Crosstalk between Lys63- and Lys11-polyubiquitin signaling at DNA damage sites is driven by Cezanne.

The establishment of polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.

Histone H3 N-terminal mimicry drives a novel network of methyl-effector interactions.

The reader ability of PHD fingers is largely limited to the recognition of the histone H3 N-terminal tail. Distinct subsets of PHDs bind either H3K4me3 (a transcriptional activator mark) or H3K4me0 (a transcriptional repressor state). Structural studies have identified common features among the different H3K4me3 effector PHDs, including (1) removal of the initiator methionine residue of H3 to prevent steric interference, (2) a groove where arginine-2 binds, and (3) an aromatic cage that engages methylated lysine-4. We hypothesize that some PHDs might have the ability to engage with non-histone ligands, as long as they adhere to these three rules. A search of the human proteome revealed an enrichment of chromatin-binding proteins that met these criteria, which we termed H3 N-terminal mimicry proteins (H3TMs). Seven H3TMs were selected, and used to screen a protein domain microarray for potential effector domains, and they all had the ability to bind H3K4me3-interacting effector domains. Furthermore, the binding affinity between the VRK1 peptide and the PHD domain of PHF2 is ∼3-fold stronger than that of PHF2 and H3K4me3 interaction. The crystal structure of PHF2 PHD finger bound with VRK1 K4me3 peptide provides a molecular basis for stronger binding of VRK1 peptide. In addition, a number of the H3TMs peptides, in their unmethylated form, interact with NuRD transcriptional repressor complex. Our findings provide in vitro evidence that methylation of H3TMs can promote interactions with PHD and Tudor domain-containing proteins and potentially block interactions with the NuRD complex. We propose that these interactions can occur in vivo as well.

Protein domain microarrays as a platform to decipher signaling pathways and the histone code.

Signal transduction is driven by protein interactions that are controlled by posttranslational modifications (PTM). Usually, protein domains are responsible for "reading" the PTM signal deposited on the interacting partners. Protein domain microarrays have been developed as a high throughput platform to facilitate the rapid identification of protein-protein interactions, and this approach has become broadly used in biomedical research. In this review, we will summarize the history, development and applications of this technique, including the use of protein domain microarrays in identifying both novel protein-protein interactions and small molecules that block these interactions. We will focus on the approaches we use in the Protein Array and Analysis Core - the PAAC - at MD Anderson Cancer Center. We will also address the technical limitations and discuss future directions.

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.

Site-Specific Identification of Lysine Acetylation Stoichiometries in Mammalian Cells.

Functional characterization of the lysine acetylation pathway requires quantitative measurement of the modification abundance at the stoichiometry level. Here, we developed a systematic workflow for global untargeted identification of site-specific Lys acetylation stoichiometries in mammalian cells. Our strategy includes an optimized protocol for in vitro chemical labeling of unmodified lysine with stable isotope-encoded acetyl-NHS ester, deep proteomic profiling with a high resolution mass spectrometer, and a new software tool for quantitative analysis and stoichiometry determination. The workflow was validated using in vitro chemically labeled BSA and synthetic peptides with multiple Lys acetylations at various positions. In the proof-of-concept study, we applied the strategy to analyze the proteome of HeLa cells and determined the stoichiometries of over 600 acetylation sites with good reproducibility. Sodium butyrate treatment induced a significant increase of acetylation stoichiometries in HeLa cells. Analysis of site-specific stoichiometry dynamics revealed the coregulation of closely positioned acetylation sites on histones H3 and H4 upon treatment.

Study on the migration characteristics of bioaerosols and optimization of ventilation patterns in a negative pressure isolation ward considering different patient postures.

