Acid Suspends the Circadian Clock in Hypoxia through Inhibition of mTOR.

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.

Amino Acids License Kinase mTORC1 Activity and Treg Cell Function via Small G Proteins Rag and Rheb.

Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and Rheb GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or Rheb1-Rheb2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while Rheb1-Rheb2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells.

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.

Acute effects of a ketone monoester, whey protein, or their coingestion on mTOR trafficking and protein-protein colocalization in human skeletal muscle.

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their coingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their coingestion on mechanistic target of rapamycin (mTOR)-related protein-protein colocalization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2 ± 4.1 yr) ingested either: 1) 0.36 g·kg-1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET + PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120 and 300 min in the postprandial period for immunofluorescence assessment of protein translocation and colocalization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P < 0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) colocalization at 120 min versus basal; however, the decrease was sustained at 300 min versus basal (P < 0.0001) only in KET + PRO. PRO and KET + PRO increased (Interaction: P < 0.0001) mTOR-Rheb colocalization at 120 min versus basal; however, KET + PRO resulted in a sustained increase in mTOR-Rheb colocalization at 300 min that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) colocalization at 120 and 300 min (Time: P = 0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle.NEW & NOTEWORTHY We explored the effects of a ketone monoester (KET), whey protein (PRO), or their coingestion (KET + PRO) on mTOR-related protein-protein colocalization and intracellular trafficking in human muscle. All treatments decreased TSC2-Rheb colocalization at 120 minutes; however, KET + PRO sustained the decrease at 300 min. Only PRO and KET + PRO increased mTOR-Rheb colocalization; however, the increase at 300 min was greater in KET + PRO. Treatment intake increased mTOR-WGA colocalization, suggesting translocation to the fiber periphery. Ketone bodies influence the spatial regulation of mTOR.

CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

Inhibition of CIRBP represses the proliferation and migration of vascular smooth muscle cells via inhibiting Rheb/mTORC1 axis.

The excessive migration and proliferation of vascular smooth muscle cells (VSMCs) plays a vital role in vascular intimal hyperplasia. CIRBP is involved in the proliferation of various cancer cells. This study was aimed to explore the role of CIRBP in the proliferation and migration of VSMCs. Adenovirus was used to interfere with cold-inducible RNA-binding protein (CIRBP) expression, while lentivirus was used to overexpress Ras homolog enriched in brain (Rheb). Western blotting and qRT-PCR were used to evaluate the expression of CIRBP, Rheb, and mechanistic target of rapamycin complex 1 (mTORC1) activity. The cell proliferation was determined by Ki67 immunofluorescence staining and CCK-8 assay. The wound healing assay was performed to assess cell migration. Additionally, immunohistochemistry was conducted to explore the role of CIRBP in intimal hyperplasia after vascular injury. We found that silencing CIRBP inhibited the proliferation and migration of VSMCs, decreased the expression of Rheb and mTORC1 activity. Restoration of mTORC1 activity via insulin or overexpression of Rheb via lentiviral transfection both attenuated the inhibitory effects of silencing CIRBP on the proliferation and migration of VSMCs. Moreover, Rheb overexpression abolished the inhibitory effect of silencing CIRBP on mTORC1 activity in VSMCs. CIRBP was upregulated in the injured carotid artery. Silencing CIRBP ameliorated intimal hyperplasia after vascular injury. In the summary, silencing CIRBP attenuates mTORC1 activity via reducing Rheb expression, thereby supressing the proliferation and migration of VSMCs and intimal hyperplasia after vascular injury.

Rheb1 is required for limb growth through regulating chondrogenesis in growth plate.

Ras homology enriched in the brain (Rheb) is well established as a critical regulator of cell proliferation and differentiation in response to growth factors and nutrients. However, the role of Rheb1 in limb development remains unknown. Here, we found that Rheb1 was dynamically expressed during the proliferation and differentiation of chondrocytes in the growth plate. Given that Prrx1+ limb-bud-like mesenchymal cells are the source of limb chondrocytes and are essential for endochondral ossification, we conditionally deleted Rheb1 using Prrx1-Cre and found a limb dwarfism in Prrx1-Cre; Rheb1fl/fl mice. Normalized to growth plate height, the conditional knockout (cKO) mice exhibited a significant decrease in column count of proliferative zones which was increased in hypertrophic zones resulting in decreased growth plate size, indicating abnormal endochondral ossification. Interestingly, although Rheb1 deletion profoundly inhibited the transcription factor Sox9 in limb cartilage; levels of runx2 and collagen type 2 were both increased. These novel findings highlight the essential role of Rheb1 in limb growth and indicate a complex regulation of Rheb1 in chondrocyte proliferation and differentiation.

