bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒03‒21
forty-seven papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

  1. Autophagy. 2021 Mar 18. 1-19
      Increased macroautophagy/autophagy and lysosomal activity promote tumor growth, survival and chemo-resistance. During acute starvation, autophagy is rapidly engaged by AMPK (AMP-activated protein kinase) activation and MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) inhibition to maintain energy homeostasis and cell survival. TFEB (transcription factor E3) and TFE3 (transcription factor binding to IGHM enhancer 3) are master transcriptional regulators of autophagy and lysosomal activity and their cytoplasm/nuclear shuttling is controlled by MTORC1-dependent multisite phosphorylation. However, it is not known whether and how the transcriptional activity of TFEB or TFE3 is regulated. We show that AMPK mediates phosphorylation of TFEB and TFE3 on three serine residues, leading to TFEB and TFE3 transcriptional activity upon nutrient starvation, FLCN (folliculin) depletion and pharmacological manipulation of MTORC1 or AMPK. Collectively, we show that MTORC1 specifically controls TFEB and TFE3 cytosolic retention, whereas AMPK is essential for TFEB and TFE3 transcriptional activity. This dual and opposing regulation of TFEB and TFE3 by MTORC1 and AMPK is reminiscent of the regulation of another critical regulator of autophagy, ULK1 (unc-51 like autophagy activating kinase 1). Surprisingly, we show that chemoresistance is mediated by AMPK-dependent activation of TFEB, which is abolished by pharmacological inhibition of AMPK or mutation of serine 466, 467 and 469 to alanine residues within TFEB. Altogether, we show that AMPK is a key regulator of TFEB and TFE3 transcriptional activity, and we validate AMPK as a promising target in cancer therapy to evade chemotherapeutic resistance.AbbreviationsACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; AMPKi: AMPK inhibitor, SBI-0206965; CA: constitutively active; CARM1: coactivator-associated arginine methyltransferase 1; CFP: cyan fluorescent protein; CLEAR: coordinated lysosomal expression and regulation; DKO: double knock-out; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; DQ-BSA: self-quenched BODIPY® dye conjugates of bovine serum albumin; EBSS: Earle's balanced salt solution; FLCN: folliculin; GFP: green fluorescent protein; GST: glutathione S-transferases; HD: Huntington disease; HTT: huntingtin; KO: knock-out; LAMP1: lysosomal associated membrane protein 1; MEF: mouse embryonic fibroblasts; MITF: melanocyte inducing transcription factor; MTORC1: MTOR complex 1; PolyQ: polyglutamine; RPS6: ribosomal protein S6; RT-qPCR: reverse transcription quantitative polymerase chain reaction; TCL: total cell lysates; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TKO: triple knock-out; ULK1: unc-51 like autophagy activating kinase 1.
    Keywords:  AMP-activated protein kinase; autophagy; drug resistance; lysosomal biogenesis; mechanistic target of rapamycin kinase; phosphorylation; transcription factor E3; transcription factor EB
  2. Nat Commun. 2021 Mar 19. 12(1): 1782
      Pharmacological inhibition of vacuolar-type H+-ATPase (V-ATPase) by its specific inhibitor can abrogate tumor metastasis, prevent autophagy, and reduce cellular signaling responses. Bafilomycin A1, a member of macrolide antibiotics and an autophagy inhibitor, serves as a specific and potent V-ATPases inhibitor. Although there are many V-ATPase structures reported, the molecular basis of specific inhibitors on V-ATPase remains unknown. Here, we report the cryo-EM structure of bafilomycin A1 bound intact bovine V-ATPase at an overall resolution of 3.6-Å. The structure reveals six bafilomycin A1 molecules bound to the c-ring. One bafilomycin A1 molecule engages with two c subunits and disrupts the interactions between the c-ring and subunit a, thereby preventing proton translocation. Structural and sequence analyses demonstrate that the bafilomycin A1-binding residues are conserved in yeast and mammalian species and the 7'-hydroxyl group of bafilomycin A1 acts as a unique feature recognized by subunit c.
  3. Front Cell Dev Biol. 2021 ;9 642494
      During primary tumorigenesis isolated cancer cells may undergo genetic or epigenetic changes that render them responsive to additional intrinsic or extrinsic cues, so that they enter a transitional state and eventually acquire an aggressive, metastatic phenotype. Among these changes is the alteration of the cell metabolic/catabolic machinery that creates the most permissive conditions for invasion, dissemination, and survival. The lysosomal system has emerged as a crucial player in this malignant transformation, making this system a potential therapeutic target in cancer. By virtue of their ubiquitous distribution in mammalian cells, their multifaced activities that control catabolic and anabolic processes, and their interplay with other organelles and the plasma membrane (PM), lysosomes function as platforms for inter- and intracellular communication. This is due to their capacity to adapt and sense nutrient availability, to spatially segregate specific functions depending on their position, to fuse with other compartments and with the PM, and to engage in membrane contact sites (MCS) with other organelles. Here we review the latest advances in our understanding of the role of the lysosomal system in cancer progression. We focus on how changes in lysosomal nutrient sensing, as well as lysosomal positioning, exocytosis, and fusion perturb the communication between tumor cells themselves and between tumor cells and their microenvironment. Finally, we describe the potential impact of MCS between lysosomes and other organelles in propelling cancer growth and spread.
    Keywords:  cancer progression; lysosomal exocytosis; lysosomal membrane contact sites; lysosome movement; lysosome positioning
  4. Development. 2021 Mar 15. pii: dev.194258. [Epub ahead of print]
      Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2 NPC2 is a soluble lysosomal protein which functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, while reducing cellular cholesterol bioavailability. Zygotic null npc2 m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2 m/m zebrafish exhibited significant developmental defects including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes, no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2 +/m and MZnpc2 m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2 m/m zebrafish.
