bims-lypmec 2022-11-13 papers

bims-lypmec

Biomed News

on Lysosomal positioning and metabolism in cardiomyocytes

Issue of 2022‒11‒13
eight papers selected by
Satoru Kobayashi
New York Institute of Technology

How cells recognize and remove the perforated lysosome.
Lysosomal dysfunction, autophagic defects, and CLN5 accumulation underlie the pathogenesis of KCTD7-mutated neuronal ceroid lipofuscinoses.
The interdependent transport of yeast vacuole Ca2+ and H+ and the role of Phosphatidylinositol 3,5-bisphosphate.
--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.
Regulation of exosome release by lysosomal acid ceramidase in coronary arterial endothelial cells: Role of TRPML1 channel.
Spatiotemporal Control of Vascular CaV1.2 by α1C S1928 Phosphorylation.
An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.
Nanomaterials Respond to Lysosomal Function for Tumor Treatment.

Autophagy. 2022 Nov 11. 1-3

How cells recognize and remove the perforated lysosome.

Keisuke Tabata, Marika Saeki, Tamotsu Yoshimori, Maho Hamasaki.

  Macroautophagy (hereafter autophagy) is a highly conserved intracellular degradation system to maintain cellular homeostasis by degrading cellular components such as misfolded proteins, nonfunctional organelles, pathogens, and cytosol. Conversely, selective autophagy targets and degrades specific cargo, such as organelles, bacteria, etc. We previously reported that damaged lysosomes are autophagy targets, via a process called lysophagy. However, how cells target damaged lysosomes through autophagy is not known. We performed proteomics analysis followed by siRNA screening to identify genes involved in targeting damaged lysosomes and identified a new E3 ligase complex, involving CUL4A (cullin 4A), as a regulatory complex in lysophagy. We also found that this complex mediates K48-linked poly-ubiquitination on lysosome protein LAMP2 during lysosomal damage; particularly, the lumenal side of LAMP2 is important to recruit the complex to damaged lysosomes. This protein modification is thus critical to initiate the clearance of damaged lysosomes.

Keywords:  CUL4A; LAMP2; lysophagy; lysosomal membrane damage; selective autophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2022.2138686
Autophagy. 2022 Nov 11. 1-3

Lysosomal dysfunction, autophagic defects, and CLN5 accumulation underlie the pathogenesis of KCTD7-mutated neuronal ceroid lipofuscinoses.

Yalan Wang, Hongyan Wang, Chenji Wang.

  Lysosomes are essential catabolic organelles responsible for the degradation of biomacromolecules into low-molecular-weight materials for subsequent reuse. Neuronal ceroid lipofuscinoses (NCLs) are a group of fatal neurodegenerative lysosomal storage disorders characterized by the intracellular accumulation of lipoprotein aggregates (called ceroid lipofuscin) in neurons and other tissues. Mutations in KCTD7, which encodes a substrate-binding adaptor for the CUL3-RING E3 (CRL3) ubiquitin ligase complex, are categorized as a unique NCL subtype. However, the molecular mechanisms underlying the KCTD7-mutated NCLs remain unclear. In our recent study, we showed that KCTD7 deficiency leads to the accumulation of lysosomal storage deposits owing to lysosomal dysfunction and macroautophagic/autophagic defects. We identified CLN5 as an authentic substrate of CRL3-KCTD7 E3s. Wild-type KCTD7 targets CLN5 for ubiquitination and proteasomal degradation, whereas NCL patient-derived KCTD7 mutations disrupt the interaction between KCTD7-CUL3 or KCTD7-CLN5 and ultimately lead to excessive CLN5 accumulation in the endoplasmic reticulum. Accumulated CLN5 disrupts the interaction between CLN6-CLN8 and lysosomal enzymes, leading to impaired ER-to-Golgi trafficking of lysosomal enzymes. Thus, our findings indicate that KCTD7 is a key player in maintaining lysosomal and autophagic homeostasis and demonstrate that KCTD7 and CLN5, two NCL causative genes, are biochemically linked and function in a common neurodegenerative pathway.

Keywords:  Autophagy; lipofuscinoses; lysosome; neurodegeneration; trafficking; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2022.2140882
J Biol Chem. 2022 Nov 02. pii: S0021-9258(22)01115-2. [Epub ahead of print] 102672

The interdependent transport of yeast vacuole Ca2+ and H+ and the role of Phosphatidylinositol 3,5-bisphosphate.

Chi Zhang, Yilin Feng, Adam Balutowski, Gregory E Miner, David A Rivera-Kohr, Michael R Hrabak, Katherine D Sullivan, Annie Guo, Jorge D Calderin, Rutilio A Fratti.

  Yeast vacuoles are acidified by the v-type H+-ATPase (V-ATPase) that is comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme on the vacuole is stabilized in part through interactions between the VO a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). PI(3,5)P2 also affects vacuolar Ca2+ release through the channel Yvc1, and uptake through the Ca2+ pump Pmc1. Here we asked if H+ and Ca2+ transport activities were connected through PI(3,5)P2. We found that overproduction of PI(3,5)P2 by the hyperactive fab1T2250A mutant augmented vacuole acidification, whereas the kinase-inactive fab1EEE mutant attenuated the formation of a H+ gradient. Separately, we tested the effects of excess Ca2+ on vacuole acidification. Adding micromolar Ca2+ blocked vacuole acidification, whereas chelating Ca2+ accelerated acidification. The effect of adding Ca2+ on acidification was eliminated when the Ca2+/H+ antiporter Vcx1 was absent, indicating that the vacuolar H+ gradient can collapse during Ca2+ stress through Vcx1 activity. This, however was independent of PI(3,5)P2, suggesting that PI(3,5)P2 plays a role in sub-micromolar Ca2+ flux, but not under Ca2+ shock. To see if the link between Ca2+ and H+ transport was bidirectional we examined Ca2+ transport when vacuole acidification was inhibited. We found that Ca2+ transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca2+ transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P2.

Keywords:  Calcium ATPase; Calcium Transport; Fab1; Lysosome; PIKfyve; Phosphoinositide; Pmc1; Stv1; V-ATPase; Vcx1; Vph1; organellar pH homeostasis

DOI:  https://doi.org/10.1016/j.jbc.2022.102672
Autophagy. 2022 Nov 10. 1-20

--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.

Sabrina Chumpen Ramirez, Rubén Gómez-Sánchez, Pauline Verlhac, Ralph Hardenberg, Eleonora Margheritis, Katia Cosentino, Fulvio Reggiori, Christian Ungermann.

  During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9's role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.

Keywords:  Autophagosome; lipid transfer; membrane contact site; phagophore; scramblase

DOI:  https://doi.org/10.1080/15548627.2022.2136340
Curr Top Membr. 2022 ;pii: S1063-5823(22)00011-4. [Epub ahead of print]90 37-63

Regulation of exosome release by lysosomal acid ceramidase in coronary arterial endothelial cells: Role of TRPML1 channel.

Guangbi Li, Dandan Huang, Pengyang Li, Xinxu Yuan, Viktor Yarotskyy, Pin-Lan Li.

  Lysosomal acid ceramidase (AC) has been reported to determine multivesicular body (MVB) fate and exosome secretion in different mammalian cells including coronary arterial endothelial cells (CAECs). However, this AC-mediated regulation of exosome release from CAECs and associated underlying mechanism remain poorly understood. In the present study, we hypothesized that AC controls lysosomal Ca2+ release through TRPML1 channel to regulate exosome release in murine CAECs. To test this hypothesis, we isolated and cultured CAECs from WT/WT and endothelial cell-specific Asah1 gene (gene encoding AC) knockout mice. Using these CAECs, we first demonstrated a remarkable increase in exosome secretion and significant reduction of lysosome-MVB interaction in CAECs lacking Asah1 gene compared to those cells from WT/WT mice. ML-SA1, a TRPML1 channel agonist, was found to enhance lysosome trafficking and increase lysosome-MVB interaction in WT/WT CAECs, but not in CAECs lacking Asah1 gene. However, sphingosine, an AC-derived sphingolipid, was able to increase lysosome movement and lysosome-MVB interaction in CAECs lacking Asah1 gene, leading to reduced exosome release from these cells. Moreover, Asah1 gene deletion was shown to substantially inhibit lysosomal Ca2+ release through suppression of TRPML1 channel activity in CAECs. Sphingosine as an AC product rescued the function of TRPML1 channel in CAECs lacking Asah1 gene. These results suggest that Asah1 gene defect and associated deficiency of AC activity may inhibit TRPML1 channel activity, thereby reducing MVB degradation by lysosome and increasing exosome release from CAECs. This enhanced exosome release from CAECs may contribute to the development of coronary arterial disease under pathological conditions.

