Umpolung Phosphorylation of Tyrosine via 1,2-Phospha-Brook Rearrangement.

Phosphorylated tyrosine is a fundamental building block of bioactive peptides and proteins. However, the chemoselective phosphorylation of tyrosine over other nucleophilic amino acid residues in unprotected peptides remains a significant challenge. Here we report an umpolung strategy that converts the C-terminal tyrosine into an electrophilic spirolactone cyclohexadienone motif through hypervalent iodine oxidation, followed by a 1,2-phospha-Brook rearrangement using phosphite diesters as nucleophilic phosphoryl donors. This reaction proceeds chemoselectively at the tyrosine phenol and is applicable to a wide range of peptide substrates containing various nucleophilic amino acid residues, including serine and threonine.

Leuco Ethyl Violet as Self-Activating Prodrug Photocatalyst for In Vivo Amyloid-Selective Oxygenation.

Aberrant aggregates of amyloid-ß (Aß) and tau protein (tau), called amyloid, are related to the etiology of Alzheimer disease (AD). Reducing amyloid levels in AD patients is a potentially effective approach to the treatment of AD. The selective degradation of amyloids via small molecule-catalyzed photooxygenation in vivo is a leading approach; however, moderate catalyst activity and the side effects of scalp injury are problematic in prior studies using AD model mice. Here, leuco ethyl violet (LEV) is identified as a highly active, amyloid-selective, and blood-brain barrier (BBB)-permeable photooxygenation catalyst that circumvents all of these problems. LEV is a redox-sensitive, self-activating prodrug catalyst; self-oxidation of LEV through a hydrogen atom transfer process under photoirradiation produces catalytically active ethyl violet (EV) in the presence of amyloid. LEV effectively oxygenates human Aß and tau, suggesting the feasibility for applications in humans. Furthermore, a concept of using a hydrogen atom as a caging group of a reactive catalyst functional in vivo is postulated. The minimal size of the hydrogen caging group is especially useful for catalyst delivery to the brain through BBB.

Unimolecular Chemiexcited Oxygenation of Pathogenic Amyloids.

Pathogenic protein aggregates, called amyloids, are etiologically relevant to various diseases, including neurodegenerative Alzheimer disease. Catalytic photooxygenation of amyloids, such as amyloid-ß (Aß), reduces their toxicity; however, the requirement for light irradiation may limit its utility in large animals, including humans, due to the low tissue permeability of light. Here, we report that Cypridina luciferin analogs, dmCLA-Cl and dmCLA-Br, promoted selective oxygenation of amyloids through chemiexcitation without external light irradiation. Further structural optimization of dmCLA-Cl led to the identification of a derivative with a polar carboxylate functional group and low cellular toxicity: dmCLA-Cl-acid. dmCLA-Cl-acid promoted oxygenation of Aß amyloid and reduced its cellular toxicity without photoirradiation. The chemiexcited oxygenation developed in this study may be an effective approach to neutralizing the toxicity of amyloids, which can accumulate deep inside the body, and treating amyloidosis.

Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification.

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.

A chemical catalyst enabling histone acylation with endogenous acyl-CoA.

Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst, mBnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables mBnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.

Quantitative Assays for Catalytic Photo-Oxygenation of Alzheimer Disease-Related Tau Proteins.

Catalytic photo-oxygenation of tau amyloid is a potential therapeutic approach to tauopathies, including Alzheimer disease (AD). However, tau is a complex target containing great molecular size and heterogeneous isoforms/proteoforms. Although catalytic photo-oxygenation has been confirmed when using catalyst 1 and recombinant tau pretreated with heparin, its effects on tau from human patients have not yet been clarified. In this study, focusing on the histidine residues being oxygenated, we have constructed two assay systems capable of quantitatively evaluating the catalytic activity when used on human patient tau: (1) fluorescence labeling at oxygenated histidine sites and (2) LC-MS/MS analysis of histidine-containing fragments. Using these assays, we identified 2 as a promising catalyst for oxygenation of human tau. In addition, our results suggest that aggregated tau induced by heparin is different from actual AD patient tau in developing effective photo-oxygenation catalysts.

Actin filaments accumulated in the nucleus remain in the vicinity of condensing chromosomes in the zebrafish early embryo.

