Dataset Information

Internally Tagged Ubiquitin: a Tool to Identify Linear Polyubiquitin-modified Proteins by Mass Spectrometry

ABSTRACT: Even though ubiquitination controls a plethora of cellular processes, modifications by linear polyubiquitin have so far only been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (Otulin) and a very few substrates have been identified. The existing methods for the study of lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present a novel approach to discovering linear polyubiquitin-modified substrates by combining lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-Rex HEK293T cells and afterwards validated newly identified linear polyubiquitin targets. Moreover, we demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for the NFkappaB signalling. Overall, we have established a powerful method for the detection of linear polyubiquitin substrates.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Nicolas Nalpas

LAB HEAD: Koraljka Husnjak

PROVIDER: PXD005878 | Pride | 2017-03-21

REPOSITORIES: Pride

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Publications

Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.

Kliza Katarzyna K Taumer Christoph C Pinzuti Irene I Franz-Wachtel Mirita M Kunzelmann Simone S Stieglitz Benjamin B Macek Boris B Husnjak Koraljka K

Nature methods 20170320 5

Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified prote ...[more]

PMID: 28319114

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Project description:The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning and other essential developmental processes. The zinc-finger transcription factor Zelda (ZLD) plays a key role in the transcriptional activation of these earliest-expressed genes. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD prior to (mitotic cycles 8 and 9), during (mitotic cycles 13 and early 14) and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8-9 embryos, most of which remain bound through mitotic cycle 14. As expected, these ZLD-bound regions include the promoters and enhancers of the small subset of genes transcribed at this early stage. However we also observed ZLD bound at cycle 8-9 to the promoters of a large fraction of the several thousand genes whose first transcription does not occur until roughly an hour and four mitotic cycles later. These early ZLD-bound regions include virtually all of the thousands of known and presumed enhancers bound at cycle 14 by the transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription, but also plays a major role in activating the genome at the MZT. Genome-wide mapping of Zelda in wild-type Drosophila melanogaster embryos prior (mitotic cycles 8-9), during (cycles 13-14), and after (late cycle 14) maternal-to-zygotic transition

Dataset Information

Internally Tagged Ubiquitin: a Tool to Identify Linear Polyubiquitin-modified Proteins by Mass Spectrometry

Publications

Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.

Similar Datasets

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