bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–03–27
fifty papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning.
  2. RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.
  3. pH regulates potassium conductance and drives a constitutive proton current in human TMEM175.
  4. Structure of the human GlcNAc-1-phosphotransferase αβ subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation.
  5. Recycling of autophagosomal components from autolysosomes by the recycler complex.
  6. Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR.
  7. The central moTOR of metabolism.
  8. Effects of mTORC1 inhibition on proteasome activity and levels.
  9. Emerging Roles for Mammalian Target of Rapamycin (mTOR) Complexes in Bladder Cancer Progression and Therapy.
  10. Regulation of mTOR complexes in long-lived growth hormone receptor knockout and Snell dwarf mice.
  11. Blood-brain barrier delivery for lysosomal storage disorders with IgG-lysosomal enzyme fusion proteins.
  12. Activation of lysosomal mediated cell death in the course of autophagy by mTORC1 inhibitor.
  13. Acidic nanoparticles protect against α-synuclein-induced neurodegeneration through the restoration of lysosomal function.
  14. Delineating the neuropathology of lysosomal storage diseases using patient-derived induced pluripotent stem cells.
  15. Live cell microscopy of mitochondria-lysosome contact site formation and tethering dynamics.
  16. HEPES-buffering of bicarbonate-containing culture medium perturbs lysosomal glucocerebrosidase activity.
  17. TFEB- and TFE3-dependent autophagy activation supports cancer proliferation in the absence of centrosomes.
  18. Human Placental Intracellular Cholesterol Transport: A Focus on Lysosomal and Mitochondrial Dysfunction and Oxidative Stress.
  19. Mesenchymal Stem Cell-Based Therapy for Lysosomal Storage Diseases and Other Neurodegenerative Disorders.
  20. SPG15 protein deficits are at the crossroads between lysosomal abnormalities, altered lipid metabolism and synaptic dysfunction.
  21. Amino Acid Signaling for TOR in Eukaryotes: Sensors, Transducers, and a Sustainable Agricultural fuTORe.
  22. Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.
  23. Preprint Highlight: ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA- dependent STING signaling.
  24. Efficacy and Safety of a Krabbe Disease Gene Therapy.
  25. Optochemical Control of mTOR Signaling and mTOR-Dependent Autophagy.
  26. S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions.
  27. Over-Mutated Mitochondrial, Lysosomal and TFEB-Regulated Genes in Parkinson's Disease.
  28. Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.
  29. Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies.
  30. As predicted by hyperfunction theory, rapamycin treatment during development extends lifespan.
  31. The mTOR chromatin-bound interactome in prostate cancer.
  32. Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission.
  33. Dot6/Tod6 degradation fine-tunes the repression of ribosome biogenesis under nutrient-limited conditions.
  34. Spatial regulation of endosomes in growing dendrites.
  35. A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.
  36. Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles.
  37. Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis.
  38. Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment.
  39. ZFP36L2 suppresses mTORc1 through a P53-dependent pathway to prevent peri-partum cardiomyopathy in mice.
  40. Site-Specific Chemoenzymatic Conjugation of High-Affinity M6P Glycan Ligands to Antibodies for Targeted Protein Degradation.
  41. Ppp4r3a deficiency leads to depression-like behaviors in mice by modulating the synthesis of synaptic proteins.
  42. Cathepsin V: Molecular characteristics and significance in health and disease.
  43. Mice eat less and explore more after intake of certain amino acids.
  44. Intermittent and periodic fasting, longevity and disease.
  45. Clathrin coated pits as signaling platforms for Akt signaling.
  46. LAMP2A regulates the loading of proteins into exosomes.
  47. Pitfalls of X-chromosome inactivation testing in females with Fabry disease.
  48. The roles of dynein and myosin VI motor proteins in endocytosis.
  49. Large self-assembled clathrin lattices spontaneously disassemble without sufficient adaptor proteins.
  50. Hexokinase 1 cellular localization regulates the metabolic fate of glucose.
  1. Nat Commun. 2022 Mar 21. 13(1): 1540
    RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning.
    Gaurav Kumar, Prateek Chawla, Neha Dhiman, Sanya Chadha, Sheetal Sharma, Kanupriya Sethi, Mahak Sharma, Amit Tuli.
      The bidirectional movement of lysosomes on microtubule tracks regulates their whole-cell spatial arrangement. Arl8b, a small GTP-binding (G) protein, promotes lysosome anterograde trafficking mediated by kinesin-1. Herein, we report an Arl8b effector, RUFY3, which regulates the retrograde transport of lysosomes. We show that RUFY3 interacts with the JIP4-dynein-dynactin complex and facilitates Arl8b association with the retrograde motor complex. Accordingly, RUFY3 knockdown disrupts the positioning of Arl8b-positive endosomes and reduces Arl8b colocalization with Rab7-marked late endosomal compartments. Moreover, we find that RUFY3 regulates nutrient-dependent lysosome distribution, although autophagosome-lysosome fusion and autophagic cargo degradation are not impaired upon RUFY3 depletion. Interestingly, lysosome size is significantly reduced in RUFY3 depleted cells, which could be rescued by inhibition of the lysosome reformation regulatory factor PIKFYVE. These findings suggest a model in which the perinuclear cloud arrangement of lysosomes regulates both the positioning and size of these proteolytic compartments.
    DOI:  https://doi.org/10.1038/s41467-022-29077-y
  2. Nat Commun. 2022 Mar 21. 13(1): 1506
    RUFY3 and RUFY4 are ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin.
    Tal Keren-Kaplan, Amra Sarić, Saikat Ghosh, Chad D Williamson, Rui Jia, Yan Li, Juan S Bonifacino.
      The small GTPase ARL8 associates with endolysosomes, leading to the recruitment of several effectors that couple endolysosomes to kinesins for anterograde transport along microtubules, and to tethering factors for eventual fusion with other organelles. Herein we report the identification of the RUN- and FYVE-domain-containing proteins RUFY3 and RUFY4 as ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin for retrograde transport along microtubules. Using various methodologies, we find that RUFY3 and RUFY4 interact with both GTP-bound ARL8 and dynein-dynactin. In addition, we show that RUFY3 and RUFY4 promote concentration of endolysosomes in the juxtanuclear area of non-neuronal cells, and drive redistribution of endolysosomes from the axon to the soma in hippocampal neurons. The function of RUFY3 in retrograde transport contributes to the juxtanuclear redistribution of endolysosomes upon cytosol alkalinization. These studies thus identify RUFY3 and RUFY4 as ARL8-dependent, dynein-dynactin adaptors or regulators, and highlight the role of ARL8 in the control of both anterograde and retrograde endolysosome transport.
    DOI:  https://doi.org/10.1038/s41467-022-28952-y
  3. Sci Adv. 2022 Mar 25. 8(12): eabm1568
    pH regulates potassium conductance and drives a constitutive proton current in human TMEM175.
    Wang Zheng, Chen Shen, Longfei Wang, Shaun Rawson, Wen Jun Xie, Carl Nist-Lund, Jason Wu, Zhangfei Shen, Shiyu Xia, Jeffrey R Holt, Hao Wu, Tian-Min Fu.
      Human TMEM175, a noncanonical potassium (K+) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson's disease (PD). Structurally, the TMEM175 family exhibits an architecture distinct from canonical potassium channels, as it lacks the typical TVGYG selectivity filter. Here, we show that human TMEM175 not only exhibits pH-dependent structural changes that reduce K+ permeation at acidic pH but also displays proton permeation. TMEM175 constitutively conducts K+ at pH 7.4 but displays reduced K+ permeation at lower pH. In contrast, proton current through TMEM175 increases with decreasing pH because of the increased proton gradient. Molecular dynamics simulation, structure-based mutagenesis, and electrophysiological analysis suggest that K+ ions and protons share the same permeation pathway. The M393T variant of human TMEM175 associated with PD shows reduced function in both K+ and proton permeation. Together, our structural and electrophysiological analysis reveals a mechanism of TMEM175 regulation by pH.
    DOI:  https://doi.org/10.1126/sciadv.abm1568
  4. Nat Struct Mol Biol. 2022 Mar 24.
    Structure of the human GlcNAc-1-phosphotransferase αβ subunits reveals regulatory mechanism for lysosomal enzyme glycan phosphorylation.
    Hua Li, Wang-Sik Lee, Xiang Feng, Lin Bai, Benjamin C Jennings, Lin Liu, Balraj Doray, William M Canfield, Stuart Kornfeld, Huilin Li.
      Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an α2β2γ2 heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates. Here we report a cryo-EM structure of the catalytic core of human PTase and the identification of a hockey stick-like motif that controls activation of the enzyme. Movement of this motif out of the catalytic pocket is associated with a rearrangement of part of the peripheral domains that unblocks hydrolase glycan access to the catalytic site, thereby activating PTase. We propose that PTase fluctuates between inactive and active states in solution, and selective substrate binding of a lysosomal hydrolase through its protein-binding determinant to PTase locks the enzyme in the active state to permit glycan phosphorylation. This mechanism would help ensure that only N-linked glycans of lysosomal enzymes are phosphorylated.
