Huixing Feng

Metabolic engineering for enhanced fatty acids synthesis in Saccharomyces cerevisiae

Microbial production of biofuel has attracted significant attention in recent years. The fatty acids are important precursors for the production of fuels and chemicals, and its biosynthesis is initiated by the conversion of acetyl-CoA to malonyl-CoA which requires acetyl-CoA as key substrate. Herein, the yeast Saccharomyces cerevisiae was proposed to be metabolically engineered for cytosol acetyl-CoA enhancement for fatty acid synthesis. By gene disruption strategy, idh1 and idh2 genes involved in citrate turnover in tricarboxylic acid cycle (TCA cycle) were disrupted and the citrate production level was increased to 4- and 5-times in mutant yeast strains. In order to convert accumulated citrate to cytosol acetyl-CoA, a heterologous ATP-citrate lyase (ACL) was overexpressed in yeast wild type and idh1,2 disrupted strains. The wild type strain expressing acl mainly accumulated saturated fatty acids: C14:0, C16:0 and C18:0 at levels about 20%, 14% and 27%, respectively. Additionally, the idh1,2 disrupted strains expressing acl mainly accumulated unsaturated fatty acids. Specifically in Δidh1 strain expressing acl, 80% increase in C16:1 and 60% increase in C18:1 was detected. In Δidh2 strain expressing acl, 60% increase in C16:1 and 45% increase in C18:1 was detected. In Δidh1/2 strain expressing acl, there was 92% increase in C16:1 and 77% increase in C18:1, respectively. The increased fatty acids from our study may well be potential substrates for the production of hydrocarbon molecules as potential biofuels.

HBx genotype D represses GSTP1 expression and increases the oxidative level and apoptosis in HepG2 cells

Epigenetics has been implicated in human cancer development. Epigenetic factors include HBx protein, which is able to induce hypermethylation and suppresses tumor suppressor genes. One of such tumor suppressor genes, GSTP1, shows reduced expression in many human cancers. Hypermethylation of GSTP1 is the most studied mechanism of its silence. In the present study, we reported that GSTP1 expression was completely depleted in HBV integrated HepG2.2.15 cells due to the hypermethylation in its promoter region. And it was HBx, especially HBx genotype D, that played the key role in repressing GSTP1 expression. Further functional studies like ROS assay and apoptosis detection were also used to confirm this repression. Our findings should facilitate the understanding of HBV and their influences on the epigenetic modulations for epigenetic tumorigenesis during HBV‐mediated hepatocellular carcinogenesis.

iTRAQ-coupled 2-D LC-MS/MS analysis of protein profile associated with HBV-modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.

Protein profile in HBx transfected cells: A comparative iTRAQ-coupled 2D LC-MS/MS analysis

The x protein of HBV (HBx) has been involved in the development of hepatocellular carcinoma (HCC), with a possible link to individual genotypes. Nevertheless, the underlying mechanism remains obscure. In this study, we aim to identify the HBx-induced protein profile in HepG2 cells by LC-MS/MS proteomics analysis. Our results indicated that proteins were differentially expressed in HepG2 cells transfected by HBx of various genotypes. Proteins associated with cytoskeleton were found to be either up-regulated (MACF1, HMGB1, Annexin A2) or down-regulated (Lamin A/C). These may in turn result in the decrease of focal adhesion and increase of cell migration in response to HBx. Levels of other cellular proteins with reported impact on the function of extracellular matrix (ECM) proteins and cell migration, including Ca(2+)-binding proteins (S100A11, S100A6, and S100A4) and proteasome protein (PSMA3), were affected by HBx. The differential protein profile identified in this study was also supported by our functional assay which indicated that cell migration was enhanced by HBx. Our preliminary study provided a new platform to establish a comprehensive cellular protein profile by LC-MS/MS proteomics analysis. Further downstream functional assays, including our reported cell migration assay, should provide new insights in the association between HCC and HBx.

Proteomic analysis of HBV-associated HCC: Insights on mechanisms of disease onset and biomarker discovery

The development of hepatocellular carcinoma (HCC) can be considered as an end-stage outcome of chronic hepatitis B virus (HBV) infection. Early prognostic markers are needed to allow effective treatments and prevent HCC from developing. Proteomics analysis has been used to identify markers from clinical samples from HCC patients. This approach can be further improved by identifying early biomarkers before the onset of HCC. One way would be to use the cell-based HBV replication system, which is reflective of the early stage of virus infection and thus secreted proteins identified at this stage may have relevance in HCC prognosis. In this review, we focus the discussion on the current status of proteomics analysis of cellular proteins and HCC biomarker identification, with a special highlight on the potential of the cell-based HBV replication system for the identification of prognostic HCC biomarkers.

