bims-lysosi 2021-06-06 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–06–06
thirty papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Lipid-mediated impairment of axonal lysosome transport contributing to autophagic stress.
TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy.
CLN5 and CLN3 function as a complex to regulate endolysosome function.
MTOR Signaling and Metabolism in Early T Cell Development.
Loss of PKA regulatory subunit 1α aggravates cardiomyocyte necrosis and myocardial ischemia/reperfusion injury.
Thyroid hormone receptor-α regulates autophagy, mitochondrial biogenesis, and fatty acid utilization in skeletal muscle.
Multiplexed suppression of TOR complex 1 induces autophagy during starvation.
mTOR Signaling in Metabolic Stress Adaptation.
The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.
Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.
FKBP39 controls nutrient dependent Nprl3 expression and TORC1 activity in Drosophila.
mTORC1 induces eukaryotic translation initiation factor 4E interaction with TOS-S6 kinase 1 and its activation.
Endosomal mTORC2 Is Required for Phosphoinositide-Dependent AKT Activation in Platelet-Derived Growth Factor-Stimulated Glioma Cells.
An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA.
Cathepsin K: A Novel Diagnostic and Predictive Biomarker for Renal Tumors.
Inhibition of the mTOR pathway and reprogramming of protein synthesis by MDM4 reduce ovarian cancer metastatic properties.
Codon Bias Can Determine Sorting of a Potassium Channel Protein.
Extracellular Acidification Induces Lysosomal Dysregulation.
Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis.
Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2.
Metabolic remodeling precedes mTORC1-mediated cardiac hypertrophy.
Glucosylceramide and galactosylceramide, small glycosphingolipids with significant impact on health and disease.
The ESCRT-III complex contributes to macromitophagy in yeast.
Membrane contact site-dependent cholesterol transport regulates Na+/K+-ATPase polarization and spermiogenesis in Caenorhabditis elegans.
Surrogate Cerebrospinal Fluid Biomarkers for Assessing the Efficacy of Gene Therapy in Hurler Syndrome.
Generation of an induced pluripotent stem cell line (TRNDi030-A) from a patient with Farber disease carrying a homozygous p. Y36C (c. 107 A>G) mutation in ASAH1.
Robustness and innovation along the endocytic route: Lessons from darkness.
Non-invasive and high-throughput interrogation of exon-specific isoform expression.
Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly.
A Comprehensive Review: Sphingolipid Metabolism and Implications of Disruption in Sphingolipid Homeostasis.

Autophagy. 2021 Jun 04.

Lipid-mediated impairment of axonal lysosome transport contributing to autophagic stress.

Joseph C Roney, Sunan Li, Tamar Farfel-Becker, Ning Huang, Tao Sun, Yuxiang Xie, Xiu-Tang Cheng, Mei-Yao Lin, Frances M Platt, Zu-Hang Sheng.

  Efficient degradation of autophagic vacuoles (AVs) generated at axon terminals by mature lysosomes enriched in the cell body represents an exceptional challenge that neurons face in maintaining cellular homeostasis. Here, we discuss our recent findings revealing a lipid-mediated impairment of lysosome transport to distal axons contributing to axonal AV accumulation in the neurodegenerative lysosomal storage disorder Niemann-Pick disease type C (NPC). Using transmission electron microscopy, we observed a striking buildup of endocytic and autophagic organelles in NPC dystrophic axons, indicating defects in the clearance of organelles destined for lysosomal degradation. We further revealed that elevated cholesterol on NPC lysosome membranes abnormally sequesters motor-adaptors of axonal lysosome delivery, resulting in impaired anterograde lysosome transport into distal axons that disrupts maturation of axonal AVs during their retrograde transport route. Together, our study demonstrates a mechanism by which altered membrane lipid composition compromises axonal lysosome trafficking and positioning and shows that lowering lysosomal lipid levels rescues lysosome transport into NPC axons, thus reducing axonal autophagic stress at early stages of NPC disease.

Keywords:  Niemann-Pick disease type C; autophagy; axonal dystrophy; axonal transport; cholesterol; kinesin; lipid; lysosomal storage disorder; lysosome; neurodegeneration

DOI:  https://doi.org/10.1080/15548627.2021.1938916
Int J Mol Sci. 2021 May 23. pii: 5494. [Epub ahead of print]22(11):

TFEB Overexpression, Not mTOR Inhibition, Ameliorates RagCS75Y Cardiomyopathy.

Maengjo Kim, Linghui Lu, Alexey V Dvornikov, Xiao Ma, Yonghe Ding, Ping Zhu, Timothy M Olson, Xueying Lin, Xiaolei Xu.

  A de novo missense variant in Rag GTPase protein C (RagCS75Y) was recently identified in a syndromic dilated cardiomyopathy (DCM) patient. However, its pathogenicity and the related therapeutic strategy remain unclear. We generated a zebrafish RragcS56Y (corresponding to human RagCS75Y) knock-in (KI) line via TALEN technology. The KI fish manifested cardiomyopathy-like phenotypes and poor survival. Overexpression of RagCS75Y via adenovirus infection also led to increased cell size and fetal gene reprogramming in neonatal rat ventricle cardiomyocytes (NRVCMs), indicating a conserved mechanism. Further characterization identified aberrant mammalian target of rapamycin complex 1 (mTORC1) and transcription factor EB (TFEB) signaling, as well as metabolic abnormalities including dysregulated autophagy. However, mTOR inhibition failed to ameliorate cardiac phenotypes in the RagCS75Y cardiomyopathy models, concomitant with a failure to promote TFEB nuclear translocation. This observation was at least partially explained by increased and mTOR-independent physical interaction between RagCS75Y and TFEB in the cytosol. Importantly, TFEB overexpression resulted in more nuclear TFEB and rescued cardiomyopathy phenotypes. These findings suggest that S75Y is a pathogenic gain-of-function mutation in RagC that leads to cardiomyopathy. A primary pathological step of RagCS75Y cardiomyopathy is defective mTOR-TFEB signaling, which can be corrected by TFEB overexpression, but not mTOR inhibition.

