Interactome Analysis Reveals the Novel Function of N-Myristoyl Transferase (NMT) 1 and 2 in DNA Damage Response Open Access
Hu, Jiaxuan (Spring 2024)
Abstract
DNA damage response (DDR), which plays a pivotal role in maintaining genomic integrity, has been emerging as a critical mechanism to target as novel cancer therapies. Recent advancements have spotlighted the significance of post-translational modifications (PTMs), such as phosphorylation, acetylation, ubiquitylation, and SUMOylation in activating and coordinating cellular processes that detect and repair DNA damage. N-myristoylation, which is a less-studied PTM catalyzed by N-myristoyl transferase (NMT) 1 and 2, involves attaching a 14-carbon saturated fatty acid (myristic acid) to N-terminal glycine or lysine residues. This lipid modification significantly impacts protein characteristics, dynamics, and functions, thereby regulating various cellular processes including signal transduction pathways, apoptosis, and immune system response. Despite the significant role of N-myristoylation in biological functions, there remains a significant gap in our understanding of the interplay between N-myristoylation and DDR. In this study, we aim to delineate the involvement of NMT1 and NMT2 interactome in DDR. Utilizing affinity purification with EGFP-NMT1 or EGFP-NMT2 coupled with quantitative proteomics to systematically characterize the cellular interacting proteome of NMT1 and NMT2 in HEK293T cells, we identified 641 and 678 interacting proteins of NMT1 and NMT2, respectively. Gene ontology enrichment analysis revealed the interactome of NMT1 and NMT2 ubiquitously expressed not only in cytoplasm, but also in nucleus. Interestingly, process network analysis by MetaCore database showed the interactome of NMT1 and NMT2 significantly involving in DNA damage response. Further analysis determined 31 and 33 proteins associated with DNA repair in the NMT1 and NMT2 interactome, respectively. Validation study has confirmed the physiological interaction of NMT1 and NMT2 with the DDR interactors such as RecQ-like DNA helicase (BLM), Poly [ADP-ribose] polymerase 1 (PARP1), and RNA helicase aquarius (AQR). Overall, we established a foundational understanding of NMT1 and NMT2's involvement in DDR, highlighting the significance of NMT1 and NMT2 involvement in DNA repair. Furthermore, the identification of DNA repair proteins in NMT1 and NMT2 interactomes established a platform for future studies to unravel the mechanistic details of these interactions and their implications for cancer biology and therapy.
Table of Contents
Table of Contents
Chapter 1: INTRODUCTION
Chapter 2: MATERIALS AND METHODS
2.1 Materials
2.2 Bacteria transformation
2.3 Plasmid purification
2.4 Cell culture and treatment
2.5 Transfection
2.6 Protein lysate preparation
2.7 Bicinchoninate (BCA) protein assay
2.8 Co-immunoprecipitation
2.9 Western blotting
2.10 Mass spectrometry
2.11 Data analysis
2.12 Bioinformatics tool
2.13 Graph representation
Chapter 3: RESULTS
3.1 Identification of interactome of NMT1 and NMT2
3.2 NMT1 and NMT2 interactome involve in various cellular component and has a wide range of molecular functions
3.3 NMT1 and NMT2 interactome have functions related to DNA damage response
3.4 Identification of proteins involved in DNA damage response in NMT1 and NMT2 interactome
3.5 Validation of proteins involved in DNA damage repair pathways
Chapter 4: DISCUSSIONS
REFERENCES
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