bims-lysosi 2021-02-28 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–02–28
33 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

mTORC1 activity is supported by spatial association with focal adhesions.
BLOC1S1/GCN5L1/BORCS1 is a critical mediator for the initiation of autolysosomal tubulation.
C9orf72 ALS-FTD: recent evidence for dysregulation of the autophagy-lysosome pathway at multiple levels.
The role of lysosomal ion channels in lysosome dysfunction.
The Expression of TRIM6 Activates the mTORC1 Pathway by Regulating the Ubiquitination of TSC1-TSC2 to Promote Renal Fibrosis.
Maintenance of neural stem-progenitor cells by the lysosomal biosynthesis regulators TFEB and TFE3 in the embryonic mouse telencephalon.
Electroacupuncture ameliorates beta-amyloid pathology and cognitive impairment in Alzheimer disease via a novel mechanism involving activation of TFEB (transcription factor EB).
β-propeller proteins WDR45 and WDR45B regulate autophagosome maturation into autolysosomes in neural cells.
Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia.
Ambroxol increases glucocerebrosidase (GCase) activity and restores GCase translocation in primary patient-derived macrophages in Gaucher disease and Parkinsonism.
Overcoming Chemoresistance: Altering pH of Cellular Compartments by Chloroquine and Hydroxychloroquine.
Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain.
Sunitinib treatment promotes metastasis of drug-resistant renal cell carcinoma via TFE3 signaling pathway.
The important role of TFEB in autophagy-lysosomal pathway and autophagy-related diseases: a systematic review.
Evaluation of HIV-1 derived lentiviral vectors as transductors of Mucopolysaccharidosis type IV A fibroblasts.
The role of glucosylsphingosine as an early indicator of disease progression in early symptomatic type 1 Gaucher disease.
FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity.
Role of the V1G1 subunit of V-ATPase in breast cancer cell migration.
STRADA-mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation.
Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.
Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.
Bladder cancer cell-intrinsic PD-L1 signals promote mTOR and autophagy activation that can be inhibited to improve cytotoxic chemotherapy.
Full Activation of Kinase Protein Kinase B by Phosphoinositide-Dependent Protein Kinase-1 and Mammalian Target of Rapamycin Complex 2 Is Required for Early Natural Killer Cell Development and Survival.
Prognostic and Therapeutic Potentials of OncomiRs Modulating mTOR Pathways in Virus-Associated Hepatocellular Carcinoma.
Loss of optineurin drives cancer immune evasion via palmitoylation-dependent IFNGR1 lysosomal sorting and degradation.
WDR35 is involved in subcellular localization of acetylated tubulin in 293T cells.
In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.
Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors.
Endoplasmic Reticulum-Plasma Membrane Contact Sites: Regulators, Mechanisms, and Physiological Functions.
Pathogenesis of Diabesity-induced Kidney Disease: Role of Kidney Nutrient Sensing.
The vacuolar amino acid transport system is a novel, direct target of GATA transcription factors.
The major cap-binding protein eIF4E regulates lipid homeostasis and diet-induced obesity.
A plant plasma-membrane H+-ATPase promotes yeast TORC1 activation via its carboxy-terminal tail.

J Cell Biol. 2021 May 03. pii: e202004010. [Epub ahead of print]220(5):

mTORC1 activity is supported by spatial association with focal adhesions.

Yoana Rabanal-Ruiz, Adam Byron, Alexander Wirth, Ralitsa Madsen, Lucia Sedlackova, Graeme Hewitt, Glyn Nelson, Julian Stingele, Jimi C Wills, Tong Zhang, André Zeug, Reinhard Fässler, Bart Vanhaesebroeck, Oliver D K Maddocks, Evgeni Ponimaskin, Bernadette Carroll, Viktor I Korolchuk.

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.

DOI: https://doi.org/10.1083/jcb.202004010
Autophagy. 2021 Feb 25.

BLOC1S1/GCN5L1/BORCS1 is a critical mediator for the initiation of autolysosomal tubulation.

Kaiyuan Wu, Allen Seylani, Jing Wu, Xufeng Wu, Christopher K E Bleck, Michael N Sack.

  The mechanisms orchestrating recycling of lysosomes through autophagic lysosome reformation (ALR) is incompletely understood. Previous data show that genetic depletion of BLOC1S1/GCN5L1/BORCS1 increases autolysosome (AL) accumulation. We postulated that this phenotype may manifest due to perturbed ALR. We explored this in control and bloc1s1 liver-specific knockout (LKO) mouse hepatocytes, showing that in response to nutrient-deprivation LKO's fail to initiate ALR due to blunted lysosomal tubulation. As kinesin motor proteins and the intracellular cytoskeleton are requirements for tubular formation from ALs, we explored the interaction of BLOC1S1 with motor proteins and cytoskeletal factors. BLOC1S1 interacts with the ARL8B-KIF5B (GTPase and kinesin motor protein) complex to recruit KIF5B to ALs. Furthermore, BLOC1S1 interacts with the actin nucleation promoting factor WHAMM, which is an essential structural protein in the initiation of lysosomal tubulation (LT). Interestingly, the genetic reintroduction of BLOC1S1 rescues LT in LKO hepatocytes, but not when KIF5B is concurrently depleted. Finally, given the central role of MTORC1 signaling in ALR initiation, it was interesting that MTORC1 activity was increased despite the absence of LT in LKO hepatocytes. Concurrently, inhibition of MTORC1 abolished BLOC1S1 reconstitution-mediated rescue of LT in LKO hepatocytes. Taken together these data demonstrate that the functional interaction of BLOC1S1 with the kinesin binding complex and the actin cytoskeleton are a requirement for LT which, in parallel with MTORC1 signaling, initiate lysosome recycling via ALR.

Keywords:  Autophagic lysosome reformation; GCN5L1; MTORC1; autophagy; hepatocyte; lysosomal tubulation; lysosome

DOI:  https://doi.org/10.1080/15548627.2021.1894759
Autophagy. 2021 Feb 26. 1-17

C9orf72 ALS-FTD: recent evidence for dysregulation of the autophagy-lysosome pathway at multiple levels.

Jimmy Beckers, Arun Kumar Tharkeshwar, Philip Van Damme.

  Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two clinically distinct classes of neurodegenerative disorders. Yet, they share a range of genetic, cellular, and molecular features. Hexanucleotide repeat expansions (HREs) in the C9orf72 gene and the accumulation of toxic protein aggregates in the nervous systems of the affected individuals are among such common features. Though the mechanisms by which HREs cause toxicity is not clear, the toxic gain of function due to transcribed HRE RNA or dipeptide repeat proteins (DPRs) produced by repeat-associated non-AUG translation together with a reduction in C9orf72 expression are proposed as the contributing factors for disease pathogenesis in ALS and FTD. In addition, several recent studies point toward alterations in protein homeostasis as one of the root causes of the disease pathogenesis. In this review, we discuss the effects of the C9orf72 HRE in the autophagy-lysosome pathway based on various recent findings. We suggest that dysfunction of the autophagy-lysosome pathway synergizes with toxicity from C9orf72 repeat RNA and DPRs to drive disease pathogenesis.Abbreviation: ALP: autophagy-lysosome pathway; ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ASO: antisense oligonucleotide; C9orf72: C9orf72-SMCR8 complex subunit; DENN: differentially expressed in normal and neoplastic cells; DPR: dipeptide repeat protein; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; ER: endoplasmic reticulum; FTD: frontotemporal dementia; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HRE: hexanucleotide repeat expansion; iPSC: induced pluripotent stem cell; ISR: integrated stress response; M6PR: mannose-6-phosphate receptor, cation dependent; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: neurodegenerative disorder; RAN: repeat-associated non-ATG; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SLC66A1/PQLC2: solute carrier family 66 member 1; SMCR8: SMCR8-C9orf72 complex subunit; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; WDR41: WD repeat domain 41.

