bims-lysosi 2021-08-08 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–08–08
27 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Arginine-selective modulation of the lysosomal transporter PQLC2 through a gate-tuning mechanism.
S-Palmitoylation determines TMEM55B-dependent positioning of lysosomes.
Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4.
Rethinking Lysosomes and Lysosomal Disease.
Differences in MPS I and MPS II Disease Manifestations.
Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1.
Blastocyst trophectoderm endocytic activation, a marker of adverse developmental programming.
TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.
TFEB Signalling-Related MicroRNAs and Autophagy.
Endolysosomal Cation Channels and MITF in Melanocytes and Melanoma.
Branched-chain Amino Acids: Catabolism in Skeletal Muscle and Implications for Muscle and Whole-body Metabolism.
Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration.
Exendin-4 stimulates autophagy in pancreatic β-cells via the RAPGEF/EPAC-Ca2+-PPP3/calcineurin-TFEB axis.
Two-Pore Channels Regulate Expression of Various Receptors and Their Pathway-Related Proteins in Multiple Ways.
Lsm12 is an NAADP receptor and a two-pore channel regulatory protein required for calcium mobilization from acidic organelles.
Inhibition of Extracellular Cathepsin D Reduces Hepatic Lipid Accumulation and Leads to Mild Changes in Inflammationin NASH Mice.
mTOR Activity and Autophagy in Senescent Cells, a Complex Partnership.
TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins.
Gene therapy for Fabry disease: Progress, challenges, and outlooks on gene-editing.
Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization.
Mammalian Target of Rapamycin Signaling Pathway Regulates Mitochondrial Quality Control of Brown Adipocytes in Mice.
Pharmacological rescue of impaired mitophagy in Parkinson's disease-related LRRK2 G2019S knock-in mice.
The Interactome of the VAP Family of Proteins: An Overview.
Exocyst protein subnetworks integrate Hippo and mTOR signaling to promote virus detection and cancer.
The Coordination of Local Translation, Membranous Organelle Trafficking, and Synaptic Plasticity in Neurons.
Lipid regulation of NLRP3 inflammasome activity through organelle stress.
Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains.

Proc Natl Acad Sci U S A. 2021 Aug 10. pii: e2025315118. [Epub ahead of print]118(32):

Arginine-selective modulation of the lysosomal transporter PQLC2 through a gate-tuning mechanism.

Xavier Leray, Rossella Conti, Yan Li, Cécile Debacker, Florence Castelli, François Fenaille, Anselm A Zdebik, Michael Pusch, Bruno Gasnier.

  Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; "picklock two") is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge ("trans") compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter's cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners.

Keywords:  PQLC2; arginine-sensing; gating; lysosome; transporter

DOI:  https://doi.org/10.1073/pnas.2025315118
J Cell Sci. 2021 Aug 05. pii: jcs.258566. [Epub ahead of print]

S-Palmitoylation determines TMEM55B-dependent positioning of lysosomes.

Sönke Rudnik, Saskia Heybrock, Paul Saftig, Markus Damme.

  The spatio-temporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule-motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo: TMEM55B, as an integral lysosomal membrane protein, is a component of such a complex mediating the retrograde transport of lysosomes by establishing an interaction with the cytosolic scaffold protein JIP4 and dynein/dynactin. Here we show that TMEM55B and its paralog TMEM55A are S-palmitoylated proteins and lipidated at multiple cysteine-residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes the retention of the mutated protein in the Golgi-apparatus. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor-protein-dependent sorting of TMEM55B. Our data suggest a critical role of S-palmitoylation on the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

Keywords:  Acyl-RAC; Lysosomal positioning; S-palmitoylation; TMEM55A; TMEM55B

DOI:  https://doi.org/10.1242/jcs.258566
J Cell Sci. 2021 Aug 01. pii: jcs258459. [Epub ahead of print]134(15):

Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4.

Zachary N Wilson, Dalton Buysse, Matt West, Daniel Ahrens, Greg Odorizzi.

  Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion and the sequestration of ubiquitylated transmembrane protein cargos within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitylates ILV cargos. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in sorting of a subset of ILV cargos. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, a subunit of vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance, and disrupts vacuolar localization of Ypt7 and Vps41, two crucial mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates this fusion and rescues the ILV cargo-sorting defects in yeast that lack PI(3,5)P2 or Doa4 activity, our results suggest that the V-ATPase has a role in coordinating ILV cargo sorting with the membrane fusion machinery. This article has an associated First Person interview with the first author of the paper.

Keywords:  Endosomes; Intralumenal vesicles; Membrane fusion; V-ATPase

DOI:  https://doi.org/10.1242/jcs.258459
Neurosci Lett. 2021 Aug 03. pii: S0304-3940(21)00533-4. [Epub ahead of print] 136155

Rethinking Lysosomes and Lysosomal Disease.

Steven U Walkley.

