A Stress-Induced Pathway for the Degradation of Misfolded Proteins in S. cerevisiae

The integrity of the proteome is fundamental for cell viability. Proteins can misfold due to genetic mutations or environmental stress. These misfolded proteins have a high tendency to accumulate as toxic protein aggregates which are associated with several well-known pathologies like Alzheimer’s, Huntington’s or Parkinson’s disease. To prevent protein misfolding, cells have evolved several protein quality control mechanisms that monitor and preserve the integrity of the proteome. In this PhD thesis we have uncovered and characterized a stress-inducible protein degradation pathway in budding yeast (Saccharomyces cerevisiae) that targets misfolded but also native proteins in the cytosol and the endoplasmic reticulum (ER) membrane for degradation. We employed an ER membrane-anchored reporter protein harbouring a misfolded cytosolic domain that was selectively degraded by the proteasome under stress conditions. A genetic screen, performed prior to the start of the PhD project, found the ubiquitin E3 ligase Ubr1, the serine protease Ynm3 and an uncharacterized protein (Yjl144W, named Roq1 by us) to be required for the stressinduced degradation of this misfolded model substrate. The three identified proteins act together in novel a linear protein degradation pathway, which we termed Stressinduced Homeostatically Regulated Degradation (SHRED). Mechanistic analysis elucidated that the ROQ1 gene is transcriptionally upregulated during various stresses. The resulting Roq1 protein is cleaved by Ynm3, which uncovers a positively charged arginine residue on its N-terminus. Subsequently, cleaved Roq1 through its new Nterminus interacts with Ubr1 and modulates its substrate specificity. Modified substrate recognition by Ubr1 enhances the proteasomal degradation of certain cytosolic and ER membrane proteins. Furthermore, a genetic screen and mass spectrometry analysis revealed endogenous candidate substrates of SHRED proposing that this pathway is not only implicated in quality control but also in quantity control of proteins

The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved

Examination of the Virulence of <i>Actinobacillus pleuropneumoniae</i> Serovar 16 in Pigs

Different virulence variants of A. pleuropneumoniae are involved in the etiology of porcine pleuropneumonia. The purpose of the present trial was examination of the virulence of the Actinobacillus pleuropneumoniae A-85/14 strain, the type strain of serovar 16, in an animal challenge experiment. Thirty 12-week-old piglets seronegative for A. pleuropneumoniae were allocated into three trial groups each of 10 animals, and they were infected intranasally with 106, 107, or 108 colony forming units (cfu) of the strain, respectively. Clinical signs were recorded twice a day, and the animals were euthanized 6 days after the infection. Typical clinical signs and postmortem lesions of porcine pleuropneumonia were seen in the animals of each trial group; however, they were generally mild, and no significant differences could be seen between the three groups. Even 106 colony forming units of A. pleuropneumoniae A-85/14 strain could induce clinical signs and lesions. Based on these results, the type strain of serovar 16 of A. pleuropneumoniae must be regarded as a typical pathogenic strain of the species

DataSheet1_The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.pdf

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved. </p

Table2_The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.xlsx

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved. </p

Table1_The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae.xlsx

Prolonged cell cycle arrests occur naturally in differentiated cells and in response to various stresses such as nutrient deprivation or treatment with chemotherapeutic agents. Whether and how cells survive prolonged cell cycle arrests is not clear. Here, we used S. cerevisiae to compare physiological cell cycle arrests and genetically induced arrests in G1-, meta- and anaphase. Prolonged cell cycle arrest led to growth attenuation in all studied conditions, coincided with activation of the Environmental Stress Response (ESR) and with a reduced ribosome content as determined by whole ribosome purification and TMT mass spectrometry. Suppression of the ESR through hyperactivation of the Ras/PKA pathway reduced cell viability during prolonged arrests, demonstrating a cytoprotective role of the ESR. Attenuation of cell growth and activation of stress induced signaling pathways also occur in arrested human cell lines, raising the possibility that the response to prolonged cell cycle arrest is conserved. </p

nucGEMs probe the biophysical properties of the nucleoplasm

Abstract The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeast and in mammalian cell lines. We found differences in particle motility between the nucleus and the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm