A Bacterial Quorum Sensing Molecule Elicits a General Stress Response in Saccharomyces cerevisiae.

Bacteria assess their population density through a chemical communication mechanism termed quorum sensing, in order to coordinate group behavior. Most research on quorum sensing has focused primarily on its role as an intraspecies chemical signaling mechanism that enables the regulation of certain phenotypes through targeted gene expression. However, in recent years several seminal studies have revealed important phenomena in which quorum sensing molecules appear to serve additional roles as interspecies signals that may regulate microbial ecology. In this study, we asked whether the budding yeast Saccharomyces cerevisiae can sense chemical signals from prokaryotes. When exposed to a variety of quorum sensing molecules from different bacterial species and from Candida albicans we found that N-(3-oxododecanoyl)-L-homoserine lactone (C12) from the opportunistic human pathogen Pseudomonas aeruginosa induces a remarkable stress response in yeast. Microarray experiments confirmed and aided in interpreting these findings, showing a unique and specific expression pattern that differed significantly from the response to previously described stress factors. We further characterized this response and report preliminary findings on the molecular basis for the recognition of C12 by the yeast.

Human SIRT1 Multispecificity Is Modulated by Active-Site Vicinity Substitutions during Natural Evolution.

Many enzymes that catalyze protein post-translational modifications can specifically modify multiple target proteins. However, little is known regarding the molecular basis and evolution of multispecificity in these enzymes. Here, we used a combined bioinformatics and experimental approaches to investigate the evolution of multispecificity in the sirtuin-1 (SIRT1) deacetylase. Guided by bioinformatics analysis of SIRT1 orthologs and substrates, we identified and examined important amino acid substitutions that have occurred during the evolution of sirtuins in Metazoa and Fungi. We found that mutation of human SIRT1 at these positions, based on sirtuin orthologs from Fungi, could alter its substrate specificity. These substitutions lead to reduced activity toward K382 acetylated p53 protein, which is only present in Metazoa, without affecting the high activity toward the conserved histone substrates. Results from ancestral sequence reconstruction are consistent with a model in which ancestral sirtuin proteins exhibited multispecificity, suggesting that the multispecificity of some metazoan sirtuins, such as hSIRT1, could be a relatively ancient trait.

Marker-free genetic manipulations in yeast using CRISPR/CAS9 system.

The budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applications. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.

Directed evolution of SIRT6 for improved deacylation and glucose homeostasis maintenance.

Mammalian SIRT6 is a well-studied histone deacetylase that was recently shown to exhibit high protein deacylation activity enabling the removal of long chain fatty acyl groups from proteins. SIRT6 was shown to play key roles in cellular homeostasis by regulating a variety of cellular processes including DNA repair and glucose metabolism. However, the link between SIRT6 enzymatic activities and its cellular functions is not clear. Here, we utilized a directed enzyme evolution approach to generate SIRT6 mutants with improved deacylation activity. We found that while two mutants show increased deacylation activity at high substrate concentration and improved glucose metabolism they exhibit no improvement and even abolished deacetylation activity on H3K9Ac and H3K56Ac in cells. Our results demonstrate the separation of function between SIRT6 catalytic activities and suggest that SIRT6 deacylation activity in cells is important for glucose metabolism and can be mediated by still unknown acylated cellular proteins.

The evolution of a G1/S transcriptional network in yeasts.

The G1-to-S cell cycle transition is promoted by the periodic expression of a large set of genes. In Saccharomyces cerevisiae G1/S gene expression is regulated by two transcription factor (TF) complexes, the MBF and SBF, which bind to specific DNA sequences, the MCB and SCB, respectively. Despite extensive research little is known regarding the evolution of the G1/S transcription regulation including the co-evolution of the DNA binding domains with their respective DNA binding sequences. We have recently examined the co-evolution of the G1/S TF specificity through the systematic generation and examination of chimeric Mbp1/Swi4 TFs containing different orthologue DNA binding domains in S. cerevisiae (Hendler et al. in PLoS Genet 13:e1006778. doi: 10.1371/journal.pgen.1006778 , 2017). Here, we review the co-evolution of G1/S transcriptional network and discuss the evolutionary dynamics and specificity of the MBF-MCB and SBF-SCB interactions in different fungal species.

Gene duplication and co-evolution of G1/S transcription factor specificity in fungi are essential for optimizing cell fitness.

Transcriptional regulatory networks play a central role in optimizing cell survival. How DNA binding domains and cis-regulatory DNA binding sequences have co-evolved to allow the expansion of transcriptional networks and how this contributes to cellular fitness remains unclear. Here we experimentally explore how the complex G1/S transcriptional network evolved in the budding yeast Saccharomyces cerevisiae by examining different chimeric transcription factor (TF) complexes. Over 200 G1/S genes are regulated by either one of the two TF complexes, SBF and MBF, which bind to specific DNA binding sequences, SCB and MCB, respectively. The difference in size and complexity of the G1/S transcriptional network across yeast species makes it well suited to investigate how TF paralogs (SBF and MBF) and DNA binding sequences (SCB and MCB) co-evolved after gene duplication to rewire and expand the network of G1/S target genes. Our data suggests that whilst SBF is the likely ancestral regulatory complex, the ancestral DNA binding element is more MCB-like. G1/S network expansion took place by both cis- and trans- co-evolutionary changes in closely related but distinct regulatory sequences. Replacement of the endogenous SBF DNA-binding domain (DBD) with that from more distantly related fungi leads to a contraction of the SBF-regulated G1/S network in budding yeast, which also correlates with increased defects in cell growth, cell size, and proliferation.

Harnessing the oxidation susceptibility of deubiquitinases for inhibition with small molecules.

Deubiquitinases (DUBs) counteract ubiquitination by removing or trimming ubiquitin chains to alter the signal. Their diverse role in biological processes and involvement in diseases have recently attracted great interest with regard to their mechanism and inhibition. It has been shown that some DUBs are regulated by reactive oxygen species (ROS) in which the catalytic Cys residue undergoes reversible oxidation, hence modulating DUBs activity under oxidative stress. Reported herein for the first time, the observation that small molecules, which are capable of generating ROS efficiently, inhibit DUBs by selective and nonreversible oxidation of the catalytic Cys residue. Interestingly, the small molecule beta-lapachone, which is currently in clinical trials for cancer, is among the potent inhibitors, thus suggesting possible new cellular targets for its therapeutic effects. Our study describes a novel mechanism of DUBs inhibition and opens new opportunities in exploiting them for cancer therapy.