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Streptavidin biotin-CCW16 pulldown to determine CCW16-modified proteins in HeLa lysate

ABSTRACT: The SUMO-targeted E3 ubiquitin ligase RNF4 plays an important role in safeguarding genome and proteome integrity. RNF4 recognizes polySUMO modified proteins and induces their proteolytic or non-proteolytic ubiquitylation. Based on the key functions of RNF4 maintaining proteome and genome stability, control of DNA damage response and DNA-protein crosslink repair, we explored its role in cancer cell survival and as a potential drug target. We found that RNF4 is overexpressed in acute myeloid leukemia and expression levels correlate with poor survival. To develop PROTACs for targeted degradation of RNF4, we aimed to exploit the recently developed cysteine-reactive RNF4 ligand CCW16, and synthesized a set of CCW16-derived degrader molecules using established VHL- and CRBN E3 ligands. To determine CCW16 specificity and binding partners, we performed a Streptavidin Biotin-CCW16 pull down of HeLa cell lysate. We detected around 2000 proteins, which showed significant increase in binding to CCW16 biotin, compared to Biotin only. Interestingly, we found proteins of the peroxiredoxin family covalently modified with CCW16 at the catalytically active cysteine residues, indicating an induction of oxidative stress.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Stefan Müller

LAB HEAD: Stefan Müller

PROVIDER: PXD056954 | Pride | 2025-08-21

REPOSITORIES: Pride

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			Action	DRS
	20240330_HF_LC1_MF_GGO_185_GG_1.msf	Msf
	20240330_HF_LC1_MF_GGO_Biotin-CCW16_Rep1.raw	Raw
	20240330_HF_LC1_MF_GGO_Biotin-CCW16_Rep2.raw	Raw
	20240330_HF_LC1_MF_GGO_Biotin-CCW16_Rep3.raw	Raw
	20240330_HF_LC1_MF_GGO_Biotin_Rep1.raw	Raw

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