    DOI:  https://doi.org/10.1038/s41594-022-00748-0
  5. Nat Cell Biol. 2022 Mar 24.
    Recycling of autophagosomal components from autolysosomes by the recycler complex.
    Chuchu Zhou, Zhe Wu, Wanqing Du, Huilin Que, Yufen Wang, Qinqin Ouyang, Fenglei Jian, Weigang Yuan, Yuan Zhao, Rui Tian, Ying Li, Yang Chen, Shuaixin Gao, Catherine C L Wong, Yueguang Rong.
      Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed 'recycler'. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.
    DOI:  https://doi.org/10.1038/s41556-022-00861-8
  6. Biochim Biophys Acta Mol Basis Dis. 2022 Mar 19. pii: S0925-4439(22)00069-2. [Epub ahead of print] 166399
    Adipose deficiency and aberrant autophagy in a Drosophila model of MPS VII is corrected by pharmacological stimulators of mTOR.
    Indrani Basu, Sudipta Bar, Mohit Prasad, Rupak Datta.
      Mucopolysaccharidosis type VII (MPS VII) is a recessively inherited lysosomal storage disorder caused due to β-glucuronidase (β-GUS) enzyme deficiency. Prominent clinical symptoms include hydrops fetalis, musculoskeletal deformities, neurodegeneration and hepatosplenomegaly leading to premature death in most cases. Apart from these, MPS VII is also characterized as adipose storage deficiency disorder although the underlying mechanism of this lean phenotype in the patients or β-GUS-deficient mice still remains a mystery. We addressed this issue using our recently developed Drosophila model of MPS VII (the CG2135-/- fly), which also exhibited a significant loss of body fat. We report here that the lean phenotype of the CG2135-/- larvae is due to fewer number of adipocytes, smaller lipid droplets and reduced adipogenesis. Our data further revealed that there is an abnormal accumulation of autophagosomes in the CG2135-/- larvae due to autophagosome-lysosome fusion defect. Decreased lysosome-mediated turnover also led to attenuated mTOR activity in the CG2135-/- larvae. Interestingly, treatment of the CG2135-/- larvae with mTOR stimulators, 3BDO or glucose, led to the restoration of mTOR activity with simultaneous correction of the autophagy defect and adipose storage deficiency. Our finding thus established a hitherto unknown mechanistic link between autophagy dysfunction, mTOR downregulation and reduced adiposity in MPS VII.
    Keywords:  Adipose deficiency; Atg8a; Drosophila; Impaired autophagy; MPS VII; mTORC1
    DOI:  https://doi.org/10.1016/j.bbadis.2022.166399
  7. Dev Cell. 2022 Mar 15. pii: S1534-5807(22)00126-5. [Epub ahead of print]
    The central moTOR of metabolism.
    Judith Simcox, Dudley W Lamming.
      The protein kinase mechanistic target of rapamycin (mTOR) functions as a central regulator of metabolism, integrating diverse nutritional and hormonal cues to control anabolic processes, organismal physiology, and even aging. This review discusses the current state of knowledge regarding the regulation of mTOR signaling and the metabolic regulation of the four macromolecular building blocks of the cell: carbohydrate, nucleic acid, lipid, and protein by mTOR. We review the role of mTOR in the control of organismal physiology and aging through its action in key tissues and discuss the potential for clinical translation of mTOR inhibition for the treatment and prevention of diseases of aging.
    Keywords:  amino acids; lipids; mTOR; mTORC1; mTORC2; metabolism; protein; rapamycin
    DOI:  https://doi.org/10.1016/j.devcel.2022.02.024
  8. BMB Rep. 2022 Mar 24. pii: 5583. [Epub ahead of print]
    Effects of mTORC1 inhibition on proteasome activity and levels.
    Seo Hyeong Park, Won Hoon Choi, Min Jae Lee.
      The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenance of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance.
  9. Cancers (Basel). 2022 Mar 18. pii: 1555. [Epub ahead of print]14(6):
    Emerging Roles for Mammalian Target of Rapamycin (mTOR) Complexes in Bladder Cancer Progression and Therapy.
    Jianya Huan, Petros Grivas, Jasmine Birch, Donna E Hansel.
      The mammalian target of rapamycin (mTOR) pathway regulates important cellular functions. Aberrant activation of this pathway, either through upstream activation by growth factors, loss of inhibitory controls, or molecular alterations, can enhance cancer growth and progression. Bladder cancer shows high levels of mTOR activity in approximately 70% of urothelial carcinomas, suggesting a key role for this pathway in this cancer. mTOR signaling initiates through upstream activation of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKT) and results in activation of either mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2). While these complexes share several key protein components, unique differences in their complex composition dramatically alter the function and downstream cellular targets of mTOR activity. While significant work has gone into analysis of molecular alterations of the mTOR pathway in bladder cancer, this has not yielded significant benefit in mTOR-targeted therapy approaches in urothelial carcinoma to date. New discoveries regarding signaling convergence onto mTOR complexes in bladder cancer could yield unique insights the biology and targeting of this aggressive disease. In this review, we highlight the functional significance of mTOR signaling in urothelial carcinoma and its potential impact on future therapy implications.
    Keywords:  bladder cancer; inhibitor; invasion; mTOR; progression; targeted therapy; urothelial carcinoma
    DOI:  https://doi.org/10.3390/cancers14061555
  10. Aging (Albany NY). 2022 Mar 19. 14(undefined):
    Regulation of mTOR complexes in long-lived growth hormone receptor knockout and Snell dwarf mice.
    Xiaofang Shi, S Joseph Endicott, Richard A Miller.
      Downregulation of mTOR (mechanistic target of rapamycin) can extend lifespan in multiple species, including mice. Growth hormone receptor knockout mice (GHRKO) and Snell dwarf mice have 40% or greater lifespan increase, and have lower mTORC1 function, which might reflect alteration in mTORC1 components or alteration of upstream proteins that modulate mTOR activity. Here we report reduction of mTORC components DEPTOR and PRAS40 in liver of these long-lived mice; these changes are opposite in direction to those that would be expected to lead to lower mTORC1 function. In contrast, levels of the upstream regulators TSC1 and TSC2 are elevated in GHRKO and Snell liver, kidney and skeletal muscle, and the ratio of phosphorylated TSC2 to total TSC2 is lower in the tissues of the long-lived mutant mice. In addition, knocking down TSC2 in GHRKO fibroblasts reversed the effects of the GHRKO mutation on mTORC1 function. Thus increased amounts of unphosphorylated, active, inhibitory TSC may contribute to lower mTORC1 function in these mice.
    Keywords:  TSC; aging; growth hormone receptor; lifespan extension; mTOR
    DOI:  https://doi.org/10.18632/aging.203959
  11. Adv Drug Deliv Rev. 2022 Mar 17. pii: S0169-409X(22)00124-7. [Epub ahead of print]184 114234
    Blood-brain barrier delivery for lysosomal storage disorders with IgG-lysosomal enzyme fusion proteins.
    William M Pardridge.
      The majority of lysosomal storage diseases affect the brain. Treatment of the brain with intravenous enzyme replacement therapy is not successful, because the recombinant lysosomal enzymes do not cross the blood-brain barrier (BBB). Biologic drugs, including lysosomal enzymes, can be re-engineered for BBB delivery as IgG-enzyme fusion proteins. The IgG domain of the fusion protein is a monoclonal antibody directed against an endogenous receptor-mediated transporter at the BBB, such as the insulin receptor or the transferrin receptor. This receptor transports the IgG across the BBB, in parallel with the endogenous receptor ligand, and the IgG acts as a molecular Trojan horse to ferry into brain the lysosomal enzyme genetically fused to the IgG. The IgG-enzyme fusion protein is bi-functional and retains both high affinity binding for the BBB receptor, and high lysosomal enzyme activity. IgG-lysosomal enzymes are presently in clinical trials for treatment of the brain in Mucopolysaccharidosis.
    Keywords:  Cerebrospinal fluid; Drug targeting; Endothelium; Fusion proteins; Mathematical model; Receptor-mediated transport
    DOI:  https://doi.org/10.1016/j.addr.2022.114234
  12. Sci Rep. 2022 Mar 23. 12(1): 5052
    Activation of lysosomal mediated cell death in the course of autophagy by mTORC1 inhibitor.
    Sameer Ullah Khan, Anup Singh Pathania, Abubakar Wani, Kaneez Fatima, Mubashir Javed Mintoo, Baseerat Hamza, Masroor Ahmad Paddar, Wadhwa Bhumika, Loveleena Kour Anand, Mir Shahid Maqbool, Sameer Ahmad Mir, Jaspreet Kour, Vunnam Venkateswarlu, Dilip Manikrao Mondhe, Sanghapal D Sawant, Fayaz Malik.
