bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022‒04‒03
thirty-six papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing
  1. Folliculin promotes substrate-selective mTORC1 activity by activating RagC to recruit TFE3.
  2. Quantitative phosphoproteomic analyses identify STK11IP as a lysosome-specific substrate of mTORC1 that regulates lysosomal acidification.
  3. ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.
  4. Age-dependent formation of TMEM106B amyloid filaments in human brains.
  5. Amyloid fibrils in disease FTLD-TDP are composed of TMEM106B not TDP-43.
  6. Mass spectrometry-based proteomics in neurodegenerative lysosomal storage disorders.
  7. Cryo-EM structures of the human GATOR1-Rag-Ragulator complex reveal a spatial-constraint regulated GAP mechanism.
  8. Current methods to analyse lysosome morphology, positioning, motility and function.
  9. The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.
  10. Organelle transporters and inter-organelle communication as drivers of metabolic regulation and cellular homeostasis.
  11. A Fluorogenic Far Red-Emitting Molecular Viscometer for Ascertaining Lysosomal Stress in Live Cells and Caenorhabditis elegans.
  12. Chemical Biology Screening Identifies a Vulnerability to Checkpoint Kinase Inhibitors in TSC2-Deficient Renal Angiomyolipomas.
  13. Hgs Deficiency Caused Restrictive Cardiomyopathy via Disrupting Proteostasis.
  14. Anabolic Function Downstream of TOR Controls Trade-offs Between Longevity and Reproduction at the Level of Specific Tissues in C. elegans.
  15. Metabolomic Study Using Time-of-Flight Mass Spectrometry Reveals Novel Urinary Biomarkers for Gaucher Disease Type 1.
  16. The mTORC1 Signaling Support Cellular Metabolism to Dictate Decidual NK Cells Function in Early Pregnancy.
  17. Advancing Diagnosis and Treatment of Niemann-Pick C disease through Biomarker Discovery.
  18. Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation.
  19. TET2-mediated epigenetic reprogramming of breast cancer cells impairs lysosome biogenesis.
  20. Lipidomic analysis identifies age-disease-related changes and potential new biomarkers in brain-derived extracellular vesicles from metachromatic leukodystrophy mice.
  21. EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.
  22. Protein kinase C: release from quarantine by mTORC2.
  23. Ca2+ regulation of constitutive vesicle trafficking.
  24. Safety and effectiveness of resistance training in patients with late onset Pompe disease - a pilot study.
  25. Structural and biochemical insights into lipid transport by VPS13 proteins.
  26. Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.
  27. Ras and Rab Interactor 3: From Cellular Mechanisms to Human Diseases.
  28. Membrane trafficking: ESCRTs act here, there, and everywhere.
  29. Ultrasensitive profiling of UV mutations identifies thousands of subclinical facial tumors in tuberous sclerosis complex.
  30. Neuronal Ceroid Lipofuscinosis: The Multifaceted Approach to the Clinical Issues, an Overview.
  31. Cell senescence, rapamycin and hyperfunction theory of aging.
  32. From telomere to telomere: The transcriptional and epigenetic state of human repeat elements.
  33. Sphingolipid changes in Parkinson L444P GBA mutation fibroblasts promote α-synuclein aggregation.
  34. Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization.
  35. The solute carrier SLC15A4 is required for optimal trafficking of nucleic acid-sensing TLRs and ligands to endolysosomes.
  36. Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis.
  1. PLoS Biol. 2022 Mar 31. 20(3): e3001594
    Folliculin promotes substrate-selective mTORC1 activity by activating RagC to recruit TFE3.
    Kristina Li, Shogo Wada, Bridget S Gosis, Chelsea Thorsheim, Paige Loose, Zolt Arany.
      Mechanistic target of rapamycin complex I (mTORC1) is central to cellular metabolic regulation. mTORC1 phosphorylates a myriad of substrates, but how different substrate specificity is conferred on mTORC1 by different conditions remains poorly defined. Here, we show how loss of the mTORC1 regulator folliculin (FLCN) renders mTORC1 specifically incompetent to phosphorylate TFE3, a master regulator of lysosome biogenesis, without affecting phosphorylation of other canonical mTORC1 substrates, such as S6 kinase. FLCN is a GTPase-activating protein (GAP) for RagC, a component of the mTORC1 amino acid (AA) sensing pathway, and we show that active RagC is necessary and sufficient to recruit TFE3 onto the lysosomal surface, allowing subsequent phosphorylation of TFE3 by mTORC1. Active mutants of RagC, but not of RagA, rescue both phosphorylation and lysosomal recruitment of TFE3 in the absence of FLCN. These data thus advance the paradigm that mTORC1 substrate specificity is in part conferred by direct recruitment of substrates to the subcellular compartments where mTORC1 resides and identify potential targets for specific modulation of specific branches of the mTOR pathway.
    DOI:  https://doi.org/10.1371/journal.pbio.3001594
  2. Nat Commun. 2022 Apr 01. 13(1): 1760
    Quantitative phosphoproteomic analyses identify STK11IP as a lysosome-specific substrate of mTORC1 that regulates lysosomal acidification.
    Zhenzhen Zi, Zhuzhen Zhang, Qiang Feng, Chiho Kim, Xu-Dong Wang, Philipp E Scherer, Jinming Gao, Beth Levine, Yonghao Yu.
      The evolutionarily conserved serine/threonine kinase mTORC1 is a central regulator of cell growth and proliferation. mTORC1 is activated on the lysosome surface. However, once mTORC1 is activated, it is unclear whether mTORC1 phosphorylates local lysosomal proteins to regulate specific aspects of lysosomal biology. Through cross-reference analyses of the lysosome proteome with the mTORC1-regulated phosphoproteome, we identify STK11IP as a lysosome-specific substrate of mTORC1. mTORC1 phosphorylates STK11IP at Ser404. Knockout of STK11IP leads to a robust increase of autophagy flux. Dephosphorylation of STK11IP at Ser404 represses the role of STK11IP as an autophagy inhibitor. Mechanistically, STK11IP binds to V-ATPase, and regulates the activity of V-ATPase. Knockout of STK11IP protects mice from fasting or Methionine/Choline-Deficient Diet (MCD)-induced fatty liver. Thus, our study demonstrates that STK11IP phosphorylation represents a mechanism for mTORC1 to regulate lysosomal acidification and autophagy, and points to STK11IP as a promising therapeutic target for the amelioration of diseases with aberrant autophagy signaling.
    DOI:  https://doi.org/10.1038/s41467-022-29461-8
  3. Life Sci Alliance. 2022 Jul;pii: e202101239. [Epub ahead of print]5(7):
    ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.
    Marta Wróbel, Jarosław Cendrowski, Ewelina Szymańska, Malwina Grębowicz-Maciukiewicz, Noga Budick-Harmelin, Matylda Macias, Aleksandra Szybińska, Michał Mazur, Krzysztof Kolmus, Krzysztof Goryca, Michalina Dąbrowska, Agnieszka Paziewska, Michał Mikula, Marta Miączyńska.
      Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.
    DOI:  https://doi.org/10.26508/lsa.202101239
  4. Nature. 2022 Mar 28.
    Age-dependent formation of TMEM106B amyloid filaments in human brains.
    Manuel Schweighauser, Diana Arseni, Mehtap Bacioglu, Melissa Huang, Sofia Lövestam, Yang Shi, Yang Yang, Wenjuan Zhang, Abhay Kotecha, Holly J Garringer, Ruben Vidal, Grace I Hallinan, Kathy L Newell, Airi Tarutani, Shigeo Murayama, Masayuki Miyazaki, Yuko Saito, Mari Yoshida, Kazuko Hasegawa, Tammaryn Lashley, Tamas Revesz, Gabor G Kovacs, John van Swieten, Masaki Takao, Masato Hasegawa, Bernardino Ghetti, Maria Grazia Spillantini, Benjamin Ryskeldi-Falcon, Alexey G Murzin, Michel Goedert, Sjors H W Scheres.
      Many age-dependent neurodegenerative diseases, like Alzheimer's and Parkinson's, are characterised by abundant inclusions of amyloid filaments. Filamentous inclusions of the proteins tau, amyloid-β (Aβ), α-synuclein and TDP-43 are the most common1,2. Here, we used electron cryo-microscopy (cryo-EM) structure determination to show that residues 120-254 of the lysosomal type II transmembrane protein 106B (TMEM106B) also form amyloid filaments in human brains. We determined the cryo-EM structures of TMEM106B filaments from a number of brain regions of 22 individuals with abundant amyloid deposits, including sporadic and inherited tauopathies, Aβ-amyloidoses, synucleinopathies and TDP-43 proteinopathies, as well as from the frontal cortex of 3 neurologically normal individuals with no or only few amyloid deposits. We observed three TMEM106B folds, with no clear relationships between folds and diseases. TMEM106B filaments correlated with the presence of a 29 kDa sarkosyl-insoluble fragment and globular cytoplasmic inclusions, as detected by an antibody specific for the C-terminal region of TMEM106B. The identification of TMEM106B filaments in the brains of older, but not younger, neurologically normal individuals indicates that they form in an age-dependent manner.
    DOI:  https://doi.org/10.1038/s41586-022-04650-z
  5. Nature. 2022 Mar 28.
    Amyloid fibrils in disease FTLD-TDP are composed of TMEM106B not TDP-43.
    Yi Xiao Jiang, Qin Cao, Michael R Sawaya, Romany Abskharon, Peng Ge, Michael DeTure, Dennis W Dickson, Janine Y Fu, Rachel R Ogorzalek Loo, Joseph A Loo, David S Eisenberg.
      Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition, following only Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45-64-year-olds with behavioral changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions detected by TAR DNA-binding protein (TDP-43) immunoreactivity3. Here, we extracted amyloid fibrils from brains of four patients, representing four out of five FTLD-TDP subclasses and determined their near-atomic resolution structures by cryogenic electron-microscopy (cryo-EM). Unexpectedly, all amyloid fibrils examined are composed of a 135-residue C-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, abundant non-fibrillar aggregated TDP-43 is present, as revealed by immunogold labeling. Our observations confirm that FTLD-TDP is an amyloid-involved disease and suggest that amyloid involvement in FTLD-TDP is of protein TMEM106B, rather than of TDP-43.
    DOI:  https://doi.org/10.1038/s41586-022-04670-9
  6. Mol Omics. 2022 Mar 28.
    Mass spectrometry-based proteomics in neurodegenerative lysosomal storage disorders.
    Wenping Li, Stephanie M Cologna.
      The major function of the lysosome is to degrade unwanted materials such as lipids, proteins, and nucleic acids; therefore, deficits of the lysosomal system can result in improper degradation and trafficking of these biomolecules. Diseases associated with lysosomal failure can be lethal and are termed lysosomal storage disorders (LSDs), which affect 1 in 5000 live births collectively. LSDs are inherited metabolic diseases caused by mutations in single lysosomal and non-lysosomal proteins and resulting in the subsequent accumulation of macromolecules within. Most LSD patients present with neurodegenerative clinical symptoms, as well as damage in other organs. The discovery of new biomarkers is necessary to understand and monitor these diseases and to track therapeutic progress. Over the past ten years, mass spectrometry (MS)-based proteomics has flourished in the biomarker studies in many diseases, including neurodegenerative, and more specifically, LSDs. In this review, biomarkers of disease pathophysiology and monitoring of LSDs revealed by MS-based proteomics are discussed, including examples from Niemann-Pick disease type C, Fabry disease, neuronal ceroid-lipofuscinoses, mucopolysaccharidosis, Krabbe disease, mucolipidosis, and Gaucher disease.
    DOI:  https://doi.org/10.1039/d2mo00004k
  7. Mol Cell. 2022 Mar 21. pii: S1097-2765(22)00211-8. [Epub ahead of print]
    Cryo-EM structures of the human GATOR1-Rag-Ragulator complex reveal a spatial-constraint regulated GAP mechanism.
    Shawn B Egri, Christna Ouch, Hui-Ting Chou, Zhiheng Yu, Kangkang Song, Chen Xu, Kuang Shen.
      mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.
    Keywords:  GAP; GATOR1; Rag GTPase; enzyme mechanism; mTOR complex 1; mTORC1; nutrient sensing
    DOI:  https://doi.org/10.1016/j.molcel.2022.03.002
  8. Traffic. 2022 Mar 28.
    Current methods to analyse lysosome morphology, positioning, motility and function.
    Duarte C Barral, Leopoldo Staiano, Cláudia Guimas Almeida, Dan F Cutler, Emily R Eden, Clare E Futter, Antony Galione, André R A Marques, Diego Luis Medina, Gennaro Napolitano, Carmine Settembre, Otília V Vieira, Johannes M F G Aerts, Peace Atakpa-Adaji, Gemma Bruno, Antonella Capuozzo, Elvira De Leonibus, Chiara Di Malta, Cristina Escrevente, Alessandra Esposito, Paolo Grumati, Michael J Hall, Rita O Teodoro, Susana S Lopes, J Paul Luzio, Jlenia Monfregola, Sandro Montefusco, Frances M Platt, Roman Polishuck, Maria De Risi, Irene Sambri, Chiara Soldati, Miguel C Seabra.
      Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has revealed that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling, and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyse lysosome morphology, positioning, motility, and function. We highlight the principles behind these methods, the methodological strategies, and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.
    Keywords:  Endolysosomes; Lysosomal storage diseases; Lysosome biogenesis; Lysosome exocytosis; Lysosome-related organelles; Lysosomes; Membrane contact sites; TFEB; mTOR
    DOI:  https://doi.org/10.1111/tra.12839
  9. J Cell Sci. 2022 Mar 28. pii: jcs.259421. [Epub ahead of print]
    The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.