  Lysosomal storage diseases were recognized and defined over a century ago as a class of disorders affecting mostly children and causing systemic disease often accompanied by major neurological consequences. Since their discovery, research focused on understanding their causes has been an important driver of our ever-expanding knowledge of cell biology and the central role that lysosomes play in cell function. Today we recognize over 50 so-called storage diseases, with most understood at the level of gene, protein and pathway involvement, but few fully clarified in terms of how the defective lysosomal function causes brain disease; even fewer have therapies that can effectively rescue brain function. Importantly, we also recognize that storage diseases are not simply a class of lysosomal disorders all by themselves, as increasingly a critical role for the greater lysosomal system with its endosomal, autophagosomal and salvage streams has also emerged in a host of neurodevelopmental and neurodegenerative diseases. Despite persistent challenges across all aspects of these complex disorders, and as reflected in this and other articles focused on lysosomal storage diseases in this special issue of Neuroscience Letters, the progress and promise to both understand and effectively treat these conditions has never been greater.

Keywords:  Autophagosome; Endosome; Lysosomal storage disorders; Lysosome; Neurodegenerative disorders; Neurodevelopmental disorders; TFEB; mTOR

DOI:  https://doi.org/10.1016/j.neulet.2021.136155
Int J Mol Sci. 2021 Jul 23. pii: 7888. [Epub ahead of print]22(15):

Differences in MPS I and MPS II Disease Manifestations.

Christiane S Hampe, Brianna D Yund, Paul J Orchard, Troy C Lund, Jacob Wesley, R Scott McIvor.

  Mucopolysaccharidosis (MPS) type I and II are two closely related lysosomal storage diseases associated with disrupted glycosaminoglycan catabolism. In MPS II, the first step of degradation of heparan sulfate (HS) and dermatan sulfate (DS) is blocked by a deficiency in the lysosomal enzyme iduronate 2-sulfatase (IDS), while, in MPS I, blockage of the second step is caused by a deficiency in iduronidase (IDUA). The subsequent accumulation of HS and DS causes lysosomal hypertrophy and an increase in the number of lysosomes in cells, and impacts cellular functions, like cell adhesion, endocytosis, intracellular trafficking of different molecules, intracellular ionic balance, and inflammation. Characteristic phenotypical manifestations of both MPS I and II include skeletal disease, reflected in short stature, inguinal and umbilical hernias, hydrocephalus, hearing loss, coarse facial features, protruded abdomen with hepatosplenomegaly, and neurological involvement with varying functional concerns. However, a few manifestations are disease-specific, including corneal clouding in MPS I, epidermal manifestations in MPS II, and differences in the severity and nature of behavioral concerns. These phenotypic differences appear to be related to different ratios between DS and HS, and their sulfation levels. MPS I is characterized by higher DS/HS levels and lower sulfation levels, while HS levels dominate over DS levels in MPS II and sulfation levels are higher. The high presence of DS in the cornea and its involvement in the arrangement of collagen fibrils potentially causes corneal clouding to be prevalent in MPS I, but not in MPS II. The differences in neurological involvement may be due to the increased HS levels in MPS II, because of the involvement of HS in neuronal development. Current treatment options for patients with MPS II are often restricted to enzyme replacement therapy (ERT). While ERT has beneficial effects on respiratory and cardiopulmonary function and extends the lifespan of the patients, it does not significantly affect CNS manifestations, probably because the enzyme cannot pass the blood-brain barrier at sufficient levels. Many experimental therapies, therefore, aim at delivery of IDS to the CNS in an attempt to prevent neurocognitive decline in the patients.

Keywords:  dermatan sulfate; glycosaminoglycans; heparin sulfate; mucopolysaccharidosis type I; mucopolysaccharidosis type II

DOI:  https://doi.org/10.3390/ijms22157888
Genes (Basel). 2021 Jul 15. pii: 1076. [Epub ahead of print]12(7):

Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1.

Annie Brown, Jiayi Zhang, Brendan Lawler, Biao Lu.

   BACKGROUND: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1).
RESULTS: CRISPR-Cas9 targeting to coding sequences of β-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA.
CONCLUSIONS: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.

Keywords:  CRISPR-Cas9; Gaucher disease; disease model; lysosomal storage disorder; β-glucocerebrosidase

DOI:  https://doi.org/10.3390/genes12071076
Reproduction. 2021 Aug 01. pii: REP-21-0234.R1. [Epub ahead of print]

Blastocyst trophectoderm endocytic activation, a marker of adverse developmental programming.

Laura Caetano, Judith Eckert, David Johnston, David Chatelet, David Tumbarello, Neil Smyth, Sue Ingamells, Anthony Price, Tom Fleming.

The mouse preimplantation embryo is sensitive to its environment including maternal dietary protein restriction which can alter the developmental programme and affect lifetime health. Previously, we have shown maternal low protein diet (LPD) causes reduction in blastocyst mTORC1 signalling coinciding with reduced availability of branched-chain amino acids (BCAAs) in surrounding uterine fluid. BCAA deficiency leads to increased endocytosis and lysosome biogenesis in blastocyst trophectoderm (TE), a response to promote compensatory histotrophic nutrition. Here, we first investigated the induction mechanism by individual variation in BCAA deficiency in an in vitro quantitative model of TE responsiveness. We found isoleucine (ILE) deficiency as the most effective activator of TE endocytosis and lysosome biogenesis, with less potent roles for other BCAAs and insulin; cell volume was also influential. TE response to low ILE included upregulation of vesicles comprising megalin receptor and cathepsin-B and the response was activated from blastocyst formation. Second, we identified the transcription factor TFEB as mediating the histotrophic response by translocation from cytoplasm to nucleus during ILE deficiency and in response to mTORC1 inhibition. Lastly, we investigated whether a similar mechanism responsive to maternal nutritional status was found in human blastocysts. Blastocysts from women with high body-mass index, but not the method of fertilisation, revealed stimulated lysosome biogenesis and TFEB nuclear migration. We propose TE lysosomal phenotype as an early biomarker of environmental nutrient stress that may associate with long-term health outcome.

