bims-lysosi 2021-09-19 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒09‒19
thirty-one papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Regulation of human mTOR complexes by DEPTOR.
Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace.
Loss of Christianson Syndrome Na+/H+ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons.
Dynamic modelling of the PI3K/MTOR signalling network uncovers biphasic dependence of mTORC1 activity on the mTORC2 subunit SIN1.
The Development and Survival of Thymic Epithelial Cells Require TSC1-Dependent Negative Regulation of mTORC1 Activity.
Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cells homeostasis.
UBE2S exerts oncogenic activities in urinary bladder cancer by ubiquitinating TSC1.
A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy.
Role of mTor signaling for tubular function and disease.
Glucocerebrosidase dysfunction in neurodegenerative disease.
Alpha-Synuclein defects autophagy by impairing SNAP29-mediated autophagosome-lysosome fusion.
TFEB Insufficiency Promotes Cardiac Hypertrophy by Blocking Autophagic Degradation of GATA4.
Membrane trafficking: Retrofusion as an escape route out of the endosome.
The ingenious ULKs: expanding the repertoire of the ULK complex with phosphoproteomics.
Proteomics of autism and Alzheimer's mouse models reveal common alterations in mTOR signaling pathway.
A glutamine sensor that directly activates TORC1.
A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling.
Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer.
Activation of mTORC1 and c-Jun by Prohibitin1 loss in Schwann cells may link mitochondrial dysfunction to demyelination.
Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.
Protein structural features predict responsiveness to pharmacological chaperone treatment for three lysosomal storage disorders.
Cross-Species and Human Inter-Tissue Network Analysis of Genes Implicated in Longevity and Aging Reveal Strong Support for Nutrient Sensing.
Lysosomal amino acid transporters as key players in inflammatory diseases.
An active tethering mechanism controls the fate of vesicles.
Molecular mechanisms of dietary restriction promoting health and longevity.
Treatment of GM2 Gangliosidosis in Adult Sandhoff Mice using an Intravenous Self-Complementary Hexosaminidase Vector.
Increased fidelity of protein synthesis extends lifespan.
De novo design of tyrosine and serine kinase-driven protein switches.
GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.
RORα is critical for mTORC1 activity in T cell-mediated colitis.
Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders.

Elife. 2021 09 14. pii: e70871. [Epub ahead of print]10

Regulation of human mTOR complexes by DEPTOR.

Matthias Wälchli, Karolin Berneiser, Francesca Mangia, Stefan Imseng, Louise-Marie Craigie, Edward Stuttfeld, Michael N Hall, Timm Maier.

  The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.

Keywords:  DEPTOR; cancer; cryo-EM; human; mTOR; metabolism; molecular biophysics; signaling; structural biology

DOI:  https://doi.org/10.7554/eLife.70871
Elife. 2021 09 14. pii: e68799. [Epub ahead of print]10

Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace.

Maren Heimhalt, Alex Berndt, Jane Wagstaff, Madhanagopal Anandapadamanaban, Olga Perisic, Sarah Maslen, Stephen McLaughlin, Conny Wing-Heng Yu, Glenn R Masson, Andreas Boland, Xiaodan Ni, Keitaro Yamashita, Garib N Murshudov, Mark Skehel, Stefan M Freund, Roger L Williams.

  The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.

Keywords:  DEPTOR; biochemistry; cancer; chemical biology; cryo-EM; human; mTOR; molecular biophysics; partial inhibition; signal transduction; structural biology

DOI:  https://doi.org/10.7554/eLife.68799
J Neurosci. 2021 Sep 07. pii: JN-RM-1244-20. [Epub ahead of print]

Loss of Christianson Syndrome Na+/H+ Exchanger 6 (NHE6) Causes Abnormal Endosome Maturation and Trafficking Underlying Lysosome Dysfunction in Neurons.

Matthew F Pescosolido, Qing Ouyang, Judy S Liu, Eric M Morrow.

Loss-of-function mutations in endosomal Na+/H+ exchanger 6 (NHE6) cause the X-linked neurologic disorder Christianson syndrome (CS). Patients exhibit symptoms associated with both neurodevelopmental and neurodegenerative abnormalities. While loss of NHE6 has been shown to over-acidify the endosome lumen, and is associated with endolysosome neuropathology, NHE6-mediated mechanisms in endosome trafficking and lysosome function have been understudied. Here, we show that NHE6-null mouse neurons demonstrate worsening lysosome function with time in culture, likely as a result of defective endosome trafficking. NHE6-null neurons exhibit overall reduced lysosomal proteolysis despite over-acidification of the endosome and lysosome lumen. Akin to Nhx1 mutants in Saccharomyces cerevisiae, we observe decreased endosome-lysosome fusion in NHE6-null neurons. Also, we find premature activation of pH-dependent cathepsin D (CatD) in endosomes. While active CatD is increased in endosomes, CatD activation and CatD protein levels are reduced in the lysosome. Protein levels of another mannose 6-phosphate receptor (M6PR)-dependent enzyme, β-N-acetylglucosaminidase, were also decreased in lysosomes of NHE6-null neurons. M6PRs accumulate in late endosomes, suggesting defective M6PR recycling and retromer function in NHE6-null neurons. Finally, coincident with decreased endosome-lysosome fusion, using total internal reflection fluorescence (TIRF), we also find a prominent increase in fusion between endosomal multivesicular bodies (MVBs) and the plasma membrane (PM), indicating enhanced exosome secretion from NHE6-null neurons. In summary, in addition to over-acidification of endosomes and lysosomes, loss of NHE6 leads to defects in endosome maturation and trafficking, including enhanced exosome release, contributing to lysosome deficiency and potentially leading to neurodegenerative disease.SIGNIFICANCE STATEMENTLoss-of-function mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome (CS), an X-linked neurological disorder. Loss of NHE6 has been shown to over-acidify endosomes; however, endosome trafficking mechanisms have been understudied, and the mechanisms leading to neurodegeneration are largely unknown. In NHE6-null mouse neurons in vitro, we find worsening lysosome function with days in culture. Notably, pH-dependent lysosome enzymes, such as cathepsin D (CatD), have reduced activity in lysosomes yet increased, precocious activity in endosomes in NHE6-null neurons. Further, endosomes show reduced fusion to lysosomes, and increased fusion to the plasma membrane with increased exosome release. This study identifies new mechanisms involving defective endosome maturation and trafficking that impair lysosome function in CS, likely contributing to neurodegeneration.

