Laboratory Evolution of a Saccharomyces cerevisiae × S. eubayanus Hybrid Under Simulated Lager-Brewing Conditions.

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilization and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae × S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Identification of catechols as histone-lysine demethylase inhibitors.

Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity in the low µM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS cells.

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 µm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.

Inhibitors of histone demethylases.

Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the inhibitors are either previously reported inhibitors of related enzymes or compounds derived from these. Development in terms of selectivity and potency is still pertinent. Several reports on the development of functional assays have been published.

Enzyme kinetic studies of histone demethylases KDM4C and KDM6A: towards understanding selectivity of inhibitors targeting oncogenic histone demethylases.

To investigate ligand selectivity between the oncogenic KDM4C and tumor repressor protein KDM6A histone demethylases, KDM4C and KDM6A were enzymatically characterized, and subsequently, four compounds were tested for inhibitory effects. 2,4-dicarboxypyridine and (R)-N-oxalyl-O-benzyltyrosine (3) are both known to bind to a close KDM4C homolog and 3 binds in the part of the cavity that accommodates the side chain in position 11 of histone 3. The inhibition measurements showed significant selectivity between KDM4C and KDM6A. This demonstrates that despite very similar active site topologies, selectivity between Jumonji family histone demethylases can be obtained even with small molecule ligands.