IndraLab
Statements
sparser
"MTOR-dependent mechanisms include activation of AMPK/ TSC2 axis by energy depletion [68], activation of TSC2 by regulated in development and DNA damage responses 1 (REDD1) [69] and promyelocytic leukemiamediated (PML) inhibition of mTOR-Rheb-GTP interaction, and consequent nuclear translocation of mTOR [70]."
reach
"Mechanistically, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, an intracellular vesicular trafficking system, resulting in the induction of endoplasmic reticulum stress and the suppression of both lipid raft mediated PI3K activation and mTOR and RheB complex formation, which collectively led to an effective inhibition of mTOR and AKT signaling."
sparser
"It was also described that lysosomal MTOR localization brings it in close vicinity to its main regulator, RHEB (Ras homolog, mTORC1 binding), and that as a result, the MTOR-RHEB interaction can activate MTOR kinase activity leading to the phosphorylation of downstream effectors [ xref ]."
sparser
"However, Rag interaction with mTORC1 does not directly activate the mTOR kinase; rather Rag proteins modulate cellular translocation of mTORC1 to the lysosome that facilitates an mTOR and RHEB interaction, which in the presence of elevated AA concentrations, results in RHEB-induced activation of mTORC1."
sparser
"The results showed that
mTOR ΔN bound with RHEB by a K D value of 7.27 μM ( xref C), corresponding to the value reported by
the BLI analysis ( K D ∼ 6.5 μM). xref Then, we evaluated the effect of P1_WT to inhibit RHEB-mTOR ΔN interaction by preincubating
RHEB with a series of different concentration of P1_WT before adding mTOR ΔN . As a result, P1_WT inhibited RHEB binding to mTOR ΔN with an IC 50 value similar to that of mTORC1 activity inhibition (∼0.3
μM; xref B–C and xref D)."
sparser
"Previously,
we studied the molecular interactions of RHEB with full-length mTOR
and mTOR ΔN , yielding K D values of ∼2.4 and 6.4 μM, respectively. xref In the RHEB-mTOR ΔN interface,
α5- and α7-helices of mTOR ΔN stabilize
the mTORC1-RHEB complexation followed by the kinase activations. xref The residues (aa 63–79) of RHEB in the
switch II region lie between α5- and α7-helices, where
α5 interacts with Q72, T73, S75, I76, and D77 residues of RHEB
( xref D), while
α7 interacts with the same residues with the extension to bind
with N79 and N-terminal resides (K5, S6, R7, and K8; xref E). xref "
sparser
"Furthermore, we assayed the inhibitory effect of P1_WT for the
PPI of RHEB-mTOR ΔN using the AlphaLISA system (PerkinElmer,
USA). xref We used the 6xHis tagged RHEB (∼20
kDa) and mTOR ΔN (∼13 kDa) prepared by the
BL21(DE3) E. coli overexpression system. xref RHEB was first de-tagged by thrombin and then
labeled with biotin using a NHS-(PEG) 24 -biotin reagent."
sparser
"To evaluate the effect of P1_WT to inhibit RHEB-mTOR ΔN interaction,
we used the AlphaLISA-based assay as previously described. xref , xref First, we evaluated RHEB-mTOR ΔN interaction by
mixing different concentrations of 6xHis-mTOR ΔN with
1 μM biotinylated RHEB in a 384-well OptiPlateTM (PerkinElmer,
USA) followed by adding 100 μg/mL streptavidin-coated donor
beads and 200 μg/mL anti-6xHis-coated acceptor beads."
sparser
"Sancak and coworkers had previously suggested that Rheb localizes to late endosomes, and that amino acid-stimulated mTORC1 translocation facilitates mTOR-Rheb interactions. xref We find that in TSC2 −/− cells overexpressing Rab5CA, endogenous Rheb is hyperactivated but still cannot activate mTORC1."