DOI: https://doi.org/10.1530/REP-21-0234
Cell Stem Cell. 2021 Jul 28. pii: S1934-5909(21)00288-5. [Epub ahead of print]

TFEB-mediated endolysosomal activity controls human hematopoietic stem cell fate.

Laura García-Prat, Kerstin B Kaufmann, Florin Schneiter, Veronique Voisin, Alex Murison, Jocelyn Chen, Michelle Chan-Seng-Yue, Olga I Gan, Jessica L McLeod, Sabrina A Smith, Michelle C Shoong, Darrien Parris, Kristele Pan, Andy G X Zeng, Gabriela Krivdova, Kinam Gupta, Shin-Ichiro Takayanagi, Elvin Wagenblast, Weijia Wang, Mathieu Lupien, Timm Schroeder, Stephanie Z Xie, John E Dick.

  It is critical to understand how human quiescent long-term hematopoietic stem cells (LT-HSCs) sense demand from daily and stress-mediated cues and then transition into bioenergetically active progeny to differentiate and meet these cellular needs. However, the demand-adapted regulatory circuits of these early steps of hematopoiesis are largely unknown. Here we show that lysosomes, sophisticated nutrient-sensing and signaling centers, are regulated dichotomously by transcription factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSCs and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, promoting quiescence and self-renewal, and governing erythroid-myeloid commitment. In contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism, driving LT-HSC activation. Our study identifies TFEB-mediated control of lysosomal activity as a central regulatory hub for proper and coordinated stem cell fate determination.

Keywords:  MYC; TFEB; TfR1; anabolism; endocytosis; erythropoiesis; long-term HSC; lysosomes; myelopoiesis; self-renewal

DOI:  https://doi.org/10.1016/j.stem.2021.07.003
Biomolecules. 2021 Jul 04. pii: 985. [Epub ahead of print]11(7):

TFEB Signalling-Related MicroRNAs and Autophagy.

Davide Corà, Federico Bussolino, Gabriella Doronzo.

  The oncogenic Transcription Factor EB (TFEB), a member of MITF-TFE family, is known to be the most important regulator of the transcription of genes responsible for the control of lysosomal biogenesis and functions, autophagy, and vesicles flux. TFEB activation occurs in response to stress factors such as nutrient and growth factor deficiency, hypoxia, lysosomal stress, and mitochondrial damage. To reach the final functional status, TFEB is regulated in multimodal ways, including transcriptional rate, post-transcriptional regulation, and post-translational modifications. Post-transcriptional regulation is in part mediated by miRNAs. miRNAs have been linked to many cellular processes involved both in physiology and pathology, such as cell migration, proliferation, differentiation, and apoptosis. miRNAs also play a significant role in autophagy, which exerts a crucial role in cell behaviour during stress or survival responses. In particular, several miRNAs directly recognise TFEB transcript or indirectly regulate its function by targeting accessory molecules or enzymes involved in its post-translational modifications. Moreover, the transcriptional programs triggered by TFEB may be influenced by the miRNA-mediated regulation of TFEB targets. Finally, recent important studies indicate that the transcription of many miRNAs is regulated by TFEB itself. In this review, we describe the interplay between miRNAs with TFEB and focus on how these types of crosstalk affect TFEB activation and cellular functions.

Keywords:  TFEB; autophagy; miRNA

DOI:  https://doi.org/10.3390/biom11070985
Biomolecules. 2021 Jul 13. pii: 1021. [Epub ahead of print]11(7):

Endolysosomal Cation Channels and MITF in Melanocytes and Melanoma.

Carla Abrahamian, Christian Grimm.

  Microphthalmia-associated transcription factor (MITF) is the principal transcription factor regulating pivotal processes in melanoma cell development, growth, survival, proliferation, differentiation and invasion. In recent years, convincing evidence has been provided attesting key roles of endolysosomal cation channels, specifically TPCs and TRPMLs, in cancer, including breast cancer, glioblastoma, bladder cancer, hepatocellular carcinoma and melanoma. In this review, we provide a gene expression profile of these channels in different types of cancers and decipher their roles, in particular the roles of two-pore channel 2 (TPC2) and TRPML1 in melanocytes and melanoma. We specifically discuss the signaling cascades regulating MITF and the relationship between endolysosomal cation channels, MAPK, canonical Wnt/GSK3 pathways and MITF.

Keywords:  MCOLN; MITF; TFEB; TPC; TPC1; TPC2; TRPML; TRPML1; calcium; lysosome; mTOR; melanocytes; melanoma; mucolipin; two-pore

DOI:  https://doi.org/10.3390/biom11071021
Front Physiol. 2021 ;12 702826

Branched-chain Amino Acids: Catabolism in Skeletal Muscle and Implications for Muscle and Whole-body Metabolism.

Gagandeep Mann, Stephen Mora, Glory Madu, Olasunkanmi A J Adegoke.