    Keywords:  Cholesterol; Niemann-Pick type C; Niemann-Pick type C2; Notch3; Npc2; Zebrafish
  5. Talanta. 2021 May 15. pii: S0039-9140(21)00129-6. [Epub ahead of print]227 122208
      A lysosome-targeting ratiometric fluorescent nanoprobe based on cell-penetrating peptides (CPPs) and quantum dots (QDs) has been developed for monitoring pH fluctuation in living cells. The as-prepared nanoprobe is constructed by Rhodamine B labeled R9RGD CPPs as H+ response unit and the red fluorescent QDs as reference unit to achieve ratiometric pH measurement. With the help of RhB-R9RGD CPPs, the nanoprobe efficiently stains lysosomes and enables discernment of lysosomal pH fluctuation in cells treated with different pH buffers and drug stimulation. The method of using dye labeled CPPs to realize functionalization of nanoparticle in one-step reported herein is expected to obtain wider applications in the detection of subcellular active substances by combining different small molecular probes and functional peptides.
    Keywords:  Cell-penetrating peptides; Lysosome-targeting; Quantum dots; Ratiometric; pH nanoprobe
  6. Drugs. 2021 Mar 15.
      Fabry disease (FD) is a rare X-linked lysosomal storage disease based on a deficiency of α-galactosidase A (AGAL) caused by mutations in the α-galactosidase A gene (GLA). The lysosomal accumulation of glycosphingolipids, especially globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3, deacylated form), leads to a multisystemic disease with progressive renal failure, cardiomyopathy with potentially malignant cardiac arrhythmias, and strokes, which considerably limits the life expectancy of affected patients. Diagnostic confirmation in male patients is based on the detection of AGAL deficiency in blood leukocytes, whereas in women, due to the potentially high residual enzymatic activity, molecular genetic detection of a causal mutation is required. Current treatment options for FD include recombinant enzyme replacement therapy (ERT) with intravenous agalsidase-alfa (0.2 mg/kg body weight) or agalsidase-beta (1 mg/kg body weight) every 2 weeks and oral chaperone therapy with migalastat (123 mg every other day), which selectively and reversibly binds to the active site of AGAL, thereby correcting the misfolding of the enzyme and allowing it to traffic to the lysosome. These therapies enable cellular Gb3 clearance and improve the burden of disease. However, in about 40% of all ERT-treated men, ERT can lead to infusion-associated reactions and the formation of neutralizing antidrug antibodies, which reduces the efficacy of therapy. In chaperone therapy, there are carriers of amenable mutations that show limited clinical success. This article provides a brief overview of the clinical picture in FD patients, diagnostic confirmation, and interdisciplinary clinical management of FD. The focus is on current and future therapeutic options.
  7. J Extracell Vesicles. 2021 Mar;10(5): e12058
      In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.
    Keywords:  Fabry disease; N‐sulfoglucosamine sulfohydrolase; Sanfilippo syndrome; alpha‐galactosidase A; drug delivery; enzyme replacement therapy; lysosomal storage disorders
  8. Cell Rep. 2021 Mar 16. pii: S2211-1247(21)00144-3. [Epub ahead of print]34(11): 108830
      Rab1A is a small GTPase known for its role in vesicular trafficking. Recent evidence indicates that Rab1A is essential for amino acids (aas) sensing and signaling to regulate mTORC1 in normal and cancer cells. However, Rab1A's in vivo function in mammals is not known. Here, we report the generation of tamoxifen (TAM)-induced whole body Rab1A knockout (Rab1A-/-) in adult mice. Rab1A-/- mice are viable but become hyperglycemic and glucose intolerant due to impaired insulin transcription and β-cell proliferation and maintenance. Mechanistically, Rab1A mediates AA-mTORC1 signaling, particularly branched chain amino acids (BCAA), to regulate the stability and localization of the insulin transcription factor Pdx1. Collectively, these results reveal a physiological role of aa-Rab1A-mTORC1 signaling in the control of whole-body glucose homeostasis in mammals. Intriguingly, Rab1A expression is reduced in β-cells of type 2 diabetes (T2D) patients, which is correlated with loss of insulin expression, suggesting that Rab1A downregulation contributes to T2D progression.
    Keywords:  PDX1; Rab1A; alpha-cell; amino acids; beta-cell; diabetes; glucose homeostasis; insulin; mTOR; trans-differentiation
  9. Brain Commun. 2021 ;3(1): fcaa148
      Acetyl-dl-leucine is a derivative of the branched chain amino acid leucine. In observational clinical studies, acetyl-dl-leucine improved symptoms of ataxia, in particular in patients with the lysosomal storage disorder, Niemann-Pick disease type C1. Here, we investigated acetyl-dl-leucine and its enantiomers acetyl-l-leucine and acetyl-d-leucine in symptomatic Npc1-/- mice and observed improvement in ataxia with both individual enantiomers and acetyl-dl-leucine. When acetyl-dl-leucine and acetyl-l-leucine were administered pre-symptomatically to Npc1-/- mice, both treatments delayed disease progression and extended life span, whereas acetyl-d-leucine did not. These data are consistent with acetyl-l-leucine being the neuroprotective enantiomer. Altered glucose and antioxidant metabolism were implicated as one of the potential mechanisms of action of the l-enantiomer in Npc1-/- mice. When the standard of care drug miglustat and acetyl-dl-leucine were used in combination significant synergy resulted. In agreement with these pre-clinical data, when Niemann-Pick disease type C1 patients were evaluated after 12 months of acetyl-dl-leucine treatment, rates of disease progression were slowed, with stabilization or improvement in multiple neurological domains. A beneficial effect of acetyl-dl-leucine on gait was also observed in this study in a mouse model of GM2 gangliosidosis (Sandhoff disease) and in Tay-Sachs and Sandhoff disease patients in individual-cases of off-label-use. Taken together, we have identified an unanticipated neuroprotective effect of acetyl-l-leucine and underlying mechanisms of action in lysosomal storage diseases, supporting its further evaluation in clinical trials in lysosomal disorders.
