Utilizing selenocysteine for expressed protein ligation and bioconjugations
From JACS (February 2017)
Expressed protein ligation (EPL) is a method for protein semi-synthesis, which enables the site-specific incorporation of post-translational modifications, of non-natural groups or, in biotechnological applications, the expression of proteins which are cytotoxic. In EPL, an N-terminal cysteine in a protein fragment reacts under mild aqueous conditions with a C-terminal thioester in a second fragment, resulting in the formation of a native peptide bond. EPL is however limited by moderate efficiency and slow kinetics, in particular for native proteins. Further, a cysteine remains at the ligation site, potentially introducing a mutation. Ligations of selenocysteine (Sec) overcome these limitations as Sec is a better nucleophile than cysteine. This study presents a heterologous expression method of Sec-containing proteins, combined with Sec-ligation to generate a series of protein products with high efficiency. Importantly, the authors demonstrate that post-ligation, the Sec residue remaining in the protein sequence can selectively be converted into alanine or serine, even in the presence of other cysteine residues. Moreover, Sec can be used as a synthetic handle to introduce a series of post-translational modifications, including analogues of phosphorylation or ubiquitylation. Together, this study showcases the power of Sec-ligation in protein engineering.
An allosteric PRC2 inhibitor targeting the H3K27me3 binding pocket of EED
From Nature Chemical Biology (July 2016)
Tri-methylation of lysine 27 in histone H3 (H3K27me3) is a key histone modification which controls critical developmental processes. Importantly, in many cancers the responsible methyltransferase, polycomb repressive complex 2 (PRC2), is found to be overexpressed, leading to gene dysregulation. Therefore, PRC2 and in particular its catalytic subunit EZH2 is an important drug target. Recently, several inhibitors against the binding site of the methyltransferase cofactor s-adenosylmethionine (SAM) have been developed. Treated cells however acquire resistance mutations. Thus, in the current study a team from the Novartis Institute of BioMedical Research in Shanghai developed an allosteric inhibitor of PRC2 with nanomolar efficacy which is non-competitive with SAM. Their new compound, EED226, targets the EED subunit which is important for allosteric activation of PRC2. Structure-activity relationships, X-ray analysis of the enzyme-inhibitor complex, in vitro and in vivo xenograft studies demonstrate the mechanism of action as well as the potency of this novel class of epigenetic modulator compounds. Moreover, the novel compounds from this study and from a companion paper in the same issue of Nature Chemical Biology act synergistically with already available PRC2 inhibitors, providing a novel avenue of targeting PRC2 in future applications of correcting dysregulated epigenetic states in cancers.
Single-molecule decoding of combinatorially modified nucleosomes
From Science (May 2016)
In chromatin, individual nucleosomes are decorated by distinct combinations of histone modifications. The genomic-locus specific elucidation of these modification patterns on a molecular level has however been highly challenging. A team led by Bradley Bernstein at Harvard has addressed this problem by isolating individual nucleosomes from a range of cell types. These nucleosomes were then immobilized on a microscopy slide, and their co-existing modification pattern were interrogated using fluorescently labeled antibodies under single-molecule observation. After establishing the modification patterns, the nucleosomal DNA was sequenced in situ using single-molecule sequencing protocols. This allowed precise mapping of complex modification patterns to individual defined genomic loci. Importantly, employing this method the authors could detect a stem-cell specific modification pattern, bivalent chromatin, and determine its genomic localization with high precision. Given that current limitations regarding genomic coverage can be solved, this true single-molecule method thus has the potential to push epigenomic analysis to the single-cell and single-locus levels.
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