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.

Altered Mitochondrial Morphology and Bioenergetics in a New Yeast Model Expressing Aβ42

“…The preincubation of cells with the mitochondria-targeted antioxidant SkQ1 substantially diminished mROS. Most importantly, the mitochondrial fragmentation induced by mROS preceded the development of the generalized oxidative stress [ 92 ]. The main rather optimistic conclusions can be drawn from these and the above described results.…”

“…The plasmid also has an ampicillin-resistance gene and a prototrophic factor for uracil URA3 from the Y. lipolytica genome. Insertion of the PCR products into the pZ-express++ vector was performed as described in [ 92 ]. The sequences of Aβ42, Aβ40, eGFP-Aβ42, eGFP-Aβ40, and eGFP were inserted into the vector.…”

“…The transfection of Y. lipolytica Po1f cells by electroporation was carried out as described in [ 92 ].…”

“…ROS production was determined with dihydroethidium, and yeast cell viability was detected with Sytox Green Dead Cell Stain [ 94 ], as described in [ 92 ]. Where indicated, the cells were preincubated for 1 h with 250 nM SkQThy, a mitochondria-targeted consisting of a lipophilic cation triphenylphosphonium linked via a C 10 aliphatic chain with an antioxidant thymoquinone (Thy) with versatile healing abilities [ 61 ].…”

“…Oxygen consumption by yeast mitochondria was monitored amperometrically using a closed Clark-type oxygen electrode in a continuously stirred, thermostatically controlled 1 mL cell as described in [ 92 ]. Respiratory rates of mitochondria were expressed as ng-atoms O/min/mg protein.…”