The human telomeric nucleosome displays distinct structural and dynamic properties.

Telomeres protect the ends of our chromosomes and are key to maintaining genomic integrity during cell division and differentiation. However, our knowledge of telomeric chromatin and nucleosome structure at the molecular level is limited. Here, we aimed to define the structure, dynamics as well as properties in solution of the human telomeric nucleosome. We first determined the 2.2 Å crystal structure of a human telomeric nucleosome core particle (NCP) containing 145 bp DNA, which revealed the same helical path for the DNA as well as symmetric stretching in both halves of the NCP as that of the 145 bp '601' NCP. In solution, the telomeric nucleosome exhibited a less stable and a markedly more dynamic structure compared to NCPs containing DNA positioning sequences. These observations provide molecular insights into how telomeric DNA forms nucleosomes and chromatin and advance our understanding of the unique biological role of telomeres.

An efficient proteome-wide strategy for discovery and characterization of cellular nucleotide-protein interactions.

Metabolite-protein interactions define the output of metabolic pathways and regulate many cellular processes. Although diseases are often characterized by distortions in metabolic processes, efficient means to discover and study such interactions directly in cells have been lacking. A stringent implementation of proteome-wide Cellular Thermal Shift Assay (CETSA) was developed and applied to key cellular nucleotides, where previously experimentally confirmed protein-nucleotide interactions were well recaptured. Many predicted, but never experimentally confirmed, as well as novel protein-nucleotide interactions were discovered. Interactions included a range of different protein families where nucleotides serve as substrates, products, co-factors or regulators. In cells exposed to thymidine, a limiting precursor for DNA synthesis, both dose- and time-dependence of the intracellular binding events for sequentially generated thymidine metabolites were revealed. Interactions included known cancer targets in deoxyribonucleotide metabolism as well as novel interacting proteins. This stringent CETSA based strategy will be applicable for a wide range of metabolites and will therefore greatly facilitate the discovery and studies of interactions and specificities of the many metabolites in human cells that remain uncharacterized.

Assessing the Efficacy of Mdm2/Mdm4-Inhibiting Stapled Peptides Using Cellular Thermal Shift Assays.

Previous publications on stapled peptide inhibitors against Mdm2/Mdm4-p53 interactions have established that this new class of drugs have the potential to be easily optimised to attain high binding affinity and specificity, but the mechanisms controlling their cellular uptake and target engagement remain elusive and controversial. To aid in understanding the rules of peptide and staple design, and to enable rapid optimisation, we employed the newly-developed cellular thermal shift assay (CETSA). CETSA was able to validate stapled peptide binding to Mdm2 and Mdm4, and the method was also used to determine the extent of cellular uptake, cellular availability, and intracellular binding of the endogenous target proteins in its native environment. Our data suggest that while the stapled peptides engage their targets intracellularly, more work is needed to improve their cellular entry and target engagement efficiency in vivo. CETSA now provides a valuable tool to optimize such in vivo properties of stapled peptides.

Hexamethylene bisacetamide (HMBA) simultaneously targets AKT and MAPK pathway and represses NF kappaB activity: implications for cancer therapy.

Hexamethylene Bisacetamide (HMBA) is a hybrid polar compound originally developed as a differentiation inducing agent. We show in this study that HMBA can inhibit activation of several NF kappaB target genes in both lung and breast cancer cell lines. Furthermore, consistent with its ability to inhibit NF kappaB function, HMBA can also sensitize cells to apoptosis. We show that HMBA mediates inhibition of the Akt and ERK/MAPK cascade, both of which are critical for cell survival and proliferation and are well known regulators of NF kappaB activation. We also show that PTEN negative breast cancer cells which have hyper activation of the PI3K/Akt pathway show increased sensitivity to growth inhibitory effects of combination of HMBA and TNFalpha. Furthermore, HMBA can decrease the kinase activity of the IKK complex leading to defective phosphorylation of I kappaB alpha and Ser536 of p65. This study gives mechanistic insight into the mechanism of action of HMBA, provides the rationale for the potential use of HMBA in combination with various existing kinase inhibitors in combination therapy and also suggests useful biomarkers for monitoring tumor response to HMBA.