    Keywords:  GM2 gangliosidosis; Niemann-Pick disease type C; acetyl-leucine; lysosomal storage diseases; miglustat
  10. Front Cell Dev Biol. 2021 ;9 613336
      In recent decades, compelling evidence has emerged showing that organelles are not static structures but rather form a highly dynamic cellular network and exchange information through membrane contact sites. Although high-throughput techniques facilitate identification of novel contact sites (e.g., organelle-organelle and organelle-vesicle interactions), little is known about their impact on cellular physiology. Moreover, even less is known about how the dysregulation of these structures impacts on cellular function and therefore, disease. Particularly, cancer cells display altered signaling pathways involving several cell organelles; however, the relevance of interorganelle communication in oncogenesis and/or cancer progression remains largely unknown. This review will focus on organelle contacts relevant to cancer pathogenesis. We will highlight specific proteins and protein families residing in these organelle-interfaces that are known to be involved in cancer-related processes. First, we will review the relevance of endoplasmic reticulum (ER)-mitochondria interactions. This section will focus on mitochondria-associated membranes (MAMs) and particularly the tethering proteins at the ER-mitochondria interphase, as well as their role in cancer disease progression. Subsequently, the role of Ca2+ at the ER-mitochondria interphase in cancer disease progression will be discussed. Members of the Bcl-2 protein family, key regulators of cell death, also modulate Ca2+ transport pathways at the ER-mitochondria interphase. Furthermore, we will review the role of ER-mitochondria communication in the regulation of proteostasis, focusing on the ER stress sensor PERK (PRKR-like ER kinase), which exerts dual roles in cancer. Second, we will review the relevance of ER and mitochondria interactions with other organelles. This section will focus on peroxisome and lysosome organelle interactions and their impact on cancer disease progression. In this context, the peroxisome biogenesis factor (PEX) gene family has been linked to cancer. Moreover, the autophagy-lysosome system is emerging as a driving force in the progression of numerous human cancers. Thus, we will summarize our current understanding of the role of each of these organelles and their communication, highlighting how alterations in organelle interfaces participate in cancer development and progression. A better understanding of specific organelle communication sites and their relevant proteins may help to identify potential pharmacological targets for novel therapies in cancer control.
    Keywords:  cancer; endoplasmic reticulum; interorganelle communication; lysosome; mitochondria; peroxisome
  11. J Inherit Metab Dis. 2021 Mar 19.
      S-palmitoylation is a reversible posttranslational modification in which a 16-carbon saturated fatty acid (generally palmitate) is attached to specific cysteine residues in polypeptides via thioester linkage. Dynamic S-palmitoylation (palmitoylation-depalmitoylation), like phosphorylation-dephosphorylation, regulates the function of numerous proteins, especially in the brain. While a family of 23 palmitoyl-acyl transferases (PATS), commonly known as ZDHHCs, catalyze S-palmitoylation of proteins, the thioesterases, localized either in the cytoplasm (e.g. APT1) or in the lysosome (e.g. PPT1) mediate depalmitoylation. Previously, we reported that APT1 requires dynamic S-palmitoylation for shuttling between the cytosol and the plasma membrane. APT1 depalmitoylated H-Ras to regulate its signaling pathway that stimulates cell proliferation. Although we demonstrated that APT1 catalyzed its own depalmitoylation, the ZDHHC(s) that S-palmitoylated APT1 had remained unidentified. We report here that ZDHHC5 and ZDHHC23 catalyze APT1 S-palmitoylation. Intriguingly, lysosomal Ppt1-deficiency in Cln1-/- mouse, a reliable animal model of INCL, markedly reduced ZDHHC5 and ZDHHC23 levels. Remarkably, in the brain of these mice decreased ZDHHC5 and ZDHHC23 levels suppressed membrane-bound APT1, thereby, increasing plasma membrane-localized H-Ras, which activated its signaling pathway stimulating microglia proliferation. Increased inflammatory cytokines produced by microglia together with increased complement C1q level contributed to the transformation of astrocytes to neurotoxic A1 phenotype. Importantly, neuroinflammation was ameliorated by treatment of Cln1-/- mice with a PPT1-mimetic small molecule, N-tert(Butyl)hydroxylamine (NtBuHA). Our results revealed a novel pathway to neuropathology in an INCL mouse model and uncovered a previously unrecognized mechanism of the neuroprotective actions of NtBuHA and its potential as a drug target. This article is protected by copyright. All rights reserved.
    Keywords:  Infantile neuronal ceroid lipofuscinosis; Lysosomal storage disease; Neuroinflammation; Palmitoyl-protein thioesterases-1; S-palmitoylation
  12. Transl Psychiatry. 2021 Mar 15. 11(1): 165
      The mechanistic target of rapamycin (mTOR) is a ubiquitously expressed kinase that acts through two complexes, mTORC1 and mTORC2, to regulate protein homeostasis, as well as long lasting forms of synaptic and behavioral plasticity. Alteration of the mTOR pathway is classically involved in neurodegenerative disorders, and it has been linked to dysregulation of cognitive functions and affective states. However, information concerning the specific involvement of the p70 S6 kinase 1 (S6K1), a downstream target of the mTORC1 pathway, in learning and memory processes and in the regulation of affective states remains scant. To fill this gap, we exposed adult male mice lacking S6K1 to a battery of behavioral tests aimed at measuring their learning and memory capabilities by evaluating reference memory and flexibility with the Morris water maze, and associative memory using the contextual fear conditioning task. We also studied their anxiety-like and depression-like behaviors by, respectively, performing elevated plus maze, open field, light-dark emergence tests, and sucrose preference and forced swim tests. We found that deleting S6K1 leads to a robust anxious phenotype concomitant with associative learning deficits; these symptoms are associated with a reduction of adult neurogenesis and neuronal atrophy in the hippocampus. Collectively, these results provide grounds for the understanding of anxiety reports after treatments with mTOR inhibitors and will be critical for developing novel compounds targeting anxiety.
  13. Bio Protoc. 2021 Jan 20. 11(2): e3888
      ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020 ) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.
    Keywords:  ATP13A2; P5 ATPase; Polyamine; Reconstitution; Spermine; Transport assay; Yeast membranes
  14. Biochem Biophys Res Commun. 2021 Mar 16. pii: S0006-291X(21)00416-2. [Epub ahead of print]552 1-8
      The degradation of nucleolar proteins - nucleophagy - is elicited by nutrient starvation or the inactivation of target of rapamycin complex 1 (TORC1) protein kinase in budding yeast. Prior to nucleophagy, nucleolar proteins migrate to the nucleus-vacuole junction (NVJ), where micronucleophagy occurs, whereas rDNA (rRNA gene) repeat regions are condensed and escape towards NVJ-distal sites. This suggests that the NVJ controls nucleolar dynamics from outside of the nucleus after TORC1 inactivation, but its molecular mechanism is unclear. Here, we show that sorting nexin (SNX) Mdm1, an inter-organelle tethering protein at the NVJ, mediates TORC1 inactivation-induced nucleolar dynamics. Furthermore, Mdm1 was required for proper nucleophagic degradation of nucleolar proteins after TORC1 inactivation, where it was dispensable for the induction of nucleophagic flux itself. This indicated that nucleophagy and nucleolar dynamics are independently regulated by TORC1 inactivation. Finally, Mdm1 was critical for survival during nutrient starvation conditions. Mutations of SNX14, a human Mdm1 homolog, cause neurodevelopmental disorders. This study provides a novel insight into relationship between sorting nexin-mediated microautophagy and neurodevelopmental disorders.
    Keywords:  Mdm1; NVJ; Nucleophagy; SNX14; TORC1; rDNA
  15. FEBS J. 2021 Mar 17.
      Autophagy is a highly conserved catabolic process cells use to maintain their homeostasis by degrading misfolded, damaged, and excessive proteins, non-functional organelles, foreign pathogens, and other cellular components. Hence, autophagy can be non-selective, where bulky portions of the cytoplasm are degraded upon stress, or a highly selective process, where pre-selected cellular components are degraded. To distinguish between different cellular components, autophagy employs selective autophagy receptors, which will link the cargo to the autophagy machinery, thereby sequestering it in the autophagosome for its subsequent degradation in the lysosome. Autophagy receptors undergo post-translational and structural modifications to fulfil their role in autophagy, or upon executing their role, for their own degradation. We highlight the four most prominent protein modifications - phosphorylation, ubiquitination, acetylation, and oligomerisation - that are essential for autophagy receptor recruitment, function, and turnover. Understanding the regulation of selective autophagy receptors will provide deeper insights into the pathway and open up potential therapeutic avenues.
    Keywords:  Autophagy; oligomerisation; phosphorylation; receptor; ubiquitination
  16. Cancer Cell. 2021 Mar 10. pii: S1535-6108(21)00118-5. [Epub ahead of print]
      Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.
    Keywords:  NRF2; RAS-driven cancer; autophagy; macropinocytosis; p62/SQSTM1
  17. Front Cell Dev Biol. 2021 ;9 643769
      Lysosomal degradation of ubiquitinated transmembrane protein receptors (cargo) relies on the function of Endosomal Sorting Complex Required for Transport (ESCRT) protein complexes. The ESCRT machinery is comprised of five unique oligomeric complexes with distinct functions. Target of Myb1 (TOM1) is an ESCRT protein involved in the initial steps of endosomal cargo sorting. To exert its function, TOM1 associates with ubiquitin moieties on the cargo via its VHS and GAT domains. Several ESCRT proteins, including TOLLIP, Endofin, and Hrs, have been reported to form a complex with TOM1 at early endosomal membrane surfaces, which may potentiate the role of TOM1 in cargo sorting. More recently, it was found that TOM1 is involved in other physiological processes, including autophagy, immune responses, and neuroinflammation, which crosstalk with its endosomal cargo sorting function. Alteration of TOM1 function has emerged as a phosphoinositide-dependent survival mechanism for bacterial infections and cancer progression. Based on current knowledge of TOM1-dependent cellular processes, this review illustrates how TOM1 functions in coordination with an array of protein partners under physiological and pathological scenarios.
    Keywords:  ESCRT; Endofin; TOL; TOLLIP; TOM1; endosome; phosphoinositides
  18. EMBO Mol Med. 2021 Mar 18. e13560
      Rosacea is a chronic inflammatory skin disorder whose pathogenesis is unclear. Here, several lines of evidence were provided to demonstrate that mTORC1 signaling is hyperactivated in the skin, especially in the epidermis, of both rosacea patients and a mouse model of rosacea-like skin inflammation. Both mTORC1 deletion in epithelium and inhibition by its specific inhibitors can block the development of rosacea-like skin inflammation in LL37-induced rosacea-like mouse model. Conversely, hyperactivation of mTORC1 signaling aggravated rosacea-like features. Mechanistically, mTORC1 regulates cathelicidin through a positive feedback loop, in which cathelicidin LL37 activates mTORC1 signaling by binding to Toll-like receptor 2 (TLR2) and thus in turn increases the expression of cathelicidin itself in keratinocytes. Moreover, excess cathelicidin LL37 induces both NF-κB activation and disease-characteristic cytokine and chemokine production possibly via mTORC1 signaling. Topical application of rapamycin improved clinical symptoms in rosacea patients, suggesting mTORC1 inhibition can serve as a novel therapeutic avenue for rosacea.
