Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

Epigenetic meets metabolism: novel vulnerabilities to fight cancer.

Histones undergo a plethora of post-translational modifications (PTMs) that regulate nucleosome and chromatin dynamics and thus dictate cell fate. Several evidences suggest that the accumulation of epigenetic alterations is one of the key driving forces triggering aberrant cellular proliferation, invasion, metastasis and chemoresistance pathways. Recently a novel class of histone "non-enzymatic covalent modifications" (NECMs), correlating epigenome landscape and metabolic rewiring, have been described. These modifications are tightly related to cell metabolic fitness and are able to impair chromatin architecture. During metabolic reprogramming, the high metabolic flux induces the accumulation of metabolic intermediate and/or by-products able to react with histone tails altering epigenome homeostasis. The accumulation of histone NECMs is a damaging condition that cancer cells counteracts by overexpressing peculiar "eraser" enzymes capable of removing these modifications preserving histones architecture. In this review we explored the well-established NECMs, emphasizing the role of their corresponding eraser enzymes. Additionally, we provide a parterre of drugs aiming to target those eraser enzymes with the intent to propose novel routes of personalized medicine based on the identification of epi-biomarkers which might be selectively targeted for therapy. Video Abstract.

pH-Responsive and Recyclable Hydrogels for Gas Releasing and Scavenging.

Gas-releasing/scavenging hydrogels have wide applications in biomedical and industrial fields. However, the covalently crosslinked nature of these existing materials makes them difficult to degrade or recycle, leading to a waste of raw materials and aggravating environmental pollution. Herein, a new class of pH-responsive and recyclable hydrogels with versatile gas-releasing and scavenging properties is reported, utilizing pH changes to reversibly control disassembly and reassembly of the hydrogel network. The initial hydrogels are constructed via the one-pot radical polymerization and contain dynamic molecular networks based on hydrophobic interactions, which can disassemble when the materials are placed in low pH solutions. The disassembled copolymer chains can reform hydrogels, following supplementation with fresh mineral salts and micelle monomers in neutral solutions. Moreover, the mineral salts used to reform hydrogels can function as gas donors or scavengers, endowing these hydrogels with versatile gas-releasing and consuming properties. Overall, this research provides a facile and environmentally friendly method to recycle hydrogels with gas-releasing and gas-scavenging properties, which have potential applications in diverse fields, including wound healing, wastewater management, and gas therapy for diseases.

The Tale of DJ-1 (PARK7): A Swiss Army Knife in Biomedical and Psychological Research.

DJ-1 (also known as PARK7) is a multifunctional enzyme in human beings that is highly conserved and that has also been discovered in diverse species (ranging from prokaryotes to eukaryotes). Its complex enzymatic and non-enzymatic activities (such as anti-oxidation, anti-glycation, and protein quality control), as well as its role as a transcriptional coactivator, enable DJ-1 to serve as an essential regulator in multiple cellular processes (e.g., epigenetic regulations) and make it a promising therapeutic target for diverse diseases (especially cancer and Parkinson's disease). Due to its nature as a Swiss army knife enzyme with various functions, DJ-1 has attracted a large amount of research interest, from different perspectives. In this review, we give a brief summary of the recent advances with respect to DJ-1 research in biomedicine and psychology, as well as the progress made in attempts to develop DJ-1 into a druggable target for therapy.

Tunable Toxicity of Bufadienolides is Regulated through a Configuration Inversion Catalyzed by a Short-Chain Dehydrogenase/Reductase.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14ß-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(ß→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

Chemical and Synthetic Biology Approaches for Cancer Vaccine Development.

