bims-lysosi 2022-02-27 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022–02–27
thirty-two papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Inositol triphosphate-triggered calcium release from the endoplasmic reticulum induces lysosome biogenesis via TFEB/ TFE3.
Low level lysosomal membrane permeabilization for limited release and sub-lethal functions of cathepsin proteases in the cytosol and nucleus.
The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB.
Imatinib disturbs lysosomal function and morphology and impairs the activity of mTORC1 in human hepatocyte cell lines.
Lysosomes at the Crossroads of Cell Metabolism, Cell Cycle, and Stemness.
The role of lysosomal cathepsins in neurodegeneration: Mechanistic insights, diagnostic potential and therapeutic approaches.
Transport of lysosomes decreases in the perinuclear region: Insights from changepoint analysis.
Lysosomes: Multifunctional compartments ruled by a complex regulatory network.
Glucocerebrosidase-associated Parkinson disease: Pathogenic mechanisms and potential drug treatments.
Loss of Slc38a4 imprinting is a major cause of mouse placenta hyperplasia in somatic cell nuclear transferred embryos at late gestation.
YAP regulates an SGK1/mTORC1/SREBP-dependent lipogenic program to support proliferation and tissue growth.
Gene Therapy Developments for Pompe Disease.
ER-phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum.
Saturation variant interpretation using CRISPR prime editing.
Loss of legumain induces premature senescence and mediates aging-related renal fibrosis.
Role of Lysosomal Acidification Dysfunction in Mesenchymal Stem Cell Senescence.
The mTOR/4E-BP1/eIF4E signalling pathway as source of cancer drug targets.
PARK2 regulates eIF4B-driven lymphomagenesis.
Deletion of Slc6a14 reduces cancer growth and metastatic spread and improves survival in KPC mouse model of spontaneous pancreatic cancer.
MON-2, a Golgi protein, promotes longevity by upregulating autophagy through mediating inter-organelle communications.
Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates.
Interaction of TOR and PKA Signaling in S. cerevisiae.
Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.
Innate Immunity in Mucopolysaccharide Diseases.
Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation.
Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation.
Low-dose metformin targets the lysosomal AMPK pathway through PEN2.
Tissue-specific alternative splicing separates the catalytic and cell signaling functions of human leucyl-tRNA synthetase.
The role of SHMT2 in modulating lipid metabolism in hepatocytes via glycine-mediated mTOR activation.
Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.
Galactocerebrosidase deficiency induces an increase in lactosylceramide content: a new hallmark of Krabbe disease?
CHML is an NRF2 target gene that regulates mTOR function.

J Biol Chem. 2022 Feb 16. pii: S0021-9258(22)00180-6. [Epub ahead of print] 101740

Inositol triphosphate-triggered calcium release from the endoplasmic reticulum induces lysosome biogenesis via TFEB/ TFE3.

Mouhannad Malek, Anna M Wawrzyniak, Michael Ebner, Dmytro Puchkov, Volker Haucke.

  Lysosomes serve as dynamic regulators of cell and organismal physiology by integrating the degradation of macromolecules with receptor and nutrient signaling. Previous studies have established that activation of the transcription factors TFEB and TFE3 induces the expression of lysosomal genes and proteins in signaling-inactive starved cells, that is, under conditions when activity of the master regulator of nutrient-sensing signaling mTORC1 is repressed. How lysosome biogenesis is triggered in signaling-active cells is incompletely understood. Here we identify a role for calcium release from the lumen of the endoplasmic reticulum (ER) in the control of lysosome biogenesis that is independent of mTORC1. We show using functional imaging that calcium efflux from ER stores induced by inositol-triphosphate [IP3] accumulation upon depletion of INPP5A, an inositol 5-phosphatase downregulated in cancer and defective in spinocerebellar ataxia, or receptor-mediated phospholipase C activation leads to the induction of lysosome biogenesis. This mechanism involves calcineurin and the nuclear translocation and elevated transcriptional activity of TFEB/ TFE3. Our findings reveal a crucial function for INPP5A-mediated IP3 hydrolysis in the control of lysosome biogenesis via TFEB/ TFE3, thereby contributing to our understanding how cells are able to maintain their lysosome content under conditions of active receptor and nutrient signaling.

Keywords:  calcium; imaging; inositol-triphosphate; lysosome biogenesis; signaling

DOI:  https://doi.org/10.1016/j.jbc.2022.101740
FEBS Open Bio. 2022 Feb 24.

Low level lysosomal membrane permeabilization for limited release and sub-lethal functions of cathepsin proteases in the cytosol and nucleus.

Thomas Reinheckel, Martina Tholen.

  For a long time, lysosomes were purely seen as organelles in charge of garbage disposal within the cell. They destroy any cargo delivered into their lumen with a plethora of highly potent hydrolytic enzymes, including various proteases. In case of damage to their limiting membranes, the lysosomes release their soluble content with detrimental outcomes for the cell. In recent years however, this view of the lysosome changed towards acknowledging it as a platform for integration of manifold intra- and extracellular signals. Even impaired lysosomal membrane integrity is no longer considered to be a one-way street to cell death. Increasing evidence suggests that lysosomal enzymes, mainly cathepsin proteases, can be released in a spatially and temporarily restricted manner that is compatible with cellular survival. This way, cathepsins can act in the cytosol and the nucleus, where they affect important cellular processes such as cell division. Here, we review this evidence and discuss the routes and molecular mechanisms by which the cathepsins may reach their unusual destination.

Keywords:  Cathepsin; Cell Cycle; Cell Death; Lysosome; Protease

DOI:  https://doi.org/10.1002/2211-5463.13385
J Biol Chem. 2022 Feb 17. pii: S0021-9258(22)00184-3. [Epub ahead of print] 101744

The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB.

Tetsuya Kimura, Yoshitomo Hayama, Daisuke Okuzaki, Shigeyuki Nada, Masato Okada.

  The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by ten-fold in the Ragulator-deficient clone compared to that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.

