Hsp90 interaction networks in fungi-tools and techniques

Abstract

Heat-shock protein 90 (Hsp90) is a central regulator of cellular proteostasis. It stabilizes numerous proteins that are involved in fundamental processes of life, including cell growth, cell-cycle progression and the environmental response. In addition to stabilizing proteins, Hsp90 governs gene expression and controls the release of cryptic genetic variation. Given its central role in evolution and development, it is important to identify proteins and genes that interact with Hsp90. This requires sophisticated genetic and biochemical tools, including extensive mutant collections, suitable epitope tags, proteomics approaches and Hsp90-specific pharmacological inhibitors for chemogenomic screens. These usually only exist in model organisms, such as the yeast Saccharomyces cerevisiae. Yet, the importance of other fungal species, such as Candida albicans and Cryptococcus neoformans, as serious human pathogens accelerated the development of genetic tools to study their virulence and stress response pathways. These tools can also be exploited to map Hsp90 interaction networks. Here, we review tools and techniques for Hsp90 network mapping available in different fungi and provide a summary of existing mapping efforts. Mapping Hsp90 networks in fungal species spanning >500 million years of evolution provides a unique vantage point, allowing tracking of the evolutionary history of eukaryotic Hsp90 networks.

Keywords: Aspergillus fumigatus; Candida; Cryptococcus neoformans; Hsp90; chemogenomics; mutant libraries; protein–protein interactions; proteomics; synthetic lethality.

Figure 1.

Phylogenetic relationships amongst fungal species with available mutant libraries. Divergence times for branches leading to C. albicans, S. cerevisiae and C. glabrata (all Saccharomycotina), A. fumigatus (Pezizomycotina) and C. neoformans (Basidiomycota) are indicated by colored circles.

Figure 2.

Synthetic lethality identifies genes acting in the same pathway or complex. Yeast cells are viable when experiencing either sub-lethal depletion of Hsp90 function or loss of function of ‘your favourite gene’ (YFG). The combination of both, however, is not tolerated and yeast cells are either ‘sick’ (reduced growth) or dead. Hsp90 function can be reduced by either pharmacological inhibition or the use of hypomorphic alleles and loss-of-function mutations can be achieved as described in the text.

Figure 3.

Candida albicans mutant libraries sizes and overlaps. Upset R-plot depicting the size of each library on the left (set size) and the overlap between different libraries on the right. The Homann library covers 166 TF gene deletions (Homann et al. 2009). The Mitchell library consists of 703 genes disrupted by transposon insertions (Davis et al. 2002). The Noble library comprises 674 clean gene deletion mutants (Noble et al. 2010). The GRACE library provides repressible mutants for 2357 genes (Roemer et al. 2003). Vertical bars represent the number of genes shared between each of the libraries, the libraries sharing these genes are indicated by the connected dots. There is little overlap in genes represented between libraries, together these libraries allow disruption of 2603 genes, covering 42% of the C. albicans genome.

Figure 4.

Set-up of the original yeast two-hybrid system for use in S. cerevisiae and its adaption to Candida two-hybrid. (A) The original yeast two-hybrid system uses LacZ as the reporter gene (Fields and Song 1989). When the Gal4 DBD-tagged bait protein interacts with the Gal4 AD-tagged prey protein, Gal4 induces the expression of LacZ via the GAL1 promoter. Colonies where bait and prey proteins interact will appear blue when grown on X-gal media. (B) The Candida two-hybrid system uses C. albicans optimized genes (Stynen, van Dijck and Tournu ; Legrand et al. ; Schoeters et al. 2018). The background strain, SC2H3, has two reporter genes, Streptococcus thermophilus LacZ and C. albicans HIS1. Each reporter gene is under the C. albicans ADH1 promoter and five copies of the Staphylococcus aureus LexA operon. The LacZ reporter cassette is integrated into chromosome 1 and the HIS1 reporter cassette is integrated into chromosome 4. When S. aureus LexA DBD-tagged bait interacts with viral VP16 AD-tagged prey, expression of LacZ and HIS1 is induced. Strains where bait and prey proteins interact will grow on histidine deficient media and have increased β-galactosidase activity, measurable via assay.