    Prado Vargas Duarte, Ralph Hardenberg, Muriel Mari, Stefan Walter, Fulvio Reggiori, Florian Fröhlich, Ayelén González Montoro, Christian Ungermann.
      Lysosomes mediate degradation of macromolecules to their precursors for their cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. The Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein, and an effector of the minor Rab5 isoform Ypt52. Strikingly, the bph1▵ mutant has lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy inducing conditions. In agreement, proteomics of bph1▵ vacuoles reveal an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1▵ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.
    Keywords:  Atg8; Autophagy; Endosome; LYST; Rab5; Ypt52
    DOI:  https://doi.org/10.1242/jcs.259421
  10. Mol Metab. 2022 Mar 25. pii: S2212-8778(22)00050-3. [Epub ahead of print] 101481
    Organelle transporters and inter-organelle communication as drivers of metabolic regulation and cellular homeostasis.
    Aakriti Jain, Roberto Zoncu.
      Spatial compartmentalization of metabolic pathways within membrane-separated organelles is key to the ability of eukaryotic cells to precisely regulate their biochemical functions. Membrane-bound organelles such as mitochondria, endoplasmic reticulum (ER) and lysosomes enable the concentration of metabolic precursors within optimized chemical environments, greatly accelerating the efficiency of both anabolic and catabolic reactions, enabling division of labor and optimal utilization of resources. However, metabolic compartmentalization also poses a challenge to cells because it creates spatial discontinuities that must be bridged for reaction cascades to be connected and completed. To do so, cells employ different methods to coordinate metabolic fluxes occurring in different organelles, such as membrane-localized transporters to facilitate regulated metabolite exchange between mitochondria and lysosomes, non-vesicular transport pathways via physical contact sites connecting the ER with both mitochondria and lysosomes, as well as localized regulatory signaling processes that coordinately regulate the activity of all these organelles. Effective communication among these systems is essential to cellular health and function, whereas disruption of inter-organelle communication is an emerging driver in a multitude of diseases, from cancer to neurodegeneration.
    Keywords:  Contact sites; Lysosome; Metabolism; Mitochondria; Transporters; mTORC1
    DOI:  https://doi.org/10.1016/j.molmet.2022.101481
  11. Front Chem. 2022 ;10 840297
    A Fluorogenic Far Red-Emitting Molecular Viscometer for Ascertaining Lysosomal Stress in Live Cells and Caenorhabditis elegans.
    Akshay Silswal, Ashutosh Kanojiya, Apurba Lal Koner.
      The cellular physiochemical properties such as polarity, viscosity, and pH play a critical role in cellular homeostasis. The dynamic change of lysosomal viscosity in live cells associated with different environmental stress remains enigmatic and needs to be explored. We have developed a new class of Julolidine-based molecular viscometers with an extended π-conjugation to probe the lysosomal viscosity in live cells. High biocompatibility, pH tolerance, and the fluorogenic response with far red-emission (>600 nm) properties make these molecular viscometers suitable for live-cell fluorescence imaging in Caenorhabditis elegans. Among these probes, JIND-Mor is specifically designed to target lysosomes via simple modification. The real-time monitoring of lysosomal viscosity change under cellular stress was achieved. We believe that such a class of molecule viscometers has the potential to monitor lysosomal health in pathogenic conditions.
    Keywords:  cancer; cellular viscometry; far red-emitting fluoroprobe; lysosomal probe; molecular rotor
    DOI:  https://doi.org/10.3389/fchem.2022.840297
  12. Front Oncol. 2022 ;12 852859
    Chemical Biology Screening Identifies a Vulnerability to Checkpoint Kinase Inhibitors in TSC2-Deficient Renal Angiomyolipomas.
    Robert M Vaughan, Jennifer J Kordich, Chun-Yuan Chan, Nanda K Sasi, Stephanie L Celano, Kellie A Sisson, Megan Van Baren, Matthew G Kortus, Dean J Aguiar, Katie R Martin, Jeffrey P MacKeigan.
      The tuberous sclerosis complex (TSC) is a rare genetic syndrome and multisystem disease resulting in tumor formation in major organs. A molecular hallmark of TSC is a dysregulation of the mammalian target of rapamycin (mTOR) through loss-of-function mutations in either tumor suppressor TSC1 or TSC2. Here, we sought to identify drug vulnerabilities conferred by TSC2 tumor-suppressor loss through cell-based chemical biology screening. Our small-molecule chemical screens reveal a sensitivity to inhibitors of checkpoint kinase 1/2 (CHK1/2), regulators of cell cycle, and DNA damage response, in both in vitro and in vivo models of TSC2-deficient renal angiomyolipoma (RA) tumors. Further, we performed transcriptional profiling on TSC2-deficient RA cell models and discovered that these recapitulate some of the features from TSC patient kidney tumors compared to normal kidneys. Taken together, our study provides a connection between mTOR-dependent tumor growth and CHK1/2, highlighting the importance of CHK1/2 inhibition as a potential antitumor strategy in TSC2-deficient tumors.
    Keywords:  AZD7762; CHEK1/2; Chk1/2; TSC2; checkpoint kinase inhibitors; mTOR; tuberous sclerosis complex; tumor xenografts
    DOI:  https://doi.org/10.3389/fonc.2022.852859
  13. Int J Biol Sci. 2022 ;18(5): 2018-2031
    Hgs Deficiency Caused Restrictive Cardiomyopathy via Disrupting Proteostasis.
    Zhenhua Li, Tianle Wang, Chong Xin, Yao Song, Jingyi Kong, Jingping Xu, Qiqi Liu, Yan Teng, Ning Hou, Xuan Cheng, Guan Yang, Wenjia Liu, Bin Zhou, Youyi Zhang, Xiao Yang, Jian Wang.
      The molecular mechanisms underlying restrictive cardiomyopathy (RCM) are not fully understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) is a vital element of Endosomal sorting required for transport (ESCRT), which mediates protein sorting for degradation and is crucial for protein homeostasis (proteostasis) maintenance. However, the physiological function and underlying mechanisms of HGS in RCM are unexplored. We hypothesized that HGS may play vital roles in cardiac homeostasis. Cardiomyocyte-specific Hgs gene knockout mice were generated and developed a phenotype similar to human RCM. Proteomic analysis revealed that Hgs deficiency impaired lysosomal homeostasis in cardiomyocytes. Loss of Hgs disrupted cholesterol transport and lysosomal integrity, resulting in lysosomal storage disorder (LSD) with aberrant autophagosome accumulation and protein aggregation. Suppression of protein aggregation by doxycycline treatment attenuated cardiac fibrosis, and diastolic dysfunction in Hgs-knockout mice. These findings uncovered a novel physiological role of HGS in regulating cardiac lysosomal homeostasis and proteostasis, suggesting that the deficient HGS contributes to LSD-associated RCM-like cardiomyopathy.
