The First Yarrowia lipolytica Yeast Models Expressing Hepatitis B Virus X Protein: Changes in Mitochondrial Morphology and Functions

Abstract

Chronic hepatitis B virus infection is the dominant cause of hepatocellular carcinoma, the main cause of cancer death. HBx protein, a multifunctional protein, is essential for pathogenesis development; however, the underlying mechanisms are not fully understood. The complexity of the system itself, and the intricate interplay of many factors make it difficult to advance in understanding the mechanisms underlying these processes. The most obvious solution is to use simpler systems by reducing the number of interacting factors. Yeast cells are particularly suitable for studying the relationships between oxidative stress, mitochondrial dynamics (mitochondrial fusion and fragmentation), and mitochondrial dysfunction involved in HBx-mediated pathogenesis. For the first time, genetically modified yeast, Y. lipolytica, was created, expressing the hepatitis B virus core protein HBx, as well as a variant fused with eGFP at the C-end. It was found that cells expressing HBx experienced stronger oxidative stress than the control cells. Oxidative stress was alleviated by preincubation with the mitochondria-targeted antioxidant SkQThy. Consistent with these data, in contrast to the control cells (pZ-0) containing numerous mitochondrial forming a mitochondrial reticulum, in cells expressing HBx protein, mitochondria were fragmented, and preincubation with SkQThy partially restored the mitochondrial reticulum. Expression of HBx had a significant influence on the bioenergetic function of mitochondria, making them loosely coupled with decreased respiratory rate and reduced ATP formation. In sum, the first highly promising yeast model for studying the impact of HBx on bioenergy, redox-state, and dynamics of mitochondria in the cell and cross-talk between these parameters was offered. This fairly simple model can be used as a platform for rapid screening of potential therapeutic agents, mitigating the harmful effects of HBx.

Keywords: HBx; Yarrowia lipolytica; hepatitis B virus; hepatocellular carcinoma; heterologous expression; mitochondria-targeted antioxidants; mitochondrial dysfunction; oxidative stress; yeast.

Figure 1

Western blot analysis of eGFP fusion proteins from pZ-0, pZ-eGFP, and pZ-HBx-eGFP strains of Y. lipolytica. The eGFP and HBx-eGFP signals are indicated by arrows. On the right, the molecular weights of the markers are shown.

Figure 2

Mitochondrial morphology of Y. lipolytica mutants. Effect of SkQThy. eGFP fluorescence shown in green. Bars are 10 µm. Cells were preincubated without SkQThy (left panel) or with 250 nM SkQThy (right panel) for 1 h, then washed with 50 mM PBS, pH 5.5, and stained with 500 nM MitoTracker Red CmxRos (shown in red) for 30 min.

Figure 3

Oxidative stress and cell death in Y. lipolytica strains. Panel (A)—flow cytometry measurement; Panel (B)—the same results are presented in the form of histograms summarizing results from three independent experiments.

Figure 4

Amperometric recording of oxygen consumption by Y. lipolytica mitochondria. (A) Y. lipolytica Po1f; (B) Y. lipolytica Po1f pZ-HBx. The basic incubation medium contained 0.6 M mannitol, 2 mM Tris-phosphate, pH 7.2, 1 mM EDTA, 20 mM Tris-pyruvate + 5 mM Tris-malate and mitochondria (0.5 mg protein/mL). Respiratory control ratios upon successive ADP additives were: (A) 4.3, 5.2; (B) 2.3, 2.2.

Figure 5

Oxygen consumption rates by Y. lipolytica mitochondria in states 3 and 4, and uncoupled states. When indicated, 2 µM CCCP was added. The statistical analyses were carried out by the one-way ANOVA test. ***: p < 0.001; *: 0.01 < p < 0.05.

Figure 6

Amperometric recording of oxygen consumption by Y. lipolytica mitochondria in state 3 respiration as affected by respiratory inhibitors: rotenone (A) and antimycin (B). Incubation medium was supplemented with 1 mM ADP and mitochondria (0.5 mg protein/mL).

Figure 7

Membrane depolarization of Y. lipolytica mitochondria by palmitate. The basic incubation medium was supplemented with 20 mM safranine O and mitochondria (0.5 mg protein/mL).

Figure 8

Swelling of Y. lipolytica mitochondria. The basic incubation medium was supplemented with 40 mM KCl and mitochondria (0.2 mg protein/mL). The statistical analyses were carried out by the one-way ANOVA test. ***: p < 0.001.

Figure 9

ATP production by Y. lipolytica mitochondria. (A) The basic incubation medium was supplemented with 5 µM Phenol Red, 6 μM Ap5A (an inhibitor of adenylate kinase), and mitochondria (0.2 mg protein/mL); (B) The basic incubation medium was supplemented with 6 μM Ap5A, 1 mM glucose, 1 mM NADP, hexokinase (10 U/mL), glucose-6-phosphate dehydrogenase (3 U/mL), and mitochondria (0.2 mg protein/mL). The statistical analyses were carried out by the one-way ANOVA test. ***: p < 0.001.

Figure 10

H₂O₂ generation by Y. lipolytica mitochondria. The statistical analyses were carried out by the one-way ANOVA test. *: 0.01 < p < 0.05.