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Liver fibrosis is an urgent clinical problem without effective therapies. Here we conducted a high-content screening on a natural Euphorbiaceae diterpenoid library to identify a potent anti-liver fibrosis lead, 12-deoxyphorbol 13-palmitate (DP). Leveraging a photo-affinity labeling approach, apolipoprotein L2 (APOL2), an endoplasmic reticulum (ER)-rich protein, was identified as the direct target of DP. Mechanistically, APOL2 is induced in activated hepatic stellate cells upon transforming growth factor-ß1 (TGF-ß1) stimulation, which then binds to sarcoplasmic/ER calcium ATPase 2 (SERCA2) to trigger ER stress and elevate its downstream protein kinase R-like ER kinase (PERK)-hairy and enhancer of split 1 (HES1) axis, ultimately promoting liver fibrosis. As a result, targeting APOL2 by DP or ablation of APOL2 significantly impairs APOL2-SERCA2-PERK-HES1 signaling and mitigates fibrosis progression. Our findings not only define APOL2 as a novel therapeutic target for liver fibrosis but also highlight DP as a promising lead for treatment of this symptom.
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Electrophilic small molecules with novel reactivity are powerful tools that enable activity-based protein profiling and covalent inhibitor discovery. Here, we report a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine (CPzP) for selective modification of proteins via a nucleophilic aromatic substitution (SNAr) mechanism. Chemoproteomic profiling reveals that CPzPs engage cysteines within functionally diverse protein sites including ribosomal protein S5 (RPS5), inosine monophosphate dehydrogenase 2 (IMPDH2), and heat shock protein 60 (HSP60). Through the optimization of appended recognition elements, we demonstrate the utility of CPzP for covalent inhibition of prolyl endopeptidase (PREP) by targeting a noncatalytic active-site cysteine. This study suggests that the proteome reactivity of CPzPs can be modulated by both electronic and steric features of the ring system, providing a new tunable electrophile for applications in chemoproteomics and covalent inhibitor design.
Assuntos
Cisteína , Pirazóis , Piridinas , Piridinas/química , Piridinas/farmacologia , Cisteína/química , Pirazóis/química , Pirazóis/farmacologia , Humanos , Ligantes , Descoberta de DrogasRESUMO
The human acetyltransferase paralogues EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potencies of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize the binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of the relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.
Assuntos
Acetiltransferases , Lisina , Humanos , Preparações Farmacêuticas , Proteína p300 Associada a E1A , Proteína de Ligação a CREB/químicaRESUMO
Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.
Assuntos
Proteoma , Humanos , Acetilcoenzima A/química , LigantesRESUMO
The human acetyltransferase paralogs EP300 and CREBBP are master regulators of lysine acetylation whose activity has been implicated in various cancers. In the half-decade since the first drug-like inhibitors of these proteins were reported, three unique molecular scaffolds have taken precedent: an indane spiro-oxazolidinedione (A-485), a spiro-hydantoin (iP300w), and an aminopyridine (CPI-1612). Despite increasing use of these molecules to study lysine acetylation, the dearth of data regarding their relative biochemical and biological potencies makes their application as chemical probes a challenge. To address this gap, here we present a comparative study of drug-like EP300/CREBBP acetyltransferase inhibitors. First, we determine the biochemical and biological potencies of A-485, iP300w, and CPI-1612, highlighting the increased potency of the latter two compounds at physiological acetyl-CoA concentrations. Cellular evaluation shows that inhibition of histone acetylation and cell growth closely aligns with the biochemical potencies of these molecules, consistent with an on-target mechanism. Finally, we demonstrate the utility of comparative pharmacology by using it to investigate the hypothesis that increased CoA synthesis caused by knockout of PANK4 can competitively antagonize binding of EP300/CREBBP inhibitors and demonstrate proof-of-concept photorelease of a potent inhibitor molecule. Overall, our study demonstrates how knowledge of relative inhibitor potency can guide the study of EP300/CREBBP-dependent mechanisms and suggests new approaches to target delivery, thus broadening the therapeutic window of these preclinical epigenetic drug candidates.
RESUMO
Various proteins bind to chromatin to regulate DNA and its associated processes such as replication, transcription, and damage repair. The identification and characterization of these chromatin-associating proteins remain a challenge, as their interactions with chromatin often occur within the context of the local nucleosome or chromatin structure, which makes conventional peptide-based strategies unsuitable. Here, we developed a simple and robust protein labeling chemistry to prepare synthetic multifunctional nucleosomes that carry a photoreactive group, a biorthogonal handle, and a disulfide moiety to examine chromatin-protein interactions in a nucleosomal context. Using the prepared protein- and nucleosome-based photoaffinity probes, we examined a number of protein-protein and protein-nucleosome interactions. In particular, we (i) mapped the binding sites for the HMGN2-nucleosome interaction, (ii) provided the evidence for transition between the active and poised states of DOT1L in recognizing H3K79 within the nucleosome, and (iii) identified OARD1 and LAP2α as nucleosome acidic patch-associating proteins. This study provides powerful and versatile chemical tools for interrogating chromatin-associating proteins.
Assuntos
Cromatina , Nucleossomos , DNA/química , Fatores de Transcrição/metabolismo , Sítios de LigaçãoRESUMO
Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.
Assuntos
Epigênese Genética , Histonas , Lisina , Nucleossomos , Proteínas Proto-Oncogênicas , Cromatina/metabolismo , Microscopia Crioeletrônica , Histonas/química , Histonas/metabolismo , Metilação , Nucleossomos/química , Nucleossomos/metabolismo , Lisina/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Humanos , Animais , Sondas Moleculares/química , Processamento de Proteína Pós-TraducionalRESUMO
Covalent modifications of DNA and histones are key cellular epigenetic marks to regulate gene functions. Most of these epigenetic marks are added or removed by corresponding enzymes known as writers and erasers, whose catalytic activities normally rely on the presence of cellular metabolites as cofactors. Epigenetic marks can either directly alter the chromatin structure and dynamics through changing the intra-/internucleosomal histone-histone and histone-DNA interactions or recruit readers that further bring in other proteins with chromatin-modifying/remodeling activities to reshape the local and regional chromatin organization. In these two ways, epigenetic modifications modulate diverse DNA-templated processes, such as gene transcription, DNA replication, and DNA damage repair. Therefore, elucidation of the regulatory mechanisms and biological significance of epigenetic marks requires the identification and characterization of the protein-protein, protein-nucleic acid, and protein-small molecule interactions that control the underlying epigenetic processes. Here, we review the recent advances in using photo-cross-linking strategies to interrogate the epigenetic interactome, focusing on the protein-protein interactions mediated by epigenetic marks in histone tails. We also discuss future directions of developing photo-cross-linking-based tools and methods toward the investigation of the binding events in nucleosomal/chromatinic contexts, and toward the in situ capture of the epigenetic interactome in live cells or even organisms.
Assuntos
Epigênese Genética , Histonas , Histonas/química , Cromatina , Nucleossomos , DNA/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Posttranslational modifications (PTMs) of histones have been demonstrated to be the key regulating mechanism of nucleosome dynamics and chromatin structure. Lysine succinylation is a recently discovered PTM that plays critical roles in metabolism, epigenetic signaling, and is correlated with several diseases. One significant challenge in studying the effects of this modification on nucleosome dynamics is to obtain site-specifically modified histones. Here, we report the rapid site-specific incorporation of a succinylation mimic into histones, which facilitates the characterization of its impact on nucleosome dynamics with a Förster resonance energy transfer (FRET) approach.