Keywords:  Ragulator; lysosome; mammalian target of rapamycin (mTOR); nuclear translocation; scaffold protein; transcription factor EB

DOI:  https://doi.org/10.1016/j.jbc.2022.101744
Food Chem Toxicol. 2022 Feb 16. pii: S0278-6915(22)00066-7. [Epub ahead of print] 112869

Imatinib disturbs lysosomal function and morphology and impairs the activity of mTORC1 in human hepatocyte cell lines.

Noëmi Johanna Roos, Riccardo Vincenzo Mancuso, Gerda Mawududzi Sanvee, Jamal Bouitbir, Stephan Krähenbühl.

  The tyrosine kinase inhibitors (TKIs) imatinib and lapatinib are associated with severe hepatotoxicity, whose mechanisms are currently under investigation. As amphiphilic drugs, imatinib and lapatinib enrich in lysosomes. In the present study, we investigated their effects on lysosomal morphology and function in HepG2 and HuH-7 cells and explored possible links between lysosomal dysfunction and hepatotoxicity. Both TKIs increased the lysosomal volume time and concentration-dependently in HepG2 and HuH-7 cells. In HepG2 cells, lapatinib and imatinib raised the lysosomal pH and destabilized the lysosomal membrane, thereby impairing lysosomal proteolytic activity such as cathepsin B processing. Imatinib activated the transcription factor EB (TFEB), a regulator of lysosomal biogenesis and function, as demonstrated by nuclear TFEB accumulation and increased expression of TFEB-target genes. Because of lysosomal dysfunction, imatinib impaired mTORC1 activation, a protein complex activated on the lysosomal surface, which explained TFEB activation. HepG2 cells treated with imatinib showed increased levels of MAP1LC3A/B-II and of ATG13 (S318) phosphorylation, indicating induction of autophagy due to TFEB activation. Finally, imatinib induced apoptosis in HepG2 cells in a time and concentration-dependent manner, explained by lysosomal and mitochondrial toxicity. Our findings provide a new lysosome-centered mechanism for imatinib-induced hepatotoxicity that could be extended to other lysosomotropic drugs.

Keywords:  Autophagy; HepG2 cells; Hepatotoxicity; TFEB; Tyrosine kinase inhibitors (TKI); mTORC1

DOI:  https://doi.org/10.1016/j.fct.2022.112869
Int J Mol Sci. 2022 Feb 18. pii: 2290. [Epub ahead of print]23(4):

Lysosomes at the Crossroads of Cell Metabolism, Cell Cycle, and Stemness.

Ada Nowosad, Arnaud Besson.

  Initially described as lytic bodies due to their degradative and recycling functions, lysosomes play a critical role in metabolic adaptation to nutrient availability. More recently, the contribution of lysosomal proteins to cell signaling has been established, and lysosomes have emerged as signaling hubs that regulate diverse cellular processes, including cell proliferation and cell fate. Deciphering these signaling pathways has revealed an extensive crosstalk between the lysosomal and cell cycle machineries that is only beginning to be understood. Recent studies also indicate that a number of lysosomal proteins are involved in the regulation of embryonic and adult stem cell fate and identity. In this review, we will focus on the role of the lysosome as a signaling platform with an emphasis on its function in integrating nutrient sensing with proliferation and cell cycle progression, as well as in stemness-related features, such as self-renewal and quiescence.

Keywords:  CDK; cell cycle; cell metabolism; cell signaling; lysosome; mTOR; nutrient sensing; quiescence; self-renewal; stemness

DOI:  https://doi.org/10.3390/ijms23042290
Biochim Biophys Acta Mol Cell Res. 2022 Feb 22. pii: S0167-4889(22)00034-9. [Epub ahead of print] 119243

The role of lysosomal cathepsins in neurodegeneration: Mechanistic insights, diagnostic potential and therapeutic approaches.

Alice Drobny, Susy Prieto Huarcaya, Jan Dobert, Annika Kluge, Josina Bunk, Theresia Schlothauer, Friederike Zunke.

  Lysosomes are ubiquitous organelles with a fundamental role in maintaining cellular homeostasis by mediating degradation and recycling processes. Cathepsins are the most abundant lysosomal hydrolyses and responsible for the bulk degradation of various substrates. A correct autophagic function is essential for neuronal survival, as most neurons are post-mitotic and thus susceptible to accumulate cellular components. Increasing evidence suggest a crucial role of the lysosome in neurodegeneration as a key regulator of aggregation-prone and disease-associated proteins, such as α-synuclein, β-amyloid and huntingtin. Particularly, alterations in lysosomal cathepsins CTSD, CTSB and CTSL can contribute to the pathogenesis of neurodegenerative diseases as seen for neuronal ceroid lipofuscinosis, synucleinopathies (Parkinson's disease, Dementia with Lewy Body and Multiple System Atrophy) as well as Alzheimer's and Huntington disease. In this review, we provide an overview of recent evidence implicating CTSD, CTSB and CTSL in neurodegeneration, with a special focus on the role of these enzymes in α-synuclein metabolism. In addition, we summarize the potential role of lysosomal cathepsins as clinical biomarkers in neurodegenerative diseases and discuss potential therapeutic approaches by targeting lysosomal function.

Keywords:  Alzheimer's disease; Cathepsins; Huntington disease; Lysosome; Neurodegeneration; Neuronal ceroid lipofuscinosis; Parkinson's disease; Synucleinopathies; α-Synuclein

DOI:  https://doi.org/10.1016/j.bbamcr.2022.119243
Biophys J. 2022 Feb 21. pii: S0006-3495(22)00156-4. [Epub ahead of print]

Transport of lysosomes decreases in the perinuclear region: Insights from changepoint analysis.

Nathan T Rayens, Keisha J Cook, Scott A McKinley, Christine K Payne.

