Name: putative K29-linked substrates identification using sAB-K29
Keywords: K29-linked ubiquitination

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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 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

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

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

Identification of HP0244 regulon at different pH conditions

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

PPARgamma ubiquitination sites in HEK293T cells

Project description:Peroxisome proliferator–activated receptor γ (PPARγ) is the central regulator of adipogenesis, and its dysregulation is linked to obesity and metabolic diseases. Identification of the factors that regulate PPARγ expression and activity is therefore crucial for combating obesity. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with a known role in xenobiotic detoxification. Recent studies have suggested that AhR also plays essential roles in energy metabolism. However, the detailed mechanisms remain unclear. We previously reported that experiments with adipocyte-specific Cullin 4b (Cul4b)-knockout mice showed that CUL4B suppresses adipogenesis by targeting PPARγ. Here, using immunoprecipitation, ubiquitination, real-time PCR and Gst-pulldown assays, we report that AhR functions as the substrate receptor in CUL4B–RING E3 ubiquitin ligase (CRL4B) complex and is required for recruiting PPARγ. AhR overexpression reduced PPARγ stability and suppressed adipocyte differentiation, and AhR knockdown stimulated adipocyte differentiation in 3T3-L1 cells. Furthermore, we found that two lysine sites on residues 268 and 293 in PPARγ are targeted for CRL4B-mediated ubiquitination, indicating cross-talk between acetylation and ubiquitination. Our findings establish a critical role of AhR in regulating PPARγ stability and suggest that the AhR–PPARγ interaction may represent a potential therapeutic target for managing metabolic diseases arising from PPARγ dysfunction.

2019-11-01 | PXD015830 | Pride

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

Interplay between acetylation and ubiquitination of Isw1 confers multidrug resistance in Cryptococcus neoformans

Project description:Lysine acetylation and ubiquitination are one of many protein modifications and play a crucial role in the biological regulation of many organisms, but little is known about the relationship between acetylation and ubiquitination few. Here, the Isw1 protein is an important member of the chromatin remodeling complex, and we performed single-protein modification mass spectrometry detection of the C. neoformans Isw1 protein and site mutations for both detected modifications. The data showed that the two modifications of Cryptococcus neoformans Isw1 protein have a balance of each other. Acetylation can maintain protein stability and maintain protein function, while ubiquitination can reduce protein level and maintain Isw1 protein expression. The expression level of Isw1 protein leads to resistance to antifungal drugs. These results reveal the resistance mechanism of Isw1 protein of Cryptococcus neoformans to antifungal drugs.

2022-12-26 | PXD037150 | Pride

Cyclophilin A isomerization of Septin 2 mediates abscission during cytokinesis

Project description:The isomerase activity of Cyclophilin A is important for midbody abscission during cell division, however to date, midbody substrates remain unknown. In this study, we report that the GTP-binding protein Septin 2 interacts with Cyclophilin A. We highlight a dynamic series of Septin 2 phenotypes at the midbody, previously undescribed in human cells. Furthermore, Cyclophilin A depletion or loss of isomerase activity is sufficient to induce phenotypic Septin 2 defects at the midbody. Structural and molecular analysis reveals that Septin 2 proline 259 is important for interaction with Cyclophilin A. Moreover, an isomerisation-deficient EGFP-Septin 2 proline 259 mutant displays defective midbody localisation, and undergoes impaired abscission, consistent with data from cells with loss of Cyclophilin A expression or activity. Collectively, this data reveals Septin 2 as a novel interacting partner and isomerase substrate of Cyclophilin A at the midbody that is required for abscission during cytokinesis in cancer cells.

2023-10-24 | PXD042822 | Pride

Transcriptional characterization of maize roots in response to urea and nitrate

Project description:Although urea is the most used nitrogen fertilizer worldwide, little is known on the capacity of crop plants to use urea per se as a nitrogen source for development and growth. To date, the molecular and physiological bases of its transport have been investigated only in a limited number of species. In particular, up to date only one study reported the transcriptomic modulation induced by urea treatment in the model plant Arabidopsis (MM-CM-)rigout et al., 2008 doi: 10.1104/pp.108.119339). In maize, one of crops using huge amount of urea, only a physiological characterization of uptake and assimilation of the N-source has been conducted. General aim of the present work was the comprehension of the molecular basis of urea uptake and assimilation in maize plants, using a transcriptomic approach. In addition, the work focused on the possible interactions between the two main N-sources, conceivably occurring concomitantly in the soil, urea and nitrate. 5 dd-old maize plants were treated for 8 hours with nutrient solution containing nitrogen in form of urea; nitrate; urea and nitrate; or not exposed to any form of nitrogen. Three different biological replicates were used for each sample repeating the experiment three times. All samples were obtained pooling roots of six plants.

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