    Keywords:  LL37; Rapamycin; mTOR; rosacea; skin inflammation
  19. Front Oncol. 2021 ;11 632524
      Transcription factor EB (TFEB), a member of the MiT family, is dysregulated in different cancers and exerts specific biological functions within the tumor microenvironment. Downregulation of TFEB induces macrophage polarization in the TME and promotes tumor progression. However, the biological role and clinical significance of TFEB in prostate cancer (PCa) remain unknown. This study aimed to identify the role of TFEB in PCa and its potential clinical value. We explored TFEB expression in PCa using public databases and verified its prognostic value using immunohistochemistry in PCa tissue samples. The results revealed that TFEB expression was up-regulated in PCa tissues and was associated with cancer metastasis. Next, overexpression of TFEB promoted PCa cell malignant behavior in in vivo and in vitro experiments. RNA-sequencing and bioinformatics analysis showed high expression of TFEB promoted lysosomal biogenesis and knockdown of TFEB expression decreased the number of lysosomes. Furthermore, the ATP-binding cassette transporter A2 (ABCA2) was identified as a target gene of TFEB, which was verified using the cleavage under targets and release using nuclease (CUT&RUN) assay and qRT-PCR. Silencing of ABCA2 reduced lysosomal biogenesis and decreased matrix metalloproteinases expression, which reduced PCa cell invasion and migration in the tumor microenvironment. Our study suggests that TFEB promotes PCa progression by regulating ABCA2 through lysosomal biogenesis and may serve as a prognostic factor or as a potential therapeutic target of PCa.
    Keywords:  ABCA2; TFEB; biochemical recurrence; lysosomal biogenesis; metastasis; prostate cancer; tumor microenvironment
  20. Front Cell Dev Biol. 2021 ;9 630279
      Autophagy is involved in many physiological processes. Transcription factor EB (TFEB) is a master regulator of autophagy and coordinates the expression of autophagic proteins, lysosomal hydrolases, and lysosomal membrane proteins. Though autophagy has been implicated in several human diseases, little is known regarding TFEB gene expression and regulation in the process. Since dysfunctional autophagy plays critical roles in acute myocardial infarction (AMI), dysregulated TFEB gene expression may be associated with AMI by regulating autophagy. In this study, the TFEB gene promoter was genetically and functionally analyzed in AMI patients (n = 352) and ethnic-matched controls (n = 337). A total of fifteen regulatory variants of the TFEB gene, including eight single-nucleotide polymorphisms (SNPs), were identified in this population. Among these, six regulatory variants [g.41737274T>C (rs533895008), g.41737144A>G, g.41736987C > T (rs760293138), g.41736806C > T (rs748537297), g.41736635T > C (rs975050638), and g.41736544C > T] were only identified in AMI patients. These regulatory variants significantly altered the transcriptional activity of the TFEB gene promoter. Further electrophoretic mobility shift assay revealed that three of the variants evidently affected the binding of transcription factors. Therefore, this study identified novel TFEB gene regulatory variants which affect the gene expression. These TFEB gene regulatory variants may contribute to AMI development as a rare risk factor.
    Keywords:  TFEB; acute myocardial infarction; autophagy; genetics; promoter
  21. Nat Commun. 2021 03 15. 12(1): 1678
      Protein homeostasis is modulated by stress response pathways and its deficiency is a hallmark of aging. The integrated stress response (ISR) is a conserved stress-signaling pathway that tunes mRNA translation via phosphorylation of the translation initiation factor eIF2. ISR activation and translation initiation are finely balanced by eIF2 kinases and by the eIF2 guanine nucleotide exchange factor eIF2B. However, the role of the ISR during aging remains poorly understood. Using a genomic mutagenesis screen for longevity in Caenorhabditis elegans, we define a role of eIF2 modulation in aging. By inhibiting the ISR, dominant mutations in eIF2B enhance protein homeostasis and increase lifespan. Consistently, full ISR inhibition using phosphorylation-defective eIF2α or pharmacological ISR inhibition prolong lifespan. Lifespan extension through impeding the ISR occurs without a reduction in overall protein synthesis. Instead, we observe changes in the translational efficiency of a subset of mRNAs, of which the putative kinase kin-35 is required for lifespan extension. Evidently, lifespan is limited by the ISR and its inhibition may provide an intervention in aging.
  22. Cancer Discov. 2021 Mar 19.
      The membrane repair factor MYOF prevented lysosome membrane damage in pancreatic cancer cells.
  23. EMBO Rep. 2021 Mar 19. e51740
      Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.
    Keywords:  DYRK3; FUS-ALS; Hsp90; phase separation; stress granules
  24. Front Cell Dev Biol. 2021 ;9 629522
      In the last few years, metabolism has been shown to be controlled by cross-organelle communication. The relationship between the endoplasmic reticulum and mitochondria/lysosomes is the most studied; here, inositol 1,4,5-triphosphate (IP3) receptor (IP3R)-mediated calcium (Ca2+) release plays a central role. Recent evidence suggests that IP3R isoforms participate in synthesis and degradation pathways. This minireview will summarize the current findings in this area, emphasizing the critical role of Ca2+ communication on organelle function as well as catabolism and anabolism, particularly in cancer.
    Keywords:  IP3Rs; calcium; endoplasmic reticulum; inositol triphosphate (IP3) receptors; lysosome; metabolism; mitochondria
  25. Dev Cell. 2021 Mar 09. pii: S1534-5807(21)00162-3. [Epub ahead of print]
      Neuronal activity increases energy consumption and requires balanced production to maintain neuronal function. How activity is coupled to energy production remains incompletely understood. Here, we report that Rheb regulates mitochondrial tricarboxylic acid cycle flux of acetyl-CoA by activating pyruvate dehydrogenase (PDH) to increase ATP production. Rheb is induced by synaptic activity and lactate and dynamically trafficked to the mitochondrial matrix through its interaction with Tom20. Mitochondria-localized Rheb protein is required for activity-induced PDH activation and ATP production. Cell-type-specific gain- and loss-of-function genetic models for Rheb reveal reciprocal changes in PDH phosphorylation/activity, acetyl-CoA, and ATP that are not evident with genetic or pharmacological manipulations of mTORC1. Mechanistically, Rheb physically associates with PDH phosphatase (PDP), enhancing its activity and association with the catalytic E1α-subunit of PDH to reduce PDH phosphorylation and increase its activity. Findings identify Rheb as a nodal point that balances neuronal activity and neuroenergetics via Rheb-PDH axis.