Chondrocyte autophagy mediated by T-2 toxin via AKT/TSC/Rheb/mTOR signaling pathway and protective effect of CSA-SeNP.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic, degenerative, and cartilage-damaging disease for which low selenium and T-2 toxins are considered environmental pathogenic factors. This study aimed to investigate the molecular mechanisms of autophagy in cartilage damage caused by T-2 toxin and the protective effect of chondroitin sulfate A nano-elemental selenium (CSA-SeNP) on the cartilage. METHODS: KBD chondrocytes and C28/I2 human chondrocyte cell lines were used. T-2 toxin, AKT inhibitor, and CSA-SeNP treatment experiments were conducted separately, with a treatment time of 24 h. Autophagy was monitored using MDC staining, and mRFP-GFP-LC3 adenovirus, respectively. RT-qPCR and western blotting were used to detect the expression of the relevant genes and proteins. RESULTS: The suppression of autophagy observed in KBD chondrocytes was replicated by applying 10 ng/mL T-2 toxin to C28/I2 chondrocytes for 24 h. The AKT/TSCR/Rheb/mTOR signaling pathway was activated by T-2 toxin, which inhibits autophagy. The supplementation with CSA-SeNP alleviated the inhibition of autophagy by T-2 toxin through the AKT/TSCR/Rheb/mTOR signaling pathway. CONCLUSIONS: Loss of autophagy regulated by the AKT/TSCR/Rheb/mTOR signaling pathway plays an important role in cartilage damage caused by T-2 toxin. CSA-SeNP supplementation attenuated inhibition of autophagy in chondrocytes by T-2 toxin by modulating this signaling pathway. These findings provide promising new targets for the prevention and treatment of cartilage disease.

Unraveling the Role of Ras Homolog Enriched in Brain (Rheb1 and Rheb2): Bridging Neuronal Dynamics and Cancer Pathogenesis through Mechanistic Target of Rapamycin Signaling.

Ras homolog enriched in brain (Rheb1 and Rheb2), small GTPases, play a crucial role in regulating neuronal activity and have gained attention for their implications in cancer development, particularly in breast cancer. This study delves into the intricate connection between the multifaceted functions of Rheb1 in neurons and cancer, with a specific focus on the mTOR pathway. It aims to elucidate Rheb1's involvement in pivotal cellular processes such as proliferation, apoptosis resistance, migration, invasion, metastasis, and inflammatory responses while acknowledging that Rheb2 has not been extensively studied. Despite the recognized associations, a comprehensive understanding of the intricate interplay between Rheb1 and Rheb2 and their roles in both nerve and cancer remains elusive. This review consolidates current knowledge regarding the impact of Rheb1 on cancer hallmarks and explores the potential of Rheb1 as a therapeutic target in cancer treatment. It emphasizes the necessity for a deeper comprehension of the molecular mechanisms underlying Rheb1-mediated oncogenic processes, underscoring the existing gaps in our understanding. Additionally, the review highlights the exploration of Rheb1 inhibitors as a promising avenue for cancer therapy. By shedding light on the complicated roles between Rheb1/Rheb2 and cancer, this study provides valuable insights to the scientific community. These insights are instrumental in guiding the identification of novel targets and advancing the development of effective therapeutic strategies for treating cancer.

Translation initiation factor eIF3a regulates glucose metabolism and cell proliferation via promoting small GTPase Rheb synthesis and AMPK activation.

Eukaryotic translation initiation factor 3 subunit A (eIF3a), the largest subunit of the eIF3 complex, has been shown to be overexpressed in malignant cancer cells, potentially making it a proto-oncogene. eIF3a overexpression can drive cancer cell proliferation but contributes to better prognosis. While its contribution to prognosis was previously shown to be due to its function in suppressing synthesis of DNA damage repair proteins, it remains unclear how eIF3a regulates cancer cell proliferation. In this study, we show using genetic approaches that eIF3a controls cell proliferation by regulating glucose metabolism via the phosphorylation and activation of AMP-activated protein kinase alpha (AMPKα) at Thr172 in its kinase activation loop. We demonstrate that eIF3a regulates AMPK activation mainly by controlling synthesis of the small GTPase Rheb, largely independent of the well-known AMPK upstream liver kinase B1 and Ca2+/calmodulin-dependent protein kinase kinase 2, and also independent of mammalian target of rapamycin signaling and glucose levels. Our findings suggest that glucose metabolism in and proliferation of cancer cells may be translationally regulated via a novel eIF3a-Rheb-AMPK signaling axis.