      Lysosomal biogenesis plays a vital role in cell fate. Under certain conditions, excessive lysosomal biogenesis leads to susceptibility for lysosomal membrane permeabilization resulting in various pathological conditions including cell death. In cancer cells apoptosis machinery becomes dysregulated during the course of treatment, thus allows cancer cells to escape apoptosis. So it is therefore imperative to identify cytotoxic agents that exploit non-apoptotic mechanisms of cell death. Our study showed that pancreatic cancer cells treated with SDS-203 triggered an incomplete autophagic response and a nuclear translocation of transcriptional factor TFEB. This resulted in abundant biosynthesis and accumulation of autophagosomes and lysosomes into the cells leading to their death. It was observed that the silencing of autophagy genes didn't alter the cell fate, whereas siRNA-mediated silencing of TFEB subdued SDS-203 mediated lysosomal biogenesis and associated cell death. Further mouse tumors treated with SDS-203 showed a significant reduction in tumor burden and increased expression of lysosomal markers. Taken together this study demonstrates that SDS-203 treatment triggers non-apoptotic cell death in pancreatic cancer cells through a mechanism of lysosome over accumulation.
    DOI:  https://doi.org/10.1038/s41598-022-07955-1
  13. Aging Cell. 2022 Mar 23. e13584
    Acidic nanoparticles protect against α-synuclein-induced neurodegeneration through the restoration of lysosomal function.
    Marie-Laure Arotcarena, Federico N Soria, Anthony Cunha, Evelyne Doudnikoff, Geoffrey Prévot, Jonathan Daniel, Mireille Blanchard-Desce, Philippe Barthélémy, Erwan Bezard, Sylvie Crauste-Manciet, Benjamin Dehay.
      Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, associated with the accumulation of misfolded α-synuclein and lysosomal impairment, two events deemed interconnected. Protein aggregation is linked to defects in degradation systems such as the autophagy-lysosomal pathway, while lysosomal dysfunction is partly related to compromised acidification. We have recently proven that acidic nanoparticles (aNPs) can re-acidify lysosomes and ameliorate neurotoxin-mediated dopaminergic neurodegeneration in mice. However, no lysosome-targeted approach has yet been tested in synucleinopathy models in vivo. Here, we show that aNPs increase α-synuclein degradation through enhancing lysosomal activity in vitro. We further demonstrate in vivo that aNPs protect nigral dopaminergic neurons from cell death, ameliorate α-synuclein pathology, and restore lysosomal function in mice injected with PD patient-derived Lewy body extracts carrying toxic α-synuclein aggregates. Our results support lysosomal re-acidification as a disease-modifying strategy for the treatment of PD and other age-related proteinopathies.
    Keywords:  Parkinson's disease; acidic nanoparticles; alpha-synuclein, neurodegeneration, therapeutics; in vivo; lysosomal restoration
    DOI:  https://doi.org/10.1111/acel.13584
  14. Stem Cells Dev. 2022 Mar 22.
    Delineating the neuropathology of lysosomal storage diseases using patient-derived induced pluripotent stem cells.
    K R Sabitha, Divya Chandran, Ashok K Shetty, Dinesh Upadhya.
      Lysosomal storage diseases (LSD) are inherited metabolic diseases caused due to deficiency of lysosomal enzymes, essential for the normal development of the brain and other organs. Approximately two-thirds of the patients suffering from LSD exhibit neurological deficits and impose an escalating challenge to the medical and scientific field. The advent of iPSC technology has aided researchers in efficiently generating functional neuronal and non-neuronal cells through directed differentiation protocols, as well as in decoding the cellular, subcellular and molecular defects associated with LSDs using two-dimensional cultures and cerebral organoid models. This review highlights the information assembled from patient-derived iPSCs on neurodevelopmental and neuropathological defects identified in LSDs. Multiple studies have identified neural progenitor cell migration and differentiation defects, substrate accumulation, axon growth and myelination defects, impaired calcium homeostasis and altered electrophysiological properties, using patient-derived iPSCs. In addition, these studies have also uncovered defective lysosomes, mitochondria, endoplasmic reticulum, Golgi complex, autophagy and vesicle trafficking and signaling pathways, oxidative stress, neuroinflammation, blood brain barrier dysfunction, neurodegeneration, gliosis, altered transcriptomes in LSDs. The review also discusses the therapeutic applications such as drug discovery, repurposing of drugs, synergistic effects of drugs, targeted molecular therapies, gene therapy, and transplantation applications of mutation corrected lines identified using patient-derived iPSCs for different LSDs.
    DOI:  https://doi.org/10.1089/scd.2021.0304
  15. STAR Protoc. 2022 Jun 17. 3(2): 101262
    Live cell microscopy of mitochondria-lysosome contact site formation and tethering dynamics.
    Tayler B Belton, Eric D Leisten, Jasmine Cisneros, Yvette C Wong.
      Mitochondria-lysosome contact sites are critical for maintaining cellular homeostasis by regulating mitochondrial and lysosomal network dynamics and mediating metabolite exchange. Here, we present a protocol to quantitatively analyze the formation and tethering duration of mitochondria-lysosome contact sites by using time-lapse live confocal microscopy of LAMP1 and TOMM20. Although this protocol focuses on mammalian HeLa cells, it can be applied to other cell types for further studies on mitochondria-lysosome contact regulation and function, and elucidation of their role in human disorders. For complete details on the use and execution of this protocol, please refer to Wong et al. (2018) and Wong et al. (2019b).
    Keywords:  Cell Biology; Cell culture; Microscopy
    DOI:  https://doi.org/10.1016/j.xpro.2022.101262
  16. J Cell Biochem. 2022 Mar 21.
    HEPES-buffering of bicarbonate-containing culture medium perturbs lysosomal glucocerebrosidase activity.
    Martijn J C van der Lienden, Jan Aten, Rolf G Boot, Marco van Eijk, Johannes M F G Aerts, Chi-Lin Kuo.
      Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N-glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent activity-based probes (ABPs) that covalently label GCase in reaction-based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage-like RAW264.7 cells. Specific attention was paid to the impact of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate-buffered medium. Here, we report how HEPES-buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES-containing medium was also found to reduce maturation of other lysosomal enzymes (α-glucosidase and β-glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD-patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.
    Keywords:  Gaucher disease; cell culture medium; diagnosis; glucocerebrosidase; glucosylsphingosine; lysosome
    DOI:  https://doi.org/10.1002/jcb.30234
  17. Autophagy. 2022 Mar 22. 1-21
    TFEB- and TFE3-dependent autophagy activation supports cancer proliferation in the absence of centrosomes.
    Chien-Han Kao, Ting-Yu Su, Wei-Syun Huang, Xin-Ying Lu, Wann-Neng Jane, Chien-Yung Huang, Hung-Hsiang Huang, Won-Jing Wang.
      Centrosome amplification is a phenomenon frequently observed in human cancers, so centrosome depletion has been proposed as a therapeutic strategy. However, despite being afflicted with a lack of centrosomes, many cancer cells can still proliferate, implying there are impediments to adopting centrosome depletion as a treatment strategy. Here, we show that TFEB- and TFE3-dependent autophagy activation contributes to acentrosomal cancer proliferation. Our biochemical analyses uncover that both TFEB and TFE3 are novel PLK4 (polo like kinase 4) substrates. Centrosome depletion inactivates PLK4, resulting in TFEB and TFE3 dephosphorylation and subsequent promotion of TFEB and TFE3 nuclear translocation and transcriptional activation of autophagy- and lysosome-related genes. A combination of centrosome depletion and inhibition of the TFEB-TFE3 autophagy-lysosome pathway induced strongly anti-proliferative effects in cancer cells. Thus, our findings point to a new strategy for combating cancer.
    Keywords:  Anti-cancer therapy; PLK4; autophagy; centrosome; lysosomal biogenesis; transcription factor E3; transcription factor EB
    DOI:  https://doi.org/10.1080/15548627.2022.2051880
  18. Antioxidants (Basel). 2022 Mar 04. pii: 500. [Epub ahead of print]11(3):
    Human Placental Intracellular Cholesterol Transport: A Focus on Lysosomal and Mitochondrial Dysfunction and Oxidative Stress.
    Maria Jose Yañez, Andrea Leiva.
      The placenta participates in cholesterol biosynthesis and metabolism and regulates exchange between the maternal and fetal compartments. The fetus has high cholesterol requirements, and it is taken up and synthesized at elevated rates during pregnancy. In placental cells, the major source of cholesterol is the internalization of lipoprotein particles from maternal circulation by mechanisms that are not fully understood. As in hepatocytes, syncytiotrophoblast uptake of lipoprotein cholesterol involves lipoprotein receptors such as low-density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI). Efflux outside the cells requires proteins such as the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. However, mechanisms associated with intracellular traffic of cholesterol in syncytiotrophoblasts are mostly unknown. In hepatocytes, uptaken cholesterol is transported to acidic late endosomes (LE) and lysosomes (LY). Proteins such as Niemann-Pick type C 1 (NPC1), NPC2, and StAR related lipid transfer domain containing 3 (STARD3) are required for cholesterol exit from the LE/LY. These proteins transfer cholesterol from the lumen of the LE/LY into the LE/LY-limiting membrane and then export it to the endoplasmic reticulum, mitochondria, or plasma membrane. Although the production, metabolism, and transport of cholesterol in placental cells are well explored, there is little information on the role of proteins related to intracellular cholesterol traffic in placental cells during physiological or pathological pregnancies. Such studies would be relevant for understanding fetal and placental cholesterol management. Oxidative stress, induced by generating excess reactive oxygen species (ROS), plays a critical role in regulating various cellular and biological functions and has emerged as a critical common mechanism after lysosomal and mitochondrial dysfunction. This review discusses the role of cholesterol, lysosomal and mitochondrial dysfunction, and ROS in the development and progression of hypercholesterolemic pregnancies.
