A MOZ-TIF2 leukemia mouse model displays KAT6-dependent H3K23 propionylation and overexpression of a set of active developmental genes.

Aberrant regulation of chromatin modifiers is a common occurrence across many cancer types, and a key priority is to determine how specific alterations of these proteins, often enzymes, can be targeted therapeutically. MOZ, a histone acyltransferase, is recurrently fused to coactivators CBP, p300, and TIF2 in cases of acute myeloid leukemia (AML). Using either pharmacological inhibition or targeted protein degradation in a mouse model for MOZ-TIF2-driven leukemia, we show that KAT6 (MOZ/MORF) enzymatic activity and the MOZ-TIF2 protein are necessary for indefinite proliferation in cell culture. MOZ-TIF2 directly regulates a small subset of genes encoding developmental transcription factors, augmenting their high expression. Furthermore, transcription levels in MOZ-TIF2 cells positively correlate with enrichment of histone H3 propionylation at lysine 23 (H3K23pr), a recently appreciated histone acylation associated with gene activation. Unexpectedly, we also show that MOZ-TIF2 and MLL-AF9 regulate transcription of unique gene sets, and their cellular models exhibit distinct sensitivities to multiple small-molecule inhibitors directed against AML pathways. This is despite the shared genetic pathways of wild-type MOZ and MLL. Overall, our data provide insight into how aberrant regulation of MOZ contributes to leukemogenesis. We anticipate that these experiments will inform future work identifying targeted therapies in the treatment of AML and other diseases involving MOZ-induced transcriptional dysregulation.

Structural Basis of Sirtuin 6-Catalyzed Nucleosome Deacetylation.

The reversible acetylation of histone lysine residues is controlled by the action of acetyltransferases and deacetylases (HDACs), which regulate chromatin structure and gene expression. The sirtuins are a family of NAD-dependent HDAC enzymes, and one member, sirtuin 6 (Sirt6), influences DNA repair, transcription, and aging. Here, we demonstrate that Sirt6 is efficient at deacetylating several histone H3 acetylation sites, including its canonical site Lys9, in the context of nucleosomes but not free acetylated histone H3 protein substrates. By installing a chemical warhead at the Lys9 position of histone H3, we trap a catalytically poised Sirt6 in complex with a nucleosome and employ this in cryo-EM structural analysis. The structure of Sirt6 bound to a nucleosome reveals extensive interactions between distinct segments of Sirt6 and the H2A/H2B acidic patch and nucleosomal DNA, which accounts for the rapid deacetylation of nucleosomal H3 sites and the disfavoring of histone H2B acetylation sites. These findings provide a new framework for understanding how HDACs target and regulate chromatin.

Semisynthetic Approach to the Analysis of Tumor Suppressor PTEN Ubiquitination.

Phosphatase and tensin homologue (PTEN) tumor suppressor protein is a PIP3 lipid phosphatase that is subject to multifaceted post-translational modifications. One such modification is the monoubiquitination of Lys13 that may alter its cellular localization but is also positioned in a manner that could influence several of its cellular functions. To explore the regulatory influence of ubiquitin on PTEN's biochemical properties and its interaction with ubiquitin ligases and a deubiquitinase, the generation of a site-specifically and stoichiometrically ubiquitinated protein could be beneficial. Here, we describe a semisynthetic method that relies upon sequential expressed protein ligation steps to install ubiquitin at a Lys13 mimic in near full-length PTEN. This approach permits the concurrent installation of C-terminal modifications in PTEN, thereby facilitating an analysis of the interplay between N-terminal ubiquitination and C-terminal phosphorylation. We find that the N-terminal ubiquitination of PTEN inhibits its enzymatic function, reduces its binding to lipid vesicles, modulates its processing by NEDD4-1 E3 ligase, and is efficiently cleaved by the deubiquitinase, USP7. Our ligation approach should motivate related efforts for uncovering the effects of ubiquitination of complex proteins.

KATs off: Biomedical insights from lysine acetyltransferase inhibitors.

Lysine acetyltransferase (KAT) enzymes including the p300, MYST, and GCN5 families play major roles in modulating the structure of chromatin and regulating transcription. Because of their dysregulation in various disease states including cancer, efforts to develop inhibitors of KATs have steadily gained momentum. Here we provide an overview of recent progress on the development of high quality chemical probes of the p300 and MYST family of KATs and how they are emerging as useful tools for basic and translational investigation.

Distinct biochemical properties of the class I histone deacetylase complexes.

Classical histone deacetylases (HDACs) are enzymes that can hydrolytically cleave acetyl-Lys in histones and other proteins and serve as established drug targets in some forms of cancer. Class I HDACs 1-3 typically exist in a range of multiprotein complexes inside cells and show distinct biological functions in modulating gene expression. In recent years, it has become possible to purify and analyze the structure and enzymatic properties of several of these HDAC complexes, including CoREST, MiDAC, NuRD, Sin3, SMRT, MIER, and RERE. Here, we summarize what is experimentally established and/or computationally predicted about the structure of these complexes to describe their particular catalytic activities and site-specificities with modified nucleosome substrates.

Histone H2B Deacylation Selectivity: Exploring Chromatin's Dark Matter with an Engineered Sortase.

We describe a new method to produce histone H2B by semisynthesis with an engineered sortase transpeptidase. N-Terminal tail site-specifically modified acetylated, lactylated, and ß-hydroxybutyrylated histone H2Bs were incorporated into nucleosomes and investigated as substrates of histone deacetylase (HDAC) complexes and sirtuins. A wide range of rates and site-specificities were observed by these enzyme forms suggesting distinct biological roles in regulating chromatin structure and epigenetics.

