Ethanol Exposure to Ethanol-Oxidizing HEPG2 Cells Induces Intracellular Protein Aggregation.
Ethanol Exposure to Ethanol-Oxidizing HEPG2 Cells Induces Intracellular Protein Aggregation.
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背景：攻击体是细胞内蛋白质聚集体的集合。在酒精性肝炎患者的肝细胞中，侵袭性小体在组织学上表现为称为Mallory-Denk（M-D）小体的细胞内含物。蛋白酶体是一种多催化细胞内蛋白酶，其催化正常（天然）和异常（错误折叠和/或受损）蛋白质的降解。该酶通过在异常蛋白形成聚集体之前快速降解异常蛋白来最小化细胞内蛋白聚集体的形成。当蛋白酶体活性被特异性抑制剂或细胞内氧化剂（例如，过氧亚硝酸盐、乙醛），增强了攻击体形成。在这里，我们试图验证是否通过乙醇暴露抑制蛋白酶体活性增强VL-17 A细胞中蛋白质聚集体的形成，VL-17 A细胞是重组的乙醇氧化HepG 2细胞，表达乙醇脱氢酶（ADH）和细胞色素P450 2 E1（CYP 2 E1）。研究方法：我们将乙醇非氧化性HepG 2细胞（ADH−/CYP 2 E1 −）或乙醇氧化性VL-17 A（ADH+/CYP 2 E1+）暴露于不同水平的乙醇中24小时或72小时。在这些处理之后，我们对细胞的侵袭体进行染色（显微镜下检测）并量化它们的数量和大小。我们还进行了流式细胞术分析，以证实我们的显微镜下发现。此外，对酒精性肝炎患者肝细胞中的攻击组含量进行定量。结果如下：在我们将VL-17 A细胞暴露于增加剂量的乙醇24小时或72小时后，20 S蛋白酶体活性随着乙醇浓度的升高而下降。在乙醇暴露24小时后，VL-17 A细胞中的攻击性基因组数量在所有使用的乙醇浓度下比其未处理的对照高1.8倍。乙醇暴露72小时后，平均攻击基因组数比未暴露的对照细胞高2.5倍。所有乙醇暴露的VL-17 A细胞的平均聚集体大小显著高于未暴露的对照细胞，但不受乙醇暴露持续时间的影响。将细胞共暴露于EtOH和雷帕霉素（后者是自噬激活剂）完全防止EtOH诱导的侵袭体形成。在酒精诱导的肝炎（AH）患者的肝脏中，攻击体的染色强度是无酒精使用障碍（AUD）患者肝脏的2.2倍。结论：我们的结论是，乙醇诱导的蛋白酶体抑制乙醇代谢的VL-17 A肝癌细胞的蛋白质聚集体的积累。值得注意的是，自噬激活去除了这些聚集体。这些发现的意义进行了讨论。
Background: Aggresomes are collections of intracellular protein aggregates. In liver cells of patients with alcoholic hepatitis, aggresomes appear histologically as cellular inclusions known as Mallory–Denk (M–D) bodies. The proteasome is a multicatalytic intracellular protease that catalyzes the degradation of both normal (native) and abnormal (misfolded and/or damaged) proteins. The enzyme minimizes intracellular protein aggregate formation by rapidly degrading abnormal proteins before they form aggregates. When proteasome activity is blocked, either by specific inhibitors or by intracellular oxidants (e.g., peroxynitrite, acetaldehyde), aggresome formation is enhanced. Here, we sought to verify whether inhibition of proteasome activity by ethanol exposure enhances protein aggregate formation in VL-17A cells, which are recombinant, ethanol-oxidizing HepG2 cells that express both alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Methods: We exposed ethanol-non-oxidizing HepG2 cells (ADH−/CYP2E1−) or ethanol-oxidizing VL-17A (ADH+/CYP2E1+) to varying levels of ethanol for 24 h or 72 h. After these treatments, we stained cells for aggresomes (detected microscopically) and quantified their numbers and sizes. We also conducted flow cytometric analyses to confirm our microscopic findings. Additionally, aggresome content in liver cells of patients with alcohol-induced hepatitis was quantified. Results: After we exposed VL-17A cells to increasing doses of ethanol for 24 h or 72 h, 20S proteasome activity declined in response to rising ethanol concentrations. After 24 h of ethanol exposure, aggresome numbers in VL-17A cells were 1.8-fold higher than their untreated controls at all ethanol concentrations employed. After 72 h of ethanol exposure, mean aggresome numbers were 2.5-fold higher than unexposed control cells. The mean aggregate size in all ethanol-exposed VL-17A cells was significantly higher than in unexposed control cells but was unaffected by the duration of ethanol exposure. Co-exposure of cells to EtOH and rapamycin, the latter an autophagy activator, completely prevented EtOH-induced aggresome formation. In the livers of patients with alcohol-induced hepatitis (AH), the staining intensity of aggresomes was 2.2-fold higher than in the livers of patients without alcohol use disorder (AUD). Conclusions: We conclude that ethanol-induced proteasome inhibition in ethanol-metabolizing VL-17A hepatoma cells causes accumulation of protein aggregates. Notably, autophagy activation removes such aggregates. The significance of these findings is discussed.
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DOI：
10.1083/jcb.143.7.1883
发表时间：
1998-12-28
期刊：
The Journal of cell biology
影响因子：
--
作者：
Johnston JA;Ward CL;Kopito RR
通讯作者：
Kopito RR
影响因子：
13.5
作者：
MAUCH, TJ;DONOHUE, TM;TUMA, DJ
通讯作者：
TUMA, DJ
影响因子：
3.2
作者：
Thomes, Paul G.;Trambly, Casey S.;Donohue, Terrence M., Jr.
通讯作者：
Donohue, Terrence M., Jr.
影响因子：
3.6
作者：
Bardag-Gorce, F;French, BA;French, SW
通讯作者：
French, SW
DOI：
10.1016/j.biocel.2012.10.002
发表时间：
2013-02
影响因子：
4
作者：
Thomes, Paul G.;Osna, Natalia A.;Davis, John S.;Donohue, Terrence M., Jr.
通讯作者：
Donohue, Terrence M., Jr.