Browsing by Author "Schiemann AH"

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    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.
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    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.
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    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.
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    Pathogenicity assessment of seven RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia in the Netherlands.
    (Elsevier Ltd on behalf of British Journal of Anaesthesia, 2025-01-30) van den Bersselaar LR; Schiemann AH; Yang C-Y; Voermans NC; Malagon I; Scheffer G-J; Bjorksten AR; Gillies R; Hellblom A; Kamsteeg E-J; Snoeck MMJ; Stowell KM; Hemmings HC
    Background Malignant hyperthermia (MH) susceptibility is associated with variants in RYR1, the gene encoding the skeletal muscle ryanodine receptor-1 (RyR1), in 70–75% of patients. Functional characterisation demonstrating an increased sensitivity to RyR1 agonists is necessary among other criteria for inclusion in the European Malignant Hyperthermia Group list of MH susceptibility diagnostic variants. Methods Seven variants in the RYR1 gene, p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys, identified in MH-susceptible individuals were introduced into the cDNA for the human RYR1 gene. These variants were tested in cultured human embryonic kidney HEK293 cells for their effect on calcium release in response to the RyR1 agonist 4-chloro-m-cresol. Calcium release of each variant was compared with wild-type and benign and pathogenic controls. Each variant was subjected to curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 Variant Curation Expert Panel guidelines. Results Six of seven RYR1 variants (p.Glu342Lys, p.Leu2288Ser, p.Phe2340Leu, p.Arg2676Trp, p.Val3324Ala, p.Phe4076Leu) showed hypersensitivity to 4-chloro-m-cresol compared with wild-type. The p.Trp5020Cys variant did not release calcium in response to 4-chloro-m-cresol. All variants had minor allele frequencies <0.1%. Rare exome variant ensemble learner scores of p.Glu342Lys, p.Leu2288Ser, p.Phe4076Leu, and p.Trp5020Cys were >0.85, supporting pathogenicity. Conclusions The variants p.Glu342Lys, p.Leu2288Ser p.Phe2340Leu, and p.Arg2676Trp are pathogenic or likely pathogenic for MH and can be used for presymptomatic testing for MH susceptibility. As current knowledge on the p.Val3324Ala, p.Phe4076Leu, and p.Trp5020Cys variants remains insufficient, they are still classified as variants of uncertain significance.
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    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.