Person: Manning, Brendan
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Manning
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Brendan
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Manning, Brendan
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Publication Ex Vivo and in Vivo Stable Isotope Labelling of Central Carbon Metabolism and Related Pathways With Analysis by LC–MS/MS(Springer Science and Business Media LLC, 2019-02) Yuan, Min; Kremer, Daniel M.; Huang, He; Breitkopf, Susanne B.; Ben-Sahra, Issam; Manning, Brendan; Lyssiotis, Costas A.; Asara, JohnTargeted tandem mass spectrometry (LC–MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6–7 h for a single sample from experimental labelling and metabolite extraction to peak integration.Publication mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer(2017) Zabala-Letona, Amaia; Arruabarrena-Aristorena, Amaia; Martín-Martín, Natalia; Fernandez-Ruiz, Sonia; Sutherland, James D.; Clasquin, Michelle; Tomas-Cortazar, Julen; Jimenez, Jose; Torres, Ines; Quang, Phong; Ximenez-Embun, Pilar; Bago, Ruzica; Ugalde-Olano, Aitziber; Loizaga-Iriarte, Ana; Lacasa-Viscasillas, Isabel; Unda, Miguel; Torrano, Verónica; Cabrera, Diana; van Liempd, Sebastiaan M.; Cendon, Ylenia; Castro, Elena; Murray, Stuart; Revandkar, Ajinkya; Alimonti, Andrea; Zhang, Yinan; Barnett, Amelia; Lein, Gina; Pirman, David; Cortazar, Ana R.; Arreal, Leire; Prudkin, Ludmila; Astobiza, Ianire; Valcarcel-Jimenez, Lorea; Zuñiga-García, Patricia; Fernandez-Dominguez, Itziar; Piva, Marco; Caro-Maldonado, Alfredo; Sánchez-Mosquera, Pilar; Castillo-Martín, Mireia; Serra, Violeta; Beraza, Naiara; Gentilella, Antonio; Thomas, George; Azkargorta, Mikel; Elortza, Felix; Farràs, Rosa; Olmos, David; Efeyan, Alejo; Anguita, Juan; Muñoz, Javier; Falcón-Pérez, Juan M.; Barrio, Rosa; Macarulla, Teresa; Mato, Jose M.; Martinez-Chantar, Maria L.; Cordon-Cardo, Carlos; Aransay, Ana M.; Marks, Kevin; Baselga, José; Tabernero, Josep; Nuciforo, Paolo; Manning, Brendan; Marjon, Katya; Carracedo, ArkaitzActivation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation1,2. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model3 and human biopsies4 of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus5 exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.Publication Improved detection of synthetic lethal interactions in Drosophila cells using variable dose analysis (VDA)(National Academy of Sciences, 2017) Housden, Benjamin E.; Li, Zhongchi; Kelley, Colleen; Wang, Yuanli; Hu, Yanhui; Valvezan, Alexander; Manning, Brendan; Perrimon, NorbertSynthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells, but this approach generally suffers from low consistency between screens. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called Variable Dose Analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC), and generated a SS/L network for TSC. Using this network, we identified four Food and Drug Administration-approved drugs that selectively affect viability of TSC-deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.Publication Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signaling to suppress tumorigenesis(2013) Liu, Pengda; Gan, Wenjian; Inuzuka, Hiroyuki; Lazorchak, Adam S; Gao, Daming; Arojo, Omotooke; Liu, Dou; Wan, Lixin; Zhai, Bo; Yu, Yonghao; Yuan, Min; Kim, Byeong Mo; Shaik, Shavali; Menon, Suchithra; Gygi, Steven; Lee, Tae Ho; Asara, John; Manning, Brendan; Blenis, John; Su, Bing; Wei, WenyiThe mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at T86 and T398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin/IGF-1-mediated, but also PDGF or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Lastly, a cancer patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-mTORC2 activation via bypassing this negative regulation. Together, our work reveals a Sin1 phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1/S6K/Sin1 signaling axis might cause aberrant hyper-activation of mTORC2/Akt that facilitates tumorigenesis.Publication The