Keywords:  Amyotrophic lateral sclerosis (ALS); autophagy; axonal transport; c9orf72; dipeptide repeat protein (DPR); frontotemporal dementia (FTD); lysosome; smcr8; wdr41

DOI:  https://doi.org/10.1080/15548627.2021.1872189
Inhal Toxicol. 2021 Feb 25. 1-14

The role of lysosomal ion channels in lysosome dysfunction.

Rebekah L Kendall, Andrij Holian.

  Lysosomes offer a unique arrangement of degradative, exocytic, and signaling capabilities that make their continued function critical to cellular homeostasis. Lysosomes owe their function to the activity of lysosomal ion channels and transporters, which maintain concentration gradients of H+, K+, Ca2+, Na+, and Cl- across the lysosomal membrane. This review examines the contributions of lysosomal ion channels to lysosome function, showing how ion channel function is integral to degradation and autophagy, maintaining lysosomal membrane potential, controlling Ca2+ signaling, and facilitating exocytosis. Evidence of lysosome dysfunction in a variety of disease pathologies creates a need to understand how lysosomal ion channels contribute to lysosome dysfunction. For example, the loss of function of the TRPML1 Ca2+ lysosome channel in multiple lysosome storage diseases leads to lysosome dysfunction and disease pathogenesis while neurodegenerative diseases are marked by lysosome dysfunction caused by changes in ion channel activity through the TRPML1, TPC, and TMEM175 ion channels. Autoimmune disease is marked by dysregulated autophagy, which is dependent on the function of multiple lysosomal ion channels. Understanding the role of lysosomal ion channel activity in lysosome membrane permeability and NLRP3 inflammasome activation could provide valuable mechanistic insight into NLRP3 inflammasome-mediated diseases. Finally, this review seeks to show that understanding the role of lysosomal ion channels in lysosome dysfunction could give mechanistic insight into the efficacy of certain drug classes, specifically those that target the lysosome, such as cationic amphiphilic drugs.

Keywords:  BK; Lysosome; NLRP3; TMEM175; TRPML1; autophagy; crystalline silica; lysosomal membrane permeability

DOI:  https://doi.org/10.1080/08958378.2021.1876188
Front Cell Dev Biol. 2020 ;8 616747

The Expression of TRIM6 Activates the mTORC1 Pathway by Regulating the Ubiquitination of TSC1-TSC2 to Promote Renal Fibrosis.

Weiwei Liu, Yang Yi, Chuanfu Zhang, Baojuan Zhou, Lin Liao, Wenrui Liu, Jing Hu, Qiming Xu, Jie Chen, Jianrao Lu.

  Renal fibrosis is considered as the final pathway of all types of kidney diseases, which can lead to the progressive loss of kidney functions and eventually renal failure. The mechanisms behind are diversified, in which the mammalian target of rapamycin (mTOR) pathway is one of the most important regulatory pathways that accounts for the disease. Several processes that are regulated by the mTOR pathway, such as autophagy, epithelial-mesenchymal transition (EMT), and endoplasmic reticulum (ER) stress, are tightly associated with renal fibrosis. In this study, we have reported that the expression of tripartite motif-containing (TRIM) protein 6, a member of TRIM family protein, was highly expressed in renal fibrosis patients and positively correlated with the severity of renal fibrosis. In our established in vitro and in vivo renal fibrosis models, its expression was upregulated by the Angiotensin II-induced nuclear translocation of nuclear factor-κB (NF-κB) p50 and p65. In HK2 cells, the expression of TRIM6 promoted the ubiquitination of tuberous sclerosis proteins (TSC) 1 and 2, two negative regulators of the mTORC1 pathway. Moreover, the knockdown of TRIM6 was found efficient for alleviating renal fibrosis and inhibiting the downstream processes of EMT and ER in both HK2 cells and 5/6-nephrectomized rats. Clinically, the level of TRIM6, TSC1/2, and NF-κB p50 was found closely related to renal fibrosis. As a result, we have presented the first study on the role of TRIM6 in the mTORC1 pathway in renal fibrosis models and our findings suggested that TRIM6 may be a potential target for the treatment of renal fibrosis.

Keywords:  TRIM6; TSC1; TSC2; angiotensin II; mTOR; renal fibrosis

DOI:  https://doi.org/10.3389/fcell.2020.616747
Stem Cells. 2021 Feb 20.

Maintenance of neural stem-progenitor cells by the lysosomal biosynthesis regulators TFEB and TFE3 in the embryonic mouse telencephalon.

Naoya Yuizumi, Yujin Harada, Takaaki Kuniya, Takehiko Sunabori, Masato Koike, Masaki Wakabayashi, Yasushi Ishihama, Yutaka Suzuki, Daichi Kawaguchi, Yukiko Gotoh.

  Lysosomes have recently been implicated in regulation of quiescence in adult neural stem cells (NSCs). Whether lysosomes regulate the differentiation of neural stem-progenitor cells (NPCs) in the embryonic brain has remained unknown, however. We here show that lysosomes are more abundant in rapidly dividing NPCs than in differentiating neurons in the embryonic mouse neocortex and ganglionic eminence. The genes for TFEB and TFE3, master regulators of lysosomal biosynthesis, as well as other lysosome-related genes were also expressed at higher levels in NPCs than in differentiating neurons. Anatomic analysis revealed accumulation of lysosomes at the apical and basal endfeet of NPCs. Knockdown of TFEB and TFE3, or that of the lysosomal transporter Slc15a4, resulted in premature differentiation of neocortical NPCs. Conversely, forced expression of an active form of TFEB (TFEB-AA) suppressed neuronal differentiation of NPCs in association with up-regulation of NPC-related genes. These results together point to a previously unappreciated role for TFEB and TFE3, and possibly for lysosomes, in maintenance of the undifferentiated state of embryonic NPCs. We further found that lysosomes are even more abundant in an NPC subpopulation that rarely divides and includes the embryonic origin of adult NSCs than in the majority of NPCs that divide frequently for construction of the embryonic brain, and that overexpression of TFEB-AA also suppressed the cell cycle of neocortical NPCs. Our results thus also implicate lysosomes in establishment of the slowly dividing, embryonic origin of adult NSCs. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Regulation of adult tissue stem cells by organelles has recently received much attention, but the developmental functions of lysosomes have been less studied. We now reveal the importance of lysosomes for suppression of premature differentiation of mouse embryonic neural stem-progenitor cells (NPCs). Our data also implicate Slc15a4, a lysosomal histidine and short-peptide transporter that is associated with systemic lupus erythematosus (SLE), in NPC maintenance. These findings shed light on the role of lysosomes in regulation of neural development and tissue stem cells, and they may therefore provide insight into the pathogenesis of lysosome-related diseases such as SLE.