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting α-amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly.

Live Cell Synthetic Histone Acetylation by Chemical Catalyst.

Posttranslational modifications (PTMs) of histones, such as lysine acetylation and ubiquitination, regulate chromatin structure and gene expression. In living organisms, histone PTMs are catalyzed by histone-modifying enzymes. Here, we describe an entirely chemical method to introduce histone modifications in living cells without genetic manipulation. The chemical catalyst PEG-LANA-DSSMe activates a thioester acetyl donor, N,S-diacetylcysteamine (NAC-Ac), and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells.

Improving Drug Sensitivity of HIV-1 Protease Inhibitors by Restriction of Cellular Efflux System in a Fission Yeast Model.

Fission yeast can be used as a cell-based system for high-throughput drug screening. However, higher drug concentrations are often needed to achieve the same effect as in mammalian cells. Our goal here was to improve drug sensitivity so reduced drugs could be used. Three different methods affecting drug uptakes were tested using an FDA-approved HIV-1 protease inhibitor (PI) drug Darunavir (DRV). First, we tested whether spheroplasts without cell walls increase the drug sensitivity. Second, we examined whether electroporation could be used. Although small improvements were observed, neither of these two methods showed significant increase in the EC50 values of DRV compared with the traditional method. In contrast, when DRV was tested in a mutant strain PR836 that lacks key proteins regulating cellular efflux, a significant increase in the EC50 was observed. A comparison of nine FDA-approved HIV-1 PI drugs between the wild-type RE294 strain and the mutant PR836 strain showed marked enhancement of the drug sensitivities ranging from an increase of 0.56 log to 2.48 logs. Therefore, restricting cellular efflux through the adaption of the described fission yeast mutant strain enhances the drug sensitivity, reduces the amount of drug used, and increases the chance of success in future drug discovery.

Live-Cell Protein Modification by Boronate-Assisted Hydroxamic Acid Catalysis.

Selective methods for introducing protein post-translational modifications (PTMs) within living cells have proven valuable for interrogating their biological function. In contrast to enzymatic methods, abiotic catalysis should offer access to diverse and new-to-nature PTMs. Herein, we report the boronate-assisted hydroxamic acid (BAHA) catalyst system, which comprises a protein ligand, a hydroxamic acid Lewis base, and a diol moiety. In concert with a boronic acid-bearing acyl donor, our catalyst leverages a local molarity effect to promote acyl transfer to a target lysine residue. Our catalyst system employs micromolar reagent concentrations and affords minimal off-target protein reactivity. Critically, BAHA is resistant to glutathione, a metabolite which has hampered many efforts toward abiotic chemistry within living cells. To showcase this methodology, we installed a variety of acyl groups in E. coli dihydrofolate reductase expressed within human cells. Our results further establish the well-known boronic acid-diol complexation as a bona fide bio-orthogonal reaction with applications in chemical biology and in-cell catalysis.

Live-cell epigenome manipulation by synthetic histone acetylation catalyst system.

Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome's acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.

Hydroxamic Acid-Piperidine Conjugate is an Activated Catalyst for Lysine Acetylation under Physiological Conditions.

Lysine acylation of proteins is an essential chemical reaction for posttranslational modification and as a means of protein modification in various applications. N,N-Dimethyl-4-aminopyridine (DMAP) derivatives are widely-used catalysts for lysine acylation of proteins; however, the DMAP moiety mostly exists in a protonated, and thus deactivated, form under physiological conditions due to its basicity. An alternative catalytic motif furnishing higher acylation activity would further broaden the possible applications of chemical lysine acylation. We herein report that the hydroxamic acid-piperidine conjugate Ph-HXA is a more active catalytic motif for lysine acetylation than DMAP under physiological conditions. In contrast to DMAP, the hydroxamic acid moiety is mostly deprotonated under aqueous neutral pH, resulting in a higher concentration of the activated form. The Ph-HXA catalyst is also more tolerant of deactivation by a high concentration of glutathione than DMAP. Therefore, Ph-HXA might be a suitable catalytic motif for target protein-selective and site-selective acetylation in cells.

Synthetic hyperacetylation of nucleosomal histones.