LncRNA-HOTAIRM1 promotes aerobic glycolysis and proliferation in osteosarcoma via the miR-664b-3p/Rheb/mTOR pathway.

Osteosarcoma (OS), which is a common and aggressive primary bone malignancy, occurs mainly in children and adolescent. Long noncoding RNAs (lncRNAs) are reported to play a pivotal role in various cancers. Here, we found that the lncRNA HOTAIRM1 is upregulated in OS cells and tissues. A set of functional experiments suggested that HOTAIRM1 knockdown attenuated the proliferation and stimulated the apoptosis of OS cells. A subsequent mechanistic study revealed that HOTAIRM1 functions as a competing endogenous RNA to elevate ras homologue enriched in brain (Rheb) expression by sponging miR-664b-3p. Immediately afterward, upregulated Rheb facilitates proliferation and suppresses apoptosis by promoting the mTOR pathway-mediated Warburg effect in OS. In summary, our findings demonstrated that HOTAIRM1 promotes the proliferation and suppresses the apoptosis of OS cells by enhancing the Warburg effect via the miR-664b-3p/Rheb/mTOR axis. Understanding the underlying mechanisms and targeting the HOTAIRM1/miR-664b-3p/Rheb/mTOR axis are essential for OS clinical treatment.

Oncolytic Avian Reovirus p17-Modulated Inhibition of mTORC1 by Enhancement of Endogenous mTORC1 Inhibitors Binding to mTORC1 To Disrupt Its Assembly and Accumulation on Lysosomes.

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.

Cardiac decompensation and promiscuous prenylation of small GTPases in cardiomyocytes in response to local mevalonate pathway disruption.

Investigations of major mevalonate pathway enzymes have demonstrated the importance of local isoprenoid synthesis in cardiac homeostasis. Farnesyl diphosphate synthase (FPPS) synthesizes isoprenoid precursors needed for cholesterol biosynthesis and protein prenylation. Wang, Zhang, Chen et al, in a recently published article in The Journal of Pathology, elegantly elucidated the pathological outcomes of FPPS deficiency in cardiomyocytes, which paradoxically resulted in increased prenylation of the small GTPases Ras and Rheb. Cardiomyocyte FPPS depletion caused severe dilated cardiomyopathy that was associated with enhanced GTP-loading and abundance of Ras and Rheb in lipidated protein-enriched cardiac fractions and robust activation of downstream hypertrophic ERK1/2 and mTOR signaling pathways. Cardiomyopathy and activation of ERK1/2 and mTOR caused by loss of FPPS were ameliorated by inhibition of farnesyltransferase, suggesting that impairment of FPPS activity results in promiscuous activation of Ras and Rheb through non-canonical actions of farnesyltransferase. Here, we discuss the findings and adaptive signaling mechanisms in response to disruption of local cardiomyocyte mevalonate pathway activity, highlighting how alteration in a key branch point in the mevalonate pathway affects cardiac biology and function and perturbs protein prenylation, which might unveil novel strategies and intricacies of targeting the mevalonate pathway to treat cardiovascular diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Spatial regulation of mTORC1 signalling: Beyond the Rag GTPases.

The mechanistic (or mammalian) Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism. By integrating mitogenic signals, mTORC1-dependent phosphorylation of substrates dictates the balance between anabolic, pro-growth and catabolic, recycling processes in the cell. The discovery that amino acids activate mTORC1 by promoting its translocation to the lysosome was a fundamental advance in the understanding of mTORC1 signalling. It has since become clear that the lysosome-cytoplasm shuttling of mTORC1 represents just one layer of spatial control of this signalling pathway. This review will focus on exploring the subcellular localisation of mTORC1 and its regulators to multiple sites within the cell. We will discuss how these spatially distinct regions such as endoplasmic reticulum, plasma membrane and the endosomal pathway co-operate to transduce nutrient availability to mTORC1, allowing for tight control of cell growth.

