bims-lysosi 2021-11-28 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒11‒28
37 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.
The 40S-LARP1 complex reprograms the cellular translatome upon mTOR inhibition to preserve the protein synthetic capacity.
L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation.
Golgi requires a new casting in the screenplay of mucopolysaccharidosis II cytopathology.
Loss of Function of Mutant IDS Due to Endoplasmic Reticulum-Associated Degradation: New Therapeutic Opportunities for Mucopolysaccharidosis Type II.
Presenilin/γ-secretase activity is located in acidic compartments of live neurons.
Patient-Specific iPSC-Derived Neural Differentiated and Hepatocyte-like Cells, Carrying the Compound Heterozygous Mutation p.V1023Sfs*15/p.G992R, Present the "Variant" Biochemical Phenotype of Niemann-Pick Type C1 Disease.
Pharmacological Chaperones for β-Galactosidase Related to GM1 -Gangliosidosis and Morquio B: Recent Advances.
LAMP3 inhibits autophagy and contributes to cell death by lysosomal membrane permeabilization.
Possible Involvement of the Nutrient and Energy Sensors mTORC1 and AMPK in Cell Fate Diversification in a Non-Metazoan Organism.
Sestrin2 as a Potential Target for Regulating Metabolic-Related Diseases.
NAADP-binding proteins find their identity.
The fission yeast FLCN/FNIP complex augments TORC1 repression or activation in response to amino acid (AA) availability.
Lysosomal dysfunction impairs mitochondrial quality control and is associated with neurodegeneration in TBCK encephaloneuronopathy.
RNF13 Dileucine Motif Variants L311S and L312P Interfere with Endosomal Localization and AP-3 Complex Association.
Comprehensive-targeted lipidomic analysis in Niemann-Pick C disease.
Reduction of glutamate neurotoxicity: A novel therapeutic approach for Niemann-Pick disease, type C1.
Mechanistic Target of Rapamycin Complex 2 Regulation of the Primary Human Trophoblast Cell Transcriptome.
Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins.
A Comprehensive, Targeted NGS Approach to Assessing Molecular Diagnosis of Lysosomal Storage Diseases.
Benign tumors in TSC are amenable to treatment by GD3 CAR T cells in mice.
Post-transcriptional regulation of ATG1 is a critical node that modulates autophagy during distinct nutrient stresses.
Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model.
New insights and therapeutic opportunities for progranulin-deficient frontotemporal dementia.
iPSC-Derived Gaucher Macrophages Display Growth Impairment and Activation of Inflammation-Related Cell Death.
Activation Mechanisms of the VPS34 Complexes.
The evolutionary conserved TLDc domain defines a new class of (H+)V-ATPase interacting proteins.
Innate host defense mechanisms SAC bacteria by regulating phosphoinositide kinases and phosphatases.
Detection of Mosaic Variants in Mothers of MPS II Patients by Next Generation Sequencing.
Golgi membrane protein Erd1 Is essential for recycling a subset of Golgi glycosyltransferases.
The mTOR Pathway in Pluripotent Stem Cells: Lessons for Understanding Cancer Cell Dormancy.
PI4P/PS countertransport by ORP10 at ER-endosome membrane contact sites regulates endosome fission.
Development of a versatile HPLC-based method to evaluate the activation status of small GTPases.
Characterization of a spontaneous cell line from primary mouse fibroblasts as a model to study Sanfilippo syndrome.
Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway.
The lysosomal trafficking regulator is necessary for normal wound healing.
TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

PLoS One. 2021 ;16(11): e0259313

Reactive oxygen species prevent lysosome coalescence during PIKfyve inhibition.

Golam T Saffi, Evan Tang, Sami Mamand, Subothan Inpanathan, Aaron Fountain, Leonardo Salmena, Roberto J Botelho.

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.

DOI: https://doi.org/10.1371/journal.pone.0259313
Sci Adv. 2021 Nov 26. 7(48): eabg9275

The 40S-LARP1 complex reprograms the cellular translatome upon mTOR inhibition to preserve the protein synthetic capacity.

Pedro Fuentes, Joffrey Pelletier, Carolina Martinez-Herráez, Virginia Diez-Obrero, Flavia Iannizzotto, Teresa Rubio, Marta Garcia-Cajide, Sandra Menoyo, Victor Moreno, Ramón Salazar, Albert Tauler, Antonio Gentilella.

[Figure: see text].

DOI: https://doi.org/10.1126/sciadv.abg9275
Cells. 2021 Nov 11. pii: 3122. [Epub ahead of print]10(11):

L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation.

Beatriz Castejón-Vega, Alejandro Rubio, Antonio J Pérez-Pulido, José L Quiles, Jon D Lane, Beatriz Fernández-Domínguez, María Begoña Cachón-González, Carmen Martín-Ruiz, Alberto Sanz, Timothy M Cox, Elísabet Alcocer-Gómez, Mario D Cordero.

  AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by β-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases.RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities.
INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases.
CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy.

Keywords:  GM2 gangliosidosis; L-arginine; autophagy; mTOR

DOI:  https://doi.org/10.3390/cells10113122
Biol Futur. 2021 Nov 27.

Golgi requires a new casting in the screenplay of mucopolysaccharidosis II cytopathology.

Kinga Molnár, Julianna Kobolák, András Dinnyés.

  Lysosome (L), a hydrolytic compartment of the endo-lysosomal system (ELS), plays a central role in the metabolic regulation of eukaryotic cells. Furthermore, it has a central role in the cytopathology of several diseases, primarily in lysosomal storage diseases (LSDs). Mucopolysaccharidosis II (MPS II, Hunter disease) is a rare LSD caused by idunorate-2-sulphatase (IDS) enzyme deficiency. To provide a new platform for drug development and clarifying the background of the clinically observed cytopathology, we established a human in vitro model, which recapitulates all cellular hallmarks of the disease. Some of our results query the traditional concept by which the storage vacuoles originate from the endosomal system and suggest a new concept, in which endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and RAB2/LAMP positive Golgi (G) vesicles play an initiative role in the vesicle formation. In this hypothesis, Golgi is not only an indirectly affected organelle but enforced to be the main support of vacuole formation. The purposes of this minireview are to give a simple guide for understanding the main relationships in ELS, to present the storage vacuoles and their relation to ELS compartments, to recommend an alternative model for vacuole formation, and to place the Golgi in spotlight of MPS II cytopathology.

