Mitochondrial E3 Ligase, MARCH5 degrades Hepatitis B Virus X protein on Mitochondria
C. MARCH5 attenuates Cycloocygenase-2 induction by HBx
Previously our lab reported that the expression of COX-2 mRNA was significantly elevated in cells transfected with HBV replicon but not in cells transfected with HBV genome lacking the HBx gene. Cyclooxygenase-2 (COX-2) is known to be an important enzyme mainly responsible for causing inflammation. Whether MARCH5-WT overexpression affacts to HBx-dependent COX-2 luciferase activity. A COX-2-Luc reporter plasmid was transfected with expression vectors encoding HBx and HBx-mito into Huh7 cells. HBx and mitochondrial targeting HBx increased COX-2 luciferase activity in a dose dependent manner. (Fig. 6A, B). Next, we examined the effect of MARCH5 on HBx mediated inflammatory responses. MARCH5 wild type inhibited HBx induced COX-2 luciferase activity (Fig. 6C) and also block the mitochondrial targeting HBx-mediated COX-2 activity (Fig. 6D). Moreover, mitochondria associated HBx has been known to activate inflammatory signal transduction by reactive oxygen species (ROS). COX-2 induction was also correlated with the ability of HBx to increase ROS levels. Thus, we analyzed whether MARCH5 is involved in the HBx mediated ROS production. We transfected Huh7 cells with the HBx and evaluated ROS levels using DCF-DA that became high fluorescent upon oxidation by intracellular ROS. We found that ROS levels were increased by overexpression of the HBx. And we confirmed that overexpression of MARCH5WT reduced the increased ROS in Hun7 cells expressing HBx, but MARCH5HW does not affect ROS production (Fig.
6E). Furthermore, HBx-mito mediated stimulation of ROS release was significantly inhibited by MARCH5WT. But MARCH5HW does not affect ROS release (Fig. 6F). Collectively, MARCH5 inhibits the HBx-induced COX-2 activity and ROS production through its degradation in a MARCH5 ubiquitin ligase activity dependent manner, indicating that MARCH5 abrogates HBx dependent inflammatory signaling.
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Fig. 6. MARCH5 reduces the HBx-induced cytotoxicity. (A, B) The activation of COX-2 promoter by HBx was examined by luciferase reporter gene assay. Huh7 cells were transfected with 0.5 ug of human COX-2 promoter linked to luciferase reporter with various concentration of Flag-HBx (A) or Flag-HBx-Mito (B). At 36h post-transfection, luciferase activity was determined. (C, D) The reduction of HBx-mediated COX-2 promoter activity by MARCH5 was examined by reporter gene assay. COX-2 reporter and HBx (C) or Flag-HBX-Mito (D) plasmids were co-transfected into Huh7 cells with Myc-MARCH5WT. Graphs
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represent fold induction relative to luciferase activity in control cells. Data represent the average of three independent experiments. Error bars, mean ± SEM **P<0.005. *P<0.05. (E, F) HBx-induced ROS levels were decreased by MARCH5. Flag-HBx (E) or Flag-HBx-Mito (F) plasmids were introduced into Huh7 cells with Myc-MARCH5WT or Myc-MARCH5H43W. After incubation of 36h, the cells were stained with DCF-DA, intracellular ROS levels were determined by FACS analysis. Graphs represent fold induction relative to luciferase activity in control cells. Data represent the average of three independent experiments. Error bars, means ± SEM **P<0.005. *P<0.05.
55 IV. Discussion
HBV X protein (HBx) plays a crucial role in HCC development via various pathways, such as its transactivation effect, DNA repair, and epigenetic changes, as well as by influencing apoptosis. HBx up-regulates cellular ROS levels through perturbing mitochondrial function.
Therefore, cellular regulatory mechanism is required to reduce the HBx-induced cellular toxicity. In this study, we demonstrate a regulatory role for MARCH5 in HBx-mediated HCC development. These data show that the expression level of MARCH5 is crucial factor for HCC development as well as HBx regulation. (i) the elevated MARCH5 either binds (Figure 4) or subsequently degrades HBx through ubiquitination in the mitochondria (Figure 5).