  Branched-chain amino acids (BCAAs) are critical for skeletal muscle and whole-body anabolism and energy homeostasis. They also serve as signaling molecules, for example, being able to activate mammalian/mechanistic target of rapamycin complex 1 (mTORC1). This has implication for macronutrient metabolism. However, elevated circulating levels of BCAAs and of their ketoacids as well as impaired catabolism of these amino acids (AAs) are implicated in the development of insulin resistance and its sequelae, including type 2 diabetes, cardiovascular disease, and of some cancers, although other studies indicate supplements of these AAs may help in the management of some chronic diseases. Here, we first reviewed the catabolism of these AAs especially in skeletal muscle as this tissue contributes the most to whole body disposal of the BCAA. We then reviewed emerging mechanisms of control of enzymes involved in regulating BCAA catabolism. Such mechanisms include regulation of their abundance by microRNA and by post translational modifications such as phosphorylation, acetylation, and ubiquitination. We also reviewed implications of impaired metabolism of BCAA for muscle and whole-body metabolism. We comment on outstanding questions in the regulation of catabolism of these AAs, including regulation of the abundance and post-transcriptional/post-translational modification of enzymes that regulate BCAA catabolism, as well the impact of circadian rhythm, age and mTORC1 on these enzymes. Answers to such questions may facilitate emergence of treatment/management options that can help patients suffering from chronic diseases linked to impaired metabolism of the BCAAs.

Keywords:  branched-chain amino acids; catabolism; mTORC1; protein synthesis; skeletal muscle

DOI:  https://doi.org/10.3389/fphys.2021.702826
Mol Neurodegener. 2021 Aug 03. 16(1): 51

Processing of progranulin into granulins involves multiple lysosomal proteases and is affected in frontotemporal lobar degeneration.

Swetha Mohan, Paul J Sampognaro, Andrea R Argouarch, Jason C Maynard, Mackenzie Welch, Anand Patwardhan, Emma C Courtney, Jiasheng Zhang, Amanda Mason, Kathy H Li, Eric J Huang, William W Seeley, Bruce L Miller, Alma Burlingame, Mathew P Jacobson, Aimee W Kao.

   BACKGROUND: Progranulin loss-of-function mutations are linked to frontotemporal lobar degeneration with TDP-43 positive inclusions (FTLD-TDP-Pgrn). Progranulin (PGRN) is an intracellular and secreted pro-protein that is proteolytically cleaved into individual granulin peptides, which are increasingly thought to contribute to FTLD-TDP-Pgrn disease pathophysiology. Intracellular PGRN is processed into granulins in the endo-lysosomal compartments. Therefore, to better understand the conversion of intracellular PGRN into granulins, we systematically tested the ability of different classes of endo-lysosomal proteases to process PGRN at a range of pH setpoints.
RESULTS: In vitro cleavage assays identified multiple enzymes that can process human PGRN into multi- and single-granulin fragments in a pH-dependent manner. We confirmed the role of cathepsin B and cathepsin L in PGRN processing and showed that these and several previously unidentified lysosomal proteases (cathepsins E, G, K, S and V) are able to process PGRN in distinctive, pH-dependent manners. In addition, we have demonstrated a new role for asparagine endopeptidase (AEP) in processing PGRN, with AEP having the unique ability to liberate granulin F from the pro-protein. Brain tissue from individuals with FTLD-TDP-Pgrn showed increased PGRN processing to granulin F and increased AEP activity in degenerating brain regions but not in regions unaffected by disease.
CONCLUSIONS: This study demonstrates that multiple lysosomal proteases may work in concert to liberate multi-granulin fragments and granulins. It also implicates both AEP and granulin F in the neurobiology of FTLD-TDP-Pgrn. Modulating progranulin cleavage and granulin production may represent therapeutic strategies for FTLD-Pgrn and other progranulin-related diseases.

Keywords:  Asparagine endopeptidase; Frontotemporal lobar degeneration; Granulin; Lysosome; Progranulin; Protease; pH

DOI:  https://doi.org/10.1186/s13024-021-00472-1
Autophagy. 2021 Aug 02. 1-17

Exendin-4 stimulates autophagy in pancreatic β-cells via the RAPGEF/EPAC-Ca2+-PPP3/calcineurin-TFEB axis.

Francesco P Zummo, Stanislaus I Krishnanda, Merilin Georgiou, Finbarr Pm O'Harte, Vadivel Parthsarathy, Kirsty S Cullen, Minna Honkanen-Scott, James Am Shaw, Penny E Lovat, Catherine Arden.

  Macroautophagy/autophagy is critical for the regulation of pancreatic β-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic β-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux.Pancreatic β-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic β-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D.Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5' AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB.

Keywords:  Autophagy; GLP1R agonists; PPP3/calcineurin; RAPGEF4; TFEB; diabetes; pancreatic β-cell

DOI:  https://doi.org/10.1080/15548627.2021.1956123
Cells. 2021 Jul 16. pii: 1807. [Epub ahead of print]10(7):

Two-Pore Channels Regulate Expression of Various Receptors and Their Pathway-Related Proteins in Multiple Ways.

Sonja Grossmann, Robert Theodor Mallmann, Norbert Klugbauer.

