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Putative K29-linked substrates identification using sAB-K29

ABSTRACT: We have identified K29-linked ubiquitination is involved in cell cycle regulation and enriched in midbody formation. To identify the substrates of K29-linked ubiquitination in the midbody, we soughtto perform immunoprecipitation (IP) under denaturing conditions to minimize the presence of noncovalently bound proteins. we synchronized HeLa cells to telophase to maximize the abundance of the midbody and lysed the cells using 8 M urea. The lysate was further diluted in 1 M urea, followed by IP using biotinylated sAB-K29. The enriched proteins were separated on an SDS-PAGE gel and subjected to label-free quantitative mass spectrometry analysis.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Qingyun Zheng

LAB HEAD: Lei Liu

PROVIDER: PXD024425 | Pride | 2021-07-11

REPOSITORIES: Pride

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			Action	DRS
	checksum.txt	Txt
	denatureIP_beads_C1.raw	Raw
	denatureIP_beads_C2.raw	Raw
	denatureIP_beads_C3.raw	Raw
	denatureIP_beads_C4.raw	Raw

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Publications

K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.

Yu Yuanyuan Y Zheng Qingyun Q Erramilli Satchal K SK Pan Man M Park Seongjin S Xie Yuan Y Li Jingxian J Fei Jingyi J Kossiakoff Anthony A AA Liu Lei L Zhao Minglei M

Nature chemical biology 20210708 8

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can sp ...[more]

PMID: 34239127

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Hepatitis B Core Protein is Posttranslationally Modified through K29-linked Ubiquitination

Project description:Hepatitis B virus (HBV) core protein (HBc) plays many roles in the HBV life cycle such as regulation of transcription, RNA encapsidation, reverse transcription and viral release. To accomplish these functions, HBc interacts with many host proteins and undergoes different posttranslational modifications (PTMs). One of the most common PTM is ubiquitination that was shown to change function, stability, and intracellular localization of different viral proteins, but the role of HBc ubiquitination in the HBV life cycle remains unknown. Here, we found that HBc protein is posttranslationally modified through K29-linked ubiquitination. We performed a series of co-immunoprecipitation experiments with wild type HBc, lysine to arginine HBc mutants and wild type ubiquitin, single lysine to arginine ubiquitin mutants or single ubiquitin-accepting lysine constructs. We observed that HBc protein could be modified by ubiquitination in transfected as well as infected hepatoma cells. In addition, ubiquitination predominantly occurred on HBc lysine 7 and the preferred ubiquitin chain linkage was through ubiquitin-K29. Mass spectrometry (MS) analyses detected UBR5 as a potential E3 ubiquitin ligase involved in K29-linked ubiquitination. These findings emphasize that ubiquitination of HBc may play an important role in HBV life cycle.

2020-12-01 | PXD021712 | Pride

The CLASP2 Protein Interaction Network in Adipocytes Links CLIP2 to AGAP3, CLASP2 to G2L1, MARK2, and SOGA1, and Identifies SOGA1 as a Microtubule-Associated Protein

Project description:CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 co-immunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and also revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and also with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.

2017-06-01 | PXD003674 | Pride

The midbody acts as a translationally active membraneless organelle

Project description:mRNAs associated with microtubules during interphase, metaphase and the midbody stage of cytokinesis were sequenced. Selective midbody-localized RNAs were identified and their translational characteristics were studied.

2020-07-18 | GSE134476 | GEO

Analysis of the midbody interactome reveals new roles for PP1 phosphatases in late cytokinesis

Project description:The midbody is an organelle assembled at the intercellular bridge connecting the two daughter cells at the end of mitosis. It is essential for the final separation of the daughter cells and involved in other important processes, including cell fate, pluripotency, apical-basal polarity, and cilium/lumen formation. The midbody is composed of numerous proteins with diverse functions distributed in a precise pattern. Here we report the first characterization of the midbody protein-protein interaction network (interactome), which provides an extremely valuable resource for understanding its multiple roles. Analysis of this midbody interactome led to the discovery that PP1/MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis through de-phosphorylation of the kinesin MKLP1/KIF23, a finding that unexpectedly expands the temporal window of activity of this phosphatase during mitosis.

2019-08-29 | PXD012374 | Pride

Ni Deprivation in Synechococcus WH8102

Project description:Two sets of microarray experiments examining the transcriptional response to Ni deprivation. One set was for growth on ammonium and the other for growth on urea Pairwise replicated comparisons (Ni deprivation in urea and ammonia)

2012-08-08 | E-GEOD-39972 | biostudies-arrayexpress

YBX1-IP in mouse bone marrw myeloma cells

Project description:The total protein of bone marrw myeloma cells was extracted with cell lysis buffer for IP. YBX1 antibody was used to perform Immunoprecipitation.

2023-04-19 | PXD039386 | Pride

The deubiquitinase TRABID stabilises the K29/K48-specific E3 ubiquitin ligase HECTD1

Project description:The OTU deubiquitinase TRABID is highly tuned for the recognition and cleavage of K29 and K33-linked ubiquitin chains. Yet the cellular functions of these atypical ubiquitin signals remain unclear. Here, we compared the interactome of two different catalytically-dead TRABID constructs, one that lacks the OTU domain and the other a single point mutant that is catalytically inactive. Since both constructs also act as ubiquitin trapping mutants, this approach was used to differentiate between interactors and substrates. The E3 ubiquitin ligase HECTD1 was confirmed as a TRABID substrate, and using UbiCREST and Ubiquitin-AQUA proteomics, we determined that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that in contrast to UBE3C, HECTD1 requires the formation of branched K29/K48-linked chains for its full activity. Finally, we used transient knockdown and genetic knock out of TRABID in mammalian cells to show that TRABID stabilises HECTD1 protein levels. This study identifies TRABID-HECTD1 as a K29-specific DUB/E3 pair and provides novel insights on the assembly of branched ubiquitin signals.

2021-06-23 | PXD022703 | Pride

Transcription profiling of Arabidopsis wild type seedlings grown with NH4NO3 or urea as nitrogen sources

Project description:wild-type seedlings were grown on NH4NO3 or Urea as nitrogen source

2009-03-01 | E-MEXP-1771 | biostudies-arrayexpress

Identification of HP0244 regulon at different pH conditions

Project description:Transcriptional profiling of Helicobacter pylori comparing 26695 wild-type strain and a HP0244-deficient mutant 26695/âHP0244::km treated at three different pH conditions (pH 7.4, pH 4.5 without urea, or pH 2.5 with 30 mM urea) for 30 min to define the HP0244 acid-responsive regulon Keywords: Genetic modification and stress response Wild type vs HP0244-deficient mutant at three different pH conditions (pH7.4, pH4.5 without urea, and pH2.5 with 30 mM urea). Three biological replicates for each pH condition: 3 wild type and 3 mutant, independently grown, pH treated, and harvested.

2010-05-05 | E-GEOD-12900 | biostudies-arrayexpress

Co-immunoprecipitation coupled with mass for RNR10 in rice&Identification of ubiquitination sites in rice DNR1

Project description:RNR10 is a key factor that regulates the response of rice roots to nitrogen.In an attempt to determine the target(s) of RNR10, we performed co-immunoprecipitation (Co-IP) coupled with mass spectrometry analysis to search for RNR10 interactors.DNR1 is a downstream target gene of RNR10.Previous studies have shown that rice DNR1 is subjected to ubiquitination modification. In order to identify the ubiquitination modification sites of DNR1,we performed mass spectrometry analyses.

2023-09-30 | PXD043400 | Pride

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