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Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCFβTRCP-dependent ubiquitylation of the actin-organizing protein Dbn1

ABSTRACT: Ubiquitin widely modifies proteins, thereby regulating most cellular functions. The complexity of ubiquitin signalling necessitates unbiased methods enabling global detection of dynamic protein ubiquitylation. Here, we describe UBIMAX (UBiquitin target Identification by Mass spectrometry in Xenopus egg extracts), which enriches ubiquitin-conjugated proteins and quantifies regulation of protein ubiquitylation under precise and adaptable conditions. We benchmark UBIMAX by investigating DNA double-strand break-responsive ubiquitylation events, identifying previously known targets and revealing the actin-organising protein Dbn1 as a novel major target of DNA damage-induced ubiquitylation. We find that Dbn1 is targeted for proteasomal degradation by the SCFβ-Trcp1 ubiquitin ligase, in a conserved mechanism driven by ATM-mediated phosphorylation of a previously uncharacterized β-Trcp1 degron containing an SQ motif. We further show that this degron is sufficient to induce DNA-damage dependent protein degradation of a model substrate. Collectively, we demonstrate UBIMAX’s ability to identify novel targets of stimulus-regulated ubiquitylation and reveal an SCFβ-Trcp1-mediated ubiquitylation mechanism controlled directly by the apical DNA damage response kinases.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Exploris 480

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

SUBMITTER: Camilla Colding-Christensen

LAB HEAD: Michael Lund Nielsen

PROVIDER: PXD042086 | Pride | 2023-12-14

REPOSITORIES: Pride

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			Action	DRS
	1_DSB-UBIMAX_search.tar.gz	Other
	20180308_QE6_nLC12_CSC_SA_HSS_C1_F1.raw	Raw
	20180308_QE6_nLC12_CSC_SA_HSS_C1_F2.raw	Raw
	20180308_QE6_nLC12_CSC_SA_HSS_C1_F3.raw	Raw
	20180308_QE6_nLC12_CSC_SA_HSS_C1_F4.raw	Raw

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Project description:Ubiquitylation is a common post translational modification of eukaryotic proteins. Protein ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite in the asexual blood stage cycle of the human malaria parasite, Plasmodium falciparum (Pf). Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified. 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor, MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We constructed a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

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Profiling ubiquitin signaling with UBIMAX reveals DNA damage- and SCFβTRCP-dependent ubiquitylation of the actin-organizing protein Dbn1

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