Keywords:  ERGIC; Endo-lysosomal system; Golgi; LAMP; Lysosomal storage disease; RAB2

DOI:  https://doi.org/10.1007/s42977-021-00107-y
Int J Mol Sci. 2021 Nov 12. pii: 12227. [Epub ahead of print]22(22):

Loss of Function of Mutant IDS Due to Endoplasmic Reticulum-Associated Degradation: New Therapeutic Opportunities for Mucopolysaccharidosis Type II.

Koji Matsuhisa, Kazunori Imaizumi.

  Mucopolysaccharidosis type II (MPS II) results from the dysfunction of a lysosomal enzyme, iduronate-2-sulfatase (IDS). Dysfunction of IDS triggers the lysosomal accumulation of its substrates, glycosaminoglycans, leading to mental retardation and systemic symptoms including skeletal deformities and valvular heart disease. Most patients with severe types of MPS II die before the age of 20. The administration of recombinant IDS and transplantation of hematopoietic stem cells are performed as therapies for MPS II. However, these therapies either cannot improve functions of the central nervous system or cause severe side effects, respectively. To date, 729 pathogenetic variants in the IDS gene have been reported. Most of these potentially cause misfolding of the encoded IDS protein. The misfolded IDS mutants accumulate in the endoplasmic reticulum (ER), followed by degradation via ER-associated degradation (ERAD). Inhibition of the ERAD pathway or refolding of IDS mutants by a molecular chaperone enables recovery of the lysosomal localization and enzyme activity of IDS mutants. In this review, we explain the IDS structure and mechanism of activation, and current findings about the mechanism of degradation-dependent loss of function caused by pathogenetic IDS mutation. We also provide a potential therapeutic approach for MPS II based on this loss-of-function mechanism.

Keywords:  endoplasmic reticulum-associated degradation; iduronate-2-sulfatase; lysosomal storage disorder; mucopolysaccharidosis type II

DOI:  https://doi.org/10.3390/ijms222212227
J Neurosci. 2021 Nov 19. pii: JN-RM-1698-21. [Epub ahead of print]

Presenilin/γ-secretase activity is located in acidic compartments of live neurons.

Masato Maesako, Mei Cq Houser, Yuliia Turchyna, Michael S Wolfe, Oksana Berezovska.

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of β-amyloid (Aβ) peptides. Yet, exactly where within the neuron γ-secretase processes APP C99 to generate Aβ and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly "visualize" the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTrackerTM-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aβ from C99, we also show that intracellular Aβ is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aβ being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.Significance Statement:Alzheimer's disease (AD) genetics and histopathology highlight the importance of APP processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly "visualize" that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular Aβ is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aβ in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.

DOI: https://doi.org/10.1523/JNEUROSCI.1698-21.2021
Int J Mol Sci. 2021 Nov 10. pii: 12184. [Epub ahead of print]22(22):

Patient-Specific iPSC-Derived Neural Differentiated and Hepatocyte-like Cells, Carrying the Compound Heterozygous Mutation p.V1023Sfs*15/p.G992R, Present the "Variant" Biochemical Phenotype of Niemann-Pick Type C1 Disease.

Christin Völkner, Maik Liedtke, Robert Untucht, Andreas Hermann, Moritz J Frech.

  Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder caused by autosomal recessive mutations in the NPC1 gene. Patients display a wide spectrum on the clinical as well as on the molecular level, wherein a so-called "variant" biochemical phenotype can be observed. Here, we report an in vitro analysis of fibroblasts obtained from an NP-C1 patient carrying the undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R. Since NP-C1 is a neurovisceral disease and the patient suffers from severe neurological as well as hepatic symptoms, we extended our study to neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells. We detected slightly increased intracellular cholesterol levels compared to the control cell line in fibroblasts, neural differentiated and hepatocyte-like cells, suggesting a "variant" biochemical phenotype. Furthermore, the total NPC1 protein, as well as post-ER glycoforms of the NPC1 protein, tended to be reduced. In addition, colocalization analysis revealed a mild reduction of the NPC1 protein in the lysosomes. The patient was diagnosed with NP-C1 at the age of 34 years, after an initial misdiagnosis of schizophrenia. After years of mild and unspecific symptoms, such as difficulties in coordination and concentration, symptoms progressed and the patient finally presented with ataxia, dysarthria, dysphagia, vertical supranuclear gaze palsy, and hepatosplenomegaly. Genetic testing finally pointed towards an NP-C1 diagnosis, revealing the so-far undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R in the NPC1 gene. In light of these findings, this case provides support for the p.G992R mutation being causative for a "variant" biochemical phenotype leading to an adult-onset type of NP-C1 disease.

Keywords:  NP-C1; NPC1; cholesterol; filipin; iPSCs; induced pluripotent stem cells

DOI:  https://doi.org/10.3390/ijms222212184
Chem Rec. 2021 Nov;21(11): 2980-2989

Pharmacological Chaperones for β-Galactosidase Related to GM1 -Gangliosidosis and Morquio B: Recent Advances.

Arnold E Stütz, Martin Thonhofer, Patrick Weber, Andreas Wolfsgruber, Tanja M Wrodnigg.

  A short survey on selected β-galactosidase inhibitors as potential pharmacological chaperones for GM1 -gangliosidosis and Morquio B associated mutants of human lysosomal β-galactosidase is provided highlighting recent developments in this particular area of lysosomal storage disorders and orphan diseases.

Keywords:  Carbohydrates; Inhibitors; Lysosomal storage disease; Lysosomal β-Galactosidase; Pharmacological chaperone

DOI:  https://doi.org/10.1002/tcr.202100269
Autophagy. 2021 Nov 22. 1-19

LAMP3 inhibits autophagy and contributes to cell death by lysosomal membrane permeabilization.

Tsutomu Tanaka, Blake M Warner, Drew G Michael, Hiroyuki Nakamura, Toshio Odani, Hongen Yin, Tatsuya Atsumi, Masayuki Noguchi, John A Chiorini.

