| Popis: |
Histone proteins are extremely important biological targets that are heavily post-translationally modified to control DNA related processes. To study these proteins in vitro, we have developed a number of chemical techniques that allow for the synthesis of selectively modified histones. These proteins can be refolded into nucleosomes and the stability, dynamics, and structure of the nucleosome can be probed using biochemical and biophysical techniques. Here we present an optimized peptide-synthesis strategy for the generation of peptide thioesters, the biophysical characterization of two modified histones, and explore the use of histones as potent cell-penetrating peptides as a new method to study histone cytoplasmic maturation.In order to generate the reactive peptides to generate fully-synthetic histone proteins, we utilize a masked thioester. We discovered that 3,4-diaminobenzoic acid, our standard thioester precursor, would accumulate number side-products where peptide chain extension occurred on both amines when Gly-rich peptides were synthesized. To remedy this, we developed an alternative protection scheme that allowed for the selective protection and deprotection of both amines using Fmoc and alloxycarbonyl (Alloc) protecting groups. This allowed for the synthesis of peptides containing multiple glycines in a row and a 44-amino acid peptide in high yield. This has since been used for the total synthesis of 4 different core histone proteins ranging in size from 101-214 amino acids in size. While many post-translational modifications of histones exist on the unstructured histone tails, modifications to the folded histone core are also important for control of nucleosome dynamics and stability. Here we present data showing that H4-K91ac and H3-T118ph destabilize nucleosomes, but in different manners. H4-K91ac is a modification that occurs between the tetramer and dimer subunits and weakens dimer interactions in nucleosomes. We find that this modification completely inhibits the formation of histone octamers in solution, presumably because of the weakened interface. H4-K91ac nucleosomes are destabilized when compared to wild-type in thermal denaturation and Nap1 chaperone-based reconstitutions. H3-T118ph has previously been shown to be greatly destabilizing, and here we investigated an alternative nucleosome complex formed during reconstitution. We characterized these structures using atomic force microscopy, fluorescence quantitation, and chaperone-based nucleosome reconstitutions. We demonstrate that these structures contain two histone octamers are packed side-by-side by a single strand of DNA.Finally, we show a method that allows for the uptake of exogenous fluorescently labeled histone proteins into a variety of eukaryotic cells, including human cancer cell lines such as HeLa and MDA-231 triple-negative breast cancer cells. We find that folded histones have an innate cell-penetrating ability. We hypothesized that histones bind to the cell surface through a charge-charge interaction and are brought into the cell utilizing a caveolin-dependent mechanism. This approach presents a novel tool to explore initial steps in histone processing and chromatin assembly, and here we look at the role of the cytoplasmic histone modifications added by histone acetyltransferase 1 (Hat1). |