Cancer vaccines have been considered promising therapeutic strategies and are often constructed from whole cells, attenuated pathogens, carbohydrates, peptides, nucleic acids, etc. However, the use of whole organisms or pathogens can elicit unwanted immune responses arising from unforeseen reactions to the vaccine components. On the other hand, synthetic vaccines, which contain antigens that are conjugated, often with carrier proteins, can overcome these issues. Therefore, in this review we have highlighted the synthetic approaches and discussed several bioconjugation strategies for developing antigen-based cancer vaccines. In addition, the major synthetic biology approaches that were used to develop genetically modified cancer vaccines and their progress in clinical research are summarized here. Furthermore, to boost the immune responses of any vaccines, the addition of suitable adjuvants and a proper delivery system are essential. Hence, this review also mentions the synthesis of adjuvants and utilization of biomaterial scaffolds, which may facilitate the design of future cancer vaccines.

An Azidoribose Probe to Track Ketoamine Adducts in Histone Ribose Glycation.

Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose (5-AR), as well as the synthesis of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe's utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.

Synthesis of an Alkynyl Methylglyoxal Probe to Investigate Nonenzymatic Histone Glycation.

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite that modifies histones in vivo and induces changes in chromatin structure and function. Here we report the synthesis and application of a chemical probe for investigating MGO-glycation. A two-step synthesis of a Cu-click compatible alkynyl oxoaldehyde probe (AlkMGO) via sequential Dess-Martin and Riley oxidations is presented. This synthesis elevates the accessibility and utility of an important tool for tracking, enriching, and studying MGO-glycation to aid in understanding its underlying biochemical functions.

(De)Toxifying the Epigenetic Code.

Cells are continuously subjected to an array of reactive/toxic chemical species which are produced both endogenously through metabolic pathways and taken up exogenously by diet and exposure to drugs or toxins. As a result, proteins often undergo non-enzymatic covalent modifications (NECMs) by these species, which can alter protein structure, function, stability, and binding partner affinity. NECMs accumulate over time and are linked to various diseases such as Alzheimer's disease, cancer, and diabetes. In the cellular proteome, histones have some of the longest half-lives, making them prime targets for NECMs. In addition, histones have emerged as key regulators of transcription, a function that is primarily controlled by modification of their tails. These modifications are usually installed or removed enzymatically, but recent evidence suggests that some may also occur non-enzymatically. Despite the vast knowledge detailing the relationship between histone modifications and gene regulation, NECMs on histones remain poorly explored. A major reason for this difference stems from the fact that, unlike their enzymatically installed counterparts, NECMs are difficult to both control and test in vivo. Here, we review advances in our understanding of the effect non-enzymatic covalent modifications (NECMs) have on the epigenetic landscape, cellular fate, and their implications in disease. Cumulatively, this illustrates how the epigenetic code is directly toxified by chemicals and detoxified by corresponding eraser enzymes.

Insights into the thioamidation of thiopeptins to enhance the understanding of the biosynthetic logic of thioamide-containing thiopeptides.

Thiopeptins are a complex of thiopeptide antibiotics similar in structure to thiostrepton and harboring a thioamide, a rare moiety among natural products. Here, we illustrate through a series of in vivo experiments that the thioamide moiety of thiopeptins is generated posttranslationally by a TfuA-YcaO pair, encoded in the thiopeptin biosynthetic gene cluster, before the maturation of the thiopeptide bicyclic scaffold, enhancing the understanding of the biosynthetic logic of thioamide-containing thiopeptides.

An α/ß-hydrolase fold protein in the biosynthesis of thiostrepton exhibits a dual activity for endopeptidyl hydrolysis and epoxide ring opening/macrocyclization.