Keywords:  RagCS75Y; Rags; TFEB; cardiomyopathy; mTOR

DOI:  https://doi.org/10.3390/ijms22115494
Biochem J. 2021 Jun 01. pii: BCJ20210171. [Epub ahead of print]

CLN5 and CLN3 function as a complex to regulate endolysosome function.

Seda Yasa, Etienne Sauvageau, Graziana Modica, Stephane Lefrancois.

  CLN5 is a soluble endolysosomal protein whose function is poorly understood. Mutations in this protein cause a rare neurodegenerative disease, Neuronal Ceroid Lipofuscinosis. We previously found that depletion of CLN5 leads to dysfunctional retromer, resulting in the degradation of the lysosomal sorting receptor, sortilin. However, how a soluble lysosomal protein can modulate the function of a cytosolic protein, retromer, is not known. In this work, we show that deletion of CLN5 not only results in retromer dysfunction, but also in impaired endolysosome fusion events. This results in delayed degradation of endocytic proteins and in defective autophagy. CLN5 modulates these various pathways by regulating downstream interactions between CLN3, an endolysosomal integral membrane protein whose mutations also result in Neuronal Ceroid Lipofuscinosis, RAB7A, and a subset of RAB7A effectors. Our data supports a model where CLN3 and CLN5 function as an endolysosomal complex regulating various functions.

Keywords:  CLN3; CLN5; Rab7A; endosomes; palmitoylation; retromer

DOI:  https://doi.org/10.1042/BCJ20210171
Genes (Basel). 2021 May 13. pii: 728. [Epub ahead of print]12(5):

MTOR Signaling and Metabolism in Early T Cell Development.

Guy Werlen, Ritika Jain, Estela Jacinto.

  The mechanistic target of rapamycin (mTOR) controls cell fate and responses via its functions in regulating metabolism. Its role in controlling immunity was unraveled by early studies on the immunosuppressive properties of rapamycin. Recent studies have provided insights on how metabolic reprogramming and mTOR signaling impact peripheral T cell activation and fate. The contribution of mTOR and metabolism during early T-cell development in the thymus is also emerging and is the subject of this review. Two major T lineages with distinct immune functions and peripheral homing organs diverge during early thymic development; the αβ- and γδ-T cells, which are defined by their respective TCR subunits. Thymic T-regulatory cells, which have immunosuppressive functions, also develop in the thymus from positively selected αβ-T cells. Here, we review recent findings on how the two mTOR protein complexes, mTORC1 and mTORC2, and the signaling molecules involved in the mTOR pathway are involved in thymocyte differentiation. We discuss emerging views on how metabolic remodeling impacts early T cell development and how this can be mediated via mTOR signaling.

Keywords:  T lymphocytes; T-cell metabolism; early T cell development; mTOR; mTORC1; mTORC2; thymocytes

DOI:  https://doi.org/10.3390/genes12050728
J Biol Chem. 2021 Jun 01. pii: S0021-9258(21)00648-7. [Epub ahead of print] 100850

Loss of PKA regulatory subunit 1α aggravates cardiomyocyte necrosis and myocardial ischemia/reperfusion injury.

Yuening Liu, Jingrui Chen, Peng Xia, Constantine A Stratakis, Zhaokang Cheng.

  Reperfusion therapy, the standard treatment for acute myocardial infarction, can trigger necrotic death of cardiomyocytes and provoke ischemia/reperfusion (I/R) injury. However, signaling pathways that regulate cardiomyocyte necrosis remain largely unknown. Our recent genome-wide RNAi screen has identified a potential necrosis suppressor gene PRKAR1A, which encodes PKA regulatory subunit 1α (R1α). R1α is primarily known for regulating PKA activity by sequestering PKA catalytic subunits in the absence of cAMP. Here, we showed that depletion of R1α augmented cardiomyocyte necrosis in vitro and in vivo, resulting in exaggerated myocardial I/R injury and contractile dysfunction. Mechanistically, R1α loss downregulated the Nrf2 antioxidant transcription factor and aggravated oxidative stress following I/R. Degradation of the endogenous Nrf2 inhibitor Keap1 through p62-dependent selective autophagy was blocked by R1α depletion. Phosphorylation of p62 at Ser349 by mammalian target of rapamycin complex 1 (mTORC1), a critical step in p62-Keap1 interaction, was induced by I/R, but diminished by R1α loss. Activation of PKA by forskolin or isoproterenol almost completely abolished hydrogen peroxide-induced p62 phosphorylation. In conclusion, R1α loss induces unrestrained PKA activation and impairs the mTORC1-p62-Keap1-Nrf2 antioxidant defense system, leading to aggravated oxidative stress, necrosis and myocardial I/R injury. Our findings uncover a novel role of PKA in oxidative stress and necrosis, which may be exploited to develop new cardioprotective therapies.

