Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones.

Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

Non-reducible disulfide bond replacement implies that disulfide exchange is not required for hepcidin-ferroportin interaction.

Previous studies have led to opposing hypotheses about the requirement of intermolecular disulfide exchange in the binding of the iron regulatory peptide hepcidin to its receptor ferroportin. To clarify this issue, we used the diaminodiacid approach to replace the disulfide bonds in hepcidin with non-reducible thioether bonds. Our results implied that disulfide exchange is not required for the interaction between hepcidin and ferroportin. This theory is further supported by our development of biologically active minihepcidins that do not show activity dependence on cysteine.