Keywords:  TFE3; TFEB; adult neural stem cell; lysosome; neural stem-progenitor cell; neurogenesis

DOI:  https://doi.org/10.1002/stem.3359
Autophagy. 2021 Feb 23. 1-15

Electroacupuncture ameliorates beta-amyloid pathology and cognitive impairment in Alzheimer disease via a novel mechanism involving activation of TFEB (transcription factor EB).

Xiaoyan Zheng, Wenjia Lin, Yimin Jiang, Kejia Lu, Wenjing Wei, Qingwei Huo, Shaoyang Cui, Xifei Yang, Min Li, Nenggui Xu, Chunzhi Tang, Ju-Xian Song.

  Alzheimer disease (AD) is the most prevalent neurodegenerative disorder leading to dementia in the elderly. Unfortunately, no cure for AD is available to date. Increasing evidence has proved the roles of misfolded protein aggregation due to impairment of the macroautophagy/autophagy-lysosomal pathway (ALP) in the pathogenesis of AD, and thus making TFEB (transcription factor EB), which orchestrates ALP, as a promising target for treating AD. As a complementary therapy, acupuncture or electroacupuncture (EA) has been commonly used for treating human diseases. Although the beneficial effects of acupuncture for AD have been primarily studied both pre-clinically and clinically, the real efficacy of acupuncture on AD remains inconclusive and the underlying mechanisms are largely unexplored. In this study, we demonstrated the cognitive-enhancing effect of three-needle EA (TNEA) in an animal model of AD with beta-amyloid (Aβ) pathology (5xFAD). TNEA reduced APP (amyloid beta (A4) precursor protein), C-terminal fragments (CTFs) of APP and Aβ load, and inhibited glial cell activation in the prefrontal cortex and hippocampus of 5xFAD. Mechanistically, TNEA activated TFEB via inhibiting the AKT-MAPK1-MTORC1 pathway, thus promoting ALP in the brains. Therefore, TNEA represents a promising acupuncture therapy for AD, via a novel mechanism involving TFEB activation.Abbreviations Aβ: β-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; AKT1: thymoma viral proto-oncogene 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta (A4) precursor protein; BACE1: beta-site APP cleaving enzyme 1; CQ: chloroquine; CTFs: C-terminal fragments; CTSD: cathepsin D; EA: electroacupuncture; FC: fear conditioning; GFAP: glial fibrillary acidic protein; HI: hippocampus; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPT: microtubule-associated protein tau; MTORC1: mechanistic target of rapamycin kinase complex 1; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TNEA: three-needle electroacupuncture.

Keywords:  Alzheimer disease; autophagy-lysosomal pathway; electroacupuncture; transcription factor EB

DOI:  https://doi.org/10.1080/15548627.2021.1886720
Curr Biol. 2021 Feb 17. pii: S0960-9822(21)00146-9. [Epub ahead of print]

β-propeller proteins WDR45 and WDR45B regulate autophagosome maturation into autolysosomes in neural cells.

Cuicui Ji, Hongyu Zhao, Di Chen, Hong Zhang, Yan G Zhao.

  Mutations in WDR45 and WDR45B cause the human neurological diseases β-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B, along with WIPI1 and WIPI2, belong to a WD40 repeat-containing phosphatidylinositol-3-phosphate (PI(3)P)-binding protein family. Their yeast homolog Atg18 forms a complex with Atg2 and is required for autophagosome formation in part by tethering isolation membranes (IMs) (autophagosome precursor) to the endoplasmic reticulum (ER) to supply lipid for IM expansion in the autophagy pathway. The exact functions of WDR45/45B are unclear. We show here that WDR45/45B are specifically required for neural autophagy. In Wdr45/45b-depleted cells, the size of autophagosomes is decreased, and this is rescued by overexpression of ATG2A, providing in vivo evidence for the lipid transfer activity of ATG2-WIPI complexes. WDR45/45B are dispensable for the closure of autophagosomes but essential for the progression of autophagosomes into autolysosomes. WDR45/45B interact with the tether protein EPG5 and target it to late endosomes/lysosomes to promote autophagosome maturation. In the absence of Wdr45/45b, formation of the fusion machinery, consisting of SNARE proteins and EPG5, is dampened. BPAN- and ID-related mutations of WDR45/45B fail to rescue the autophagy defects in Wdr45/45b-deficient cells, possibly due to their impaired binding to EPG5. Promoting autophagosome maturation by inhibiting O-GlcNAcylation increases SNARE complex formation and facilitates the fusion of autophagosomes with late endosomes/lysosomes in Wdr45/45b double knockout (DKO) cells. Thus, our results uncover a novel function of WDR45/45B in autophagosome-lysosome fusion and provide molecular insights into the development of WDR45/WDR45B mutation-associated diseases.

Keywords:  BPAN; ID; WDR45; WDR45B; autophagy

DOI:  https://doi.org/10.1016/j.cub.2021.01.081
Nat Commun. 2021 02 24. 12(1): 1158

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia.

Alessio Colombo, Lina Dinkel, Stephan A Müller, Laura Sebastian Monasor, Martina Schifferer, Ludovico Cantuti-Castelvetri, Jasmin König, Lea Vidatic, Tatiana Bremova-Ertl, Andrew P Lieberman, Silva Hecimovic, Mikael Simons, Stefan F Lichtenthaler, Michael Strupp, Susanne A Schneider, Sabina Tahirovic.

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1-/- microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential.

DOI: https://doi.org/10.1038/s41467-021-21428-5
Parkinsonism Relat Disord. 2021 Feb 10. pii: S1353-8020(21)00044-4. [Epub ahead of print]84 112-121

Ambroxol increases glucocerebrosidase (GCase) activity and restores GCase translocation in primary patient-derived macrophages in Gaucher disease and Parkinsonism.

A E Kopytova, G N Rychkov, M A Nikolaev, G V Baydakova, A A Cheblokov, K A Senkevich, D A Bogdanova, O I Bolshakova, I V Miliukhina, V A Bezrukikh, G N Salogub, S V Sarantseva, T C Usenko, E Y Zakharova, A K Emelyanov, S N Pchelina.

  Mutations in the glucocerebrosidase gene (GBA) encoding the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease (GD) and are the most commonly known genetic risk factor for Parkinson disease (PD). Ambroxol is one of the most effective pharmacological chaperones of GCase. Fourteen GD patients, six PD patients with mutations in the GBA gene (GBA-PD), and thirty controls were enrolled. GCase activity and hexosylsphingosine (HexSph) concentration were measured in dried blood and macrophage spots using liquid chromatography coupled with tandem mass spectrometry. The effect of ambroxol on GCase translocation to lysosomes was assessed using confocal microscopy. The results showed that ambroxol treatment significantly increased GCase activity in cultured macrophages derived from patient blood monocytic cell (PBMC) of GD (by 3.3-fold) and GBA-PD patients (by 3.5-fold) compared to untreated cells (p < 0.0001 and p < 0.0001, respectively) four days after cultivation. Ambroxol treatment significantly reduced HexSph concentration in GD (by 2.1-fold) and GBA-PD patients (by 1.6-fold) (p < 0.0001 and p < 0.0001, respectively). GD macrophage treatment resulted in increased GCase level and increased enzyme colocalization with the lysosomal marker LAMP2. The possible binding modes of ambroxol to mutant GCase carrying N370S amino acid substitution at pH 4.7 were examined using molecular docking and molecular dynamics simulations. The ambroxol position characterized by minimal binding free energy was observed in close vicinity to the residue, at position 370. Taken together, these data showed that PBMC-derived macrophages could be used for assessing ambroxol therapy response for GD patients and also for GBA-PD patients.

