In the budding yeast Saccharomyces cerevisiae the protein phosphatase Sit4 and four associated proteins (Sap4, Sap155, Sap185, and Sap190) mediate G(1) to S cell cycle progression and a number of signaling events controlled by the target of rapamycin TOR signaling cascade. Sit4 and the Sap proteins are ubiquitously conserved and their human orthologs, PP6 and three PP6R proteins, share significant sequence identity with their yeast counterparts. However, relatively little is known about the functions of the PP6 and PP6R proteins in mammalian cells. Here we demonstrate that the human PP6R proteins physically interact with Sit4 when expressed in yeast cells. Remarkably, expression of PP6R2 and PP6R3 but not expression of PP6R1 rescues the growth defect and rapamycin hypersensitivity of yeast cells lacking all four Saps, and these effects require Sit4. Moreover, PP6R2 and PP6R3 enhance cyclin G(1) gene expression and DNA synthesis, and partially abrogate the G(1) cell cycle delay and the budding defect of the yeast quadruple sap mutant strain. In contrast, the human PP6R proteins only modestly support nitrogen catabolite gene expression and are unable to restore normal levels of eIF2alpha phosphorylation in the quadruple sap mutant strain. These results illustrate that the human PP6-associated proteins are capable of providing distinct rapamycin-sensitive and Sit4-dependent Sap functions in the heterologous context of the yeast cell. We hypothesize that the human Saps may play analogous roles in mTORC1-PP6 signaling events in metazoans.

Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH).

Candida auris is a pathogen of urgent threat level as marked by the CDC. The formation of biofilms is an essential property of this fungus to establish infection and escape drug treatment. However, our understanding of pathogenesis through biofilm is hampered by heterogeneity in C. auris biofilms observed in different studies. It is imperative to replicate in vivo conditions for studying C. auris biofilm formation in vitro. Different methods are standardized, but the surface used to form biofilms lacks consistency as well as the architecture of a typical biofilm. Here, we report an in vitro technique to grow C. auris biofilms on gelatin-coated coverslips. Interestingly, C. auris cells grown on gelatin-coated coverslips either on modified synthetic sweat media or RPMI 1640 resulted in similar multilayer biofilm formation with extracellular polymeric substances (EPS). This method is also consistent with the biofilm formation of other Candida species, such as Candida glabrata and Candida albicans. Biofilms of C. glabrata developed through this method show pseudohyphae and EPS. This method can be used to understand the molecular basis of biofilm formation, associated pathogenesis, and drug tolerance. The technique is cost-effective and would thus serve in rightful screening and repurposing drug libraries for designing new therapeutics against the less-studied high-alarm pathogen C. auris. IMPORTANCE Heterogeneity is seen when multidrug-resistant C. auris biofilm is cultured using different reported methods. Biofilm formed on the gelatin surface mimics the condition of a host environment that has multilayers and EPS. This method has feasibility for drug screening and analyzing biofilms through three-dimensional (3D) reconstruction. This in vitro biofilm formation technique is also exploited to study the formation of biofilm of other Candida species. The biofilms of C. glabrata and C. albicans can also be correctly mimicked using gelatin in the biofilm-forming environment. Thus, the novel in vitro method for biofilm formation reported here can be widely used to understand the mechanism of biofilm formation, related virulence properties, and drug tolerance of C. auris and other Candida species. This simple and low-cost technique is highly suitable for screening novel inhibitors and repurposed libraries and to design new therapeutics against Candida species.