sparser
"Although S100A8/A9 is known to be secreted mainly by myeloid cells such as neutrophils and macrophages, xref , xref multiple lines of evidence suggest that S100A8/A9 derived from BADs, but not myeloid cells, may play a major role in the suppression of osteogenesis, at least in the initiating stage. (1) Rheb BAD and Rheb AD KO mice displayed markedly enhanced S100A8/A9 expression and secretion in primary BADs in comparison with their control littermates. (2) Rheb BAD mice exhibited markedly higher S100A8/A9 level in mature BAD fraction than in the SVF fraction of BAT, which mainly includes myeloid cells, lymphocytes, preadipocytes, and endothelial cells. xref , xref (3) Both primary BADs and induced C2C12 BADs with Rheb-mTOR inactivation showed markedly increased S100A8/A9 secretion in vitro . (4) Macrophages with Rheb knockout in vitro did not display significant alteration in S100A8/A9 expression. (5) There were far less infiltrating macrophages than BADs in the BAT (less than 5% of CD45 + leukocytes and even lesser macrophages), which does not change during aging. xref Additionally, no significant differences were observed in the number of either total, M1, or M2 macrophages between the control and Rheb knockout mice. (6) We further revealed whitened BAT of aged mice secrete much more S100A8 than bone marrow per se."
sparser
"Mice with high-fat diet-induced obesity is associated with hyperactivation of Rheb-mTOR signaling. xref Paradoxically, mice with ectopic Rheb expression in pancreatic β cells showed improved glucose tolerance and resistance to hyperglycemia induced by obesity. xref While Rheb activates mTOR signaling to suppress lipolysis, it inhibits white-beige transformation of fat and thermogenesis in an mTORC1-independent but PDE45-CAMP-dependent mechanism, xref further complicating the regulatory role of Rheb-mTOR in metabolism."
reach
"In addition to the interactions between Rheb, Rag GTPases, and mTOR, several protein complexes have been found to act upstream of Rheb and Rag GTPases to control their nucleotide loading status : either by promoting the hydrolysis of GTP and increasing the ratio of GDP/GTP by working as the GAP, or by stimulating the exchange between GDP and GTP by working as the GEF."
reach
"However, in terms of leucine stimulation, the latest report by Suryawan et al. found that neonatal pigs fed with a low protein diet supplemented with leucine or its metabolite beta-hydroxy-beta-methylbutyrate (HMbeta) failed to promote the Rheb and mTOR complex formation, but this effect was not observed in pigs fed a high protein diet combined with leucine supplementation."
sparser
"The lead compound they developed is a dimeric quinacrine (DQ661), which is derived from Lys05, has improved lysosomal targeting capability and impairs the activity of palmitoyl-protein thioesterase (PPT1), which is required for mTOR interaction with Rheb at the lysosome [ xref ]; inhibition of PPT1 also prevents mTORC1 from associating with the lysosomal membrane, causing mTOR inhibition [ xref ]."
sparser
"We found that TET can enhance the interaction of SQSTM1 with MAP1LC3-II and ubiquitinated proteins due to NRF2-mediated SQSTM1 transcription and Rheb-mTOR signaling activation, thus dramatically inducing SQSTM1-selective autophagy and directly leading to Col-I degradation in lysosome ( xref )."
sparser
"For instance, similar to Rheb binding to mTOR, binding of DNA/Ku70/80 to DNA-PKcs (which together form the DNA-PK complex) has been proposed to stimulate DNA-PKcs activity via an allosteric mechanism that involves the movement of the N-HEAT towards the FAT domain, leading to conformational changes in the KD [ xref ]."