DOI: https://doi.org/10.1523/JNEUROSCI.1244-20.2021
PLoS Comput Biol. 2021 Sep 16. 17(9): e1008513

Dynamic modelling of the PI3K/MTOR signalling network uncovers biphasic dependence of mTORC1 activity on the mTORC2 subunit SIN1.

Milad Ghomlaghi, Guang Yang, Sungyoung Shin, David E James, Lan K Nguyen.

The PI3K/MTOR signalling network regulates a broad array of critical cellular processes, including cell growth, metabolism and autophagy. The mechanistic target of rapamycin (MTOR) kinase functions as a core catalytic subunit in two physically and functionally distinct complexes mTORC1 and mTORC2, which also share other common components including MLST8 (also known as GβL) and DEPTOR. Despite intensive research, how mTORC1 and 2 assembly and activity are coordinated, and how they are functionally linked remain to be fully characterized. This is due in part to the complex network wiring, featuring multiple feedback loops and intricate post-translational modifications. Here, we integrate predictive network modelling, in vitro experiments and -omics data analysis to elucidate the emergent dynamic behaviour of the PI3K/MTOR network. We construct new mechanistic models that encapsulate critical mechanistic details, including mTORC1/2 coordination by MLST8 (de)ubiquitination and the Akt-to-mTORC2 positive feedback loop. Model simulations validated by experimental studies revealed a previously unknown biphasic, threshold-gated dependence of mTORC1 activity on the key mTORC2 subunit SIN1, which is robust against cell-to-cell variation in protein expression. In addition, our integrative analysis demonstrates that ubiquitination of MLST8, which is reversed by OTUD7B, is regulated by IRS1/2. Our results further support the essential role of MLST8 in enabling both mTORC1 and 2's activity and suggest MLST8 as a viable therapeutic target in breast cancer. Overall, our study reports a new mechanistic model of PI3K/MTOR signalling incorporating MLST8-mediated mTORC1/2 formation and unveils a novel regulatory linkage between mTORC1 and mTORC2.

DOI: https://doi.org/10.1371/journal.pcbi.1008513
J Immunol. 2021 Sep 17. pii: ji2100463. [Epub ahead of print]

The Development and Survival of Thymic Epithelial Cells Require TSC1-Dependent Negative Regulation of mTORC1 Activity.

Zhanfeng Liang, Qian Zhang, Zhaoqi Zhang, Lina Sun, Xue Dong, Tianxiu Li, Liang Tan, Xubiao Xie, Liguang Sun, Yong Zhao.

Thymic epithelial cells (TECs) are critical for the development and generation of functionally competent T cells. Until now, the mechanism that regulates the survival of TECs is poorly understood. In the current study, we found that Tsc1 controls the homeostasis of medullary TECs (mTECs) by inhibiting lysosomal-mediated apoptosis pathway in mice. TEC-specific deletion of Tsc1 predominately decreased the cell number of mTECs and, to a lesser content, affected the development cortical TECs. The defect of mTECs caused by Tsc1 deficiency in mice impaired thymocyte development and peripheral T cell homeostasis. Mechanistically, Tsc1 deficiency did not affect the cell proliferation of mTECs but increased the apoptosis of mTECs significantly. RNA-sequencing analysis showed that pathways involved in lysosomal biogenesis, cell metabolism, and apoptosis were remarkably elevated in Tsc1-deficient mTECs compared with their wild-type counterparts. Tsc1-deficient mTECs exhibited overproduction of reactive oxygen species and malfunction of lysosome, with lysosome membrane permeabilization and the release of cathepsin B and cathepsin L to the cytosol, which then lead to Bid cleaved into active truncated Bid and subsequently intrinsic apoptosis. Finally, we showed that the impaired development of mTECs could be partially reversed by decreasing mTORC1 activity via haploinsufficiency of Raptor Thus, Tsc1 is essential for the homeostasis of mTECs by inhibiting lysosomal-mediated apoptosis through mTORC1-dependent pathways.

DOI: https://doi.org/10.4049/jimmunol.2100463
J Cell Sci. 2021 Sep 16. pii: jcs.254631. [Epub ahead of print]

Cholesteryl hemiazelate causes lysosome dysfunction impacting vascular smooth muscle cells homeostasis.

Liliana S Alves, André R A Marques, Nuno Padrão, Filomena A Carvalho, José Ramalho, Catarina Lopes, Maria I L Soares, Clare E Futter, Teresa M V D Pinho E Melo, Nuno C Santos, Otília V Vieira.