    Keywords:  cholesterol traffic; lysosome; mitochondria; oxidative stress; placenta
    DOI:  https://doi.org/10.3390/antiox11030500
  19. Front Pharmacol. 2022 ;13 859516
    Mesenchymal Stem Cell-Based Therapy for Lysosomal Storage Diseases and Other Neurodegenerative Disorders.
    Shaza S Issa, Alisa A Shaimardanova, Victor V Valiullin, Albert A Rizvanov, Valeriya V Solovyeva.
      Lysosomal storage diseases (LSDs) are a group of approximately 50 genetic disorders caused by mutations in genes coding enzymes that are involved in cell degradation and transferring lipids and other macromolecules. Accumulation of lipids and other macromolecules in lysosomes leads to the destruction of affected cells. Although the clinical manifestations of different LSDs vary greatly, more than half of LSDs have symptoms of central nervous system neurodegeneration, and within each disorder there is a considerable variation, ranging from severe, infantile-onset forms to attenuated adult-onset disease, sometimes with distinct clinical features. To date, treatment options for this group of diseases remain limited, which highlights the need for further development of innovative therapeutic approaches, that can not only improve the patients' quality of life, but also provide full recovery for them. In many LSDs stem cell-based therapy showed promising results in preclinical researches. This review discusses using mesenchymal stem cells for different LSDs therapy and other neurodegenerative diseases and their possible limitations.
    Keywords:  cell therapy; clinical trials; lysosomal storage diseases; mesenchymal stem cell transplantation; mesenchymal stem cells; neurodegenerative diseases
    DOI:  https://doi.org/10.3389/fphar.2022.859516
  20. Hum Mol Genet. 2022 Mar 21. pii: ddac063. [Epub ahead of print]
    SPG15 protein deficits are at the crossroads between lysosomal abnormalities, altered lipid metabolism and synaptic dysfunction.
    Lara Marrone, Paolo M Marchi, Christopher P Webster, Raffaele Marroccella, Ian Coldicott, Steven Reynolds, João Alves-Cruzeiro, Zih-Liang Yang, Adrian Higginbottom, Mukhran Khundadze, Pamela J Shaw, Christian A Hübner, Matthew R Livesey, Mimoun Azzouz.
      Hereditary Spastic Paraplegia type 15 (HSP15) is a neurodegenerative condition caused by the inability to produce SPG15 protein, which leads to lysosomal swelling. However, the link between lysosomal aberrations and neuronal death is poorly explored. To uncover the functional consequences of lysosomal aberrations in disease pathogenesis, we analyze human dermal fibroblasts from HSP15 patients as well as primary cortical neurons derived from an SPG15 knockout (KO) mouse model. We find that SPG15 protein loss induces defective anterograde transport, impaired neurite outgrowth, axonal swelling and reduced autophagic flux in association with the onset of lysosomal abnormalities. Additionally, we observe lipid accumulation within the lysosomal compartment, suggesting that distortions in cellular lipid homeostasis are intertwined with lysosomal alterations. We further demonstrate that SPG15 KO neurons exhibit synaptic dysfunction, accompanied by augmented vulnerability to glutamate-induced excitotoxicity. Overall, our study establishes an intimate link between lysosomal aberrations, lipid metabolism and electrophysiological impairments, suggesting that lysosomal defects are at the core of multiple neurodegenerative disease processes in HSP15.
    DOI:  https://doi.org/10.1093/hmg/ddac063
  21. Biomolecules. 2022 Mar 02. pii: 387. [Epub ahead of print]12(3):
    Amino Acid Signaling for TOR in Eukaryotes: Sensors, Transducers, and a Sustainable Agricultural fuTORe.
    Nanticha Lutt, Jacob O Brunkard.
      Eukaryotic cells monitor and regulate metabolism through the atypical protein kinase target of rapamycin (TOR) regulatory hub. TOR is activated by amino acids in animals and fungi through molecular signaling pathways that have been extensively defined in the past ten years. Very recently, several studies revealed that TOR is also acutely responsive to amino acid metabolism in plants, but the mechanisms of amino acid sensing are not yet established. In this review, we summarize these discoveries, emphasizing the diversity of amino acid sensors in human cells and highlighting pathways that are indirectly sensitive to amino acids, i.e., how TOR monitors changes in amino acid availability without a bona fide amino acid sensor. We then discuss the relevance of these model discoveries to plant biology. As plants can synthesize all proteinogenic amino acids from inorganic precursors, we focus on the possibility that TOR senses both organic metabolites and inorganic nutrients. We conclude that an evolutionary perspective on nutrient sensing by TOR benefits both agricultural and biomedical science, contributing to ongoing efforts to generate crops for a sustainable agricultural future.
    Keywords:  Arabidopsis thaliana; Castor1; GATOR; GCN2; Ragulator; Sestrin2; amino acid signaling; mTOR; metabolism; target of rapamycin
    DOI:  https://doi.org/10.3390/biom12030387
  22. Cells. 2022 Mar 08. pii: 921. [Epub ahead of print]11(6):
    Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters.
    Margherita Festa, Velia Minicozzi, Anna Boccaccio, Laura Lagostena, Antonella Gradogna, Tianwen Qi, Alex Costa, Nina Larisch, Shin Hamamoto, Emanuela Pedrazzini, Stefan Milenkovic, Joachim Scholz-Starke, Matteo Ceccarelli, Alessandro Vitale, Petra Dietrich, Nobuyuki Uozumi, Franco Gambale, Armando Carpaneto.
      A distinct set of channels and transporters regulates the ion fluxes across the lysosomal membrane. Malfunctioning of these transport proteins and the resulting ionic imbalance is involved in various human diseases, such as lysosomal storage disorders, cancer, as well as metabolic and neurodegenerative diseases. As a consequence, these proteins have stimulated strong interest for their suitability as possible drug targets. A detailed functional characterization of many lysosomal channels and transporters is lacking, mainly due to technical difficulties in applying the standard patch-clamp technique to these small intracellular compartments. In this review, we focus on current methods used to unravel the functional properties of lysosomal ion channels and transporters, stressing their advantages and disadvantages and evaluating their fields of applicability.
    Keywords:  ion channels; lysosomes; patch-clamp; plant vacuole; transporters
    DOI:  https://doi.org/10.3390/cells11060921
  23. Mol Biol Cell. 2022 Apr 01. 33(4): mbcP22021003
    Preprint Highlight: ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA- dependent STING signaling.
    Luis Bonet-Ponce.
      
    DOI:  https://doi.org/10.1091/mbc.P22-02-1003
  24. Hum Gene Ther. 2022 Mar 22.
    Efficacy and Safety of a Krabbe Disease Gene Therapy.
    Juliette Hordeaux, Brianne A Jeffrey, Jinlong Jian, Gourav R Choudhury, Kristofer Michalson, Thomas W Mitchell, Elizabeth L Buza, Jessica Chichester, Cecilia Dyer, Jessica Bagel, Charles H Vite, Allison M Bradbury, James M Wilson.
      Krabbe disease is a lysosomal storage disease caused by mutations in the gene that encodes galactosylceramidase, in which galactosylsphingosine (psychosine) accumulation drives demyelination in the central and peripheral nervous systems, ultimately progressing to death in early childhood. Gene therapy, alone or in combination with transplant, has been developed for almost two decades in mouse models, with increasing therapeutic benefit paralleling the improvement of next-generation adeno-associated virus (AAV) vectors. This effort has recently shown remarkable efficacy in the canine model of the disease by two different groups that used either systemic or cerebrospinal fluid (CSF) administration of AAVrh10 or AAV9. Building on our experience developing CSF-delivered, AAV-based drug products for a variety of neurodegenerative disorders, we conducted efficacy, pharmacology, and safety studies of AAVhu68 delivered to the CSF in two relevant natural Krabbe animal models, and in nonhuman primates. In newborn Twitcher mice, the highest dose (1 × 1011 genome copies [GC]) of AAVhu68.hGALC injected into the lateral ventricle led to a median survival of 130 days compared to 40.5 days in vehicle-treated mice. When this dose was administered intravenously, the median survival was 49 days. A single intracisterna magna injection of AAVhu68.cGALC at 3 × 1013 GC into presymptomatic Krabbe dogs increased survival for up to 85 weeks compared to 12 weeks in controls. It prevented psychosine accumulation in the CSF, preserved peripheral nerve myelination, ambulation, and decreased brain neuroinflammation and demyelination, although some regions remained abnormal. In a Good Laboratory Practice-compliant toxicology study, we administered the clinical candidate into the cisterna magna of 18 juvenile rhesus macaques at 3 doses that displayed efficacy in mice. We observed no dose-limiting toxicity and sporadic minimal degeneration of dorsal root ganglia (DRG) neurons. Our studies demonstrate the efficacy, scalability, and safety of a single cisterna magna AAVhu68 administration to treat Krabbe disease. ClinicalTrials.Gov ID: NCT04771416.
    Keywords:  AAV; Krabbe; Twitcher mouse; cisterna magna; demyelination; galactosylceramidase (GALC); galactosylceramide; lysosomal storage disease; psychosine
    DOI:  https://doi.org/10.1089/hum.2021.245
  25. ACS Pharmacol Transl Sci. 2022 Mar 11. 5(3): 149-155
    Optochemical Control of mTOR Signaling and mTOR-Dependent Autophagy.
    Tianyi Wang, Kaiqi Long, Yang Zhou, Xiaoding Jiang, Jinzhao Liu, John H C Fong, Alan S L Wong, Wai-Lung Ng, Weiping Wang.
      As an important regulator of cell metabolism, proliferation, and survival, mTOR (mammalian target of rapamycin) signaling provides both a potential target for cancer treatment and a research tool for investigation of cell metabolism. One inhibitor for both mTORC1 and mTORC2 pathways, OSI-027, exhibited robust anticancer efficacy but induced side effects. Herein, we designed a photoactivatable OSI-027 prodrug, which allowed the release of OSI-027 after light irradiation to inhibit the mTOR signaling pathway, triggering autophagy and leading to cell death. This photoactivatable prodrug can provide novel strategies for mTOR-targeting cancer therapy and act as a new tool for investigating mTOR signaling and its related biological processes.
    DOI:  https://doi.org/10.1021/acsptsci.1c00230
  26. Nat Commun. 2022 Mar 22. 13(1): 1548
    S6K1-mediated phosphorylation of PDK1 impairs AKT kinase activity and oncogenic functions.
    Qiwei Jiang, Xiaomei Zhang, Xiaoming Dai, Shiyao Han, Xueji Wu, Lei Wang, Wenyi Wei, Ning Zhang, Wei Xie, Jianping Guo.
      Functioning as a master kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1) plays a fundamental role in phosphorylating and activating protein kinases A, B and C (AGC) family kinases, including AKT. However, upstream regulation of PDK1 remains largely elusive. Here we report that ribosomal protein S6 kinase beta 1 (S6K1), a member of AGC kinases and downstream target of mechanistic target of rapamycin complex 1 (mTORC1), directly phosphorylates PDK1 at its pleckstrin homology (PH) domain, and impairs PDK1 interaction with and activation of AKT. Mechanistically, S6K1-mediated phosphorylation of PDK1 augments its interaction with 14-3-3 adaptor protein and homo-dimerization, subsequently dissociating PDK1 from phosphatidylinositol 3,4,5 triphosphate (PIP3) and retarding its interaction with AKT. Pathologically, tumor patient-associated PDK1 mutations, either attenuating S6K1-mediated PDK1 phosphorylation or impairing PDK1 interaction with 14-3-3, result in elevated AKT kinase activity and oncogenic functions. Taken together, our findings not only unravel a delicate feedback regulation of AKT signaling via S6K1-mediated PDK1 phosphorylation, but also highlight the potential strategy to combat mutant PDK1-driven cancers.
    DOI:  https://doi.org/10.1038/s41467-022-28910-8
  27. J Clin Med. 2022 Mar 21. pii: 1749. [Epub ahead of print]11(6):
    Over-Mutated Mitochondrial, Lysosomal and TFEB-Regulated Genes in Parkinson's Disease.
    Eulàlia Segur-Bailach, Olatz Ugarteburu, Frederic Tort, Laura Texido, Celia Painous, Yaroslau Compta, Maria José Martí, Antonia Ribes, Laura Gort.
      The association between Parkinson's disease (PD) and mutations in genes involved in lysosomal and mitochondrial function has been previously reported. However, little is known about the involvement of other genes or cellular mechanisms. We aim to identify novel genetic associations to better understand the pathogenesis of PD. We performed WES in a cohort of 32 PD patients and 30 age-matched controls. We searched for rare variants in 1667 genes: PD-associated, related to lysosomal function and mitochondrial function and TFEB-regulated. When comparing the PD patient cohort with that of age matched controls, a statistically significant burden of rare variants in the previous group of genes were identified. In addition, the Z-score calculation, using the European population database (GnomAD), showed an over-representation of particular variants in 36 genes. Interestingly, 11 of these genes are implicated in mitochondrial function and 18 are TFEB-regulated genes. Our results suggest, for the first time, an involvement of TFEB-regulated genes in the genetic susceptibility to PD. This is remarkable as TFEB factor has been reported to be sequestered inside Lewy bodies, pointing to a role of TFEB in the pathogenesis of PD. Our data also reinforce the involvement of lysosomal and mitochondrial mechanisms in PD.
    Keywords:  Parkinson’s disease; TFEB; WES; lysosomal; lysosomal diseases; mitochondrial; mitochondrial function; risk-factor
    DOI:  https://doi.org/10.3390/jcm11061749
  28. J Lipid Res. 2022 Mar 18. pii: S0022-2275(22)00032-3. [Epub ahead of print] 100199
    Consequences of excessive glucosylsphingosine in glucocerebrosidase-deficient zebrafish.
    Lindsey T Lelieveld, Sophie Gerhardt, Saskia Maas, Kimberley C Zwiers, Claire de Wit, Ernst H Beijk, Maria J Ferraz, Marta Artola, Annemarie H Meijer, Christian Tudorache, Daniela Salvatori, Rolf G Boot, Johannes M F G Aerts.
      In Gaucher disease (GD), the deficiency of glucocerebrosidase (GCase/ GBA1) causes lysosomal accumulation of glucosylceramide (GlcCer), which is partly converted by acid ceramidase (ACase) to glucosylsphingosine (GlcSph) in the lysosome. Chronically elevated blood and tissue GlcSph is thought to contribute to symptoms in GD patients as well as to increased risk for Parkinson's disease. On the other hand, formation of GlcSph may be beneficial since the water soluble sphingoid base is excreted via urine and bile. To study the role of excessive GlcSph formation during glucocerebrosidase deficiency we studied zebrafish that have two orthologues of acid ceramidase, Asah1a and Asah1b. Only the latter is involved in formation of GlcSph in glucocerebrosidase-deficient zebrafish as revealed by knockouts of Asah1a or Asah1b with GCase deficiency (either pharmacologically induced or genetic). Comparison of zebrafish with excessive GlcSph (gba1-/- fish) and without GlcSph (gba1-/-:asah1b-/- fish) allowed us to study consequences of chronic high levels of GlcSph. Prevention of excessive GlcSph in gba1-/-:asah1b-/- fish did not restrict storage cells, GlcCer accumulation or neuroinflammation. However, GD fish lacking excessive GlcSph show an ameliorated course of disease reflected by significantly increased lifespan, delayed locomotor abnormality, and delayed development of an abnormal curved back posture. The loss of tyrosine hydroxylase 1 (th1) mRNA, a marker of dopaminergic neurons, is slowed down in brain of GD fish lacking excessive GlcSph. In conclusion, in the zebrafish GD model, excess GlcSph has little impact on (neuro)inflammation or the presence of GlcCer-laden macrophages, but rather seems harmful to th1-positive dopaminergic neurons.
    Keywords:  Gaucher disease; acid ceramidase; dopaminergic neurons; glucosylceramide; lipid metabolism; lysosphingolipids; sphingolipids; zebrafish
    DOI:  https://doi.org/10.1016/j.jlr.2022.100199
  29. Life Sci Alliance. 2022 Mar;pii: e202101269. [Epub ahead of print]5(7):
    Retrograde transport of CDMPR depends on several machineries as analyzed by sulfatable nanobodies.
    Dominik P Buser, Gaétan Bader, Martin Spiess.
      Retrograde protein transport from the cell surface and endosomes to the TGN is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. We used a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN for the cation-dependent MPR (CDMPR) with and without rapid or gradual inactivation of candidate machinery proteins. Although knockdown of retromer (Vps26), epsinR, or Rab9a reduced CDMPR arrival to the TGN, no effect was observed upon silencing of TIP47. Strikingly, when retrograde transport was analyzed by rapamycin-induced rapid depletion (knocksideways) or long-term depletion by knockdown of the clathrin adaptor AP-1 or of the GGA machinery, distinct phenotypes in sulfation kinetics were observed, suggesting a potential role of GGA adaptors in retrograde and anterograde transport. Our study illustrates the usefulness of derivatized, sulfation-competent nanobodies, reveals novel insights into CDMPR trafficking biology, and further outlines that the selection of machinery inactivation is critical for phenotype analysis.
    DOI:  https://doi.org/10.26508/lsa.202101269
  30. Aging (Albany NY). 2022 Mar 05. 14(5): 2020-2024
    As predicted by hyperfunction theory, rapamycin treatment during development extends lifespan.
    Mikhail V Blagosklonny.
      
    Keywords:  growth; mTOR; quasi-programmed aging; reprogramming; sirolimus
    DOI:  https://doi.org/10.18632/aging.203937
  31. Cell Rep. 2022 Mar 22. pii: S2211-1247(22)00275-3. [Epub ahead of print]38(12): 110534
    The mTOR chromatin-bound interactome in prostate cancer.