Keywords:  Allosteric binding site; Ambroxol; Binding free energy; GCase; Gaucher disease; Parkinson's disease; Pharmacological chaperones

DOI:  https://doi.org/10.1016/j.parkreldis.2021.02.003
Front Cell Dev Biol. 2021 ;9 627639

Overcoming Chemoresistance: Altering pH of Cellular Compartments by Chloroquine and Hydroxychloroquine.

Peter W Halcrow, Jonathan D Geiger, Xuesong Chen.

  Resistance to the anti-cancer effects of chemotherapeutic agents (chemoresistance) is a major issue for people living with cancer and their providers. A diverse set of cellular and inter-organellar signaling changes have been implicated in chemoresistance, but it is still unclear what processes lead to chemoresistance and effective strategies to overcome chemoresistance are lacking. The anti-malaria drugs, chloroquine (CQ) and its derivative hydroxychloroquine (HCQ) are being used for the treatment of various cancers and CQ and HCQ are used in combination with chemotherapeutic drugs to enhance their anti-cancer effects. The widely accepted anti-cancer effect of CQ and HCQ is their ability to inhibit autophagic flux. As diprotic weak bases, CQ and HCQ preferentially accumulate in acidic organelles and neutralize their luminal pH. In addition, CQ and HCQ acidify the cytosolic and extracellular environments; processes implicated in tumorigenesis and cancer. Thus, the anti-cancer effects of CQ and HCQ extend beyond autophagy inhibition. The present review summarizes effects of CQ, HCQ and proton pump inhibitors on pH of various cellular compartments and discuss potential mechanisms underlying their pH-dependent anti-cancer effects. The mechanisms considered here include their ability to de-acidify lysosomes and inhibit autophagosome lysosome fusion, to de-acidify Golgi apparatus and secretory vesicles thus affecting secretion, and to acidify cytoplasm thus disturbing aerobic metabolism. Further, we review the ability of these agents to prevent chemotherapeutic drugs from accumulating in acidic organelles and altering their cytosolic concentrations.

Keywords:  Golgi pH; chemoresistance; chloroquine; cytosolic pH; endolysosome pH; hydroxychloroquine

DOI:  https://doi.org/10.3389/fcell.2021.627639
Sci Rep. 2021 Feb 22. 11(1): 4342

Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain.

Thanida Laopanupong, Pinidphon Prombutara, Phongthon Kanjanasirirat, Salisa Benjaskulluecha, Atsadang Boonmee, Tanapat Palaga, Stephane Méresse, Jiraporn Paha, Tegar Adriansyah Putra Siregar, Tanawadee Khumpanied, Suparerk Borwornpinyo, Angkana Chaiprasert, Pongsak Utaisincharoen, Marisa Ponpuak.

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

DOI: https://doi.org/10.1038/s41598-021-83835-4
Cell Death Dis. 2021 Feb 26. 12(2): 220

Sunitinib treatment promotes metastasis of drug-resistant renal cell carcinoma via TFE3 signaling pathway.

Luchao Li, Shuo Zhao, Zhengfang Liu, Nianzhao Zhang, Shuo Pang, Jikai Liu, Cheng Liu, Yidong Fan.

Receptor tyrosine kinase (RTK) inhibitors, such as sunitinib and sorafenib, remain the first-line drugs for the treatment of mRCC. Acquired drug resistance and metastasis are the main causes of treatment failure. However, in the case of metastasis Renal Cell Cancer (mRCC), which showed a good response to sunitinib, we found that long-term treatment with sunitinib could promote lysosome biosynthesis and exocytosis, thereby triggering the metastasis of RCC. By constructing sunitinib-resistant cell lines in vivo, we confirmed that TFE3 plays a key role in the acquired resistance to sunitinib in RCC. Under the stimulation of sunitinib, TFE3 continued to enter the nucleus, promoting the expression of endoplasmic reticulum (ER) protein E-Syt1. E-Syt1 and the lysosomal membrane protein Syt7 form a heterodimer, which induces ER fragmentation, Ca2+ release, and lysosomal exocytosis. Lysosomal exocytosis has two functions: pumping sunitinib out from the cytoplasm, which promotes resistance to sunitinib in RCC, releasing cathepsin B (CTSB) into the extracellular matrix (ECM), which can degrade the ECM to enhance the invasion and metastasis ability of RCC. Our study found that although sunitinib is an effective drug for the treatment of mRCC, once RCC has acquired resistance to sunitinib, sunitinib treatment will promote metastasis.

DOI: https://doi.org/10.1038/s41419-021-03511-3
Eur Rev Med Pharmacol Sci. 2021 Feb;pii: 24875. [Epub ahead of print]25(3): 1641-1649

The important role of TFEB in autophagy-lysosomal pathway and autophagy-related diseases: a systematic review.

T-T Song, R-S Cai, R Hu, Y-S Xu, B-N Qi, Y-A Xiong.

Autophagy is a main metabolic process in which eukaryotic cells use lysosomes to eliminate abnormal proteins and damaged organelles to maintain cell homeostasis. Studies have revealed that neurodegenerative diseases, tumor, hepatic diseases, etc. are related to abnormal autophagy processes in recent years. Recent studies have shown that TFEB is a major transcription regulator of autophagy-lysosomal pathway (ALP) transcriptional regulation, which positively regulates the expression of autophagy and lysosomal biogenesis-related genes, thereby promoting autophagosome formation, autophagosome-lysosome fusion, and degradation of autophagy substrates. It has also been found that TFEB promotes clearance of intracellular substrates through lysosomal exocytosis. Therefore, the study of biological functions and related regulatory mechanisms of TFEB will provide important clues and theoretical basis for further explaining its physiological pathogenesis and the treatment of related diseases.

DOI: https://doi.org/10.26355/eurrev_202102_24875
Gene. 2021 Feb 22. pii: S0378-1119(21)00121-9. [Epub ahead of print] 145527

Evaluation of HIV-1 derived lentiviral vectors as transductors of Mucopolysaccharidosis type IV A fibroblasts.

María Alejandra Puentes-Tellez, Oscar F Sánchez, Felipe Rojas-Rodriguez, Eliana Benincore-Flóre, Hector Barbosa, Carlos J Alméciga Díaz.

  Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease produced by the deficiency of the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycans (GAGs) accumulation. Since currently available treatments remain limited and unspecific, novel therapeutic approaches are essential for the disease treatment. In an attempt to reduce treatment limitations, gene therapy rises as a more effective and specific alternative. We present in this study the delivery assessment of GALNS and sulfatase-modifying factor 1 (SUMF1) genes via HIV-1 derived lentiviral vectors into fibroblasts from MPS IVA patients. After transduction, we determined GALNS enzymatic activity, lysosomal mass change, and autophagy pathway impairment. Additionally, we computationally assessed the effect of mutations over the enzyme-substrate interaction and phenotypic effects. The results showed that the co-transduction of MPS IVA fibroblasts with GALNS and SUMF1 cDNAs led to a significant increase in GALNS enzyme activity and a reduction of lysosomal mass. We show that patient-specific differences in cellular response are directly associated with the set of mutations on each patient. Lastly, we present new evidence supporting autophagy impairment in MPS IVA due to the presence and changes in autophagy proteins in treated MPS IVA fibroblasts. Our results offer new evidence that demonstrate the potential of lentiviral vectors as a strategy to correct GALNS deficiency.

Keywords:  GALNS; Gene therapy; MPS IVA; SUMF1; lentiviral vector

DOI:  https://doi.org/10.1016/j.gene.2021.145527
Mol Genet Metab Rep. 2021 Jun;27 100729

The role of glucosylsphingosine as an early indicator of disease progression in early symptomatic type 1 Gaucher disease.

Ashlee R Stiles, Erin Huggins, Luca Fierro, Seung-Hye Jung, Manisha Balwani, Priya S Kishnani.

  Gaucher disease (GD), a lysosomal storage disorder caused by β-glucocerebrosidase deficiency, results in the accumulation of glucosylceramide and glucosylsphingosine. Glucosylsphingosine has emerged as a sensitive and specific biomarker for GD and treatment response. However, limited information exists on its role in guiding treatment decisions in pre-symptomatic patients identified at birth or due to a positive family history. We present two pediatric patients with GD1 and highlight the utility of glucosylsphingosine monitoring in guiding treatment initiation.

Keywords:  Glucosylsphingosine; Lyso-Gb1; Monitoring; Pediatric; Type 1 Gaucher disease; p.N409S

DOI:  https://doi.org/10.1016/j.ymgmr.2021.100729
Front Cell Dev Biol. 2021 ;9 625715

FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity.

Marianna Kapetanou, Tobias Nespital, Luke S Tain, Andre Pahl, Linda Partridge, Efstathios S Gonos.

  Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic β5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

Keywords:  FOXO factors; aging; insulin signaling; longevity; proteasome; proteostasis

DOI:  https://doi.org/10.3389/fcell.2021.625715
Sci Rep. 2021 Feb 25. 11(1): 4615

Role of the V1G1 subunit of V-ATPase in breast cancer cell migration.

Maria De Luca, Roberta Romano, Cecilia Bucci.

V-ATPase is a large multi-subunit complex that regulates acidity of intracellular compartments and of extracellular environment. V-ATPase consists of several subunits that drive specific regulatory mechanisms. The V1G1 subunit, a component of the peripheral stalk of the pump, controls localization and activation of the pump on late endosomes and lysosomes by interacting with RILP and RAB7. Deregulation of some subunits of the pump has been related to tumor invasion and metastasis formation in breast cancer. We observed a decrease of V1G1 and RAB7 in highly invasive breast cancer cells, suggesting a key role of these proteins in controlling cancer progression. Moreover, in MDA-MB-231 cells, modulation of V1G1 affected cell migration and matrix metalloproteinase activation in vitro, processes important for tumor formation and dissemination. In these cells, characterized by high expression of EGFR, we demonstrated that V1G1 modulates EGFR stability and the EGFR downstream signaling pathways that control several factors required for cell motility, among which RAC1 and cofilin. In addition, we showed a key role of V1G1 in the biogenesis of endosomes and lysosomes. Altogether, our data describe a new molecular mechanism, controlled by V1G1, required for cell motility and that promotes breast cancer tumorigenesis.

DOI: https://doi.org/10.1038/s41598-021-84222-9
Dev Neurobiol. 2021 Feb 22.

STRADA-mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation.

Louis T Dang, Shivanshi Vaid, Grace Lin, Preethi Swaminathan, Jordan Safran, Anna Loughman, Monica Lee, Trevor Glenn, Fernanda Majolo, Peter B Crino, Jack M Parent.

  Genetic diseases involving overactivation of the mechanistic target of rapamycin (mTOR) pathway, so-called "mTORopathies," often manifest with malformations of cortical development (MCDs), epilepsy, and cognitive impairment. How mTOR pathway hyperactivation results in abnormal human cortical development is poorly understood. To study the effect of mTOR hyperactivity on early stages of cortical development, we focused on Pretzel Syndrome (polyhydramnios, megalencephaly, symptomatic epilepsy; PMSE syndrome), a rare mTORopathy caused by homozygous germline mutations in the STRADA gene. We developed a human cortical organoid (hCO) model of PMSE and examined morphology and size for the first 2 weeks of organoid growth, and cell type composition at weeks 2, 8, and 12 of differentiation. In the second week, PMSE hCOs enlarged more rapidly than controls and displayed an abnormal Wnt pathway-dependent increase in neural rosette structures. PMSE hCOs also exhibited delayed neurogenesis, decreased subventricular zone progenitors, increased proliferation and cell death, and an abnormal architecture of primary cilia. At week 8, PMSE hCOs had fewer deep layer neurons. By week 12, neurogenesis recovered in PMSE organoids, but they displayed increased outer radial glia, a cell type thought to contribute to expansion of the human cerebral cortex. Together, these findings suggest that megalencephaly in PMSE arises from expansion of neural stem cells in early corticogenesis and potentially also from increased outer radial glial at later gestational stages. The delayed neuronal differentiation in PMSE organoids demonstrates the important role the mTOR pathway plays in maintenance and expansion of the stem cell pool.

Keywords:  Pretzel Syndrome; STRADA; and symptomatic epilepsy; cerebral organoids; corticogenesis; induced pluripotent stem cells; mTOR; mechanistic target of rapamycin; megalencephaly; neurogenesis; polyhydramnios; stem cell model

DOI:  https://doi.org/10.1002/dneu.22816
Autophagy. 2021 Feb 22.

Mouse models for hereditary spastic paraplegia uncover a role of PI4K2A in autophagic lysosome reformation.

Mukhran Khundadze, Federico Ribaudo, Adeela Hussain, Henry Stahlberg, Nahal Brocke-Ahmadinejad, Patricia Franzka, Rita-Eva Varga, Milena Zarkovic, Thanakorn Pungsrinont, Miriam Kokal, Ian G Ganley, Christian Beetz, Marc Sylvester, Christian A Hübner.

