Amino Acids Enhance Polyubiquitination of Rheb and Its Binding to mTORC1 by Blocking Lysosomal ATXN3 Deubiquitinase Activity.

Amino-acid-induced lysosomal mechanistic target of rapamycin complex 1 (mTORC1) localization through the Rag GTPases is a critical step for its activation by Rheb GTPase. However, how the mTORC1 interacts with Rheb on the lysosome remains elusive. We report that amino acids enhance the polyubiquitination of Rheb (Ub-Rheb), which shows a strong binding preference for mTORC1 and supports its activation, while the Ub-Rheb is subjected to subsequent degradation. Mechanistically, we identified ATXN3 as a Ub-Rheb deubiquitinase whose lysosomal localization is blocked by active Rag heterodimer in response to amino acid stimulation. Consistently, cells lacking functional Rag heterodimer on the lysosome accumulate Ub-Rheb, and blockade of its degradation instigates robust lysosomal mTORC1 localization and its activation without the Ragulator-Rag system. Thus, polyubiquitination of Rheb is an important post-translational modification, which facilitates the binding of mTORC1 to Rheb on the lysosome and is another crosstalk between the amino acid and growth factor signaling for mTORC1 activation.

Mitochondrial NIX Promotes Tumor Survival in the Hypoxic Niche of Glioblastoma.

Cancer cells rely on mitochondrial functions to regulate key survival and death signals. How cancer cells regulate mitochondrial autophagy (mitophagy) in the tumor microenvironment as well as utilize mitophagy as a survival signal is still not well understood. Here, we elucidate a key survival mechanism of mitochondrial NIX-mediated mitophagy within the hypoxic region of glioblastoma, the most malignant brain tumor. NIX was overexpressed in the pseudopalisading cells that envelop the hypoxic-necrotic regions, and mitochondrial NIX expression was robust in patient-derived glioblastoma tumor tissues and glioblastoma stem cells. NIX was required for hypoxia and oxidative stress-induced mitophagy through NFE2L2/NRF2 transactivation. Silencing NIX impaired mitochondrial reactive oxygen species clearance, cancer stem cell maintenance, and HIF/mTOR/RHEB signaling pathways under hypoxia, resulting in suppression of glioblastoma survival in vitro and in vivo. Clinical significance of these findings was validated by the compelling association between NIX expression and poor outcome for patients with glioblastoma. Taken together, our findings indicate that the NIX-mediated mitophagic pathway may represent a key therapeutic target for solid tumors, including glioblastoma. SIGNIFICANCE: NIX-mediated mitophagy regulates tumor survival in the hypoxic niche of glioblastoma microenvironment, providing a potential therapeutic target for glioblastoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5218/F1.large.jpg.

Weak membrane interactions allow Rheb to activate mTORC1 signaling without major lysosome enrichment.

Stable localization of the Rheb GTPase to lysosomes is thought to be required for activation of mTOR complex 1 (mTORC1) signaling. However, the lysosome targeting mechanisms for Rheb remain unclear. We therefore investigated the relationship between Rheb subcellular localization and mTORC1 activation. Surprisingly, we found that Rheb was undetectable at lysosomes. Nonetheless, functional assays in knockout human cells revealed that farnesylation of the C-terminal CaaX motif on Rheb was essential for Rheb-dependent mTORC1 activation. Although farnesylated Rheb exhibited partial endoplasmic reticulum (ER) localization, constitutively targeting Rheb to ER membranes did not support mTORC1 activation. Further systematic analysis of Rheb lipidation revealed that weak, nonselective, membrane interactions support Rheb-dependent mTORC1 activation without the need for a specific lysosome targeting motif. Collectively, these results argue against stable interactions of Rheb with lysosomes and instead that transient membrane interactions optimally allow Rheb to activate mTORC1 signaling.

Rheb (Ras Homolog Enriched in Brain 1) Deficiency in Mature Macrophages Prevents Atherosclerosis by Repressing Macrophage Proliferation, Inflammation, and Lipid Uptake.

