Significance to metallomicsDithiol gliotoxin is a near-terminal biosynthetic intermediate from the gliotoxin biosynthetic pathway in the human pathogen Aspergillus fumigatus. Chemically reduced gliotoxin, dithiol gliotoxin (DTG), is revealed as a biological zinc chelator, and conversely, zinc can relieve the hitherto cryptic fungal autotoxicity of DTG. There is a systems-wide impact of zinc chelation by DTG on the fungal proteome, and we suggest it is DTG, as opposed to gliotoxin, which chelates zinc from metalloproteins. Since gliotoxin can be sequestered by both fungi and bacteria, our findings infer a new avenue to interfere with, and exploit, cellular zinc homeostasis in microorganisms. IntroductionGliotoxin and holomycin are microbial natural products, which are produced by fungal and bacterial spp., respectively (Fig. 1). [1][2][3] Both are low molecular mass metabolites, and each contains a disulphide bridge formed by the action of oxidoreductases, namely GliT and HlmI, on the respective dithiol precursor (Fig. 1). [4][5][6] Disulphide bridge formation is essential for microbial self-protection against these reactive dithiol intermediates, and is a pre-requisite for the Major Facilitator Superfamily transporter GliA-mediated secretion of gliotoxin by Aspergillus fumigatus. 5-8Both metabolites are also present as bis-thiomethylated forms, and gliotoxin bis-thiomethyltransferase GtmA converts dithiol gliotoxin (DTG) to bis-dethiobis(methylthio)gliotoxin (BmGT) in A. fumigatus, whereas the origin of the cognate activity against dithiol holomycin in Streptomyces clavuligeris is unknown (Fig. 1). 9,10Bernardo et al. have shown that upon uptake by eukaryotic cells,

Conflicting reports exist in the literature regarding the role of wild-type huntingtin in determining the toxicity of the aggregated, mutant huntingtin in Huntington's disease (HD). Some studies report the amelioration of toxicity of the mutant protein in the presence of the wild-type protein, while others indicate sequestration of the wild-type protein by mutant huntingtin. Over the years, yeast has been established as a valid model organism to study molecular changes associated with HD, especially at the protein level. We have used an inducible system to express human huntingtin fragments harboring normal (25Q) and pathogenic (103Q) polyglutamine lengths under the control of a galactose promoter in a yeast model of HD. We show that the relative expression level of each allele (wild-type/mutant) decides the cellular phenotype. When the expression level of wild-type huntingtin is high, an increase in the solubility of the mutant protein is observed. Fluorescence-recovery-after-photobleaching (FRAP) studies show that solubility reaches ∼94% in these cells. This leads to reduction in oxidative stress and cytotoxicity, and increases cell viability. In-cell FRET studies show that interaction between these proteins does not require the presence of a mediator. When the expression of wild-type huntingtin is low, it is sequestered into aggregates by the mutant protein. Even under these conditions, cytotoxicity is attenuated. Our findings indicate that the presence of wild-type huntingtin has a beneficial role even when its relative expression level is lower than that of the mutant protein.

Maintenance of cellular redox homoeostasis forms an important part of the cellular defence mechanism and continued cell viability. Despite extensive studies, the role of the chaperone Hsp104 (heat-shock protein of 102 kDa) in propagation of misfolded protein aggregates in the cell and generation of oxidative stress remains poorly understood. Expression of RNQ1-RFP in Saccharomyces cerevisiae cells led to the generation of the prion form of the protein and increased oxidative stress. In the present study, we show that disruption of Hsp104 in an isogenic yeast strain led to solubilization of RNQ1-RFP. This reduced the oxidative stress generated in the cell. The higher level of oxidative stress in the Hsp104-containing (parental) strain correlated with lower activity of almost all of the intracellular antioxidant enzymes assayed. Surprisingly, this did not correspond with the gene expression analysis data. To compensate for the decrease in protein translation induced by a high level of reactive oxygen species, transcriptional up-regulation takes place. This explains the discrepancy observed between the transcription level and functional enzymatic product. Our results show that in a ΔHsp104 strain, due to lower oxidative stress, no such mismatch is observed, corresponding with higher cell viability. Thus Hsp104 is indirectly responsible for enhancing the oxidative stress in a prion-rich environment.

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