Keywords:  Carney complex; PKA; adenylyl cyclase; adrenergic receptor; cardiomyopathy; catecholamine; mitochondrial permeability transition; myocardial infarction; necrosis; oxidative stress

DOI:  https://doi.org/10.1016/j.jbc.2021.100850
Endocrinology. 2021 Jun 04. pii: bqab112. [Epub ahead of print]

Thyroid hormone receptor-α regulates autophagy, mitochondrial biogenesis, and fatty acid utilization in skeletal muscle.

Jin Zhou, Karine Gauthier, Jia Pei Ho, Andrea Lim, Xu-Guang Zhu, Cho Rong Han, Rohit Anthony Sinha, Sheue-Yann Cheng, Paul Michael Yen.

  Skeletal muscle (SM) weakness occurs in hypothyroidism and resistance to thyroid hormone alpha (RTHα) syndrome. However, the cell signaling and molecular mechanism(s) underlying muscle weakness under these conditions is not well understood. We thus examined the role of thyroid hormone receptor alpha (TRα), the predominant TR isoform in SM, on autophagy, mitochondrial biogenesis and metabolism to demonstrate the molecular mechanism(s) underlying muscle weakness in these two conditions.Two genetic mouse models, TRα1 PV/+ mice which expresses mutant Thra1PV gene ubiquitously, and SM-TRα1 L400R/+ mice, which expresses TRα1 L400R in a muscle-specific manner, were used in this study. Gastrocnemius muscle from TRα1 PV/+, SM-TRα1 L400R/+, and their control mice was harvested for analyses. We demonstrated that loss of TRα1 signaling in gastrocnemius muscle from both the genetic mouse models led to decreased autophagy as evidenced by accumulation of p62 and decreased expression of lysosomal markers (LAMP1, and LAMP2) and lysosomal proteases (cathepsin B and cathepsin D). The expression of PGC1α, TFAM, and ERRα, key factors contributing to mitochondrial biogenesis as well as mitochondrial proteins were decreased, suggesting that there was reduced mitochondrial biogenesis due to the expression of mutant TRα1. Transcriptomic and metabolomic analyses of SM suggested that lipid catabolism was impaired, and was associated with decreased acylcarnitines and tricarboxylic acid cycle (TCA cycle) intermediates in the SM from the mouse line expressing SM-specific mutant TRα1. Our results provide new insight into TRα1-mediated cell signaling, molecular, and metabolic changes that occur in SM when TR action is impaired.

Keywords:  TRα1 mutation; autophagy; lipid metabolism; mitochondrial function; muscle

DOI:  https://doi.org/10.1210/endocr/bqab112
Autophagy. 2021 Jun 04.

Multiplexed suppression of TOR complex 1 induces autophagy during starvation.

Tomoyuki Fukuda, Kazuhiro Shiozaki.

  Target of rapamycin complex 1 (TORC1) promotes cellular anabolism and suppresses macroautophagy/autophagy. In mammalian cells starved of amino acid, the GATOR1 complex, a negative regulator of TORC1, is released from its inhibitor GATOR2 and inactivates TORC1. We have recently identified the evolutionarily conserved GATOR2 components in fission yeast including Sea3, an ortholog of mammalian WDR59, but, unexpectedly, Sea3 acts as a part of GATOR1 to suppress TORC1. Moreover, fission yeast GATOR1 is not required for the amino-acid starvation-induced TORC1 attenuation, which is instead mediated by the Gcn2 pathway. Conversely, absence of a nitrogen source suppresses TORC1 in a manner dependent on GATOR1 as well as the Tsc1-Tsc2 complex, whose mammalian equivalent functions as a growth-factor sensitive TORC1 inhibitor. Thus, the evolutionarily conserved signaling modules are utilized differently between fission yeast and mammals to control TORC1 activity and autophagy.

Keywords:  GATOR complex; Gcn2; Rag GTPase; TOR; TSC complex; autophagy

DOI:  https://doi.org/10.1080/15548627.2021.1938915
Biomolecules. 2021 May 01. pii: 681. [Epub ahead of print]11(5):

mTOR Signaling in Metabolic Stress Adaptation.

Cheng-Wei Wu, Kenneth B Storey.

  The mechanistic target of rapamycin (mTOR) is a central regulator of cellular homeostasis that integrates environmental and nutrient signals to control cell growth and survival. Over the past two decades, extensive studies of mTOR have implicated the importance of this protein complex in regulating a broad range of metabolic functions, as well as its role in the progression of various human diseases. Recently, mTOR has emerged as a key signaling molecule in regulating animal entry into a hypometabolic state as a survival strategy in response to environmental stress. Here, we review current knowledge of the role that mTOR plays in contributing to natural hypometabolic states such as hibernation, estivation, hypoxia/anoxia tolerance, and dauer diapause. Studies across a diverse range of animal species reveal that mTOR exhibits unique regulatory patterns in an environmental stressor-dependent manner. We discuss how key signaling proteins within the mTOR signaling pathways are regulated in different animal models of stress, and describe how each of these regulations uniquely contribute to promoting animal survival in a hypometabolic state.

Keywords:  Akt; TOR; anoxia; cell signaling; dauer; environmental stress; estivation; hibernation; hypoxia; metabolism; protein translation

DOI:  https://doi.org/10.3390/biom11050681
PLoS Biol. 2021 Jun 02. 19(6): e3001281

The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.