  In atherosclerotic lesions, vascular smooth muscle cells (VSMCs) represent half of the foam cell population, characterized by an aberrant accumulation of undigested lipids within lysosomes. Loss of lysosome function impacts VSMCs homeostasis and disease progression. Understanding the molecular mechanisms underlying lysosome dysfunction in these cells is, therefore, crucial. We identify cholesteryl hemiazelate (ChA), a stable oxidation end-product of cholesteryl-polyunsaturated fatty acid esters, as an inducer of lysosome malfunction in VSMCs. ChA-treated VSMCs acquire a foam cell-like phenotype, characterized by enlarged lysosomes full of ChA and neutral lipids. The lysosomes are perinuclear and exhibit degradative capacity and cargo exit defects. Lysosome luminal pH is also altered. Even though, the transcriptional response machinery and autophagy are not activated by ChA, the addition of recombinant lysosomal acid lipase (LAL) is able to rescue lysosome dysfunction. ChA significantly affects VSMCs proliferation and migration impacting atherosclerosis. In sum, this work shows that: 1) ChA is sufficient to induce lysosomal dysfunction in VSMCs; 2) In ChA-treated VSMCs, neither lysosome biogenesis nor autophagy are triggered; and 3) Recombinant LAL can be a therapeutic approach for lysosomal dysfunction.

Keywords:  Atherosclerosis; Lysosome adaptation; Lysosome dysfunction; Oxidized lipids; Vascular smooth muscle cells

DOI:  https://doi.org/10.1242/jcs.254631
Biochem Biophys Res Commun. 2021 Sep 06. pii: S0006-291X(21)01240-7. [Epub ahead of print]578 7-14

UBE2S exerts oncogenic activities in urinary bladder cancer by ubiquitinating TSC1.

Hao Tang, Tong Fang, Meng Ji, Jun-Ping Wang, Le-Le Song, Qiu-Yan Zhang, Jin-Sheng Wu.

  Ubiquitin-conjugating enzyme E2S (UBE2S), an important E2 enzyme in the process of ubiquitination, has exhibited oncogenic activities in various malignant tumors. However, it remains unknown whether UBE2S plays a role in urinary bladder cancer (UBC) development. In the current study, our data confirmed UBE2S upregulation in UBC. In vitro and in vivo experiments demonstrated that UBE2S knockdown resulted in attenuated proliferation and enhanced apoptosis, which was inverse to the phenotypes with UBE2S overexpression. Gain and loss of function assays confirmed that UBE2S exerts oncogenic activities in UBC by mediating the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Furthermore, we discovered that this UBE2S-modulated carcinogenic mechanism was in the consequence of directly targeting tuberous sclerosis 1 (TSC1), which is the upstream inhibitor of mTOR signaling for ubiquitous degradation. Taken together, this study demonstrated that UBE2S is a carcinogen in UBC and promotes UBC progression by ubiquitously degrading TSC1. This consequently mediates the activation of the mTOR pathway, suggesting a potential therapeutic regimen for UBC by targeting the newly identified UBE2S/TSC1/mTOR axis.

Keywords:  Bladder cancer; TSC1; UBE2S; Ubiquitination

DOI:  https://doi.org/10.1016/j.bbrc.2021.08.057
Neurobiol Dis. 2021 Sep 09. pii: S0969-9961(21)00256-4. [Epub ahead of print] 105507

A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy.

Mali Cosden, Sarah Jinn, Lihang Yao, Cheryl A Gretzula, Monika Kandebo, Dawn Toolan, Nathan G Hatcher, Lei Ma, Wei Lemaire, Gregory C Adam, Christine Burlein, Christina Minnick, Rose Flick, Marla L Watt, James Mulhearn, Mark Fraley, Robert E Drolet, Jacob N Marcus, Sean M Smith.

  Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.

Keywords:  Glucocerebrosidase; Glucosylceramide synthase inhibitor; Glycosphingolipids; Lysosomal biology; Parkinson's disease; Preformed fibril model

DOI:  https://doi.org/10.1016/j.nbd.2021.105507
Physiology (Bethesda). 2021 Sep 13.

Role of mTor signaling for tubular function and disease.

Florian Grahammer, Tobias B Huber, Ferruh Artunc.

  The mechanistic target of rapamycin (mTOR) forms two distinct intracellular multiprotein complexes that control a multitude of intracellular processes linked to metabolism, proliferation, actin cytoskeleton and survival. Recent studies have identified the importance of these complexes for transport regulation of ions and nutrients along the entire nephron. First reports could link altered activity of these complexes to certain disease entities i.e. diabetic nephropathy, AKI or hyperkalemia.

Keywords:  ENaC; RomK; endocytosis; mTOR; proximal tubule

DOI:  https://doi.org/10.1152/physiol.00021.2021
Essays Biochem. 2021 Sep 16. pii: EBC20210018. [Epub ahead of print]

Glucocerebrosidase dysfunction in neurodegenerative disease.

Sarah M Brooker, Dimitri Krainc.