Assuntos
Histonas , Nucleossomos , Transferência Ressonante de Energia de Fluorescência , Histonas/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
The nucleosome, the basic repeating unit of chromatin, is a dynamic structure that consists of DNA and histones. Insights derived from biochemical and biophysical approaches have revealed that histones posttranslational modifications (PTMs) are key regulators of nucleosome structure and dynamics. Mounting evidence suggests that the newly identified negatively charged histone lysine acylations play significant roles in altering nucleosome and chromatin dynamics, subsequently affecting downstream DNA-templated processes including gene transcription and DNA damage repair. Here, we present an overview of the dynamic changes of nucleosome and chromatin structures in response to negatively charged histone lysine acylations, including lysine malonylation, lysine succinylation, and lysine glutarylation.
RESUMO
Lysine malonylation is a recently characterized post-translational modification involved in the regulation of energy metabolism and gene expression. One unique feature of this post-translational modification is its potential susceptibility to decarboxylation, which poses possible challenges to its study. As a step towards addressing these challenges, we report the synthesis and evaluation of a stable isostere of malonyllysine. First, we find that synthetic substitution of the malonyl group with a tetrazole isostere results in amino acid's resistant to thermal decarboxylation. Next, we demonstrate that protected variants of this amino acid are readily incorporated into peptides. Finally, we show that tetrazole isosteres of malonyllysine can be recognized by anti-malonyllysine antibodies and histone deacylases, validating their ability to mimic features of the endogenous lysine modification. Overall, this study establishes a new chemical strategy for stably mimicking a metabolite-derived post-translational modification, providing a foothold for tool development and functional analyses.
Assuntos
Lisina/química , Tetrazóis/síntese química , Lisina/análogos & derivados , Conformação Molecular , Tetrazóis/químicaRESUMO
Lysine succinylation (Ksucc) is a novel posttranslational modification that frequently occurs on chromatin proteins including histones and non-histone proteins. Histone Ksucc affects nucleosome dynamics by increasing the DNA unwrapping rate and accessibility. However, very little is known about the regulation and functions of Ksucc located on non-histone chromosomal proteins. Here, we site-specifically installed a succinyl lysine analogue (Kcsucc) onto the non-histone chromosomal protein HMG-17 (HMGN2) to mimic the natural succinylated protein. We found that the incorporation of Kcsucc into HMGN2 at the K30 site (HMGN2Kc30succ), which is located within the nucleosome-binding domain (NBD), leads to significantly decreased HMGN2 binding to the mononucleosome. HMGN2Kc30succ also increased the nucleosomal DNA accessibility by promoting nucleosomal DNA unwrapping in the entry/exit region. This study reveals a novel mechanism of non-histone protein succinylation on altering chromatin recruitment, which can further affect nucleosome and chromatin dynamics.
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Remodelin is a putative small molecule inhibitor of the RNA acetyltransferase NAT10 which has shown preclinical efficacy in models of the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Here we evaluate remodelin's assay interference characteristics and effects on NAT10-catalyzed RNA cytidine acetylation. We find the remodelin chemotype constitutes a cryptic assay interference compound, which does not react with small molecule thiols but demonstrates protein reactivity in ALARM NMR and proteome-wide affinity profiling assays. Biophysical analyses find no direct evidence for interaction of remodelin with the NAT10 acetyltransferase active site. Cellular studies verify that N4-acetylcytidine (ac4C) is a nonredundant target of NAT10 activity in human cell lines and find that this RNA modification is not affected by remodelin treatment in several orthogonal assays. These studies display the potential for remodelin's chemotype to interact with multiple protein targets in cells and indicate remodelin should not be applied as a specific chemical inhibitor of NAT10-catalyzed RNA acetylation.
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Lysine succinylation is a recently discovered posttranslational modification that plays critical roles in metabolism, epigenetic signaling, and human diseases. To investigate the effects of site-specific histone lysine succinylation on nucleosome dynamics requires the generation of homogeneously modified histones, which is a significant challenge. Here, we report a protocol for the rapid site-specific installation of a succinyl lysine analog onto histone. We then use a Förster resonance energy transfer approach to characterize the impact on nucleosome dynamics. For complete details on the use and execution of this protocol, please refer to Jing et al. (2018).
Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Ácido Succínico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Chromatin-modifying lysine acetyltransferases employ multiple protein domains to regulate transcription. In this issue, Vannam et al. (2021) describe dCBP-1, a small molecule degrader of the multidomain acetyltransferases EP300 and CREBBP. This provides a new tool for interrogating EP300/CREBBP function and also suggests a strategy for pharmacological differentiation of acetyltransferase paralogs.
Assuntos
Acetiltransferases , Proteína de Ligação a CREB , Acetiltransferases/metabolismo , Proteína de Ligação a CREB/genética , Cromatina , Domínios ProteicosRESUMO
Chemical proteomics provides a powerful strategy for the high-throughput assignment of enzyme function or inhibitor selectivity. However, identifying optimized probes for an enzyme family member of interest and differentiating signal from the background remain persistent challenges in the field. To address this obstacle, here we report a physiochemical discernment strategy for optimizing chemical proteomics based on the coenzyme A (CoA) cofactor. First, we synthesize a pair of CoA-based sepharose pulldown resins differentiated by a single negatively charged residue and find this change alters their capture properties in gel-based profiling experiments. Next, we integrate these probes with quantitative proteomics and benchmark analysis of "probe selectivity" versus traditional "competitive chemical proteomics." This reveals that the former is well-suited for the identification of optimized pulldown probes for specific enzyme family members, while the latter may have advantages in discovery applications. Finally, we apply our anionic CoA pulldown probe to evaluate the selectivity of a recently reported small molecule N-terminal acetyltransferase inhibitor. These studies further validate the use of physical discriminant strategies in chemoproteomic hit identification and demonstrate how CoA-based chemoproteomic probes can be used to evaluate the selectivity of small molecule protein acetyltransferase inhibitors, an emerging class of preclinical therapeutic agents.
Assuntos
Acetiltransferases/química , Sondas Moleculares/química , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Reprodutibilidade dos Testes , Especificidade por Substrato , Espectrometria de Massas em Tandem/métodosRESUMO
Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.
Assuntos
DNA/metabolismo , Histonas/síntese química , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatina/química , Cromatina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histonas/genética , Lisina/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Posttranslational modifications of histones play key roles in the dynamic regulation of chromatin structure. Lysine succinylation is a new type of histone modification, but its biological significance in chromatin structure and dynamics remains unknown. Here we develop a chemical approach to site-specifically install a succinyl lysine analog into histones. This analog serves as an ideal structural and functional mimic to natural succinyl lysine. The incorporation of this succinylation mimic into histone H2B at lysine 34, a succinylation site at the nucleosomal DNA-histone interface, leads to significant decrease in nucleosome stability in vitro, which is consistent with the defects in chromatin structure of a budding yeast strain containing a lysine-to-glutamate mutation at the corresponding residue of yeast histone H2B. This study provides a simple method for the rapid generation of histones with site-specific succinylation mimics, and reveals novel regulatory mechanisms of histone succinylation in the dynamic organization of chromatin.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Succinatos/metabolismo , Cromatina/química , Cromatina/metabolismo , Histonas/química , Humanos , Lisina/química , Saccharomyces cerevisiae/metabolismo , Succinatos/químicaRESUMO
Polyethersulfone (PES) membranes with improved hemocompatibility were prepared via solid-liquid interface aminolysis and heparinization. Reactive amino groups were generated by immersing solid PES membranes in proper diamine solution. Heparin was covalent immobilized on the surface via amide bond. The feasibility of surface aminolysis for the introduction of amino groups and the effectiveness for further heparin immobilization were confirmed by surface group analysis. The effect of aminolysis time on surface amino group concentration and bulk mechanical properties was investigated. The surface amino group concentration determined the amount and bioactivity of immobilized heparin chains. SEM images suggested that both the aminolysis and heparinization reaction had little effect on the surface morphology of PES membranes. Contact angle measurement, surface charge analysis, protein and platelet adsorption/adhesion experiment were applied to study the surface properties. The results showed that the heparinized PES membranes displayed enhanced hydrophilicity and hemocompatibility, indicating potential application in blood purification and other blood contacting fields.