Due to the serious global harm caused by the outbreak of various viral infectious diseases, how to improve indoor air quality and contain the spread of infectious bioaerosols has become a popular research subject. Negative pressure isolation ward is a key place to prevent the spread of aerosol particles. However, there is still limited knowledge available regarding airflow patterns and bioaerosol diffusion behavior in the ward, which is not conducive to reducing the risk of cross-infection between health care workers (HCWs) and patients. In addition, ventilation layout and patient posture have important effects on aerosol distribution. In this study, the spatial and temporal characteristics as well as dispersion patterns of bioaerosols under different ventilation patterns in the ward were investigated using the computational fluid dynamics (CFD) technique. It is concluded that changes in the location of droplet release source due to different body positions of the patient have a significant effect on the bioaerosol distribution. After optimizing the layout arrangements of exhaust air, the aerosol concentration in the ward with the patient in both supine and sitting positions is significantly reduced with particle removal efficiencies exceeding 95%, that is, the ventilation performance is improved. Meanwhile, the proportion of aerosol deposition on all surfaces of the ward is decreased, especially the deposition on both the patient's body and the bed is less than 1%, implying that the risk of HCWs being infected through direct contact is reduced.

SPINDOC binds PARP1 to facilitate PARylation.

SPINDOC is tightly associated with the histone H3K4me3 effector protein SPIN1. To gain a better understanding of the biological roles of SPINDOC, we identified its interacting proteins. Unexpectedly, SPINDOC forms two mutually exclusive protein complexes, one with SPIN1 and the other with PARP1. Consistent with its ability to directly interact with PARP1, SPINDOC expression is induced by DNA damage, likely by KLF4, and recruited to DNA lesions with dynamics that follows PARP1. In SPINDOC knockout cells, the levels of PARylation are reduced, in both the absence and presence of DNA damage. The SPINDOC/PARP1 interaction promotes the clearance of PARP1 from damaged DNA, and also impacts the expression of known transcriptional targets of PARP1. To address the in vivo roles of SPINDOC in PARP1 regulation, we generate SPINDOC knockout mice, which are viable, but slightly smaller than their wildtype counterparts. The KO mice display reduced levels of PARylation and, like PARP1 KO mice, are hypersensitive to IR-induced DNA damage. The findings identify a SPIN1-independent role for SPINDOC in the regulation of PARP1-mediated PARylation and the DNA damage response.

Psychological effects of cognitive behavioral therapy on internet addiction in adolescents: A systematic review protocol.

BACKGROUND: In this study, we aim to assess the psychological effects of cognitive behavioral therapy (CBT) on internet addiction (IA) in adolescents. METHODS: This study will search the following databases of Cochrane Library, PUBMED, EMBASE, Scopus, Web of Science, PsycINFO, Chinese Biomedical Literature Database, and China National Knowledge Infrastructure. All these electronic databases will be searched from inception to the September 30, 2019 without any language limitation. Two authors will conduct study selection, data extraction, and study quality assessment, respectively. Any disagreements between 2 authors will be solved by a third author through discussion. Statistical analysis will be performed using RevMan 5.3 software. RESULTS: This study will investigate the psychological effects of CBT on IA in adolescents by measuring psychopathological symptoms, depression, anxiety, time spent on the internet (hours/day), and health-related quality of life. CONCLUSION: This study summarizes current evidence of CBT on IA in adolescents and may provide guidance for both intervention and future researches.PROSPERO registration number: PROSPERO CRD42019153290.

Inhalation exposure to cigarette smoke and inflammatory agents induces epigenetic changes in the lung.

Smoking-related lung tumors are characterized by profound epigenetic changes including scrambled patterns of DNA methylation, deregulated histone acetylation, altered gene expression levels, distorted microRNA profiles, and a global loss of cytosine hydroxymethylation marks. Here, we employed an enhanced version of bisulfite sequencing (RRBS/oxRRBS) followed by next generation sequencing to separately map DNA epigenetic marks 5-methyl-dC and 5-hydroxymethyl-dC in genomic DNA isolated from lungs of A/J mice exposed whole-body to environmental cigarette smoke for 10 weeks. Exposure to cigarette smoke significantly affected the patterns of cytosine methylation and hydroxymethylation in the lungs. Differentially hydroxymethylated regions were associated with inflammatory response/disease, organismal injury, and respiratory diseases and were involved in regulation of cellular development, function, growth, and proliferation. To identify epigenetic changes in the lung associated with exposure to tobacco carcinogens and inflammation, A/J mice were intranasally treated with the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the inflammatory agent lipopolysaccharide (LPS), or both. NNK alone caused minimal epigenetic alterations, while exposure either to LPS or NNK/LPS in combination led to increased levels of global cytosine methylation and formylation, reduced cytosine hydroxymethylation, decreased histone acetylation, and altered expression levels of multiple genes. Our results suggest that inflammatory processes are responsible for epigenetic changes contributing to lung cancer development.

