DeepHBV: a deep learning model to predict hepatitis B virus (HBV) integration sites.

BACKGROUND: The hepatitis B virus (HBV) is one of the main causes of viral hepatitis and liver cancer. HBV integration is one of the key steps in the virus-promoted malignant transformation. RESULTS: An attention-based deep learning model, DeepHBV, was developed to predict HBV integration sites. By learning local genomic features automatically, DeepHBV was trained and tested using HBV integration site data from the dsVIS database. Initially, DeepHBV showed an AUROC of 0.6363 and an AUPR of 0.5471 for the dataset. The integration of genomic features of repeat peaks and TCGA Pan-Cancer peaks significantly improved model performance, with AUROCs of 0.8378 and 0.9430 and AUPRs of 0.7535 and 0.9310, respectively. The transcription factor binding sites (TFBS) were significantly enriched near the genomic positions that were considered. The binding sites of the AR-halfsite, Arnt, Atf1, bHLHE40, bHLHE41, BMAL1, CLOCK, c-Myc, COUP-TFII, E2A, EBF1, Erra, and Foxo3 were highlighted by DeepHBV in both the dsVIS and VISDB datasets, revealing a novel integration preference for HBV. CONCLUSIONS: DeepHBV is a useful tool for predicting HBV integration sites, revealing novel insights into HBV integration-related carcinogenesis.

DeepEBV: a deep learning model to predict Epstein-Barr virus (EBV) integration sites.

MOTIVATION: Epstein-Barr virus (EBV) is one of the most prevalent DNA oncogenic viruses. The integration of EBV into the host genome has been reported to play an important role in cancer development. The preference of EBV integration showed strong dependence on the local genomic environment, which enables the prediction of EBV integration sites. RESULTS: An attention-based deep learning model, DeepEBV, was developed to predict EBV integration sites by learning local genomic features automatically. First, DeepEBV was trained and tested using the data from the dsVIS database. The results showed that DeepEBV with EBV integration sequences plus Repeat peaks and 2-fold data augmentation performed the best on the training dataset. Furthermore, the performance of the model was validated in an independent dataset. In addition, the motifs of DNA-binding proteins could influence the selection preference of viral insertional mutagenesis. Furthermore, the results showed that DeepEBV can predict EBV integration hotspot genes accurately. In summary, DeepEBV is a robust, accurate and explainable deep learning model, providing novel insights into EBV integration preferences and mechanisms. AVAILABILITYAND IMPLEMENTATION: DeepEBV is available as open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepEBV.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

HACE1 is essential for astrocyte mitochondrial function and influences Huntington disease phenotypes in vivo.

Oxidative stress is a prominent feature of Huntington disease (HD), and we have shown previously that reduced levels of hace1 (HECT domain and Ankyrin repeat containing E3 ubiquitin protein ligase 1) in patient striatum may contribute to the pathogenesis of HD. Hace1 promotes the stability of Nrf2 and thus plays an important role in antioxidant response mechanisms, which are dysfunctional in HD. Moreover, hace1 overexpression mitigates mutant huntingtin (mHTT)-induced oxidative stress in vitro through promotion of the Nrf2 antioxidant response. Here, we show that the genetic ablation of hace1 in the YAC128 mouse model of HD accelerates motor deficits and exacerbates cognitive and psychiatric phenotypes in vivo. We find that both the expression of mHTT and the ablation of hace1 alone are sufficient to cause deficits in astrocytic mitochondrial respiration. We confirm the crucial role of hace1 in astrocytes in vivo, since its ablation is sufficient to cause dramatic astrogliosis in wild-type FVB/N mice. Astrogliosis is not observed in the presence of mHTT but a strong dysregulation in the expression of astrocytic markers in HACE1-/- x YAC128 striatum suggests an additive effect of mHTT expression and hace1 loss on this cell type. HACE1-/- x YAC128 mice and primary cells derived from these animals therefore provide model systems that will allow for the further dissection of Nrf2 pathways and astrocyte dysfunction in the context of HD.

Laquinimod decreases Bax expression and reduces caspase-6 activation in neurons.

Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit.

A Huntingtin-based peptide inhibitor of caspase-6 provides protection from mutant Huntingtin-induced motor and behavioral deficits.

