Dataset Information

Deubiquitinases that digest Met1 chains in Rice

ABSTRACT: Met1 type ubiquitination and deubiquitination are involved in the regulation of many fundamental processes such as inflammation and innate immunity, and their interference by pathogens can suppress immune responses in human cells. However, few plant-derived deubiquitinases (DUBs) against Met1 ubiquitin chains has been reported, and the selection mechanism for linkage-type of ubiquitin chains remains elusive, which greatly limits our understanding of the functions of plant-derived DUBs. Using a dehydroalanine (DHA)-bearing Met1 diubiquitin (Met1-diUb) suicide probe, synthesized in one-pot, we captured OTUB1 from Oryza sativa (OsOTUB1) and uncovered its ability to hydrolyze Met1 ubiquitin chains (Met1 activity). Further, by resolving the apo structure of OsOTUB1 and its complex with Met1-diUb, we found that Met1-specific motifs (N-handle motif and C-handle motif) in the S1’ pocket of OsOTUB1 confers its Met1 activity. Through large-scale sequence alignment and hydrolysis experiments, Met1-specific motifs in the S1' pocket of the OTUB subfamily (OTUBs) were found to determine the Met1 ability of OTUBs, regardless of species. Furthermore, by analyzing the species distribution of OTUBs capable of hydrolyzing Met1 ubiquitin chains, we found that whereas this activity does not exist in metazoans, it is conserved in green plants (Viridiplantae). This discovery may inform studies of the differentiation between primitive plants and animals.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Oryza Sativa (rice)

TISSUE(S): Leaf

SUBMITTER: Li-Ning Lu

LAB HEAD: Lining Lu

PROVIDER: PXD032822 | Pride | 2022-10-15

REPOSITORIES: pride

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Dataset's files

Source:

			Action	DRS
	19-076_LLN0329_Lu-1.msf	Msf
	19-076_LLN0329_Lu-1.mzML	Mzml
	19-076_LLN0329_Lu-1.mzid.gz	Mzid
	19-076_LLN0329_Lu-1.raw	Raw
	19-076_LLN0329_Lu-2.msf	Msf

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Publications

Met1-specific motifs conserved in OTUB subfamily of green plants enable rice OTUB1 to hydrolyse Met1 ubiquitin chains.

Lu Lining L Zhai Xiaoguo X Li Xiaolong X Wang Shuansuo S Zhang Lijun L Wang Luyang L Jin Xi X Liang Lujun L Deng Zhiheng Z Li Zichen Z Wang Yanfeng Y Fu Xiangdong X Hu Honggang H Wang Jiawei J Mei Ziqing Z He Zhengguo Z Wang Feng F

Nature communications 20220809 1

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ...[more]

PMID: 35945250

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Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.