sparser
"The gene products of TSC1 (called hamartin) and TSC2 (called tuberin) assemble the TSC1/2 complex, which physiologically suppresses mTOR by inhibiting Rheb, an essential activator of the mTORC. xref Patients with TSC present with epilepsy early in life and have the characteristic presence of “tubers,” tumorlike focal lesions all over the body including in brains. xref Children with TSC can present with sleep abnormalities such as increased incidences of night waking, parasomnias, severe difficult waking up early in the morning, and daytime sleepiness. xref These abnormalities were not significantly different between epileptic and nonepileptic subgroups, representing inherent sleep and circadian disruptions in TSC patients. xref In a polysomnography study on children with TSC, sleep architecture was found to be severely disrupted, with sleep fragmentation, a shorter total sleep time, reduced sleep efficiency, and decreased rapid eye movement sleep. xref Interestingly, in this study, nocturnal seizures were recorded in some patients and associated with a more severe sleep fragmentation. xref In an adult cohort of TSC patients, similar sleep abnormalities were found, namely insomnia and excessive daytime sleepiness, which, unlike in the pediatric group, was positively correlated with their seizure history and antiseizure drug use. xref To illustrate the scale of sleep abnormalities in TSC, a large natural history study of more than 2000 patients with TSC reported sleep abnormalities as the second most reported behavioral problem in about 40% of the patients. xref Thus, it is clear that patients with TSC present with chronic sleep abnormalities, regardless of their seizure history, which indicates an intrinsic circadian dysfunction."
sparser
"Contrary to expectation, distinct from TSC2, IL-37d inhibited both the interactions of Rheb-mTOR and mTOR-S6K, suggesting that the binding of IL-37d to Switch II of Rheb leads to mTOR disassociations from Rheb and its substrate, thereby facilitating mTOR signal termination (Fig. xref )."
sparser
"However, in terms of leucine stimulation, the latest report by Suryawan et al. found that neonatal pigs fed with a low-protein diet supplemented with leucine or its metabolite β-hydroxy-β-methylbutyrate (HMβ) failed to promote the Rheb–mTOR complex formation, but this effect was not observed in pigs fed a high-protein diet combined with leucine supplementation ( xref )."
sparser
"In view of the fact that the human experimental studies by xref and xref have observed consistent results, there seems to be reason to believe that both aerobic exercise and resistance exercise can downregulate the expression of TSC2, promote the dissociation of TSC2-Rheb, and enhance the lysosomal co-localization of mTOR-Rheb, which subsequently inhibits the conversion of Rheb to inactive state loaded with GDP and promotes the activation of mTORC1."
sparser
"Rheb-GTP binds to mTOR complex 1 (mTORC1) to activate its kinase
activity toward the S6Ks, 4E-BP1, and other substrates, leading to enhancement of
multiple anabolic biosynthetic pathways that enable production of the building
blocks (e.g. nucleotides) and macromolecules (e.g. ribosomes) required for cell size
increase and mitosis ( xref )."
reach
"Studies have shown that FMDV-infected PK15 cells promote the transfer of mTOR to lysosomes to enhance the interaction between mTOR and Rheb and activate the PI3K/AKT/TSC2/Rheb/mTOR/p70S6K1 pathway to promote viral replication [52], which is consistent with the results of RNA‒seq in this study.Interleukin, interferon, the tumour necrosis factor superfamily and other cytokines act in concert with specific cytokine inhibitors and soluble cytokine receptors to regulate the human immune response [53]."
sparser
"A number of studies have looked at the involvement of Rheb in the cellular response to amino acids (Garami et al , xref ; Inoki et al , xref ; Tee et al , xref ; Zhang et al , xref ; Long et al , xref ; Smith et al , xref ; Roccio et al , xref ; Bai et al , xref ; Sancak et al , xref ), with some disagreement on whether amino acids affect Rheb GTP‐loading or Rheb‐mTOR binding."
sparser
"Previous studies demonstrated that the interaction between Rheb and mTOR was independent of Rheb’s GTP/GDP-loading status in vivo ( xref ; xref ), and the GDP-bound Rheb D60I mutant displayed a higher binding affinity to mTOR compared to wild-type or GTP-bound Rheb mutants in vivo ."
reach
"The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time."
reach
"Hence the dynamic aspects of the interaction between mTOR, Rheb and raptor in living cells requires new approaches for the detection of interaction, such as provided by the fluorescence resonance energy transfer - fluorescence lifetime imaging (FRET-FLIM) technique for observation of appropriately labelled materials in an active environment [XREF_BIBR - XREF_BIBR]."
