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1.
J Proteomics ; 215: 103669, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31987925

RESUMO

The selection of a data processing method for use in mass spectrometry-based label-free proteome quantification contributes significantly to its accuracy and precision. In this study, we comprehensively evaluated 7 commonly-used label-free quantification methods (MaxQuant-Spectrum count, MaxQuant-iBAQ, MaxQuant-LFQ, MaxQuant-LFAQ, Proteome Discoverer, MetaMorpheus, TPP-StPeter) with a focus on missing values, precision, accuracy, selectivity, and reproducibility of low abundance protein quantification in both single shot and fractionation. Our results showed that among the tested strategies, MaxQuant in MaxLFQ mode outperformed other strategies in terms of accuracy and precision in both whole proteome and low abundance proteome quantification, whereas the Proteome Discoverer (PD) strategy using SEQUEST as a search engine performed better in terms of quantifiable low abundance proteome coverage. We subsequently applied the PD and MaxLFQ strategies in a blood proteomic dataset and found that many FDA-approved tumor prognostic biomarkers could be identified as well as quantified using the PD strategy, indicating the potential advantage of PD in label-free quantification studies. These results provide a reference for method choice in label-free quantification data analysis. SIGNIFICANCE: Mass spectrometry-based label-free quantification methods play an important role in label-free proteome data analysis. In this study, we evaluated 7 commonly-used label-free quantification methods with respect to the following aspects: missing values, precision, accuracy, selectivity, and reproducibility for low abundance protein quantification. The results showed that, among the strategies evaluated, the PD strategy with SEQUEST as a search engine performed better in terms of low abundance protein coverage. This study provides a reference for method choice in label-free quantification data analysis.

2.
Rapid Commun Mass Spectrom ; 34(2): e8573, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31484223

RESUMO

RATIONALE: Lys-N, also known as lysine-specific metalloendopeptidase, functions as the "sister" enzyme of lysyl endopeptidase (Lys-C) in proteomic research. Its digestion specificity at the N-terminal lysine residue makes it a very useful tool in proteomics analysis, especially in mass spectrometry (MS)-based de novo sequencing of proteins. METHODS: Here we present a complete production process of highly purified Lys-N from dry fruit of Grifola frondosa (maitake mushroom). The purification process includes one step of microfiltration plus one step of UF/DF (ultrafiltration used in tandem with a diafiltration method) recovery and four steps of chromatographic purification. RESULTS: The overall yield of the process was approximately 6.7 mg Lys-N protein/kg dry fruit of G. frondosa. The assay data demonstrated that the purified Lys-N exhibited high enzymatic activity and specificity. CONCLUSIONS: The novel production process provides for the first time the extraction of Lys-N from dry fruit of G. frondosa. The process is also stable and scalable, and provides an economic way of producing the enzyme in large quantities for MS-based proteomics and other biological studies.

3.
J Proteomics ; 210: 103545, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31626998

RESUMO

Targeting specific ubiquitin E3 ligase for degradation of disease-driven protein has recently been an important concept for cancer therapy, as exemplified by the case of thalidomide for the treatment of multiple myeloma. E7070, an aryl sulfonamide drug, exhibited anticancer activity by targeting the E3 ligase receptor DCAF15, with RBM39 as the only known substrate. Exploration of additional substrates of E7070 would facilitate elucidation of its mechanism of actions. To this end, we used a strategy combing pSILAC method with two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase) to accurately monitor the protein turnover and increase the depth of proteome profiling. Systematically, we showed that E7070 treatment changed turnover rates of 868 proteins (1.5 fold change and p-value <.05). Several proteins displayed accelerated turnover indicating they were potential new substrates of E7070, among which, pre-mRNA splicing factor 39 (PRPF39) had been reported to be overexpressed in certain cancers. We further demonstrated that PRPF39 was ubiquitinated and degraded by E7070 in a DCAF15-dependent manner, and represented a new bona fide substrate of E7070. The degradation of PRPF39 might also be contributed to the anticancer activity of E7070. SIGNIFICANCE: Identification of degraded substrates is difficult because protein abundance is a comprehensive result regulated by protein production and degradation at the same time. Pulsed SILAC (pSILAC), a method to measure protein turnover, would provide higher sensitivity than total protein quantification. In addition, some peptide sequences are not amenable to MS analysis after LysC-Trypsin digestion. LysN-LysargiNase, as a mirror protease combination of LysC-Trypsin, can be complementary for peptide identification with LysC-Trypsin. By combining pSILAC with two complementary digestion approaches (LysC-Trypsin and LysN-LysArgiNase), we systematically investigated E7070-dependent protein degradation. As a result, we found several potential degradation substrates of E7070 including PRPF39. Further, by exploiting a series of biological assays, we demonstrated that E7070 can lead to the ubiquitination and proteasomal degradation of PRPF39 by promoting the recruitment of PRPF39 to the CUL4-DCAF15 E3 ubiquitin ligase.

