115 research outputs found
Upstream regulation of yeast TOR complexes
Full text linkNitrogen is an essential component of living organism. Protein synthesis and biosynthesis of nitrogen containing molecules essential for growth, such as amino acids and NAD, strictly depend on the availability of the nitrogen source. Limiting amount of nitrogen limit cell growth. Exogenous amino acids and other nitrogenous compounds such as ammonium constitute the nitrogen source. The budding yeast S. cerevisiae can sense and utilize a total of 21 different nitrogenous compounds to sustain growth. The uptake and utilization of different nitrogen sources is hierarchical and subject to strict and complex regulation at the transcriptional, translation and post-translational level. In general, preferred nitrogen sources inhibit the uptake and catabolism of non-preferred nitrogen sources.
The target of rapamycin (TOR) is a conserved Ser/Thr protein kinase among eukaryotes controlling growth in response to nutrients and growth factors. TOR is found in two essential conserved multiprotein complexes named TOR complex 1 (TORC1) and TORC2. In yeast, TORC1 signaling is sensitive to nutrients, particularly to availability of the nitrogen source. Thus, TORC1 by promoting anabolic processes, such as protein synthesis and ribosome biogenesis, couples growth to the availability of the nitrogen source.
The sensing mechanism activating TORC1 in response to nitrogen source sufficiency is only poorly understood. Recent studies identified the EGO complex as intermediary component of the amino acid sensing pathway. In the presence of amino acids, particularly leucine, the EGO complex is activated and promotes TORC1 activity. The small GTP binding proteins Gtr1 and Gtr2 are part of the EGO complex. Gtr1 and Gtr2 form a heterodimeric complex. In response to amino acids Gtr1 is loaded with GTP and Gtr2 with GDP, leading to activation of the EGO complex. Several factors determine EGO complex activation by dictating the Gtr1/2 guanosine loading. Vam6 was proposed to act as guanosine exchange factor (GEF) towards Gtr1. The Npr2/Npr3/Iml1 complex was reported to act as GTPase activating protein (GAP) for Gtr1. Therefore, amino acid stimulation of TORC1 is decreased in the absence of Vam6 and increased in the absence of Iml1. Several observations point out that EGO complex signaling, alone, is not sufficient to explain TORC1 activation by the nitrogen source. For instance, 1) components of the EGO complex signaling are not essential; 2) ammonium starvation down-regulates growth even in cells where EGO complex signaling is hyperactivated. Therefore, nitrogen source and amino acids sufficiency might signal to TORC1 via distinct mechanisms.
In this study we analyze the effect of different nitrogen sources on TORC1 activity. We use the phosphorylation state of the direct TORC1 target Sch9 as readout for TORC1 activity. We describe that preferred nitrogen sources activate TORC1 signaling stronger and better than non-preferred nitrogen source. TORC1 activation by preferred nitrogen sources is paralleled by an increase in glutamine synthesis and accumulation. Growth is increased in the presence of preferred nitrogen sources in a glutamine synthesis dependent way. Therefore, glutamine constitutes a metabolic input linking TORC1 activation in response to the quality of the nitrogen source to growth capacity. We find that EGO complex signaling is dispensable when a preferred nitrogen source is provided. TORC1 activation and growth increase are still induced in cells compromised for EGO complex signaling. Taken together, we demonstrate that nitrogen source and amino acid sufficiency act via discrete mechanisms to activate TORC1
New insights into the "in vivo" and "in vitro" functions of mammalian TOR complex 2
Full text linkTarget of rapamycin (TOR) is the main controller of cell growth and metabolism in response to nutrients, growth factors and the cellular energy status. TOR is a serine/threonine kinase conserved from yeast to mammals and is found in two functionally and structurally distinct multi-protein complexes named TOR complex 1 (TORC1) and TORC2. Mammalian TORC1 (mTORC1) contains mTOR, mLST8, raptor and PRAS40, while mTORC2 contains mTOR, mLST8, rictor, mSin1, and PRR5. mTORC2 is activated in response to growth factors, such as insulin and insulin-like growth factor 1 (IGF1), and its main functions involve the regulation of actin cytoskeleton dynamics and phosphorylation of several AGC kinases in their hydrophobic motif. TORC1 is directly inhibited by the immunosuppressant and anti-cancer drug rapamycin, whereas TORC2 is not. Thus, use of rapamycin provides a simple and straightforward method to specifically study the TORC1 signaling branch. There is no known TORC2-specific inhibitor, so genetic manipulation is required to study its biological function(s).