Development of a versatile HPLC-based method to evaluate the activation status of small GTPases.

Small GTPases cycle between an inactive GDP-bound and an active GTP-bound state to control various cellular events, such as cell proliferation, cytoskeleton organization, and membrane trafficking. Clarifying the guanine nucleotide-bound states of small GTPases is vital for understanding the regulation of small GTPase functions and the subsequent cellular responses. Although several methods have been developed to analyze small GTPase activities, our knowledge of the activities for many small GTPases is limited, partly because of the lack of versatile methods to estimate small GTPase activity without unique probes and specialized equipment. In the present study, we developed a versatile and straightforward HPLC-based assay to analyze the activation status of small GTPases by directly quantifying the amounts of guanine nucleotides bound to them. This assay was validated by analyzing the RAS-subfamily GTPases, including HRAS, which showed that the ratios of GTP-bound forms were comparable with those obtained in previous studies. Furthermore, we applied this assay to the investigation of psychiatric disorder-associated mutations of RHEB (RHEB/P37L and RHEB/S68P), revealing that both mutations cause an increase in the ratio of the GTP-bound form in cells. Mechanistically, loss of sensitivity to TSC2 (a GTPase-activating protein for RHEB) for RHEB/P37L, as well as both decreased sensitivity to TSC2 and accelerated guanine-nucleotide exchange for RHEB/S68P, is involved in the increase of their GTP-bound forms, respectively. In summary, the HPLC-based assay developed in this study provides a valuable tool for analyzing small GTPases for which the activities and regulatory mechanisms are less well understood.

The non-canonical nuclear functions of key players of the PI3K-AKT-MTOR pathway.

The PI3K-AKT-MTOR signal transduction pathway is one of the essential signalling cascades within the cell due to its involvement in many vital functions. The pathway initiates with the recruitment of phosphatidylinositol-3 kinases (PI3Ks) onto the plasma membrane, generating phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3 ] and subsequently activating AKT. Being the central node of the PI3K network, AKT activates the mechanistic target of rapamycin kinase complex 1 (MTORC1) via Tuberous sclerosis complex 2 inhibition in the cytoplasm. Although the cytoplasmic role of the pathway has been widely explored for decades, we now know that most of the effector molecules of the PI3K axis diverge from the canonical route and translocate to other cell organelles including the nucleus. The presence of phosphoinositides (PtdIns) inside the nucleus itself indicates the existence of a nuclear PI3K signalling. The nuclear localization of these signaling components is evident in regulating many nuclear processes like DNA replication, transcription, DNA repair, maintenance of genomic integrity, chromatin architecture, and cell cycle control. Here, our review intends to present a comprehensive overview of the nuclear functions of the PI3K-AKT-MTOR signaling biomolecules.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

TSC-insensitive Rheb mutations induce oncogenic transformation through a combination of constitutively active mTORC1 signalling and proteome remodelling.

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1 that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, tuberous sclerosis complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving an increased rate of anaerobic glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through differential interactions with 5' AMP-activated protein kinase (AMPK) which modulate its activity. Our findings suggest that unique, personalized, combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are high value agents used for disease therapy ("biologic drugs") or as diagnostic tools which are widely used in the healthcare sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1, a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here, we show that certain constitutively active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum-free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb[T23M] markedly (2-3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value.

mTOR Senses Intracellular pH through Lysosome Dispersion from RHEB.

Acidity, generated in hypoxia or hypermetabolic states, perturbs homeostasis and is a feature of solid tumors. That acid peripherally disperses lysosomes is a three-decade-old observation, yet one little understood or appreciated. However, recent work has recognized the inhibitory impact this spatial redistribution has on mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of metabolism. This finding argues for a paradigm shift in localization of mTORC1 activator Ras homolog enriched in brain (RHEB), a conclusion several others have now independently reached. Thus, mTORC1, known to sense amino acids, mitogens, and energy to restrict biosynthesis to times of adequate resources, also senses pH and, via dampened mTOR-governed synthesis of clock proteins, regulates the circadian clock to achieve concerted responses to metabolic stress. While this may allow cancer to endure metabolic deprivation, immune cell mTOR signaling likewise exhibits pH sensitivity, suggesting that suppression of antitumor immune function by solid tumor acidity may additionally fuel cancers, an obstacle potentially reversible through therapeutic pH manipulation.