Proper Sterol Distribution Is Required for Candida albicans Hyphal Formation and Virulence.

Candida albicans is an opportunistic fungus responsible for the majority of systemic fungal infections. Multiple factors contribute to C. albicans pathogenicity. C. albicans strains lacking CaArv1 are avirulent. Arv1 has a conserved Arv1 homology domain (AHD) that has a zinc-binding domain containing two cysteine clusters. Here, we explored the role of the CaAHD and zinc-binding motif in CaArv1-dependent virulence. Overall, we found that the CaAHD was necessary but not sufficient for cells to be virulent, whereas the zinc-binding domain was essential, as Caarv1/Caarv1 cells expressing the full-length zinc-binding domain mutants, Caarv1C3S and Caarv1C28S, were avirulent. Phenotypically, we found a direct correlation between the avirulence of Caarv1/Caarv1, Caarrv1AHD , Caarv1C3S , and Caarv1C28S cells and defects in bud site selection, septa formation and localization, and hyphal formation and elongation. Importantly, all avirulent mutant strains lacked the ability to maintain proper sterol distribution. Overall, our results have established the importance of the AHD and zinc-binding domain in fungal invasion, and have correlated an avirulent phenotype with the inability to maintain proper sterol distribution.

Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d.

AMP kinase is a heterotrimeric serine/threonine protein kinase that regulates a number of metabolic processes, including lipid biosynthesis and metabolism. AMP kinase activity is regulated by phosphorylation, and the kinases involved have been uncovered. The particular phosphatases counteracting these kinases remain elusive. Here we discovered that the protein phosphatase 2A heterotrimer, PP2A(Ppp2r2d), regulates the phosphorylation state of AMP kinase by dephosphorylating Thr-172, a residue that activates kinase activity when phosphorylated. Co-immunoprecipitation and co-localization studies indicated that PP2A(Ppp2r2d) directly interacted with AMP kinase. PP2A(Ppp2r2d) dephosphorylated Thr-172 in rat aortic and human vascular smooth muscle cells. A positive correlation existed between decreased phosphorylation, decreased acetyl-CoA carboxylase Acc1 phosphorylation, and sterol response element-binding protein 1c-dependent gene expression. PP2A(Ppp2r2d) protein expression was up-regulated in the aortas of mice fed a high fat diet, and the increased expression correlated with increased blood lipid levels. Finally, we found that the aortas of mice fed a high fat diet had decreased AMP kinase Thr-172 phosphorylation, and contained an Ampk-PP2A(Ppp2r2d) complex. Thus, PP2A(Ppp2r2d) may antagonize the aortic AMP kinase activity necessary for maintaining normal aortic lipid metabolism. Inhibiting PP2A(Ppp2r2d) or activating AMP kinase represents a potential pharmacological treatment for many lipid-related diseases.

Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake.

Dysregulation of cholesterol homeostasis is associated with various metabolic diseases, including atherosclerosis and type 2 diabetes. The sterol response element binding protein (SREBP)-2 transcription factor induces the expression of genes involved in de novo cholesterol biosynthesis and low density lipoprotein (LDL) uptake, thus it plays a crucial role in maintaining cholesterol homeostasis. Here, we found that overexpressing microRNA (miR)-185 in HepG2 cells repressed SREBP-2 expression and protein level. miR-185-directed inhibition caused decreased SREBP-2-dependent gene expression, LDL uptake, and HMG-CoA reductase activity. In addition, we found that miR-185 expression was tightly regulated by SREBP-1c, through its binding to a single sterol response element in the miR-185 promoter. Moreover, we found that miR-185 expression levels were elevated in mice fed a high-fat diet, and this increase correlated with an increase in total cholesterol level and a decrease in SREBP-2 expression and protein. Finally, we found that individuals with high cholesterol had a 5-fold increase in serum miR-185 expression compared with control individuals. Thus, miR-185 controls cholesterol homeostasis through regulating SREBP-2 expression and activity. In turn, SREBP-1c regulates miR-185 expression through a complex cholesterol-responsive feedback loop. Thus, a novel axis regulating cholesterol homeostasis exists that exploits miR-185-dependent regulation of SREBP-2 and requires SREBP-1c for function.

Non-invasive detection of a small number of bioluminescent cancer cells in vivo.

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.