  Hereditary spastic paraplegia (HSP) denotes genetically heterogeneous disorders (SPGs) characterized by leg spasticity due to degeneration of corticospinal axons. SPG11 and SPG15 have a similar clinical course and together are the most prevalent autosomal recessive HSP entity. The respective proteins play a role for macroautophagy/autophagy and autophagic lysosome reformation (ALR). Here, we report that spg11 and zfyve26 KO mice developed motor impairments within the same course of time. This correlated with enhanced accumulation of autofluorescent material in neurons and progressive neuron loss. In agreement with defective ALR, tubulation events were diminished in starved KO mouse embryonic fibroblasts (MEFs) and lysosomes decreased in neurons of KO brain sections. Confirming that both proteins act in the same molecular pathway, the pathologies were not aggravated upon simultaneous disruption of both. We further show that PI4K2A (phosphatidylinositol 4-kinase type 2 alpha), which phosphorylates phosphatidylinositol to phosphatidylinositol-4-phosphate (PtdIns4P), accumulated in autofluorescent deposits isolated from KO but not WT brains. Elevated PI4K2A abundance was already found at autolysosomes of neurons of presymptomatic KO mice. Immunolabelings further suggested higher levels of PtdIns4P at LAMP1-positive structures in starved KO MEFs. An increased association with LAMP1-positive structures was also observed for clathrin and DNM2/dynamin 2, which are important effectors of ALR recruited by phospholipids. Because PI4K2A overexpression impaired ALR in control cells, while its knockdown increased tubulation, we conclude that PI4K2A modulates phosphoinositide levels at autolysosomes and thus the recruitment of downstream effectors of ALR. Therefore, PI4K2A may play an important role in the pathogenesis of SPG11 and SPG15.

Keywords:  SPG11; SPG15; autophagy; lysosome; neurodegeneration

DOI:  https://doi.org/10.1080/15548627.2021.1891848
Cell Metab. 2021 Feb 17. pii: S1550-4131(21)00057-7. [Epub ahead of print]

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.

Abigail S Krall, Peter J Mullen, Felicia Surjono, Milica Momcilovic, Ernst W Schmid, Christopher J Halbrook, Apisadaporn Thambundit, Steven D Mittelman, Costas A Lyssiotis, David B Shackelford, Simon R V Knott, Heather R Christofk.

  Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

Keywords:  asparaginase; asparagine; cancer metabolism; cancer treatment; dietary restriction; metformin; respiration

DOI:  https://doi.org/10.1016/j.cmet.2021.02.001
Cancer Med. 2021 Feb 24.

Bladder cancer cell-intrinsic PD-L1 signals promote mTOR and autophagy activation that can be inhibited to improve cytotoxic chemotherapy.

Deyi Zhang, Ryan M Reyes, Erica Osta, Suresh Kari, Harshita B Gupta, Alvaro S Padron, Anand V R Kornepati, Aravind Kancharla, Xiujie Sun, Yilun Deng, Bogang Wu, Ratna Vadlamudi, Rong Li, Robert S Svatek, Tyler J Curiel.

  Tumor cell-intrinsic programmed death-ligand 1 (PD-L1) signals mediate immunopathologic effects in breast, colon, and ovarian cancers and in melanomas, but bladder cancer (BC) effects are unreported. We show here that BC cell-intrinsic PD-L1 signals in mouse MB49 and human RT4, UM-UC3, and UM-UC-14 BC cells regulate important pathologic pathways and processes, including effects not reported in other cancers. α-PD-L1 antibodies reduced BC cell proliferation in vitro, demonstrating direct signaling effects. BC cell-intrinsic PD-L1 promoted mammalian target of rapamycin complex 1 (mTORC1) signals in vitro and augmented in vivo immune-independent cell growth and metastatic cancer spread, similar to effects we reported in melanoma and ovarian cancer. BC cell-intrinsic PD-L1 signals also promoted basal and stress-induced autophagy, whereas these signals inhibited autophagy in melanoma and ovarian cancer cells. BC cell-intrinsic PD-L1 also mediated chemotherapy resistance to the commonly used BC chemotherapy agents cis-platinum and gemcitabine and to the mTORC1 inhibitor, rapamycin. Thus, BC cell-intrinsic PD-L1 signals regulate important virulence and treatment resistance pathways that suggest novel, actionable treatment targets meriting additional studies. As a proof-of-concept, we showed that the autophagy inhibitor chloroquine improved cis-platinum treatment efficacy in vivo, with greater efficacy in PD-L1 null versus PD-L1-replete BC.

Keywords:  PD-L1; autophagy; bladder cancer; chemotherapy; mTOR

DOI:  https://doi.org/10.1002/cam4.3739
Front Immunol. 2020 ;11 617404

Full Activation of Kinase Protein Kinase B by Phosphoinositide-Dependent Protein Kinase-1 and Mammalian Target of Rapamycin Complex 2 Is Required for Early Natural Killer Cell Development and Survival.

Junming He, Jun Zhao, Yuhe Quan, Xinlei Hou, Meixiang Yang, Zhongjun Dong.

  The role of PI3K-mTOR pathway in regulating NK cell development has been widely reported. However, it remains unclear whether NK cell development depends on the protein kinase B (PKB), which links PI3K and mTOR, perhaps due to the potential redundancy of PKB. PKB has two phosphorylation sites, threonine 308 (T308) and serine 473 (S473), which can be phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK1) and mTORC2, respectively. In this study, we established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. We found that the single deletion of PDK1 or Rictor could lead to a significant defect in NK cell development, while combined deletion of PDK1 and Rictor severely hindered NK cell development at the early stage. Notably, ectopic expression of myristoylated PKB significantly rescued this defect. In terms of mechanism, in PDK1/Rictor-deficient NK cells, E4BP4, a transcription factor for NK cell development, was less expressed, and the exogenous supply of E4BP4 could alleviate the developmental defect of NK cell in these mice. Besides, overexpression of Bcl-2 also helped the survival of PDK1/Rictor-deficient NK cells, suggesting an anti-apoptotic role of PKB in NK cells. In summary, complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development, and PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis.

Keywords:  development; mammalian target of rapamycin complex 2; natural killer (NK) cell; phosphoinositide-dependent protein kinase-1 (PDK1); protein kinase B (PKB); survival

DOI:  https://doi.org/10.3389/fimmu.2020.617404
Front Oncol. 2020 ;10 604540

Prognostic and Therapeutic Potentials of OncomiRs Modulating mTOR Pathways in Virus-Associated Hepatocellular Carcinoma.

Neeti Nadda, Shashi Bala Paul, Dawesh P Yadav, Sonu Kumar, Vishnubhatla Sreenivas, Anoop Saraya, Shivanand Gamanagatti, Subrat Kumar Acharya, Shalimar, Baibaswata Nayak.

   Background: Dysregulated oncomiRs are attributed to hepatocellular carcinoma (HCC) through targeting mTOR signaling pathway responsible for cell growth and proliferation. The potential of these oncomiRs as biomarker for tumor response or as target for therapy needs to be evaluated.
AIM: Tumor response assessment by OncomiR changes following locoregional therapy (LRT) and targeting of these oncomiRs modulating pathway.
Methods: All consecutive viral-HCC patients of BCLC stage-A/B undergoing LRT were included. OncomiRs (miR-21, -221, and -16) change in circulation and AFP-ratio at 1-month post-LRT to baseline was estimated to differentiate various categories of response as per mRECIST criteria. OncomiR modulating mTOR pathway was studied by generating miR-21 and miR-221 overexpressing Huh7 stable cell lines.
Results: Post-LRT tumor response was assessed in 90 viral-HCC patients (CR, 40%; PR, 31%, and PD, 29%). Significant increase of miRNA-21 and -221 expression was observed in PD (p = 0.040, 0.047) and PR patients (miR-21, p = 0.045). Fold changes of miR-21 can differentiate response in group (CR from PR+PD) at AUROC 0.718 (95% CI, 0.572-0.799) and CR from PD at AUROC 0.734 (95% CI, 0.595-0.873). Overexpression of miR-21 in hepatoma cell line had shown increased phosphorylation p70S6K, the downstream regulator of cell proliferation in mTOR pathway. Upregulation of AKT, mTOR, and RPS6KB1 genes were found significant (P < 0.005) and anti-miR-21 specifically reduced mTOR gene (P = 0.02) expression.
Conclusions: The miR-21 fold change correlates well with imaging in predicting tumor response. Overexpression of miR-21 has a role in HCC through mTOR pathway activation and can be targeted.

