In vitro and in vivo interaction of AtRma2 E3 ubiquitin ligase and auxin binding protein 1

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1

Blood ◽

10.1182/blood.2020006528 ◽

2021 ◽

Author(s):

Roger Belizaire ◽

Sebastian Hassan John Koochaki ◽

Namrata D. Udeshi ◽

Alexis Vedder ◽

Lei Sun ◽

...

Keyword(s):

Cell Lines ◽

Ubiquitin Ligase ◽

Therapeutic Potential ◽

Mutant Cell ◽

E3 Ubiquitin Ligase ◽

Akt Signaling ◽

Allelic Series ◽

Genetic Ablation

CBL encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and non-receptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10-20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, PIK3R1 recruitment, and downstream PI3K/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine-kinase binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

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E3 ubiquitin ligase isolated by differential display regulates cervical cancer growth in vitro and in vivo via microRNA-143

Experimental and Therapeutic Medicine ◽

10.3892/etm.2016.3429 ◽

2016 ◽

Vol 12 (2) ◽

pp. 676-682 ◽

Cited By ~ 3

Author(s):

Jibin Li ◽

Xinling Wang ◽

Yanshang Zhang ◽

Yan Zhang

Keyword(s):

Cervical Cancer ◽

Ubiquitin Ligase ◽

Differential Display ◽

E3 Ubiquitin Ligase ◽

Cancer Growth

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Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression

Cancers ◽

10.3390/cancers12051340 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1340 ◽

Cited By ~ 2

Author(s):

Olaia Martinez-Iglesias ◽

Alba Casas-Pais ◽

Raquel Castosa ◽

Andrea Díaz-Díaz ◽

Daniel Roca-Lema ◽

...

Keyword(s):

Small Molecule ◽

Ubiquitin Ligase ◽

Epithelial To Mesenchymal Transition ◽

Tumour Progression ◽

E3 Ubiquitin Ligase ◽

Therapeutic Drug ◽

Mesenchymal Transition ◽

E Cadherin

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression.

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Interaction of auxin-binding protein 1 with maize coleoptile plasma membranes in vitro

Planta ◽

10.1007/s004250050091 ◽

1997 ◽

Vol 201 (4) ◽

pp. 470-476 ◽

Cited By ~ 3

Author(s):

Christine Schiebl ◽

Antje Walther ◽

Ursula Rescher ◽

Dieter Klämbt

Keyword(s):

Plasma Membranes ◽

Binding Protein ◽

Maize Coleoptile ◽

Auxin Binding ◽

Auxin Binding Protein

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Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1811891116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1319-1324 ◽

Cited By ~ 7

Author(s):

Xing Wang ◽

Yifei Zhang ◽

Seth S. Blair

Keyword(s):

Ubiquitin Ligase ◽

Hippo Pathway ◽

E3 Ubiquitin Ligase ◽

Growth Control ◽

Adaptor Protein ◽

Domain Growth ◽

C Terminus ◽

The Hippo Pathway

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat’s regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, we find that Dlish binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. We further show that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.

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JAC1 suppresses proliferation of breast cancer through the JWA/p38/SMURF1/HER2 signaling

Cell Death Discovery ◽

10.1038/s41420-021-00426-y ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Yanlin Ren ◽

Dongyin Chen ◽

Zurong Zhai ◽

Junjie Chen ◽

Aiping Li ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Ubiquitin Ligase ◽

Breast Cancer Cells ◽

E3 Ubiquitin Ligase ◽

Her2 Positive ◽

Her2 Positive Breast Cancer ◽

Positive Breast Cancer

AbstractThe overexpression of HER2 is associated with a malignant proliferation of breast cancer. In this study, we developed a non-cytotoxic JWA gene activating compound 1 (JAC1) to inhibit the proliferation of HER2-positive breast cancer cells in vitro and in vivo experimental models. JAC1 increased the ubiquitination of HER2 at the K716 site through the E3 ubiquitin ligase SMURF1 which was due to the decreased expression of NEDD4, the E3 ubiquitin ligase of SMURF1. In conclusion, JAC1 suppresses the proliferation of HER2-positive breast cancer cells through the JWA triggered HER2 ubiquitination signaling. JAC1 may serve as a potential therapeutic agent for HER2-positive breast cancer.

