TAGC 2020
TAGC 2020
Matthew Berg(1), Yanrui Zhu(1), Bianca Ruiz(2), Joshua Isaacson(1), Julie Genereaux(1), Raphael Loll-Krippleber(3), Bryan-Joseph San Luis(3), Charles Boone(3), Grant Brown(3), Judit Villen(2), Christopher Brandl(1)
1) Department of Biochemistry, University of Western Ontario, London, ON, Canada; 2) Department of Genome Sciences, University of Washington, Seattle, WA, USA; 3) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada.
Life does not require a perfectly accurate proteome. In fact, errors occur at a rate of one mis-incorporated amino acid in every 104 to 105 codons. Mistranslation, or the mis-incorporation of an amino acid that differs from what is specified by the “standard” genetic code, can also occur due to mutations in the translation machinery. Cells therefore have mechanisms to cope with the resulting errors in protein folding and aggregation. Defects in these pathways may contribute to disease due to a loss of proteostasis. Our goal was to examine how different types of mistranslation affect cells. Using three tRNA variants that mistranslate the genetic code, we investigated genetic interactions and effects of mistranslation on the proteome in Saccharomyces cerevisiae. The tRNA variants mistranslate alanine at proline codons, serine at proline codons or serine at arginine codons with frequencies of 2.9%, 4.7% and 2.8% respectively. The alanine at proline and serine at arginine mistranslating tRNAs cause ~10% increase in doubling time as measured by growth in liquid media, while the more severe serine at proline mistranslating tRNA causes ~20% increase. All mistranslating tRNAs induce a heat shock response. Synthetic genetic array analysis of the tRNAs against the yeast temperature sensitive collection revealed that all the tRNAs had negative genetic interactions with genes involved in protein folding. Interestingly, however, we found distinct differences in the genetic interactions of each tRNA. Similarly, proteome analysis using mass spectrometry identified different subsets of up and down regulated proteins, depending on the type of mistranslation. We conclude that while protein quality control mechanisms are required for all types of mistranslation, the specific amino acid substitutions effect cells in different ways. We previously found variants in human tRNAs that have the potential to mistranslate. Based on the unique genetic and proteomic responses observed for different mistranslating tRNAs, we believe that in addition to exacerbating diseases caused by protein mis-folding, naturally occurring mistranslating tRNAs have the potential to negatively influence a wider range of diseases, depending on the specific amino acid substitution caused by the mistranslation.
Malene Urbanus(1), Harley Mount(1), Dylan Valleau(1), Eleanor Latomanski(4), Frederick Roth(1,3), Hayley Newton(4), Alexei Savchenko(2), Alexander Ensminger(1)
1) University of Toronto, Toronto, ON Canada; 2) University of Calgary, Calgary, AB Canada; 3) Lunenfeld-Tanenbaum Research Institute, Mt. Sinai Hospital, Toronto, ON Canada; 4) University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC Australia.
A central pillar of molecular pathogenesis is that bacteria inject proteins (effectors) into the
host cell in order to modulate host proteins. An extreme example of this comes from Legionella pneumophila, an intracellular bacterial pathogen that injects over 300 proteins into the host cell during infection. Using yeast as a genetically tractable proxy for the eukaryotic host, we recently uncovered a novel class of effectors in Legionella pneumophila: “metaeffectors” (or “effectors of effectors”) that break the rules by targeting other effectors rather than host proteins. This is a dimension of microbial biology that was not widely considered prior to our work and may be one explanation for the large size of L. pneumophila’s effector arsenal. Having first established metaeffector-discovery methodologies in L. pneumophila (300+ effectors), we have recently extended our search to other bacterial pathogens (Coxiella, Chlamydia, Yersinia) with promising results.
We will present our data resulting from two systems-level approaches to identifying metaeffectors and other effector-effector interactions. In the first ever comprehensive screen for metaeffectors, we used yeast to screen all 112,000 possible pairwise genetic interactions between a library of 330×330 L. pneumophila effectors. We identified all previously described instances of effector-effector antagonism as well as nine previously unknown metaeffectors. In a second approach, we have modified a high-throughput protein-protein interaction assay (BFG-Y2H) to directly measure all possible pairwise physical interactions between effectors. These studies have already informed several detailed structure-function studies of previously uncharacterized effectors, revealing new activities against the host and a diversity of effector regulatory mechanisms. These discovery pipelines are now primed to extend this work to a variety of other pathogens.
Charles Hoffman(1), Jeremy Eberhard(1), Sheng Xiang Huang(1), Juliane Dessalines(1), Nicholas Ollila(1), Harrison Silva(1), Patricia Dranchak(2), James Inglese(2)
1) Boston College, Chestnut Hill, MA; 2) National Center for Advancing Translational Sciences, NIH, Rockville, MD.
The fission yeast Schizosaccharomyces pombe is an ideal host for high throughput screens (HTSs) to identify inhibitors of heterologously-expressed mammalian proteins involved in cyclic nucleotide metabolism. This is due to the following: 1) PKA is not essential in S. pombe, 2) PKA activity dramatically affects growth, mating and transcription of the fbp1 gene, for which several reporters have been developed, 3) phenotypic screens in S. pombe are inexpensive and readily identify compounds that are both active against the target protein and are permeable to mammalian cells, 4) target identification is relatively straightforward using strains that express the fission yeast homolog of the target protein. Our previous HTSs using S. pombe strains expressing mammalian phosphodiesterases, identified compounds that are biologically active in mammalian cell culture and can be used as tool compounds or lead compounds for drug development. We have now successful deployed both GFP- and luciferase-based screens for mammalian adenylyl cyclase (AC) inhibitors using S. pombe strains that express mammalian ACs together with a mutationally-activated human GNAS GS protein [1]. These screens were carried out in 1536-well microtiter dishes, allowing for quantitative HTSs in which compounds are tested at multiple concentrations. By recording GFP signals with an Acumen laser cytometer, we avoided the significant background generated by soluble fluorescent compounds. Successful screens of 100,000 to 125,000 compounds (the NCATS Genesis library) were completed with strains expressing human AC4, AC7, and AC9. This library is composed of collections of molecules with shared scaffolds to aid in the identification of lead compounds that are amenable to medicinal chemistry to develop more effective and drug-like molecules. Follow-up assays of cAMP production in response to compound treatment have identified two functional scaffolds that are likely to act as direct inhibitors of mammalian transmembrane ACs. Given the challenge of using biochemical approaches to inhibitor development for these integral membrane proteins, this work represents a significant break-through in the discovery of AC inhibitors that are likely to be effective in mammalian cells.
[1] Getz, R.A. et al. Cell Signal, 2019. 60: p. 114-121.
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