Englerin A Rewires Phosphosignaling via Hsp27 Hyperphosphorylation to Induce Cytotoxicity in Renal Cancer Cells.

Englerin A (EA) is a small-molecule natural product with selective cytotoxicity against renal cancer cells. EA has been shown to induce apoptosis and cell death through cell-cycle arrest and/or insulin signaling pathways. However, its biological mode of action or targets in renal cancer remains enigmatic. In this study, we employed advanced mass spectrometry-based phosphoproteomics approaches to identify EA's functional roles in renal cancer. We identified 10,940 phosphorylation sites, of which 706 sites exhibited EA-dependent phosphorylation changes. Integrated analysis of motifs and interaction networks suggested activation of stress-activated kinases including p38 upon EA treatment. Of note, a downstream target of p38, Hsp27, was found to be hyperphosphorylated on multiple sites upon EA treatment. Among these, a novel site Ser65 on Hsp27, which was further validated by targeted proteomics, was shown to be crucial for EA-induced cytotoxicity in renal cancer cells. Taken together, these data reveal the complex signaling cascade that is induced upon EA treatment and importantly provide insights into its effects on downstream molecular signaling.

Phosphoproteomics Unravel HBV Triggered Rewiring of Host Phosphosignaling Events.

Hepatitis B virus (HBV) infection persists as a major global health problem despite the availability of HBV vaccines for disease prevention. However, vaccination rates remains low in some regions of the world, driving the need for novel strategies to minimise infections and prevent disease progression. Thus, understanding of perturbed molecular signaling events during early phases of HBV infection is required. Phosphosignaling is known to be involved in the HBV infection processes, yet systems-level changes in phosphosignaling pathways in the host during infection remain unclear. To this end, we performed phosphoproteome profiling on HBV-infected HepG2-NTCP cells. Our results showed that HBV infection drastically altered the host phosphoproteome and its associated proteins, including kinases. Computational analysis of this phosphoproteome revealed dysregulation of the pathways involved in immune responses, cell cycle processes, and RNA processing during HBV infection. Kinase Substrate Enrichment Analysis (KSEA) identified the dysregulated activities of important kinases, including those from CMGC (CDK, MAPK, GSK, and CLK), AGC (protein kinase A, G, and C), and TK (Tyrosine Kinase) families. Of note, the inhibition of CLKs significantly reduced HBV infection in HepG2-NTCP cells. In all, our study unravelled the aberrated phosphosignaling pathways and the associated kinases, presenting potential entry points for developing novel therapeutic strategies for HBV treatment.

Mapping the Interactions of HBV cccDNA with Host Factors.

Hepatitis B virus (HBV) infection is a major health problem affecting about 300 million people globally. Although successful administration of a prophylactic vaccine has reduced new infections, a cure for chronic hepatitis B (CHB) is still unavailable. Current anti-HBV therapies slow down disease progression but are not curative as they cannot eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The cccDNA minichromosome persists in the nuclei of infected hepatocytes where it forms the template for all viral transcription. Interactions between host factors and cccDNA are crucial for its formation, stability, and transcriptional activity. Here, we summarize the reported interactions between HBV cccDNA and various host factors and their implications on HBV replication. While the virus hijacks certain cellular processes to complete its life cycle, there are also host factors that restrict HBV infection. Therefore, we review both positive and negative regulation of HBV cccDNA by host factors and the use of small molecule drugs or sequence-specific nucleases to target these interactions or cccDNA directly. We also discuss several reporter-based surrogate systems that mimic cccDNA biology which can be used for drug library screening of cccDNA-targeting compounds as well as identification of cccDNA-related targets.

Direct visualization of Agrobacterium-delivered VirE2 in recipient cells.

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single-stranded DNA molecules (T-DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split-GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2-GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1-10) was expressed in recipient cells. Upon delivery of VirE2-GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2-GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium-delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf-infiltration assay; most of VirE2 moved at a speed of 1.3-3.1 µm sec⁻¹ in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2-GFP formed filamentous structures of different lengths, even in the absence of T-DNA. As a non-natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium-delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T-DNA transformation for a non-natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non-natural host recipient. The split-GFP approach could enable the real-time visualization of VirE2 trafficking inside recipient cells.

Polygenic sex determination system in zebrafish.

BACKGROUND: Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based "blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. CONCLUSIONS/SIGNIFICANCE: Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system.

FluoMEP: a new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphism.

PCR-based identification of differences between two unknown genomes often requires complex manipulation of the templates prior to amplification and/or gel electrophoretic separation of a large number of samples with manual methods. Here, we describe a new genotyping method, called fluorescent motif enhanced polymorphism (fluoMEP). The fluoMEP method is based on random amplified polymorphic DNA (RAPD) assay, but combines the advantages of the large collection of unlabelled 10mer primers (ca. 5000) from commercial sources and the power of the automated CE devices used for the detection of amplified fragment length polymorphism (AFLP) patterns. The link between these two components is provided by a fluorescently labeled "common primer" that is used in a two-primer PCR together with an unlabeled RAPD primer. By using the same "common primer" and a series of RAPD primers, DNA templates can be screened quickly and effectively for polymorphisms. Our manuscript describes the optimization of the method and its characterization on different templates. We demonstrate by using several different approaches that the addition of the "common primer" to the PCR changes the profile of amplified fragments, allowing for screening various parts of the genome with the same set of unlabeled primers. We also present an in silico analysis of the genomic localization of fragments amplified by a RAPD primer with two different "common primers" and alone.