Thiostrepton (TSR), an archetypal bimacrocyclic thiopeptide antibiotic that arises from complex posttranslational modifications of a genetically encoded precursor peptide, possesses a quinaldic acid (QA) moiety within the side-ring system of a thiopeptide-characteristic framework. Focusing on selective engineering of the QA moiety, i.e., by fluorination or methylation, we have recently designed and biosynthesized biologically more active TSR analogs. Using these analogs as chemical probes, we uncovered an unusual indirect mechanism of TSR-type thiopeptides, which are able to act against intracellular pathogens through host autophagy induction in addition to direct targeting of bacterial ribosome. Herein, we report the accumulation of 6'-fluoro-7', 8'-epoxy-TSR, a key intermediate in the preparation of the analog 6'-fluoro-TSR. This unexpected finding led to unveiling of the TSR maturation process, which involves an unusual dual activity of TsrI, an α/ß-hydrolase fold protein, for cascade C-N bond cleavage and formation during side-ring system construction. These two functions of TsrI rely on the same catalytic triad, Ser72-His200-Asp191, which first mediates endopeptidyl hydrolysis that occurs selectively between the residues Met-1 and Ile1 for removal of the leader peptide and then triggers epoxide ring opening for closure of the QA-containing side-ring system in a regio- and stereo-specific manner. The former reaction likely requires the formation of an acyl-Ser72 enzyme intermediate; in contrast, the latter is independent of Ser72. Consequently, C-6' fluorination of QA lowers the reactivity of the epoxide intermediate and, thereby, allows the dissection of the TsrI-associated enzymatic process that proceeds rapidly and typically is difficult to be realized during TSR biosynthesis.

An enzymatic [4+2] cyclization cascade creates the pentacyclic core of pyrroindomycins.

The [4+2] cycloaddition remains one of the most intriguing transformations in synthetic and natural products chemistry. In nature, however, there are remarkably few enzymes known to have this activity. We herein report an unprecedented enzymatic [4+2] cyclization cascade that has a central role in the biosynthesis of pyrroindomycins, which are pentacyclic spirotetramate natural products. Beginning with a linear intermediate that contains two pairs of 1,3-diene and alkene groups, the dedicated cyclases PyrE3 and PyrI4 act in tandem to catalyze the formation of two cyclohexene rings in the dialkyldecalin system and the tetramate spiro-conjugate of the molecules. The two cyclizations are completely enzyme dependent and proceed in a regio- and stereoselective manner to establish the enantiomerically pure pentacyclic core. Analysis of a related spirotetronate pathway confirms that homologs are functionally exchangeable, establishing the generality of these findings and explaining how nature creates diverse active molecules with similar rigid scaffolds.

Post-translational modifications involved in the biosynthesis of thiopeptide antibiotics.

Thiopeptide antibiotics are a class of typical ribosomally synthesized and post-translationally modified peptides (RiPPs) with complex chemical structures that are difficult to construct via chemical synthesis. To date, more than 100 thiopeptides have been discovered, and most of these compounds exhibit remarkable biological activities, such as antibacterial, antitumor and immunosuppressive activities. Therefore, studies of the biosynthesis of thiopeptides can contribute to the development of new drug leads and facilitate the understanding of the complex post-translational modifications (PTMs) of peptides and/or proteins. Since the biosynthetic gene clusters of thiopeptides were first discovered in 2009, several research studies regarding the biochemistry and enzymology of thiopeptide biosyntheses have been reported, indicating that their characteristic framework is constructed via a cascade of common PTMs and that additional specific PTMs diversify the molecules. In this review, we primarily summarize recent advances in understanding the biosynthesis of thiopeptide antibiotics and propose some potential applications based on our insights into the biosynthetic logic and machinery.

Isolation and identification of l/d-lactate-conjugated bufadienolides from toad eggs revealing lactate racemization in amphibians.

Three pairs of bufadienolide l/d-lactate epimers (1-6) were isolated from the eggs of the toad Bufo bufo gargarizans. The structures were elucidated by using spectroscopic methods, X-ray diffraction analysis and a modified Mosher's method. Compounds 1-6 represent the first occurrence of lactate-conjugated bufadienolides in nature, and illustrate the existence of an enzyme-controlled epimerization from l- to d-lactate in amphibians. The biosynthetic pathways, in which two key enzymes might be involved (i.e., lactate racemase and acyltransferase), were proposed. In addition, the biological assays revealed that compounds 1-4 are potent cytotoxic agents against human gastric cancer cells BGC-823 and human lung cancer cells A549 with IC50 values in a range of 8.0 to 80.0 nM.

Bufospirostenin A and Bufogargarizin C, Steroids with Rearranged Skeletons from the Toad Bufo bufo gargarizans.

