Induction of the Unfolded Protein Response at High Temperature in Saccharomyces cerevisiae.

Ire1 is an endoplasmic reticulum (ER)-located endoribonuclease that is activated in response to ER stress. In yeast Saccharomyces cerevisiae cells, Ire1 promotes HAC1-mRNA splicing to remove the intron sequence from the HAC1u mRNA ("u" stands for "uninduced"). The resulting mRNA, which is named HAC1i mRNA ("i" stands for "induced"), is then translated into a transcription factor that is involved in the unfolded protein response (UPR). In this study, we designed an oligonucleotide primer that specifically hybridizes to the exon-joint site of the HAC1i cDNA. This primer allowed us to perform real-time reverse transcription-PCR to quantify HAC1i mRNA abundance with high sensitivity. Using this method, we detected a minor induction of HAC1-mRNA splicing in yeast cells cultured at their maximum growth temperature of 39 °C. Based on our analyses of IRE1-gene mutant strains, we propose that when yeast cells are cultured at or near their maximum growth temperature, protein folding in the ER is disturbed, leading to a minor UPR induction that supports cellular growth.

Architecture of human Rag GTPase heterodimers and their complex with mTORC1.

The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.

The Central Role of Proprotein Convertase Subtilisin/Kexin Type 9 in Septic Pathogen Lipid Transport and Clearance.

Microbial cell walls contain pathogenic lipids, including LPS in gram-negative bacteria, lipoteichoic acid in gram-positive bacteria, and phospholipomannan in fungi. These pathogen lipids are major ligands for innate immune receptors and figure prominently in triggering the septic inflammatory response. Alternatively, pathogen lipids can be cleared and inactivated, thus limiting the inflammatory response. Accordingly, biological mechanisms for sequestering and clearing pathogen lipids from the circulation have evolved. Pathogen lipids released into the circulation are initially bound by transfer proteins, notably LPS binding protein and phospholipid transfer protein, and incorporated into high-density lipoprotein particles. Next, LPS binding protein, phospholipid transfer protein, and other transfer proteins transfer these lipids to ApoB-containing lipoproteins, including low-density (LDL) and very-low-density lipoproteins and chylomicrons. Pathogen lipids within these lipoproteins and their remnants are then cleared from the circulation by the liver. Hepatic clearance involves the LDL receptor (LDLR) and possibly other receptors. Once absorbed by the liver, these lipids are then excreted in the bile. Recent evidence suggests pathogen lipid clearance can be modulated. Importantly, reduced proprotein convertase subtilisin/kexin type 9 activity increases recycling of the LDLR and thereby increases LDLR on the surface of hepatocytes, which increases clearance by the liver of pathogen lipids transported in LDL. Increased pathogen lipid clearance, which can be achieved by inhibiting proprotein convertase subtilisin/kexin type 9, may decrease the systemic inflammatory response to sepsis and improve clinical outcomes.

Immunoproteomic profiling of Saccharomyces cerevisiae systemic infection in a murine model.

Saccharomyces cerevisiae is considered a safe microorganism widely used as a dietary supplement. However, in the latest decades several cases of S. cerevisiae infections have been reported. Recent studies in a murine model of systemic infection have also revealed the virulence of some S. cerevisiae dietary strains. Here we use an immunoproteomic approach based on protein separation by 2D-PAGE followed by Western-blotting to compare the serological response against a virulent dietary and a non-virulent laboratory strains leading to the identification of highly different patterns of antigenic proteins. Thirty-six proteins that elicit a serological response in mice have been identified. Most of them are involved in stress responses and metabolic pathways. Their selectivity as putative biomarkers for S. cerevisiae infections was assessed by testing sera from S. cerevisiae-infected mice against Candida albicans and C. glabrata proteins. Some chaperones and metabolic proteins showed cross-reactivity. We also compare the S. cerevisiae immunodetected proteins with previously described C. albicans antigens. The results point to the stress-related proteins Ahp1, Yhb1 and Oye2, as well as the glutamine synthetase Gln1 and the oxysosterol binding protein Kes1 as putative candidates for being evaluated as biomarkers for diagnostic assays of S. cerevisiae infections. BIOLOGICAL SIGNIFICANCE: S. cerevisiae can cause opportunistic infections, and therefore, a precise diagnosis of fungal infections is necessary. This immunoproteomic analysis of sera from a model murine infection with a virulent dietary S. cerevisiae strain has been shown to be a source of candidate proteins for being evaluated as biomarkers to develop assays for diagnosis of S. cerevisiae infections. To our knowledge, this is the first study devoted to the identification of S. cerevisiae immunogenic proteins and the results allowed the proposal of five antigens to be further investigated.