Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Systematic analysis of nuclear deubiquitylases – insight into BRCC3-regulated signaling (#792)

Gabriela Prus 1 , Shanka Sapathy 1 , Magdalena Budzowska 1 , Thomas Wild 1 , Chuna Ram Choudhary 1
  1. Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen N, DK, Denmark

BACKGROUND: Ubiquitination is intimately involved in key nuclear processes such as gene transcription, DNA replication, and DNA damage repair. Therefore, many nuclear deubiquitylating enzymes (DUBs) play important roles in regulating these processes. For example, the deubiquitylase BRCC36 (BRCC3) is part of the BRCA1 (breast cancer type 1 susceptibility protein) complex, which includes BRCA1, Rap80, MERIT40, BRE, and the scaffolding protein Abraxas. The BRCA1 complex plays an important role in DNA double strand-break repair and facilitates error-free repair through homologous recombination. It binds to ubiquitylated histones H2A and H2AX at DNA lesion, promoting DNA end resection. Intriguing, BRCC36 is thought to prevent exceeded DNA resection via counteracting ubiquitylation of H2A. However, whether BRCC36 plays another role in BRCA1 complex as well as its site-specificity remains unclear, leaving a considerable gap in understanding the relationship between the DUB and its substrates.

METHODS: Here, we apply a combination of CRISPR-based genome engineering and quantitative proteomics to obtain a systematic and comprehensive picture of DUB-regulated signaling in the nucleus on the endogenous level. Our multi-step workflow provides a new depth to ubiquitylome studies and allows for comprehensive analysis of DUB site-specificity.

RESULTS AND CONCLUSIONS: In our ongoing work we have generated KO cell lines for 8 nuclear DUBs, including BRCC36-KO cell line. Using SILAC-based proteomics, we mapped ubiquitylation sites regulated upon BRCC36 deletion. Our preliminary data shows the significant decrease in several ubiquitylation sites, including the components of BRCA1 complex: MERIT40 and BRE.  These preliminary results indicate a role of BRCC36 in regulating ubiquitylation of the BRCA1 complex members.