    DOI:  https://doi.org/10.7150/ijbs.69024
  14. Front Aging. 2021 Sep;pii: 725068. [Epub ahead of print]2
    Anabolic Function Downstream of TOR Controls Trade-offs Between Longevity and Reproduction at the Level of Specific Tissues in C. elegans.
    Amber C Howard, Dilawar Mir, Santina Snow, Jordan Horrocks, Hussein Sayed, Zhengxin Ma, Aric N Rogers.
      As the most energetically expensive cellular process, translation must be finely tuned to environmental conditions. Dietary restriction attenuates signaling through the nutrient sensing mTOR pathway, which reduces translation and redirects resources to preserve the soma. These responses are associated with increased lifespan but also anabolic impairment, phenotypes also observed when translation is genetically suppressed. Here, we restricted translation downstream of mTOR separately in major tissues in C. elegans to better understand their roles in systemic adaptation and whether consequences to anabolic impairment were separable from positive effects on lifespan. Lowering translation in neurons, hypodermis, or germline tissue led to increased lifespan under well-fed conditions and improved survival upon withdrawal of food, indicating that these are key tissues coordinating enhanced survival when protein synthesis is reduced. Surprisingly, lowering translation in body muscle during development shortened lifespan while accelerating and increasing reproduction, a reversal of phenotypic trade-offs associated with systemic translation suppression. Suppressing mTORC1 selectively in body muscle also increased reproduction while slowing motility during development. In nature, this may be indicative of reduced energy expenditure related to foraging, acting as a "GO!" signal for reproduction. Together, results indicate that low translation in different tissues helps direct distinct systemic adaptations and suggest that unknown endocrine signals mediate these responses. Furthermore, mTOR or translation inhibitory therapeutics that target specific tissues may achieve desired interventions to aging without loss of whole-body anabolism.
    Keywords:  adaptive response; aging; cell non-autonomous; lifespan; mRNA translation; optimal foraging theory; reproduction; trade-offs
    DOI:  https://doi.org/10.3389/fragi.2021.725068
  15. J Proteome Res. 2022 Mar 29.
    Metabolomic Study Using Time-of-Flight Mass Spectrometry Reveals Novel Urinary Biomarkers for Gaucher Disease Type 1.
    Iskren Menkovic, Michel Boutin, Abdulfatah Alayoubi, Filipa Curado, Peter Bauer, François E Mercier, Christiane Auray-Blais.
      Gaucher disease (GD) is a lysosomal storage disorder resulting from a biallelic mutation in the gene GBA1, leading to deficiencies in the enzyme β-glucocerebrosidase (Gcase). Inabilities of the Gcase to catabolize its substrate result in the accumulation of sphingolipids in macrophages, which impairs the cell functions and ultimately leads to multisystemic clinical manifestations. Important variability in symptoms and manifestations may lead to challenging diagnosis and patient care. Plasma glucosylsphingosine (lyso-Gb1) is a biomarker frequently used for prognosis, monitoring, and patient follow-up. While lyso-Gb1 appears to be a valid biomarker, few studies have investigated other matrices for potential GD biomarkers. The main objective of this study was to investigate the urine matrix as a potential source of new GD biomarkers by performing a metabolomic study using time-of-flight mass spectrometry. Our study highlighted a significant increase of eight urinary lyso-Gb1 analogues. Moreover, a novel class of biomarkers, named polycyclic lyso-Gb1 analogues, was identified. These four new molecules were more elevated than lyso-Gb1 and related analogues in urine specimens of GD patients. Further investigations are warranted to validate the efficiency of these newly found biomarkers on a larger cohort of Gaucher patients and to compare them with plasma biomarkers currently quantified in clinical laboratories.
    Keywords:  Gaucher disease; biomarkers; glucosylsphingosine; lyso-Gb1; lyso-Gb1 analogues; metabolomic; polycyclic lyso-Gb1 analogues; time-of-flight mass spectrometry; urine
    DOI:  https://doi.org/10.1021/acs.jproteome.2c00068
  16. Front Immunol. 2022 ;13 771732
    The mTORC1 Signaling Support Cellular Metabolism to Dictate Decidual NK Cells Function in Early Pregnancy.
    Song Yan, Jie Dong, Chenxi Qian, Shuqiang Chen, Qian Xu, Hui Lei, Xiaohong Wang.
      Cellular metabolism plays an important role in regulating both human and murine NK cell functions. However, it remains unclear whether cellular metabolic process impacts on the function of decidual NK cells (dNK), essential tissue-resident immune cells maintaining the homeostasis of maternal-fetal interface. Remarkably, we found that glycolysis blockage enhances dNK VEGF-A production but restrains its proliferation. Furthermore, levels of IFN-γ and TNF-α secreted by dNK get decreased when glycolysis or oxidative phosphorylation (OXPHOS) is inhibited. Additionally, glycolysis, OXPHOS, and fatty acid oxidation disruption has little effects on the secretion and the CD107a-dependent degranulation of dNK. Mechanistically, we discovered that the mammalian target of rapamycin complex 1 (mTORC1) signaling inhibition leads to decreased glycolysis and OXPHOS in dNK. These limited metabolic processes are associated with attenuated dNK functions, which include restricted production of cytokines including IFN-γ and TNF-α, diminished CD107a-dependent degranulation, and restrained dNK proliferation. Finally, we reported that the protein levels of several glycolysis-associated enzymes are altered and the mTORC1 activity is significantly lower in the decidua of women with recurrent pregnancy loss (RPL) compared with normal pregnancy, which might give new insights about the pathogenesis of RPL. Collectively, our data demonstrate that glucose metabolism and mTORC1 signaling support dNK functions in early pregnancy.
    Keywords:  RPL; cytokines; cytotoxicity; decidual NK cells; mTORC1; metabolism
    DOI:  https://doi.org/10.3389/fimmu.2022.771732
  17. Explor Neuroprotective Ther. 2021 Dec 30. 1(3): 146-158
    Advancing Diagnosis and Treatment of Niemann-Pick C disease through Biomarker Discovery.
    Xuntian Jiang, Daniel S Ory.
      Niemann-Pick C is a rare neurodegenerative, lysosomal storage disease caused by accumulation of unesterified cholesterol. Diagnosis of the disease is often delayed due to its rarity, the heterogeneous presentation and the early non-specific symptoms. The discovery of disease-specific biomarkers - cholestane-3β,5α,6β-triol (C-triol), trihydroxycholanic acid glycinate (TCG) and N-palmitoyl-O-phosphocholineserine (PPCS, initially referred to as lysoSM-509) - has led to development of non-invasive, blood-based diagnostics. Dissemination of these rapid, sensitive, and specific clinical assays has accelerated diagnosis. Moreover, the superior receiver operating characteristic of the TCG bile acid biomarker and its detection in dried blood spots has also facilitated development of a newborn screen for NPC, which is currently being piloted in New York state. The C-triol, TCG and PPCS biomarkers have also proven useful for monitoring treatment response in peripheral tissues, but are uninformative with respect to treatment efficacy in the central nervous system (CNS). A major gap for the field is the lack of a validated, non-invasive biomarker to monitor the course of disease and CNS response to therapy.