Jung Mi Park, Seung Wook Yang, Wei Zhuang, Asim K Bera, Yan Liu, Deepak Gurbani, Sergei J von Hoyningen-Huene, Sadie Miki Sakurada, Haiyun Gan, Shondra M Pruett-Miller, Kenneth D Westover, Malia B Potts.

Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.

DOI: https://doi.org/10.1371/journal.pbio.3001281
BMC Biol. 2021 Jun 04. 19(1): 117

Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase.

Cheng-Wen He, Xue-Fei Cui, Shao-Jie Ma, Qin Xu, Yan-Peng Ran, Wei-Zhi Chen, Jun-Xi Mu, Hui Li, Jing Zhu, Qingqiu Gong, Zhiping Xie.

   BACKGROUND: The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways.
RESULTS: Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation.
CONCLUSIONS: Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

Keywords:  Atg8; ESCRT; Hfl1; Microautophagy; Vacuole; Yeast

DOI:  https://doi.org/10.1186/s12915-021-01048-7
Cell Death Dis. 2021 Jun 02. 12(6): 571

FKBP39 controls nutrient dependent Nprl3 expression and TORC1 activity in Drosophila.

Ying Zhou, Jian Guo, Xinyu Wang, Yang Cheng, Jianwen Guan, Priyam Barman, Ming-An Sun, Yuanyuan Fu, Wanhong Wei, Congjing Feng, Mary A Lilly, Youheng Wei.

Target of Rapamycin Complex 1 (TORC1) is a master regulator that coordinates nutrient status with cell metabolism. The GTPase-activating protein towards Rags complex 1 (GATOR1) inhibits TORC1 activity and protects cells from damage during periods of stress. Here we characterize multiple pathways that regulate the expression of the GATOR1 component Nprl3 in Drosophila. We determine that the stability of Nprl3 is impacted by the Unassembled Soluble Complex Proteins Degradation (USPD) pathway. In addition, we find that FK506 binding protein 39 (FKBP39)-dependent proteolytic destruction maintains Nprl3 at low levels in nutrient replete conditions. Nutrient starvation abrogates the degradation of the Nprl3 protein and rapidly promotes Nprl3 accumulation. Consistent with a role in promoting the stability of a TORC1 inhibitor, mutations in fkbp39 decrease TORC1 activity and increase autophagy. Finally, we show that the 5'UTR of nprl3 transcripts contain a functional upstream open reading frame (uORF) that inhibits main ORF translation. In summary, our work has uncovered novel mechanisms of Nprl3 regulation and identifies an important role for FKBP39 in the control of cellular metabolism.

DOI: https://doi.org/10.1038/s41419-021-03860-z
Cell Cycle. 2021 May;20(9): 839-854

mTORC1 induces eukaryotic translation initiation factor 4E interaction with TOS-S6 kinase 1 and its activation.

Sheikh Tahir Majeed, Asiya Batool, Rabiya Majeed, Nadiem Nazir Bhat, Muhammad Afzal Zargar, Khurshid Iqbal Andrabi.

  Eukaryotic translation initiation factor 4E was recently shown to be a substrate of mTORC1, suggesting it may be a mediator of mTORC1 signaling. Here, we present evidence that eIF4E phosphorylated at S209 interacts with TOS motif of S6 Kinase1 (S6K1). We also show that this interaction is sufficient to overcome rapamycin sensitivity and mTORC1 dependence of S6K1. Furthermore, we show that eIF4E-TOS interaction relieves S6K1 from auto-inhibition due to carboxy terminal domain (CTD) and primes it for hydrophobic motif (HM) phosphorylation and activation in mTORC1 independent manner. We conclude that the role of mTORC1 is restricted to engaging eIF4E with S6K1-TOS motif to influence its state of HM phosphorylation and inducing its activation.

Keywords:  S6 Kinase 1; eIF4E; mTOR

DOI:  https://doi.org/10.1080/15384101.2021.1901038
Cancers (Basel). 2021 May 16. pii: 2405. [Epub ahead of print]13(10):

Endosomal mTORC2 Is Required for Phosphoinositide-Dependent AKT Activation in Platelet-Derived Growth Factor-Stimulated Glioma Cells.

Suree Kim, Sukyeong Heo, Joseph Brzostowski, Dongmin Kang.

  The serine/threonine kinase AKT is a major effector during phosphatidylinositol 3-kinase (PI3K)-driven cell signal transduction in response to extracellular stimuli. AKT activation mechanisms have been extensively studied; however, the mechanism underlying target of rapamycin complex 2 (mTORC2) phosphorylation of AKT at Ser473 in the cellular endomembrane system remains to be elucidated. Here, we demonstrate that endocytosis is required for AKT activation through phosphorylation at Ser473 via mTORC2 using platelet-derived growth factor-stimulated U87MG glioma cells. mTORC2 components are localized to early endosomes during growth factor activation, and the association of mTORC2 with early endosomes is responsible for the local activation of AKT, which is critical for specific signal transduction through glycogen synthase kinase-3 beta and forkhead box O1/O3 phosphorylation. Furthermore, endosomal phosphoinositide, represented by PtdIns(3,4)P2, provides a binding platform for mTORC2 to phosphorylate AKT Ser473 in endosomes through mammalian Sty1/Spc1-interacting protein (mSIN), a pleckstrin homology domain-containing protein, and is dispensable for AKT phosphorylation at Thr308. This PtdIns(3,4)P2-mediated endosomal AKT activation provides a means to integrate PI3K activated by diverse stimuli to mTORC2 assembly. These early endosomal events induced by endocytosis, together with the previously identified AKT activation by PtdIns(3,4,5)P3, contribute to the strengthening of the transduction of AKT signaling through phosphoinositide.