Therefore, elevated MARCH5 reduces the cellular toxicity induced by HBx (Figure 6).
HBx shows various intracellular localization, nucleus, cytoplasm or mitochondria.
We found HBx changed the localization depending on expression time. HBx initially localized to nucleus and moved to cytoplasm, finally showed translocation to mitochondria at the highest expression time point. The high expression level of HBx resulted in abnormal mitochondrial morphology and function. Mitochondrial HBx forms HBx-mitochondria aggregates at the perinuclear region induced by the HBx-mediated modulation of the microtubule network. HBx-associated mitochondrial aggregates deregulates mitochondrial function via induction of reactive oxygen species (ROS), targeting VDAC, activation of NF-kB, STAT3 or translocation of Raf1 to mitochondria. HBV/HBx also promotes mitophagy to remove the damaged mitochondria to facilitate the persistent virus infection. However, though mitochondria are crucial organelle for cell life and death with their own quality control system, regulatory mechanism for HBx or HCC in the mitochondria has not been still reported.
Ubiquitin proteasome system (UPS) is essential to maintain the cellular homeostasis through regulation of the protein quality control; right protein, right amount, right time. UPS recognizes and cleans up the non-self in cells. HBx regulatory UPS has been poorly understood in cells. E3 ligase, siah-1 is just known to down-regulate HBx via ubiquitination in a p53 dependent manner in cytoplasm. MARCH5 is the mitochondria E3 ligase which has
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multi-functions to maintain the mitochondrial quality control in cells. On the mitochondria outermembrane, MARCH5 tunes up the target proteins to preserve the right amount for needs of cells or to function at the right time. MARCH5 controls mitochondrial morphology by ubiquitination of Mfn1 under stress and regulates tethering ER to mitochondria by ubiquitination of Mfn2. Additionally, MARCH5 removes abnormal protein to maintain the right protein in cells. For an instance, MARCH5 get rid of abnormally mutated or aggregated proteins, polyQ or mutated SOD. In HBV-infected cells, MARCH5 recognized and removed mitochondria-associated HBx aggregates by ubiquitination and reduced cellular toxicity. In the absence of MARCH5 in cells, HBx induced cell proliferation rate was significantly increased and showed high expression level of HBx. Consistently, human HCC tissue analysis showed differential expression pattern of MARCH5 in tumor tissues. The tumor tissues in early stage showed higher expression than non-tumor tissue whereas, later stage HCC tissues showed decreased MARCH5 expression levels. These are statistically related to the tumor multiplicity. Therefore, the expression levels of MARCH5 in tumors could be involved in tumor progression. This is the first mention that MARCH5 is involved in tumor progression.
Mitochondrial dysfunction has been implicated in tumorigenesis. Mitochondria consume less oxygen and produce decreased ATP in tumor cells. Finally, tumor cells come to be glycolytic because of suppressive mitochondrial energy production. Mitochondrial morphology is involved in cellular functions, cell death and survival. Mitochondrial network structure is maintained by balanced fusion and fission events which are controlled by mitochondrial dynamics controlling molecules, fission proteins; Drp1, hFis1 and fusion proteins; Mfns (Mfn1 and 2). Recent study reported that imbalanced mitochondrial network structure affects tumorigenesis.
Impaired mitochondrial fusion or enhanced fission by Drp1/Mfns (Mfn1 and 2) promote lung adenocarcinoma cells or breast cancer metastasis. Persistent mitochondrial hyperfusion by Drp1 loss also induces chromosomal instability. Additionally, their fusion or fission activities are regulated by MARCH5 mediated ubiquitination. A few somatic mutations in MARCH5 gene are found in cancer cell lines and tissues (sanger ref;
http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/). Notably, Substitution of
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Asparagine for Histidine 43 [p.H43N, c.127C>A] in RING domain of MARCH5 was reported. MARCH5 has a C3HC5 zinc finger motif in RING domain in which the histidine is a conserved residue for zinc coordination and also have an important role for ligase activity.