4.
J Mass Spectrom ; 55(1): e4441, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31840882

RESUMO

Column heating strategy is often applied in nano-high-performance liquid chromatography-mass spectrometer (nanoHPLC-MS) platform for enhancing the analytical efficiency of peptides or proteins. Nonetheless, the influence effects of column heating in peptides or proteins identification still lack of deep understanding. In this study, a systematic comparison of room temperature (RT) and column heating of nanoHPLC was done. Based on the data, under column heating condition, the backpressure of nanoHPLC can be decreased. Due to the increase of resolution, the peak widths of precursor ion were narrowed. As a result, in MS/MS data acquisition part, more time was spared for MS1 detecting and MS2 fragmenting, which eventually resulted in increased identification of peptides and proteins. Moreover, we also proposed the application scope of column heating by evaluating its influence on sample detection. On one hand, column heating significantly increased the identification of membrane proteins due to more efficient elution of highly hydrophobic peptides compared with RT. On the other hand, heating was not suitable for analyzing short or/and hydrophilic peptides with low retention time, which would be eluted out during sample loading process under high temperature and missed by mass spectrometric detection. In conclusion, our study provides a reference for rational application of column heating in proteomics research.

5.
J Proteomics ; 213: 103614, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31846764

RESUMO

Lysine methylation is a widespread protein post-translational modification showing essentialities in versatile cellular process. EZH2, a methyltransferase specifically trimethylates the lysine 27 of histone H3 and its aberrance in several cancers promotes the development of its inhibitors against hematological tumors. In this study, we presented a deep exploration of lysine mono-, di- and trimethylomes in EZH2 wild-type and Y641 mutant lymphoma cell lines. Our results showed that several substrates were modified in different methylation levels. Moreover, these methylated lysine residues could also undergo other types of PTMs. Combined with the differences proved in protein expression, lysine acetylation, lysine ubiquitylation and protein N-termianl acetylation level, our study underlined the substrate specificity of lysine methylation and its crosstalk with other types of PTMs. Totally, our study raised new insights into the global cellular methylation features in hematological cell lines, which provided further inspects into the distribution and function of lysine methylation. SIGNIFICANCE: Our study showed the global landscape of mono-, di- and trimethylomes in the EZH2-aberrant DLBCL cell lines, revealing the molecular characteristics of lysine methylation. Combined with the protein abundance and potential crosstalk among different types of PTMs, our study raised new insights into the global cellular methylation features in hematological tumors and provided further inspects into the distribution and function of lysine methylation.

6.
Anal Chem ; 91(22): 14522-14529, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31634432

RESUMO

Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.

7.
Proc Natl Acad Sci U S A ; 116(43): 21732-21738, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31594848

RESUMO

Endoplasmic reticulum (ER) stress plays an important role in metabolic diseases like obesity and type 2 diabetes mellitus (T2DM), although the underlying mechanisms and regulatory pathways remain to be elucidated. Here, we induced chronic low-grade ER stress in lean mice to levels similar to those in high-fat diet (HFD)-fed obese mice and found that it promoted hyperglycemia due to enhanced hepatic gluconeogenesis. Mechanistically, sustained ER stress up-regulated the deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14), which increased the stability and levels of 3',5'-cyclic monophosphate-responsive element binding (CREB) protein (CBP) to enhance glucagon action and hepatic gluconeogenesis. Exogenous overexpression of USP14 in the liver significantly increased hepatic glucose output. Consistent with this, liver-specific knockdown of USP14 abrogated the effects of ER stress on glucose metabolism, and also improved hyperglycemia and glucose intolerance in obese mice. In conclusion, our findings show a mechanism underlying ER stress-induced disruption of glucose homeostasis, and present USP14 as a potential therapeutic target against T2DM.