This thesis describes new in vivo and in vitro functions of mTORC2. The first part deals with the in vivo function of mTORC2 in adipose tissue. The adipose tissue, in addition to its function as a long-term fat storage depot, also has endocrine functions, plays an important role in the regulation of whole body glucose and lipid metabolism and is one of the most insulin-responsive tissues in the body. To study mTORC2 function in adipose tissue, we have generated mice that lack the mTORC2-essential component rictor specifically in adipose tissue. Phenotypic characterization revealed the unexpected finding that these mice were larger due to an increase in lean tissue mass and that they had elevated serum IGF1 levels. Furthermore, the knockout mice were hyperinsulinemic, but glucose tolerant. Overall, these findings suggest an important role for adipose mTORC2 in controlling full body growth and whole body glucose metabolism.
The second part of this thesis describes a new in vitro function of mTORC2 in fibroblasts. We have taken advantage of the raptor and rictor floxed mice to isolate mouse embryonic fibroblasts (MEFs), which were then used to establish inducible raptor and rictor knockout MEF cell lines. After initial characterization of these two cell lines, a deeper analysis of the role of mTORC2 in the actin-mediated process of cell migration was performed. We have found that mTORC2 is required for cell migration and for regulating the activity of the Rho GTPases Rac1, Cdc42, and RhoA. We have extended this study by showing that mTORC2-dependent cell migration is also required in oncogenic cells, which suggests that mTORC2 could have an important function in the development of cancer and metastasis
Quantitative phosphoproteomics reveal that mTOR regulates cell growth and proliferation by phosphorylating a functionally diverse set of substrates
Full text linkThe atypical Ser/Thr kinase target of rapamycin (TOR) is a central controller of cell growth and proliferation. TOR forms two distinct multiprotein complexes, TORC1 and TORC2, which are structurally and functionally conserved from yeast to humans. Four major inputs control mammalian TOR (mTOR): growth factors, such as insulin; cellular energy levels, such as the AMP:ATP ratio; stress, such as hypoxia; and nutrients, such as amino acids. mTOR controls cell growth by the positive and negative regulation of several anabolic and catabolic processes, respectively, that collectively regulate cell size and proliferation. These cellular processes include autophagy, cytoskeleton rearrangement, glycolysis, lipogenesis, nutrient transport, ribosome biogenesis, and translation. Dysregulation of the mTOR signaling network has been associated with aging, and a multitude of diseases including cancer, cardiovascular disease, diabetes, inflammation, immune dysfunctions, and neurodegeneration. However, relatively few direct substrates of either one of the two mTOR complexes, mTORC1 and mTORC2, are known.
To determine downstream effectors of mammalian TOR (mTOR), we applied a functional, quantitative phosphoproteomics workflow to identify novel mTORC1 or mTORC2 regulated phosphorylations. Raptor and Rictor are essential components of mTORC1 and mTORC2, respectively. To distinguish phosphorylations regulated by mTORC1 or mTORC2, we specifically deleted Raptor or Rictor using an inducible gene knockout system in mouse embryonic fiberblasts (MEFs). We detected 4584 phosphorylation sites on 1398 proteins, and identified 335 novel mTOR effectors. Many of the novel effectors are implicated in cancer and metabolic diseases, but have no known links to mTOR. To distinguish direct mTOR substrates from indirect effectors, we combined peptide array in vitro kinase assays with phosphorylation motif analysis. This revealed that mTORC1 phosphorylates CAD in vivo and in vitro. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) is the initial and rate limiting enzyme in de novo pyrimidine synthesis. The macrolide rapamycin, which forms a complex with FKBP12, binds and acutely inhibits mTORC1 but not mTORC2. Rapamycin treatment inhibited growth factor stimulated CAD phosphorylation and oligomerization, decreased de novo pyrimidine synthesis, and delayed progression of S-phase where CAD activity is essential. Thus mTORC1 phosphorylates CAD and thereby stimulates de novo pyrimidine synthesis to promote cell proliferation.
