Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

Battu, Srikanth; Afroz, Sumbul; Giddaluru, Jeevan; Naz, Sumera; Huang, Weishan; Khumukcham, Saratchandra Singh; Khan, Rafiq Ahmad; Bhat, Saleem Yousuf; Qureshi, Insaf Ahmed; Manavathi, Bramanandam; Khan, Aleem Ahmed; August, Avery; Hasnain, Seyed E.; Khan, Nooruddin

doi:10.1371/journal.pbio.2005317

Cited by 31 publications

(38 citation statements)

References 64 publications

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“…As expected following the undocking of mTOR from the lysosomes and in line with recent works [39,41], we observed the induction of ATG by HF in all the analyzed cell lines. On long term sustained AAR and autophagy, as induced by HF, led to apoptosis.…”

Section: Discussionsupporting

confidence: 92%

“…Here, we found that HF initially induces autophagosome formation and later impairs the autophagosome-lysosome fusion step, much alike chloroquine. Of note, our work is the first to show a dual-effect of HF on autophagy as the other studies investigating HF-induced autophagy reported only the induction of autophagy by the HF and not an impairment of the autophagosome-lysosome fusion step [39,41]. We hypothesize that the effect of HF on the autophagosomelysosome fusion step may differ depending on the cell line, HF concentration and incubation time.…”

Section: Discussionmentioning

confidence: 50%

“…HF has been previously reported to induce autophagy [39][40][41]. In nutrient-rich conditions, HF induces autophagy Fig.…”

Section: Discussionmentioning

confidence: 86%

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Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

et al. 2019

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Background In the event of amino acid starvation, the cell activates two main protective pathways: Amino Acid starvation Response (AAR), to inhibit global translation, and autophagy, to recover the essential substrates from degradation of redundant self-components. Whether and how AAR and autophagy (ATG) are cross-regulated and at which point the two regulatory pathways intersect remain unknown. Here, we provide experimental evidence that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) specifically located at the lysosome level links the AAR with the autophagy pathway. Methods As an inducer of the AAR, we used halofuginone (HF), an alkaloid that binds to the prolyl-tRNA synthetase thus mimicking the unavailability of proline (PRO). Induction of AAR was determined assessing the phosphorylation of the eukaryotic translation initiation factor (eIF) 2α. Autophagy was monitored by assessing the processing and accumulation of microtubule-associated protein 1 light chain 3 isoform B (LC3B) and sequestosome-1 (p62/SQSTM1) levels. The activity of mTORC1 was monitored through assessment of the phosphorylation of mTOR, (rp)S6 and 4E-BP1. Global protein synthesis was determined by puromycin incorporation assay. mTORC1 presence on the membrane of the lysosomes was monitored by cell fractionation and mTOR expression was determined by immunoblotting. Results In three different types of human cancer cells (thyroid cancer WRO cells, ovarian cancer OAW-42 cells, and breast cancer MCF-7 cells), HF induced both the AAR and the autophagy pathways time-dependently. In WRO cells, which showed the strongest induction of autophagy and of AAR, global protein synthesis was little if any affected. Consistently, 4E-BP1 and (rp)S6 were phosphorylated. Concomitantly, mTOR expression and activation declined along with its detachment from the lysosomes and its degradation by the proteasome, and with the nuclear translocation of transcription factor EB (TFEB), a transcription factor of many ATG genes. The extra supplementation of proline rescued all these effects. Conclusions We demonstrate that the AAR and autophagy are mechanistically linked at the level of mTORC1, and that the lysosome is the central hub of the cross-talk between these two metabolic stress responses.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 50%

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Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

et al. 2019

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“…Surprisingly, the levels of phospho-eIF2α were not altered, except under UV-stress in GCN2 depleted cells, where it was abolished. Furthermore, GCN2 was shown to induce autophagy that clear the SG (Battu et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi

Malvezzi

Aricó

Souza-Melo

et al. 2020

Front. Cell. Infect. Microbiol.

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The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (Tck1, Tck2, and Tck3) are found in the Trypanosoma cruzi genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several T. cruzi strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of T. cruzi devoid of TcK1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative T. cruzi epimastigotes depleted of TcK1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in TcK1 depleted line. However, eIF2α is still phosphorylated in TcK1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of Tck1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the TcK1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in T. cruzi affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.

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“…Later, cells were washed gently with ice‐cold 1XPBS and lysed in lysis buffer (20 mM HEPES,150 mM NaCl, pH 7.5, 1% Triton‐X 100, 100 mM NaF, 1 mM EDTA, 17.5 mM β‐glycerophosphate, 10% glycerol, 1mM phenyl methyl sulphonyl fluoride, 2 mg/mL pepstatin, and 4 mg/mL aprotinin, supplemented with protease inhibitor cocktail tablets (Sigma–Aldrich) were added dropwise to cells on culture dish. Cells were incubated on ice for 30 min in cell lysis buffer with occasional vortexing as described earlier . Cells were scraped and lysed in cell lysis buffer.…”

Section: Methodsmentioning

confidence: 99%

Activation of integrated stress response pathway regulates IL‐1β production through posttranscriptional and translational reprogramming in macrophages

et al. 2019

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Immune cells sense and programme its cellular machinery appropriately to the environmental changes through the activation of cytoprotective adaptive pathway so-called the "integrated stress response (ISR)". However, the mechanisms implicated in ISR-induced protective responses are poorly understood. Here, we show that ISR activation by arsenite (Ar) results in suppression of IL-1β production in macrophages and inhibition of DSSinduced colitis in a murine model through a novel posttranscriptional and translation regulatory (PTR) mechanism. Ar triggers PTR events through eIF2α-phosphorylation, which results in the attenuation of active polysome formation leading to the accumulation of translationally stalled IL-1β mRNAs. Translationally stalled IL-1β mRNAs recruit RNAbinding proteins (TIA-1/TIAR), resulting in the formation of RBP-RNA complexes known as stress granules (SGs). The SGs bound IL-1β mRNAs might undergo degradation through induction of autophagy. Also, we show that Ar posttranslationally impairs processing and secretion of IL-1β by diminishing inflammasome activation. Altogether, this study unveils a novel mechanism of IL-1β regulation and further suggests that pharmacological activation of cytoprotective ISR pathway might provide an effective therapeutic intervention against inflammatory diseases.Keywords: arsenite r autophagy r integrated stress response (ISR) pathway r stress granules r TIA-1/TIAR Additional supporting information may be found online in the Supporting Information section at the end of the article.

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Amino acid starvation sensing dampens IL-1β production by activating riboclustering and autophagy

Cited by 31 publications

References 64 publications

Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

GCN2-Like Kinase Modulates Stress Granule Formation During Nutritional Stress in Trypanosoma cruzi

Activation of integrated stress response pathway regulates IL‐1β production through posttranscriptional and translational reprogramming in macrophages

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