Dataset Information

Identifying the Ubiquitination Targets of E6AP by Orthogonal Ubiquitin Transfer

ABSTRACT: E3 ubiquitin (UB) ligases, the ending module of the E1-E2-E3 cascades, transmit diverse signals in the cell by attaching UB to cellular proteins. Identifying E3 substrates holds the key to elucidate the roles of E3s in cell regulation. We constructed an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in neurodevelopmental disorders and cancer. We used yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells afforded 131 potential E6AP targets. Among them we verified MAPK1, CDK1 and CDK4, and PRMT5 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E6AP substrates and would guide the engineering of OUT cascades with other E3s to interrogate their biological functions.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Duc Duong

LAB HEAD: Jun Yin

PROVIDER: PXD005584 | Pride | 2018-02-07

REPOSITORIES: Pride

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Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer.

Wang Yiyang Y Liu Xianpeng X Zhou Li L Duong Duc D Bhuripanyo Karan K Zhao Bo B Zhou Han H Liu Ruochuan R Bi Yingtao Y Kiyokawa Hiroaki H Yin Jun J

Nature communications 20171220 1

E3 ubiquitin (UB) ligases are the ending modules of the E1-E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively t ...[more]

PMID: 29263404

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Project description:Post translational protein modifications (PTMs) including small chemical groups and small proteins, belonging to the ubiquitin family, are essential for virtually all cellular processes. In addition to modification by a single PTM, proteins can be modified by a combination of different modifiers, which are able to influence each other. Since little is known about crosstalk between different ubiquitin family members, we developed an improved method enabling identification of co-modified proteins on a system-wide level using mass spectrometry. We focused on the role of crosstalk between SUMO and ubiquitin during proteasomal degradation. Using two complementary approaches, we identified 498 proteins to be significantly co-modified by SUMO and ubiquitin upon MG132 treatment. These targets included many enzymatic components of PTM machinery, involved in SUMOylation and ubiquitylation, but also phosphorylation, methylation and acetylation, revealing a highly complex interconnected network of crosstalk between different PTMs. In addition various other biological processes were found to be significantly enriched within the group of co-modified proteins, including transcription, DNA repair and the cell cycle. Interestingly, the latter group mostly consisted of proteins involved in mitosis, including a subset of chromosome segregation regulators. We hypothesize that group modification by SUMO-targeted ubiquitin ligases regulates the stability of the identified subset of mitotic proteins, which ensures proper chromosome segregation. The mitotic regulators KIF23 and MIS18BP1 were verified to be co-modified by SUMO and ubiquitin upon inhibition of the proteasome and subsequently identified as novel RNF4 targets. Both modifications on MIS18BP1 were observed to increase simultaneously during late mitosis, while the total protein level decreased immediately afterwards. These results confirm the regulation of MIS18BP1 via SUMO-ubiquitin crosstalk during mitosis. Combined, our work highlights extensive crosstalk between SUMO and ubiquitin, providing a resource for further unraveling of SUMO-ubiquitin crosstalk.

			Action	DRS
	HUMAN.fasta	Fasta
	ctl_01.msf	Msf
	ctl_01.pdResult	Other
	ctl_01.raw	Raw
	ctl_2and3.msf	Msf