  Sjögren syndrome (SS) is a chronic and progressive autoimmune disease characterized by dry mouth and dry eyes, and characteristic autoantibodies. Evidence of altered macroautophagy/autophagy and apoptosis has been associated with SS, but a mechanistic understanding of the gene expression changes associated with these abnormal processes has not been realized. Recently, increased LAMP3 (lysosomal associated membrane protein 3) expression was found in a subset of SS patients and was associated with increased apoptosis and autoantigen accumulation and release. To better understand how LAMP3 expression might modulate apoptosis, cell biology, and biochemical studies were used to examine the effect of LAMP3 expression in minor salivary gland cells. LAMP3 expression resulted in degradation of LAMP1 increasing lysosomal membrane permeabilization and relocalization of cathepsins to the cytoplasm, resulting in destabilizing autophagic flux and caspase activation. These findings highlight the central role of LAMP3 expression in the pathogenesis of SS.

Keywords:  Apoptosis; Sjögren syndrome; autophagy; lysosomal membrane permeabilization; lysosome-associated membrane protein 3; salivary gland

DOI:  https://doi.org/10.1080/15548627.2021.1995150
Front Cell Dev Biol. 2021 ;9 758317

Possible Involvement of the Nutrient and Energy Sensors mTORC1 and AMPK in Cell Fate Diversification in a Non-Metazoan Organism.

Julian D Gross, Catherine J Pears.

  mTORC1 and AMPK are mutually antagonistic sensors of nutrient and energy status that have been implicated in many human diseases including cancer, Alzheimer's disease, obesity and type 2 diabetes. Starved cells of the social amoeba Dictyostelium discoideum aggregate and eventually form fruiting bodies consisting of stalk cells and spores. We focus on how this bifurcation of cell fate is achieved. During growth mTORC1 is highly active and AMPK relatively inactive. Upon starvation, AMPK is activated and mTORC1 inhibited; cell division is arrested and autophagy induced. After aggregation, a minority of the cells (prestalk cells) continue to express much the same set of developmental genes as during aggregation, but the majority (prespore cells) switch to the prespore program. We describe evidence suggesting that overexpressing AMPK increases the proportion of prestalk cells, as does inhibiting mTORC1. Furthermore, stimulating the acidification of intracellular acidic compartments likewise increases the proportion of prestalk cells, while inhibiting acidification favors the spore pathway. We conclude that the choice between the prestalk and the prespore pathways of cell differentiation may depend on the relative strength of the activities of AMPK and mTORC1, and that these may be controlled by the acidity of intracellular acidic compartments/lysosomes (pHv), cells with low pHv compartments having high AMPK activity/low mTORC1 activity, and those with high pHv compartments having high mTORC1/low AMPK activity. Increased insight into the regulation and downstream consequences of this switch should increase our understanding of its potential role in human diseases, and indicate possible therapeutic interventions.

Keywords:  AMP-dependent protein kinase (AMPK); DIF-1; Dictyostelium discoideum; acidic vesicles; ammonia; cell fate decision; mechanistic target of rapamycin complex 1 (mTORC1)

DOI:  https://doi.org/10.3389/fcell.2021.758317
Front Endocrinol (Lausanne). 2021 ;12 751020

Sestrin2 as a Potential Target for Regulating Metabolic-Related Diseases.

Linan Gong, Zanzan Wang, Zhenggui Wang, Zhiguo Zhang.

  Sestrin2 is a highly conserved protein that can be induced under a variety of stress conditions, including DNA damage, oxidative stress, endoplasmic reticulum (ER) stress, and metabolic stress. Numerous studies have shown that the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway has a crucial role in the regulation of metabolism. Sestrin2 regulates metabolism via a number of pathways, including activation of AMPK, inhibition of the mTOR complex 1 (mTORC1), activation of mTOR complex 2 (mTORC2), inhibition of ER stress, and promotion of autophagy. Therefore, modulation of Sestrin2 activity may provide a potential therapeutic target for the prevention of metabolic diseases such as insulin resistance, diabetes, obesity, non-alcoholic fatty liver disease, and myocardial ischemia/reperfusion injury. In this review, we examined the regulatory relationship between Sestrin2 and the AMPK/mTOR signaling pathway and the effects of Sestrin2 on energy metabolism.

Keywords:  AMPK; Sestrin2; mTOR; metabolic diseases; metabolism

DOI:  https://doi.org/10.3389/fendo.2021.751020
Trends Biochem Sci. 2021 Nov 20. pii: S0968-0004(21)00234-6. [Epub ahead of print]

NAADP-binding proteins find their identity.

Jonathan S Marchant, Gihan S Gunaratne, Xinjiang Cai, James T Slama, Sandip Patel.

  Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.

Keywords:  calcium signaling; cancer; endosomes; lysosomes; photoaffinity probes

DOI:  https://doi.org/10.1016/j.tibs.2021.10.008
iScience. 2021 Nov 19. 24(11): 103338

The fission yeast FLCN/FNIP complex augments TORC1 repression or activation in response to amino acid (AA) availability.

Isabel A Calvo, Shalini Sharma, Joao A Paulo, Alexander O D Gulka, Andras Boeszoermenyi, Jingyu Zhang, Jose M Lombana, Christina M Palmieri, Laura A Laviolette, Haribabu Arthanari, Othon Iliopoulos, Steven P Gygi, Mo Motamedi.

  The target of Rapamycin complex1 (TORC1) senses and integrates several environmental signals, including amino acid (AA) availability, to regulate cell growth. Folliculin (FLCN) is a tumor suppressor (TS) protein in renal cell carcinoma, which paradoxically activates TORC1 in response to AA supplementation. Few tractable systems for modeling FLCN as a TS are available. Here, we characterize the FLCN-containing complex in Schizosaccharomyces pombe (called BFC) and show that BFC augments TORC1 repression and activation in response to AA starvation and supplementation, respectively. BFC co-immunoprecipitates V-ATPase, a TORC1 modulator, and regulates its activity in an AA-dependent manner. BFC genetic and proteomic networks identify the conserved peptide transmembrane transporter Ptr2 and the phosphoribosylformylglycinamidine synthase Ade3 as new AA-dependent regulators of TORC1. Overall, these data ascribe an additional repressive function to Folliculin in TORC1 regulation and reveal S. pombe as an excellent system for modeling the AA-dependent, FLCN-mediated repression of TORC1 in eukaryotes.

Keywords:  Cell biology; Cellular physiology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2021.103338
Brain Commun. 2021 ;3(4): fcab215

Lysosomal dysfunction impairs mitochondrial quality control and is associated with neurodegeneration in TBCK encephaloneuronopathy.

Jesus A Tintos-Hernández, Adrian Santana, Kierstin N Keller, Xilma R Ortiz-González.