  Two-pore channels (TPCs) constitute a small family of ion channels within membranes of intracellular acidic compartments, such as endosomes and lysosomes. They were shown to provide transient and locally restricted Ca2+-currents, likely responsible for fusion and/or fission events of endolysosomal membranes and thereby for intracellular vesicle trafficking. Genetic deletion of TPCs not only affects endocytosis, recycling, and degradation of various surface receptors but also uptake and impact of bacterial protein toxins and entry and intracellular processing of some types of viruses. This review points to important examples of these trafficking defects on one part but mainly focuses on the resulting impact of the TPC inactivation on receptor expression and receptor signaling. Thus, a detailed RNA sequencing analysis using TPC1-deficient fibroblasts uncovered a multitude of changes in the expression levels of surface receptors and their pathway-related signaling proteins. We refer to several classes of receptors such as EGF, TGF, and insulin as well as proteins involved in endocytosis.

Keywords:  RNA sequence analysis; endolysosomal system; intracellular trafficking; receptor endocytosis; two-pore channel

DOI:  https://doi.org/10.3390/cells10071807
Nat Commun. 2021 Aug 06. 12(1): 4739

Lsm12 is an NAADP receptor and a two-pore channel regulatory protein required for calcium mobilization from acidic organelles.

Jiyuan Zhang, Xin Guan, Kunal Shah, Jiusheng Yan.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. However, the molecular identity of the NAADP receptor remains unknown. Given the necessity of the endolysosomal two-pore channel (TPC1 or TPC2) in NAADP signaling, we performed affinity purification and quantitative proteomic analysis of the interacting proteins of NAADP and TPCs. We identified a Sm-like protein Lsm12 complexed with NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling.

DOI: https://doi.org/10.1038/s41467-021-24735-z
Front Immunol. 2021 ;12 675535

Inhibition of Extracellular Cathepsin D Reduces Hepatic Lipid Accumulation and Leads to Mild Changes in Inflammationin NASH Mice.

Tulasi Yadati, Tom Houben, Albert Bitorina, Yvonne Oligschlaeger, Marion J Gijbels, Ronny Mohren, Dieter Lütjohann, Princy Khurana, Sandeep Goyal, Aditya Kulkarni, Jan Theys, Berta Cillero-Pastor, Ronit Shiri-Sverdlov.

   Background & Aims: The lysosomal enzyme, cathepsin D (CTSD) has been implicated in the pathogenesis of non-alcoholic steatohepatitis (NASH), a disease characterised by hepatic steatosis and inflammation. We have previously demonstrated that specific inhibition of the extracellular CTSD leads to improved metabolic features in Sprague-Dawley rats with steatosis. However, the individual roles of extracellular and intracellular CTSD in NASH are not yet known. In the current study, we evaluated the underlying mechanisms of extracellular and intracellular CTSD fractions in NASH-related metabolic inflammation using specific small-molecule inhibitors.
Methods: Low-density lipoprotein receptor knock out (Ldlr-/-) mice were fed a high-fat, high cholesterol (HFC) diet for ten weeks to induce NASH. Further, to investigate the effects of CTSD inhibition, mice were injected either with an intracellular (GA-12) or extracellular (CTD-002) CTSD inhibitor or vehicle control at doses of 50 mg/kg body weight subcutaneously once in two days for ten weeks.
Results: Ldlr-/- mice treated with extracellular CTSD inhibitor showed reduced hepatic lipid accumulation and an associated increase in faecal bile acid levels as compared to intracellular CTSD inhibitor-treated mice. Furthermore, in contrast to intracellular CTSD inhibition, extracellular CTSD inhibition switched the systemic immune status of the mice to an anti-inflammatory profile. In line, label-free mass spectrometry-based proteomics revealed that extra- and intracellular CTSD fractions modulate proteins belonging to distinct metabolic pathways.
Conclusion: We have provided clinically translatable evidence that extracellular CTSD inhibition shows some beneficial metabolic and systemic inflammatory effects which are distinct from intracellular CTSD inhibition. Considering that intracellular CTSD inhibition is involved in essential physiological processes, specific inhibitors capable of blocking extracellular CTSD activity, can be promising and safe NASH drugs.

Keywords:  NASH; extracellular cathepsin D; inflammation; lipoprotein metabolism; lysosomal enzymes; small-compound inhibitors

DOI:  https://doi.org/10.3389/fimmu.2021.675535
Int J Mol Sci. 2021 Jul 29. pii: 8149. [Epub ahead of print]22(15):

mTOR Activity and Autophagy in Senescent Cells, a Complex Partnership.

Angel Cayo, Raúl Segovia, Whitney Venturini, Rodrigo Moore-Carrasco, Claudio Valenzuela, Nelson Brown.