Lysosomes are membrane-bound organelles that serve as the endpoint for endocytosis, phagocytosis, and autophagy, degrading the molecules, pathogens, and organelles localized within them. These cellular functions require intracellular transport. We use fluorescence microscopy to characterize the motion of lysosomes as a function of intracellular region, perinuclear or periphery, and lysosome diameter. Single particle tracking data is complemented by changepoint identification and analysis of a mathematical model for state-switching. We first classify lysosomal motion as motile or stationary. We then study how lysosome location and diameter affects the proportion of time spent in each state and quantify the speed during motile periods. We find that the proportion of time spent stationary is strongly region-dependent, with significantly decreased motility in the perinuclear region. Increased lysosome diameter only slightly decreases speed. Overall, these results demonstrate the importance of decomposing particle trajectories into qualitatively different behaviors before conducting population-wide statistical analysis. Our results suggest that intracellular region is an important factor to consider in studies of intracellular transport.

DOI: https://doi.org/10.1016/j.bpj.2022.02.032
FEBS Open Bio. 2022 Feb 25.

Lysosomes: Multifunctional compartments ruled by a complex regulatory network.

Jonathan Martinez-Fabregas, Joaquin Tamargo-Azpilicueta, Irene Diaz-Moreno.

  More than fifty years have passed since Nobel laureate Cristian de Duve described for the first time the presence of tiny subcellular compartments filled with hydrolytic enzymes: the lysosome. For a long time, lysosomes were deemed simple waste bags exerting a plethora of hydrolytic activities involved in the recycling of biopolymers, and lysosomal genes were considered to just be simple housekeeping genes, transcribed in a constitutive fashion. However, lysosomes are emerging as multifunctional signalling hubs involved in multiple aspects of cell biology, both under homeostatic and pathological conditions. Lysosomes are involved in the regulation of cell metabolism through the mTOR/TFEB axis. They are also key players in the regulation and onset of the immune response. Furthermore, it is becoming clear that lysosomal hydrolases can regulate several biological processes outside of the lysosome. They are also implicated in a complex communication network among subcellular compartments that involves intimate organelle-to-organelle contacts. Furthermore, lysosomal dysfunction is nowadays accepted as the causative event behind several human pathologies: low frequency inherited diseases, cancer, or neurodegenerative, metabolic, inflammatory, and autoimmune diseases. Recent advances in our knowledge of the complex biology of lysosomes have established them as promising therapeutic targets for the treatment of different pathologies. Although recent discoveries have started to highlight that lysosomes are controlled by a complex web of regulatory networks, that in some cases seem to be cell- and stimuli-dependent, to harness the full potential of lysosomes as therapeutic targets we need a deeper understanding of the little-known signalling pathways regulating this subcellular compartment and its functions.

Keywords:  STATs; TFEB; lysosomes; mTORC1; signalling pathways; transcriptional regulation

DOI:  https://doi.org/10.1002/2211-5463.13387
Neurobiol Dis. 2022 Feb 17. pii: S0969-9961(22)00054-7. [Epub ahead of print]166 105663

Glucocerebrosidase-associated Parkinson disease: Pathogenic mechanisms and potential drug treatments.

Matthew E Gegg, Elisa Menozzi, Anthony H V Schapira.

  Dysfunction of the endolysosomal system is implicated in the pathogenesis of both sporadic and familial Parkinson disease (PD). Variants in genes encoding lysosomal proteins have been estimated to be associated with more than half of PD cases. The most common genetic risk factor for PD are variants in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), which is involved in sphingolipid metabolism. In this review we will describe the clinical symptoms and pathology of GBA-PD, and how this might be affected by the type of GBA variant. The putative mechanisms by which GCase deficiency in neurons and glia might contribute to PD pathogenesis will then be discussed, with particular emphasis on the accumulation of α-synuclein aggregates and the spread of pathogenic α-synuclein species between the cell types. The dysregulation of not only sphingolipids, but also phospholipids and cholesterol in the misfolding of α-synuclein is reviewed, as are neuroinflammation and the interaction of GCase with LRRK2 protein, another important contributor to PD pathogenesis. Study of both non-manifesting GBA carriers and GBA-PD cohorts provides an opportunity to identify robust biomarkers for PD progression as well as clinical trials for potential treatments. The final part of this review will describe preclinical studies and clinical trials for increasing GCase activity or reducing toxic substrate accumulation.

Keywords:  Autophagy; Glucocerebrosidase; Lipid; Lysosome; Neuroinflammation; Parkinson disease; α-synuclein

DOI:  https://doi.org/10.1016/j.nbd.2022.105663
Cell Rep. 2022 Feb 22. pii: S2211-1247(22)00131-0. [Epub ahead of print]38(8): 110407

Loss of Slc38a4 imprinting is a major cause of mouse placenta hyperplasia in somatic cell nuclear transferred embryos at late gestation.

Zhenfei Xie, Wenhao Zhang, Yi Zhang.

  Placenta hyperplasia is commonly observed in cloned animals and is believed to impede the proper development of cloned embryos. However, the mechanism underlying this phenomenon is largely unknown. Here, we show that placenta hyperplasia of cloned mouse embryos occurs in both middle and late gestation. Interestingly, restoring paternal-specific expression of an amino acid transporter Slc38a4, which loses maternal H3K27me3-dependent imprinting and becomes biallelically expressed in cloned placentae, rescues the overgrowth of cloned placentae at late gestation. Molecular analyses reveal that loss of Slc38a4 imprinting leads to over-activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in cloned placentae, which is likely due to the increased amino acids transport by SLC38A4. Collectively, our study not only reveals loss of Slc38a4 imprinting is responsible for overgrowth of cloned placentae at late gestation but also suggests the underlying mechanism involves increased amino acid transport and over-activation of mTORC1.

Keywords:  H3K27me3 imprinting; Slc38a4; amino acid; cloning; late gestation; mTORC1; metabolism; placenta hyperplasia; placenta overgrowth; somatic cell nuclear transfer

DOI:  https://doi.org/10.1016/j.celrep.2022.110407
Dev Cell. 2022 Feb 15. pii: S1534-5807(22)00070-3. [Epub ahead of print]

YAP regulates an SGK1/mTORC1/SREBP-dependent lipogenic program to support proliferation and tissue growth.