Trimethylation of the R5 Silica-Precipitating Peptide Increases Silica Particle Size by Redirecting Orthosilicate Binding.

The unmodified R5 peptide from silaffin in the diatom Cylindrotheca fusiformis rapidly precipitates silica particles from neutral aqueous solutions of orthosilicic acid. A range of post-translational modifications found in R5 contribute toward tailoring silica morphologies in a species-specific manner. We investigated the specific effect of R5 lysine side-chain trimethylation, which adds permanent positive charges, on silica particle formation. Our studies revealed that a doubly trimethylated R5K3,4me3 peptide has reduced maximum activity yet, surprisingly, generates larger silica particles. Molecular dynamics simulations of R5K3,4me3 binding by the precursor orthosilicate anion revealed that orthosilicate preferentially associates with unmodified lysine side-chain amines and the peptide N terminus. Thus, larger silica particles arise from reduced orthosilicate association with trimethylated lysine side chains and their redirection to the N terminus of the R5 peptide.

A clickable glutamine (CliQ) derivative for the traceless reversible modification of peptides and proteins.

The Cu(i)-mediated click reaction of proteins with affinity tags enables their selective isolation from complex mixtures. However, irreversible protein modification limits the interpretation of results from subsequent biophysical and biochemical assays. We report a facile and modular chemical strategy to reversibly modify peptides and proteins with biotin and FLAG affinity tags at a clickable glutamine (CliQ) residue.

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity.

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non-naturally occurring AMPs. We have used a backbone-cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta-hairpin-like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram-negative, Escherichia coli but improved the activity against gram-positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram-negative versus gram-positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram-positive bacterium but less active against gram-negative bacterium. Our results show that there is not always a correlation between improved hairpin-structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Hairpin structure stability plays a role in the activity of two antimicrobial peptides.

Many naturally occurring antimicrobial peptides (AMPs) are amphipathic with a ß-hairpin conformation stabilized by cross-strand disulfides across the associated ß-strands. Here, we show that the disulfides are not essential. Other structuring means such as better ß-turns and noncovalent cross-strand interactions can, with proper design, replace the disulfides with no loss in antimicrobial activity. Our results also demonstrate that the hairpin turn region may play a role in membrane recognition for at least one member of this class, since a homodimeric turnless ß-sheet analog showed no antimicrobial activity. We also examined the effects of N-terminal fatty acid adducts on AMPs. Surprisingly, the large hydrophobic carboxylic moieties examined completely eliminated the antimicrobial activity of previously active ß-hairpin peptides.

Selenocysteine as a Latent Bioorthogonal Electrophilic Probe for Deubiquitylating Enzymes.

Deubiquitylating enzymes (DUBs) remove ubiquitin (Ub) from various cellular proteins and render eukaryotic ubiquitylation a dynamic process. The misregulation of protein ubiquitylation is associated with many human diseases, and there is an urgent need to identify specific DUBs associated with therapeutically relevant targets of Ub. We report the development of two facile selenocysteine-based strategies to generate the DUB probe dehydroalanine (Dha). Optimized oxidative or alkylative elimination of Se yielded Dha at the C-terminus of Ub. The high utility of alkylative elimination, which is compatible with multiple thiols in Ub targets, was demonstrated by generating a probe derived from the Ub ligase tripartite motif protein 25 (TRIM-25). Successful capture of the TRIM-25-associated DUB, ubiquitin-specific protease 15, demonstrated the versatility of our chemical strategy for identifying target-specific DUBs.

Aromatic thiol-mediated cleavage of N-O bonds enables chemical ubiquitylation of folded proteins.

Access to protein substrates homogenously modified by ubiquitin (Ub) is critical for biophysical and biochemical investigations aimed at deconvoluting the myriad biological roles for Ub. Current chemical strategies for protein ubiquitylation, however, employ temporary ligation auxiliaries that are removed under harsh denaturing conditions and have limited applicability. We report an unprecedented aromatic thiol-mediated N-O bond cleavage and its application towards native chemical ubiquitylation with the ligation auxiliary 2-aminooxyethanethiol. Our interrogation of the reaction mechanism suggests a disulfide radical anion as the active species capable of cleaving the N-O bond. The successful semisynthesis of full-length histone H2B modified by the small ubiquitin-like modifier-3 (SUMO-3) protein further demonstrates the generalizability and compatibility of our strategy with folded proteins.

A time-resolved Förster resonance energy transfer assay to measure activity of the deamidase of the prokaryotic ubiquitin-like protein.

The modification of proteins in Mycobacterium tuberculosis (Mtb) by the prokaryotic ubiquitin-like protein (Pup) targets them for degradation by mycobacterial proteasomes. Although functionally similar to eukaryotic deubiquitylating enzymes, the deamidase of Pup, called Dop, has no known mammalian homologs. Because Dop is necessary for persistent infection by Mtb, its selective inhibition holds potential for tuberculosis therapy. To facilitate high-throughput screens for Dop inhibitors, we developed a time-resolved Förster resonance energy transfer (TR-FRET)-based assay for Dop function. The TR-FRET assay was successfully applied to determine the Michaelis constant for adenosine triphosphate (ATP) binding and to test the cofactor tolerance of Dop.