The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas.

More than 90% of chondroblastomas contain a heterozygous mutation replacing lysine-36 with methionine-36 (K36M) in the histone H3 variant H3.3. Here we show that H3K36 methylation is reduced globally in human chondroblastomas and in chondrocytes harboring the same genetic mutation, due to inhibition of at least two H3K36 methyltransferases, MMSET and SETD2, by the H3.3K36M mutant proteins. Genes with altered expression as well as H3K36 di- and trimethylation in H3.3K36M cells are enriched in cancer pathways. In addition, H3.3K36M chondrocytes exhibit several hallmarks of cancer cells, including increased ability to form colonies, resistance to apoptosis, and defects in differentiation. Thus, H3.3K36M proteins reprogram the H3K36 methylation landscape and contribute to tumorigenesis, in part through altering the expression of cancer-associated genes.

Thrombolytic and Antiplatelet Effects of a Novel Plasminogen Activator from the Venom of Gloydius Brevicaudus Viper.

AIM: To investigate the thrombolytic and antiplatelet effects of a novel plasminogen activator from the venom of the Gloydius brevicaudus viper (GBV-PA) in vitro and in vivo. METHODS: Thrombolytic experiments were performed in rabbit models of ear vein thrombosis and carotid artery thrombosis and in dog model of acute cerebral infarction. Inhibition of thrombus formation was evaluated in rat inferior vena cava thrombosis model and ferric chloride-induced arterial thrombosis. In vitro, we assayed the antithrombotic effect of GBV-PA on rabbit blood clots, euglobulin lysis time (ELT) of rabbit plasma, and ADP-induced platelet aggregation. RESULTS: GBV-PA intravenous administration significantly reduced vascular recanalization times of rabbit ear veins thrombosis and thrombus weight of rabbit carotid artery thrombosis. The arterial recanalization rates were dose- and time-dependently improved after the administration of GBV-PA in canine acute cerebral infarction model. Thrombus length and weight were significantly reduced by GBV-PA both in rat inferior vena cava and ferric chloride-induced arterial thrombosis models. Thrombus formation in the blood of rabbits that were administered of GBV-PA was also inhibited. GBV-PA radically reduced plasma ELT of the rabbit's blood clots. ADP-induced platelet aggregation was inhibited by GBV-PA in a dose-dependent manner with a half-maximal inhibitory concentration of 19.9 µg/mL. CONCLUSION: This study demonstrates that GBV-PA is a thrombolytic and antiplatelet agent. It has significant antithrombotic effects on various in vitro and in vivo experimental models of thrombosis. The mechanisms that underline its antithrombotic effects were related to GBV-PA's capabilities of increasing fibrinolytic activity and inhibition of platelet aggregation.

Development of a model for predicting reaction rate constants of organic chemicals with ozone at different temperatures.

To assess the persistence and fate of volatile organic compounds in the troposphere, the rate constants for the reaction with ozone (kO3) are needed. As kO3 values are only available for hundreds of compounds, and experimental determination of kO3 is costly and time-consuming, it is of importance to develop predictive models on kO3. In this study, a total of 379 logkO3 values at different temperatures were used to develop and validate a model for the prediction of kO3, based on quantum chemical descriptors, Dragon descriptors and structural fragments. Molecular descriptors were screened by stepwise multiple linear regression, and the model was constructed by partial least-squares regression. The cross validation coefficient QCUM(2) of the model is 0.836, and the external validation coefficient Qext(2) is 0.811, indicating that the model has high robustness and good predictive performance. The most significant descriptor explaining logkO3 is the BELm2 descriptor with connectivity information weighted atomic masses. kO3 increases with increasing BELm2, and decreases with increasing ionization potential. The applicability domain of the proposed model was visualized by the Williams plot. The developed model can be used to predict kO3 at different temperatures for a wide range of organic chemicals, including alkenes, cycloalkenes, haloalkenes, alkynes, oxygen-containing compounds, nitrogen-containing compounds (except primary amines) and aromatic compounds.