Impaired methylation as a novel mechanism for proteasome suppression in liver cells.
Impaired methylation as a novel mechanism for proteasome suppression in liver cells.
复制标题
甲基化受损是肝细胞中蛋白酶体抑制的新机制。
DOI：
10.1016/j.bbrc.2009.12.074
复制
发表时间：
2010
影响因子：
3.1
通讯作者：
Kharbanda,KusumK
中科院分区：
文献类型：
--
作者：
Osna,NataliaA;White,RondaL;DonohueJr,TerrenceM;Beard,MichaelR;Tuma,DeanJ;Kharbanda,KusumK
蛋白酶体是一种多催化蛋白降解酶，受乙醇诱导的氧化应激调节;这种抑制作用归因于CYP 2 E1产生的代谢产物。然而，在某些条件下，似乎除了氧化应激，其他机制也参与蛋白酶体调节。本研究调查了在肝细胞暴露于乙醇期间发生的受损蛋白质甲基化是否可能有助于抑制蛋白酶体活性。我们测量了在维持或防止蛋白质甲基化的条件下，Huh 770细胞、肝细胞、肝细胞质和核提取物或纯化的20 S蛋白酶体中的糜蛋白酶样蛋白酶体活性。同时用S-腺苷甲硫氨酸（SAM）处理，可防止乙醇或杀结核菌素降低肝癌细胞和肝细胞的蛋白酶体活性。此外，结核菌素诱导的蛋白酶体活性下降发生在核和胞质组分。细胞胞质组分或高度纯化的20 S蛋白酶体在缓冲液中暴露于低SAM：S-腺苷高半胱氨酸（SAH）比率也抑制蛋白酶体功能，表明一种或多种甲基转移酶可能与蛋白酶体亚基相关。免疫印迹纯化的20 S兔红细胞蛋白酶体制备使用甲基赖氨酸特异性抗体揭示了一个25 kDa的蛋白酶体亚基，显示出与抗甲基赖氨酸的阳性反应。当20 S蛋白酶体暴露于不同的SAM：SAH比率时，这种反应性被修改。我们的结论是，受损的甲基化蛋白酶体亚基抑制肝细胞中的蛋白酶体活性，表明一个额外的，但新的机制，蛋白酶体活性调节乙醇。
The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.