Srimayee Vaidyanathan, Talhah M Salmi, Rasan M Sathiqu, Malcolm J McConville, Andrew G Cox, Kristin K Brown.

  The coordinated regulation of growth control and metabolic pathways is required to meet the energetic and biosynthetic demands associated with proliferation. Emerging evidence suggests that the Hippo pathway effector Yes-associated protein 1 (YAP) reprograms cellular metabolism to meet the anabolic demands of growth, although the mechanisms involved are poorly understood. Here, we demonstrate that YAP co-opts the sterol regulatory element-binding protein (SREBP)-dependent lipogenic program to facilitate proliferation and tissue growth. Mechanistically, YAP stimulates de novo lipogenesis via mechanistic target of rapamcyin (mTOR) complex 1 (mTORC1) signaling and subsequent activation of SREBP. Importantly, YAP-dependent regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1) is required to activate mTORC1/SREBP and stimulate de novo lipogenesis. We also find that the SREBP target genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are conditionally required to support YAP-dependent proliferation and tissue growth. These studies reveal that de novo lipogenesis is a metabolic vulnerability that can be targeted to disrupt YAP-dependent proliferation and tissue growth.

Keywords:  SGK1; SREBP; YAP; cell metabolism; growth; lipogenesis; mTORC1; proliferation

DOI:  https://doi.org/10.1016/j.devcel.2022.02.004
Biomedicines. 2022 Jan 28. pii: 302. [Epub ahead of print]10(2):

Gene Therapy Developments for Pompe Disease.

Zeenath Unnisa, John K Yoon, Jeffrey W Schindler, Chris Mason, Niek P van Til.

  Pompe disease is an inherited neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The most severe form is infantile-onset Pompe disease, presenting shortly after birth with symptoms of cardiomyopathy, respiratory failure and skeletal muscle weakness. Late-onset Pompe disease is characterized by a slower disease progression, primarily affecting skeletal muscles. Despite recent advancements in enzyme replacement therapy management several limitations remain using this therapeutic approach, including risks of immunogenicity complications, inability to penetrate CNS tissue, and the need for life-long therapy. The next wave of promising single therapy interventions involves gene therapies, which are entering into a clinical translational stage. Both adeno-associated virus (AAV) vectors and lentiviral vector (LV)-mediated hematopoietic stem and progenitor (HSPC) gene therapy have the potential to provide effective therapy for this multisystemic disorder. Optimization of viral vector designs, providing tissue-specific expression and GAA protein modifications to enhance secretion and uptake has resulted in improved preclinical efficacy and safety data. In this review, we highlight gene therapy developments, in particular, AAV and LV HSPC-mediated gene therapy technologies, to potentially address all components of the neuromuscular associated Pompe disease pathology.

Keywords:  Pompe disease; adeno-associated viral vector; enzyme replacement therapy; hematopoietic stem cell; lentiviral vectors

DOI:  https://doi.org/10.3390/biomedicines10020302
Physiol Rev. 2022 Feb 21.

ER-phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum.

Fulvio Reggiori, Maurizio Molinari.

  ER-phagy (reticulo-phagy) defines the degradation of portions of the endoplasmic reticulum (ER) within lysosomes or vacuoles. It is part of the self-digestion (i.e., auto-phagic) programs recycling cytoplasmic material and organelles, which rapidly mobilize metabolites in cells confronted with nutrient shortage. Moreover, selective clearance of ER subdomains participates to the control of ER size and activity during ER stress, the re-establishment of ER homeostasis after ER stress resolution and the removal of ER parts, in which aberrant and potentially cytotoxic material has been segregated. ER-phagy relies on the individual and/or concerted activation of the ER-phagy receptors, ER peripheral or integral membrane proteins that share the presence of LC3/Atg8-binding motifs in their cytosolic domains. ER-phagy involves the physical separation of portions of the ER from the bulk ER network, and their delivery to the endolysosomal/vacuolar catabolic district. This last step is accomplished by a variety of mechanisms including macro-ER-phagy (in which ER fragments are sequestered by double-membrane autophagosomes that eventually fuse with lysosomes/vacuoles), micro-ER-phagy (in which ER fragments are directly engulfed by endosomes/lysosomes/vacuoles), or direct fusion of ER-derived vesicles with lysosomes/vacuoles. ER-phagy is dysfunctional in specific human diseases and its regulators are subverted by pathogens, highlighting its crucial role for cell and organism life.

Keywords:  Autophagy; Disease; ER-phagy; Endoplasmic Reticulum; Lysosomal degradation

DOI:  https://doi.org/10.1152/physrev.00038.2021
Nat Biotechnol. 2022 Feb 21.

Saturation variant interpretation using CRISPR prime editing.

Steven Erwood, Teija M I Bily, Jason Lequyer, Joyce Yan, Nitya Gulati, Reid A Brewer, Liangchi Zhou, Laurence Pelletier, Evgueni A Ivakine, Ronald D Cohn.

High-throughput functional characterization of genetic variants in their endogenous locus has so far been possible only with methods that rely on homology-directed repair, which are limited by low editing efficiencies. Here, we adapted CRISPR prime editing for high-throughput variant classification and combined it with a strategy that allows for haploidization of any locus, which simplifies variant interpretation. We demonstrate the utility of saturation prime editing (SPE) by applying it to the NPC intracellular cholesterol transporter 1 gene (NPC1), mutations in which cause the lysosomal storage disorder Niemann-Pick disease type C. Our data suggest that NPC1 is very sensitive to genetic perturbation, with 410 of 706 assayed missense mutations being classified as deleterious, and that the derived function score of variants is reflective of diverse molecular defects. We further applied our approach to the BRCA2 gene, demonstrating that SPE is translatable to other genes with an appropriate cellular assay. In sum, we show that SPE allows for efficient, accurate functional characterization of genetic variants.

DOI: https://doi.org/10.1038/s41587-021-01201-1
Aging Cell. 2022 Feb 23. e13574

Loss of legumain induces premature senescence and mediates aging-related renal fibrosis.