  Biallelic variants in the TBCK gene cause intellectual disability with remarkable clinical variability, ranging from static encephalopathy to progressive neurodegeneration (TBCK-Encephaloneuronopathy). The biological factors underlying variable disease penetrance remain unknown. Since previous studies had suggested aberrant autophagy, we tested whether mitophagy and mitochondrial function are altered in TBCK -/- fibroblasts derived from patients exhibiting variable clinical severity. Our data show significant accumulation of mitophagosomes, reduced mitochondrial respiratory capacity and mitochondrial DNA content, suggesting impaired mitochondrial quality control. Furthermore, the degree of mitochondrial dysfunction correlates with a neurodegenerative clinical course. Since mitophagy ultimately depends on lysosomal degradation, we also examined lysosomal function. Our data show that lysosomal proteolytic function is significantly reduced in TBCK -/- fibroblasts. Moreover, acidifying lysosomal nanoparticles rescue the mitochondrial respiratory defects in fibroblasts, suggesting impaired mitochondrial quality control secondary to lysosomal dysfunction. Our data provide insight into the disease mechanisms of TBCK Encephaloneuronopathy and the potential relevance of mitochondrial function as a biomarker beyond primary mitochondrial disorders. It also supports the benefit of lysosomal acidification strategies for disorders of impaired lysosomal degradation affecting mitochondrial quality control.

Keywords:  intellectual disability; lysosome; mitochondria; mitophagy; neurodegeneration

DOI:  https://doi.org/10.1093/braincomms/fcab215
Cells. 2021 Nov 06. pii: 3063. [Epub ahead of print]10(11):

RNF13 Dileucine Motif Variants L311S and L312P Interfere with Endosomal Localization and AP-3 Complex Association.

Valérie C Cabana, Antoine Y Bouchard, Audrey M Sénécal, Kim Ghilarducci, Saïd Kourrich, Laurent Cappadocia, Marc P Lussier.

  Developmental and epileptic encephalopathies (DEE) are rare and serious neurological disorders characterized by severe epilepsy with refractory seizures and a significant developmental delay. Recently, DEE73 was linked to genetic alterations of the RNF13 gene, which convert positions 311 or 312 in the RNF13 protein from leucine to serine or proline, respectively (L311S and L312P). Using a fluorescence microscopy approach to investigate the molecular and cellular mechanisms affected by RNF13 protein variants, the current study shows that wild-type RNF13 localizes extensively with endosomes and lysosomes, while L311S and L312P do not extensively colocalize with the lysosomal marker Lamp1. Our results show that RNF13 L311S and L312P proteins affect the size of endosomal vesicles along with the temporal and spatial progression of fluorescently labeled epidermal growth factor, but not transferrin, in the endolysosomal system. Furthermore, GST-pulldown and co-immunoprecipitation show that RNF13 variants disrupt association with AP-3 complex. Knockdown of AP-3 complex subunit AP3D1 alters the lysosomal localization of wild-type RNF13 and similarly affects the size of endosomal vesicles. Importantly, our study provides a first step toward understanding the cellular and molecular mechanism altered by DEE73-associated genetic variations of RNF13.

Keywords:  AP-3 complex; RNF13; developmental and epileptic encephalopathy-73; endosomes; genetic mutations; intracellular trafficking; lysosomes

DOI:  https://doi.org/10.3390/cells10113063
Mol Genet Metab. 2021 Nov 16. pii: S1096-7192(21)00823-4. [Epub ahead of print]

Comprehensive-targeted lipidomic analysis in Niemann-Pick C disease.

Sara Boenzi, Giulio Catesini, Elisa Sacchetti, Francesco Tagliaferri, Carlo Dionisi-Vici, Federica Deodato.

  Niemann-Pick C disease (NPC) is a lysosomal disease caused by mutations in NPC1 or NPC2 genes responsible for intracellular accumulation of free cholesterol and glycosphingolipids in a variety of tissues. We collected plasma samples from 15 NPC1 patients and 15 age-matched controls to analyze the impairment of lipid metabolism. Comprehensive-targeted quantitative lipidomic analysis was per-formed by Ion Mobility Mass Spectrometry, while oxysterols and lyso-sphingolipids, the classical NPC biomarkers, were analyzed by LC-MS/MS. Lipidomic analysis allowed the quantitation of ~1100 lipid species, belonging to 13 different classes. Statistical analysis of collected data showed a significant differentiation between NPC patients and controls. Lipid profiling showed an elevation of arachidonic acid and total diacylglycerols. Conversely, sphingomyelins, phosphatidylethano-lamines, phosphatidylcholines, cholesterylesters, and lactosylceramides were decreased. Indeed, the lipid imbalance was consistent with the increased concentrations of oxysterols and lyso-sphingolipids. Our study revealed a novel disease biosignature suggesting new potential diagnostic biomarkers. The alteration in key lipids molecules involved in inflammatory pathways and in oxidative stress regulation, provides new insights in the complex pathophysiology of the disease, still largely un-known.

Keywords:  Arachidonic acid; LC-MS/MS; Lipidomic analysis; Lyso-sphingolipids; Niemann-Pick disease C; Oxysterols

DOI:  https://doi.org/10.1016/j.ymgme.2021.11.005
Mol Genet Metab. 2021 Nov 16. pii: S1096-7192(21)00826-X. [Epub ahead of print]

Reduction of glutamate neurotoxicity: A novel therapeutic approach for Niemann-Pick disease, type C1.

Antony Cougnoux, Julia C Yerger, Mason Fellmeth, Jenny Serra-Vinardell, Fatemeh Navid, Christopher A Wassif, Niamh X Cawley, Forbes D Porter.