  Cellular senescence is a form of proliferative arrest triggered in response to a wide variety of stimuli and characterized by unique changes in cell morphology and function. Although unable to divide, senescent cells remain metabolically active and acquire the ability to produce and secrete bioactive molecules, some of which have recognized pro-inflammatory and/or pro-tumorigenic actions. As expected, this "senescence-associated secretory phenotype (SASP)" accounts for most of the non-cell-autonomous effects of senescent cells, which can be beneficial or detrimental for tissue homeostasis, depending on the context. It is now evident that many features linked to cellular senescence, including the SASP, reflect complex changes in the activities of mTOR and other metabolic pathways. Indeed, the available evidence indicates that mTOR-dependent signaling is required for the maintenance or implementation of different aspects of cellular senescence. Thus, depending on the cell type and biological context, inhibiting mTOR in cells undergoing senescence can reverse senescence, induce quiescence or cell death, or exacerbate some features of senescent cells while inhibiting others. Interestingly, autophagy-a highly regulated catabolic process-is also commonly upregulated in senescent cells. As mTOR activation leads to repression of autophagy in non-senescent cells (mTOR as an upstream regulator of autophagy), the upregulation of autophagy observed in senescent cells must take place in an mTOR-independent manner. Notably, there is evidence that autophagy provides free amino acids that feed the mTOR complex 1 (mTORC1), which in turn is required to initiate the synthesis of SASP components. Therefore, mTOR activation can follow the induction of autophagy in senescent cells (mTOR as a downstream effector of autophagy). These functional connections suggest the existence of autophagy regulatory pathways in senescent cells that differ from those activated in non-senescence contexts. We envision that untangling these functional connections will be key for the generation of combinatorial anti-cancer therapies involving pro-senescence drugs, mTOR inhibitors, and/or autophagy inhibitors.

Keywords:  autophagy; mTOR; senescence

DOI:  https://doi.org/10.3390/ijms22158149
Proteins. 2021 Aug 04.

TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins.

Tim P Levine.

  TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its over-expression being associated with familial frontotemporal lobar degeneration, and point mutation linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a non-standard way to show that the lumenal portion of TMEM106B is a member of the LEA-2 domain superfamily. More sensitive tools (HMMER, HHpred and trRosetta) extended this to predict LEA-2 domains in two yeast proteins. One is Vac7, a regulator of PI(3,5)P2 production in the degradative vacuole, equivalent to the lysosome, which has a LEA-2 domain in its lumenal domain. The other is Tag1, another vacuolar protein, which signals to terminate autophagy and has three LEA-2 domains in its lumenal domain. Further analysis of LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid binding groove. This implies that TMEM106B, Vac7 and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles. This article is protected by copyright. All rights reserved.

Keywords:  Endosome; LEA_2; Lipid transfer protein; Lysosome; Structural bioinformatics; TMEM106B; Tag1; Vac7; Vacuole; YLR173W

DOI:  https://doi.org/10.1002/prot.26201
Mol Genet Metab. 2021 Jul 21. pii: S1096-7192(21)00757-5. [Epub ahead of print]

Gene therapy for Fabry disease: Progress, challenges, and outlooks on gene-editing.

Jakob M Domm, Sarah K Wootton, Jeffrey A Medin, Michael L West.

  Gene therapy is the delivery of a therapeutic gene for endogenous cellular expression with the goal of rescuing a disease phenotype. It has been used to treat an increasing number of human diseases with many strategies proving safe and efficacious in clinical trials. Gene delivery may be viral or non-viral, performed in vivo or ex vivo, and relies on gene integration or transient expression; all of these techniques have been applied to the treatment of Fabry disease. Fabry disease is a genetic disorder of the α-galactosidase A gene, GLA, that causes an accumulation of glycosphingolipids in cells leading to cardiac, renal and cerebrovascular damage and eventually death. Currently, there are no curative treatments available, and the therapies that are used have significant drawbacks. These treatment concerns have led to the advent of gene therapies for Fabry disease. The first Fabry patients to receive gene therapy were treated with recombinant lentivirus targeting their hematopoietic stem/progenitor cells. Adeno-associated virus treatments have also begun. Alternatively, the field of gene-editing is a new and rapidly growing field. Gene-editing has been used to repair disease-causing mutations or insert genes into cellular DNA. These techniques have the potential to be applied to the treatment of Fabry disease provided the concerns of gene-editing technology, such as safety and efficiency, were addressed. This review focuses on the current state of gene therapy as it is being developed for Fabry disease, including progresses and challenges as well as an overview of gene-editing and how it may be applied to correct Fabry disease-causing mutations in the future.

Keywords:  Fabry disease; Gene editing; Gene therapy; Lysosomal storage disease

DOI:  https://doi.org/10.1016/j.ymgme.2021.07.006
Chem Sci. 2021 Jul 21. 12(28): 9630-9644

Strategic engineering of alkyl spacer length for a pH-tolerant lysosome marker and dual organelle localization.

Suprakash Biswas, Tanoy Dutta, Akshay Silswal, Rohit Bhowal, Deepak Chopra, Apurba L Koner.

Long-term visualization of lysosomal properties is extremely crucial to evaluate diseases related to their dysfunction. However, many of the reported lysotrackers are less conducive to imaging lysosomes precisely because they suffer from fluorescence quenching and other inherent drawbacks such as pH-sensitivity, polarity insensitivity, water insolubility, slow diffusibility, and poor photostability. To overcome these limitations, we have utilized an alkyl chain length engineering strategy and synthesized a series of lysosome targeting fluorescent derivatives namely NIMCs by attaching a morpholine moiety at the peri position of the 1,8-naphthalimide (NI) ring through varying alkyl spacers between morpholine and 1,8-naphthalimide. The structural and optical properties of the synthesized NIMCs were explored by 1H-NMR, single-crystal X-ray diffraction, UV-Vis, and fluorescence spectroscopy. Afterward, optical spectroscopic measurements were carefully performed to identify a pH-tolerant, polarity sensitive, and highly photostable fluoroprobes for further live-cell imaging applications. NIMC6 displayed excellent pH-tolerant and polarity-sensitive properties. Consequently, all NIMCs were employed in kidney fibroblast cells (BHK-21) to investigate their applicability for lysosome targeting and probing lysosomal micropolarity. Interestingly, a switching of localization from lysosomes to the endoplasmic reticulum (ER) was also achieved by controlling the linker length and this phenomenon was subsequently applied in determining ER micropolarity. Additionally, the selected probe NIMC6 was also employed in BHK-21 cells for 3-D spheroid imaging and in Caenorhabditis elegans (C. elegans) for in vivo imaging, to evaluate its efficacy for imaging animal models.