8.
J Proteome Res ; 18(10): 3762-3769, 2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31483678

RESUMO

Lysine succinylation (Ksu) is a novel identified post-translational modification that is conserved from prokaryotes to eukaryotes. As a kind of acylation, Ksu was reported to have different functions than other acylations at lysine residues. However, recent studies on Ksu have mainly focused on plants and bacteria. Ksu studies in vertebrates are still rare; thus, the biological function of Ksu in mammals needs to be studied further. In this study, we performed global Ksu mapping in Danio rerio (zebrafish) using mass spectrometry-based proteomics with the enrichment of Ksu peptides by immunoprecipitation technology. As a result, we identified 552 Ksu sites in 164 proteins. The raw data are available via ProteomeXchange with the identifier PXD013173. Compared with our previous studies on lysine acetylation and crotonylation, Ksu plays a major role in diverse metabolic processes such as carbon metabolism and the tricarboxylic acid circle. In addition, we defined five new succinylation motifs: (su)KA, (su)KxxxxA, (su)KxxxxL, (su)KxA, and (su)KxV. In conclusion, our results provide a proteome-wide database to study Ksu in zebrafish, and our bioinformatics results facilitated the understanding of the role of Ksu in central metabolism.

9.
EMBO J ; 38(18): e100948, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31418899

RESUMO

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.


Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Sirtuínas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , GMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Análise Serial de Proteínas/métodos , Proteômica/métodos , Sistemas do Segundo Mensageiro
10.
J Med Chem ; 62(16): 7473-7488, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31335138

RESUMO

Alterations of fibroblast growth factor receptors (FGFRs) play key roles in numerous cancer progression and development, which makes FGFRs attractive targets in the cancer therapy. In the present study, based on a newly devised FGFR target-specific scoring function, a novel FGFR inhibitor hit was identified through virtual screening. Hit-to-lead optimization was then performed by integrating molecular docking and site-of-metabolism predictions with an array of in vitro evaluations and X-ray cocrystal structure determination, leading to a covalent FGFR inhibitor 15, which showed a highly selective and potent FGFR inhibition profile. Pharmacokinetic assessment, protein kinase profiling, and hERG inhibition evaluation were also conducted, and they confirmed the value of 15 as a lead for further investigation. Overall, this study exemplifies the importance of the integrative use of computational methods and experimental techniques in drug discovery.

11.
Haematologica ; 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289198

RESUMO

Aberrant expression of CDK9/cyclin T1 has been found in diffuse large B-cell lymphoma, and suggest that CDK9 seems to be a potential therapeutic target for diffuse large B-cell lymphoma. Here, we firstly demonstrated that CDKI-73, a novel CDK inhibitor, potently blocks CDK9, triggered apoptosis and dramatically repressed diffuse large B-cell lymphoma cell growth owing to CDK9 inhibition. CDK9 inhibitors specifically elevated the trimethylation of H3K27, which we speculate was due to reduced expression of JMJD3/UTX. Considering the important role of the trimethylation of H3K27 in tumor progression, the synergistic effect of the combination therapy of CDK9 inhibitors with EZH2 inhibitors was investigated. EZH2 inhibitors reversed the upregulation of trimethylation of H3K27, and synergistically inhibited diffuse large B-cell lymphoma and other solid tumors growth in vitro and in vivo. These findings provide a rational basis for the application of CDK9 inhibitors in combination with EZH2 inhibitors in clinical trials.

12.
Nat Commun ; 10(1): 2701, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221965

RESUMO

One of the biggest hurdles for the development of metabolism-targeted therapies is to identify the responsive tumor subsets. However, the metabolic vulnerabilities for most human cancers remain unclear. Establishing the link between metabolic signatures and the oncogenic alterations of receptor tyrosine kinases (RTK), the most well-defined cancer genotypes, may precisely direct metabolic intervention to a broad patient population. By integrating metabolomics and transcriptomics, we herein show that oncogenic RTK activation causes distinct metabolic preference. Specifically, EGFR activation branches glycolysis to the serine synthesis for nucleotide biosynthesis and redox homeostasis, whereas FGFR activation recycles lactate to fuel oxidative phosphorylation for energy generation. Genetic alterations of EGFR and FGFR stratify the responsive tumors to pharmacological inhibitors that target serine synthesis and lactate fluxes, respectively. Together, this study provides the molecular link between cancer genotypes and metabolic dependency, providing basis for patient stratification in metabolism-targeted therapies.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Mutação com Ganho de Função , Perfilação da Expressão Gênica/métodos , Glicólise/efeitos dos fármacos , Glicólise/genética , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Metabolômica/métodos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Seleção de Pacientes , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Serina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Cell Mol Med ; 23(6): 4301-4312, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30993883