    DOI:  https://doi.org/10.37349/ent.2021.00012
  18. Sci Adv. 2022 Apr;8(13): eabm5149
    Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation.
    Sudeep Banjade, Lu Zhu, Jeffrey R Jorgensen, Sho W Suzuki, Scott D Emr.
      The general mechanisms by which ESCRTs (Endosomal Sorting Complexes Required for Transport) are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin are critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.
    DOI:  https://doi.org/10.1126/sciadv.abm5149
  19. Life Sci Alliance. 2022 Jul;pii: e202101283. [Epub ahead of print]5(7):
    TET2-mediated epigenetic reprogramming of breast cancer cells impairs lysosome biogenesis.
    Audrey Laurent, Thierry Madigou, Maud Bizot, Marion Turpin, Gaëlle Palierne, Elise Mahé, Sarah Guimard, Raphaël Métivier, Stéphane Avner, Christine Le Péron, Gilles Salbert.
      Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression by controlling the chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs, and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. The TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes that, interestingly, differed in part from the one observed upon treatment with the hypomethylating agent decitabine. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC-binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function. Thus, an extensive cross-talk between TET2 and the oncogenic transcription factor MYC establishes a lysosomal storage disease-like state that contributes to an exacerbated sensitivity to autophagy inducers.
    DOI:  https://doi.org/10.26508/lsa.202101283
  20. Lipids Health Dis. 2022 Mar 27. 21(1): 32
    Lipidomic analysis identifies age-disease-related changes and potential new biomarkers in brain-derived extracellular vesicles from metachromatic leukodystrophy mice.
    Melissa R Pergande, Christina Kang, Diann George, Pearl A Sutter, Stephen J Crocker, Stephanie M Cologna, Maria I Givogri.
      BACKGROUND: Recent findings show that extracellular vesicle constituents can exert short- and long-range biological effects on neighboring cells in the brain, opening an exciting avenue for investigation in the field of neurodegenerative diseases. Although it is well documented that extracellular vesicles contain many lipids and are enriched in sphingomyelin, cholesterol, phosphatidylserines and phosphatidylinositols, no reports have addressed the lipidomic profile of brain derived EVs in the context of Metachromatic Leukodystrophy, a lysosomal storage disease with established metabolic alterations in sulfatides.METHODS: In this study, we isolated and characterized the lipid content of brain-derived EVs using the arylsulfatase A knockout mouse as a model of the human condition.
    RESULTS: Our results suggest that biogenesis of brain-derived EVs is a tightly regulated process in terms of size and protein concentration during postnatal life. Our lipidomic analysis demonstrated that sulfatides and their precursors (ceramides) as well as other lipids including fatty acids are altered in an age-dependent manner in EVs isolated from the brain of the knockout mouse.
    CONCLUSIONS: In addition to the possible involvement of EVs in the pathology of Metachromatic Leukodystrophy, our study underlines that measuring lipid signatures in EVs may be useful as biomarkers of disease, with potential application to other genetic lipidoses.
    Keywords:  Extracellular vesicles; Lipidomic; Mass spectrometry; Metachromatic leukodystrophy
    DOI:  https://doi.org/10.1186/s12944-022-01644-8
  21. Cancer Lett. 2022 Mar 23. pii: S0304-3835(22)00124-0. [Epub ahead of print] 215641
    EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.
    Jie Wang, Jonathan D Wren, Yingjun Ding, Junxiong Chen, Nikhil Mittal, Chao Xu, Xing Li, Cengxi Zeng, Meng Wang, Jing Shi, Yanhui H Zhang, Sangyoon J Han, Xin A Zhang.
      As a partner of tetraspanins, EWI2 suppresses glioblastoma, melanoma, and prostate cancer; but its role in lung cancer has not been investigated. Bioinformatics analysis reveals that EWI2 gene expression is up regulated in lung adenocarcinoma and higher expression of EWI2 mRNA may predict poorer overall survival. However, experimental analysis shows that EWI2 protein is actually downregulated constantly in the tissues of lung adenocarcinoma and lung squamous cell carcinoma. Forced expression of EWI2 in human lung adenocarcinoma cells reduces total cellular and cell surface levels of various integrins and growth factor receptors, which initiates the outside-in motogenic and mitogenic signaling. These reductions result in the decreases in 1) cell-matrix adhesion, cell movement, and cell transformation in vitro and 2) tumor growth, burden, and metastasis in vivo, and result from the increases in lysosomal trafficking and proteolytic degradation of theses membrane receptors. EWI2 elevates lysosome formation by promoting nuclear retention of TFEB, the master transcription factor driving lysosomogenesis. In conclusion, EWI2 as a lung cancer suppressor attenuates lung cancer cells in a comprehensive fashion by inhibiting both tumor growth and tumor metastasis; EWI2 as an endolysosome regulator promotes lysosome activity to enhance lysosomal degradation of growth factor receptors and integrins and then reduce their levels and functions; and EWI2 can become a promising therapeutic candidate given its accessibility at the cell surface, dual inhibition on growth factor receptors and integrins, and broad-spectrum anti-cancer activity. More importantly, our observations also provide a novel therapeutic strategy to bypass the resistance to EGFR inhibitors.
    Keywords:  EGFR; Integrin; Lysosome; TFEB; Tumor metastasis
    DOI:  https://doi.org/10.1016/j.canlet.2022.215641
  22. Trends Biochem Sci. 2022 Mar 28. pii: S0968-0004(22)00062-7. [Epub ahead of print]
    Protein kinase C: release from quarantine by mTORC2.
    Timothy R Baffi, Alexandra C Newton.
      Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation.
    Keywords:  PKC; autophosphorylation; mTORC2; phosphorylation
    DOI:  https://doi.org/10.1016/j.tibs.2022.03.003
  23. Fac Rev. 2022 ;11 6
    Ca2+ regulation of constitutive vesicle trafficking.
    John Sargeant, Jesse C Hay.
      Constitutive vesicle trafficking is the default pathway used by all cells for movement of intracellular cargoes between subcellular compartments and in and out of the cell. Classically, constitutive trafficking was thought to be continuous and unregulated, in contrast to regulated secretion, wherein vesicles are stored intracellularly until undergoing synchronous membrane fusion following a Ca2+ signal. However, as shown in the literature reviewed here, many continuous trafficking steps can be up- or down-regulated by Ca2+, including several steps associated with human pathologies. Notably, we describe a series of Ca2+ pumps, channels, Ca2+-binding effector proteins, and their trafficking machinery targets that together regulate the flux of cargo in response to genetic alterations as well as baseline and agonist-dependent Ca2+ signals. Here, we review the most recent advances, organized by organellar location, that establish the importance of these components in trafficking steps. Ultimately, we conclude that Ca2+ regulates an expanding series of distinct mechanistic steps. Furthermore, the involvement of Ca2+ in trafficking is complex. For example, in some cases, the same Ca2+ effectors regulate surprisingly distinct trafficking steps, or even the same trafficking step with opposing influences, through binding to different target proteins.