Dekun Wang, Lichun Kang, Chuan'ai Chen, Jiasen Guo, Lingfang Du, Donghui Zhou, Gang Li, Yuying Zhang, Xue Mi, Mianzhi Zhang, Shuxia Liu, Xiaoyue Tan.

  Aging is an independent risk factor for acute kidney injury and subsequent chronic kidney diseases, while the underlying mechanism is still elusive. Here, we found that renal tubules highly express a conserved lysosomal endopeptidase, legumain, which is significantly downregulated with the growing of age. Tubule-specific legumain-knockout mice exhibit spontaneous renal interstitial fibrosis from the 3rd month. In the tubule-specific legumain-knockout mice and the cultured legumain-knockdown HK-2 cells, legumain deficiency induces the activation of tubular senescence and thus increases the secretion of profibrotic senescence-associated cytokines, which in turn accelerates the activation of fibroblasts. Blockage of senescence mitigates the fibrotic lesion caused by legumain deficiency. Mechanistically, we found that silencing down of legumain leads to the elevated lysosome pH value, enlargement of lysosome size, and increase of lysosomal voltage dependent membrane channel proteins. Either legumain downregulation or aging alone induces the activation of nuclear transcription factors EB (TFEB) while it fails to further upregulate in the elderly legumain-knockdown tubules, accompanied with impaired mitophagy and increased mitochondrial ROS (mtROS) accumulation. Therapeutically, supplementation of exosomal legumain ameliorated fibronectin and collagen I production in an in vitro coculture system of tubular cells and fibroblasts. Altogether, our data demonstrate that loss of legumain in combined with aging dysregulates lysosomal homeostasis, although either aging or legumain deficiency alone induces lysosome adaptation via stimulating lysosomal biogenesis. Consequently, impaired mitophagy leads to mtROS accumulation and therefore activates tubular senescence and boosts the interstitial fibrosis.

Keywords:  aging-related renal fibrosis; autophagy; legumain (asparagine endopetidase); premature senescence

DOI:  https://doi.org/10.1111/acel.13574
Front Cell Dev Biol. 2022 ;10 817877

Role of Lysosomal Acidification Dysfunction in Mesenchymal Stem Cell Senescence.

Weijun Zhang, Jinwu Bai, Kai Hang, Jianxiang Xu, Chengwei Zhou, Lijun Li, Zhongxiang Wang, Yibo Wang, Kanbin Wang, Deting Xue.

  Mesenchymal stem cell (MSC) transplantation has been widely used as a potential treatment for a variety of diseases. However, the contradiction between the low survival rate of transplanted cells and the beneficial therapeutic effects has affected its clinical use. Lysosomes as organelles at the center of cellular recycling and metabolic signaling, play essential roles in MSC homeostasis. In the first part of this review, we summarize the role of lysosomal acidification dysfunction in MSC senescence. In the second part, we summarize some of the potential strategies targeting lysosomal proteins to enhance the therapeutic effect of MSCs.

Keywords:  V-ATPase; lysosomal acidification; mesenchymal stem cells; pH; senescence

DOI:  https://doi.org/10.3389/fcell.2022.817877
Curr Med Chem. 2022 Feb 24.

The mTOR/4E-BP1/eIF4E signalling pathway as source of cancer drug targets.

Cristina Maracci, Stefano Motta, Alice Romagnoli, Matteo Costantino, Paola Perego, Daniele Di Marino.

  The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4E-BPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signaling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1 and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds.

Keywords:  eIF4F; mTOR allosteric inhibitors; rapamycin; translational control

DOI:  https://doi.org/10.2174/0929867329666220224112042
Mol Cancer Res. 2022 Feb 01. pii: molcanres.MCR-21-0729-A.2021. [Epub ahead of print]

PARK2 regulates eIF4B-driven lymphomagenesis.

Bandish B Kapadia, Anirban Roychowdhury, Forum Kayastha, Nahid Nanaji, Ronald B Gartenhaus.

Patients with high-risk diffuse large B-cell lymphoma (DLBCL) have poor outcomes following first-line cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab (R-CHOP); thus, treatment of this fatal disease remains an area of unmet medical need and requires identification of novel therapeutic approaches. Dysregulation of protein translation initiation has emerged as a common downstream node in several malignancies, including lymphoma. Ubiquitination, a prominent post-translational modification associated with substrate degradation, has recently been shown to be a key modulator of nascent peptide synthesis by limiting several translational initiation factors. While a few deubiquitinases have been identified, the E3-ligase responsible for the critical ubiquitination of these translational initiation factors is still unknown. In this study, using complementary cellular models along with clinical readouts, we establish that PARK2 ubiquitinates eIF4B and consequently regulates overall protein translational activity. The formation of this interaction depends on upstream signaling, which is negatively regulated at the protein level of PARK2. Through biochemical, mutational, and genetic studies, we identified PARK2 as a mTORC1 substrate. mTORC1 phosphorylates PARK2 at Ser127, which blocks its cellular ubiquitination activity, thereby hindering its tumor suppressor effect on eIF4B's stability. This resultant increase of eIF4B protein level helps drive enhanced overall protein translation. These data support a novel paradigm in which PARK2-generated eIF4B ubiquitination serves as an anti-oncogenic intracellular inhibitor of protein translation, attenuated by mTORC1 signaling. Implications: Our data implicates the FASN/mTOR-PARK2-eIF4B axis as a critical driver of enhanced oncogene expression contributing to lymphomagenesis.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0729
Biochem J. 2022 Feb 25. pii: BCJ20210855. [Epub ahead of print]

Deletion of Slc6a14 reduces cancer growth and metastatic spread and improves survival in KPC mouse model of spontaneous pancreatic cancer.

Bradley Schniers, Mitchell Wachtel, Meenu Sharma, Ksenija Korac, Devaraja Rajasekaran, Shengping Yang, Tyler Sniegowski, Vadivel Ganapathy, Yangzom Doma Bhutia.