Keywords:  AKT; endocytosis; endosome; mTORC2; phosphatidylinositol

DOI:  https://doi.org/10.3390/cancers13102405
Int J Mol Sci. 2021 May 31. pii: 5948. [Epub ahead of print]22(11):

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA.

Alon M Douek, Mitra Amiri Khabooshan, Jason Henry, Sebastian-Alexander Stamatis, Florian Kreuder, Georg Ramm, Minna-Liisa Änkö, Donald Wlodkowic, Jan Kaslin.

  Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgshΔex5-6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

Keywords:  CRISPR/Cas9; Sanfilippo syndrome; animal disease model; childhood dementia; heparan sulfate; lysosomal storage disorder; mucopolysaccharidosis; neuroinflammation; zebrafish

DOI:  https://doi.org/10.3390/ijms22115948
Cancers (Basel). 2021 May 18. pii: 2441. [Epub ahead of print]13(10):

Cathepsin K: A Novel Diagnostic and Predictive Biomarker for Renal Tumors.

Anna Caliò, Matteo Brunelli, Stefano Gobbo, Pedram Argani, Enrico Munari, George Netto, Guido Martignoni.

  Cathepsin K is a papain-like cysteine protease with high matrix-degrading activity. Among several cathepsins, cathepsin K is the most potent mammalian collagenase, mainly expressed by osteoclasts. This review summarizes most of the recent findings of cathepsin K expression, highlighting its role in renal tumors for diagnostic purposes and as a potential molecular target. Indeed, cathepsin K is a recognized diagnostic tool for the identification of TFE3/TFEB-rearranged renal cell carcinoma, TFEB-amplified renal cell carcinoma, and pure epithelioid PEComa/epithelioid angiomyolipoma. More recently, its expression has been observed in a subgroup of eosinophilic renal neoplasms molecularly characterized by TSC/mTOR gene mutations. Interestingly, both TSC mutations or TFE3 rearrangement have been reported in pure epithelioid PEComa/epithelioid angiomyolipoma. Therefore, cathepsin K seems to be a downstream marker of TFE3/TFEB rearrangement, TFEB amplification, and mTOR pathway activation. Given the established role of mTOR inhibitors as a pharmacological option in renal cancers, cathepsin K could be of use as a predictive marker of therapy response and as a potential target. In the future, uropathologists may implement the use of cathepsin K to establish a diagnosis among renal tumors with clear cells, papillary architecture, and oncocytic features.

Keywords:  PEComa; TSC1/TSC2; angiomyolipoma; cathepsin K; differential diagnosis; mTOR pathway; predictive markers; renal cancers; translocation renal cell carcinoma

DOI:  https://doi.org/10.3390/cancers13102441
Cell Death Dis. 2021 May 29. 12(6): 558

Inhibition of the mTOR pathway and reprogramming of protein synthesis by MDM4 reduce ovarian cancer metastatic properties.

Rossella Lucà, Maria Rita Assenza, Fabio Maiullari, Luisa Pieroni, Silvia Maiullari, Giulia Federici, Federica Marini, Roberto Rizzi, Andrea Urbani, Silvia Soddu, Fabiola Moretti.

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

DOI: https://doi.org/10.1038/s41419-021-03828-z
Cells. 2021 May 07. pii: 1128. [Epub ahead of print]10(5):

Codon Bias Can Determine Sorting of a Potassium Channel Protein.

Anja J Engel, Marina Kithil, Markus Langhans, Oliver Rauh, Matea Cartolano, James L Van Etten, Anna Moroni, Gerhard Thiel.

  Due to the redundancy of the genetic code most amino acids are encoded by multiple synonymous codons. It has been proposed that a biased frequency of synonymous codons can affect the function of proteins by modulating distinct steps in transcription, translation and folding. Here, we use two similar prototype K+ channels as model systems to examine whether codon choice has an impact on protein sorting. By monitoring transient expression of GFP-tagged channels in mammalian cells, we find that one of the two channels is sorted in a codon and cell cycle-dependent manner either to mitochondria or the secretory pathway. The data establish that a gene with either rare or frequent codons serves, together with a cell-state-dependent decoding mechanism, as a secondary code for sorting intracellular membrane proteins.

Keywords:  codon usage; dual sorting; effect of synonymous codon exchange; membrane protein sorting

DOI:  https://doi.org/10.3390/cells10051128
Cells. 2021 May 13. pii: 1188. [Epub ahead of print]10(5):

Extracellular Acidification Induces Lysosomal Dysregulation.

Bryce Ordway, Robert J Gillies, Mehdi Damaghi.