    Keywords:  Golgi; apoptosis-linked gene 2 (ALG-2); calcium; calcium channel; calcium signaling; endoplasmic reticulum; late endosomes; lysosomes; secretion; vesicle coat; vesicle trafficking
    DOI:  https://doi.org/10.12703/r/11-6
  24. Neuromuscul Disord. 2022 Feb 17. pii: S0960-8966(22)00035-9. [Epub ahead of print]
    Safety and effectiveness of resistance training in patients with late onset Pompe disease - a pilot study.
    Caleb Bhatnagar, Jeet Shah, Bhumi Ramani, Abhilasha Surampalli, Mahima Avanti, Shlomit Radom-Aizik, Margaret Knight, Lan Weiss, Vince Caiozzo, Virginia Kimonis.
      Pompe disease is a progressive myopathy resulting from deficiency in lysosomal enzyme acid α-glucosidase (GAA), which leads to glycogen accumulation in lysosomes primarily in skeletal and cardiac muscle. Enzyme replacement therapy (ERT) with recombinant human (rh) GAA works well in alleviating the cardiomyopathy; however, many patients continue to have progressive muscle weakness. The purpose of this study was to evaluate the effectiveness of a respiratory training combined with 24-week supervised resistance training program on muscle strength (measured by Biodex)), and respiratory function including maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP) in subjects with late onset Pompe disease receiving ERT. Ten subjects participated in a 24-week resistance exercise program, three times per week, in addition to respiratory muscle exercise training six days per week. Overall, at the end of the resistance training program, as measured by Biodex dynamometry, the leg extensor strength improved by 10.5 ± 3.2Nm. (<p 0.01), leg flexors improved by 12.1 ± 4.1Nm (p < 0.01), the elbow flexors improved by 5.1 ± 2.3Nm (p = 0.03), and the elbow extensor strength improved by a mean of 4.5 ± 1.9Nm. (p = 0.02). MIP improved by 8.5 ± 3.7 cm H2O (p = 0.03) and the MEP by 6.4 ± 4.4 (p = 0.16). The exercise training significantly improved the trajectories of MIP and 6 MWT outcomes but not FVC when compared with the natural history data available in 6 individuals. These pilot results indicate that resistance training combined with respiratory training and ERT had a positive effect on muscular strength, functional capacity, and respiratory function in patients with late-onset Pompe disease and might be considered as a potential adjunct therapy in this population.
    Keywords:  Exercise; Neuromuscular; Pompe disease; Resistance
    DOI:  https://doi.org/10.1016/j.nmd.2022.02.002
  25. J Cell Biol. 2022 Jun 06. pii: e202202030. [Epub ahead of print]221(5):
    Structural and biochemical insights into lipid transport by VPS13 proteins.
    Jyoti Adlakha, Zhouping Hong, PeiQi Li, Karin M Reinisch.
      VPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are anchored between membranes via interactions with receptors, including both peripheral and integral membrane proteins. Here we present the crystal structure of VPS13s adaptor binding domain (VAB) complexed with a Pro-X-Pro peptide recognition motif present in one such receptor, the integral membrane protein Mcp1p, and show biochemically that other Pro-X-Pro motifs bind the VAB in the same site. We further demonstrate that Mcp1p and another integral membrane protein that interacts directly with human VPS13A, XK, are scramblases. This finding supports an emerging paradigm of a partnership between bulk lipid transport proteins and scramblases. Scramblases can re-equilibrate lipids between membrane leaflets as lipids are removed from or inserted into the cytosolic leaflet of donor and acceptor organelles, respectively, in the course of protein-mediated transport.
    DOI:  https://doi.org/10.1083/jcb.202202030
  26. Brain. 2022 Mar 31. pii: awab376. [Epub ahead of print]
    Profiling PI3K-AKT-MTOR variants in focal brain malformations reveals new insights for diagnostic care.
    Filomena Pirozzi, Matthew Berkseth, Rylee Shear, Lorenzo Gonzalez, Andrew E Timms, Josef Sulc, Emily Pao, Nora Oyama, Francesca Forzano, Valerio Conti, Renzo Guerrini, Emily S Doherty, Sulagna C Saitta, Christina M Lockwood, Colin C Pritchard, William B Dobyns, Edward Novotny, Jason N N Wright, Russell P Saneto, Seth Friedman, Jason Hauptman, Jeffrey Ojemann, Raj P Kapur, Ghayda M Mirzaa.
      Focal malformations of cortical development including focal cortical dysplasia, hemimegalencephaly and megalencephaly, are a spectrum of neurodevelopmental disorders associated with brain overgrowth, cellular and architectural dysplasia, intractable epilepsy, autism and intellectual disability. Importantly, focal cortical dysplasia is the most common cause of focal intractable paediatric epilepsy. Gain and loss of function variants in the PI3K-AKT-MTOR pathway have been identified in this spectrum, with variable levels of mosaicism and tissue distribution. In this study, we performed deep molecular profiling of common PI3K-AKT-MTOR pathway variants in surgically resected tissues using droplet digital polymerase chain reaction (ddPCR), combined with analysis of key phenotype data. A total of 159 samples, including 124 brain tissue samples, were collected from 58 children with focal malformations of cortical development. We designed an ultra-sensitive and highly targeted molecular diagnostic panel using ddPCR for six mutational hotspots in three PI3K-AKT-MTOR pathway genes, namely PIK3CA (p.E542K, p.E545K, p.H1047R), AKT3 (p.E17K) and MTOR (p.S2215F, p.S2215Y). We quantified the level of mosaicism across all samples and correlated genotypes with key clinical, neuroimaging and histopathological data. Pathogenic variants were identified in 17 individuals, with an overall molecular solve rate of 29.31%. Variant allele fractions ranged from 0.14 to 22.67% across all mutation-positive samples. Our data show that pathogenic MTOR variants are mostly associated with focal cortical dysplasia, whereas pathogenic PIK3CA variants are more frequent in hemimegalencephaly. Further, the presence of one of these hotspot mutations correlated with earlier onset of epilepsy. However, levels of mosaicism did not correlate with the severity of the cortical malformation by neuroimaging or histopathology. Importantly, we could not identify these mutational hotspots in other types of surgically resected epileptic lesions (e.g. polymicrogyria or mesial temporal sclerosis) suggesting that PI3K-AKT-MTOR mutations are specifically causal in the focal cortical dysplasia-hemimegalencephaly spectrum. Finally, our data suggest that ultra-sensitive molecular profiling of the most common PI3K-AKT-MTOR mutations by targeted sequencing droplet digital polymerase chain reaction is an effective molecular approach for these disorders with a good diagnostic yield when paired with neuroimaging and histopathology.