    Catherine R Dufour, Charlotte Scholtes, Ming Yan, Yonghong Chen, Lingwei Han, Ting Li, Hui Xia, Qiyun Deng, Mathieu Vernier, Vincent Giguère.
      A growing number of studies support a direct role for nuclear mTOR in gene regulation and chromatin structure. Still, the scarcity of known chromatin-bound mTOR partners limits our understanding of how nuclear mTOR controls transcription. Herein, comprehensive mapping of the mTOR chromatin-bound interactome in both androgen-dependent and -independent cellular models of prostate cancer (PCa) identifies a conserved 67-protein interaction network enriched for chromatin modifiers, transcription factors, and SUMOylation machinery. SUMO2/3 and nuclear pore protein NUP210 are among the strongest interactors, while the androgen receptor (AR) is the dominant androgen-inducible mTOR partner. Further investigation reveals that NUP210 facilitates mTOR nuclear trafficking, that mTOR and AR form a functional transcriptional module with the nucleosome remodeling and deacetylase (NuRD) complex, and that androgens specify mTOR-SUMO2/3 promoter-enhancer association. This work identifies a vast network of mTOR-associated nuclear complexes advocating innovative molecular strategies to modulate mTOR-dependent gene regulation with conceivable implications for PCa and other diseases.
    Keywords:  AR; CP: Cancer; CP: Molecular biology; ChIP-MS; ESRRA; FOXA1; HDAC2; HOXB13; PML; ZNF618
    DOI:  https://doi.org/10.1016/j.celrep.2022.110534
  32. EMBO J. 2022 Mar 22. e109352
    Endosomal phosphatidylinositol 3-phosphate controls synaptic vesicle cycling and neurotransmission.
    Guan-Ting Liu, Gaga Kochlamazashvili, Dmytro Puchkov, Rainer Müller, Carsten Schultz, Albert I Mackintosh, Dennis Vollweiter, Volker Haucke, Tolga Soykan.
      Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration.
    Keywords:  endocytosis; endosomes; neurotransmission; phosphatidylinositol 3-phosphate; synaptic vesicle
    DOI:  https://doi.org/10.15252/embj.2021109352
  33. iScience. 2022 Mar 18. 25(3): 103986
    Dot6/Tod6 degradation fine-tunes the repression of ribosome biogenesis under nutrient-limited conditions.
    Kino Kusama, Yuta Suzuki, Ena Kurita, Tomoyuki Kawarasaki, Keisuke Obara, Fumihiko Okumura, Takumi Kamura, Kunio Nakatsukasa.
      Ribosome biogenesis (Ribi) is a complex and energy-consuming process, and should therefore be repressed under nutrient-limited conditions to minimize unnecessary cellular energy consumption. In yeast, the transcriptional repressors Dot6 and Tod6 are phosphorylated and inactivated by the TORC1 pathway under nutrient-rich conditions, but are activated and repress ∼200 Ribi genes under nutrient-limited conditions. However, we show that in the presence of rapamycin or under nitrogen starvation conditions, Dot6 and Tod6 were readily degraded by the proteasome in a SCFGrr1 and Tom1 ubiquitin ligase-dependent manner, respectively. Moreover, promiscuous accumulation of Dot6 and Tod6 excessively repressed Ribi gene expression as well as translation activity and caused a growth defect in the presence of rapamycin. Thus, we propose that degradation of Dot6 and Tod6 is a novel mechanism to ensure an appropriate level of Ribi gene expression and thereby fine-tune the repression of Ribi and translation activity for cell survival under nutrient-limited conditions.
    Keywords:  Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2022.103986
  34. Dev Biol. 2022 Mar 17. pii: S0012-1606(22)00047-1. [Epub ahead of print]486 5-14
    Spatial regulation of endosomes in growing dendrites.
    Chan Choo Yap, Bettina Winckler.
      Many membrane proteins are highly enriched in either dendrites or axons. This non-uniform distribution is a critical feature of neuronal polarity and underlies neuronal function. The molecular mechanisms responsible for polarized distribution of membrane proteins has been studied for some time and many answers have emerged. A less well studied feature of neurons is that organelles are also frequently non-uniformly distributed. For instance, EEA1-positive early endosomes are somatodendritic whereas synaptic vesicles are axonal. In addition, some organelles are present in both axons and dendrites, but not distributed uniformly along the processes. One well known example are lysosomes which are abundant in the soma and proximal dendrite, but sparse in the distal dendrite and the distal axon. The mechanisms that determine the spatial distribution of organelles along dendrites are only starting to be studied. In this review, we will discuss the cell biological mechanisms of how the distribution of diverse sets of endosomes along the proximal-distal axis of dendrites might be regulated. In particular, we will focus on the regulation of bulk homeostatic mechanisms as opposed to local regulation. We posit that immature dendrites regulate organelle motility differently from mature dendrites in order to spatially organize dendrite growth, branching and sculpting.
    Keywords:  Dendrite; Dendritogenesis; Directional Transport; Endosome; Lysosome; Organelle Positioning
    DOI:  https://doi.org/10.1016/j.ydbio.2022.03.004
  35. Cells. 2022 Mar 17. pii: 1018. [Epub ahead of print]11(6):
    A Phosphosite Mutant Approach on LRRK2 Links Phosphorylation and Dephosphorylation to Protective and Deleterious Markers, Respectively.
    Antoine Marchand, Alessia Sarchione, Panagiotis S Athanasopoulos, Hélène Bauderlique-Le Roy, Liesel Goveas, Romain Magnez, Matthieu Drouyer, Marco Emanuele, Franz Y Ho, Maxime Liberelle, Patricia Melnyk, Nicolas Lebègue, Xavier Thuru, R Jeremy Nichols, Elisa Greggio, Arjan Kortholt, Thierry Galli, Marie-Christine Chartier-Harlin, Jean-Marc Taymans.
      The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.
    Keywords:  LRRK2; Parkinson’s disease; RABs; lysosome; phosphorylation
    DOI:  https://doi.org/10.3390/cells11061018
  36. Methods Mol Biol. 2022 ;2442 353-365
    Visualization of Cytosolic Galectin Accumulation Around Damaged Vesicles and Organelles.
    Ming-Hsiang Hong, I-Chun Weng, Fang-Yen Li, Fu-Tong Liu.
      Galectins are animal lectins that recognize β-galactoside and bind glycans. Recent studies have indicated that cytosolic galectins recognize cytosolically exposed glycans and accumulate around endocytic vesicles or organelles damaged by various disruptive substances. Accumulated galectins engage other cytosolic proteins toward damaged vesicles, leading to cellular responses, such as autophagy. Disruptive substances include bacteria, viruses, particulate matters, and protein aggregates; thus, this process is implicated in the pathogenesis of various diseases. In this chapter, we describe methods for studying three disruptive substances: photosensitizers, Listeria monocytogenes, and Helicobacter pylori. We summarize the tools used for the detection of cytosolic galectin accumulation around damaged vesicles.
    Keywords:  APEX2-enhanced electron microscopy; Endosomes; Fluorescence microscopy; Galectins; Glycans; Helicobacter pylori; Immunofluorescence microscopy; Immunogold electron microscopy; Listeria monocytogenes; Lysosomes; Phagosomes; Photosensitizers
    DOI:  https://doi.org/10.1007/978-1-0716-2055-7_19
  37. EMBO Rep. 2022 Mar 23. e54278
    Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis.
    Sota Kuno, Hiroaki Fujita, Yu-Ki Tanaka, Yasumitsu Ogra, Kazuhiro Iwai.
      Iron is not only essential but also a toxic trace element. Under iron repletion, ferritin maintains cellular iron homeostasis by storing iron to avoid iron toxicity. Under iron depletion, the ferritin-specific autophagy adaptor NCOA4 delivers ferritin to lysosomes via macroautophagy to enable cells to use stored iron. Here, we show that NCOA4 also plays crucial roles in the regulation of ferritin fate under iron repletion. NCOA4 forms insoluble condensates via multivalent interactions generated by the binding of iron to its intrinsically disordered region. This sequesters NCOA4 away from ferritin and allows ferritin accumulation in the early phase of iron repletion. Under prolonged iron repletion, NCOA4 condensates can deliver ferritin to lysosomes via a TAX1BP1-dependent non-canonical autophagy pathway, thereby preventing relative iron deficiency due to excessive iron storage and reduced iron uptake. Together, these observations suggest that the NCOA4-ferritin axis modulates intracellular iron homeostasis in accordance with cellular iron availability.
    Keywords:  NCOA4; autophagy; ferritin; iron metabolism; phase separation
    DOI:  https://doi.org/10.15252/embr.202154278
  38. Cells. 2022 Mar 11. pii: 962. [Epub ahead of print]11(6):
    Early Endosomal Vps34-Derived Phosphatidylinositol-3-Phosphate Is Indispensable for the Biogenesis of the Endosomal Recycling Compartment.
    Marina Marcelić, Hana Mahmutefendić Lučin, Antonija Jurak Begonja, Gordana Blagojević Zagorac, Pero Lučin.
