Consistent mutational paths predict eukaryotic thermostability

peer-reviewedBackground: Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published. Results: Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations. Conclusions: The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability

Cytotoxic activities of new iron(III) and nickel(II) chelates of some S-methyl-thiosemicarbazones on K562 and ECV304 cells

The S-methyl-thiosemicarbazones of the 2- hydroxy-R-benzaldehyde (R= H, 3-OH 3-OCH3 or 4-OCH3) reacted with the corresponding aldehydes in the presence of FeCl3 and NiCl2. New ONNO chelates of iron(III) and nickel (II) with hydroxy- or methoxy-substitued N1,N4-diarylidene-Smethyl- thiosemicarbazones were characterized by means of elemental analysis, conductivity and magnetic measurements, UV-Vis, IR and 1H-NMR spectroscopies. Cytotoxic activities of the compounds were determined using K562 chronic myeloid leukemia and ECV304 human endothelial cell lines by MTT assay. It was determined that monochloro N1-4- methoxysalicylidene-N4-4-methoxysalicylidene-S-methylthiosemicarbazidato- iron(III) complex showed selective anti-leukemic effects in K562 cells while has no effect in ECV304 cells in the 0.53 μg/ml (IC50) concentrations. Also, some methoxy-substitued nickel(II) chelates exhibit high cytotoxic activitiy against both of these cell lines in low concentrations. Cytotoxicity data were evaluated depending on cell lines origin and position of the substituents on aromatic rings

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin

Maturation of iron-sulphur (Fe/S) proteins involves complex biosynthetic machinery. In vivo synthesis of [2Fe-2S] clusters on the mitochondrial scaffold protein Isu1 requires the cysteine desulphurase complex Nfs1-Isd11, frataxin, ferredoxin Yah1 and its reductase Arh1. The roles of Yah1-Arh1 have remained enigmatic, because they are not required for in vitro Fe/S cluster assembly. Here, we reconstitute [2Fe-2S] cluster synthesis on Isu1 in a reaction depending on Nfs1-Isd11, frataxin, Yah1, Arh1 and NADPH. Unlike in the bacterial system, frataxin is an essential part of Fe/S cluster biosynthesis and is required simultaneously and stoichiometrically to Yah1. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1-apo-Isu1. Nuclear magnetic resonance structural studies identify the Yah1-apo-Isu1 interaction surface and suggest a pathway for electron flow from reduced ferredoxin to Isu1. Together, our study defines the molecular function of the ferredoxin Yah1 and its human orthologue FDX2 in mitochondrial Fe/S cluster synthesis