58 V. Conclusion
In this study, we propose a new role of MARCH5 in mitochondrial regulatory mechanism during hepatocarcinogenesis via degradation of HBx. MARCH5 ubiquitinates mitochondrial HBx and subsequent protesasomal degradation. Furthermore, HBV mediated hepatocellular carcinoma patients’ liver tissue analysis showed differential expression of MARCH5 in a solid tumor stages; increased MARCH5 expression in early stages tumors and remarkable reduction in malignant tumor stage. Our data suggested that MARCH5 mediated cellular homeostasis have a pivotal role in tumorigenesis as well as hepatocarcinogenesis.
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Proceedings of the National Academy of Sciences of the United States of America 109, 2382-2387, doi:10.1073/pnas.1114985109 (2012).
62 -국문 요약-
바이러스 감염시 미토콘드리아 E3 ligase 인 MARCH5의 새로운 생체 기능 아주대학교 의생명과학과
유영석
(지도 교수 : 조 혜 성)
미토콘드리아는 세포의 항상성을 유지하는데 중요한 역할을 하는 방어막 시스템을 가지고 있다. UPS(Ubiquitin proteasome system)는 분자단위 조절 시스템으로 미토콘드리아 외막에 존재하는 첫번째 방어막으로 작용한다. 본 연구에서는 미토콘드리아 외막에 존재하는 UPS 의 하나인 MARCH5의 기능을 연구하고자 하였다. MAVS 단백질은 바이러스가 들어왔을 때 aggregation을 형성하여 인터페론과 사이토카인 분비를 증가시키지만, 이러한 반응이 지속될 경우에는 여러 종류의 면역질환이 야기된다. 하지만 이러한 aggregated 단백질을 풀어주는 연구에 대해서는 아직 알려지지 않았다. 이 연구를 통해 미토콘드리아 E3 ubiquitin ligase인 MARCH5가 이러한 aggregated MAVS 단백질을 풀어주어
조절한다는 것을 확인하였다. 먼저 MARCH5+/- 마우스와 MARCH5가 결손 된
여러 면역 세포에서 확인한 결과 RNA 바이러스에 감염되었을 때 분비된 IFN의 양이 증가하는 것을 관찰할 수 있었고, 이와 함께 바이러스의 양 자체도 감소하였다. MARCH5는 세포에 바이러스가 감염되었을 때 MAVS와 결합하는 것으로 보아 활성화된 형태의 aggregated MAVS와 결합한다는 것을 알 수 있었다.
MARCH5가 aggregation 된 MAVS를 제거할 때 Lys 7, 500의 잔기에 ubiquitination을 시켜 MAVS를 제거한다는 것을 확인할 수 있었다. 두 번째로, HBx 단백질은 HBV에 의해 만들어지는 단백질로 구조를 가지고 있지 않는 단백질이다. 높은 발현을 보이는 HBx에 의해 미토콘드리아가 기능을 상실하고, 이러한 미토콘드리아의 기능 상실은 간암형성에 영향을 준다. MARCH5와 HBx가
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결합을 하여 HBx를 유비퀴티네이션 시키는 것을 확인할 수 있었으나, ligase activity가 없는 MARCH5에 의해서는 HBx가 ubiquitination되지 않는 다는 것을 확인할 수 있었다. 또한 In vitro 와 in vivo 유비퀴티네이션 실험을 통해 MARCH5가 HBx 를 proteasome dependent 한 degradation을 통해 유비퀴티네이션을 촉진한다는 것을 확인하였다. 이것으로 보아 MARCH5는 aggregated MAVS나 HBx의 단백질이 미토콘드리아에 accumulation 되는것을 막아주는 조절자라는 것을 확인할 수 있었다.
ARTICLE
Received 19 Dec 2014|Accepted 25 Jun 2015|Published 6 Aug 2015