  Parkinson's disease (PD) and related neurodegenerative disorders, termed the synucleinopathies, are characterized pathologically by the accumulation of protein aggregates containing α-synuclein (aSyn), resulting in progressive neuronal loss. There is considerable need for the development of neuroprotective strategies to halt or slow disease progression in these disorders. To this end, evaluation of genetic mutations associated with the synucleinopathies has helped to elucidate crucial mechanisms of disease pathogenesis, revealing key roles for lysosomal and mitochondrial dysfunction. The GBA1 gene, which encodes the lysosomal hydrolase β-glucocerebrosidase (GCase) is the most common genetic risk factor for PD and is also linked to other neurodegenerative disorders including dementia with Lewy bodies (DLB). Additionally, homozygous mutations in GBA1 are associated with the rare lysosomal storage disorder, Gaucher's disease (GD). In this review, we discuss the current knowledge in the field regarding the diverse roles of GCase in neurons and the multifactorial effects of loss of GCase enzymatic activity. Importantly, GCase has been shown to have a bidirectional relationship with aSyn, resulting in a pathogenic feedback loop that can lead to progressive aSyn accumulation. Alterations in GCase activity have furthermore been linked to multiple distinct pathways involved in neurodegeneration, and therefore GCase has emerged as a promising target for therapeutic drug development for PD and related neurodegenerative disorders, particularly DLB.

Keywords:  GBA1; Lewy Body dementia; Parkinsons disease; alpha synuclein

DOI:  https://doi.org/10.1042/EBC20210018
Cell Death Dis. 2021 Sep 17. 12(10): 854

Alpha-Synuclein defects autophagy by impairing SNAP29-mediated autophagosome-lysosome fusion.

Qilin Tang, Pan Gao, Thomas Arzberger, Matthias Höllerhage, Jochen Herms, Günter Höglinger, Thomas Koeglsperger.

Dopaminergic (DA) cell death in Parkinson's disease (PD) is associated with the gradual appearance of neuronal protein aggregates termed Lewy bodies (LBs) that are comprised of vesicular membrane structures and dysmorphic organelles in conjunction with the protein alpha-Synuclein (α-Syn). Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests α-Syn-mediated alterations in the lysosomal degradation of aggregated proteins and organelles - a process termed autophagy. Here, we used a combination of molecular biology and immunochemistry to investigate the effect of α-Syn on autophagy turnover in cultured human DA neurons and in human post-mortem brain tissue. We found α-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, α-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, α-Syn decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of α-Syn on autophagy whereas SNAP29 co-expression reversed the α-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In accord with our results from cultured neurons, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human post-mortem brain tissue of Lewy body pathology (LBP) cases. In summary, our results thus demonstrate a previously unknown effect of α-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autolysosome fusion. As such, our findings will therefore support the investigation of autophagy-associated pathological changes in PD.

DOI: https://doi.org/10.1038/s41419-021-04138-0
J Biol Chem. 2021 Sep 10. pii: S0021-9258(21)00991-1. [Epub ahead of print] 101189

TFEB Insufficiency Promotes Cardiac Hypertrophy by Blocking Autophagic Degradation of GATA4.

Rui Song, Han Lei, Li Feng, Wanwen Cheng, Ying Li, Ling Ling Yao, Jie Liu.

  Autophagosome-lysosome pathway (ALP) insufficiency has been suggested to play a critical role in the pathogenesis of cardiac hypertrophy. However, the mechanisms underlying ALP insufficiency remain largely unknown, and strategies to specifically manipulate ALP insufficiency for treating cardiac hypertrophy are lacking. Transcription factor EB (TFEB), as a master regulator of ALP, regulates the generation and function of autophagosomes and lysosomes. We found TFEB was significantly decreased, while autophagosome markers were increased in phenylephrine (PE)- and transverse aortic constriction (TAC)-induced cardiomyocyte hypertrophy and failing hearts from patients with dilated cardiomyopathy. Knocking down TFEB induced ALP insufficiency, as indicated by increased autophagosome markers, decreased LC3II flux, and cardiomyocyte hypertrophy manifested through increased levels of atrial natriuretic peptide (ANP) and β-myosin heavy chain 7 (β-MHC) and enlarged cell size. The effects of TFEB knockdown were abolished by promoting autophagy. TFEB overexpression improved autophagic flux and attenuated PE-stimulated cardiomyocyte hypertrophy and TAC-induced hypertrophic remodeling, fibrosis, and cardiac dysfunction. Curcumin analog compound C1, a specific TFEB activator, similarly attenuated PE-induced ALP insufficiency and cardiomyocyte hypertrophy. TFEB knockdown increased the accumulation of GATA4, a transcription factor for several genes causing cardiac hypertrophy by blocking autophagic degradation of GATA4, whereas knocking down GATA4 attenuated TFEB downregulation-induced cardiomyocyte hypertrophy. Both TFEB overexpression and C1 promoted GATA4 autophagic degradation and alleviated PE-induced cardiomyocyte hypertrophy. In conclusion, TFEB downregulation plays a vital role in the development of pressure overload-induced cardiac hypertrophy by causing ALP insufficiency and blocking autophagic degradation. Activation of TFEB represents a potential therapeutic strategy for treating cardiac hypertrophy.

Keywords:  GATA4; TFEB; autophagosome-lysosome pathway; autophagy; heart; hypertrophy

DOI:  https://doi.org/10.1016/j.jbc.2021.101189
Curr Biol. 2021 Sep 13. pii: S0960-9822(21)01045-9. [Epub ahead of print]31(17): R1037-R1040

Membrane trafficking: Retrofusion as an escape route out of the endosome.

Emily R Eden, Clare E Futter.

Intraluminal vesicles accumulate within the endosomal lumen before lysosomal delivery or extracellular release. A new study reports the development of an elegant assay showing that these vesicles can escape from the endosomal lumen by 'back-fusion' or 'retrofusion' with the endosomal limiting membrane.

DOI: https://doi.org/10.1016/j.cub.2021.07.055
Autophagy. 2021 Sep 14. 1-3

The ingenious ULKs: expanding the repertoire of the ULK complex with phosphoproteomics.

Thomas J Mercer, Sharon A Tooze.

