The interactome and proteomic responses of ALKBH7 in cell lines by in-depth proteomics analysis

Abstract

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.

Keywords: ALKBH7; Lipid metabolism; MS/MS; Programmed necrosis.

Fig. 1

Gene Ontology and pathway enrichment analysis of proteins enriched in mitochondrion associated with ALKBH7. (a) ALKBH7 complexes in the crude mitochondrial extract from HeLa S3 cells were enriched with anti-ALKBH7 agarose. Five regions of gel with distinctive protein bands were excised for in-gel digestion, and further identified by MS. (b) Gene Ontology and pathway enrichment analysis of identified proteins detected through IP-MS/MS. The categories of biological pathway, biological process, cellular component, and molecular function are shown in blue, red, green, and purple, respectively. The percentage of genes per category is represented by the size of the node. (c) Protein-protein interaction network constructed with enriched proteins in mitochondrion identified through IP-MS/MS

Fig. 2

Venn diagram and correlation analysis of identified proteins in shALKBH7 and ALKBH7^OE cell lines through TMT-based MS/MS. (a) transient overexpressed ALKBH7-FLAG HEK293T cells and the stable ALKBH7 knockdown HeLa cell line ‘72’ and ‘73’. (b) The number of proteins unique to, or shared by, both biological replicates of TMT-based MS/MS and IP-MS/MS is shown in a Venn diagram. (c) Correlation matrix showing all the abundance of the identified proteins in two biological replicates of TMT-based MS/MS. Red boxes indicate correlations between biological duplicates

Fig. 3

Bioinformatic analysis of differentially expressed proteins (DEPs) in shALKBH7 and ALKBH7^OE cell lines through TMT-based MS/MS. (a) Hierarchical clustering of DEPs in one or two biological replicates of TMT-based MS/MS by Heatmap. Red indicates upregulated DEPs, whereas green indicates downregulated DEPs. The panel on the left shows the five different clusters. (b) GO and pathway enrichment analysis of DEPs in Clusters 3 and 5. The categories of biological pathway, biological process, cellular component, and molecular function are shown in blue, red, green, and purple, respectively. The percentage of genes per category is represented by the size of the node

Fig. 4

A comprehensive interaction network among the proteins changed in two biological TMT-MS/MS replicates. (a) Box plot denotes the fold change of DEPs in Cluster 3 and 5. Statistical outlier proteins are colored and labeled. (b) Protein-protein interaction network constructed with DEPs in two biological replicates of TMT-based MS/MS; no connective DEPs were excluded. DEPs are represented by colored nodes labeled with the gene name. The average fold changes of DEPs in two biological replicates of TMT-based MS/MS are reflected by color intensity. Upregulated and downregulated DEPs are shown in red and green, respectively. V-shape nodes denote DEPs located in mitochondrion. Node size correlates with the number of interactions

Fig. 5

Validation of TMT-based proteomics data. (a) Two DEPs (UQCRH and HMGN1) identified in proteomics data were validated by western blotting. Knockdown of ALKBH7 resulted in upregulation of UQCRH and HMGN1 expression, and the opposite pattern of expression was detected in ALKBH7 overexpression cell lines, which were consistent with our proteomics data. (b) The relative intensity of UQCRH and HMGN1 in western blotting (normalized by corresponding β-actin expression)