DOI: https://doi.org/10.1039/d1sc00542a
Front Physiol. 2021 ;12 638352

Mammalian Target of Rapamycin Signaling Pathway Regulates Mitochondrial Quality Control of Brown Adipocytes in Mice.

Bahetiyaer Huwatibieke, Wenzhen Yin, Lingchao Liu, Yuxin Jin, Xinxin Xiang, Jingyan Han, Weizhen Zhang, Yin Li.

  The mammalian target of rapamycin (mTOR) is an important protein kinase that senses changes in extracellular and intracellular energy levels and plays a key role in regulating energy metabolism. Brown adipose tissue, which can be converted to white adipose tissue, contains a large number of mitochondria and regulates energy expenditure through thermogenesis. Because obesity is a process of fat accumulation due to chronic excessive energy intake, we attempted to determine whether the mTOR signaling pathway can affect the mitochondrial quality control of brown adipocytes through sensing energy status, thereby regulating brown/white adipocyte transformation. In the present study, through activation or inhibition of mTOR signaling, we detected mitochondrial biogenesis, dynamics, and autophagy-related markers in brown adipocytes. We found that activation of mTOR signaling downregulated the expression of mitochondrial biogenesis, dynamics, and autophagy-relevant markers and inhibited the mitochondrial quality control of brown adipocytes, indicating a phenotypic transformation of brown to white adipocytes. In contrast, inhibition of mTOR signaling upregulated the expression of mitochondrial biogenesis, dynamics, and mitophagy-relevant markers and strengthened mitochondrial quality control, suggesting an inhibition of the phenotypic transformation of brown to white adipocytes. In conclusion, the mTOR signaling pathway plays an important role in modulating the transformation of adipocytes by regulating mitochondrial quality control.

Keywords:  brown adipose tissue; mammalian target of rapamycin; mitochondria; obesity; white adipose tissue

DOI:  https://doi.org/10.3389/fphys.2021.638352
Elife. 2021 Aug 03. pii: e67604. [Epub ahead of print]10

Pharmacological rescue of impaired mitophagy in Parkinson's disease-related LRRK2 G2019S knock-in mice.

Francois Singh, Alan R Prescott, Philippa Rosewell, Graeme Ball, Alastair D Reith, Ian G Ganley.

  Parkinson's disease (PD) is a major and progressive neurodegenerative disorder, yet the biological mechanisms involved in its aetiology are poorly understood. Evidence links this disorder with mitochondrial dysfunction and/or impaired lysosomal degradation - key features of the autophagy of mitochondria, known as mitophagy. Here, we investigated the role of LRRK2, a protein kinase frequently mutated in PD, in this process in vivo. Using mitophagy and autophagy reporter mice, bearing either knockout of LRRK2 or expressing the pathogenic kinase-activating G2019S LRRK2 mutation, we found that basal mitophagy was specifically altered in clinically relevant cells and tissues. Our data show that basal mitophagy inversely correlates with LRRK2 kinase activity in vivo. In support of this, use of distinct LRRK2 kinase inhibitors in cells increased basal mitophagy, and a CNS penetrant LRRK2 kinase inhibitor, GSK3357679A, rescued the mitophagy defects observed in LRRK2 G2019S mice. This study provides the first in vivo evidence that pathogenic LRRK2 directly impairs basal mitophagy, a process with strong links to idiopathic Parkinson's disease, and demonstrates that pharmacological inhibition of LRRK2 is a rational mitophagy-rescue approach and potential PD therapy.

Keywords:  LRRK2; Mitophagy; cell biology; kinase inhibitor; mito-QC; mouse; neuroscience; parkinson's disease

DOI:  https://doi.org/10.7554/eLife.67604
Cells. 2021 Jul 14. pii: 1780. [Epub ahead of print]10(7):

The Interactome of the VAP Family of Proteins: An Overview.

Christina James, Ralph H Kehlenbach.

  Membrane contact sites (MCS) are sites of close apposition of two organelles that help in lipid transport and synthesis, calcium homeostasis and several other biological processes. The VAMP-associated proteins (VAPs) VAPA, VAPB, MOSPD2 and the recently described MOSPD1 and MOSPD3 are tether proteins of MCSs that are mainly found at the endoplasmic reticulum (ER). VAPs interact with various proteins with a motif called FFAT (two phenylalanines in an acidic tract), recruiting the associated organelle to the ER. In addition to the conventional FFAT motif, the recently described FFNT (two phenylalanines in a neutral tract) and phospho-FFAT motifs contribute to the interaction with VAPs. In this review, we summarize and compare the recent interactome studies described for VAPs, including in silico and proximity labeling methods. Collectively, the interaction repertoire of VAPs is very diverse and highlights the complexity of interactions mediated by the different FFAT motifs to the VAPs.