OBJECTIVE: Macrophage foam cell formation is an important process in atherosclerotic plaque development. The small GTPase Rheb (Ras homolog enriched in brain 1) regulates endocytic trafficking that is critical for foam cell formation. However, it is unclear whether and how macrophage Rheb regulates atherogenesis, which are the focuses of the current study. Approach and Results: Immunofluorescence study confirmed the colocalization of Rheb in F4/80 and Mac-2 (galectin-3)-labeled lesional macrophages. Western blot and fluorescence-activated cell sorting analysis showed that Rheb expression was significantly increased in atherosclerotic lesions of atherosclerosis-prone (apoE-/- [apolipoprotein E deficient]) mice fed with Western diet. Increased Rheb expression was also observed in oxidized LDL (low-density lipoprotein)-treated macrophages. To investigate the in vivo role of macrophage Rheb, we established mature RhebmKO (macrophage-specific Rheb knockout) mice by crossing the Rheb floxed mice with F4/80-cre mice. Macrophage-specific knockout of Rheb in mice reduced Western diet-induced atherosclerotic lesion by 32%, accompanied with a decrease in macrophage content in plaque. Mechanistically, loss of Rheb in macrophages repressed oxidized LDL-induced lipid uptake, inflammation, and macrophage proliferation. On the contrary, lentivirus-mediated overexpression of Rheb in macrophages increased oxidized LDL-induced lipid uptake and inflammation, and the stimulatory effect of Rheb was suppressed by the mTOR (mammalian target of rapamycin) inhibitor rapamycin or the PKA (protein kinase A) activator forskolin. CONCLUSIONS: Macrophage Rheb plays important role in Western diet-induced atherosclerosis by promoting macrophage proliferation, inflammation, and lipid uptake. Inhibition of expression and function of Rheb in macrophages is beneficial to prevent diet-induced atherosclerosis.

Effects of antiepileptic drugs in a new TSC/mTOR-dependent epilepsy mouse model.

OBJECTIVE: An epilepsy mouse model for Tuberous Sclerosis Complex (TSC) was developed and validated to investigate the mechanisms underlying epileptogenesis. Furthermore, the possible antiepileptogenic properties of commonly used antiepileptic drugs (AEDs) and new compounds were assessed. METHODS: Tsc1 deletion was induced in CAMK2A-expressing neurons of adult mice. The antiepileptogenic properties of commonly used AEDs and inhibitors of the mTOR pathways were assessed by EEG recordings and by molecular read outs. RESULTS: Mice developed epilepsy in a narrow time window (10 ± 2 days) upon Tsc1 gene deletion. Seizure frequency but not duration increased over time. Seizures were lethal within 18 days, were unpredictable, and did not correlate to seizure onset, length or frequency, reminiscent of sudden unexpected death in epilepsy (SUDEP). Tsc1 gene deletion resulted in a strong activation of the mTORC1 pathway, and both epileptogenesis and lethality could be entirely prevented by RHEB1 gene deletion or rapamycin treatment. However, other inhibitors of the mTOR pathway such as AZD8055 and PF4708671 were ineffective. Except for ketogenic diet, none of commonly used AEDs showed an effect on mTORC1 activity. Vigabatrin and ketogenic diet treatment were able to significantly delay seizure onset. In contrast, survival was shortened by lamotrigine. INTERPRETATION: This novel Tsc1 mouse model is highly suitable to assess the efficacy of antiepileptic and -epileptogenic drugs to treat mTORC1-dependent epilepsy. Additionally, it allows us to study the mechanisms underlying mTORC1-mediated epileptogenesis and SUDEP. We found that early treatment with vigabatrin was not able to prevent epilepsy, but significantly delayed seizure onset.

mTORc1 activity is necessary and sufficient for phosphorylation of eNOSS1177.

Nitric oxide, produced by eNOS, plays critical roles in the regulation of vascular function and maintenance. Chronic PI3K signaling has recently been associated with vascular malformations. A well described substrate downstream of PI3K signaling is eNOS. Another critical downstream target of PI3K is the metabolic regulator, mTORc1. The relationship between mTORc1 and eNOS regulation, has not been determined. We generated cells with manipulated PI3K signaling by expressing the activating mutation, PIK3CAH1047R , or knocking down PTEN expression. We investigated eNOSS1177 phosphorylation, a major activating regulatory site, following mTORC1 inhibition. We also tested the sufficiency of mTORc1 activation to stimulate eNOSS1177 phosphorylation. Our data indicate mTORc1 activity is required for the phosphorylation of eNOSS1177 , even in the presence of robust AKT activation. Moreover, we found that expression of RHEB, which functions in the absence of AKT activation to activate mTORc1, is sufficient to phosphorylate this site. Our data indicate that mTORc1, rather than AKT, may be the critical determinant of eNOSS1177 phosphorylation. As mTORc1 is a central regulator of cellular metabolism, the finding that this regulatory complex can directly participate in the regulation of eNOS provides new insights into metabolic uncoupling and vascular disease that often accompanies diabetes, high fat diets, and aging.