    Keywords:  Rheb; mTORC1; mitochondria; neuroenergetics; neuronal activity; pyruvate dehydrogenase
  26. Genet Med. 2021 Mar 17.
      PURPOSE: Variants in NUS1 are associated with a congenital disorder of glycosylation, developmental and epileptic encephalopathies, and are possible contributors to Parkinson disease pathogenesis. How the diverse functions of the NUS1-encoded Nogo B receptor (NgBR) relate to these different phenotypes is largely unknown. We present three patients with de novo heterozygous variants in NUS1 that cause a complex movement disorder, define pathogenic mechanisms in cells and zebrafish, and identify possible therapy.METHODS: Comprehensive functional studies were performed using patient fibroblasts, and a zebrafish model mimicking NUS1 haploinsufficiency.
    RESULTS: We show that de novo NUS1 variants reduce NgBR and Niemann-Pick type C2 (NPC2) protein amount, impair dolichol biosynthesis, and cause lysosomal cholesterol accumulation. Reducing nus1 expression 50% in zebrafish embryos causes abnormal swim behaviors, cholesterol accumulation in the nervous system, and impaired turnover of lysosomal membrane proteins. Reduction of cholesterol buildup with 2-hydroxypropyl-ß-cyclodextrin significantly alleviates lysosomal proteolysis and motility defects.
    CONCLUSION: Our results demonstrate that these NUS1 variants cause multiple lysosomal phenotypes in cells. We show that the movement deficits associated with nus1 reduction in zebrafish arise in part from defective efflux of cholesterol from lysosomes, suggesting that treatments targeting cholesterol accumulation could be therapeutic.
  27. Commun Biol. 2021 Mar 19. 4(1): 373
      Proximal tubular cells (PTCs) are crucial for maintaining renal homeostasis, and tubular injuries contribute to progression of diabetic kidney disease (DKD). However, the roles of visceral adipose tissue-derived serine protease inhibitor (vaspin) in the development of DKD is not known. We found vaspin maintains PTCs through ameliorating ER stress, autophagy impairment, and lysosome dysfunction in DKD. Vaspin-/- obese mice showed enlarged and leaky lysosomes in PTCs associated with increased apoptosis, and these abnormalities were also observed in the patients with DKD. During internalization into PTCs, vaspin formed a complex with heat shock protein family A (Hsp70) member 1 like (HSPA1L) as well as 78 kDa glucose-regulated protein (GRP78). Both vaspin-partners bind to clathrin heavy chain and involve in the endocytosis. Notably, albumin-overload enhanced extracellular release of HSPA1L and overexpression of HSPA1L dissolved organelle stresses, especially autophagy impairment. Thus, vapsin/HSPA1L-mediated pathways play critical roles in maintaining organellar function of PTCs in DKD.
  28. JIMD Rep. 2021 Mar;58(1): 100-103
      Quantifying lymphocyte vacuolization in peripheral blood smears (PBSs) serves as a measure for disease severity in CLN3 disease-a lysosomal storage disorder of childhood-onset. However, thus far quantification methods are based on labor-intensive manual assessment of PBSs. As machine learning techniques like convolutional neural networks (CNNs) have been deployed quite successfully in detecting pathological features in PBSs, we explored whether these techniques could be utilized to automate quantification of lymphocyte vacuolization. Here, we present and validate a deep learning pipeline that automates quantification of lymphocyte vacuolization. By using two CNNs in succession, trained for cytoplasm-segmentation and vacuolization-detection, respectively, we obtained an excellent correlation with manual quantification of lymphocyte vacuolization (r = 0.98, n = 40). These results show that CNNs can be utilized to automate the otherwise cumbersome task of manually quantifying lymphocyte vacuolization, thereby aiding prompt clinical decisions in relation to CLN3 disease, and potentially beyond.
    Keywords:  CLN3 disease; lymphocyte vacuolization; machine learning; neuronal ceroid lipofuscinosis
  29. J Cell Sci. 2021 Mar 18. pii: jcs.254185. [Epub ahead of print]
      Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch where Rab5 is gradually exchanged with Rab7a on the same endosome. Here we provide new insight into the mechanism of endosomal maturation where we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrate a diffusion-like exchange between cytosol and endosomal membrane, and a second phase where Rab5 converges into specific domains that detaches as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5 to Rab7a switch induce the formation of new fully functional rab5 positive early endosomes. Progression through a stepwise early endosomal maturation regulates the direction of the transport and concomitantly regulates the homeostasis of early endosomes.
    Keywords:  Endosomal biogenesis; Endosome maturation; FRAP; Rab5; Rab7
  30. J Invest Dermatol. 2021 Mar 16. pii: S0022-202X(21)00995-7. [Epub ahead of print]
      Epidermal keratinocytes rapidly proliferate to repair the skin barrier and a strict control of division is necessary for healthy tissue homeostasis. However, the pathways that restrain proliferation after epidermal stress are not known. AMP-activated protein kinase (AMPK) is an important signaling mediator of energy metabolism previously associated with skin stress and cancer, yet its explicit impact on keratinocyte growth is not known. To examine the requirement of epidermal AMPK in physiologic skin repair, we genetically deleted AMPK within all adult, Keratin 14-expressing keratinocytes of mice. AMPK loss resulted in hyper-proliferation and hyperactive mTOR signaling following acute wounding, UVB exposure, and phorbol ester application. This excessive division could be completely blocked by the mTORC1 inhibitor rapamycin. Moreover, we establish that the diabetes drug metformin depends on AMPK to suppress stress-induced keratinocyte proliferation. Collectively, these findings show that keratinocyte AMPK restrains mTORC1 to control epidermal proliferation after tissue injury.
