iTRAQ-based analysis for the identification of MARCH8 targets in human esophageal squamous cell carcinoma
iTRAQ-based analysis for the identification of MARCH8 targets in human esophageal squamous cell carcinoma
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DOI：
10.1016/j.jprot.2021.104125
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发表时间：
2021-02-08
影响因子：
3.3
通讯作者：
Sharma, Rinu
中科院分区：
文献类型：
--
作者：
Singh, Shivam;Bano, Arjumand;Sharma, Rinu
MARCH 8是一种E3连接酶，主要参与免疫调节。最近，我们报道了它在人食管鳞状细胞癌中的异常表达。然而，它调节癌症的确切机制却知之甚少。我们应用高通量定量蛋白质组学方法来鉴定MARCH 8的下游蛋白质靶点。ESCC细胞中内源性MARCH 8的沉默，随后进行LC-MS/MS分析，鉴定出1，029种独特的蛋白质，其显示MARCH 8敲低后表达改变。几个先前报道的MARCH 8靶蛋白，即TFR 1，syntaxin-4，e-cadherin和CD 44被发现上调。此外，在本研究中鉴定了MARCH 8的新推定靶点，包括β 2 M。我们证明了MARCH 8与CDH 1和β 2 M相互作用并泛素化。用MG 132抑制蛋白酶体活性阻止了CDH 1和β 2 M降解，表明MARCH 8可能靶向CDH 1和β 2 M进行蛋白酶体降解。此外，ESCC组织中pm和CDH 1表达的缺失与MARCH 8表达显着负相关（r分别= -0.737和-0.651; p < 0.01）。总之，我们目前的研究已经导致识别新的目标MARCH 8，并建议MARCH 8在调节CDH 1和β 2 M营业额在食管癌cells.Significance的作用：使用定量蛋白质组学进行了识别MARCH 8的新目标。目前的研究提供了一个广泛的理解后，在ESCC的MARCH 8敲低分子重塑。该研究还巩固了MARCH 8的作用不仅限于免疫调节的观点，因为MARCH 8的沉默会影响其他各种过程，如蛋白质加工和定位。这项研究可能有助于了解MARCH 8在ESCC中的调节以及MARCH 8可能促进癌细胞逃避免疫监视的机制。
MARCH8 is an E3 ligase, primarily involved in immune-modulation. Recently, we reported its aberrant expression in human esophageal squamous cell carcinoma. However, exact mechanisms by which it regulates cancer have been poorly understood. We applied high-throughput quantitative proteomics approach to identify downstream protein targets of MARCH8. Silencing of endogenous MARCH8 in ESCC cells followed by LC-MS/MS analysis led to identification of 1,029 unique proteins showing altered expression post MARCH8 knockdown. Several previously reported MARCH8 target proteins viz. TFR1, syntaxin-4, e-cadherin and CD44 were found to be upregulated. Furthermore, new putative targets of MARCH8, including beta 2M, were identified in the present study. We demonstrated that MARCH8 interacts with and ubiquitinates CDH1 and beta 2M. Inhibiting proteasome activity with MG132 prevented CDH1 and beta 2M degradation, indicating that MARCH8 might be targeting CDH1 and beta 2M for proteasomal degradation. Further, loss of pm and CDH1 expression significantly and inversely correlated with MARCH8 expression in ESCC tissues (r = -0.737 and - 0.651, respectively; p < 0.01). In conclusion, our present study has led to identification of new targets of MARCH8 and suggests the role of MARCH8 in regulating CDH1 and beta 2M turnover in esophageal cancer cells.Significance: The use of quantitative proteomics carried out has led to the recognition of new targets of MARCH8. The present study gives a broad understanding of the molecular remodeling arising in the ESCC after MARCH8 knockdown. The study also solidifies the idea that role of MARCH8 is not just limited to immunomodulation as silencing of MARCH8 affects various other processes such as protein processing and localization. This study might help in understanding the regulation of MARCH8 in ESCCs and the mechanism by which MARCH8 might be facilitating cancer cells to evade immune surveillance.