Keywords:  Acid ceramidase; Coronary arterial endothelial cell; Dynein; Exosome; Lysosome; TRPML1 channel

DOI:  https://doi.org/10.1016/bs.ctm.2022.09.002
Circ Res. 2022 Nov 08.

Spatiotemporal Control of Vascular CaV1.2 by α1C S1928 Phosphorylation.

Miguel Martín-Aragón Baudel, Victor A Flores-Tamez, Junyoung Hong, Gopyreddy R Reddy, Pauline Maillard, Abby E Burns, Kwun Nok Mimi Man, Kent C Sasse, Sean M Ward, William A Catterall, Donald M Bers, Johannes W Hell, Madeline Nieves-Cintrón, Manuel F Navedo.

  BACKGROUND: L-type CaV1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the CaV1.2 pore-forming subunit α1C at S1928 mediates vascular CaV1.2 cooperativity during diabetic hyperglycemia.METHODS: A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, pressure myography, and Laser Speckle imaging was implemented to examine CaV1.2 cooperativity, α1C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels.
RESULTS: CaV1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1C clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y11 inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. CaV1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca2+ influx and contractility and in vivo reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice.
CONCLUSIONS: These results suggest that PKA-dependent pS1928 promotes the spatial reorganization of vascular α1C into "superclusters" upon hyperglycemia and diabetes. This triggers CaV1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct CaV1.2 and arterial function during diabetic hyperglycemia.

Keywords:  clustering; cooperative gating; diabetes; hyperglycemia; vascular dysfunction

DOI:  https://doi.org/10.1161/CIRCRESAHA.122.321479
Nat Commun. 2022 Nov 10. 13(1): 6808

An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation in renal cells with TSC2 loss.

Kaushal Asrani, Juhyung Woo, Adrianna A Mendes, Ethan Schaffer, Thiago Vidotto, Clarence Rachel Villanueva, Kewen Feng, Lia Oliveira, Sanjana Murali, Hans B Liu, Daniela C Salles, Brandon Lam, Pedram Argani, Tamara L Lotan.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.

DOI: https://doi.org/10.1038/s41467-022-34617-7
Cells. 2022 Oct 24. pii: 3348. [Epub ahead of print]11(21):

Nanomaterials Respond to Lysosomal Function for Tumor Treatment.

Xuexia Tian, Anhua Shi, Hang Yin, Yutian Wang, Qiaoyan Liu, Wenling Chen, Junzi Wu.

  The safety and efficacy of tumor treatment are difficult problems to address. Recently, lysosomes have become an important target for tumor treatment because of their special environment and function. Nanoparticles have unique physicochemical properties which have great advantages in tumor research. Therefore, in recent years, researchers have designed various types of nanoparticles to treat tumors based on lysosomal function and environment. In this review, we summarize and analyze different perspectives of tumor treatment, including direct destruction of lysosomes or lysosomal escape, drug delivery by nanoparticles, response to endogenous or exogenous stimuli, and the targeting of tumor cells or other cells. We describe the advantages and disadvantages of these approaches as well as the developmental prospects in this field. We hope to provide new ideas for better tumor treatment.

Keywords:  lysosomal 2; nanomaterials 1; tumor 3

DOI:  https://doi.org/10.3390/cells11213348