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The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown in S. cerevisiae

10.1101/673657 ◽

2019 ◽

Author(s):

Vineeth Vengayil ◽

Sunil Laxman

Keyword(s):

Ubiquitin Ligase ◽

Glucose Repression ◽

E3 Ubiquitin Ligase ◽

Proteasomal Degradation ◽

Ubiquitin E3 Ligase ◽

Nutrient Environment ◽

The Stability ◽

Glucose Availability

AbstractCells use multiple mechanisms to regulate their metabolic states depending on changes in their nutrient environment. A well-known example is the response of cells to glucose availability. In S. cerevisiae cells growing in glucose-limited medium, the re-availability of glucose leads to the downregulation of gluconeogenesis, the activation of glycolysis, and robust ‘glucose repression’. However, our knowledge of the initial mechanisms mediating this glucose-dependent downregulation of the gluconeogenic transcription factors is incomplete. We used the gluconeogenic transcription factor Rds2 as a candidate with which to discover regulators of early events leading to glucose repression. Here, we identify a novel role for the E3 ubiquitin ligase Pib1 in regulating the stability and degradation of Rds2. Glucose addition to glucose-limited cells results in rapid ubiquitination of Rds2, followed by its proteasomal degradation. Through in vivo and in vitro experiments, we establish Pib1 as a ubiquitin E3 ligase that regulates Rds2 ubiquitination and stability. Notably, this Pib1 mediated Rds2 ubiquitination, followed by proteasomal degradation, is specific to the presence of glucose. Pib1 is required for complete glucose repression, and enables cells to optimally grow in competitive environments when glucose becomes re-available. Our results reveal the existence of a Pib1 E3-ubiquitin ligase mediated regulatory program that mediates glucose-repression when glucose availability is restored.

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TRIM25 Regulates Oxaliplatin Resistance in Colorectal Cancer by Promoting EZH2 Stability

10.21203/rs.3.rs-60545/v1 ◽

2020 ◽

Author(s):

Sha Zhou ◽

Jianhong Peng ◽

Liuniu Xiao ◽

Caixia Zhou ◽

Yujing Fang ◽

...

Keyword(s):

Colorectal Cancer ◽

Stem Cell ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Disease Free Survival ◽

Annexin V ◽

Vital Role ◽

Kaplan Meier

Abstract Background Resistance to chemotherapy remains the major cause of treatment failure in patients with colorectal cancer (CRC). TRIM25, an E3-ubiquitin ligase, has been reported to play a vital role in tumorigenesis. This project aims to explore the function and mechanism of TRIM25 in regulating oxaliplatin resistance in colorectal cancer.Methods The expression of TRIM25 in colorectal cancer tissues were examined by publicly available dataset, Immunohistochemistry and western blot. Further survival analysis was conducted using Kaplan-Meier method. CCK8 assay, colony-formation assay, Annexin V-FITC /PI staining and xenograft tumor models were used for evaluating sensitivity of CRC cells to oxaliplatin. Sphere-formation assay, RT-PCR and limiting dilution assay were used to evaluate the influence of TRIM25 on stem cell properties of CRC cells. Co-immunoprecipitation, polyubiquitination assay and western bolt elucidate the mechanism by which TRIM25 regulates EZH2.Results Patients with high expression of TRIM25 have significantly higher recurrence rate (28.9% vs. 15.0%, P = 0.012) and worse disease-free survival (P = 0.006) than those with low TRIM25 expression. Downregulation of TRIM25 dramatically inhibited while TRIM25 overexpression enhanced CRC cells survival after oxaliplatin treatment. In addition, TRIM25 promotes stem cell properties of CRC cells both in vitro and in vivo (8 mice per group). Importantly, we demonstrated that TRIM25 inhibits the binding of E3-ubiquitin ligase TRAF6 to EZH2, thus stabilizing and upregulating EZH2 and promoting oxaliplatin resistance. Conclusions Our study provides evidence that TRIM25 is a novel epigenetic regulator of oxaliplatin resistance. Targeting TRIM25 might be a promising strategy for CRC treatment.

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