Separately, we characterize the autophosphorylation of mTOR on Ser2481. Insulin stimulates the phosphorylation of mTOR at Ser2481 specifically in mTORC2. Knockout of Rictor, but not Raptor, abolished mTOR autophosphorylation at Ser2481. Prolonged treatment with rapamycin, which indirectly inhibits mTORC2 complex formation, inhibited Ser2481 phosphorylation. Surprisingly, mTORC2 autophosphorylation at Ser2481 temporally occurs after the insulin-induced phosphorylation of Akt/PKB and the SGK1 substrate NDGR1. Mutation of Ser2481 to aspartic acid rendered mTOR unable to phosphorylate Akt/PKB in vitro. However the function of mTOR-Ser2481 phosphorylation in vivo remains elusive, as mutation of mTOR-Ser2481 did not alter Akt/PKB phosphorylation in vivo.
In summary, mTORC1 and mTORC2 regulate the phosphorylation of a functionally diverse set of substrates to control several anabolic and catabolic processes that determine cell size and proliferation. As a central controller of cell growth and proliferation, mTOR plays a key role in regulating development, whereas dysregulation of mTOR signaling has been linked to aging and diseases such as cancer and metabolic disorders
Heraclitus on Apollo's signs and his own: contemplating oracles and philosophical inquiry
Full text linkThis chapter will examine one of Heraclitus’ most arresting theological pronouncements (B93): The lord whose oracle is the one in Delphi neither says nor conceals but gives a sign. What kind of theology and theologising are at play in this abstracted and generalised reflection about Delphic Apollo and how does this reflection relate to and engage with the more implicit theological conceptions of Apollo conveyed in the Delphic traditions (as we find them for example in Herodotus)? What exactly is Heraclitus saying about Apollo's mode of communication and what is he implying thereby about his own? If Heraclitus is appropriating here the theology of Apolline divination as a framework within which to understand his own philosophical inquiry, why does he do so, what is involved in this appropriation and what does Apolline philosophical inquiry look like? Scholarly discussions of B93 share the mostly implicit assumption that what Heraclitus says about Apollo is in itself pretty much clear and unproblematic. Commentators take it that interpretive difficulties and dilemmas arise once we come to ask what, if anything, the comment implies about Heraclitus himself or about anything else. I will argue that Heraclitus’ comment about Apollo is pointedly difficult and paradoxical, not only in its implicit ramifications, but already on the most rudimentary and literal level of its interpretation. We are meant to be puzzled by what Heraclitus says about Apollo and it is an interpretive failure on our part if we are not so puzzled. I will suggest that, consequently, Heraclitus’ encapsulation of Apollo's modus operandi is not an unchallenging assertion of a widely familiar point but, on the most rudimentary level of its interpretation, a case of creative, involved and unobvious theological abstraction. Arguing further that Apollo does indeed serve in B93 as a paradigm of emulation for Heraclitus, I will ask how the paradoxical nature of Heraclitus’ theological remark bears on his understanding of his own use of language (and on other, related aspects of his thought) and examine the motivations, scope and limits of the implicit analogy in B93 between god (Apollo) and mortal (Heraclitus).</p
Honorary Members of the Polish Society of Nephrology. Part X — Professor Shaul G. Massry
Full text linkNiniejsza publikacja jest już dziesiątą częścią serii Członkowie Honorowi Polskiego Towarzystwa Nefrologicznego, którą autorzy przedstawiają na gościnnych łamach „Forum Nefrologicznego” od początku 2013 roku. Warto przypomnieć, że dotychczas zaprezentowano życiorysy dziewięciu Członków Honorowych, z czego sześciu pochodzi z Polski, a trzech z Niemiec, Stanów Zjednoczonych i Francji. Pierwszą postacią był prof. Tadeusz Orłowski, a w ostatnim numerze zaprezentowano postać niespodziewanie i przedwcześnie zmarłego prof. Olgierda Smoleńskiego. W obecnym numerze przyszedł czas na przedstawienie postaci Profesora Shaula Massry, znanego na całym świecie internisty i nefrologa, autora setek oryginalnych publikacji i wydań książkowych, członka honorowego wielu narodowych towarzystw naukowych, doktora honoris causa licznych uczelni medycznych na wielu kontynentach, redaktora wielu renomowanych czasopism medycznych, zwłaszcza nefrologicznych. Profesor Shaul Massry wielokrotnie przebywał w Polsce na zaproszenie wielu uczelni medycznych i Polskiej Akademii Nauk, biorąc udział w konferencjach naukowych. W klinice Profesora w Stanach Zjednoczonych przebywali też na stażach naukowych liczni młodzi polscy lekarze. Między innymi z tego powodu można Profesora nazwać przyjacielem Polski i polskich lekarzy.This is the tenth publication in the series of the Honorary Members of the Polish Society of Nephrology which has been kindly hosted by Forum