  31. Br J Cancer. 2021 Mar 15.
      BACKGROUND: Activation of mTORC1 plays a significant role in cancer development and progression. However, the metabolic mechanisms to sustain mTORC1 activation of cancer cells within stressed environments are still under-appreciated. We recently revealed high autophagy activity in tumour cells with mTORC1 hyper-activation. Nevertheless, the functions and mechanisms of autophagy in regulating mTORC1 in glioma are not studied.METHODS: Using glioma patient database and human glioma cells, we assessed the mechanisms and function of selective autophagy to sustain mTORC1 hyper-activation in glioma.
    RESULTS: We revealed a strong association of altered mRNA levels in mTORC1 upstream and downstream genes with prognosis of glioma patients. Our results indicated that autophagy-mediated lipid catabolism was essential to sustain mTORC1 activity in glioma cells under energy stresses. We found that autophagy inhibitors or fatty acid oxidation (FAO) inhibitors in combination with 2-Deoxy-D-glucose (2DG) decreased energy production and survival of glioma cells in vitro. Consistently, inhibition of autophagy or FAO inhibitors with 2DG effectively suppressed the progression of xenografted glioma with hyper-activated mTORC1.
    CONCLUSIONS: This study established an autophagy/lipid degradation/FAO/ATP generation pathway, which might be used in brain cancer cells under energy stresses to maintain high mTORC1 signalling for tumour progression.
  32. FASEB J. 2021 Apr;35(4): e21223
      The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.
    Keywords:  Sidt2; autophagy; mitochondria; myopathy; quality control
  33. Front Immunol. 2021 ;12 636078
      Following phagocytosis, the nascent phagosome undergoes maturation to become a phagolysosome with an acidic, hydrolytic, and often oxidative lumen that can efficiently kill and digest engulfed microbes, cells, and debris. The fusion of phagosomes with lysosomes is a principal driver of phagosomal maturation and is targeted by several adapted intracellular pathogens. Impairment of this process has significant consequences for microbial infection, tissue inflammation, the onset of adaptive immunity, and disease. Given the importance of phagosome-lysosome fusion to phagocyte function and the many virulence factors that target it, it is unsurprising that multiple molecular pathways have evolved to mediate this essential process. While the full range of these pathways has yet to be fully characterized, several pathways involving proteins such as members of the Rab GTPases, tethering factors and SNAREs have been identified. Here, we summarize the current state of knowledge to clarify the ambiguities in the field and construct a more comprehensive phagolysosome formation model. Lastly, we discuss how other cellular pathways help support phagolysosome biogenesis and, consequently, phagocyte function.
    Keywords:  homeostasis; lysosome; membrane fusion; microbial clearance; phagocyte; phagosome; phagosome maturation; phagosome-lysosome fusion
  34. Mol Neurodegener. 2021 Mar 19. 16(1): 17
      The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.
  35. Proc Natl Acad Sci U S A. 2021 Mar 30. pii: e2102173118. [Epub ahead of print]118(13):
  36. Aging (Albany NY). 2021 Mar 14. 13
      Caloric restriction has been shown to robustly ameliorate age-related diseases and to prolong lifespan in several model organisms, and these beneficial effects are dependent on the stimulation of autophagy. Autophagy dysfunction contributes to the accumulation of altered macromolecules, and is a key mechanism of promoting aging and age-related disorders, as neurodegenerative ones. We have previously shown that caloric restriction (CR), and CR mimetics Neuropeptide Y (NPY) and ghrelin, stimulate autophagy in rat cortical neurons, however by unknown molecular mechanisms. Overall, we show that CR, NPY, and ghrelin stimulate autophagy through PI3K/AKT/MTOR inhibition and ERK1/2-MAPK activation. The knowledge of these kinases in autophagy regulation and the contribution to the understanding of molecular mechanism facilitates the discovery of more targeted therapeutic strategies to stimulate autophagy, which is relevant in the context of age-related disorders.
    Keywords:  aging; autophagy; caloric restriction mimetics; cortical neurons
  37. Elife. 2021 Mar 19. pii: e58565. [Epub ahead of print]10
      G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/Igf signalling is reduced in fly models of C9orf72 repeat expansion using RNA-sequencing of adult brain. We further demonstrate that activation of insulin/Igf signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/Igf signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/Igf signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.
    Keywords:  D. melanogaster; neuroscience
  38. Dev Cell. 2021 Mar 15. pii: S1534-5807(21)00159-3. [Epub ahead of print]
      Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. Here, we show that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients.
    Keywords:  Chediak-Higashi syndrome; Drosophila; LYST; centrosomes; endosomal vesicle trafficking; lysosome-related organelles; mauve; microtubule nucleation; minispindles/Ch-TOG
  39. Biochem J. 2021 Mar 26. 478(6): 1199-1225
      PI3Ks are important lipid kinases that produce phosphoinositides phosphorylated in position 3 of the inositol ring. There are three classes of PI3Ks: class I PI3Ks produce PIP3 at plasma membrane level. Although D. melanogaster and C. elegans have only one form of class I PI3K, vertebrates have four class I PI3Ks called isoforms despite being encoded by four different genes. Hence, duplication of these genes coincides with the acquisition of coordinated multi-organ development. Of the class I PI3Ks, PI3Kα and PI3Kβ, encoded by PIK3CA and PIK3CB, are ubiquitously expressed. They present similar putative protein domains and share PI(4,5)P2 lipid substrate specificity. Fifteen years after publication of their first isoform-selective pharmacological inhibitors and genetically engineered mouse models (GEMMs) that mimic their complete and specific pharmacological inhibition, we review the knowledge gathered in relation to the redundant and selective roles of PI3Kα and PI3Kβ. Recent data suggest that, further to their redundancy, they cooperate for the integration of organ-specific and context-specific signal cues, to orchestrate organ development, physiology, and disease. This knowledge reinforces the importance of isoform-selective inhibitors in clinical settings.