      Phosphatidylinositol-3-phosphate (PI3P), a major identity tag of early endosomes (EEs), provides a platform for the recruitment of numerous cellular proteins containing an FYVE or PX domain that is required for PI3P-dependent maturation of EEs. Most of the PI3P in EEs is generated by the activity of Vps34, a catalytic component of class III phosphatidylinositol-3-phosphate kinase (PI3Ks) complex. In this study, we analyzed the role of Vps34-derived PI3P in the EE recycling circuit of unperturbed cells using VPS34-IN1 (IN1), a highly specific inhibitor of Vps34. IN1-mediated PI3P depletion resulted in the rapid dissociation of recombinant FYVE- and PX-containing PI3P-binding modules and endogenous PI3P-binding proteins, including EEA1 and EE sorting nexins. IN1 treatment triggered the rapid restructuring of EEs into a PI3P-independent functional configuration, and after IN1 washout, EEs were rapidly restored to a PI3P-dependent functional configuration. Analysis of the PI3P-independent configuration showed that the Vps34-derived PI3P is not essential for the pre-EE-associated functions and the fast recycling loop of the EE recycling circuit but contributes to EE maturation toward the degradation circuit, as previously shown in Vps34 knockout and knockdown studies. However, our study shows that Vps34-derived PI3P is also essential for the establishment of the Rab11a-dependent pathway, including recycling cargo sorting in this pathway and membrane flux from EEs to the pericentriolar endosomal recycling compartment (ERC). Rab11a endosomes of PI3P-depleted cells expanded and vacuolized outside the pericentriolar area without the acquisition of internalized transferrin (Tf). These endosomes had high levels of FIP5 and low levels of FIP3, suggesting that their maturation was arrested before the acquisition of FIP3. Consequently, Tf-loaded-, Rab11a/FIP5-, and Rab8a-positive endosomes disappeared from the pericentriolar area, implying that PI3P-associated functions are essential for ERC biogenesis. ERC loss was rapidly reversed after IN1 washout, which coincided with the restoration of FIP3 recruitment to Rab11a-positive endosomes and their dynein-dependent migration to the cell center. Thus, our study shows that Vps34-derived PI3P is indispensable in the recycling circuit to maintain the slow recycling pathway and biogenesis of the ERC.
    Keywords:  Rab11a endosomes; VPS34-IN1; Vps34; endosomal recycling compartment; phosphatidylinositol-3-phosphate
    DOI:  https://doi.org/10.3390/cells11060962
  39. J Clin Invest. 2022 Mar 22. pii: e154491. [Epub ahead of print]
    ZFP36L2 suppresses mTORc1 through a P53-dependent pathway to prevent peri-partum cardiomyopathy in mice.
    Hidemichi Kouzu, Yuki Tatekoshi, Hsiang-Chun Chang, Jason S Shapiro, Warren A McGee, Adam De Jesus, Issam Ben-Sahra, Zoltan Arany, Jonathan Leor, Chunlei Chen, Perry J Blackshear, Hossein Ardehali.
      Pregnancy is associated with substantial physiological changes of the heart, and disruptions in these processes can lead to peripartum-cardiomyopathy (PPCM). The molecular processes that cause physiological and pathological changes in the heart during pregnancy are not well characterized. Here, we show that mTORc1 was activated in pregnancy to facilitate cardiac enlargement that was reversed after delivery in mice. mTORc1 activation in pregnancy was negatively regulated by the mRNA-destabilizing-protein ZFP36L2 through its degradation of Mdm2 mRNA and P53 stabilization, leading to increased SESN2 and REDD1 expression. This pathway impeded uncontrolled cardiomyocytes hypertrophy during pregnancy, and mice with cardiac-specific Zfp36l2 deletion developed rapid cardiac dysfunction after delivery, while prenatal treatment of these mice with rapamycin improved post-partum cardiac function. Collectively, these data provide a novel pathway for the regulation of mTORc1 through mRNA stabilization of a P53 ubiquitin ligase. This pathway was critical for normal cardiac growth during pregnancy, and its reduction led to PPCM-like adverse remodeling in mice.
    Keywords:  Cardiology; Cardiovascular disease; Cell Biology; Heart failure; p53
    DOI:  https://doi.org/10.1172/JCI154491
  40. ACS Chem Biol. 2022 Mar 22.
    Site-Specific Chemoenzymatic Conjugation of High-Affinity M6P Glycan Ligands to Antibodies for Targeted Protein Degradation.
    Xiao Zhang, Huiying Liu, Jia He, Chong Ou, Thomas C Donahue, Musleh M Muthana, Lishan Su, Lai-Xi Wang.
      Lysosome-targeting chimeras (LYTACs) offer an opportunity for the degradation of extracellular and membrane-associated proteins of interest. Here, we report an efficient chemoenzymatic method that enables a single-step and site-specific conjugation of high-affinity mannose-6-phosphate (M6P) glycan ligands to antibodies without the need of protein engineering and conventional click reactions that would introduce "unnatural" moieties, yielding homogeneous antibody-M6P glycan conjugates for targeted degradation of membrane-associated proteins. Using trastuzumab and cetuximab as model antibodies, we showed that the wild-type endoglycosidase S (Endo-S) could efficiently perform the antibody deglycosylation and simultaneous transfer of an M6P-glycan from a synthetic M6P-glycan oxazoline to the deglycosylated antibody in a one-pot manner, giving structurally well-defined antibody-M6P glycan conjugates. A two-step procedure, using wild-type Endo-S2 for deglycosylation followed by transglycosylation with an Endo-S2 mutant (D184M), was also efficient to provide M6P glycan-antibody conjugates. The chemoenzymatic approach was highly specific for Fc glycan remodeling when both Fc and Fab domains were glycosylated, as exemplified by the selective Fc-glycan remodeling of cetuximab. SPR binding analysis indicated that the M6P conjugates possessed a nanomolar range of binding affinities for the cation-independent mannose-6-phosphate receptor (CI-MPR). Preliminary cell-based assays showed that the M6P-trastuzumab and M6P-cetuximab conjugates were able to selectively degrade the membrane-associated HER2 and EGFR, respectively. This modular glycan-remodeling strategy is expected to find wide applications for antibody-based lysosome-targeted degradation of extracellular and membrane proteins.
    DOI:  https://doi.org/10.1021/acschembio.1c00751
  41. Dis Model Mech. 2022 Mar 22. pii: dmm.049374. [Epub ahead of print]
    Ppp4r3a deficiency leads to depression-like behaviors in mice by modulating the synthesis of synaptic proteins.
    Fei Gao, Ai Liu, Xing Qi, Meitian Wang, Xiao Chen, Shijun Wei, Shang Gao, Yueqing Sun, Ping Sun, Xi Li, Wenjie Sun, Jiangxia Li, Qiji Liu.
      Chronic stress is one of the most potent risk factors for the onset of major depressive disorder. Chronic unpredictable mild stress results in reduced synaptic proteins and depression-like behaviors in rodent models. However, the upstream molecule that senses the demand for synaptic proteins and initiates their synthesis under chronic stress remains unknown. In our research, chronic unpredictable mild stress reduced the expression of PPP4R3A in the prefrontal cortex and hippocampus in mice. Selective knockout of Ppp4r3a in the cortex and hippocampus mimicked depression- and anxiety-like behavioral effects of chronic stress in mice. Notably, Ppp4r3a deficiency led to down-regulated mTORC1 signaling, which resulted in reduced synthesis of synaptic proteins and impaired synaptic functions. On the contrary, overexpression of Ppp4r3a in the cortex and hippocampus protected against behavioral and synaptic deficits induced by chronic stress in a PPP4R3A-mTORC1-dependent manner. Rapamycin treatment of Ppp4r3a-overexpressing neurons blocked the regulatory effect of Ppp4r3a on the synthesis of synaptic proteins by directly inhibiting mTORC1. Overall, our results revealed a regulatory role of Ppp4r3a in driving synaptic protein synthesis in chronic stress.
    Keywords:  Chronic unpredictable mild stress; Major depressive disorder; PPP4R3A; Synthesis of synaptic proteins; mTORC1 signaling
    DOI:  https://doi.org/10.1242/dmm.049374
  42. Mol Aspects Med. 2022 Mar 16. pii: S0098-2997(22)00028-0. [Epub ahead of print] 101086
    Cathepsin V: Molecular characteristics and significance in health and disease.
    Fabien Lecaille, Thibault Chazeirat, Ahlame Saidi, Gilles Lalmanach.
      Human cysteine cathepsins form a family of eleven proteases (B, C, F, H, K, L, O, S, V, W, X/Z) that play important roles in a considerable number of biological and pathophysiological processes. Among them, cathepsin V, also known as cathepsin L2, is a lysosomal enzyme, which is mainly expressed in cornea, thymus, heart, brain, and skin. Cathepsin V is a multifunctional endopeptidase that is involved in both the release of antigenic peptides and the maturation of MHC class II molecules and participates in the turnover of elastin fibrils as well in the cleavage of intra- and extra-cellular substrates. Moreover, there is increasing evidence that cathepsin V may contribute to the progression of diverse diseases, due to the dysregulation of its expression and/or its activity. For instance, increased expression of cathepsin V is closely correlated with malignancies (breast cancer, squamous cell carcinoma, or colorectal cancer) as well vascular disorders (atherosclerosis, aortic aneurysm, hypertension) being the most prominent examples. This review aims to shed light on current knowledge on molecular aspects of cathepsin V (genomic organization, protein structure, substrate specificity), its regulation by protein and non-protein inhibitors as well to summarize its expression (tissue and cellular distribution). Then the core biological and pathophysiological roles of cathepsin V will be depicted, raising the question of its interest as a valuable target that can open up pioneering therapeutic avenues.