Keywords:  alpha-fetoprotein; hepatitis virus; hepatocellular carcinoma; loco-regional therapy; miRNA; oncomiR

DOI:  https://doi.org/10.3389/fonc.2020.604540
Cancer Discov. 2021 Feb 24. pii: candisc.1571.2020. [Epub ahead of print]

Loss of optineurin drives cancer immune evasion via palmitoylation-dependent IFNGR1 lysosomal sorting and degradation.

Wan Du, Fang Hua, Xiong Li, Jian Zhang, Shasha Li, Weichao Wang, Jiajia Zhou, Weimin Wang, Peng Liao, Yijian Yan, Gaopeng Li, Shuang Wei, Sara Grove, Linda Vatan, Witold Zgodzinski, Marek Majewski, Grzegorz Wallner, Haoyan Chen, Ilona Kryczek, Jing-Yuan Fang, Weiping Zou.

Mutations in IFN- and MHC-signaling genes endow immunotherapy resistance. Colorectal cancer patients infrequently exhibit IFN- and MHC-signaling gene mutations, and are generally resistant to immunotherapy. In exploring the integrity of the IFN- and MHC-signaling in colorectal cancer, we found that optineurin was a shared node between the two pathways, and predicted colorectal cancer patient outcome. Loss of optineurin occurred in early stage human colorectal cancer. Immunologically, optineurin deficiency attenuated IFNGR1 and MHC-I expression, impaired T cell-immunity, and diminished immunotherapy efficacy in murine cancer models and cancer patients. Mechanistically, IFNGR1 was S-palmitoylated on Cys122, and AP3D1 bound with and sorted palmitoylated-IFNGR1 to lysosome for degradation. Unexpectedly, optineurin interacted with AP3D1 to prevent palmitoylated-IFNGR1 lysosomal sorting and degradation - thereby maintaining IFNy- and MHC-I-signaling integrity. Furthermore, pharmacologically targeting IFNGR1-palmitoylation stabilized IFNGR1, augmented tumor immunity, and sensitized checkpoint therapy. Thus, loss of optineurin drives immune evasion and intrinsic immunotherapy resistance in colorectal cancer.

DOI: https://doi.org/10.1158/2159-8290.CD-20-1571
Biochem Biophys Res Commun. 2021 Feb 18. pii: S0006-291X(21)00157-1. [Epub ahead of print]547 169-175

WDR35 is involved in subcellular localization of acetylated tubulin in 293T cells.

Takeshi Sekiguchi, Takashi Ishii, Hideki Kobayashi, Nobuaki Furuno.

  WDR35/IFT121 is an intraflagellar transport protein in primary cilia, which is associated with RagA, an mTORC1-activating protein. To elucidate the functions of the interaction between WDR35 and RagA in primary cilia, as well as mTOR signaling, we identified WDR35-interacting proteins using mass spectrometry. We found that WDR35 associates with CCT complex proteins including TCP1/CCT1, which act as molecular chaperones for α-tubulin folding. Immunostaining showed that acetylated α-tubulin was concentrated in the vicinity of primary cilia in 293T cells. In contrast, acetylated tubulin was dispersed in WDR35 partial knockout cells established from 293T cells. Similarly, scattered subcellular localization of acetylated tubulin was observed in RagA knockout cells. RagA was present in the primary cilia of NIH3T3 cells, and the GDP form of RagA exhibited strong binding to WDR35 and negative regulation of primary cilium formation. These results suggest that WDR35 is involved in the subcellular localization of acetylated tubulin in primary cilia via its interactions with TCP1 and/or RagA family proteins.

Keywords:  Primary cilia; RagA; WDR35/IFT121; mTORC1

DOI:  https://doi.org/10.1016/j.bbrc.2021.01.092
Cell. 2021 Feb 22. pii: S0092-8674(21)00171-9. [Epub ahead of print]

In vivo CRISPR screening reveals nutrient signaling processes underpinning CD8+ T cell fate decisions.

Hongling Huang, Peipei Zhou, Jun Wei, Lingyun Long, Hao Shi, Yogesh Dhungana, Nicole M Chapman, Guotong Fu, Jordy Saravia, Jana L Raynor, Shaofeng Liu, Gustavo Palacios, Yong-Dong Wang, Chenxi Qian, Jiyang Yu, Hongbo Chi.

  How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.

Keywords:  GDP-fucose; Notch; T cell memory; cell cycle exit; immunometabolism; in vivo pooled CRISPR screening; metabolic heterogeneity; nutrient signaling; systems immunology; terminal effector cell

DOI:  https://doi.org/10.1016/j.cell.2021.02.021
Nat Commun. 2021 02 23. 12(1): 1248

Facilitative lysosomal transport of bile acids alleviates ER stress in mouse hematopoietic precursors.

Avinash K Persaud, Sreenath Nair, Md Fazlur Rahman, Radhika Raj, Brenna Weadick, Debasis Nayak, Craig McElroy, Muruganandan Shanmugam, Sue Knoblaugh, Xiaolin Cheng, Rajgopal Govindarajan.

Mutations in human equilibrative nucleoside transporter 3 (ENT3) encoded by SLC29A3 results in anemia and erythroid hypoplasia, suggesting that ENT3 may regulate erythropoiesis. Here, we demonstrate that lysosomal ENT3 transport of taurine-conjugated bile acids (TBA) facilitates TBA chemical chaperone function and alleviates endoplasmic reticulum (ER) stress in expanding mouse hematopoietic stem and progenitor cells (HSPCs). Slc29a3-/- HSPCs accumulate less TBA despite elevated levels of TBA in Slc29a3-/- mouse plasma and have elevated basal ER stress, reactive oxygen species (ROS), and radiation-induced apoptosis. Reintroduction of ENT3 allows for increased accumulation of TBA into HSPCs, which results in TBA-mediated alleviation of ER stress and erythroid apoptosis. Transplanting TBA-preconditioned HSPCs expressing ENT3 into Slc29a3-/- mice increase bone marrow repopulation capacity and erythroid pool size and prevent early mortalities. Together, these findings suggest a putative role for a facilitative lysosomal transporter in the bile acid regulation of ER stress in mouse HSPCs which may have implications in erythroid biology, the treatment of anemia observed in ENT3-mutated human genetic disorders, and nucleoside analog drug therapy.

