Dataset Information

The AMBRA1 E3 ligase adaptor regulates Cyclin D protein stability

ABSTRACT: Ubiquitylation proteomics experiment for HEK293T cells treated with either non-targeting shRNA, or shRNA knockdown of AMBRA1 by two independent shRNA's (shAMBRA1-TRC20, and shAMBRA1-TRC36).

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Danielle Swaney

LAB HEAD: Nevan Krogan

PROVIDER: PXD022111 | Pride | 2021-04-22

REPOSITORIES: Pride

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The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

Chaikovsky Andrea C AC Li Chuan C Jeng Edwin E EE Loebell Samuel S Lee Myung Chang MC Murray Christopher W CW Cheng Ran R Demeter Janos J Swaney Danielle L DL Chen Si-Han SH Newton Billy W BW Johnson Jeffrey R JR Drainas Alexandros P AP Shue Yan Ting YT Seoane Jose A JA Srinivasan Preethi P He Andy A Yoshida Akihiro A Hipkins Susan Q SQ McCrea Edel E Poltorack Carson D CD Krogan Nevan J NJ Diehl J Alan JA Kong Christina C Jackson Peter K PK Curtis Christina C Petrov Dmitri A DA Bassik Michael C MC Winslow Monte M MM Sage Julien J

Nature 20210414 7856

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication<sup>1</sup>. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unch ...[more]

PMID: 33854239

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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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	MQ_results.zip	Other
	MSandHPLCmethods.xlsx	Xlsx
	MaxQuant_1.6.10.43.zip	Other
	QE20140918-04.raw	Raw
	QE20140918-05.raw	Raw