背景:线粒体和内质网(ER)通过称为线粒体相关膜(MAMs)的接触位点进行通信。许多重要的细胞功能,如生物能学,线粒体自噬,凋亡,钙信号由MAMs调节,被认为与缺血再灌注损伤(IRI)密切相关。然而,在解决这些细胞过程之间的关系的系统蛋白质组学研究中存在空白。
方法:在正常(N)和缺氧/再灌注(HR)条件下,对来自人肾近端小管上皮细胞系(HK-2细胞)的线粒体相关膜(MAMs)进行了基于4D无标记质谱的蛋白质组学分析。随后的差异蛋白质分析旨在表征疾病相关信号分子。基因本体论(GO)和京都基因和基因组百科全书(KEGG)分析应用于总蛋白和差异表达蛋白,包括生物过程(BP),单元组件(CC),分子功能(MF),和KEGG途径。Further,进行了蛋白质-蛋白质相互作用网络(PPI)探索,导致从差异表达的蛋白质中鉴定hub基因。值得注意的是,鉴定了有丝分裂融合蛋白2(MFN2)和BCL2/腺病毒E1B19-kDa相互作用蛋白3(BNIP3),随后在体外和体内进行了验证。最后,通过调节基因表达来探讨MFN2在缺氧/复氧过程中对MAMs的影响。随后,在OE-MFN2和正常HK-2细胞之间进行了比较蛋白质组学分析,提供对潜在机制的进一步见解。
结果:共鉴定出4489种蛋白质,3531成功量化。GO/KEGG分析显示MAM蛋白主要与线粒体功能和能量代谢有关。两组间的差异分析显示,HRHK-2细胞中688个蛋白的表达水平表现出显著变化,以P值<0.05和HR/N>1.5或HR/N<0.66为阈值标准。差异表达蛋白的富集分析揭示了mRNA剪接等生物过程,凋亡调节,细胞分裂,而分子功能主要与能量代谢活动有关。这些蛋白质在HR期间的细胞反应中起关键作用,提供对IRI机制和潜在治疗靶点的见解。hub基因MFN2和BNIP3在体外和体内的验证与蛋白质组发现一致。MFN2在维持线粒体相关膜(MAMs)的完整性和减轻缺氧/复氧损伤后的线粒体损伤方面具有保护作用。这种保护作用可能与PI3K/AKT通路的激活有关.
结论:位于线粒体相关膜(MAMs)中的蛋白质在肾脏缺血再灌注损伤(IRI)中具有重要作用,MFN2在这方面发挥着关键的调节作用。
The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.
A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.
A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.
The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.