  Many invasive cancers emerge through a years-long process of somatic evolution, characterized by an accumulation of heritable genetic and epigenetic changes and the emergence of increasingly aggressive clonal populations. In solid tumors, such as breast ductal carcinoma, the extracellular environment for cells within the nascent tumor is harsh and imposes different types of stress on cells, such as hypoxia, nutrient deprivation, and cytokine inflammation. Acidosis is a constant stressor of most cancer cells due to its production through fermentation of glucose to lactic acid in hypoxic or normoxic regions (Warburg effect). Over a short period of time, acid stress can have a profound effect on the function of lysosomes within the cells exposed to this environment, and after long term exposure, lysosomal function of the cancer cells can become completely dysregulated. Whether this dysregulation is due to an epigenetic change or evolutionary selection has yet to be determined, but understanding the mechanisms behind this dysregulation could identify therapeutic opportunities.

Keywords:  Warburg effect; breast cancer; cancer acidosis; cancer metastasis; intracellular pH; lysosomal localization; lysosome dysregulation; targeted therapy; tumor microenvironment

DOI:  https://doi.org/10.3390/cells10051188
J Lipid Res. 2021 Jun 01. pii: S0022-2275(21)00071-7. [Epub ahead of print] 100089

Inhibition of lysosomal phospholipase A2 predicts drug-induced phospholipidosis.

Vania Hinkovska-Galcheva, Taylour Treadwell, Jonathan M Shillingford, Angela Lee, Akira Abe, John J G Tesmer, James A Shayman.

  Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome specific phospholipase A2 (PLA2G15), and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme. Here, we assayed a library of 163 drugs was assayed for inhibition of PLA2G15 to determine whether this phospholipase was the cellular target for therapeutics other than amiodarone that cause phospholipidosis. We observed that 144 compounds inhibited PLA2G15 activity. Thirty-six compounds not previously reported to cause phospholipidosis inhibited PLA2G15 with IC50 values less than 1 mM, and were confirmed to cause phospholipidosis in an in vitro assay. Within this group, fosinopril was the most potent inhibitor (IC50 0.18 μM). Additional characterization of the inhibition of PLA2G15 by fosinopril was consistent with interference of PLA2G15 binding to liposomes. PLA2G15 inhibition was more accurate in predicting phospholipidosis compared to in silico models based on pKa and ClogP, measures of protonation and transport-independent distribution in the lysosome, respectively. In summary, PLA2G15 is a primary target for cationic amphiphilic drugs that cause phospholipidosis, and PLA2G15 inhibition by cationic amphiphilic compounds provides a potentially robust screening platform for potential toxicity during drug development.

Keywords:  1-O-acylceramide; Acyltransferase; amiodarone; cationic amphiphilic drugs; drug development; drug toxicity; drug-induced phospholipidosis; high-throughput screening; lysosome; phospholipase A(2) group XV

DOI:  https://doi.org/10.1016/j.jlr.2021.100089
Proc Natl Acad Sci U S A. 2021 Jun 08. pii: e2100844118. [Epub ahead of print]118(23):

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2.

Sven H Schmidt, Jui-Hung Weng, Phillip C Aoto, Daniela Boassa, Sebastian Mathea, Steve Silletti, Junru Hu, Maximilian Wallbott, Elizabeth A Komives, Stefan Knapp, Friedrich W Herberg, Susan S Taylor.

  To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

Keywords:  Gaussian accelerated molecular dynamics; Parkinson’s disease; hydrogen-deuterium exchange mass spectrometry (HDX-MS); kinase regulation; leucine-rich repeat kinase 2 (LRRK2)

DOI:  https://doi.org/10.1073/pnas.2100844118
J Mol Cell Cardiol. 2021 May 31. pii: S0022-2828(21)00110-3. [Epub ahead of print]

Metabolic remodeling precedes mTORC1-mediated cardiac hypertrophy.

Giovannis E Davogustto, Rebecca L Salazar, Hernan G Vasquez, Anja Karlstaedt, William P Dillon, Patrick H Guthrie, Joseph R Martin, Heidi Vitrac, Gina De La Guardia, Deborah Vela, Aleix Ribas-Latre, Corrine Baumgartner, Kristin Eckel-Mahan, Heinrich Taegtmeyer.

   RATIONALE: The nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) and its primary inhibitor, tuberin (TSC2), are cues for the development of cardiac hypertrophy. The phenotype of mTORC1 induced hypertrophy is unknown.
OBJECTIVE: To examine the impact of sustained mTORC1 activation on metabolism, function, and structure of the adult heart.
METHODS AND RESULTS: We developed a mouse model of inducible, cardiac-specific sustained mTORC1 activation (mTORC1iSA) through deletion of Tsc2. Prior to hypertrophy, rates of glucose uptake and oxidation, as well as protein and enzymatic activity of glucose 6-phosphate isomerase (GPI) were decreased, while intracellular levels of glucose 6-phosphate (G6P) were increased. Subsequently, hypertrophy developed. Transcript levels of the fetal gene program and pathways of exercise-induced hypertrophy increased. While hypertrophy did not progress to heart failure. We therefore examined the hearts of wild-type mice subjected to voluntary physical activity and observed early changes in GPI, followed by hypertrophy. Rapamycin prevented these changes in both models.
CONCLUSION: Activation of mTORC1 in the adult heart triggers the development of a non-specific form of hypertrophy which is preceded by changes in cardiac glucose metabolism.

Keywords:  Exercise; Glycolysis; Hypertrophy; Metabolism; mTORC1

DOI:  https://doi.org/10.1016/j.yjmcc.2021.05.016
Glycobiology. 2021 May 31. pii: cwab046. [Epub ahead of print]

Glucosylceramide and galactosylceramide, small glycosphingolipids with significant impact on health and disease.