    Keywords:  ddPCR; epilepsy; focal cortical dysplasia; hemimegalencephaly; mosaicism
    DOI:  https://doi.org/10.1093/brain/awab376
  27. Front Cell Dev Biol. 2022 ;10 824961
    Ras and Rab Interactor 3: From Cellular Mechanisms to Human Diseases.
    Ruinan Shen, Caitlin J Murphy, Xiaowen Xu, Mingzheng Hu, Jianqing Ding, Chengbiao Wu.
      Ras and Rab interactor 3 (RIN3) functions as a Guanine nucleotide Exchange Factor (GEF) for some members of the Rab family of small GTPase. By promoting the activation of Rab5, RIN3 plays an important role in regulating endocytosis and endocytic trafficking. In addition, RIN3 activates Ras, another small GTPase, that controls multiple signaling pathways to regulate cellular function. Increasing evidence suggests that dysregulation of RIN3 activity may contribute to the pathogenesis of several disease conditions ranging from Paget's Disease of the Bone (PDB), Alzheimer's Disease (AD), Chronic Obstructive Pulmonary Disease (COPD) and to obesity. Recent genome-wide association studies (GWAS) identified variants in the RIN3 gene to be linked with these disease conditions. Interestingly, some variants appear to be missense mutations in the functional domains of the RIN3 protein while most variants are located in the noncoding regions of the RIN3 gene, potentially altering its gene expression. However, neither the protein structure of RIN3 nor its exact function(s) (except for its GEF activity) has been fully defined. Furthermore, how the polymorphisms/variants contribute to disease pathogenesis remain to be understood. Herein, we examine, and review published studies in an attempt to provide a better understanding of the physiological function of RIN3; More importantly, we construct a framework linking the polymorphisms/variants of RIN3 to altered cell signaling and endocytic traffic, and to potential disease mechanism(s).
    Keywords:  Alzheimer’s disease; RIN3; Rab5; endocytosis; trafficking
    DOI:  https://doi.org/10.3389/fcell.2022.824961
  28. Curr Biol. 2022 Mar 28. pii: S0960-9822(22)00265-2. [Epub ahead of print]32(6): R292-R294
    Membrane trafficking: ESCRTs act here, there, and everywhere.
    Leticia Lemus, Veit Goder.
      Endosomal sorting complex required for transport (ESCRT) proteins can promote extreme membrane deformations, including scission and sealing. New work uncovers a link between these proteins and the early secretory pathway that is functionally important for programmed autophagy during Drosophila development.
    DOI:  https://doi.org/10.1016/j.cub.2022.02.043
  29. J Clin Invest. 2022 Mar 31. pii: e155858. [Epub ahead of print]
    Ultrasensitive profiling of UV mutations identifies thousands of subclinical facial tumors in tuberous sclerosis complex.
    Katarzyna Klonowska, Joannes M Grevelink, Krinio Giannikou, Barbara A Ogorek, Zachary T Herbert, Aaron R Thorner, Thomas N Darling, Joel Moss, David J Kwiatkowski.
      BACKGROUND: Tuberous Sclerosis Complex (TSC) is a neurogenetic syndrome due to loss-of-function mutations in TSC2 or TSC1, characterized by tumors at multiple body sites, including facial angiofibroma (FAF). Here, an ultrasensitive assessment of the extent and range of UV-induced mutations in TSC facial skin was performed.METHODS: A Multiplex High-sensitivity PCR Assay (MHPA) was developed, enabling mutation detection at extremely low (<0.1%) variant allele frequencies (VAF).
    RESULTS: MHPA assays were developed for both TSC2 and TP53, and applied to 81 samples, including 66 skin biopsies. UV-induced second hit mutation causing inactivation of TSC2 was pervasive in TSC facial skin with an average of 4.8 mutations per 2 mm biopsy at median VAF 0.08%, generating >150,000 incipient facial tumors (subclinical 'micro-FAFs') in the average TSC subject. The MHPA analysis also led to the identification of a refined UV-related indel signature and a recurrent complex mutation pattern, consisting of both a single or dinucleotide variant, and a 1-9 nt deletion, in cis.
    CONCLUSION: TSC facial skin can be viewed as harboring a patchwork of clonal fibroblast proliferations (micro-FAF) with indolent growth, a small proportion of which develop into clinically observable FAF. Our observations also expand the spectrum of UV-related mutation signatures.
    FUNDING: This work was supported by the TSC Alliance, Engles Family Fund for Research in TSC and LAM, and National Institutes of Health, National Heart, Lung, and Blood Institute [U01HL131022-04; Intramural Research Program].
    Keywords:  Dermatology; Genetics; Molecular genetics; Skin cancer; Tumor suppressors
    DOI:  https://doi.org/10.1172/JCI155858
  30. Front Neurol. 2022 ;13 811686
    Neuronal Ceroid Lipofuscinosis: The Multifaceted Approach to the Clinical Issues, an Overview.
    Alessandro Simonati, Ruth E Williams.
      The main aim of this review is to summarize the current state-of-art in the field of childhood Neuronal Ceroid Lipofuscinosis (NCL), a group of rare neurodegenerative disorders. These are genetic diseases associated with the formation of toxic endo-lysosomal storage. Following a brief historical review of the evolution of NCL definition, a clinically-oriented approach is used describing how the early symptoms and signs affecting motor, visual, cognitive domains, and including seizures, may lead clinicians to a rapid molecular diagnosis, avoiding the long diagnostic odyssey commonly observed. We go on to focus on recent advances in NCL research and summarize contributions to knowledge of the pathogenic mechanisms underlying NCL. We describe the large variety of experimental models which have aided this research, as well as the most recent technological developments which have shed light on the main mechanisms involved in the cellular pathology, such as apoptosis and autophagy. The search for innovative therapies is described. Translation of experimental data into therapeutic approaches is being established for several of the NCLs, and one drug is now commercially available. Lastly, we show the importance of palliative care and symptomatic treatments which are still the main therapeutic interventions.
    Keywords:  NCL clinical features; NCL pathogenetic mechanisms; NCL review; NCL treatments; neuronal ceroid lipofuscinosis
    DOI:  https://doi.org/10.3389/fneur.2022.811686
  31. Cell Cycle. 2022 Mar 31. 1-12
    Cell senescence, rapamycin and hyperfunction theory of aging.
    Mikhail V Blagosklonny.
      A hallmark of cellular senescence is proliferation-like activity of growth-promoting pathways (such as mTOR and MAPK) in non-proliferating cells. When the cell cycle is arrested, these pathways convert arrest to senescence (geroconversion), rendering cells hypertrophic, beta-Gal-positive and hyperfunctional. The senescence-associated secretory phenotype (SASP) is one of the numerous hyperfunctions. Figuratively, geroconversion is a continuation of growth in non-proliferating cells. Rapamycin, a reversible inhibitor of growth, slows down mTOR-driven geroconversion. Developed two decades ago, this model had accurately predicted that rapamycin must extend life span of animals. However, the notion that senescent cells directly cause organismal aging is oversimplified. Senescent cells contribute to organismal aging but are not strictly required. Cell senescence and organismal aging can be linked indirectly via the same underlying cause, namely hyperfunctional signaling pathways such as mTOR.