Nefrologiczne since the early 2013. The nine Honorary Members presented so far have included six figures from Poland and three from Germany, the USA and France. The series was opened with the biographical sketch of prof. Tadeusz Orłowski and its latest part depicted prof. Olgierd Smoleński. This issue offers a chance to present the figure of Professor Shaul Massry, the world-famous internist and nephrologist, the author of hundreds of original publications and books, the honorary member of numerous national scientific societies, the doctor honoris causa of many medical universities on various continents, the editor of many renowned medical journals, especially in the field of nephrology. Having been invited by many medical universities as well as the Polish Academy of Sciences Professor Shaul Massry was frequent visitor in Poland participating in many scientific conferences. His clinic hosted many young Polish doctors as trainees. For these reasons and others not mentioned here it is justified to call Shaul Massry a friend of Poland and Polish doctors
Language and doctrine in Parmenides' Way of Reality
Full text linkAs early as Plato and as recently as current scholarship, readers of Parmenides have diagnosed tensions of one sort or another between his ontological views and the language through which he expresses those views. In the first instance, this article examines earlier claims for such tensions and argues that they are predicated on problematic assumptions concerning Parmenides’ ontological commitments or his strictures regarding the use of language. In the second instance, however, it argues that Parmenides’ Way of Reality does indeed confront us with tensions between language and doctrine, that these tensions are more pointed and sustained than scholars generally recognize and that they can be identified independently of specific or determinate elaboration of Parmenides’ precise ontological views. This analysis discloses a reflective preoccupation with, and a consistent attitude towards, the scope and limitations of human language. Parmenides persistently evinces his awareness that his description of what-is proceeds through expressive measures that are imported with difficulty from a different domain and, consequently, are limited, indirect and often figurative. The article closes by pointing to a meaningful (but partial) affinity between Parmenides and those Platonists who placed their own ultimate philosophical and ontological principle beyond the expressive reach of words
Religious Experience - (Y.) Ustinova Caves and the Ancient Greek Mind. Descending Underground in the Search for Ultimate Truth. Pp. xii + 315. Oxford: Oxford University Press, 2009. Cased, £50. ISBN: 978-0-19-954856-9.
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Ancient philosophy within a global purview: Parmenides and Zhuangzi on expressing what can (and cannot) be known
No full textThis Thesis argues that widespread misconceptions about the early history of philosophy hamper engagement with ancient philosophy within a global purview. I identify and challenge an influential narrative according to which philosophy emerges through a shift from mythos to logos (Chapter 1). This framework misrepresents the history of ancient Greek philosophy and simultaneously underpins the perceived illegitimacy of ‘non-Western’ traditions as philosophical traditions. I examine how this same narrative creates major interpretive blind spots within specialist work on ancient philosophy (Chapters 2–5). I do so by exploring two consequential cases, two foundational philosophers in early Greek and classical Chinese philosophy, respectively: Parmenides and Zhuangzi.
I show how problematic assumptions inherited from mainstream macro-narratives prime us to see Parmenides as exhibiting thoroughly optimistic attitudes toward human knowledge, and Zhuangzi as either an anti-rational mystic who dismisses language or as a negative dogmatist who rejects objective knowledge (Chapter 2). I demonstrate that adopting a dialogical approach to specific questions in epistemology as they arise in Parmenides and Zhuangzi helps substantiate alternative interpretive routes which have been obscured by received historiographical frameworks. I bring Zhuangzi and Parmenides in dialogue on the issue of the limits of human cognition (Chapter 3); on the potential of analogies and indirect argumentation to push the boundaries of what can be known (Chapter 4); and finally, on whether numerical monism is defensible (Chapter 5).
From these inquiries, Parmenides emerges as someone quite other than the unmitigated epistemological optimist he is usually assumed to be, which raises fruitful questions about the status of his constructive claims. Conversely, Zhuangzi is not the anti-rationalist or negative dogmatist he is widely taken to be; instead, his own circumspect stance on the question of what can (and cannot) be known leads him to forgo all unqualified claims to knowledge
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