Inner hair cell dysfunction in Klhl18 mutant mice leads to low frequency progressive hearing loss.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

Hsp90 interaction networks in fungi-tools and techniques.

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.

Identification and Phenotypic Characterization of Hsp90 Phosphorylation Sites That Modulate Virulence Traits in the Major Human Fungal Pathogen Candida albicans.

The highly conserved, ubiquitous molecular chaperone Hsp90 is a key regulator of cellular proteostasis and environmental stress responses. In human pathogenic fungi, which kill more than 1.6 million patients each year worldwide, Hsp90 governs cellular morphogenesis, drug resistance, and virulence. Yet, our understanding of the regulatory mechanisms governing fungal Hsp90 function remains sparse. Post-translational modifications are powerful components of nature's toolbox to regulate protein abundance and function. Phosphorylation in particular is critical in many cellular signaling pathways and errant phosphorylation can have dire consequences for the cell. In the case of Hsp90, phosphorylation affects its stability and governs its interactions with co-chaperones and clients. Thereby modulating the cell's ability to cope with environmental stress. Candida albicans, one of the leading human fungal pathogens, causes ~750,000 life-threatening invasive infections worldwide with unacceptably high mortality rates. Yet, it remains unknown if and how Hsp90 phosphorylation affects C. albicans virulence traits. Here, we show that phosphorylation of Hsp90 is critical for expression of virulence traits. We combined proteomics, molecular evolution analyses and structural modeling with molecular biology to characterize the role of Hsp90 phosphorylation in this non-model pathogen. We demonstrated that phosphorylation negatively affects key virulence traits, such as the thermal stress response, morphogenesis, and drug susceptibility. Our results provide the first record of a specific Hsp90 phosphorylation site acting as modulator of fungal virulence. Post-translational modifications of Hsp90 could prove valuable in future exploitations as antifungal drug targets.