reach
"Using the sensitive advanced imaging technique of time correlated single photon counting coupled with fluorescence lifetime imaging and molecular GFP fusion technology, it was possible for the first time to probe directly the nature of the interaction between Rheb, mTOR and raptor as well as raptor and mTOR whilst providing new insights into their sub-cellular localization."
reach
"Intracellularly, schweinfurthins and their analogs arrested trans-Golgi-network trafficking, likely by binding to oxysterol-binding proteins, leading to an effective inhibition of mTOR/AKT signaling through inducing endoplasmic reticulum stress and suppressing both lipid raft-mediated PI3K activation and mTOR/RheB complex formation."
sparser
"However, the activation of this pathway is blunted in skeletal muscle of the preterm piglet, with hyperinsulinemic-euaminoa cidemic-euglycemic clamps showing lower phosphorylation of Akt and TSC2 and reduced Rheb-mTOR association in response to insulin in muscle of preterm compared to term neonates ( xref )."
reach
"However, Rag interaction with mTORC1 does not directly activate the mTOR kinase; rather Rag proteins modulate cellular translocation of mTORC1 to the lysosome that facilitates an mTOR and RHEB interaction, which in the presence of elevated AA concentrations, results in RHEB induced activation of mTORC1."
reach
"Quenching in the lifetime of the donor (EGFP-mTOR) from 2400 +/- 100 ps to 2000 +/- 100 ps in the nuclear regions indicates that EGFP-mTOR and DsRed-Rheb interact (due to excited state energy transfer) differently (i.e. they are closer) in the cell nucleus than in the cytoplasm, likely brought about by a different conformation of the mTOR and Rheb complex."
sparser
"Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60–167) or M-heat domain (∆M, aa 967–1023) with the same affinity in the absence of GTP."
| PMC
reach
"When the utilization rate of blood glucose decreases, ATP synthesis decreases, while AMP increases, increased AMP binds and activates AMPK, following serine at positions 1345 of tumor suppressor (TSC2) is phosphorylated by activated AMPK and has GTPase activity, leading to GTP hydrolyze to GDP, which weakens the binding of GTP-dependent Rheb to mTOR molecules, exerting the effect of inhibiting mTOR, thereby enhancing the autophagic activity of cells."
reach
"We observed that Rheb is associated with LEs in hippocampal neurons and recent structural studies suggest a central position of activated Rheb in the complex associating Rag and mTORC1 to the lysosome surface [24, 31, 32] and show that Rheb binds to mTOR at a site remote from its kinase active site resulting in a conformational change that favors mTOR activity [26]."
sparser
"Although it has been proposed that the GTP-bound, active from of RHEB physically binds to mTORC1 and enhances its kinase activity [ xref ], the detailed molecular mechanism of mTORC1 activation by RHEB has been elusive until recently; the cryo-electron microscopy (cryo-EM) structure of the mTORC1-RHEB complex has revealed that RHEB directly binds to mTOR distantly from the kinase catalytic site and causes a conformational change that allosterically realigns the catalytic site, thereby enhancing mTORC1 activity [ xref , xref ]."
reach
"Meanwhile, in the FMDV-infected cells, the phosphorylation of p70S6K1 and rpS6 was also decreased in the SLC38A8 cells compared to that in the WT cells (Fig 10C), suggesting that FMDV-induced the upregulation of aspartate was responsible for the increase of the phosphorylation of p70S6K1 and rpS6.As described above, amino acid stimulation enhanced interaction between Rheb and mTOR, resulting in the activation of the mTOR/p70S6K1 signaling axis."
sparser
"In response to amino acids, Raptor interacts with the lysosomal Ras-related guanosine 5’-triphosphate (GTP)-binding protein (Rag small GTPase protein complex tethering mTORC1 to the lysosomal membrane, where it encounters another small GTPase, Ras homolog enriched in brain (Rheb) that directly interacts with the mTOR kinase and stimulates the activity of mTORC1 [ xref , xref ]."