Bufospirostenin A (1) and bufogargarizin C (2), two novel steroids with rearranged A/B rings, were isolated from the toad Bufo bufo gargarizans. Compound 1 represents the first spirostanol found in animals. Compound 2 is an unusual bufadienolide with a cycloheptatriene B ring. Their structures were elucidated by spectroscopic analysis, single crystal X-ray diffraction analysis, and computational calculations.

A linear hydroxymethyl tetramate undergoes an acetylation-elimination process for exocyclic methylene formation in the biosynthetic pathway of pyrroindomycins.

We herein report the isolation and characterization of a key linear intermediate in the biosynthetic pathway of pyrroindomycins, the potent spirotetramate natural products produced by Streptomyces rugosporus. This polyene intermediate bears a γ-hydroxymethyl group that is exocyclic to the tetramate moiety, indicating that a serine residue serves as the three-carbon unit for tetramate formation and chain-elongation termination. The further conversion involves an acetylation-elimination of the exocyclic γ-hydroxymethyl group to generate a γ-methylene group, which is indispensable for intramolecular [4 + 2] cross-bridging to construct the characteristic pentacyclic core. The findings presented in this study provide new insights into the biosynthesis of pyrroindomycins, and thus suggest a common paradigm for both spirotetramates and spirotetronates in processing the exocyclic γ-hydroxymethyl group of the five-membered heterocycle.

Rational Control of Polyketide Extender Units by Structure-Based Engineering of a Crotonyl-CoA Carboxylase/Reductase in Antimycin Biosynthesis.

Bioengineering of natural product biosynthesis is a powerful approach to expand the structural diversity of bioactive molecules. However, in polyketide biosynthesis, the modification of polyketide extender units, which form the carbon skeletons, has remained challenging. Herein, we report the rational control of polyketide extender units by the structure-based engineering of a crotonyl-CoA carboxylase/reductase (CCR), in the biosynthesis of antimycin. Site-directed mutagenesis of the CCR enzyme AntE, guided by the crystal structure solved at 1.5 Šresolution, expanded its substrate scope to afford indolylmethylmalonyl-CoA by the V350G mutation. The mutant A182L selectively catalyzed carboxylation over the regular reduction. Furthermore, the combinatorial biosynthesis of heterocycle- and substituted arene-bearing antimycins was achieved by an engineered Streptomyces strain bearing AntE(V350G). These findings deepen our understanding of the molecular mechanisms of the CCRs, which will serve as versatile biocatalysts for the manipulation of building blocks, and set the stage for the rational design of polyketide biosynthesis.

Chemical proteomics approaches for protein post-translational modification studies.

The diversity and dynamics of proteins play essential roles in maintaining the basic constructions and functions of cells. The abundance of functional proteins is regulated by the transcription and translation processes, while the alternative splicing enables the same gene to generate distinct protein isoforms of different lengths. Beyond the transcriptional and translational regulations, post-translational modifications (PTMs) are able to further expand the diversity and functional scope of proteins. PTMs have been shown to make significant changes in the surface charges, structures, activation states, and interactome of proteins. Due to the functional complexity, highly dynamic nature, and low presence percentage, the study of protein PTMs remains challenging. Here we summarize and discuss the major chemical biology tools and chemical proteomics approaches to enrich and investigate the protein PTM of interest.

Chemical proteomic profiling of protein dopaminylation in colorectal cancer cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid residue of H3 ( i.e. , glutamine) and dopamine. In our previous studies, we discovered that the dynamics of this post-translational modification (including installation, removal, and replacement) were regulated by a single enzyme, transglutaminase 2 (TGM2), through reversible transamination. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of TGM2 in colon cancer cells. Due to the enzyme promiscuity of TGM2, non-histone proteins have also been identified as targets of dopaminylation on glutamine residues, however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of modification sites of these dopaminylated proteins were identified, attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that to block the installation of protein dopaminylation may become a promising anti-cancer strategy.