  Niemann-Pick disease, type C1 is a progressive, lethal, neurodegenerative disorder due to endolysosomal storage of unesterified cholesterol. Cerebellar ataxia, as a result of progressive loss of cerebellar Purkinje neurons, is a major symptom of Nieman-Pick disease, type C1. Comparing single cell RNAseq data from control (Npc1+/+) and mutant (Npc1-/-) mice, we observed significantly decreased expression of Slc1a3 in Npc1-/- astrocytes. Slc1a3 encodes a glutamate transporter (GLAST, EAAT1) which functions to decrease glutamate concentrations in the post synaptic space after neuronal firing. Glutamate is an excitatory neurotransmitter and elevated extracellular levels of glutamate can be neurotoxic. Impaired EAAT1 function underlies type-6 episodic ataxia, a rare disorder with progressive cerebellar dysfunction, thus suggesting that impaired glutamate uptake in Niemann-Pick disease, type C1 could contribute to disease progression. We now show that decreased expression of Slc1a3 in Npc1-/- mice has functional consequences that include decreased surface protein expression and decreased glutamate uptake by Npc1-/- astrocytes. To test whether glutamate neurotoxicity plays a role in Niemann-Pick disease, type C1 progression, we treated NPC1 deficient mice with ceftriaxone and riluzole. Ceftriaxone is a β-lactam antibiotic that is known to upregulate the expression of Slc1a2, an alternative glial glutamate transporter. Although ceftriaxone increased Slc1a2 expression, we did not observe a treatment effect in NPC1 mutant mice. Riluzole is a glutamate receptor antagonist that inhibits postsynaptic glutamate receptor signaling and reduces the release of glutamate. We found that treatment with riluzole increased median survival in Npc1-/- by 12%. Given that riluzole is an approved drug for the treatment of amyotrophic lateral sclerosis, repurposing of this drug may provide a novel therapeutic approach to decrease disease progression in Niemann-Pick disease type, C1 patients.

Keywords:  EAAT1; GLAST; Glutamate-mediated neurotoxicity; Lysosomal disease; NPC1; Niemann-Pick disease, type C1; Purkinje neurons; Riluzole; SLC1A3

DOI:  https://doi.org/10.1016/j.ymgme.2021.11.008
Front Cell Dev Biol. 2021 ;9 670980

Mechanistic Target of Rapamycin Complex 2 Regulation of the Primary Human Trophoblast Cell Transcriptome.

Fredrick J Rosario, Amy Catherine Kelly, Madhulika B Gupta, Theresa L Powell, Laura Cox, Thomas Jansson.

  Mechanistic Target of Rapamycin Complex 2 (mTORC2) regulates placental amino acid and folate transport. However, the role of mTORC2 in modulating other placental functions is largely unexplored. We used a gene array following the silencing of rictor to identify genes regulated by mTORC2 in primary human trophoblast (PHT) cells. Four hundred and nine genes were differentially expressed; 102 genes were down-regulated and 307 up-regulated. Pathway analyses demonstrated that inhibition of mTORC2 resulted in increased expression of genes encoding for pro-inflammatory IL-6, VEGF-A, leptin, and inflammatory signaling (SAPK/JNK). Furthermore, down-regulated genes were functionally enriched in genes involved in angiogenesis (Osteopontin) and multivitamin transport (SLC5A6). In addition, the protein expression of leptin, VEGFA, IL-6 was increased and negatively correlated to mTORC2 signaling in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In contrast, the protein expression of Osteopontin and SLC5A6 was decreased and positively correlated to mTORC2 signaling in human IUGR placentas. In conclusion, mTORC2 signaling regulates trophoblast expression of genes involved in inflammation, micronutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions necessary for fetal growth and development.

Keywords:  gene array; human; maternal-fetal exchange; nutrient sensor; placenta

DOI:  https://doi.org/10.3389/fcell.2021.670980
J Cell Biol. 2022 Jan 03. pii: e202106044. [Epub ahead of print]221(1):

Quantitative correlative microscopy reveals the ultrastructural distribution of endogenous endosomal proteins.

Jan van der Beek, Cecilia de Heus, Nalan Liv, Judith Klumperman.

The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural-functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.

DOI: https://doi.org/10.1083/jcb.202106044
Genes (Basel). 2021 Oct 30. pii: 1750. [Epub ahead of print]12(11):

A Comprehensive, Targeted NGS Approach to Assessing Molecular Diagnosis of Lysosomal Storage Diseases.

Valentina La Cognata, Sebastiano Cavallaro.

  With over 60 different disorders and a combined incidence occurring in 1:5000-7000 live births, lysosomal storage diseases (LSDs) represent a major public health problem and constitute an enormous burden for affected individuals and their families. Several reasons make the diagnosis of LSDs an arduous task for clinicians, including the phenotype and penetrance variability, the shared signs and symptoms, and the uncertainties related to biochemical enzymatic assay results. Developing a powerful diagnostic tool based on next generation sequencing (NGS) technology may help reduce the delayed diagnostic process for these families, leading to better outcomes for current therapies and providing the basis for more appropriate genetic counseling. Herein, we employed a targeted NGS-based panel to scan the coding regions of 65 LSD-causative genes. A reference group sample (n = 26) with previously known genetic mutations was used to test and validate the entire workflow. Our approach demonstrated elevated analytical accuracy, sensitivity, and specificity. We believe the adoption of comprehensive targeted sequencing strategies into a routine diagnostic route may accelerate both the identification and management of LSDs with overlapping clinical profiles, producing a significant reduction in delayed diagnostic response with beneficial results in the treatment outcome.

Keywords:  diagnosis; lysosomal storage disease (LSDs); targeted next generation sequencing (tNGS)

DOI:  https://doi.org/10.3390/genes12111750
JCI Insight. 2021 Nov 22. pii: e152014. [Epub ahead of print]6(22):

Benign tumors in TSC are amenable to treatment by GD3 CAR T cells in mice.

Ancy Thomas, Saurav Sumughan, Emilia R Dellacecca, Rohan S Shivde, Nicola Lancki, Zhussipbek Mukhatayev, Cristina C Vaca, Fei Han, Levi Barse, Steven W Henning, Jesus Zamora-Pineda, Suhail Akhtar, Nikhilesh Gupta, Jasmine O Zahid, Stephanie R Zack, Prathyaya Ramesh, Dinesh Jaishankar, Agnes Sy Lo, Joel Moss, Maria M Picken, Thomas N Darling, Denise M Scholtens, Daniel F Dilling, Richard P Junghans, I Caroline Le Poole.