    Keywords:  PI3K; genetically engineered mouse models; lipid signalling; pharmacological inhibitors
  40. Aging Cell. 2021 Mar 16. e13342
      One of the most fundamental challenges for all living organisms is to sense and respond to alternating nutritional conditions in order to adapt their metabolism and physiology to promote survival and achieve balanced growth. Here, we applied metabolomics and lipidomics to examine temporal regulation of metabolism during starvation in wild-type Caenorhabditis elegans and in animals lacking the transcription factor HLH-30. Our findings show for the first time that starvation alters the abundance of hundreds of metabolites and lipid species in a temporal- and HLH-30-dependent manner. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by supplementation of exogenous fatty acids, and that HLH-30 is required for complete oxidation of long-chain fatty acids. We further show that RNAi-mediated knockdown of the gene encoding carnitine palmitoyl transferase I (cpt-1) only impairs survival of wild-type animals and not of hlh-30 animals. Strikingly, we also find that compromised generation of peroxisomes by prx-5 knockdown renders hlh-30 animals hypersensitive to starvation, which cannot be rescued by supplementation of exogenous fatty acids. Collectively, our observations show that mitochondrial functions are compromised in hlh-30 animals and that hlh-30 animals rewire their metabolism to largely depend on functional peroxisomes during starvation, underlining the importance of metabolic plasticity to maintain survival.
    Keywords:  Caenorhabditis elegans; aging; lipidomics; metabolomics; mitochondria; peroxisome; starvation; β-oxidation
  41. Autophagy. 2021 Mar 09. 1-2
      VCP/p97 is an essential multifunctional protein implicated in a plethora of intracellular quality control systems, and abnormal function of VCP is the underlying cause of several neurodegenerative disorders. We reported that VCP regulates the levels of the macroautophagy/autophagy-inducing lipid phosphatidylinositol-3-phosphate (PtdIns3P) by modulating the activity of the BECN1 (beclin 1)-containing phosphatidylinositol 3-kinase (PtdIns3K) complex. VCP stimulates the deubiquitinase activity of ATXN3 (ataxin 3) to stabilize BECN1 protein levels and also interacts with and promotes the assembly and kinase activity of the PtdIns3K complex. Acute inhibition of VCP activity impairs autophagy induction, demonstrated by a diminished PtdIns3P production and decreased recruitment of early autophagy markers WIPI2 and ATG16L1. Thus, VCP promotes autophagosome biogenesis, in addition to its previously described role in autophagosome maturation.
    Keywords:  ATXN3; PI(3)P; PI3K; VCP/p97; autophagy initiation; beclin 1
  42. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Mar 12. pii: S1388-1981(21)00042-1. [Epub ahead of print]1866(6): 158916
    Keywords:  Bis(monoacylglycero)phosphate; Cholesterol; Exosomes; Extracellular vesicles; Lysobisphosphatidic acid; Niemann-Pick C disease; Phosphatidylglycerol
  43. Aging Cell. 2021 Mar 20. e13345
      We hypothesized that rapamycin (Rapa), acarbose (ACA), which both increase mouse lifespan, and 17α-estradiol, which increases lifespan in males (17aE2) all share common intracellular signaling pathways with long-lived Snell dwarf, PAPPA-KO, and Ghr-/- mice. The long-lived mutant mice exhibit reduction in mTORC1 activity, declines in cap-dependent mRNA translation, and increases in cap-independent translation (CIT). Here, we report that Rapa and ACA prevent age-related declines in CIT target proteins in both sexes, while 17aE2 has the same effect only in males, suggesting increases in CIT. mTORC1 activity showed the reciprocal pattern, with age-related increases blocked by Rapa, ACA, and 17aE2 (in males only). METTL3, required for addition of 6-methyl-adenosine to mRNA and thus a trigger for CIT, also showed an age-dependent increase blunted by Rapa, ACA, and 17aE2 (in males). Diminution of mTORC1 activity and increases in CIT-dependent proteins may represent a shared pathway for both long-lived-mutant mice and drug-induced lifespan extension in mice.
    Keywords:  17α-estradiol; acarbose; aging; protein translation; rapamycin; signal transduction
  44. Proc Natl Acad Sci U S A. 2021 Apr 05. pii: e2102057118. [Epub ahead of print]118(14):
  45. Proc Natl Acad Sci U S A. 2021 Mar 23. pii: e2016730118. [Epub ahead of print]118(12):
      Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson's disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.
    Keywords:  Parkinson’s disease; SNARE; Ykt6; calcineurin; conformation
  46. Aging (Albany NY). 2021 Mar 10. 13
      Because of the key role of impaired mitochondria in the progression of acute kidney injury (AKI), it is striking that peroxisome proliferator γ coactivator 1-α (PGC-1α), a transcriptional coactivator of genes involved in mitochondrial biogenesis and autophagy, protects from kidney injury. However, the specific mechanism involved in PGC-1α-mediated autophagy remains elusive. In vivo, along with the severe kidney damage, the expression of PGC-1α was decreased in cisplatin-induced AKI mice. Conversely, PGC-1α activator (ZLN005) administration could alleviate kidney injury. Consistently, in vitro overexpression of PGC-1α or ZLN005 treatment inhibited cell apoptosis and mitochondrial dysfunction induced by cisplatin. Moreover, ZLN005 treatment increased the expression of LC3-II and co-localization between LC3 and mitochondria, suggesting that the mitophagy was activated. Furthermore, PGC-1α-mediated the activation of mitophagy was reliant on the increased expression of TFEB, and the protective effects were abrogated in TFEB-knockdown cells. These data suggest that the activation of PGC-1α could alleviate mitochondrial dysfunction and kidney injury in AKI mice via TFEB-mediated autophagy.
    Keywords:  PGC-1α; TFEB; acute kidney injury; autophagy; mitochondrial dysfunction