Small molecule selectivity and specificity profiling using functional protein microarrays.

Small molecules interact with proteins to perturb their functions, a property that has been exploited both for research applications and to produce therapeutic agents for disease treatment. Commonly utilized approaches for identifying the target proteins for a small molecule have limitations in terms of throughput and resource consumption and lack a mechanism to broadly assess the selectivity profile of the small molecule. Here we describe how protein microarray technology can be applied to the study of small molecule-protein interactions using tritiated small molecules.

Cryptococcus neoformans gene expression during murine macrophage infection.

The fungal pathogen Cryptococcus neoformans survives phagocytosis by macrophages and proliferates within, ultimately establishing latent infection as a facultative intracellular pathogen that can escape macrophage control to cause disseminated disease. This process is hypothesized to be important for C. neoformans pathogenesis; however, it is poorly understood how C.

Calcium- and calcineurin-independent roles for calmodulin in Cryptococcus neoformans morphogenesis and high-temperature growth.

The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37 degrees C.

Transcriptional network of multiple capsule and melanin genes governed by the Cryptococcus neoformans cyclic AMP cascade.

Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Galpha protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Galpha protein Gpa1.

Identification of Cryptococcus neoformans temperature-regulated genes with a genomic-DNA microarray.

The ability to survive and proliferate at 37 degrees C is an essential virulence attribute of pathogenic microorganisms. A partial-genome microarray was used to profile gene expression in the human-pathogenic fungus Cryptococcus neoformans during growth at 37 degrees C. Genes with orthologs involved in stress responses were induced during growth at 37 degrees C, suggesting that a conserved transcriptional program is used by C.

Coping with stress: calmodulin and calcineurin in model and pathogenic fungi.

Calcium signaling via calmodulin and calcineurin is critical for the regulation of stress responses in fungi. The functions of calmodulin and calcineurin are largely conserved among pathogenic fungi and model fungi, however, the mechanisms of action have diverged. Saccharomyces cerevisiae is an excellent model for understanding the framework of calcium-mediated signal transduction pathways, and considerable progress has been made in understanding the details of how Ca(2+)-calmodulin and calcineurin control adaptation to environmental stress.

The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function.

Cell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S.

A PCR-based strategy to generate integrative targeting alleles with large regions of homology.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion.

Characterization of the Aspergillus nidulans 14-3-3 homologue, ArtA.

The 14-3-3 family of proteins function as small adaptors that facilitate a diverse array of cellular processes by mediating specific protein interactions. One such process is the DNA damage checkpoint, where these proteins prevent inappropriate activation of cyclin-dependent kinases. The filamentous fungus Aspergillus nidulans possesses a highly conserved 14-3-3 homologue (artA) that may function in an analogous manner to prevent septum formation.