Combining genetics and quantitative proteomics to dissect the cellular response to DNA damage. – CeLeNed

The regulation of various protein functions by Ubiquitin-like molecules modifications is of major interest in cell signalling. NEDD8 encodes the most closely related protein to ubiquitin and possesses its own set of enzymes that ensures a distinct conjugation pathway. Experiments in genetic model systems have shown a role for NEDD8 in viability, development and growth. The best characterised target for NEDD8 is the cullin family of proteins, an essential component of the Cullin-Ring E3 ligase complex. Cullin NEDDylation regulates the degradation of proteins involved in cell cycle, transcriptional regulation, O2 and centrosome as well as cytoskeletal regulation. However, recent proteomic studies have reported a wide range of other cellular targets, suggesting a crucial signalling function for NEDD8. For instance, NEDDylation of ribosomal proteins controls signalling from the nucleolus to the p53 tumour suppressor1. The NEDD8 pathway is implicated in various pathologies such as cancer and more specifically leukaemia, for which an inhibitor of the NEDD8 pathway is under clinical trial. Therefore, elucidation of the molecular targets and biological processes controlled by NEDD8 is of wide interest with clinical applications for human diseases. Our goal is to identify and characterize novel targets and biological functions for NEDDylation using C. elegans as a model organism.
C. elegans has been instrumental in the discovery of apoptosis in the context of the organism development and in response to stress stimuli such as DNA damage. We have recently discovered a role for components of the NEDD8 machinery in the DNA damage induced apoptosis in C. elegans. The ulp-3 (Ubiquitin-like protease-3) gene is orthologous to human NEDP1 de-NEDDylating enzyme. We discovered that worms deleted for the ulp-3 gene are defective in the apoptotic response following DNA damage (unpublished results). Our further genetic analysis shows that ulp-3 is either downstream of cep-1 (p53 homologue), or indeed defines an unknown apoptotic pathway parallel to cep-1. Studies in the host laboratory have recently revealed that knockdown of NEDP1 in human cells causes similar defects in the DNA damage induced apoptosis. Therefore, we have discovered a conserved role for ulp-3/NEDP1 for which the molecular mechanisms and the physiological targets are currently unknown.
We will combine innovative proteomic approaches with worm genetics to identify and validate targets of ULP-3 involved in the DNA damage response. We will exploit the novel proteomic technology we have established. Our objectives are:
1) To identify proteins targeted by the NEDDylation pathway that regulate the DNA damage response and more specifically induce germ cell apoptosis. For this, we will apply triple SILAC (Stable Isotope Labeling of Amino acid in Cell culture) approach we’ve developed in order to specifically detect protein conjugated by NEDD8. We will use C. elegans genetics as a powerful mean to compare specific genetic backgrounds. This will allow us to identify substrates that are deNEDDylated in response to DNA damage specifically by ULP-3/NEDP1.

2) To evaluate how potential NEDD8 targets impact on the DNA damage response by using C. elegans germ cells as a model system. We will investigate the effect of the corresponding gene disruption, either by analyzing the gene deletion or gene silencing by RNAi. We will combine live cell imaging, to visualize execution of apoptosis, with worm genetics.

Our proposed project should identify NEDD8 substrates involved in the DNA damage induced apoptosis. We anticipate that critical information within the signalling pathway will emerge from our studies. Given the conservation of the essential components of the DNA damage response in Eukaryotic cells, we expect that our work will have important implications relevant to human disease such as cancer.