Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from ...intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. ...Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Display omitted
•CDK9 inhibition reactivates epigenetically silenced genes in multiple cancer cells•CDK9 binds to and phosphorylates BRG1, which contributes to gene silencing•We developed a novel potent and selective CDK9 inhibitor, MC180295•MC180295 shows promising anti-tumoral and immunosensitization activity
Inhibition of a kinase typically associated with transcription elongation complexes leads to reactivation of tumor suppressor genes and increases sensitivity to immunotherapy in cancer models.
Over the last decade, numerous histone acyl post-translational modifications (acyl-PTMs) have been discovered, of which the functional significance is still under intense study. Here, we use ...high-resolution mass spectrometry to accurately quantify eight acyl-PTMs in vivo and after in vitro enzymatic assays. We assess the ability of seven histone acetyltransferases (HATs) to catalyze acylations on histones in vitro using short-chain acyl-CoA donors, proving that they are less efficient towards larger acyl-CoAs. We also observe that acyl-CoAs can acylate histones through non-enzymatic mechanisms. Using integrated metabolomic and proteomic approaches, we achieve high correlation (R
> 0.99) between the abundance of acyl-CoAs and their corresponding acyl-PTMs. Moreover, we observe a dose-dependent increase in histone acyl-PTM abundances in response to acyl-CoA supplementation in in nucleo reactions. This study represents a comprehensive profiling of scarcely investigated low-abundance histone marks, revealing that concentrations of acyl-CoAs affect histone acyl-PTM abundances by both enzymatic and non-enzymatic mechanisms.
Epigenetics has become a fundamental scientific discipline with various implications for biology and medicine. Epigenetic marks, mostly DNA methylation and histone post-translational modifications ...(PTMs), play important roles in chromatin structure and function. Accurate quantification of these marks is an ongoing challenge due to the variety of modifications and their wide dynamic range of abundance. Here we present EpiProfile 2.0, an extended version of our 2015 software (v1.0), for accurate quantification of histone peptides based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. EpiProfile 2.0 is now optimized for data-independent acquisition through the use of precursor and fragment extracted ion chromatography to accurately determine the chromatographic profile and to discriminate isobaric forms of peptides. The software uses an intelligent retention time prediction trained on the analyzed samples to enable accurate peak detection. EpiProfile 2.0 supports label-free and isotopic labeling, different organisms, known sequence mutations in diseases, different derivatization strategies, and unusual PTMs (such as acyl-derived modifications). In summary, EpiProfile 2.0 is a universal and accurate platform for the quantification of histone marks via LC–MS/MS. Being the first software of its kind, we anticipate that EpiProfile 2.0 will play a fundamental role in epigenetic studies relevant to biology and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family.
The monocytic leukemia zinc-finger protein-related factor (MORF) is a transcriptional coactivator and a catalytic subunit of the lysine acetyltransferase complex implicated in cancer and ...developmental diseases. We have previously shown that the double plant homeodomain finger (DPF) of MORF is capable of binding to acetylated histone H3. Here we demonstrate that the DPF of MORF recognizes many newly identified acylation marks. The mass spectrometry study provides comprehensive analysis of H3K14 acylation states in vitro and in vivo. The crystal structure of the MORF DPF-H3K14butyryl complex offers insight into the selectivity of this reader toward lipophilic acyllysine substrates. Together, our findings support the mechanism by which the acetyltransferase MORF promotes spreading of histone acylation.
Display omitted
•The DPF domain of the MORF KAT is a reader of global histone H3K14 acylation•The structure of the MORF DPF-H3K14bu complex offers insight into selectivity•MS analysis of H3K14 acylation states in vivo and in vitro is described•Findings support the mechanism by which the MORF KAT promotes spreading of acylation
Growing evidence suggests the important role of the lysine acylation states in metabolic pathways and epigenetic signaling. Klein et al. identified the DPF domain of the lysine acetyltransferase MORF as a reader of global histone H3K14 acylation.
Purpose
To identify the perspectives from healthcare providers about the limitations in referral, diagnosis, and treatment of lung cancer (LC) patients.
Methods
A cross-sectional study through an ...Internet-based survey was addressed to physicians of multidisciplinary teams in charge of LC patients from Cuba, Curacao, Costa Rica, Dominican Republic, El Salvador, Guatemala, Honduras, Jamaica, Panama, and Trinidad and Tobago. The questions focused on physicians’ perspectives concerning waiting times and the availability of diagnostic and staging procedures in their settings, as well as the access to systemic therapies and continuous medical education (CME).
Results
A total of 152 physicians responded to the online questionnaire (response rate 24.9%). Delays in biopsy results were the main barrier for LC diagnosis as identified by 48.2% of the respondents, followed by patients not being referred in time (31.3%), delays for staging procedures (11.4%), and time taken for biopsy (9%). Almost one-half of physicians perceived that patients are diagnosed in advanced stages. A total of 29 respondent physicians (19.1%) reported limited access to immunohistochemical or genetic analysis for common mutations. Although 73 physicians (48.0%) confirmed that their centers provided radiotherapy and systemic therapy for their patients, immunotherapy was not available in the institutions of 30 physicians (19.7%). A total of 42 practitioners (27.6%) reported that they did not have access to CME on LC topics due to working or budget restrictions.