Safoura Reza, Maciej Ugorski, Jarosław Suchański.

  Numerous clinical observations and exploitation of cellular and animal models indicate that glucosylceramide (GlcCer) and galactosylceramide (GalCer) are involved in many physiological and pathological phenomena. In many cases, the biological importance of these monohexosylcermides has been shown indirectly as the result of studies on enzymes involved in their synthesis and degradation. Under physiological conditions, GalCer plays a key role in the maintenance of proper structure and stability of myelin and differentiation of oligodendrocytes. On the other hand, GlcCer is necessary for the proper functions of epidermis. Such an important lysosomal storage disease as Gaucher disease and a neurodegenerative disorder as Parkinson's disease are characterized by mutations in the GBA1 gene, decreased activity of lysosomal GBA1 glucosylceramidase and accumulation of GlcCer. In contrast, another lysosomal disease, Krabbe disease, is associated with mutations in the GALC gene, resulting in deficiency or decreased activity of lysosomal galactosylceramidase and accumulation of GalCer and galactosylsphingosine. Little is known about the role of both monohexosylceramides in tumor progression, however, numerous studies indicate that GlcCer and GalCer play important roles in the development of multidrug-resistance by cancer cells. It was shown that GlcCer is able to provoke immune reaction and acts as a self-antigen in Gaucher disease. On the other hand, GalCer was recognized as an important cellular receptor for HIV-1. Together, these two molecules are excellent examples of how slight differences in chemical composition and molecular conformation contribute to profound differences in their physicochemical properties and biological functions.

Keywords:  Biological functions; Galactosylceramide; Glucosylceramide; Glycosphingolipids; Neurological diseases

DOI:  https://doi.org/10.1093/glycob/cwab046
Traffic. 2021 Jun 05.

The ESCRT-III complex contributes to macromitophagy in yeast.

Zulin Wu, Haiqian Xu, Junze Liu, Fan Zhou, Yongheng Liang.

  Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, GST pulldown and BiFC, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32 marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

Keywords:  Atg11; Atg32; ESCRT; Macromitophagy; Micromitophagy; Mitophagosome; Mitophagy, Snf7; Vps4

DOI:  https://doi.org/10.1111/tra.12805
Dev Cell. 2021 May 25. pii: S1534-5807(21)00402-0. [Epub ahead of print]

Membrane contact site-dependent cholesterol transport regulates Na+/K+-ATPase polarization and spermiogenesis in Caenorhabditis elegans.

Qiushi Wang, Zheng Cao, Baochen Du, Qi Zhang, Lianwan Chen, Xia Wang, Zhiheng Yuan, Peng Wang, Ruijun He, Jin Shan, Yanmei Zhao, Long Miao.

  Spermiogenesis in nematodes is a process whereby round and quiescent spermatids differentiate into asymmetric and crawling spermatozoa. The molecular mechanism underlying this symmetry breaking remains uncharacterized. In this study, we revealed that sperm-specific Na+/K+-ATPase (NKA) is evenly distributed on the plasma membrane (PM) of Caenorhabditis elegans spermatids but is translocated to and subsequently enters the invaginated membrane of the spermatozoa cell body during sperm activation. The polarization of NKA depends on the transport of cholesterol from the PM to membranous organelles (MOs) via membrane contact sites (MCSs). The inositol 5-phosphatase CIL-1 and the MO-localized PI4P phosphatase SAC-1 may mediate PI4P metabolism to drive cholesterol countertransport via sterol/lipid transport proteins through MCSs. Furthermore, the NKA function is required for C. elegans sperm motility and reproductive success. Our data imply that the lipid dynamics mediated by MCSs might play crucial roles in the establishment of cell polarity. eGraphical abstract.

Keywords:  C. elegans; Na+/K+-ATPase; cell motility and cytoskeleton; cell polarity establishment; cholesterol; major sperm protein; membrane contact sites; non-vesicular transport; phospholipid; sperm activation

DOI:  https://doi.org/10.1016/j.devcel.2021.05.002
Front Neurol. 2021 ;12 640547

Surrogate Cerebrospinal Fluid Biomarkers for Assessing the Efficacy of Gene Therapy in Hurler Syndrome.

Reiner F Haseloff, Stephanie Trudel, Ramona Birke, Michael Schümann, Eberhard Krause, Cathy Gomila, Jean-Michel Heard, Ingolf E Blasig, Jérôme Ausseil.

  Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal hydroxylase alpha-l-iduronidase (IDUA). The resulting accumulation of dermatan and heparan sulfate induces intellectual disabilities and pre-mature death, and only a few treatment options are available. In a previous study, we demonstrated the feasibility, safety, and efficacy of gene therapy by injecting recombinant adeno-associated viral vector serotype (AAV)2/5-IDUA into the brain of a canine model of MPS I. We report on a quantitative proteomic analysis of control dogs and untreated dogs with MPS I cerebrospinal fluid (CSF) that had been collected throughout the study in the MPS I dogs. Mass spectrometry (MS) analysis identified numerous proteins present at altered levels in MPS I CSF samples. Quantitative immunoblotting, performed on CSF from healthy controls, untreated MPS I dogs, and MPS I dogs early treated and late treated by gene therapy, confirmed the MS data for a subset of proteins with higher abundance (neuronal pentraxin 1, chitinase 3-like 1, monocyte differentiation antigen CD14, and insulin-like growth factor-binding protein 2). Scoring of the results shows that the expression levels of these proteins are close to those of the control group for dogs that underwent gene therapy early in life but not for older treated animals. Our results disclose four novel predictive biomarker candidates that might be valuable in monitoring the course of the neurological disease in MPS patients at diagnosis, during clinical follow-up, and after treatment.

