Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 +/- 1.28 micromolar. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Histone variants in archaea and the evolution of combinatorial chromatin complexity

Nucleosomes in eukaryotes act as platforms for the dynamic integration of epigenetic information. Posttranslational modifications are reversibly added or removed and core histones exchanged for paralogous variants, in concert with changing demands on transcription and genome accessibility. Histones are also common in archaea. Their role in genome regulation, however, and the capacity of individual paralogs to assemble into histone–DNA complexes with distinct properties remain poorly understood. Here, we combine structural modeling with phylogenetic analysis to shed light on archaeal histone paralogs, their evolutionary history, and capacity to generate combinatorial chromatin states through hetero-oligomeric assembly. Focusing on the human commensal Methanosphaera stadtmanae as a model archaeal system, we show that the heteromeric complexes that can be assembled from its seven histone paralogs vary substantially in DNA binding affinity and tetramer stability. Using molecular dynamics simulations, we go on to identify unique paralogs in M. stadtmanae and Methanobrevibacter smithii that are characterized by unstable interfaces between dimers. We propose that these paralogs act as capstones that prevent stable tetramer formation and extension into longer oligomers characteristic of model archaeal histones. Importantly, we provide evidence from phylogeny and genome architecture that these capstones, as well as other paralogs in the Methanobacteriales, have been maintained for hundreds of millions of years following ancient duplication events. Taken together, our findings indicate that at least some archaeal histone paralogs have evolved to play distinct and conserved functional roles, reminiscent of eukaryotic histone variants. We conclude that combinatorially complex histone-based chromatin is not restricted to eukaryotes and likely predates their emergence.

Histone variants in archaea and the evolution of combinatorial chromatin complexity

ABSTRACTNucleosomes in eukaryotes act as platforms for the dynamic integration of epigenetic information. Post-translational modifications are reversibly added or removed and core histones exchanged for paralogous variants, in concert with changing demands on transcription and genome accessibility. Histones are also common in archaea. Their role in genome regulation, however, and the capacity of individual paralogs to assemble into histone-DNA complexes with distinct properties remain poorly understood. Here, we combine structural modelling with phylogenetic analysis to shed light on archaeal histone paralogs, their evolutionary history and capacity to generate complex combinatorial chromatin states through hetero-oligomeric assembly. Focusing on the human commensal Methanosphaera stadtmanae as a model archaeal system, we show that the heteromeric complexes that can be assembled from its seven histone paralogs vary substantially in DNA binding affinity and tetramer stability, occupying a large but densely populated chromatin state space. Using molecular dynamics simulations, we go on to identify unique paralogs in M. stadtmanae and Methanobrevibacter smithii that are characterized by unstable dimer:dimer interfaces. We propose that these paralogs act as capstones that prevent stable tetramer formation and extension into longer oligomers characteristic of model archaeal histones. Importantly, we provide evidence from phylogeny and genome architecture that these capstones, as well as other paralogs in the Methanobacteriales, have been maintained for hundreds of millions of years following ancient duplication events. Taken together, our findings indicate that at least some archaeal histone paralogs have evolved to play distinct and conserved functional roles, reminiscent of eukaryotic histone variants. We conclude that combinatorially complex histone-based chromatin is not restricted to eukaryotes and likely predates their emergence.

Lamin A/C Assembly Defects in LMNA-Congenital Muscular Dystrophy Is Responsible for the Increased Severity of the Disease Compared with Emery–Dreifuss Muscular Dystrophy

LMNA encodes for Lamin A/C, type V intermediate filaments that polymerize under the inner nuclear membrane to form the nuclear lamina. A small fraction of Lamin A/C, less polymerized, is also found in the nucleoplasm. Lamin A/C functions include roles in nuclear resistance to mechanical stress and gene regulation. LMNA mutations are responsible for a wide variety of pathologies, including Emery–Dreifuss (EDMD) and LMNA-related congenital muscular dystrophies (L-CMD) without clear genotype–phenotype correlations. Both diseases presented with striated muscle disorders although L-CMD symptoms appear much earlier and are more severe. Seeking for pathomechanical differences to explain the severity of L-CMD mutations, we performed an in silico analysis of the UMD-LMNA database and found that L-CMD mutations mainly affect residues involved in Lamin dimer and tetramer stability. In line with this, we found increased nucleoplasmic Lamin A/C in L-CMD patient fibroblasts and mouse myoblasts compared to the control and EDMD. L-CMD myoblasts show differentiation defects linked to their inability to upregulate muscle specific nuclear envelope (NE) proteins expression. NE proteins were mislocalized, leading to misshapen nuclei. We conclude that these defects are due to both the absence of Lamin A/C from the nuclear lamina and its maintenance in the nucleoplasm of myotubes.

Covalently immobilized catalase on functionalized graphene: effect on the activity, immobilization efficiency, and tetramer stability

The different anchorage modes of catalase covalently linked to graphene affect the secondary and quaternary structures determining the biological properties.