  Mutations underlying disease in tuberous sclerosis complex (TSC) give rise to tumors with biallelic mutations in TSC1 or TSC2 and hyperactive mammalian target of rapamycin complex 1 (mTORC1). Benign tumors might exhibit de novo expression of immunogens, targetable by immunotherapy. As tumors may rely on ganglioside D3 (GD3) expression for mTORC1 activation and growth, we compared GD3 expression in tissues from patients with TSC and controls. GD3 was overexpressed in affected tissues from patients with TSC and also in aging Tsc2+/- mice. As GD3 overexpression was not accompanied by marked natural immune responses to the target molecule, we performed preclinical studies with GD3 chimeric antigen receptor (CAR) T cells. Polyfunctional CAR T cells were cytotoxic toward GD3-overexpressing targets. In mice challenged with Tsc2-/- tumor cells, CAR T cells substantially and durably reduced the tumor burden, correlating with increased T cell infiltration. We also treated aged Tsc2+/- heterozygous (>60 weeks) mice that carry spontaneous Tsc2-/- tumors with GD3 CAR or untransduced T cells and evaluated them at endpoint. Following CAR T cell treatment, the majority of mice were tumor free while all control animals carried tumors. The outcomes demonstrate a strong treatment effect and suggest that targeting GD3 can be successful in TSC.

Keywords:  Antigen; Immunology; Immunotherapy; T cells; Therapeutics

DOI:  https://doi.org/10.1172/jci.insight.152014
Autophagy. 2021 Nov 26. 1-21

Post-transcriptional regulation of ATG1 is a critical node that modulates autophagy during distinct nutrient stresses.

Vikramjit Lahiri, Shree Padma Metur, Zehan Hu, Xinxin Song, Muriel Mari, Wayne D Hawkins, Janakraj Bhattarai, Elizabeth Delorme-Axford, Fulvio Reggiori, Daolin Tang, Joern Dengjel, Daniel J Klionsky.

  Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.

Keywords:  ATG1; Amino acid starvation; DDX3; DED1; RAD53; ULK1; autophagosome; autophagy

DOI:  https://doi.org/10.1080/15548627.2021.1997305
Mol Genet Metab. 2021 Nov 17. pii: S1096-7192(21)00824-6. [Epub ahead of print]

Deficiency of the sedoheptulose kinase (Shpk) does not alter the ability of hematopoietic stem cells to rescue cystinosis in the mouse model.

Spencer Goodman, Meisha Khan, Jay Sharma, Zijie Li, Jose Cano, Carlos Castellanos, Monica V Estrada, Ilya Gertsman, Stephanie Cherqui.

  Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns-/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk-/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk-/--HSPCs into Ctns-/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

Keywords:  Cystinosis; Hematopoietic stem and progenitor cells; Macrophages; Pentose phosphate pathway; Sedopheptulose kinase

DOI:  https://doi.org/10.1016/j.ymgme.2021.11.006
Curr Opin Neurobiol. 2021 Nov 23. pii: S0959-4388(21)00113-6. [Epub ahead of print]72 131-139

New insights and therapeutic opportunities for progranulin-deficient frontotemporal dementia.

Sadaf Amin, Gillian Carling, Li Gan.

Frontotemporal dementia (FTD) is the second most common form of dementia. It affects the frontal and temporal lobes of the brain and has a highly heterogeneous clinical representation with patients presenting with a wide range of behavioral, language, and executive dysfunctions. Etiology of FTD is complex and consists of both familial and sporadic cases. Heterozygous mutations in the GRN gene, resulting in GRN haploinsufficiency, cause progranulin (PGRN)-deficient FTD characterized with cytoplasmic mislocalization of TAR DNA-binding protein 43 kDa (TDP-43) aggregates. GRN codes for PGRN, a secreted protein that is also localized in the endolysosomes and plays a critical role in regulating lysosomal homeostasis. How PGRN deficiency modulates immunity and causes TDP-43 pathology and FTD-related neurodegeneration remains an active area of intense investigation. In the current review, we discuss some of the significant progress made in the past two years that links PGRN deficiency with microglial-associated neuroinflammation, TDP-43 pathology, and lysosomal dysfunction. We also review the opportunities and challenges toward developing therapies and biomarkers to treat PGRN-deficient FTD.

DOI: https://doi.org/10.1016/j.conb.2021.10.001
Cells. 2021 Oct 21. pii: 2822. [Epub ahead of print]10(11):

iPSC-Derived Gaucher Macrophages Display Growth Impairment and Activation of Inflammation-Related Cell Death.

Daria Messelodi, Salvatore Nicola Bertuccio, Valentina Indio, Silvia Strocchi, Alberto Taddia, Salvatore Serravalle, Jessica Bandini, Annalisa Astolfi, Andrea Pession.

  Gaucher disease is a lysosomal storage disorder characterized by β-glucosidase enzyme deficiency and substrate accumulation, especially in cells of the reticuloendothelial system. Typical features of the disease are the unrestrained activation of inflammatory mechanisms, whose molecular pathways are still unclear. To investigate biological mechanisms underlying the macrophage activation in GD, we derived iPSCs from a healthy donor and a GD patient line and differentiated them into hematopoietic progenitors. While GD iPSCs are able to efficiently give rise to CD33+/CD45+ myeloid progenitors, the maturation towards the CD14+/CD163+ monocyte/macrophages fate resulted enhanced in the GD lines, that in addition displayed a decreased growth potential compared to control cells either in semisolid or in liquid culture. The GD lines growth impairment was associated with a significant upregulation of RIPK3 and MLKL, two key effectors of necroptosis, the inflammation related cell death pathway. The activation of necroptosis, which has already been linked to neuronopathic GD, may play a role in the disease proinflammatory condition and in the identified cell growth defects. Understanding the GD macrophage role in the alteration of mechanisms linked to cellular metabolism imbalance, cell death and inflammation are crucial in identifying new ways to approach the disease.

Keywords:  Gaucher disease; iPSC; inflammation; macrophages; necroptosis

DOI:  https://doi.org/10.3390/cells10112822
Cells. 2021 Nov 11. pii: 3124. [Epub ahead of print]10(11):

Activation Mechanisms of the VPS34 Complexes.

Yohei Ohashi.

  Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34-Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.

Keywords:  ATG14L; Beclin 1; PtdIns(3)P; UVRAG; VPS15; VPS34; autophagy; endocytic pathway; lipids; membranes

DOI:  https://doi.org/10.3390/cells10113124
Sci Rep. 2021 Nov 22. 11(1): 22654

The evolutionary conserved TLDc domain defines a new class of (H+)V-ATPase interacting proteins.

A F Eaton, D Brown, M Merkulova.

We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved "TLDc" domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.

DOI: https://doi.org/10.1038/s41598-021-01809-y
Autophagy. 2021 Nov 23. 1-3

Innate host defense mechanisms SAC bacteria by regulating phosphoinositide kinases and phosphatases.

Kimberly L Carey, Kai Liu, Ramnik J Xavier.

