Browsing by Author "Sattlegger E"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (poster)
    (8/09/2022) Schiemann A; Gottfried S; Sattlegger E
    The General Amino Acid Control stress signalling pathway allows cells to sense and overcome starvation. One of the major players in this pathway is the protein kinase Gcn2 found in all eukaryotic cells. Activation of Gcn2 leads to phosphorylation of the alpha subunit of eukaryotic translation initiation factor (eIF2α), which then leads to the re-programming of the cell’s gene transcription and translation profile, and ultimately allowing cells to cope with and overcome starvation. For sensing starvation, Gcn2 must directly bind to its effector protein Gcn1. This interaction is mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Overexpression of the RWDBD alone impairs Gcn2 activation through disrupting endogenous Gcn1-Gcn2 interaction, hampering eIF2α phosphorylation, and consequently cells cannot overcome starvation and fail to grow.  This dominant negative phenotype is dependent on Arg-2259 in RWDBD.  Taking advantage of this phenotype in yeast, we here found that the dominant negative phenotype was reverted by each of four amino acid substitutions, K2270A, R2289A, R2297A, and K2301A.  This correlated with increased eIF2α phosphorylation levels, suggesting their relevance for Gcn2 activation.  All but Lys-2270 are fully surface exposed, suggesting that these amino acids may directly contact Gcn2.  We also found amino acid substitutions that enhanced the dominant negative phenotype of the overexpressed RWDBD, and correlated with further reduction in eIF2α-P levels.  Our findings suggest that two helices in Gcn1 constitute at least one Gcn2 contact point.
  • Thumbnail Image
    Item
    A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation (research article)
    (PLOS, 2022-11-28) Gottfried S; Koloamatangi SMBMJ; Daube C; Schiemann AH; Sattlegger E; Lustig AJ
    The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.
  • Thumbnail Image
    Item
    Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation
    (Portland Press on behalf of the Biochemical Society, 2024-03-05) Shanmugam R; Anderson R; Schiemann AH; Sattlegger E
    The protein kinase Gcn2 and its effector protein Gcn1 are part of the General Amino Acid Control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis.  Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P).  This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation.  Xrn1 is involved in RNA decay, quality control and processing.  We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex.  Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels.  Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion doesn't enhance eIF2α de-phosphorylation.  Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly.  Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.
  • Thumbnail Image
    Item
    GCN2 in Viral Defence and the Subversive Tactics Employed by Viruses
    (Elsevier Ltd, 2024-07-01) Gibbs VJ; Lin YH; Ghuge AA; Anderson RA; Schiemann AH; Conaglen L; Sansom BJM; da Silva RC; Sattlegger E; Freed EO
    The recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host's protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.
  • Thumbnail Image
    Item
    Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein
    (Elsevier Inc, 2023-03-17) Ghuge AA; Anderson RA; Gottfried S; Daube C; Koloamatangi SMBMJ; Schiemann AH; Sattlegger E
    Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.
  • Thumbnail Image
    Item
    Re-using Criterion plastic precast gel cassettes for SDS-polyacrylamide electrophoresis.
    (2021-03-16) Sattlegger E; Anderson R
    Precast gels are made with plastic cassettes which usually are discarded after use. Here we describe how Criterion plastic gel cassettes can be re-used for making SDS-PAGE gels in-house.
  • Thumbnail Image
    Item
    Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals-Illustrated with Four Actin Cytoskeleton Proteins
    (MDPI (Basel, Switzerland), 2020-03-10) Akram Z; Ahmed I; Mack H; Kaur R; Silva RC; Castilho BA; Friant S; Sattlegger E; Munn AL
    The budding yeast Saccharomyces cerevisiae has an actin cytoskeleton that comprises a set of protein components analogous to those found in the actin cytoskeletons of higher eukaryotes. Furthermore, the actin cytoskeletons of S. cerevisiae and of higher eukaryotes have some similar physiological roles. The genetic tractability of budding yeast and the availability of a stable haploid cell type facilitates the application of molecular genetic approaches to assign functions to the various actin cytoskeleton components. This has provided information that is in general complementary to that provided by studies of the equivalent proteins of higher eukaryotes and hence has enabled a more complete view of the role of these proteins. Several human functional homologues of yeast actin effectors are implicated in diseases. A better understanding of the molecular mechanisms underpinning the functions of these proteins is critical to develop improved therapeutic strategies. In this article we chose as examples four evolutionarily conserved proteins that associate with the actin cytoskeleton: 1) yeast Hof1p/mammalian PSTPIP1, 2) yeast Rvs167p/mammalian BIN1, 3) yeast eEF1A/eEF1A1 and eEF1A2 and 4) yeast Yih1p/mammalian IMPACT. We compare the knowledge on the functions of these actin cytoskeleton-associated proteins that has arisen from studies of their homologues in yeast with information that has been obtained from in vivo studies using live animals or in vitro studies using cultured animal cell lines.