TSC-mTOR pathway regulates macrophage polarization(2013) Byles, Vanessa; Covarrubias, Anthony Joseph; Ben-Sahra, Issam; Lamming, Dudley W.; Sabatini, David M.; Manning, Brendan; Horng, TiffanyMacrophages are able to polarize to proinflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. How metabolic status regulates macrophage polarization remains not well understood, and here we examine the role of mTOR (Mechanistic Target of Rapamycin), a central metabolic pathway that couples nutrient sensing to regulation of metabolic processes. Using a mouse model in which myeloid lineage specific deletion of Tsc1 (Tsc1Δ/Δ) leads to constitutive mTOR Complex 1 (mTORC1) activation, we find that Tsc1Δ/Δ macrophages are refractory to IL-4 induced M2 polarization, but produce increased inflammatory responses to proinflammatory stimuli. Moreover, mTORC1-mediated downregulation of Akt signaling critically contributes to defective polarization. These findings highlight a key role for the mTOR pathway in regulating macrophage polarization, and suggest how nutrient sensing and metabolic status could be “hard-wired” to control of macrophage function, with broad implications for regulation of Type 2 immunity, inflammation, and allergy.Publication mTORC1 stimulates nucleotide synthesis through both transcriptional and post-translational mechanisms(BioMed Central, 2014) Ben-Sahra, Issam; Ricoult, Stephane; Howell, Jessica; Asara, John; Manning, BrendanPublication Oncogenic signaling upstream of mTORC1 drives lipogenesis and proliferation through SREBP(BioMed Central, 2014) Ricoult, Stéphane; Yecies, Jessica; Manning, BrendanPublication Mechanisms and consequences of hepatic regulation of mTORC1 by metformin(BioMed Central, 2014) Howell, Jessica; Hellberg, Kristina; Shaw, Reuben J; Manning, BrendanPublication Coordinated regulation of protein synthesis and degradation by mTORC1(Springer Science and Business Media LLC, 2014-07-13) Zhang, Yinan; Nicholatos, Justin William; Dreier, John; Ricoult, Stephane Jean Hermann; Widenmaier, Scott; Hotamisligil, Gokhan; Kwiatkowski, David; Manning, BrendanEukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis1. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.Publication Splicing factor 1 modulates dietary restriction and TORC1 pathway longevity in C. elegans(Springer Nature, 2016) Heintz, Caroline; Doktor, Thomas K.; Lanjuin, Anne; Escoubas, Caroline; Zhang, Yue; Weir, Heather; Dutta, Sneha; Silva-García, Carlos Giovanni; Bruun, Gitte H.; Morantte, Ianessa; Hoxhaj, Gerta; Manning, Brendan; Andresen, Brage S.; Mair, WilliamAgeing is driven by a loss of transcriptional and protein homeostasis1, 2, 3 and is the key risk factor for multiple chronic diseases. Interventions that attenuate or reverse systemic dysfunction associated with age therefore have the potential to reduce overall disease risk in the elderly. Precursor mRNA (pre-mRNA) splicing is a fundamental link between gene expression and the proteome, and deregulation of the splicing machinery is linked to several age-related chronic illnesses4, 5. However, the role of splicing homeostasis in healthy ageing remains unclear. Here we demonstrate that pre-mRNA splicing homeostasis is a biomarker and predictor of life expectancy in Caenorhabditis elegans. Using transcriptomics and in-depth splicing analysis in young and old animals fed ad libitum or subjected to dietary restriction, we find defects in global pre-mRNA splicing with age that are reduced by dietary restriction via splicing factor 1 (SFA-1; the C. elegans homologue of SF1, also known as branchpoint binding protein, BBP). We show that SFA-1 is specifically required for lifespan extension by dietary restriction and by modulation of the TORC1 pathway components AMPK, RAGA-1 and RSKS-1/S6 kinase. We also demonstrate that overexpression of SFA-1 is sufficient to extend lifespan. Together, these data demonstrate a role for RNA splicing homeostasis in dietary restriction longevity and suggest that modulation of specific spliceosome components may prolong healthy ageing.