  Human genetics and loss-of-function studies revealed a critical role for macroautophagy/autophagy in host defense. The autophagic delivery of intracellular pathogens to lysosomes is a central mechanism of innate immunity; thus, augmentation of host xenophagy represents a promising and powerful approach to combat infections. The precise mechanisms required for autophagosome biogenesis and maturation, however, remain unclear. Using a targeted genetic screen against phosphoinositide kinases and phosphatases, our recent work identified an essential role for the phosphoinositide phosphatase SACM1L/SAC1 in xenophagy. Re-expression of wild-type or catalytically-dead SACM1L in CRISPR knockout cells confirmed that SACM1L enzymatic activity is required to suppress replication of intracellular Salmonella. Time-dependent, quantitative and live confocal imaging demonstrated that SACM1L-deficient cells accumulate phosphatidylinositol-4-phosphate (PtdIns4P) on bacteria-containing autophagosomes, resulting in delayed fusion with degradative lysosomes and reduced bacterial killing. We further discovered that the secreted Salmonella effector protein SteA, which specifically binds PtdIns4P, exacerbates the SACM1L-dependent delay in autophagosomal maturation. These findings reveal a relationship in which the balance between host defense and bacterial survival depends upon autophagosomal membrane composition.

Keywords:  Autophagosome; PtdIns4P; SAC1; SACM1L; Salmonella; SteA; phosphatidylinositol-4-phosphate; phosphoinositide phosphatase; selective autophagy; xenophagy

DOI:  https://doi.org/10.1080/15548627.2021.2002102
Front Mol Biosci. 2021 ;8 789350

Detection of Mosaic Variants in Mothers of MPS II Patients by Next Generation Sequencing.

Alice Brinckmann Oliveira Netto, Ana Carolina Brusius-Facchin, Sandra Leistner-Segal, Francyne Kubaski, Juliana Josahkian, Roberto Giugliani.

  Mucopolysaccharidosis type II is an X-linked lysosomal storage disorder caused by mutations in the IDS gene that encodes the iduronate-2-sulfatase enzyme. The IDS gene is located on the long arm of the X-chromosome, comprising 9 exons, spanning approximately 24 kb. The analysis of carriers, in addition to detecting mutations in patients, is essential for genetic counseling, since the risk of recurrence for male children is 50%. Mosaicism is a well-known phenomenon described in many genetic disorders caused by a variety of mechanisms that occur when a mutation arises in the early development of an embryo. Sanger sequencing is limited in detecting somatic mosaicism and sequence change levels of less than 20% may be missed. The Next Generation Sequencing (NGS) has been increasingly used in diagnosis. It is a sensitive and fast method for the detection of somatic mosaicism. Compared to Sanger sequencing, which represents a cumulative signal, NGS technology analyzes the sequence of each DNA read in a sample. NGS might therefore facilitate the detection of mosaicism in mothers of MPS II patients. The aim of this study was to reanalyze, by NGS, all MPS II mothers that showed to be non-carriers by Sanger analysis. Twelve non-carriers were selected for the reanalysis on the Ion PGM and Ion Torrent S5 platform, using a custom panel that includes the IDS gene. Results were visualized in the Integrative Genomics Viewer (IGV). We were able to detected the presence of the variant previously found in the index case in three of the mothers, with frequencies ranging between 13 and 49% of the reads. These results suggest the possibility of mosaicism in the mothers. The use of a more sensitive technology for detecting low-level mosaic mutations is essential for accurate recurrence-risk estimates. In our study, the NGS analysis showed to be an effective methodology to detect the mosaic event.

Keywords:  IDS gene; carrier detection; hunter syndrome; mosaicism; mucopolysaccharidosis type II; next-generation sequencing; x-linked disease

DOI:  https://doi.org/10.3389/fmolb.2021.789350
Elife. 2021 Nov 25. pii: e70774. [Epub ahead of print]10

Golgi membrane protein Erd1 Is essential for recycling a subset of Golgi glycosyltransferases.

Richa Sardana, Carolyn M Highland, Beth E Straight, Christopher F Chavez, J Christopher Fromme, Scott D Emr.

  Protein glycosylation in the Golgi is a sequential process that requires proper distribution of transmembrane glycosyltransferase enzymes in the appropriate Golgi compartments. Some of the cytosolic machinery required for the steady-state localization of some Golgi enzymes are known but existing models do not explain how many of these enzymes are localized. Here, we uncover the role of an integral membrane protein in yeast, Erd1, as a key facilitator of Golgi glycosyltransferase recycling by directly interacting with both the Golgi enzymes and the cytosolic receptor, Vps74. Loss of Erd1 function results in mislocalization of Golgi enzymes to the vacuole/lysosome. We present evidence that Erd1 forms an integral part of the recycling machinery and ensures productive recycling of several early Golgi enzymes. Our work provides new insights on how the localization of Golgi glycosyltransferases is spatially and temporally regulated, and is finely tuned to the cues of Golgi maturation.

Keywords:  COG complex; Erd1; Glycosylation; Golgi recycling; Vps74; cell biology; cog complex; copi; erd1; glycosyl transferase; glycosylation; glycosyltransferase; golgi; golph3; recycling; vps74

DOI:  https://doi.org/10.7554/eLife.70774
Membranes (Basel). 2021 Nov 07. pii: 858. [Epub ahead of print]11(11):

The mTOR Pathway in Pluripotent Stem Cells: Lessons for Understanding Cancer Cell Dormancy.

Bashar A Alhasan, Sergei A Gordeev, Aleksandra R Knyazeva, Kseniia V Aleksandrova, Boris A Margulis, Irina V Guzhova, Irina I Suvorova.

  Currently, the success of targeted anticancer therapies largely depends on the correct understanding of the dormant state of cancer cells, since it is increasingly regarded to fuel tumor recurrence. The concept of cancer cell dormancy is often considered as an adaptive response of cancer cells to stress, and, therefore, is limited. It is possible that the cancer dormant state is not a privilege of cancer cells but the same reproductive survival strategy as diapause used by embryonic stem cells (ESCs). Recent advances reveal that high autophagy and mTOR pathway reduction are key mechanisms contributing to dormancy and diapause. ESCs, sharing their main features with cancer stem cells, have a delicate balance between the mTOR pathway and autophagy activity permissive for diapause induction. In this review, we discuss the functioning of the mTOR signaling and autophagy in ESCs in detail that allows us to deepen our understanding of the biology of cancer cell dormancy.

