Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ENVERESP (Crosstalk between nuclear envelope and DNA Damage Response: Role of nucleoporin TPR in the maintenance of genomic integrity)

Teaser

Each cell in the human body receives thousands of DNA lesions per day. To counteract risks posed by DNA damage, cells have evolved a signaling network called the DNA-damage response (DDR). This mechanism allows cells to sense DNA lesions, signal their presence and promote...

Summary

Each cell in the human body receives thousands of DNA lesions per day. To counteract risks posed by DNA damage, cells have evolved a signaling network called the DNA-damage response (DDR). This mechanism allows cells to sense DNA lesions, signal their presence and promote their repair. Inherited mutations in DNA repair genes are associated with predisposition to cancer. Moreover, defects in DDR components in early neoplastic lesions of sporadic tumors allow cancer progression.
Nucleoporins, proteins localized at the nuclear envelope, have been linked to various human diseases such as neurological disorders, autoimmune dysfunctions, and cancer. Nucleoporin TPR (Translocated Promoter Region) was previously linked to cancer since its N- terminal domain has been found fused with the protein kinase domains of various proto- oncogenes such as RAF and MET resulting in solid human tumors. TPR expression level was found deregulated in many types of human tumors such as breast and liver cancer. Importantly, amplification of TPR gene is associated with shorter survival in cancer patients, e.g., those with pediatric ependymomas (3rd most common brain tumor in children). All these findings support a critical role for TPR in the mechanism of oncogenesis and provide the prospect for accurate diagnosis and targeted cancer therapy in the future.
Importantly, although nucleoporin TPR was previously identified as one of the proteins activated in response to DNA damage, its potential role in the maintenance of genomic integrity is unknown.
The overall aim of the project was to characterize molecular crosstalk mechanism between the DNA-damage response network and nuclear envelope, which contributes to the maintenance of genome stability and prevents cancer development.

Work performed

In the first part of the project, we analyzed TPR interactome using proteomic approaches. We identified proteins interacting with TPR both in non-damaging conditions and also in the presence of replication stress causing DNA damage. Importantly, we also revealed which TPR protein interactions are modified upon DNA damage. This set of experiments allowed us to identify molecular pathways in which TPR is involved.
Next, to understand the significance of TPR protein phosphorylation (= its activation or inactivation) upon DNA damage, we used in vitro mutagenesis to replace some of the phosphorylation sites with phosphorylation/dephosphorylation-mimicking variants.
To analyze the phenotype of TPR phosphorylation mutants, we used various experimental approaches and methods including immunofluorescence, Western blot, and electron microscopy. This approach allowed us to identify particular phosphorylation site in TPR protein, which plays a crucial role in the maintenance of genomic integrity.
Then we aimed to reveal whether phosphorylation of TPR sites regulates any protein interactions upon DNA damage. Therefore, we expressed TPR phosphorylation mutants and analyzed our samples using proteomic approaches. By comparison of cells expressing either phosphorylated or dephosphorylated versions of TPR protein, we identified proteins which interaction with TPR is regulated by phosphorylation of particular sites.

Final results

Overall, by employing state-of-the-art proteomics and imaging approaches, we discovered molecular crosstalk mechanism between the DNA-damage response network and nuclear envelope.
One of the main aims of the cancer research should be to improve the health of people. We believe that from work carried out within this project will benefit not only cancer patients and their families but also society as a whole since:
1. It provides new fundamental knowledge about the role of nuclear envelope-associated proteins in the maintenance of genomic stability and prevention of cancer development.
2. It contributes to our understanding of molecular principles of response to damaged DNA.
3. Data obtained in our project will allow to other scientists start new projects, which will develop our initial observations into further details. Therefore, our project benefit to future research in the field of tumor biology.
4. It opens new possibilities for innovation of existing cancer treatments and development of new molecular targeted therapies for cancer in the future.