Keywords:  FFAT; MOSPD1; MOSPD2; MOSPD3; VAPA; VAPB; interactome

DOI:  https://doi.org/10.3390/cells10071780
Cell Rep. 2021 Aug 03. pii: S2211-1247(21)00918-9. [Epub ahead of print]36(5): 109491

Exocyst protein subnetworks integrate Hippo and mTOR signaling to promote virus detection and cancer.

Aubhishek Zaman, Xiaofeng Wu, Andrew Lemoff, Sivaramakrishna Yadavalli, Jeon Lee, Chensu Wang, Jonathan Cooper, Elizabeth A McMillan, Charles Yeaman, Hamid Mirzaei, Michael A White, Trever G Bivona.

  The exocyst is an evolutionarily conserved protein complex that regulates vesicular trafficking and scaffolds signal transduction. Key upstream components of the exocyst include monomeric RAL GTPases, which help mount cell-autonomous responses to trophic and immunogenic signals. Here, we present a quantitative proteomics-based characterization of dynamic and signal-dependent exocyst protein interactomes. Under viral infection, an Exo84 exocyst subcomplex assembles the immune kinase Protein Kinase R (PKR) together with the Hippo kinase Macrophage Stimulating 1 (MST1). PKR phosphorylates MST1 to activate Hippo signaling and inactivate Yes Associated Protein 1 (YAP1). By contrast, a Sec5 exocyst subcomplex recruits another immune kinase, TANK binding kinase 1 (TBK1), which interacted with and activated mammalian target of rapamycin (mTOR). RALB was necessary and sufficient for induction of Hippo and mTOR signaling through parallel exocyst subcomplex engagement, supporting the cellular response to virus infection and oncogenic signaling. This study highlights RALB-exocyst signaling subcomplexes as mechanisms for the integrated engagement of Hippo and mTOR signaling in cells challenged by viral pathogens or oncogenic signaling.

Keywords:  Hippo pathway; PKR; RAL; TBK1; autophagy; cancer cell survival; exocyst; innate immune signaling; mTOR; virus

DOI:  https://doi.org/10.1016/j.celrep.2021.109491
Front Cell Dev Biol. 2021 ;9 711446

The Coordination of Local Translation, Membranous Organelle Trafficking, and Synaptic Plasticity in Neurons.

Dipen Rajgor, Theresa M Welle, Katharine R Smith.

  Neurons are highly complex polarized cells, displaying an extraordinary degree of spatial compartmentalization. At presynaptic and postsynaptic sites, far from the cell body, local protein synthesis is utilized to continually modify the synaptic proteome, enabling rapid changes in protein production to support synaptic function. Synapses undergo diverse forms of plasticity, resulting in long-term, persistent changes in synapse strength, which are paramount for learning, memory, and cognition. It is now well-established that local translation of numerous synaptic proteins is essential for many forms of synaptic plasticity, and much work has gone into deciphering the strategies that neurons use to regulate activity-dependent protein synthesis. Recent studies have pointed to a coordination of the local mRNA translation required for synaptic plasticity and the trafficking of membranous organelles in neurons. This includes the co-trafficking of RNAs to their site of action using endosome/lysosome "transports," the regulation of activity-dependent translation at synapses, and the role of mitochondria in fueling synaptic translation. Here, we review our current understanding of these mechanisms that impact local translation during synaptic plasticity, providing an overview of these novel and nuanced regulatory processes involving membranous organelles in neurons.

Keywords:  LTD; LTP; endosomes; local translation; lysosomes; mitochondria; neurotransmitter receptors; synaptic plasticity

DOI:  https://doi.org/10.3389/fcell.2021.711446
Trends Immunol. 2021 Jul 29. pii: S1471-4906(21)00140-X. [Epub ahead of print]

Lipid regulation of NLRP3 inflammasome activity through organelle stress.

Jonathan J Liang, Iain D C Fraser, Clare E Bryant.

  Inflammation driven by the NLRP3 inflammasome in macrophages is an important contributor to chronic metabolic diseases that affect growing numbers of individuals. Many of these diseases involve the pathologic accumulation of endogenous lipids or their oxidation products, which can activate NLRP3. Other endogenous lipids, however, can inhibit the activation of NLRP3. The intracellular mechanisms by which these lipids modulate NLRP3 activity are now being identified. This review discusses emerging evidence suggesting that organelle stress, particularly involving mitochondria, lysosomes, and the endoplasmic reticulum, may be key in lipid-induced modification of NLRP3 inflammasome activity.

Keywords:  NLRP3; inflammasome; lipids; macrophages

DOI:  https://doi.org/10.1016/j.it.2021.07.005
Int J Mol Sci. 2021 Jul 29. pii: 8144. [Epub ahead of print]22(15):

Lipid Droplets and Their Autophagic Turnover via the Raft-Like Vacuolar Microdomains.

Muhammad Arifur Rahman, Ravinder Kumar, Enrique Sanchez, Taras Y Nazarko.

  Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs' basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.

Keywords:  autophagy; lipid droplets; lipid rafts; lipophagy; microautophagy; microlipophagy; organelle homeostasis; vacuolar microdomains; vacuole; yeast

DOI:  https://doi.org/10.3390/ijms22158144