  The mammalian ULK kinase complex is the most upstream component in the macroautophagy/autophagy signaling pathway. ULK1 and homolog ULK2, the sole serine/threonine kinases in autophagy, transduce an array of autophagy-inducing stimuli to downstream autophagic machinery, regulating autophagy from autophagosome initiation to fusion of autophagosomes with lysosomes. ULK signaling is also implicated in a diverse array of non-canonical processes from necroptosis to ER-Golgi trafficking to stress granule clearance. However, the exact mechanisms by which ULK regulates these diverse processes remain largely unknown. Most notably, the number of validated ULK substrates is surprisingly low. Our study identifies new ULK substrates from a wide array of protein families and signaling pathways and supports an expanded range of physiological roles for the ULKs. We further characterize several new substrates, including the PIK3C3/VPS34-containing complex subunit PIK3R4/VPS15 and the AMPK component PRKAG2. Finally, by analyzing PIK3R4/VPS15-deficient models we discover novel aspects of ULK signaling with potential relevance in selective autophagy.

Keywords:  AMPK; PIK3R4; PRKAG2; VPS15; VPS34; p150

DOI:  https://doi.org/10.1080/15548627.2021.1968615
Transl Psychiatry. 2021 Sep 17. 11(1): 480

Proteomics of autism and Alzheimer's mouse models reveal common alterations in mTOR signaling pathway.

Shira Mencer, Maryam Kartawy, Felix Lendenfeld, Huda Soluh, Manish Kumar Tripathi, Igor Khaliulin, Haitham Amal.

Autism spectrum disorder (ASD) and Alzheimer's disease (AD) are two different neurological disorders that share common clinical features, such as language impairment, executive functions, and motor problems. A genetic convergence has been proposed as well. However, the molecular mechanisms of these pathologies are still not well understood. Protein S-nitrosylation (SNO), the nitric oxide (NO)-mediated posttranslational modification, targets key proteins implicated in synaptic and neuronal functions. Previously, we have shown that NO and SNO are involved in the InsG3680(+/+) ASD and P301S AD mouse models. Here, we performed large-scale computational biology analysis of the SNO-proteome followed by biochemical validation to decipher the shared mechanisms between the pathologies. This analysis pointed to the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway as one of the shared molecular mechanisms. Activation of mTOR in the cortex of both mouse models was confirmed by western blots that showed increased phosphorylation of RPS6, a major substrate of mTORC1. Other molecular alterations affected by SNO and shared between the two mouse models, such as synaptic-associated processes, PKA signaling, and cytoskeleton-related processes were also detected. This is the first study to decipher the SNO-related shared mechanisms between SHANK3 and MAPT mutations. Understanding the involvement of SNO in neurological disorders and its intersection between ASD and AD might help developing an effective novel therapy for both neuropathologies.

DOI: https://doi.org/10.1038/s41398-021-01578-2
Commun Biol. 2021 Sep 17. 4(1): 1093

A glutamine sensor that directly activates TORC1.

Mirai Tanigawa, Katsuyoshi Yamamoto, Satoru Nagatoishi, Koji Nagata, Daisuke Noshiro, Nobuo N Noda, Kouhei Tsumoto, Tatsuya Maeda.

TOR complex 1 (TORC1) is an evolutionarily-conserved protein kinase that controls cell growth and metabolism in response to nutrients, particularly amino acids. In mammals, several amino acid sensors have been identified that converge on the multi-layered machinery regulating Rag GTPases to trigger TORC1 activation; however, these sensors are not conserved in many other organisms including yeast. Previously, we reported that glutamine activates yeast TORC1 via a Gtr (Rag ortholog)-independent mechanism involving the vacuolar protein Pib2, although the identity of the supposed glutamine sensor and the exact TORC1 activation mechanism remain unclear. In this study, we successfully reconstituted glutamine-responsive TORC1 activation in vitro using only purified Pib2 and TORC1. In addition, we found that glutamine specifically induced a change in the folding state of Pib2. These findings indicate that Pib2 is a glutamine sensor that directly activates TORC1, providing a new model for the metabolic control of cells.

DOI: https://doi.org/10.1038/s42003-021-02625-w
Elife. 2021 Sep 15. pii: e69883. [Epub ahead of print]10

A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling.

Sho W Suzuki, Akihiko Oishi, Nadia Nikulin, Jeff R Jorgensen, Matthew G Baile, Scott D Emr.

  Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway which is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer's disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans.

Keywords:  S. cerevisiae; cell biology; human

DOI:  https://doi.org/10.7554/eLife.69883
Gut. 2021 Sep 11. pii: gutjnl-2021-325405. [Epub ahead of print]

Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer.

Steffie Revia, Agnieszka Seretny, Lena Wendler, Ana Banito, Christoph Eckert, Kersten Breuer, Anand Mayakonda, Pavlo Lutsik, Matthias Evert, Silvia Ribback, Suchira Gallage, Ismaiel Chikh Bakri, Kai Breuhahn, Peter Schirmacher, Stefan Heinrich, Matthias M Gaida, Mathias Heikenwälder, Diego F Calvisi, Christoph Plass, Scott W Lowe, Darjus F Tschaharganeh.

  OBJECTIVE: Large-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer.DESIGN: Genetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted.
RESULTS: KDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition.
CONCLUSION: KDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition.