  Pancreatic ductal adenocarcinoma (PDAC) is lethal. There is a dire need for better therapeutic targets. Cancer cells have increased demand for sugars, amino acids, and lipids and therefore upregulate various nutrient transporters to meet this demand. In PDAC, SLC6A14 (an amino acid transporter) is upregulated, affecting overall patient survival. Previously we have shown using in vitro cell culture models and in vivo xenograft mouse models that pharmacological inhibition of SLC6A14 with a-methyl-L-tryptophan (a-MLT) attenuates PDAC growth. Mechanistically, blockade of SLC6A14-mediated amino acid transport with a-MLT leads to amino acid deprivation, eventually inhibiting mTORC1 signaling pathway, in tumor cells. Here we report on the effect of Slc6a14 deletion on various parameters of PDAC in KPC mice, a model for spontaneous PDAC. Pancreatic tumors in KPC mice show evidence of Slc6a14 upregulation. Deletion of Slc6a14 in this mouse attenuates PDAC growth, decreases metastatic spread of the tumor, reduces ascites fluid accumulation, and improves overall survival. At molecular level, we show lower proliferation index and reduced desmoplastic reaction following Slc6a14 deletion. Furthermore, we find that deletion of Slc6a14 does not lead to compensatory upregulation in any of the other amino transporters. In fact, some of the amino acid transporters are actually downregulated in response to Slc6a14 deletion, most likely related to altered mTORC1 signaling. Taken together, these results underscore the positive role SLC6A14 plays in PDAC growth and metastasis. Therefore, SLC6A14 is a viable drug target for treatment of PDAC and also for any other cancer that overexpresses this transporter.

Keywords:  KPC; Pancreatic Cancer; SLC6A14; ascites fluid; metastasis; survival

DOI:  https://doi.org/10.1042/BCJ20210855
Autophagy. 2022 Feb 19. 1-3

MON-2, a Golgi protein, promotes longevity by upregulating autophagy through mediating inter-organelle communications.

Murat Artan, Jooyeon Sohn, Cheolju Lee, Seung-Yeol Park, Seung-Jae V Lee.

  The Golgi apparatus regulates the process of modification and subcellular localization of macromolecules, including proteins and lipids. Aberrant protein sorting caused by defects in the Golgi leads to various diseases in mammals. However, the role of the Golgi apparatus in organismal longevity remained largely unknown. By employing a quantitative proteomic approach, we demonstrated that MON-2, an evolutionarily conserved Arf-GEF protein implicated in Golgi-to-endosome trafficking, promotes longevity via upregulating macroautophagy/autophagy in C. elegans. Our data using cultured mammalian cells indicate that MON2 translocates from the Golgi to the endosome under starvation conditions, subsequently increasing autophagic flux by binding LGG-1/GABARAPL2. Thus, Golgi-to-endosome trafficking appears to be an evolutionarily conserved process for the upregulation of autophagy, which contributes to organismal longevity.

Keywords:  Aging; C. elegans; Golgi; LGG-1/GABARAPL2; MON-2/MON2; autophagy; lifespan; proteomics

DOI:  https://doi.org/10.1080/15548627.2022.2039523
Nat Chem Biol. 2022 Feb 24.

Quantifying lysosomal glycosidase activity within cells using bis-acetal substrates.

Samy Cecioni, Roger A Ashmus, Pierre-André Gilormini, Sha Zhu, Xi Chen, Xiaoyang Shan, Christina Gros, Matthew C Deen, Yang Wang, Robert Britton, David J Vocadlo.

Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.

DOI: https://doi.org/10.1038/s41589-021-00960-x
Biomolecules. 2022 Jan 26. pii: 210. [Epub ahead of print]12(2):

Interaction of TOR and PKA Signaling in S. cerevisiae.

Michael Plank.

  TOR and PKA signaling are the major growth-regulatory nutrient-sensing pathways in S. cerevisiae. A number of experimental findings demonstrated a close relationship between these pathways: Both are responsive to glucose availability. Both regulate ribosome production on the transcriptional level and repress autophagy and the cellular stress response. Sch9, a major downstream effector of TORC1 presumably shares its kinase consensus motif with PKA, and genetic rescue and synthetic defects between PKA and Sch9 have been known for a long time. Further, studies in the first decade of this century have suggested direct regulation of PKA by TORC1. Nonetheless, the contribution of a potential direct cross-talk vs. potential sharing of targets between the pathways has still not been completely resolved. What is more, other findings have in contrast highlighted an antagonistic relationship between the two pathways. In this review, I explore the association between TOR and PKA signaling, mainly by focusing on proteins that are commonly referred to as shared TOR and PKA targets. Most of these proteins are transcription factors which to a large part explain the major transcriptional responses elicited by TOR and PKA upon nutrient shifts. I examine the evidence that these proteins are indeed direct targets of both pathways and which aspects of their regulation are targeted by TOR and PKA. I further explore if they are phosphorylated on shared sites by PKA and Sch9 or when experimental findings point towards regulation via the PP2ASit4/PP2A branch downstream of TORC1. Finally, I critically review data suggesting direct cross-talk between the pathways and its potential mechanism.

Keywords:  PKA; TOR; autophagy; cross-talk; kinase; nutrient sensing; ribosome production; signaling pathway interaction; stress response; substrate specificity

DOI:  https://doi.org/10.3390/biom12020210
Elife. 2022 02 22. pii: e73812. [Epub ahead of print]11

Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.

Felipe Martelli, Natalia H Hernandes, Zhongyuan Zuo, Julia Wang, Ching-On Wong, Nicholas E Karagas, Ute Roessner, Thusita Rupasinghe, Charles Robin, Kartik Venkatachalam, Trent Perry, Philip Batterham, Hugo J Bellen.

  Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit alpha 6 (nAChRα6), reducing the cholinergic response. We show that the nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.

Keywords:  D. melanogaster; antioxidant; lipid dysregulation; lysosomal dysfunction; neurodegeneration; neuroscience; oxidative stress; spinosad

DOI:  https://doi.org/10.7554/eLife.73812
Int J Mol Sci. 2022 Feb 11. pii: 1999. [Epub ahead of print]23(4):

Innate Immunity in Mucopolysaccharide Diseases.

