Zinc Finger Readers of Methylated DNA

DNA methylation is a prevalent epigenetic modification involved in regulating a number of essential cellular processes, including genomic accessibility and transcriptional outcomes. As such, aberrant alterations in global DNA methylation patterns have been associated with a growing number of disease conditions. Nevertheless, the full mechanisms by which DNA methylation information is interpreted and translated into genomic responses is not yet fully understood. Methyl-CpG binding proteins (MBPs) function as important mediators of this essential process by selectively reading DNA methylation signals and translating this information into down-stream cellular outcomes. The Cys2His2 zinc finger scaffold is one of the most abundant DNA binding motifs found within human transcription factors, yet only a few zinc finger containing proteins capable of conferring selectivity for mCpG over CpG sites have been characterized. This review summarizes our current structural understanding for the mechanisms by which the zinc finger MBPs evaluated to date read this essential epigenetic mark. Further, some of the biological implications for mCpG readout elicited by this family of MBPs are discussed

A Protein-Capsid-Based System for Cell Delivery of Selenocysteine

Selenocysteine (Sec) has received a lot of attention as a potential anticancer drug. However, its broad cytotoxicity limits its therapeutic usefulness. Thus, Sec is an attractive candidate for targeted drug delivery. Here, we demonstrate for the first time that an engineered version of the capsid formed by Aquifex aeolicus lumazine synthase (AaLS) can act as a nanocarrier for delivery of Sec to cells. Specifically, a previously reported variant of AaLS (AaLS-IC), which contains a single cysteine per subunit that projects into the capsid interior, was modified by reaction with the diselenide dimer of Sec (Sec₂) to generate a selenenylsulfide conjugate between the capsid and Sec (AaLS-IC-Sec). Importantly, it was determined that the structural context of the reactive cysteine was important for efficient capsid loading. Further, the encapsulated Sec could be quantitatively released from AaLS-IC-Sec by reducing agents such as glutathione or dithiothreitol. To assess cellular penetrance capabilities of AaLS-IC-Sec and subsequent cytotoxic response, six different cells line models were examined. Across the cell lines analyzed, cytotoxic sensitivity correlated with cellular uptake and intracellular trafficking patterns. Together these findings suggest that the engineered AaLS-IC capsid is a promising vehicle for targeted cell delivery of Sec

Chemoselective, oxidation-induced macrocyclization of tyrosine-containing peptides

Inspired by Nature’s wide range of oxidation-induced modifications to install cross-links and cycles at tyrosine (Tyr) and other phenol-containing residue side chains, we report a Tyr-selective strategy for the preparation of Tyr-linked cyclic peptides. This approach leverages N4-substituted 1,2,4-triazoline-3,5-diones (TADs) as azo electrophiles that react chemoselectively with the phenolic side chain of Tyr residues to form stable C–N1-linked cyclic peptides. In the developed method, a precursor 1,2,4-triazolidine-3,5-dione moiety, also known as a urazole, is readily constructed at any free amine revealed on a solid-supported peptide. Once prepared, the N4-substituted urazole peptide is selectively oxidized using mild, peptide-compatible conditions to generate an electrophilic N4-substituted TAD peptide intermediate that reacts selectively under aqueous conditions with internal and terminal Tyr residues to furnish Tyr-linked cyclic peptides. The approach demonstrates good tolerance of native residue side chains and enables access to cyclic peptides ranging from 3- to 11-residues in size (16- to 38-atom-containing cycles). The identity of the installed Tyr-linkage, a stable covalent C–N1 bond, was characterized using NMR spectroscopy. Finally, we applied the developed method to prepare biologically active Tyr-linked cyclic pep-tides bearing the integrin-binding RGDf epitope

Poly(Adp-ribose) binding and macroh2a mediate recruitment and functions of kdm5a at dna lesions

The histone demethylase KDM5A erases histone H3 lysine 4 methylation, which is involved in transcription and DNA damage responses (DDRs). While DDR functions of KDM5A have been identified, how KDM5A recognizes DNA lesion sites within chromatin is unknown. Here, we identify two factors that act upstream of KDM5A to promote its association with DNA damage sites. We have identified a noncanonical poly(ADP-ribose) (PAR)-binding region unique to KDM5A. Loss of the PARbinding region or treatment with PAR polymerase (PARP) inhibitors (PARPi's) blocks KDM5A-PAR interactions and DNA repair functions of KDM5A. The histone variant macroH2A1.2 is also specifically required for KDM5A recruitment and function at DNA damage sites, including homology-directed repair of DNA double-strand breaks and repression of transcription at DNA breaks. Overall, this work reveals the importance of PAR binding and macroH2A1.2 in KDM5A recognition of DNA lesion sites that drive transcriptional and repair activities at DNA breaks within chromatin that are essential for maintaining genome integrit