Keywords:  gastrointestinal cancer; hepatobiliary cancer; hepatocellular carcinoma; molecular carcinogenesis

DOI:  https://doi.org/10.1136/gutjnl-2021-325405
Elife. 2021 Sep 14. pii: e66278. [Epub ahead of print]10

Activation of mTORC1 and c-Jun by Prohibitin1 loss in Schwann cells may link mitochondrial dysfunction to demyelination.

Gustavo Della Flora Nunes, Emma R Wilson, Edward Hurley, Bin He, Bert W O'Malley, Yannick Poitelon, Lawrence Wrabetz, M Laura Feltri.

  Schwann cell (SC) mitochondria are quickly emerging as an important regulator of myelin maintenance in the peripheral nervous system (PNS). However, the mechanisms underlying demyelination in the context of mitochondrial dysfunction in the PNS are incompletely understood. We recently showed that conditional ablation of the mitochondrial protein Prohibitin 1 (PHB1) in SCs causes a severe and fast progressing demyelinating peripheral neuropathy in mice, but the mechanism that causes failure of myelin maintenance remained unknown. Here, we report that mTORC1 and c-Jun are continuously activated in the absence of Phb1, likely as part of the SC response to mitochondrial damage. Moreover, we demonstrate that these pathways are involved in the demyelination process, and that inhibition of mTORC1 using rapamycin partially rescues the demyelinating pathology. Therefore, we propose that mTORC1 and c-Jun may play a critical role as executioners of demyelination in the context of perturbations to SC mitochondria.

Keywords:  mouse; neuroscience

DOI:  https://doi.org/10.7554/eLife.66278
Dev Cell. 2021 Sep 08. pii: S1534-5807(21)00678-X. [Epub ahead of print]

Endosomal trafficking and DNA damage checkpoint kinases dictate survival to replication stress by regulating amino acid uptake and protein synthesis.

Arta Ajazi, Christopher Bruhn, Ghadeer Shubassi, Chiara Lucca, Elisa Ferrari, Angela Cattaneo, Angela Bachi, Nicola Manfrini, Stefano Biffo, Emanuele Martini, Saverio Minucci, Claudio Vernieri, Marco Foiani.

  Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress.

Keywords:  Atg6/Beclin 1; DNA damage response; Gcn2; Rad53; TORC1; amino acids; endosomal trafficking; phosphatydilinositol 3-phosphate; replication stress

DOI:  https://doi.org/10.1016/j.devcel.2021.08.019
PLoS Comput Biol. 2021 Sep 16. 17(9): e1009370

Protein structural features predict responsiveness to pharmacological chaperone treatment for three lysosomal storage disorders.

Jaie Woodard, Wei Zheng, Yang Zhang.

Three-dimensional structures of proteins can provide important clues into the efficacy of personalized treatment. We perform a structural analysis of variants within three inherited lysosomal storage disorders, comparing variants responsive to pharmacological chaperone treatment to those unresponsive to such treatment. We find that predicted ΔΔG of mutation is higher on average for variants unresponsive to treatment, in the case of datasets for both Fabry disease and Pompe disease, in line with previous findings. Using both a single decision tree and an advanced machine learning approach based on the larger Fabry dataset, we correctly predict responsiveness of three Gaucher disease variants, and we provide predictions for untested variants. Many variants are predicted to be responsive to treatment, suggesting that drug-based treatments may be effective for a number of variants in Gaucher disease. In our analysis, we observe dependence on a topological feature reporting on contact arrangements which is likely connected to the order of folding of protein residues, and we provide a potential justification for this observation based on steady-state cellular kinetics.

DOI: https://doi.org/10.1371/journal.pcbi.1009370
Front Genet. 2021 ;12 719713

Cross-Species and Human Inter-Tissue Network Analysis of Genes Implicated in Longevity and Aging Reveal Strong Support for Nutrient Sensing.

Avijit Podder, Anish Raju, Nicholas J Schork.

  Intensive research efforts have been undertaken to slow human aging and therefore potentially delay the onset of age-related diseases. These efforts have generated an enormous amount of high-throughput data covering different levels in the physiologic hierarchy, e.g., genetic, epigenetic, transcriptomic, proteomic, and metabolomic, etc. We gathered 15 independent sources of information about genes potentially involved in human longevity and lifespan (N = 5836) and subjected them to various integrated analyses. Many of these genes were initially identified in non-human species, and we investigated their orthologs in three non-human species [i.e., mice (N = 967), fruit fly (N = 449), and worm (N = 411)] for further analysis. We characterized experimentally determined protein-protein interaction networks (PPIN) involving each species' genes from 9 known protein databases and studied the enriched biological pathways among the individually constructed PPINs. We observed three important signaling pathways: FoxO signaling, mTOR signaling, and autophagy to be common and highly enriched in all four species (p-value ≤ 0.001). Our study implies that the interaction of proteins involved in the mechanistic target of rapamycin (mTOR) signaling pathway is somewhat limited to each species or that a "rewiring" of specific networks has taken place over time. To corroborate our findings, we repeated our analysis in 43 different human tissues. We investigated conserved modules in various tissue-specific PPINs of the longevity-associated genes based upon their protein expression. This analysis also revealed mTOR signaling as shared biological processes across four different human tissue-specific PPINs for liver, heart, skeletal muscle, and adipose tissue. Further, we explored our results' translational potential by assessing the protein interactions with all the reported drugs and compounds that have been experimentally verified to promote longevity in the three-comparator species. We observed that the target proteins of the FDA-approved drug rapamycin (a known inhibitor of mTOR) were conserved across all four species. Drugs like melatonin and metformin exhibited shared targets with rapamycin in the human PPIN. The detailed information about the curated gene list, cross-species orthologs, PPIN, and pathways was assembled in an interactive data visualization portal using RStudio's Shiny framework (https://agingnetwork.shinyapps.io/frontiers/).