We report combinations of a DMAP-based catalyst and phenyl acetate with optimal electron density as a new chemical system for high-yield, selective synthetic acetylation of histone lysine residues. The utility of this chemical system as a unique biologic tool is demonstrated by applying it to Xenopus laevis sperm chromatin.

Site-Selective Synthetic Acylation of a Target Protein in Living Cells Promoted by a Chemical Catalyst/Donor System.

Cell biology is tightly regulated by post-translational modifications of proteins. Methods to modulate post-translational modifications in living cells without relying on enzymes or genetic manipulation are, however, largely underexplored. We previously reported that a chemical catalyst (DSH) conjugated with a nucleosome-binding ligand can activate an acyl-CoA and promote site-selective lysine acylation of histones in test tubes. In-cell acylation by this catalyst system is challenging, however, mainly due to the low cell permeability of acyl-CoA and the propensity of DSH to form inactive disulfide. Here, we report a new catalyst system effective for in-cell acylation, comprising a cell-permeable acyl donor and pro-drugged DSH. Using E. coli dihydrofolate reductase and trimethoprim as a model protein and ligand pair, the catalyst system enabled site-selective acylation of the target protein in living cells. The findings will lead to the development of useful chemical biology tools and new therapeutic strategies capable of synthetically modulating post-translational modifications.

Bub1 kinase- and H2A phosphorylation-independent regulation of Shugoshin proteins under glucose-restricted conditions.

Shugoshin family proteins are involved in various aspects of chromatin regulations, such as chromosome segregation, chromatin structure, and gene expression. In growing yeast and mammalian cells, C-terminal phosphorylation of histone H2A by Bub1 kinase is essential for the localization of Shugoshin proteins to chromatin. Here, we show that in stationary-phase cells, Bub1-mediated H2A phosphorylation is not necessary for chromatin localization of the Shugoshin paralog Sgo2 in Schizosaccharomyces pombe, or for Sgo2-dependent suppression of gene expression in subtelomeric regions. The conserved C-terminal basic domain of Sgo2, which directly binds with phosphorylated H2A, is also dispensable for the localization of Sgo2 to chromatin at stationary phase. Instead, we found that the conserved N-terminal coiled-coil domain and the uncharacterized medial region of Sgo2 are required for Bub1-independent localization of Sgo2. Moreover, Set2-mediated H3K36 methylation was important for the regulation. Intriguingly, the chromatin localization of Sgo2 in the absence of Bub1 was also observed when cells were grown in low-glucose medium. These findings suggest a novel mechanism between nutrient availability and regulation of chromatin by Shugoshin proteins.

Structural Insights into Mdn1, an Essential AAA Protein Required for Ribosome Biogenesis.

Mdn1 is an essential AAA (ATPase associated with various activities) protein that removes assembly factors from distinct precursors of the ribosomal 60S subunit. However, Mdn1's large size (∼5,000 amino acid [aa]) and its limited homology to other well-studied proteins have restricted our understanding of its remodeling function. Here, we present structures for S. pombe Mdn1 in the presence of AMPPNP at up to ∼4 Å or ATP plus Rbin-1, a chemical inhibitor, at ∼8 Å resolution. These data reveal that Mdn1's MIDAS domain is tethered to its ring-shaped AAA domain through an ∼20 nm long structured linker and a flexible ∼500 aa Asp/Glu-rich motif. We find that the MIDAS domain, which also binds other ribosome-assembly factors, docks onto the AAA ring in a nucleotide state-specific manner. Together, our findings reveal how conformational changes in the AAA ring can be directly transmitted to the MIDAS domain and thereby drive the targeted release of assembly factors from ribosomal 60S-subunit precursors.

Kinetic analyses and structure-activity relationship studies of synthetic lysine acetylation catalysts.

Lysine acylation of proteins is a crucial chemical reaction, both as a post-translational modification and as a method for bioconjugation. We previously developed a chemical catalyst, DSH, which activates a chemically stable thioester including acyl-CoA, allowing the site-selective lysine acylation of histones under physiological conditions. However, a more active catalyst is required for efficient lysine acylation in more complex biological milieu, such as in living cells, but there are no rational guidelines for developing efficient lysine acylation catalysts for use under physiological conditions as opposed to in organic solvents. We, herein, conducted a kinetic analysis of the ability of DSH and several derivatives to mediate lysine acetylation to better understand the structural elements essential for high acetylation activity under physiological conditions. Interestingly, the obtained trend in reactivity was different from that observed in organic solvents, suggesting that a different principle is necessary for designing chemical catalysts specifically for use under physiological conditions compared to catalysts for use in organic solvents. Based on the obtained information, we identified a new catalyst scaffold with high activity and structural flexibility for further modification to improve this catalyst system.