Oriana Mandolfo, Helen Parker, Brian Bigger.

  Mucopolysaccharidoses are rare paediatric lysosomal storage disorders, characterised by accumulation of glycosaminoglycans within lysosomes. This is caused by deficiencies in lysosomal enzymes involved in degradation of these molecules. Dependent on disease, progressive build-up of sugars may lead to musculoskeletal abnormalities and multi-organ failure, and in others, to cognitive decline, which is still a challenge for current therapies. The worsening of neuropathology, observed in patients following recovery from flu-like infections, suggests that inflammation is highly implicated in disease progression. This review provides an overview of the pathological features associated with the mucopolysaccharidoses and summarises current knowledge regarding the inflammatory responses observed in the central nervous system and periphery. We propose a model whereby progressive accumulation of glycosaminoglycans elicits an innate immune response, initiated by the Toll-like receptor 4 pathway, but also precipitated by secondary storage components. Its activation induces cells of the immune system to release pro-inflammatory cytokines, such as TNF-α and IL-1, which induce progression through chronic neuroinflammation. While TNF-α is mostly associated with bone and joint disease in mucopolysaccharidoses, increasing evidence implicates IL-1 as a main effector of innate immunity in the central nervous system. The (NOD)-like receptor protein 3 inflammasome is therefore implicated in chronic neuroinflammation and should be investigated further to identify novel anti-inflammatory treatments.

Keywords:  IL-1; glycosaminoglycans; lysosomal storage disorders; mucopolysaccharidosis; neuroinflammation; neuropathology

DOI:  https://doi.org/10.3390/ijms23041999
Int J Mol Sci. 2022 Feb 10. pii: 1963. [Epub ahead of print]23(4):

Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation.

Ryuichi Mashima, Torayuki Okuyama, Mari Ohira.

  Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.

Keywords:  biosynthesis; heparan sulfate; knockout mice; lysosome

DOI:  https://doi.org/10.3390/ijms23041963
J Biol Chem. 2022 Feb 22. pii: S0021-9258(22)00190-9. [Epub ahead of print] 101750

Turnover of the mTOR inhibitor, DEPTOR, and downstream AKT phosphorylation in multiple myeloma cells, is dependent on ERK1-mediated phosphorylation.

Mario Vega, Yu Chen, Yijiang Shi, Joseph Gera, Alan Lichtenstein.

  DEPTOR is a 48kDa protein upregulated in myeloma (MM) tumor cells. DEPTOR inhibits the nutrient sensor mTOR kinase and, by repressing a negative feedback loop, promotes pro-growth AKT kinase activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here we screened for drug-induced post-translational modifications (PTMs) and identified reduced phosphorylation of DEPTOR serine-235. We show that an S-235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this PTM. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S-235 could only be expressed upon concurrent proteasome inhibition. Thus, S-235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S-235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S-235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown and an in vitro kinase assay. Inhibition of ERK1 also down-regulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this cross-talk, MM cells were transfected with wild type or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose DEPTOR-dependent cross-talk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).

Keywords:  AKT; DEPTOR; ERK; Multiple myeloma; mTOR

DOI:  https://doi.org/10.1016/j.jbc.2022.101750
Nature. 2022 Feb 23.

Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Teng Ma, Xiao Tian, Baoding Zhang, Mengqi Li, Yu Wang, Chunyan Yang, Jianfeng Wu, Xiaoyan Wei, Qi Qu, Yaxin Yu, Shating Long, Jin-Wei Feng, Chun Li, Cixiong Zhang, Changchuan Xie, Yaying Wu, Zheni Xu, Junjie Chen, Yong Yu, Xi Huang, Ying He, Luming Yao, Lei Zhang, Mingxia Zhu, Wen Wang, Zhi-Chao Wang, Mingliang Zhang, Yuqian Bao, Weiping Jia, Shu-Yong Lin, Zhiyun Ye, Hai-Long Piao, Xianming Deng, Chen-Song Zhang, Sheng-Cai Lin.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

DOI: https://doi.org/10.1038/s41586-022-04431-8
J Biol Chem. 2022 Feb 21. pii: S0021-9258(22)00197-1. [Epub ahead of print] 101757

Tissue-specific alternative splicing separates the catalytic and cell signaling functions of human leucyl-tRNA synthetase.

Max Baymiller, Benjamin Nordick, Connor M Forsyth, Susan A Martinis.

  The aminoacyl-tRNA synthetases are an ancient and ubiquitous component of all life. Many eukaryotic synthetases balance their essential function, preparing aminoacyl-tRNA for use in mRNA translation, with diverse roles in cell signaling. Herein, we use long-read sequencing to discover a leukocyte-specific exon skipping event in human leucyl-tRNA synthetase (LARS). We show that this highly expressed splice variant, LSV3, is regulated by serine-arginine rich splicing factor 1 (SRSF1) in a cell type-specific manner. LSV3 has a 71 amino acid deletion in the catalytic domain and lacks any tRNA leucylation activity in vitro. However, we demonstrate that this LARS splice variant retains its role as a leucine sensor and signal transducer for the proliferation-promoting mTOR kinase. This is despite the exon deletion in LSV3 including a portion of the previously mapped Vps34-binding domain used for one of two distinct pathways from LARS to mTOR. In conclusion, alternative splicing of LARS has separated the ancient catalytic activity of this housekeeping enzyme from its more recent evolutionary role in cell signaling, providing an opportunity for functional specificity in human immune cells.

Keywords:  Aminoacyl-tRNA synthetase; alternative splicing; mammalian target of rapamycin (mTOR); serine/arginine-rich splicing factor 1 (SRSF1); transfer RNA (tRNA); translation

DOI:  https://doi.org/10.1016/j.jbc.2022.101757
Amino Acids. 2022 Feb 25.

The role of SHMT2 in modulating lipid metabolism in hepatocytes via glycine-mediated mTOR activation.

