Genomic DNA damage is caused by endogenous or exogenous factors such as reactive metabolites, UV and ionizing radiation, chemicals, etc. Unrepaired damage can lead to genomic instability and cancer, accelerated aging, or neurodegenerative disorders. DNA damage also includes covalent DNA-protein crosslinks (DPC). In humans, the removal of DPCs involves the protease SPRTN, which breaks down covalently bound proteins into peptides, after which other mechanisms complete the repair process. In this process, proteins such as p97, Ufd1, and Npl4 also collaborate with SPRTN.
p97, also known as VCP (valosin-containing protein), is an AAA+ ATPase that uses ATP hydrolysis to extract and translocate polypeptides through its central pore. The versatility of p97’s functions is enabled by partner or adapter proteins, with more than 30 known to date. The adapters Npl4 and Ufd1 work together and associate with p97 in various biological processes, such as chromosome activity control, degradation of misfolded proteins, and chromatin regulation. Additionally, the p97-Npl4/Ufd1 complex collaborates with SPRTN in removing DPCs. Understanding the interactions between p97 and its adapters is crucial for comprehending the mechanism of action of these molecules and their potential therapeutic applications.
The aim of this master’s thesis was the preparation of recombinant Npl4 and Ufd1 proteins, their characterization, and a preliminary analysis of their interactions with p97. The work involved molecular cloning, expression of recombinant proteins in bacterial cells, protein isolation and purification using chromatographic methods, biochemical and biophysical analysis of protein purity, stability, and proper folding, as well as a preliminary characterization of protein interactions.
Npl4 and Ufd1 were successfully cloned and expressed in bacterial cells. Optimization of conditions enabled the production of both proteins in large quantities in a soluble form with a high degree of purity. Preliminary experiments demonstrated the interaction between Npl4 and Ufd1, as well as their individual interactions with p97. It was also found that Ufd1 is more thermally stable compared to Npl4. The results of this thesis will provide an important foundation for further research into the mechanism of p97 function in DPC removal in collaboration with Npl4, Ufd1, and SPRTN.
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