RESUMO

Aberrant activation of the signal transducer and activator of transcription 3 (STAT3) and the nuclear factor-κB (NF-κB) signalling pathways is associated with the development of cancer and inflammatory diseases. JAKs and IKKs are the key regulators in the STAT3 and NF-κB signalling respectively. Therefore, the two families of kinases have been the major targets for developing drugs to regulate the two signalling pathways. Here, we report a natural compound xanthatin from the traditional Chinese medicinal herb Xanthium L. as a potent inhibitor of both STAT3 and NF-κB signalling pathways. Our data demonstrated that xanthatin was a covalent inhibitor and its activities depended on its α-methylene-γ-butyrolactone group. It preferentially interacted with the Cys243 of JAK2 and the Cys412 and Cys464 of IKKß to inactivate their activities. In doing so, xanthatin preferentially inhibited the growth of cancer cell lines that have constitutively activated STAT3 and p65. These data suggest that xanthatin may be a promising anticancer and anti-inflammation drug candidate.

14.
Proteomics ; 19(9): e1800471, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30864180

RESUMO

Histidine phosphorylation is a reversible post-translational modification that is known to regulate signal transduction in prokaryotes. However, functional studies in eukaryotes have been largely neglected due to the labile nature of N-linked phosphorylated amino acids. In an effort to help elucidate the heretofore hidden vertebrate phosphoproteome, this report presents a global phosphorylation analysis of Danio rerio (zebrafish) larvae. Phosphopeptide enrichment is performed using a TiO2 affinity technique. A total of 68 unique phosphohistidine sites are detected on 63 proteins among 1076 unique phosphosites on 708 proteins. Data are available via ProteomeXchange with identifier PXD012735. This report provides the first phosphohistidine dataset obtained from zebrafish.

15.
Nucleic Acids Res ; 47(9): e52, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30805613

RESUMO

The architecture and function of chromatin are largely regulated by local interacting molecules, such as transcription factors and noncoding RNAs. However, our understanding of these regulatory molecules at a given locus is limited because of technical difficulties. Here, we describe the use of Clustered Regularly Interspaced Short Palindromic Repeats and an engineered ascorbate peroxidase 2 (APEX2) system to investigate local chromatin interactions (CAPLOCUS). We showed that with specific small-guide RNA targets, CAPLOCUS could efficiently identify both repetitive genomic regions and single-copy genomic locus with high resolution. Genome-wide sequencing revealed known and potential long-range chromatin interactions for a specific single-copy locus. CAPLOCUS also identified telomere-associated RNAs. CAPLOCUS, followed by mass spectrometry, identified both known and novel telomere-associated proteins in their native states. Thus, CAPLOCUS may be a useful approach for studying local interacting molecules at any given chromosomal location.


Assuntos
Cromatina/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , RNA Guia/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sistemas CRISPR-Cas/genética , Imunoprecipitação da Cromatina , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Endonucleases , Genoma/genética , Genômica , Células HEK293 , Humanos , Enzimas Multifuncionais , Engenharia de Proteínas , RNA Guia/química , Telômero/genética , Fatores de Transcrição/genética
16.
Mol Cell Proteomics ; 18(2): 391-405, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30420486