sparser
"Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60-167) or M-heat domain (∆M, aa 967-1023) with the same affinity in the absence of GTP."
sparser
"The site of Rheb-MTOR interaction identified by cryo-EM, accompanied by extensive biochemical verification of Rheb-GTP activation of MTORC1, invalidates the earlier conclusion that Rheb binds to the MTOR catalytic domain, which was based on the use of Rheb co-expression with MTOR fragments xref ."
sparser
"For example, the TSC1/TSC2 complex inhibits mTOR functions by inactivating Rheb; Rheb binds and activates mTOR only when the former is in its GTP-bound form. xref The phosphoinositide-3-kinase (PI3K)-Akt pathway activates mTOR by Akt-mediated phosphorylation of TSC2, followed by disassembly and inhibition of the TSC1/TSC2 complex. xref , xref The extracellular signal-related kinase (ERK) stimulates mTOR activation by either directly phosphorylating TSC2 or by inducing TSC2 phosphorylation via its two downstream effectors, ribosomal S6 kinase (RSK) and DAPK. xref – xref Finally, AMP-activated protein kinase (AMPK) phosphorylates TSC2 and stabilizes the TSC1/TSC2 complex, thereby inhibiting Rheb-mediated activation of mTOR. xref AMPK can also regulate mTOR function in a TSC-independent manner by directly phosphorylating Raptor, an essential component of mTORC1, to inhibit mTOR activity. xref Notably, TSC1 and 2, PI3K, and Akt are all subject to mutation in human tumors."
sparser
"It can be argued that the very weak affinity of Rheb for MTOR results in a loss of the Rheb-MTOR interaction upon cell extraction; however, this returns to the question of how the Rheb activation mechanism demonstrated by Yang et al . xref relates to the mechanism operative in the cell and whether a continuing association of Rheb with MTOR is required for the maintenance of MTORC1 activity in the cell."
sparser
"Fluorescence lifetime imaging of overexpressed Rheb, MTOR, and Raptor, each fused to fluorescent proteins, did detect the presence of a Rheb-MTOR complex in both the cytoplasm and the nucleus (the latter devoid of Raptor) xref ; as yet, however, the presence of endogenous Rheb in the nucleus and its localization therein await confirmation."
sparser
"As is known, Rheb interacts with mTOR directly and activates it in the GTP‐bound form. xref According to the association between HOTAIRM1 and the mTOR pathway, we tried to explore whether HOTAIRM1 promotes OS progression by modulating mTOR/c‐MYC‐mediated glycolysis in a Rheb‐dependent manner."
sparser
"Song and coworkers have also confirmed that resistance exercise in the human skeletal muscle resulted in the rapid translocation of mTOR toward the lysosome, with the concurrent dissociation of TSC2 from Rheb, which would facilitate the interaction of mTOR and GTP‐Rheb (Song et al., xref )."
sparser
"Studies have shown that FMDV-infected PK15 cells promote the transfer of mTOR to lysosomes to enhance the interaction between mTOR and Rheb and activate the PI3K/AKT/TSC2/Rheb/mTOR/p70S6K1 pathway to promote viral replication [ xref ], which is consistent with the results of RNA‒seq in this study."
reach
"Although direct interaction between Rheb and mTOR has not been ruled out, it appears more likely that Rheb-GTP recruits additional protein (s) to the mTOR complex to modulate its activity.The mTOR pathway plays a central role in nutrient and energy sensing to regulate cell growth and proliferation (reviewed in Ref."
sparser
"Proximity ligation assays (PLAs) in WT PPT1 and KO PPT1 cells established a significant loss of physical interaction between the LAMTOR1/p18 subunit of Ragulator and the ATP6V1A subunit of the vacuolar-type H+-ATPase (v-ATPase), which is critical for and results in the loss of mTOR-Rheb physical interactions ( xref ) ( xref , xref )."