Introduction and background
The enzyme NTHL1 plays a critical role in the base excision repair (BER) pathway, where it is involved in the removal of oxidized pyrimidines from DNA, thus maintaining genomic stability. NTHL1’s function is vital for the repair of oxidative DNA damage, which, if left unchecked, can lead to mutations and contribute to the development of cancer.
Interestingly, NTHL1 has recently been implicated in neuronal health and Parkinson's disease, where removal of this DNA repair protein leads to the protection of neurons from oxidative stress. Mutations or dysregulation of NTHL1 may lead to increased neuronal damage and play a role in the progression of Parkinson's disease.
While the basic enzymatic mechanism of NTHL1 in DNA repair is well characterized at molecular level, emerging evidence suggests that its role in neurodegeneration may be more complex and involves additional regulatory mechanisms. NTHL1 operates in a coordinated manner with other proteins in the BER pathway, and its activity is influenced by various factors, including cellular redox states.
Given its importance in DNA repair and its association with Parkinson's disease, further investigation into NTHL1’s function could provide new insights into the mechanisms underlying neurodegeneration and identify potential therapeutic targets for treating Parkinson's disease.
Aim of project
The aim of this project is to use structural and biochemical methods to perform an in-depth investigation of the NTHL1 enzymatic mechanism in combination with a specific inhibitor that can be used as the basis for a future intervention. Different techniques and strategies will be used to understand the molecular details of the mechanism. We will use X-ray crystallography to study structures of captured reaction intermediates, and spectroscopy to follow the reaction in real-time under different conditions, such as presence of reducing agents, different ratios of substrates and products.
Project plan and methods
Here we seek a highly motivated student to work on a project in the intersection between basic biochemical research and innovation of a potential therapeutic product.
The project will consist of the following main tasks and methods:
- Express recombinant human NTHL1 variants (both wild-type and designed mutants) using E. coli as host and purify the enzymes using chromatographic principles.
- Determine 3D atomic-resolution crystallographic structures of NTHL1 enzymes with and without inhibitory compounds.
- Measure enzymatic activity of wild-type and mutant NTHL1 enzymes under various conditions including the use of inhibitory compounds.
Additional tasks:
Upon completion of the main tasks, we offer the possibility to explore other relevant techniques such as:
- Immunoprecipitation techniques
- PAGE and Western blotting
- Cell culture techniques
Organization / collaboration
The project is a collaboration between group leader Bj?rn Dalhus at the Department of Medical Biochemistry, University of Oslo and Oslo University Hospital (OUS) and Nicola Pietro Montaldo at the Department of Microbiology, University of Oslo and Oslo University Hospital (OUS).
The work will take place at the Dalhus laboratory located at Rikshospitalet, where we have access to relevant technologies to complete the project. Dalhus is an experienced structural biologist, with interest in drug design, and Montaldo is an expert in DNA repair and mitochondrial biology. Hans-Petter Hersleth will serve as the internal supervisor at IBV and as a partner for the structural studies.