RESUMO

The open (mass tolerant) search of tandem mass spectra of peptides shows great potential in the comprehensive detection of post-translational modifications (PTMs) in shotgun proteomics. However, this search strategy has not been widely used by the community, and one bottleneck of it is the lack of appropriate algorithms for automated and reliable post-processing of the coarse and error-prone search results. Here we present PTMiner, a software tool for confident filtering and localization of modifications (mass shifts) detected in an open search. After mass-shift-grouped false discovery rate (FDR) control of peptide-spectrum matches (PSMs), PTMiner uses an empirical Bayesian method to localize modifications through iterative learning of the prior probabilities of each type of modification occurring on different amino acids. The performance of PTMiner was evaluated on three data sets, including simulated data, chemically synthesized peptide library data and modified-peptide spiked-in proteome data. The results showed that PTMiner can effectively control the PSM FDR and accurately localize the modification sites. At 1% real false localization rate (FLR), PTMiner localized 93%, 84 and 83% of the modification sites in the three data sets, respectively, far higher than two open search engines we used and an extended version of the Ascore localization algorithm. We then used PTMiner to analyze a draft map of human proteome containing 25 million spectra from 30 tissues, and confidently identified over 1.7 million modified PSMs at 1% FDR and 1% FLR, which provided a system-wide view of both known and unknown PTMs in the human proteome.


Assuntos
Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Bases de Dados de Proteínas , Humanos , Ferramenta de Busca , Software
17.
Nat Commun ; 9(1): 4770, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425250

RESUMO

Ubiquitin-specific protease 14 (USP14) is one of the major proteasome-associated deubiquitinating enzymes critical for proteome homeostasis. However, substrates of USP14 remain largely unknown, hindering the understanding of its functional roles. Here we conduct a comprehensive proteome, ubiquitinome and interactome analysis for USP14 substrate screening. Bioinformatics analysis reveals broad new potential roles of USP14, especially in lipid and carbohydrate metabolism. Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14. USP14 directly interacts with and increases FASN stability. As a result, overexpression of USP14 promotes liver triglyceride accumulation in C57BL/6 mice, whereas genetic ablation or pharmacological inhibition of USP14 ameliorates hepatosteatosis, hyperglycemia and insulin resistance in obese mice. In conclusion, our findings reveal for the first time an indispensable role of USP14 in hepatosteatosis through FASN stabilization.


Assuntos
Ácido Graxo Sintases/metabolismo , Proteoma , Ubiquitina Tiolesterase/metabolismo , Animais , Metabolismo dos Carboidratos , Biologia Computacional , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Hiperglicemia , Resistência à Insulina , Lipogênese , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Triglicerídeos/análise , Ubiquitina Tiolesterase/genética , Regulação para Cima
18.
Nat Chem Biol ; 14(12): 1118-1126, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30374165

RESUMO

SIRT6, a member of the SIRT deacetylase family, is responsible for deacetylation of histone H3 Nε-acetyl-lysines 9 (H3K9ac) and 56 (H3K56ac). As a tumor suppressor, SIRT6 has frequently been found to have low expression in various cancers. Here, we report the identification of MDL-800, a selective SIRT6 activator. MDL-800 increased the deacetylase activity of SIRT6 by up to 22-fold via binding to an allosteric site; this interaction led to a global decrease in H3K9ac and H3K56ac levels in human hepatocellular carcinoma (HCC) cells. Consequently, MDL-800 inhibited the proliferation of HCC cells via SIRT6-driven cell-cycle arrest and was effective in a tumor xenograft model. Together, these data demonstrate that pharmacological activation of SIRT6 is a potential therapeutic approach for the treatment of HCC. MDL-800 is a first-in-class small-molecule cellular SIRT6 activator that can be used to physiologically and pathologically investigate the roles of SIRT6 deacetylation.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Sirtuínas/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Cristalografia por Raios X , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Sirtuínas/química , Sirtuínas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Cell Rep ; 24(13): 3477-3487.e6, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30257209

RESUMO

Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome.


Assuntos
Histonas/metabolismo , Infertilidade Masculina/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Espermatozoides/metabolismo , Acetilação , Animais , Código das Histonas , Histonas/química , Masculino , Camundongos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Ligação Proteica , Espermatogênese , Xenopus , Fatores de Transcrição de p300-CBP/metabolismo
20.
Cell ; 175(1): 186-199.e19, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30220457

RESUMO

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Histona-Lisina N-Metiltransferase/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Animais , Carcinogênese/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Epigênese Genética/genética , Epigenômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Código das Histonas/efeitos dos fármacos , Código das Histonas/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Ativação Transcricional , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Fatores de Transcrição de p300-CBP/fisiologia
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