Keywords:  cross-species orthologs; drug targets; human aging; nutrient sensing; pathway enrichments; protein-protein interaction network

DOI:  https://doi.org/10.3389/fgene.2021.719713
Int Immunol. 2021 Sep 11. pii: dxab069. [Epub ahead of print]

Lysosomal amino acid transporters as key players in inflammatory diseases.

Noriko Toyama-Sorimachi, Toshihiko Kobayashi.

  Controlling inflammation can alleviate immune-mediated, lifestyle-related and neurodegenerative diseases. The endolysosome system plays critical roles in inflammatory responses. Endolysosomes function as signal transduction hubs to convert various environmental danger signals into gene expression, enabling metabolic adaptation of immune cells and efficient orchestration of inflammation. Solute carrier family 15 member 3 (SLC15A3) and member 4 (SLC15A4) are endolysosome-resident amino acid transporters that are preferentially expressed in immune cells. These transporters play essential roles in signal transduction through endolysosomes, and the loss of either transporter can alleviate multiple inflammatory diseases because of perturbed endolysosome-dependent signaling events, including inflammatory and metabolic signaling. Here, we summarize the findings leading to a proof-of-concept for anti-inflammatory strategies based on targeting SLC15 transporters.

Keywords:  amino acid transporter; endolysosome systems; immunometabolism; inflammatory signals; innate immune cells

DOI:  https://doi.org/10.1093/intimm/dxab069
Nat Commun. 2021 Sep 14. 12(1): 5434

An active tethering mechanism controls the fate of vesicles.

Seong J An, Felix Rivera-Molina, Alexander Anneken, Zhiqun Xi, Brian McNellis, Vladimir I Polejaev, Derek Toomre.

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.

DOI: https://doi.org/10.1038/s41467-021-25465-y
Nat Rev Mol Cell Biol. 2021 Sep 13.

Molecular mechanisms of dietary restriction promoting health and longevity.

Cara L Green, Dudley W Lamming, Luigi Fontana.

Dietary restriction with adequate nutrition is the gold standard for delaying ageing and extending healthspan and lifespan in diverse species, including rodents and non-human primates. In this Review, we discuss the effects of dietary restriction in these mammalian model organisms and discuss accumulating data that suggest that dietary restriction results in many of the same physiological, metabolic and molecular changes responsible for the prevention of multiple ageing-associated diseases in humans. We further discuss how different forms of fasting, protein restriction and specific reductions in the levels of essential amino acids such as methionine and the branched-chain amino acids selectively impact the activity of AKT, FOXO, mTOR, nicotinamide adenine dinucleotide (NAD+), AMP-activated protein kinase (AMPK) and fibroblast growth factor 21 (FGF21), which are key components of some of the most important nutrient-sensing geroprotective signalling pathways that promote healthy longevity.

DOI: https://doi.org/10.1038/s41580-021-00411-4
Curr Gene Ther. 2021 Sep 16.

Treatment of GM2 Gangliosidosis in Adult Sandhoff Mice using an Intravenous Self-Complementary Hexosaminidase Vector.

Karlaina Jl Osmon, Patrick Thompson, Evan Woodley, Subha Karumuthil-Melethil, Cliff Heindel, John G Keimel, William F Kaemmerer, Steven J Gray, Jagdeep S Walia.

  BACKGROUND: GM2 gangliosidosis is a neurodegenerative, lysosomal storage disease caused by the deficiency of β-hexosaminidase A enzyme (HexA), an α/β-subunit heterodimer. A novel variant of the human hexosaminidase α-subunit, coded by HEXM, has previously been shown to form a stable homodimer, HexM, that hydrolyzes GM2 gangliosides (GM2) in vivo.MATERIALS & METHODS: The current study assessed the efficacy of intravenous (IV) delivery of a self-complementary adeno-associated virus serotype 9 (scAAV9) vector incorporating the HEXM transgene, scAAV9/HEXM, including the outcomes based on the dosages provided to the Sandhoff (SD) mice. Six-week-old SD mice were injected with either 2.5E+12 vector genomes (low dose, LD) or 1.0E+13 vg (high dose, HD). We hypothesized that when examining the dosage comparison for scAAV9/HEXM in adult SD mice, the HD group would have more beneficial outcomes than the LD cohort. Assessments included survival, behavioral outcomes, vector biodistribution, and enzyme activity within the central nervous system.
RESULTS: Toxicity was observed in the HD cohort, with 8 of 14 mice dying within one month of the injection. As compared to untreated SD mice, which have typical survival of 16 weeks, the LD cohort and the remaining HD mice had a significant survival benefit with an average/median survival of 40.6/34.5 and 55.9/56.7 weeks, respectively. Significant behavioral, biochemical and molecular benefits were also observed. The second aim of the study was to investigate the effects of IV mannitol infusions on the biodistribution of the LD scAAV9/HEXM vector and the survival of the SD mice. Increases in both the biodistribution of the vector as well as the survival benefit (average/median of 41.6/49.3 weeks) were observed.
CONCLUSION: These results demonstrate the potential benefit and critical limitations of the treatment of GM2 gangliosidosis using IV delivered AAV vectors.

Keywords:  AAV; Adeno-associated Virus; GM2 Ganglioside ; GM2 Gangliosidosis; Gene Therapy; Hexosaminidase A; Sandhoff; Tay Sachs

DOI:  https://doi.org/10.2174/1566523221666210916153051
Cell Metab. 2021 Sep 08. pii: S1550-4131(21)00417-4. [Epub ahead of print]

Increased fidelity of protein synthesis extends lifespan.