DOI: https://doi.org/10.1038/s41467-021-21451-6
Front Cell Dev Biol. 2021 ;9 627700

Endoplasmic Reticulum-Plasma Membrane Contact Sites: Regulators, Mechanisms, and Physiological Functions.

Chenlu Li, Tiantian Qian, Ruyue He, Chun Wan, Yinghui Liu, Haijia Yu.

  The endoplasmic reticulum (ER) forms direct membrane contact sites with the plasma membrane (PM) in eukaryotic cells. These ER-PM contact sites play essential roles in lipid homeostasis, ion dynamics, and cell signaling, which are carried out by protein-protein or protein-lipid interactions. Distinct tethering factors dynamically control the architecture of ER-PM junctions in response to intracellular signals or external stimuli. The physiological roles of ER-PM contact sites are dependent on a variety of regulators that individually or cooperatively perform functions in diverse cellular processes. This review focuses on proteins functioning at ER-PM contact sites and highlights the recent progress in their mechanisms and physiological roles.

Keywords:  endoplasmic reticulum (ER); enzyme; lipid transfer; membrane contact sites (MCSs); plasma membrane; tether

DOI:  https://doi.org/10.3389/fcell.2021.627700
FEBS J. 2021 Feb 25.

Pathogenesis of Diabesity-induced Kidney Disease: Role of Kidney Nutrient Sensing.

Liad Hinden, Aviram Kogot-Levin, Joseph Tam, Gil Leibowitz.

  Diabetes kidney disease (DKD) is a major health care problem associated with increased risk for developing end-stage kidney disease (ESKD) and high mortality. It is widely accepted that DKD is primarily a glomerular disease. Recent findings however suggest that renal proximal tubule cells (KPTCs) may play a central role in the pathophysiology of DKD. In diabetes and obesity, KPTCs are exposed to nutrient overload, including glucose, free-fatty acids (FFAs) and amino acids (AAs), which dysregulate nutrient and energy sensing by mTORC1 and AMPK, with subsequent induction of tubular injury, inflammation and fibrosis. Pharmacological treatments that modulate nutrient sensing and signaling in KPTCs, including cannabinoid-1 receptor (CB1 R) antagonists and sodium glucose transporter 2 (SGLT2) inhibitors, exert robust kidney protective effects. Shedding light on how nutrients are sensed and metabolized in KPTCs and in other kidney domains, and on their effects on signal transduction pathways that mediate kidney injury, is important for understanding the pathophysiology of DKD and for the development of novel therapeutic approaches in DKD and probably also in other forms of kidney disease.

Keywords:  AMPK; CB1R; Diabetes; Obesity; SGLT2; mTORC1

DOI:  https://doi.org/10.1111/febs.15790
Biosci Biotechnol Biochem. 2021 Feb 24. 85(3): 587-599

The vacuolar amino acid transport system is a novel, direct target of GATA transcription factors.

Akane Sato, Takumi Kimura, Kana Hondo, Miyuki Kawano-Kawada, Takayuki Sekito.

  In Saccharomyces cerevisiae, Avt4 exports neutral and basic amino acids from vacuoles. Previous studies have suggested that the GATA transcription factors, Gln3 and Gat1, which are key regulators that adapt cells in response to changes in amino acid status, are involved in the AVT4 transcription. Here, we show that mutations in the putative GATA-binding sites of the AVT4 promoter reduced AVT4 expression. Consistently, a chromatin immunoprecipitation (ChIP) assay revealed that Gat1-Myc13 binds to the AVT4 promoter. Previous microarray results were confirmed that gln3∆gat1∆ cells showed a decrease in expression of AVT1 and AVT7, which also encode vacuolar amino acid transporters. Additionally, ChIP analysis revealed that the AVT6 encoding vacuolar acidic amino acid exporter represents a new direct target of the GATA transcription factor. The broad effect of the GATA transcription factors on the expression of AVT transporters suggests that vacuolar amino acid transport is integrated into cellular amino acid homeostasis.

Keywords:   Saccharomyces cerevisiae ; GATA transcription factor; amino acid transporter; vacuole

DOI:  https://doi.org/10.1093/bbb/zbaa041
Nat Metab. 2021 Feb;3(2): 244-257

The major cap-binding protein eIF4E regulates lipid homeostasis and diet-induced obesity.

Crystal S Conn, Haojun Yang, Harrison J Tom, Kenji Ikeda, Juan A Oses-Prieto, Hieu Vu, Yasuo Oguri, Supna Nair, Ryan M Gill, Shingo Kajimura, Ralph J DeBerardinis, Alma L Burlingame, Davide Ruggero.

Obesity is a global epidemic leading to increased mortality and susceptibility to comorbidities, with few viable therapeutic interventions. A hallmark of disease progression is the ectopic deposition of lipids in the form of lipid droplets in vital organs such as the liver. However, the mechanisms underlying the dynamic storage and processing of lipids in peripheral organs remain an outstanding question. Here, we show an unexpected function for the major cap-binding protein, eIF4E, in high-fat-diet-induced obesity. In response to lipid overload, select networks of proteins involved in fat deposition are altered in eIF4E-deficient mice. Specifically, distinct messenger RNAs involved in lipid metabolic processing and storage pathways are enhanced at the translation level by eIF4E. Failure to translationally upregulate these mRNAs results in increased fatty acid oxidation, which enhances energy expenditure. We further show that inhibition of eIF4E phosphorylation genetically-and by a potent clinical compound-restrains weight gain following intake of a high-fat diet. Together, our study uncovers translational control of lipid processing as a driver of high-fat-diet-induced weight gain and provides a pharmacological target to treat obesity.

DOI: https://doi.org/10.1038/s42255-021-00349-z
Sci Rep. 2021 Feb 26. 11(1): 4788

A plant plasma-membrane H+-ATPase promotes yeast TORC1 activation via its carboxy-terminal tail.

Elie Saliba, Cecilia Primo, Nadia Guarini, Bruno André.

The Target of Rapamycin Complex 1 (TORC1) involved in coordination of cell growth and metabolism is highly conserved among eukaryotes. Yet the signals and mechanisms controlling its activity differ among taxa, according to their biological specificities. A common feature of fungal and plant cells, distinguishing them from animal cells, is that their plasma membrane contains a highly abundant H+-ATPase which establishes an electrochemical H+ gradient driving active nutrient transport. We have previously reported that in yeast, nutrient-uptake-coupled H+ influx elicits transient TORC1 activation and that the plasma-membrane H+-ATPase Pma1 plays an important role in this activation, involving more than just establishment of the H+ gradient. We show here that the PMA2 H+-ATPase from the plant Nicotiana plumbaginifolia can substitute for Pma1 in yeast, to promote H+-elicited TORC1 activation. This H+-ATPase is highly similar to Pma1 but has a longer carboxy-terminal tail binding 14-3-3 proteins. We report that a C-terminally truncated PMA2, which remains fully active, fails to promote H+-elicited TORC1 activation. Activation is also impaired when binding of PMA2 to 14-3-3 s is hindered. Our results show that at least some plant plasma-membrane H+-ATPases share with yeast Pma1 the ability to promote TORC1 activation in yeast upon H+-coupled nutrient uptake.

DOI: https://doi.org/10.1038/s41598-021-83525-1