Keywords:  cerebrospinal fluid; gene therapy; mass spectrometry; mucopolysaccharidosis; surrogate marker

DOI:  https://doi.org/10.3389/fneur.2021.640547
Stem Cell Res. 2021 May;pii: S1873-5061(21)00233-6. [Epub ahead of print]53 102387

Generation of an induced pluripotent stem cell line (TRNDi030-A) from a patient with Farber disease carrying a homozygous p. Y36C (c. 107 A>G) mutation in ASAH1.

Brianna M Brooks, Charles D Yeh, Jeanette Beers, Chengyu Liu, Yu-Shan Cheng, Kirill Gorshkov, Jizhong Zou, Wei Zheng, Catherine Z Chen.

Farber disease is an ultra-rare lysosomal storage disease. Mutations in the N-acylsphingosine amidohydrolase (ASAH1) gene, which encodes for the enzyme acid ceramidase (ACDase), cause ceramides to accumulate in the body. A human induced pluripotent stem cell (iPSC) line TRNDi030-A was generated from fibroblasts of a male patient with a homozygous p. Y36C (c.107 A>G) variant in the second exon of the ASAH1 producing the alpha subunit of ACDase. This Farber disease iPSC line is a useful resource to study disease pathophysiology and to develop therapeutics for treatment of patients with Farber disease.

DOI: https://doi.org/10.1016/j.scr.2021.102387
J Cell Biol. 2021 Jul 05. pii: e202105030. [Epub ahead of print]220(7):

Robustness and innovation along the endocytic route: Lessons from darkness.

Kaela S Singleton, Victor Faundez.

What mechanisms ensure the loading of a SNARE into a nascent carrier? In this issue, Bowman et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202005173) describe an unprecedented mechanism where two sorting complexes, AP-3 and BLOC-1, the latter bound to syntaxin 13, work as a fail-safe to recognize sorting signals in VAMP7, a membrane protein required for fusion to melanosomes. Their observations define one of the first examples of distributed robustness in membrane traffic mechanisms.

DOI: https://doi.org/10.1083/jcb.202105030
Nat Cell Biol. 2021 Jun 03.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Dong-Jiunn Jeffery Truong, Teeradon Phlairaharn, Bianca Eßwein, Christoph Gruber, Deniz Tümen, Enikő Baligács, Niklas Armbrust, Francesco Leandro Vaccaro, Eva-Maria Lederer, Eva Magdalena Beck, Julian Geilenkeuser, Simone Göppert, Luisa Krumwiede, Christian Grätz, Gerald Raffl, Dominic Schwarz, Martin Zirngibl, Milica Živanić, Maren Beyer, Johann Dietmar Körner, Tobias Santl, Valentin Evsyukov, Tabea Strauß, Sigrid C Schwarz, Günter U Höglinger, Peter Heutink, Sebastian Doll, Marcus Conrad, Florian Giesert, Wolfgang Wurst, Gil Gregor Westmeyer.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

DOI: https://doi.org/10.1038/s41556-021-00678-x
Biochemistry. 2021 Jun 03.

Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly.

David L Dai, S M Naimul Hasan, Geoffrey Woollard, Yazan M Abbas, Stephanie A Bueler, Jean-Philippe Julien, John L Rubinstein, Mohammad T Mazhab-Jafari.

Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 μm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.

DOI: https://doi.org/10.1021/acs.biochem.1c00269
Int J Mol Sci. 2021 May 28. pii: 5793. [Epub ahead of print]22(11):

A Comprehensive Review: Sphingolipid Metabolism and Implications of Disruption in Sphingolipid Homeostasis.

Brianna M Quinville, Natalie M Deschenes, Alex E Ryckman, Jagdeep S Walia.

  Sphingolipids are a specialized group of lipids essential to the composition of the plasma membrane of many cell types; however, they are primarily localized within the nervous system. The amphipathic properties of sphingolipids enable their participation in a variety of intricate metabolic pathways. Sphingoid bases are the building blocks for all sphingolipid derivatives, comprising a complex class of lipids. The biosynthesis and catabolism of these lipids play an integral role in small- and large-scale body functions, including participation in membrane domains and signalling; cell proliferation, death, migration, and invasiveness; inflammation; and central nervous system development. Recently, sphingolipids have become the focus of several fields of research in the medical and biological sciences, as these bioactive lipids have been identified as potent signalling and messenger molecules. Sphingolipids are now being exploited as therapeutic targets for several pathologies. Here we present a comprehensive review of the structure and metabolism of sphingolipids and their many functional roles within the cell. In addition, we highlight the role of sphingolipids in several pathologies, including inflammatory disease, cystic fibrosis, cancer, Alzheimer's and Parkinson's disease, and lysosomal storage disorders.

Keywords:  biosynthesis; ceramide; glycosphingolipids; glycosyl hydrolase; inflammation; lysosomal storage disorder; neurodegeneration; sphingolipid; sphingosine-1-phosphate

DOI:  https://doi.org/10.3390/ijms22115793