    Keywords:  Cathepsin; Cystatin; Elastin; Marker; Pathophysiology; Protease; Protease inhibitor; Proteolysis; Proteolytic enzymes
    DOI:  https://doi.org/10.1016/j.mam.2022.101086
  43. Nature. 2022 Mar 21.
    Mice eat less and explore more after intake of certain amino acids.
      
    Keywords:  Neuroscience
    DOI:  https://doi.org/10.1038/d41586-022-00830-z
  44. Nat Aging. 2021 Jan;1(1): 47-59
    Intermittent and periodic fasting, longevity and disease.
    Valter D Longo, Maira Di Tano, Mark P Mattson, Novella Guidi.
      Intermittent and periodic fasting (IF and PF, respectively) are emerging as safe strategies to affect longevity and healthspan by acting on cellular aging and disease risk factors, while causing no or minor side effects. IF lasting from 12 to 48 hours and repeated every 1 to 7 days and PF lasting 2 to 7 days and repeated once per month or less have the potential to prevent and treat disease, but their effect on cellular aging and the molecular mechanisms involved are only beginning to be unraveled. Here, we describe the different fasting methods and their effect on longevity in organisms ranging from yeast to humans, linking them to the major nutrient-sensing signaling pathways and focusing on the benefits of the fasting and the refeeding periods. We also discuss both the therapeutic potential and side effects of IF and PF with a focus on cancer, autoimmunity, neurodegeneration and metabolic and cardiovascular disease.
    DOI:  https://doi.org/10.1038/s43587-020-00013-3
  45. J Cell Biol. 2022 Apr 04. pii: e202203026. [Epub ahead of print]221(4):
    Clathrin coated pits as signaling platforms for Akt signaling.
    Elizabeth Smythe.
      Signaling by the activated epidermal growth factor receptor (EGFR) results in diverse cell fates. In this issue, Cabral-Dias et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.201808181) demonstrate how plasma membrane clathrin coated pits can act as a signaling platform for one branch of EGFR downstream signaling.
    DOI:  https://doi.org/10.1083/jcb.202203026
  46. Sci Adv. 2022 Mar 25. 8(12): eabm1140
    LAMP2A regulates the loading of proteins into exosomes.
    João Vasco Ferreira, Ana da Rosa Soares, José Ramalho, Catarina Máximo Carvalho, Maria Helena Cardoso, Petra Pintado, Ana Sofia Carvalho, Hans Christian Beck, Rune Matthiesen, Mónica Zuzarte, Henrique Girão, Guillaume van Niel, Paulo Pereira.
      Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.
    DOI:  https://doi.org/10.1126/sciadv.abm1140
  47. Am J Med Genet A. 2022 Mar 26.
    Pitfalls of X-chromosome inactivation testing in females with Fabry disease.
    Martin Řeboun, Jakub Sikora, Martin Magner, Helena Wiederlechnerová, Alena Černá, Helena Poupětová, Gabriela Štorkánova, Dita Mušálková, Gabriela Dostálová, Lubor Goláň, Aleš Linhart, Lenka Dvořáková.
      Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding alpha-galactosidase A (AGAL). The impact of X-chromosome inactivation (XCI) on the phenotype of female FD patients remains unclear. In this study we aimed to determine pitfalls of XCI testing in a cohort of 35 female FD patients. XCI was assessed by two methylation-based and two allele-specific expression assays. The results correlated, although some variance among the four assays was observed. GLA transcript analyses identified crossing-over in three patients and detected mRNA instability in three out of four analyzed null alleles. AGAL activity correlated with XCI pattern and was not influenced by the mutation type or by reduced mRNA stability. Therefore, AGAL activity may help to detect crossing-over in patients with unstable GLA alleles. Tissue-specific XCI patterns in six patients, and age-related changes in two patients were observed. To avoid misinterpretation of XCI results in female FD patients we show that (i) a combination of several XCI assays generates more reliable results and minimizes possible biases; (ii) correlating XCI to GLA expression and AGAL activity facilitates identification of cross-over events; (iii) age- and tissue-related XCI specificities of XCI patterning should be considered.
    Keywords:  Fabry disease heterozygotes; GLA transcript expression; X chromosome inactivation assay; alpha-galactosidase A activity
    DOI:  https://doi.org/10.1002/ajmg.a.62728
  48. J Cell Sci. 2022 Mar 15. pii: jcs259387. [Epub ahead of print]135(6):
    The roles of dynein and myosin VI motor proteins in endocytosis.
    Chaithra Mayya, A Hema Naveena, Pankhuri Sinha, Christian Wunder, Ludger Johannes, Dhiraj Bhatia.
      Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking.
    Keywords:  Adaptors; Clathrin-mediated endocytosis; GL–Lect; Glycolipid–lectin hypothesis; Membrane pulling force; Molecular motors; Vesicle scission and trafficking
    DOI:  https://doi.org/10.1242/jcs.259387
  49. PLoS Comput Biol. 2022 Mar 21. 18(3): e1009969
    Large self-assembled clathrin lattices spontaneously disassemble without sufficient adaptor proteins.
    Si-Kao Guo, Alexander J Sodt, Margaret E Johnson.
      Clathrin-coated structures must assemble on cell membranes to internalize receptors, with the clathrin protein only linked to the membrane via adaptor proteins. These structures can grow surprisingly large, containing over 20 clathrin, yet they often fail to form productive vesicles, instead aborting and disassembling. We show that clathrin structures of this size can both form and disassemble spontaneously when adaptor protein availability is low, despite high abundance of clathrin. Here, we combine recent in vitro kinetic measurements with microscopic reaction-diffusion simulations and theory to differentiate mechanisms of stable vs unstable clathrin assembly on membranes. While in vitro conditions drive assembly of robust, stable lattices, we show that concentrations, geometry, and dimensional reduction in physiologic-like conditions do not support nucleation if only the key adaptor AP-2 is included, due to its insufficient abundance. Nucleation requires a stoichiometry of adaptor to clathrin that exceeds 1:1, meaning additional adaptor types are necessary to form lattices successfully and efficiently. We show that the critical nucleus contains ~25 clathrin, remarkably similar to sizes of the transient and abortive structures observed in vivo. Lastly, we quantify the cost of bending the membrane under our curved clathrin lattices using a continuum membrane model. We find that the cost of bending the membrane could be largely offset by the energetic benefit of forming curved rather than flat structures, with numbers comparable to experiments. Our model predicts how adaptor density can tune clathrin-coated structures from the transient to the stable, showing that active energy consumption is therefore not required for lattice disassembly or remodeling during growth, which is a critical advance towards predicting productive vesicle formation.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009969
  50. Mol Cell. 2022 Mar 14. pii: S1097-2765(22)00166-6. [Epub ahead of print]
    Hexokinase 1 cellular localization regulates the metabolic fate of glucose.
    Adam De Jesus, Farnaz Keyhani-Nejad, Carolina M Pusec, Lauren Goodman, Justin A Geier, Joshua S Stoolman, Paulina J Stanczyk, Tivoli Nguyen, Kai Xu, Krishna V Suresh, Yihan Chen, Arianne E Rodriguez, Jason S Shapiro, Hsiang-Chun Chang, Chunlei Chen, Kriti P Shah, Issam Ben-Sahra, Brian T Layden, Navdeep S Chandel, Samuel E Weinberg, Hossein Ardehali.
      The product of hexokinase (HK) enzymes, glucose-6-phosphate, can be metabolized through glycolysis or directed to alternative metabolic routes, such as the pentose phosphate pathway (PPP) to generate anabolic intermediates. HK1 contains an N-terminal mitochondrial binding domain (MBD), but its physiologic significance remains unclear. To elucidate the effect of HK1 mitochondrial dissociation on cellular metabolism, we generated mice lacking the HK1 MBD (ΔE1HK1). These mice produced a hyper-inflammatory response when challenged with lipopolysaccharide. Additionally, there was decreased glucose flux below the level of GAPDH and increased upstream flux through the PPP. The glycolytic block below GAPDH is mediated by the binding of cytosolic HK1 with S100A8/A9, resulting in GAPDH nitrosylation through iNOS. Additionally, human and mouse macrophages from conditions of low-grade inflammation, such as aging and diabetes, displayed increased cytosolic HK1 and reduced GAPDH activity. Our data indicate that HK1 mitochondrial binding alters glucose metabolism through regulation of GAPDH.
    Keywords:  GAPDH; S-nitrosylation; hexokinase; inflammation; innate immunity; macrophage; metabolism; mitochondria; pentose phosphate pathway; subcellular localization
    DOI:  https://doi.org/10.1016/j.molcel.2022.02.028
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