Malonylation of histone H2A at lysine 119 inhibits Bub1-dependent H2A phosphorylation and chromosomal localization of shugoshin proteins.

Post-translational modifications of histones, such as acetylation and phosphorylation, are highly conserved in eukaryotes and their combination enables precise regulation of many cellular functions. Recent studies using mass spectrometry have revealed various non-acetyl acylations in histones, including malonylation and succinylation, which change the positive charge of lysine into a negative one. However, the molecular function of histone malonylation or succinylation is poorly understood. Here, we discovered the functions of malonylation in histone H2A at lysine 119 (H2A-K119) in chromosome segregation during mitosis and meiosis. Analyses of H2A-K119 mutants in Saccharomyces cerevisiae and Schizosaccharomyces pombe showed that anionic mutations, specifically to aspartate (K119D) and glutamate (K119E), showed mis-segregation of the chromosomes and sensitivity to microtubule-destabilizing reagents in mitosis and meiosis. We found that the chromosomal localization of shugoshin proteins, which depends on Bub1-catalyzed phosphorylation of H2A at serine 121 (H2A-S121), was significantly reduced in the H2A-K119D and the H2A-K119E mutants. Biochemical analyses using K119-unmodified or -malonylated H2A-C-tail peptides showed that H2A-K119 malonylation inhibited the interaction between Bub1 and H2A, leading to a decrease in Bub1-dependent H2A-S121 phosphorylation. Our results indicate a novel crosstalk between lysine malonylation and serine/threonine phosphorylation, which may be important for fine-tuning chromatin functions such as chromosome segregation.

Leading approaches in synthetic epigenetics for novel therapeutic strategies.

In recent years, our knowledge of the epigenetic functions regulated by post-translational modifications (PTMs) of histones, and their role in various diseases, has expanded rapidly, opening the way to novel therapeutic strategies that treat epigenetic abnormalities. Many of the current approaches have been focusing on the chemical inhibition of histone-modifying enzymes to modulate histone PTM states for attaining therapeutic effects. However, recent developments in chemistry and molecular biology have contributed to the emergence of new methods that introduce histone PTMs entirely through artificial means, without reliance on endogenous enzymes. In this review article, we summarize several state-of-the-art approaches for the introduction of synthetic epigenetic modifications in cells, and discuss both their therapeutic potential and the possible challenges in developing novel therapeutic strategies utilizing them.

LC-MS/MS-based quantitative study of the acyl group- and site-selectivity of human sirtuins to acylated nucleosomes.

Chromatin structure and gene expression are dynamically regulated by posttranslational modifications of histones. Recent advance in mass spectrometry has identified novel types of lysine acylations, such as butyrylation and malonylation, whose functions and regulations are likely different from those of acetylation. Sirtuins, nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases, catalyze various deacylations. However, it is poorly understood how distinct sirtuins regulate the histone acylation states of nucleosomes that have many lysine residues. Here, we provide mass spectrometry-based quantitative information about the acyl group- and site-selectivity of all human sirtuins on acylated nucleosomes. The acyl group- and site-selectivity of each sirtuin is unique to its subtype. Sirt5 exclusively removes negatively-charged acyl groups, while Sirt1/2/3/6/7 preferentially remove hydrophobic acyl groups; Sirt1 and Sirt3 selectively remove acetyl group more than butyryl group, whereas Sirt2 and Sirt6 showed the opposite selectivity. Investigating site-selectivity for active sirtuins revealed acylated lysines on H4 tails to be poor substrates and acylated H3K18 to be a good substrate. Furthermore, we found Sirt7 to be a robust deacylase of H3K36/37, and its activity reliant on nucleosome-binding at its C-terminal basic region. All together, our quantitative dataset provides a useful resource in understanding chromatin regulations by histone acylations.