Mammalian peroxiredoxin V (PrxV) is a member of a large family of antioxidant proteins playing important roles in antioxidant defence, tumour suppression and signal transduction. However, precise functions of PrxV are not yet firmly established. This protein is expressed at high level in most of human tissues and is associated with peroxisomes, mitochondria and the nucleus. In this project the role of PrxV in mitochondrial functions, apoptotic signal transduction, DNA antioxidant defence and regulation of transcription will be investigated using specially engineered human cell lines with siRNA-mediated downregulation of PrxV or with impairment of its antioxidant activity (Objective 1, task 1). These lines are already constructed in our team using PrxV siRNA expressing plasmid and in vitro mutagenesis. Next we plan to analyse the correlation between PrxV function and NAD kinase level, PARP-1 activity, and the release of several mitochondrial proteins, essential for activation of cell death machinery (Objective 1, task 2).
Since nuclear PrxV partially co-localizes with Cajal bodies, it can affect pre-assembly of transcription complexes and induce changes of global transcription of DNA repeats, which will be studied here by real-time PCR. Using "phylogenetic footprinting" as well as other standard approaches, essential regulatory elements in the PrxV gene promoter will be identified (Objective 1, task 3), which will help to understand regulation of this gene and its integration into the cellular signalling network of antioxidant responses (Objective 2, task 4). PrxV-interacting soluble proteins in nuclear and mitochondrial extracts will be also investigated using immunoprecipitation and mass-spectrometry techniques (Objective 5, task 5). Approaches used in this project will include traditional methods of molecular biology, cell biology and genomics, as well as modern methods of bioinformatics, real-time PCR, mass-spectrometry and cell death research.
Experiments will be mainly performed by the NIS participating team 3 in St. Petersburg (headed by N. Tomilin), but advanced mitochondrial and apoptosis assays and mass-spectrometry will be performed in the laboratory of the coordinator of this project in Stockholm (Team 1, B. Zhivotovsky) and in the second participating INTAS laboratory in Bergen (team 2, M. Ziegler).
Expected results of this project:
1) elucidation of the mechanism of antiapoptotic activity of PrxV and its potential role in regulation of global gene expression and transcription of non-coding DNA (retrotransposons, tandem repeats);
2) identification of signalling pathways controlling PrxV gene expression, which will allow to decrease PrxV anti-apoptotic activity, increase efficiency of cancer chemotherapy with DNA damaging agents and may be helpful in identification of agents which can decrease ROS production by mitochondria.