DOI:  https://doi.org/10.1111/febs.15882
J Biol Chem. 2021 Apr 15. pii: S0021-9258(21)00418-X. [Epub ahead of print] 100632

Role of mTORC2 in biphasic regulation of brown fat metabolism in response to mild and severe cold.

Prasanna K R Allu, Esther Paulo, Ambre M Bertholet, Gavin Situ, Seung-Hwan Lee, Yixuan Wu, Catherine E Gleason, Bidisha Saha, Ajay Chawla, Biao Wang, David Pearce.

  Non-shivering thermogenesis is essential for mammals to maintain body temperature. According to the canonical view, temperature is sensed by cutaneous thermoreceptors and nerve impulses transmitted to the hypothalamus, which generates sympathetic signals to ß-adrenergic receptors in brown adipocytes. The energy for heat generation is primarily provided by the oxidation of fatty acids derived from triglyceride hydrolysis and cellular uptake. Fatty acids also activate the uncoupling protein, UCP1, which creates a proton leak that uncouples mitochondrial oxidative phosphorylation from ATP production, resulting in energy dissipation as heat. Recent evidence supports the idea that in response to mild cold, ß-adrenergic signals stimulate not only lipolysis and fatty acid oxidation, but also act through the mTORC2-Akt signaling module to stimulate de novo lipogenesis. This opposing anabolic effect is thought to maintain lipid fuel stores during increased catabolism. We show here, using brown fat-specific Gs-alpha knockout mice and cultured adipocytes that, unlike mild cold, severe cold directly cools brown fat and bypasses ß-adrenergic signaling to inhibit mTORC2. This cell-autonomous effect both inhibits lipogenesis and augments UCP1 expression to enhance thermogenesis. These findings suggest a novel mechanism for overriding ß-adrenergic-stimulated anabolic activities while augmenting catabolic activities to resolve the homeostatic crisis presented by severe cold.

Keywords:  UCP1; adipocytes; brown adipose tissue; cold; gene expression; lipogenesis; mTOR; thermogenesis

DOI:  https://doi.org/10.1016/j.jbc.2021.100632
Nat Commun. 2021 Apr 23. 12(1): 2431

VPS39-deficiency observed in type 2 diabetes impairs muscle stem cell differentiation via altered autophagy and epigenetics.

Cajsa Davegårdh, Johanna Säll, Anna Benrick, Christa Broholm, Petr Volkov, Alexander Perfilyev, Tora Ida Henriksen, Yanling Wu, Line Hjort, Charlotte Brøns, Ola Hansson, Maria Pedersen, Jens U Würthner, Klaus Pfeffer, Emma Nilsson, Allan Vaag, Elisabet Stener-Victorin, Karolina Pircs, Camilla Scheele, Charlotte Ling.

Insulin resistance and lower muscle quality (strength divided by mass) are hallmarks of type 2 diabetes (T2D). Here, we explore whether alterations in muscle stem cells (myoblasts) from individuals with T2D contribute to these phenotypes. We identify VPS39 as an important regulator of myoblast differentiation and muscle glucose uptake, and VPS39 is downregulated in myoblasts and myotubes from individuals with T2D. We discover a pathway connecting VPS39-deficiency in human myoblasts to impaired autophagy, abnormal epigenetic reprogramming, dysregulation of myogenic regulators, and perturbed differentiation. VPS39 knockdown in human myoblasts has profound effects on autophagic flux, insulin signaling, epigenetic enzymes, DNA methylation and expression of myogenic regulators, and gene sets related to the cell cycle, muscle structure and apoptosis. These data mimic what is observed in myoblasts from individuals with T2D. Furthermore, the muscle of Vps39+/- mice display reduced glucose uptake and altered expression of genes regulating autophagy, epigenetic programming, and myogenesis. Overall, VPS39-deficiency contributes to impaired muscle differentiation and reduced glucose uptake. VPS39 thereby offers a therapeutic target for T2D.

DOI: https://doi.org/10.1038/s41467-021-22068-5