Conclusions
This study revealed among respondents the main barriers for an appropriate management of LC patients in the Central American and Caribbean Region. Further studies must validate these findings.
Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is ...crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents.
The chromatin fiber is the control panel of eukaryotic cells. Chromatin is mostly composed of DNA, which contains the genetic instruction for cell phenotype, and histone proteins, which provide the ...scaffold for chromatin folding and part of the epigenetic inheritance. Histone writers/erasers “flag” chromatin regions by catalyzing/removing covalent histone post‐translational modifications (PTMs). Histone PTMs chemically contribute to chromatin relaxation or compaction and recruit histone readers to modulate DNA readout. The precursors of protein PTMs are mostly small metabolites. For instance, acetyl‐CoA is used for acetylation, ATP for phosphorylation, and S‐adenosylmethionine for methylation. Interestingly, PTMs such as acetylation can occur at neutral pH also without their respective enzyme when the precursor is sufficiently concentrated. Therefore, it is essential to differentially quantify the contribution of histone writers/erasers versus the effect of local concentration of metabolites to understand the primary regulation of histone PTM abundance. Aberrant phenotypes such as cancer cells have misregulated metabolism and thus the composition and the modulation of chromatin is not only driven by enzymatic tuning. In this review, the latest advances in mass spectrometry (MS) to analyze histone PTMs and the most adopted quantification methods for related metabolites, both necessary to understand PTM relative changes, are discussed.
Label-free peptide quantification in liquid chromatography–mass spectrometry (LC–MS) proteomics analyses is complicated by the presence of isobaric coeluting peptides, as they generate the same ...extracted ion chromatogram corresponding to the sum of their intensities. Histone proteins are especially prone to this, as they are heavily modified by post-translational modifications (PTMs). Their proteolytic digestion leads to a large number of peptides sharing the same mass, while carrying PTMs on different amino acid residues. We present an application of MS data-independent acquisition (DIA) to confidently determine and quantify modified histone peptides. By introducing the use of low-resolution MS/MS DIA, we demonstrate that the signals of 111 histone peptides could easily be extracted from LC–MS runs due to the relatively low sample complexity. By exploiting an LTQ-Orbitrap mass spectrometer, we parallelized MS and MS/MS scan events using the Orbitrap and the linear ion trap, respectively, decreasing the total scan time. This, in combination with large windows for MS/MS fragmentation (50 m/z) and multiple full scan events within a DIA duty cycle, led to a MS scan cycle speed of ∼45 full MS per minute, improving the definition of extracted LC–MS chromatogram profiles. By using such acquisition method, we achieved highly comparable results to our optimized acquisition method for histone peptide analysis (R 2 correlation > 0.98), which combines data-dependent acquisition (DDA) and targeted MS/MS scans, the latter targeting isobaric peptides. By using DIA, we could also remine our data set and quantify 16 additional isobaric peptides commonly not targeted during DDA experiments. Finally, we demonstrated that by performing the full MS scan in the linear ion trap, we achieve highly comparable results as when adopting high-resolution MS scans (R 2 correlation 0.97). Taken together, results confirmed that histone peptide analysis can be performed using DIA and low-resolution MS with high accuracy and precision of peptide quantification. Moreover, DIA intrinsically enables data remining to later identify and quantify isobaric peptides unknown at the time of the LC–MS experiment. These methods will open up epigenetics analyses to the proteomics community who do not have routine access to the newer generation high-resolution MS/MS generating instruments.
Context: Malaria is still a major public health problem. The biodiversity of the tropics is extremely rich and represents an invaluable source of novel bioactive molecules. For screening of this ...diversity more sensitive and economical in vitro methods are needed, Flora of Panama has been studied based on ethnomedical uses for discovering antimalarial compounds.
Objective: This review aims to provide an overview of in vitro screening methodologies for antimalarial drug discovery and to present results of this effort in Panama during the last quarter century.
Methods: A literature search in SciFinder and PubMed and original publications of Panamanian scientists was performed to gather all the information on antimalarial drug discovery from the Panamanian flora and in vitro screening methods.
Results and conclusions: A variety of colorimetric, staining, fluorometric, and mass spectrometry and radioactivity-based methods have been provided. The advantages and limitations of these methods are also discussed. Plants used in ethnomedicine for symptoms of malaria by three native Panamanian groups of Amerindians, Kuna, Ngöbe Buglé and Teribes are provided. Seven most active plants with IC50 values < 10 μg/mL were identified Talisia nervosa Radlk. (Sapindaceae), Topobea parasitica Aubl.(Melastomataceae), Monochaetum myrtoideum Naudin (Melastomataceae), Bourreria spathulata (Miers) Hemsl.(Boraginaceae), Polygonum acuminatum Kunth (Polygonaceae), Clematis campestris A. St.-Hil. (Ranunculaceae) and Terminalia triflora (Griseb.) Lillo (Combretaceae). Thirty bioactive compounds belonging to a variety of chemical classes such as spermine and isoquinoline alkaloids, glycosylflavones, phenylethanoid glycosides, ecdysteroids, quercetin arabinofuranosides, clerodane-type diterpenoids, sipandinolid, galloylquercetin derivatives, gallates, oleamide and mangiferin derivatives.
PDF