    Keywords:  Senescence; geroconversion; geroscience; gerostatics; healthspan; rapalogs; sirolimus
    DOI:  https://doi.org/10.1080/15384101.2022.2054636
  32. Science. 2022 Apr;376(6588): eabk3112
    From telomere to telomere: The transcriptional and epigenetic state of human repeat elements.
    Savannah J Hoyt, Jessica M Storer, Gabrielle A Hartley, Patrick G S Grady, Ariel Gershman, Leonardo G de Lima, Charles Limouse, Reza Halabian, Luke Wojenski, Matias Rodriguez, Nicolas Altemose, Arang Rhie, Leighton J Core, Jennifer L Gerton, Wojciech Makalowski, Daniel Olson, Jeb Rosen, Arian F A Smit, Aaron F Straight, Mitchell R Vollger, Travis J Wheeler, Michael C Schatz, Evan E Eichler, Adam M Phillippy, Winston Timp, Karen H Miga, Rachel J O'Neill.
      Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.
    DOI:  https://doi.org/10.1126/science.abk3112
  33. Brain. 2022 Apr 01. pii: awab371. [Epub ahead of print]
    Sphingolipid changes in Parkinson L444P GBA mutation fibroblasts promote α-synuclein aggregation.
    Céline Galvagnion, Frederik Ravnkilde Marlet, Silvia Cerri, Anthony H V Schapira, Fabio Blandini, Donato A Di Monte.
      Intraneuronal accumulation of aggregated α-synuclein is a pathological hallmark of Parkinson's disease. Therefore, mechanisms capable of promoting α-synuclein deposition bear important pathogenetic implications. Mutations of the glucocerebrosidase 1 (GBA) gene represent a prevalent Parkinson's disease risk factor. They are associated with loss of activity of a key enzyme involved in lipid metabolism, glucocerebrosidase, supporting a mechanistic relationship between abnormal α-synuclein-lipid interactions and the development of Parkinson pathology. In this study, the lipid membrane composition of fibroblasts isolated from control subjects, patients with idiopathic Parkinson's disease and Parkinson's disease patients carrying the L444P GBA mutation (PD-GBA) was assayed using shotgun lipidomics. The lipid profile of PD-GBA fibroblasts differed significantly from that of control and idiopathic Parkinson's disease cells. It was characterized by an overall increase in sphingolipid levels. It also featured a significant increase in the proportion of ceramide, sphingomyelin and hexosylceramide molecules with shorter chain length and a decrease in the percentage of longer-chain sphingolipids. The extent of this shift was correlated to the degree of reduction of fibroblast glucocerebrosidase activity. Lipid extracts from control and PD-GBA fibroblasts were added to recombinant α-synuclein solutions. The kinetics of α-synuclein aggregation were significantly accelerated after addition of PD-GBA extracts as compared to control samples. Amyloid fibrils collected at the end of these incubations contained lipids, indicating α-synuclein-lipid co-assembly. Lipids extracted from α-synuclein fibrils were also analysed by shotgun lipidomics. Data revealed that the lipid content of these fibrils was significantly enriched by shorter-chain sphingolipids. In a final set of experiments, control and PD-GBA fibroblasts were incubated in the presence of the small molecule chaperone ambroxol. This treatment restored glucocerebrosidase activity and sphingolipid levels and composition of PD-GBA cells. It also reversed the pro-aggregation effect that lipid extracts from PD-GBA fibroblasts had on α-synuclein. Taken together, the findings of this study indicate that the L444P GBA mutation and consequent enzymatic loss are associated with a distinctly altered membrane lipid profile that provides a biological fingerprint of this mutation in Parkinson fibroblasts. This altered lipid profile could also be an indicator of increased risk for α-synuclein aggregate pathology.
    Keywords:   GBA ; Parkinson’s disease; fibroblasts; lipidomics; α-synuclein
    DOI:  https://doi.org/10.1093/brain/awab371
  34. Nat Struct Mol Biol. 2022 Mar 28.
    Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization.
    Edward A Partlow, Kevin S Cannon, Gunther Hollopeter, Richard W Baker.
      Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.
    DOI:  https://doi.org/10.1038/s41594-022-00749-z
  35. Proc Natl Acad Sci U S A. 2022 Apr 05. 119(14): e2200544119
    The solute carrier SLC15A4 is required for optimal trafficking of nucleic acid-sensing TLRs and ligands to endolysosomes.
    Ivo Rimann, Rosana Gonzalez-Quintial, Roberto Baccala, William B Kiosses, John R Teijaro, Christopher G Parker, Xiaohong Li, Bruce Beutler, Dwight H Kono, Argyrios N Theofilopoulos.
      SignificanceA large body of evidence has indicated that recognition of self-nucleic acids by endosomal toll-like receptors (TLRs) is central to the pathogenesis of lupus-like systemic autoimmunity in spontaneous mouse models, and the solute carrier SLC15A4 is required for this recognition. Here we describe a mechanism in which SLC15A4 is a major contributor to the proper trafficking of TLRs and their ligands to endolysosomes, wherein recognition and signaling is initiated. This finding supports ongoing efforts to identify pharmacologic inhibitors for this carrier as a means to treat lupus and other inflammatory disorders.
    Keywords:  AP-3; IFN-I; SLC15 solute carriers; lupus; nucleic acid–sensing TLRs
    DOI:  https://doi.org/10.1073/pnas.2200544119
  36. Nat Commun. 2022 Apr 01. 13(1): 1732
    Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis.
    Tomasz J Nawara, Yancey D Williams, Tejeshwar C Rao, Yuesong Hu, Elizabeth Sztul, Khalid Salaita, Alexa L Mattheyses.
      Clathrin polymerization and changes in plasma membrane architecture are necessary steps in forming vesicles to internalize cargo during clathrin-mediated endocytosis (CME). Simultaneous analysis of clathrin dynamics and membrane structure is challenging due to the limited axial resolution of fluorescence microscopes and the heterogeneity of CME. This has fueled conflicting models of vesicle assembly and obscured the roles of flat clathrin assemblies. Here, using Simultaneous Two-wavelength Axial Ratiometry (STAR) microscopy, we bridge this critical knowledge gap by quantifying the nanoscale dynamics of clathrin-coat shape change during vesicle assembly. We find that de novo clathrin accumulations generate both flat and curved structures. High-throughput analysis reveals that the initiation of vesicle curvature does not directly correlate with clathrin accumulation. We show clathrin accumulation is preferentially simultaneous with curvature formation at shorter-lived clathrin-coated vesicles (CCVs), but favors a flat-to-curved transition at longer-lived CCVs. The broad spectrum of curvature initiation dynamics revealed by STAR microscopy supports multiple productive mechanisms of vesicle formation and advocates for the flexible model of CME.
    DOI:  https://doi.org/10.1038/s41467-022-29317-1
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