Keywords:  autophagy; cancer cell dormancy; embryonic diapause; embryonic stem cells; mTOR; pluripotency; pluripotent stem cells

DOI:  https://doi.org/10.3390/membranes11110858
J Cell Biol. 2022 Jan 03. pii: e202103141. [Epub ahead of print]221(1):

PI4P/PS countertransport by ORP10 at ER-endosome membrane contact sites regulates endosome fission.

Asami Kawasaki, Akiko Sakai, Hiroki Nakanishi, Junya Hasegawa, Tomohiko Taguchi, Junko Sasaki, Hiroyuki Arai, Takehiko Sasaki, Michihiro Igarashi, Fubito Nakatsu.

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER-endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER-endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER-endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

DOI: https://doi.org/10.1083/jcb.202103141
J Biol Chem. 2021 Nov 18. pii: S0021-9258(21)01237-0. [Epub ahead of print] 101428

Development of a versatile HPLC-based method to evaluate the activation status of small GTPases.

Makoto Araki, Kaho Yoshimoto, Meguri Ohta, Toshiaki Katada, Kenji Kontani.

  Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable to those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, are involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood.

Keywords:  GTPase-activating protein; RHEB; Ras protein; high-performance liquid chromatography; mammalian target of rapamycin; signal transduction; small GTPase; tuberous sclerosis complex

DOI:  https://doi.org/10.1016/j.jbc.2021.101428
Int J Biochem Cell Biol. 2021 Nov 22. pii: S1357-2725(21)00200-4. [Epub ahead of print] 106119

Characterization of a spontaneous cell line from primary mouse fibroblasts as a model to study Sanfilippo syndrome.

Noemi Veraldi, Isabelle Dentand Quadri, Ariane de Agostini.

  To evaluate a new approach to Mucopolysaccharidosis type IIIA (MPS-IIIA), work was initiated on primary fibroblasts from a well-known mouse model in which sulfamidase deficiency correlates with the accumulation of heparan sulfate - the hallmark of this disease. Once the culture of fibroblasts was established, we observed continuous proliferation with a rapid growth rate, loss of contact inhibition and late passage stability, corresponding to a spontaneously immortalized cell line. The presence of the single point D31N mutation was verified and both rapid and abundant intracellular accumulation of low molecular weight HS was observed, confirming both genotype and phenotype. This cell line is a potential in vitro model system for future studies of MPS-IIIA prior to employing animal models.

Keywords:  MPS-III; Sanfilippo; cell line; heparan sulfate; metabolic labeling

DOI:  https://doi.org/10.1016/j.biocel.2021.106119
Cells. 2021 Oct 24. pii: 2864. [Epub ahead of print]10(11):

Cathepsin K Regulates Intraocular Pressure by Modulating Extracellular Matrix Remodeling and Actin-Bundling in the Trabecular Meshwork Outflow Pathway.

Avinash Soundararajan, Sachin Anil Ghag, Sai Supriya Vuda, Ting Wang, Padmanabhan Paranji Pattabiraman.

  The homeostasis of extracellular matrix (ECM) and actin dynamics in the trabecular meshwork (TM) outflow pathway plays a critical role in intraocular pressure (IOP) regulation. We studied the role of cathepsin K (CTSK), a lysosomal cysteine protease and a potent collagenase, on ECM modulation and actin cytoskeleton rearrangements in the TM outflow pathway and the regulation of IOP. Initially, we found that CTSK was negatively regulated by pathological stressors known to elevate IOP. Further, inactivating CTSK using balicatib, a pharmacological cell-permeable inhibitor of CTSK, resulted in IOP elevation due to increased levels and excessive deposition of ECM-like collagen-1A in the TM outflow pathway. The loss of CTSK activity resulted in actin-bundling via fascin and vinculin reorganization and by inhibiting actin depolymerization via phospho-cofilin. Contrarily, constitutive expression of CTSK decreased ECM and increased actin depolymerization by decreasing phospho-cofilin, negatively regulated the availability of active TGFβ2, and reduced the levels of alpha-smooth muscle actin (αSMA), indicating an antifibrotic action of CTSK. In conclusion, these observations, for the first time, demonstrate the significance of CTSK in IOP regulation by maintaining the ECM homeostasis and actin cytoskeleton-mediated contractile properties of the TM outflow pathway.

Keywords:  actin cytoskeleton; cathepsin K; extracellular matrix; intraocular pressure; trabecular meshwork

DOI:  https://doi.org/10.3390/cells10112864
Wound Repair Regen. 2021 Nov 27.

The lysosomal trafficking regulator is necessary for normal wound healing.

Jacob C Zbinden, Gabriel J M Mirhaidari, Kevin M Blum, Andrew J Musgrave, James W Reinhardt, Christopher K Breuer, Jenny C Barker.

  Non-healing wounds are a major threat to public health throughout the United States. Tissue healing is complex multifactorial process that requires synchronicity of several cell types. Endolysosomal trafficking, which contributes to various cell functions from protein degradation to plasma membrane repair, is an understudied process in the context of wound healing. The lysosomal trafficking regulator protein (LYST) is an essential protein of the endolysosomal system through an indeterminate mechanism. In this study, we examine the impact of impaired LYST function both in vitro with primary LYST mutant fibroblasts as well as in vivo with an excisional wound model. The wound model shows that LYST mutant mice have impaired wound healing in the form of delayed epithelialization and collagen deposition, independent of macrophage infiltration and polarisation. We show that LYST mutation confers a deficit in MCP-1, IGF-1, and IGFBP-2 secretion in beige fibroblasts, which are critical factors in normal wound healing. Identifying the mechanism of LYST function is important for understanding normal wound biology, which may facilitate the development of strategies to address problem wound healing.

Keywords:  IGF-1; IGFBP-2; LYST; MCP-1; epithelialization; lysosomal trafficking regulator; wound healing

DOI:  https://doi.org/10.1111/wrr.12984
PLoS Pathog. 2021 Nov 22. 17(11): e1009820

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction.

Hataf Khan, Helena Winstone, Jose M Jimenez-Guardeño, Carl Graham, Katie J Doores, Caroline Goujon, David A Matthews, Andrew D Davidson, Suzannah J Rihn, Massimo Palmarini, Stuart J D Neil, Michael H Malim.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

DOI: https://doi.org/10.1371/journal.ppat.1009820