Uncovering the Functions of Bromodomain-Containing Proteins ATAD2A and ATAD2B in Cancer Development and Identification of their Interacting Partners

Roy, Anirban (2024) Uncovering the Functions of Bromodomain-Containing Proteins ATAD2A and ATAD2B in Cancer Development and Identification of their Interacting Partners. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Anirban Roy (18RS090)) 18RS090.pdf - Submitted Version Restricted to Repository staff only Download (21MB)

Abstract

The ATPase family AAA domain-containing protein 2 (ATAD2A/ATAD2) and its closely related paralogue, ATAD2B, are nuclear co-regulator proteins containing two AAA+ ATPase domains and bromodomain. The AAA+ ATPase domains are associated with ATP-driven chromatin remodeling, while the bromodomain recognizes acetylated lysine in histones and non-histone proteins to regulate gene expression. ATAD2A is implicated in cancer through pathways like Rb-E2F1, PI3K/AKT, and TGF-β1/Smad3, enhancing cell proliferation and cancer progression. ATAD2A is significantly upregulated in many cancer types and contributes to poor patient outcomes. Despite sharing high sequence and structural similarity with ATAD2A, little is known about the unique role of ATAD2B. The Cancer Genome Atlas (TCGA) data analysis revealed widespread overexpression of ATAD2B in breast and brain cancers. ATAD2 proteins (ATAD2A and ATAD2B) are conserved epigenetic regulators crucial for histone acetylation-related gene regulation, with their bromodomains interacting with acetyllysine (Kac) on histones. We highlighted the comparative stability, binding mode, and robustness of ATAD2A and ATAD2B bromodomains in interacting with acetylated histones and nucleosomes. Herein, we report that ATAD2 directly interacts with EMT transcription factor TWIST1 and enhances the transcriptional activation of MYC in colorectal carcinoma cells. In addition, analysis of TCGA and Clinical Proteomic Tumor Analysis Consortium (CPTAC) data suggests a correlation between ATAD2, TWIST1, and MYC overexpression and poor survival rates in colorectal carcinoma. It also highlighted the presence of the ATAD2-TWIST1-MYC axis in colorectal carcinoma. Next, we utilized a bioorthogonal chemoproteomic approach called photocrosslinking-based interactome profiling to identify the transient non-histone interacting partners of ATAD2B bromodomains. Using amber suppression mutagenesis, we successfully incorporated 4-azido-L-phenylalanine (AzF) and 4-benzoyl-L-phenylalanine (BzF) photocrosslinking unnatural amino acids into the ATAD2B bromodomain. ATAD2B-BzF mutant demonstrated superior crosslinking ability than ATAD2B-AzF mutant in capturing the cellular proteome. We found a novel interactome for ATAD2B, which includes proteins like NPM1, RELA, GATAD2A, and TRIM28, suggesting its involvement in diverse cellular processes beyond chromatin remodeling and transcriptional regulation.

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