You-Jin Choi, Geunhye Lee, Sung Ho Yun, Wonseok Lee, Jieun Yu, Sang Kyum Kim, Byung-Hoon Lee.

  Serine hydroxymethyltransferase 2 (SHMT2) converts serine into glycine in the mitochondrial matrix, transferring a methyl group to tetrahydrofolate. SHMT2 plays an important role in the maintenance of one-carbon metabolism. Previously, we found a negative correlation between the serine concentration and the progression of fatty liver disease (FLD). However, little is known about the role of SHMT2 in hepatic lipid metabolism. We established SHMT2 knockdown (KD) mouse primary hepatocytes using RNA interference to investigate the role of SHMT2 in lipid metabolism. SHMT2 KD hepatocytes showed decreased lipid accumulation with reduced glycine levels compared to the scramble cells, which was restored upon reintroducing SHMT2. SHMT2 KD hepatocytes showed downregulation of the mTOR/PPARɣ pathway with decreased gene expression related to lipogenesis and fatty acid uptake. Pharmacological activation of mTOR or PPARɣ overexpression blocked the inhibitory effect of SHMT2 KD on lipid accumulation. We also showed that glycine activated mTOR/PPARɣ signaling and identified glycine as a mediator of SHMT2-responsive lipid accumulation in hepatocytes. In conclusion, silencing SHMT2 in hepatocytes ameliorates lipid accumulation via the glycine-mediated mTOR/PPARɣ pathway. Our findings underscore the possibility of SHMT2 as a therapeutic target of FLD.

Keywords:  Glycine; Lipid metabolism; PPARɣ; SHMT2; mTOR

DOI:  https://doi.org/10.1007/s00726-022-03141-9
Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2112712119. [Epub ahead of print]119(9):

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Ranjan K Singh, Ahmed Soliman, Giambattista Guaitoli, Eliza Störmer, Felix von Zweydorf, Thomas Dal Maso, Asmaa Oun, Laura Van Rillaer, Sven H Schmidt, Deep Chatterjee, Joshua A David, Els Pardon, Thomas U Schwartz, Stefan Knapp, Eileen J Kennedy, Jan Steyaert, Friedrich W Herberg, Arjan Kortholt, Christian Johannes Gloeckner, Wim Versées.

  Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Keywords:  LRRK2; Parkinson’s disease; allosteric inhibitor; drug design; nanobody

DOI:  https://doi.org/10.1073/pnas.2112712119
Int J Biochem Cell Biol. 2022 Feb 22. pii: S1357-2725(22)00029-2. [Epub ahead of print] 106184

Galactocerebrosidase deficiency induces an increase in lactosylceramide content: a new hallmark of Krabbe disease?

Nadia Papini, Chiara Giallanza, Loredana Brioschi, Francesca Romana Ranieri, Paola Giussani, Laura Mauri, Maria Grazia Ciampa, Paola Viani, Cristina Tringali.

  Galactocerebrosidase (GALC) hydrolyses galactose residues from various substrates, including galactosylceramide, psychosine (galactosylsphingosine), and lactosylceramide. Its severe deficiency has been associated with the accumulation of psychosine, a toxic molecule with detergent-like features, which alters membrane structures and signalling pathways, inducing the death of oligodendrocytes and a sequence of events in the nervous system that explain the appearance of many clinical signs typical of Krabbe disease. Nevertheless, new evidence suggests the existence of other possible links among GALC action, myelination, and myelin stability, apart from psychosine release. In this study, we demonstrated that lactosylceramide metabolism is impaired in fibroblasts isolated from patients with Krabbe disease in the absence of psychosine accumulation. This event is responsible for the aberrant and constitutive activation of the AKT/prolin-rich AKT substrate of 40kDa (PRAS40) signalling axis, inducing B cell lymphoma 2 (BCL2) overexpression and glycogen synthase kinase 3 beta (GSK-3β) inhibition. In addition, nuclear factor E2-related factor 2 (NRF2) showed increased nuclear translocation. Due to the relevance of these molecular alterations in neurodegeneration, lactosylceramide increase should be evaluated as a novel marker of Krabbe disease, and because of its significant connections with signalling pathways.

Keywords:  Krabbe disease; galactocerebrosidase; lactosylceramide

DOI:  https://doi.org/10.1016/j.biocel.2022.106184
Mol Oncol. 2022 Feb 20.

CHML is an NRF2 target gene that regulates mTOR function.

Matthew Dodson, Wujing Dai, Annadurai Anandhan, Cody J Schmidlin, Pengfei Liu, Nathan C Wilson, Yongyi Wei, Naoya Kitamura, James J Galligan, Aikseng Ooi, Eli Chapman, Donna D Zhang.

  The transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is often highly expressed in non-small cell lung cancer (NSCLC). Through its target genes, NRF2 enhances cancer progression and chemo/radioresistance, leading to a poorer prognosis in patients with high NRF2 expression. In this study, we identified CHM like Rab escort protein (CHML; encoding Rep2) as an NRF2 target gene with an antioxidant response element (ARE) in its promoter region (-1622 to -1612). Analysis of patient data curated by The Cancer Genome Atlas (TCGA) and Oncomine databases revealed that CHML mRNA expression was elevated in lung adenocarcinoma (LUAD) patient tumor tissues and correlated with decreased patient survival. Immunohistochemistry (IHC) analysis of normal versus lung cancer patient tissues revealed that Rep2 protein levels were higher in lung tumors compared with normal tissue, which also correlated with increased levels of NRF2. Importantly, siRNA-mediated knockdown of CHML/Rep2 in A549 NSCLC cells decreased their ability to proliferate. Mechanistically, Rep2 mediates mTOR function, as loss of Rep2 inhibited, whereas overexpression enhanced, mTOR translocation and activation at the lysosome. Our findings identify a novel NRF2-Rep2-dependent regulation of mTOR function.

Keywords:  CHML/REP2; NRF2; NSCLC; mTOR

DOI:  https://doi.org/10.1002/1878-0261.13194