No abstract

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11 Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11 Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.

The number of individuals with non‐alcoholic fatty liver disease (NAFLD) is at epidemic levels worldwide. Although thought of as benign, NAFLD can progress to more severe forms of the disease that include non‐alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and in some cases hepatocellular carcinoma. Presently no therapies exist that can treat NAFLD and/or NASH even though this area of drug discovery has been highly active. The accumulation of triglycerides and fatty acids in the liver are linked to the initiation and progression of NAFLD. Intestinal monoacylglycerol acyltransferase 2 (MGAT2) catalyzes the resynthesis of triglycerides from dietary triglyceride‐derived monoacylglycerol and fatty acids. Mice lacking mMGAT2 are resistant to obesity‐dependent hepatic steatosis, a phenotype that is partially restored by expressing hMGAT2 in the intestine, suggesting that inhibiting human MGAT2 activity may be a viable option for treating NAFLD. Here, we generated transgenic hMgat2+/+ mice and characterized their responses to being fed different metabolic diets in order to test whether this cohort could be used as a model to test drug efficacy. hMgat2+/+ mice fed a steatotic diet acquired NAFLD and had elevated levels of several inflammatory cytokines in the liver, indicating the presence of ongoing NASH. Hydroxyproline and galectin‐3 levels were highly elevated, and correlated with the degree of fibrosis seen by histology. hMgat2+/+ mice fed a high fat diet became obese, were glucose intolerant, and acquired insulin resistance, suggesting the onset of diabetes. The majority of phenotypes observed with both diets were attenuated by treatment with the PPARa/d agonist, elafibranor. Thus, hMgat2+/+ mice may be an excellent “humanized” model that can be used for testing drug efficacy targeting systemic hMGAT2 activity, and undoubtedly be invaluable in furthering our understanding of how hMGAT2 activity affects overall triglyceride metabolism.