42 research outputs found
Abstract A32: The role of the ribosome in GCN2's ability to sense amino acid deprivation and initiate the integrated stress response
No full textAbstract
The ability to replicate without limit is one of the traditional hallmarks of cancer. It is therefore evident that the majority of tumor cells are operating in environments with limited nutrients. This means that tumor cells are disproportionately reliant on their stress response pathways in comparison to healthy cells. One such stress response pathway is a component of the integrated stress response pathway that responds to deprivation of amino acids. GCN2 is a serine/threonine protein kinase that is activated in response to a drop in the intracellular concentration of amino acids. Upon activation, GCN2 phosphorylates the translational initiation factor eIF2, inhibiting its activity in the translational cycle. This leads to a global inhibition of translation, whilst paradoxically upregulating the expression of certain genes involved in stress responses. It has been shown that knocking out GCN2 leads to a significant reduction in the growth of tumors in mice, suggesting that GCN2 could be a useful therapeutic target.
We have expressed and purified human GCN2 and found it to be a dimer, as has been observed for yeast GCN2. We have shown in vitro that GCN2 becomes autophosphorylated, and it catalyses phosphorylation of a putative binding partner GCN1 as well as eIF2. We have found that human GCN2 forms a stable complex with 80S ribosomes, and mammalian 80S ribosomes significantly increase GCN2 kinase activity in vitro, consistent with a previous report that an increase in ribosomal stalling leads to a rise in eIF2 phosphorylation in vivo. We are attempting to determine the structure of GCN2 bound to ribosomes in order to understand the detailed interactions and the molecular mechanisms that lead to GCN2 activation.
Citation Format: Alison J. Inglis, Sichen Shao, Glenn R. Masson, Olga Perisic, Ramanujan S. Hegde, Roger L. Williams. The role of the ribosome in GCN2's ability to sense amino acid deprivation and initiate the integrated stress response. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A32.</jats:p
Protease-activated kinase II as the mediator of epidermal growth factor-stimulated phosphorylation of ribosomal protein S6
No full textAbstractEpidermal growth factor stimulates phosphorylation of ribosomal protein S6 in serum-starved Swiss 3T3 cells, leading to the formation of highly phosphorylated derivatives containing 4–5 phosphates. Two-dimensional analysis of tryptic phosphopeptides of S6 shows an identical pattern to the ones obtained previously in other cells in response to insulin and the tumor promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate. This suggests a common intracellular mediator of S6 phosphorylation by different growth promoting agents. It is proposed that the potential mediator of this phosphorylation is the Ca2+-independent, cAMP-independent protein kinase, protease activated kinase II, as shown by the extent of phosphorylation and the tryptic phosphopeptide maps of S6 with highly purified enzyme [(1983) J. Biol. Chem. 258, 13998-14002]
Pretrial detention and alternate methods of securing the presence of the accused during the criminal procedure
No full textIn this paper, the author discusses pretrial detention and alternate methods of securing the presence of the accused during the criminal procedure. Except issues of terminology, in the introduction to this paper, the author deals with the current status of our criminal justice system, the readiness of both the state and the criminal justice system to accept innovations. Further, the author examines the accordance of pretrial detention with international sources, then its correlations with the Constitution, Criminal Procedure Codes from 2001 and 2006, and types of pretrial detention. As the paper continues, it describes institutes that are alternatives of pretrial detention: home detention and other restriction orders, and bail. The paper follows with the definition and content of the reasonable doubt concept as a mandatory condition for pronouncing pretrial detention. The major part of the paper deals is dedicated to the discussion of legal regulations of pretrial detention and its misinterpretation by judicial apparatus, and the effort to explain some undefined parts of those regulations
OVERCOMING EMPTINESS IN THE TRANSLATION OF DUBRAVKA UGREŠIĆ
No full textThe theme of this paper concerns the main aspects of translating a linguistically and culturally hybrid text, using the example of two Dubravka Ugrešić’s books (1949–2023), Karaoke culture and Europe in Sepia. The author addresses contemporary themes in a globalised and increasingly virtual world, while constantly revisiting the Soviet and Yugoslav communist past through her personal experiences. Existing between languages and interpreting such a diverse literary world requires a profound understanding of historical, linguistic, and cultural contexts, as well as translation strategies aimed at bridging the inevitable spatiotemporal gap that arises between the two versions of the text. The main research questions are: what are the criteria for including or excluding different elements of heterolinguism/ plurilingualism in a translated text? By what linguistic means is it domesticated in a culturally and linguistically hybrid text
ESCRT-II, an Endosome-Associated Complex Required for Protein Sorting Crystal Structure and Interactions with ESCRT-III and Membranes
No full textAbstractESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 Å structure of the yeast ESCRT-II core presented here reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a “winged helix” domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. We show that purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro
Crystal structure of a mammalian phosphoinositide-specific phospholipase C
No full textMammalian phosphoinositide-specific phospholipase C enzymes (PI-PLC) act as signal transducers that generate two second messengers, inositol-l,4,5-trisphosphate and diacylglycerol. The 2.4-Å structure of phospholipase Cδ1 reveals a multidomain protein incorporating modules shared by many signalling proteins. The structure suggests a mechanism for membrane attachment and Ca2+ -dependent hydrolysis of second-messenger precursors. The regulation and reversible membrane association of PI-PLC may serve as a model for understanding other multidomain enzymes involved in phospholipid signalling
Dynamics of the Phosphoinositide 3-Kinase p110δ Interaction with p85α and Membranes Reveals Aspects of Regulation Distinct from p110α
No full textSummaryPhosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes
The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity
No full textPhosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid and protein phosphatase, and both activities are necessary for its role as a tumour suppressor. PTEN activity is controlled by phosphorylation of its intrinsically disordered C-terminal tail. A recently discovered variant of PTEN, PTEN-long (PTEN-L), has a 173-residue N-terminal extension that causes PTEN-L to exhibit unique behaviour, such as movement from one cell to another. Using hydrogen/deuterium exchange mass spectrometry (HDX–MS) and biophysical assays, we show that both the N-terminal extension of PTEN-L and C-terminal tail of PTEN affect the phosphatase activity using unique mechanisms. Phosphorylation of six residues in the C-terminal tail of PTEN results in auto-inhibitory interactions with the phosphatase and C2 domains, effectively blocking both the active site and the membrane-binding interface of PTEN. Partially dephosphorylating PTEN on pThr366/pSer370 results in sufficient exposure of the active site to allow a selective activation for soluble substrates. Using HDX–MS, we identified a membrane-binding element in the N-terminal extension of PTEN-L, termed the membrane-binding helix (MBH). The MBH radically alters the membrane binding mechanism of PTEN-L compared with PTEN, switching PTEN-L to a ‘scooting ’ mode of catalysis from the ‘hopping ’ mode that is characteristic of PTEN. Key words: hydrogen/deuterium exchange mass spectrometry (HDX–MS), interfacial catalysis, intrinsically disordered protein regions, phosphatase and tensin homologue deleted on chromosome 10 (PTEN), (PTEN-L)
