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The interactome and proteomic responses of ALKBH7 in cell lines by in-depth proteomics analysis
Proteome Science ( IF 2.1 ) Pub Date : 2019-12-29 , DOI: 10.1186/s12953-019-0156-x Shu Meng 1 , Shaohua Zhan 1, 2, 3 , Wanchen Dou 4 , Wei Ge 1, 5, 6
Proteome Science ( IF 2.1 ) Pub Date : 2019-12-29 , DOI: 10.1186/s12953-019-0156-x Shu Meng 1 , Shaohua Zhan 1, 2, 3 , Wanchen Dou 4 , Wei Ge 1, 5, 6
Affiliation
Background
ALKBH7 is a mitochondrial protein, involved in programmed necrosis, fatty acid metabolism, cell cycle regulation, and prostate cancer disease. However, the exact roles of ALKBH7 and the underlying molecular mechanisms remain mysterious. Thus, investigations of the interactome and proteomic responses of ALKBH7 in cell lines using proteomics strategies are urgently required.
Methods
In the present study, we investigated the interactome of ALKBH7 in mitochondria through immunoprecipitation-mass spectrometry/mass spectrometry (IP-MS/MS). Additionally, we established the ALKBH7 knockdown and overexpression cell lines and further identified the differentially expressed proteins (DEPs) in these cell lines by TMT-based MS/MS. Two DEPs (UQCRH and HMGN1) were validated by western blotting analysis.
Results
Through bioinformatic analysis the proteomics data, we found that ALKBH7 was involved in protein homeostasis and cellular immunity, as well as cell proliferation, lipid metabolism, and programmed necrosis by regulating the expression of PTMA, PTMS, UQCRH, HMGN1, and HMGN2. Knockdown of ALKBH7 resulted in upregulation of UQCRH and HMGN1 expression, and the opposite pattern of expression was detected in ALKBH7 overexpression cell lines; these results were consistent with our proteomics data.
Conclusion
Our findings indicate that the expression of UQCRH and HMGN1 is regulated by ALKBH7, which provides potential directions for future studies of ALKBH7. Furthermore, our results also provide comprehensive insights into the molecular mechanisms and pathways associated with ALKBH7.
中文翻译：
深入蛋白质组学分析 ALKBH7 在细胞系中的相互作用组和蛋白质组学反应
背景
ALKBH7 是一种线粒体蛋白，参与程序性坏死、脂肪酸代谢、细胞周期调节和前列腺癌疾病。然而，ALKBH7 的确切作用和潜在的分子机制仍然是个谜。因此，迫切需要使用蛋白质组学策略研究 ALKBH7 在细胞系中的相互作用组和蛋白质组学反应。
方法
在本研究中，我们通过免疫沉淀-质谱/质谱（IP-MS/MS）研究线粒体中ALKBH7的相互作用组。此外，我们建立了 ALKBH7 敲低和过表达细胞系，并通过基于 TMT 的 MS/MS 进一步鉴定了这些细胞系中的差异表达蛋白 (DEP)。通过蛋白质印迹分析验证了两个 DEP（UQCRH 和 HMGN1）。
结果
通过蛋白质组学数据的生物信息学分析，我们发现ALKBH7通过调节PTMA、PTMS、UQCRH、HMGN1和HMGN2的表达参与蛋白质稳态和细胞免疫，以及细胞增殖、脂质代谢和程序性坏死。敲除 ALKBH7 导致 UQCRH 和 HMGN1 表达上调，在 ALKBH7 过表达细胞系中检测到相反的表达模式；这些结果与我们的蛋白质组学数据一致。
结论
我们的研究结果表明 UQCRH 和 HMGN1 的表达受 ALKBH7 的调节，这为 ALKBH7 的未来研究提供了潜在的方向。此外，我们的结果还为与 ALKBH7 相关的分子机制和途径提供了全面的见解。
更新日期：2019-12-29
ALKBH7 is a mitochondrial protein, involved in programmed necrosis, fatty acid metabolism, cell cycle regulation, and prostate cancer disease. However, the exact roles of ALKBH7 and the underlying molecular mechanisms remain mysterious. Thus, investigations of the interactome and proteomic responses of ALKBH7 in cell lines using proteomics strategies are urgently required.
Methods
In the present study, we investigated the interactome of ALKBH7 in mitochondria through immunoprecipitation-mass spectrometry/mass spectrometry (IP-MS/MS). Additionally, we established the ALKBH7 knockdown and overexpression cell lines and further identified the differentially expressed proteins (DEPs) in these cell lines by TMT-based MS/MS. Two DEPs (UQCRH and HMGN1) were validated by western blotting analysis.
Results
Through bioinformatic analysis the proteomics data, we found that ALKBH7 was involved in protein homeostasis and cellular immunity, as well as cell proliferation, lipid metabolism, and programmed necrosis by regulating the expression of PTMA, PTMS, UQCRH, HMGN1, and HMGN2. Knockdown of ALKBH7 resulted in upregulation of UQCRH and HMGN1 expression, and the opposite pattern of expression was detected in ALKBH7 overexpression cell lines; these results were consistent with our proteomics data.
Conclusion
Our findings indicate that the expression of UQCRH and HMGN1 is regulated by ALKBH7, which provides potential directions for future studies of ALKBH7. Furthermore, our results also provide comprehensive insights into the molecular mechanisms and pathways associated with ALKBH7.
中文翻译：
深入蛋白质组学分析 ALKBH7 在细胞系中的相互作用组和蛋白质组学反应
背景
ALKBH7 是一种线粒体蛋白，参与程序性坏死、脂肪酸代谢、细胞周期调节和前列腺癌疾病。然而，ALKBH7 的确切作用和潜在的分子机制仍然是个谜。因此，迫切需要使用蛋白质组学策略研究 ALKBH7 在细胞系中的相互作用组和蛋白质组学反应。
方法
在本研究中，我们通过免疫沉淀-质谱/质谱（IP-MS/MS）研究线粒体中ALKBH7的相互作用组。此外，我们建立了 ALKBH7 敲低和过表达细胞系，并通过基于 TMT 的 MS/MS 进一步鉴定了这些细胞系中的差异表达蛋白 (DEP)。通过蛋白质印迹分析验证了两个 DEP（UQCRH 和 HMGN1）。
结果
通过蛋白质组学数据的生物信息学分析，我们发现ALKBH7通过调节PTMA、PTMS、UQCRH、HMGN1和HMGN2的表达参与蛋白质稳态和细胞免疫，以及细胞增殖、脂质代谢和程序性坏死。敲除 ALKBH7 导致 UQCRH 和 HMGN1 表达上调，在 ALKBH7 过表达细胞系中检测到相反的表达模式；这些结果与我们的蛋白质组学数据一致。
结论
我们的研究结果表明 UQCRH 和 HMGN1 的表达受 ALKBH7 的调节，这为 ALKBH7 的未来研究提供了潜在的方向。此外，我们的结果还为与 ALKBH7 相关的分子机制和途径提供了全面的见解。
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