Mammalian Atg8 proteins and autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for response to pathogens
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Hek293t Cells Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Vojo Deretic
PROVIDER: MSV000085401 | MassIVE | Tue May 12 11:51:00 BST 2020
REPOSITORIES: MassIVE
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Project description:Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.
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Project description:Autophagy is a cell-protective and degradative process that recycles damaged and long-lived cellular components. Cancer cells are thought to take advantage of autophagy to help them to cope with the stress of tumorigenesis; thus targeting autophagy is an attractive therapeutic approach. However, there are currently no specific inhibitors of autophagy. ULK1, a serine/threonine protein kinase, is essential for the initial stages of autophagy, and here we report that two compounds, MRT67307 and MRT68921, potently inhibit ULK1 and ULK2 in vitro and block autophagy in cells. Using a drug-resistant ULK1 mutant, we show that the autophagy-inhibiting capacity of the compounds is specifically through ULK1. ULK1 inhibition results in accumulation of stalled early autophagosomal structures, indicating a role for ULK1 in the maturation of autophagosomes as well as initiation.
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Project description:Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.
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Project description:The aim of the project was to characterize changes in gene expression that are associated with induced autophagic flux. We engineered HeLa, HEK 293 and SH-SY5Y cell lines to express tandem-fluorecent LC3 (tf-LC3). The ratio of the red and green fluorophores indicated how much of the LC3 is in the acidic interior of lysosomes, which was a proxy measure for autophagic flux. RNA sequencing was performed for the cell lines at baseline and 1h, 15h and 30h after treatments. Additional untreated samples were also sequenced at some but not all time points. Autophagy was induced by amino acid starvation or mTOR inhibition (AZD8055).
2022-08-25 | E-MTAB-12020 | biostudies-arrayexpress
Project description:AimsThe mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR) occurring downstream in the PI3K/Akt/mTOR pathway, are regarded as potential prognostic markers for gastric cancer (GC). However, the prognostic value of mTOR/p-mTOR expression remains controversial. In this study, we determined the expression of mTOR, p-mTOR, p70S6k, and p-p70S6K in GC, and investigated the correlation between their overexpression, clinicopathological parameters, and overall survival (OS).MethodsThe expression of mTOR, p-mTOR, p70S6k, and p-p70S6K was examined in 120 GC patients by immunohistochemistry (IHC). The association of protein expression with clinicopathological features and OS was explored. The p-mTOR expression was detected in normal, adjacent, and GC tissues using Western blot. Eligible studies retrieved from PubMed, Ovid, Web of Science and Cochrane databases, were reviewed in this meta-analysis.ResultsIHC showed that the rates of expression of the signal transduction molecules mTOR, p-mTOR, p70S6k and p-p70S6K in GC were 60.8%, 54.2%, 53.3% and 53.3%, respectively. Overexpression of mTOR and p70S6K showed no significant association with clinical variables. Expression of p-mTOR was significantly associated with differentiation (P < 0.01), depth of invasion (P < 0.01), lymph node metastasis (P = 0.04) and TNM stage (P = 0.02). Expression of p-p70S6K was associated with differentiation (P = 0.006), depth of invasion (P < 0.001), and TNM stage (P = 0.02). In survival analysis, differentiation, depth of invasion, lymph node metastasis and TNM stage were not related to OS (all P > 0.05). Furthermore, p-mTOR and p-p70S6K expression, but not mTOR and p70S6K, were tightly associated with OS of GC patients (P = 0.006 and P < 0.001, respectively). In Western blot, p-mTOR was significantly higher in GC tissues than in normal and adjacent tissues. In the present meta-analysis, mTOR overexpression showed no relationship with any clinicopathological variables. However, p-mTOR was correlated with depth of invasion, and TNM stage (all P < 0.05), and its overexpression was associated with a shorter survival time (P < 0.001).ConclusionThe results suggest that p-mTOR is a more valuable prognostic factor than mTOR in GC.
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