HBX-Mediated Migration of HBV-Replicating HepG2 Cells: Insights on Development of Hepatocellular Carcinoma

Hepatitus B virus (HBV) is a major cause of the development of hepatpcellular carcinoma (HCC). One of the significant characteristics of tumor progression is cell migration which is reflective of cytoskeletal dynamics. The Rho GTPases contribute to a multiple cellular processes, including the cellular cytoskeletal reorganization and motility. It has been found that some Rho GTPases have oncogenic activity and can promote cancer cell invasion. Here we discuss one of the Rho GTPases, Rac1 can be activated by HBV replication and such activation results in the high motility of HBV-replicating cells. The enhanced cell motility can be interestingly alleviated by the mutation at the sites of proline rich domain located in HBX. Our findings may provide new insights on the mechanism of HCC development associated with chronic HBV infection.

Hepatitis B virus induced coupling of deadhesion and migration of HepG2 cells on thermo-responsive polymer

The unique physical property of thermo-responsive polymer (TRP) has recently prompted its increasing applications in tissue engineering. On the other hand, TRP has not been exploited for potential applications in quantitative cell screening against external stimulations. In this study, TRP is applied as a model system for elucidating the effect of HBV replication on the biophysical responses of HepG2 cells transfected by wild type HBV genome. Moreover, mutant HBV genome is designed to assess the specific activity of the SH3-binding domain of HBx during HBV replication. The adhesion contact recession and geometry transformation of HepG2 cells transfected with empty vector (pcDNA3.1 cells), wild type HBV (wtHBV cells) and mutant HBV genome (mHBV cells) are probed during the thermal transformation across lower solution critical temperature of TRP. In comparison with pcDNA3.1 cells and mHBV cells, the initial rate of reduction in degree of deformation and average adhesion energy for wtHBV cells is significantly increased. Interestingly, migration speed and persistence time of cells are found to be correlated with the cell deadhesion kinetics. Immuno-fluorescence microscopy demonstrates that HBV replication reduces the actin concentration and focal adhesions at cell periphery during the initial 30 min cell deadhesion. The results strongly suggested that HBV infection triggers the dynamic responses of HepG2 cells through the cytoskeleton remodeling and subsequent mechanochemical transduction. Overall, it is shown that TRP provides a convenient platform for quantifying biological stimulations on adherent cells.

Collective cell traction force analysis on aligned smooth muscle cell sheet between three-dimensional microwalls

During the past two decades, novel biomaterial scaffold for cell attachment and culture has been developed for applications in tissue engineering, biosensing and regeneration medicine. Tissue engineering of blood vessels remains a challenge owing to the complex three-layer histology involved. In order to engineer functional blood vessels, it is essential to recapitulate the characteristics of vascular smooth muscle cells (SMCs) inside the tunica media, which is known to be critical for vasoconstriction and vasodilation of the circulatory system. Until now, there has been a lack of understanding on the mechanotransduction of the SMC layer during the transformation from viable synthetic to quiescent contractile phenotypes. In this study, microfabricated arrays of discontinuous microwalls coated with fluorescence microbeads were developed to probe the mechanotransduction of the SMC layer. First, the system was exploited for stimulating the formation of a highly aligned orientation of SMCs in native tunica medium. Second, atomic force microscopy in combination with regression analysis was applied to measure the elastic modulus of a polyacrylamide gel layer coated on the discontinuous microwall arrays. Third, the conventional traction force assay for single cell measurement was extended for applications in three-dimensional cell aggregates. Then, the biophysical effects of discontinuous microwalls on the mechanotransduction of the SMC layer undergoing cell alignment were probed. Generally, the cooperative multiple cell–cell and cell–microwall interactions were accessed quantitatively by the newly developed assay with the aid of finite-element modelling. The results show that the traction forces of highly aligned cells lying in the middle region between two opposing microwalls were significantly lower than those lying adjacent to the microwalls. Moreover, the spatial distributions of Von Mises stress during the cell alignment process were dependent on the collective cell layer orientation. Immunostaining of the SMC sheet further demonstrated that the collective mechanotransduction induced by three-dimensional topographic cues was correlated with the reduction of actin and vinculin expression. In addition, the online two-dimensional LC–MS/MS analysis verified the modulation of focal adhesion formation under the influence of microwalls through the regulation in the expression of three key cytoskeletal proteins.

Comparative proteomics analysis of engineered Saccharomyces cerevisiae with enhanced biofuel precursor production.

The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.

HBx expression activates RhoA GTPase: impact on cell migration.

HBV (hepatitis B virus) remains a global health concern, especially in developing countries. It has been associated with the development of HCC (hepatocellular carcinoma). One of the four viral proteins, HBx, interacts with cellular proteins, which are involved in a series of cellular processes including cell migration. The Rho GTPases (guanine nucleotide triphosphatases) family of proteins is involved in the regulation of the reorganization of actin and cell migration. We have reported that HBV replication activates Rac1 through SH3 binding. Here, we reported that RhoA was activated by HBx in vitro. The cell motility was enhanced in HepG2 cells co‐transfected with HBx and RhoA, compared with those transfected with RhoA alone. Our results were consistent with the recently reported role of RhoA in promoting cell motility and may provide new insights on the mechanism of HBV‐associated HCC.