Victoria Eugenia Martinez-Miguel, Celia Lujan, Tristan Espie-Caullet, Daniel Martinez-Martinez, Saul Moore, Cassandra Backes, Suam Gonzalez, Evgeniy R Galimov, André E X Brown, Mario Halic, Kazunori Tomita, Charalampos Rallis, Tobias von der Haar, Filipe Cabreiro, Ivana Bjedov.

  Loss of proteostasis is a fundamental process driving aging. Proteostasis is affected by the accuracy of translation, yet the physiological consequence of having fewer protein synthesis errors during multi-cellular organismal aging is poorly understood. Our phylogenetic analysis of RPS23, a key protein in the ribosomal decoding center, uncovered a lysine residue almost universally conserved across all domains of life, which is replaced by an arginine in a small number of hyperthermophilic archaea. When introduced into eukaryotic RPS23 homologs, this mutation leads to accurate translation, as well as heat shock resistance and longer life, in yeast, worms, and flies. Furthermore, we show that anti-aging drugs such as rapamycin, Torin1, and trametinib reduce translation errors, and that rapamycin extends further organismal longevity in RPS23 hyperaccuracy mutants. This implies a unified mode of action for diverse pharmacological anti-aging therapies. These findings pave the way for identifying novel translation accuracy interventions to improve aging.

Keywords:  RPS23; aging; archaea; mTOR; protein synthesis; proteostasis; ribosome; translation; translation accuracy; translation fidelity

DOI:  https://doi.org/10.1016/j.cmet.2021.08.017
Nat Struct Mol Biol. 2021 Sep;28(9): 762-770

De novo design of tyrosine and serine kinase-driven protein switches.

Nicholas B Woodall, Zara Weinberg, Jesslyn Park, Florian Busch, Richard S Johnson, Mikayla J Feldbauer, Michael Murphy, Maggie Ahlrichs, Issa Yousif, Michael J MacCoss, Vicki H Wysocki, Hana El-Samad, David Baker.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

DOI: https://doi.org/10.1038/s41594-021-00649-8
EMBO J. 2021 Sep 13. e107766

GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis.

Prathyush Pothukuchi, Ilenia Agliarulo, Marinella Pirozzi, Riccardo Rizzo, Domenico Russo, Gabriele Turacchio, Julian Nüchel, Jia-Shu Yang, Charlotte Gehin, Laura Capolupo, Maria Jose Hernandez-Corbacho, Ansuman Biswas, Giovanna Vanacore, Nina Dathan, Takahiro Nitta, Petra Henklein, Mukund Thattai, Jin-Ichi Inokuchi, Victor W Hsu, Markus Plomann, Lina M Obeid, Yusuf A Hannun, Alberto Luini, Giovanni D'Angelo, Seetharaman Parashuraman.

  The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI-based retrograde transport vesicles, thus concentrating them in the trans-Golgi. In genome-edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis-Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.

Keywords:  GRASP55; Golgi apparatus; glucosylceramide synthase; glycosphingolipids; glycosylation

DOI:  https://doi.org/10.15252/embj.2021107766
Cell Rep. 2021 Sep 14. pii: S2211-1247(21)01129-3. [Epub ahead of print]36(11): 109682

RORα is critical for mTORC1 activity in T cell-mediated colitis.

Xinxin Chi, Wei Jin, Xue Bai, Xiaohong Zhao, Jing Shao, Jiaqi Li, Qinli Sun, Bing Su, Xiaohu Wang, Xuexian O Yang, Chen Dong.

  Inflammatory bowel disease (IBD) is multi-factorial chronic intestinal inflammation driven by pathogenic T cells, among which a large portion of patients are resistant to current anti-inflammatory regimes. The mechanisms underlying colitis pathogenicity and drug resistance are not fully understood. Here, we demonstrate that RORα is highly expressed in active UC patients, particularly in those non-responsive to anti-TNF treatment. Rorα deficiency in CD4+ T cells greatly reduced colitis development. Mechanistically, RORα regulated T cell infiltration in colon and inhibited T cell apoptosis. Meanwhile, genome-wide occupancy and transcriptome analysis revealed that RORα promoted mTORC1 activation. mTORC1 signaling, also hyperactivated in active UC patients, is necessary for T cell-mediated colitis. Our results thus demonstrate a crucial role of the RORα-mTORC1 axis in CD4+ T cells in promoting IBD, which may be targeted in human patients.

Keywords:  CD4(+) T cells; RORα; anti-TNF therapy; colitis; mTORC1

DOI:  https://doi.org/10.1016/j.celrep.2021.109682
Acta Neuropathol. 2021 Sep 13.

Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders.

Marzena Kurzawa-Akanbi, Seshu Tammireddy, Ivo Fabrik, Lina Gliaudelytė, Mary K Doherty, Rachel Heap, Irena Matečko-Burmann, Björn M Burmann, Matthias Trost, John M Lucocq, Anda V Gherman, Graham Fairfoul, Preeti Singh, Florence Burté, Alison Green, Ian G McKeith, Anetta Härtlova, Phillip D Whitfield, Christopher M Morris.

  Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a "pathological package" capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein-ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency.

Keywords:  Alpha-synuclein; Ceramide; Exosomes; Extracellular vesicles; Glucocerebrosidase; Lewy body disorders

DOI:  https://doi.org/10.1007/s00401-021-02367-3