Over the past decade, increasing evidence has implied a significant connection between caspase-6 activity and the pathogenesis of Huntington's disease (HD). Consequently, inhibiting caspase-6 activity was suggested as a promising therapeutic strategy to reduce mutant Huntingtin toxicity, and to provide protection from mutant Huntingtin-induced motor and behavioral deficits. Here, we describe a novel caspase-6 inhibitor peptide based on the huntingtin caspase-6 cleavage site, fused with a cell-penetrating sequence. The peptide reduces mutant Huntingtin proteolysis by caspase-6, and protects cells from mutant Huntingtin toxicity. Continuous subcutaneous administration of the peptide protected pre-symptomatic BACHD mice from motor deficits and behavioral abnormalities. Moreover, administration of the peptide in an advanced disease state resulted in the partial recovery of motor performance, and an alleviation of depression-related behavior and cognitive deficits. Our findings reveal the potential of substrate-based caspase inhibition as a therapeutic strategy, and present a promising agent for the treatment of HD.

Somatostatin receptor-2 negatively regulates ß-adrenergic receptor mediated Ca(2+) dependent signaling pathways in H9c2 cells.

In the present study, we report that somatostatin receptor 2 (SSTR2) plays a crucial role in modulation of ß1AR and ß2AR mediated signaling pathways that are associated with increased intracellular Ca(2+) and cardiac complications. In H9c2 cells, SSTR2 colocalizes with ß1AR or ß2AR in receptor specific manner. SSTR2 selective agonist inhibits isoproterenol and formoterol stimulated cAMP formation and PKA phosphorylation in concentration dependent manner. In the presence of SSTR2 agonist, the expression of PKCα and PKCß was comparable to the basal condition, however SSTR2 agonist inhibits isoproterenol or formoterol induced PKCα and PKCß expression, respectively. Furthermore, the activation of SSTR2 not only inhibits calcineurin expression and its activity, but also blocks NFAT dephosphorylation and its nuclear translocation. SSTR2 selective agonist abrogates isoproterenol mediated increase in cell size and protein content (an index of hypertrophy). Taken together, the results described here provide direct evidence in support of cardiac protective role of SSTR2 via modulation of Ca(2+) associated signaling pathways attributed to cardiac hypertrophy.

p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin.

Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy.

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling.

Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.

Receptor specific crosstalk and modulation of signaling upon heterodimerization between ß1-adrenergic receptor and somatostatin receptor-5.

In the present study we describe heterodimerization, trafficking, coupling to adenylyl cyclase and signaling in HEK-293 cells cotransfected with human-somatostatin receptor 5 (hSSTR5) and ß(1)-adrenergic receptor (ß(1)AR). hSSTR5/ß(1)AR exists as heterodimers in basal conditions which was further enhanced upon synergistic activation of both receptors. Activation of either ß(1)AR or hSSTR5 displayed dissociation of heterodimerization. In cotransfectants, ß(1)AR effect on cAMP was predominant; however, blocking ß(1)AR with antagonist resulted in 60% inhibition of forskolin-stimulated cAMP in the presence of hSSTR5 agonists. cAMP/PKA pathway in cotransfected cells was regulated in receptor-specific manner, in contrast, the status of pERK1/2 and pPI3K/AKT was predominantly regulated by hSSTR5. The expression levels of phosphorylated NFAT remained unchanged indicating blockade of calcineurin-mediated dephosphorylation and nuclear translocation of NFAT, the process predominantly regulated by pJNK in SSTR5 dependent manner. Taken together, the functional consequences of results described here might have relevance in the cardiovascular system where SSTR and AR subtypes play important roles.

Dissociation of epidermal growth factor receptor and ErbB2 heterodimers in the presence of somatostatin receptor 5 modulate signaling pathways.

Epidermal growth factor through the stimulation of epidermal growth factor receptor (EGFR) plays a critical role in the activation of MAPKs and phosphatidylinositol-3-protein kinase/AKT cell survival pathways attributed in many pathological conditions. At the cellular level, such functions involve EGFR overactivation and phosphorylation. In the present study, we describe that human embryonic kidney-293 cells transfected with somatostatin (SST) receptor 5 (SSTR5) exhibit inhibition of EGFR phosphorylation and modulate MAPK and phosphatidylinositol-3-protein kinase/AKT cell survival signaling. Furthermore, suppression of EGFR by using small interference RNA and an antagonist (AG1478) potentiates the SST effect via activation of SSTR5 on signaling molecules. In wild-type human embryonic kidney-293 cells, EGFR/ErbB2 exists as constitutive heterodimers. The presence of SSTR5 leads to the dissociation of the heteromeric complex of EGFR/ErbB2 and display preferential heterodimerization between SSTR5 and EGFR in an agonist-dependent manner. These findings highlight a new undiscovered mechanism and potential role of SSTR5 to attenuate the EGFR-mediated signaling pathways involved in tumorigenesis. Our data indicate that the activation and/or overexpression of SST receptors along with the inhibition of EGFR will serve as an important therapeutic approach in the treatment of ErbB-positive tumors.