scholarly journals The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number

Open Biology ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. 170253 ◽  
Author(s):  
Christian Arquint ◽  
Fabien Cubizolles ◽  
Agathe Morand ◽  
Alexander Schmidt ◽  
Erich A. Nigg
Keyword(s):  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
Centrosome Amplification ◽  
E3 Ubiquitin Ligases ◽  
Primary Microcephaly ◽  
Physiological Importance ◽  
Centriole Duplication ◽  
Cdk2 Activity

Deregulation of centriole duplication has been implicated in cancer and primary microcephaly. Accordingly, it is important to understand how key centriole duplication factors are regulated. E3 ubiquitin ligases have been implicated in controlling the levels of several duplication factors, including PLK4, STIL and SAS-6, but the precise mechanisms ensuring centriole homeostasis remain to be fully understood. Here, we have combined proteomics approaches with the use of MLN4924, a generic inhibitor of SCF E3 ubiquitin ligases, to monitor changes in the cellular abundance of centriole duplication factors. We identified human STIL as a novel substrate of SCF-βTrCP. The binding of βTrCP depends on a DSG motif within STIL, and serine 395 within this motif is phosphorylated in vivo . SCF-βTrCP-mediated degradation of STIL occurs throughout interphase and mutations in the DSG motif causes massive centrosome amplification, attesting to the physiological importance of the pathway. We also uncover a connection between this new pathway and CDK2, whose role in centriole biogenesis remains poorly understood. We show that CDK2 activity protects STIL against SCF-βTrCP-mediated degradation, indicating that CDK2 and SCF-βTrCP cooperate via STIL to control centriole biogenesis.

scholarly journals Decoys reveal the genetic and biochemical roles of redundant plant E3 ubiquitin ligases

2017 ◽  
Author(s):  
Chin-Mei Lee ◽  
Ann Feke ◽  
Christopher Adamchek ◽  
Kristofor Webb ◽  
José Pruneda-Paz ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Mammalian Cells ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Genetic Redundancy ◽  
Clock Period ◽  
E3 Ligases ◽  
Wide Range

AbstractThe ubiquitin proteasome system (UPS) is the main cellular route for protein degradation in plants and is important for a wide range of biological processes including daily and seasonal timing. The UPS relies on the action of E3 ubiquitin ligases to specifically recognize substrate proteins and facilitate their ubiquitylation. In plants, there are three major challenges that inhibit studies of E3 ligase function: 1) rampant genetic redundancy, 2) labile interactions between an E3 ligase and its cognate substrates, and 3) a lack of tools for rapid validation of bona fide substrates. To overcome these 3 challenges, we have developed a decoy method that allows for rapid genetic analysis of E3 ligases, in vivo identification of substrates using immunoprecipitation followed by mass spectrometry, and reconstitution of the ubiquitylation reaction in mammalian cells to rapidly validate potential substrates. We employ the strategy to study the plant F-box proteins, ZTL, LKP2, and FKF1 revealing differential genetic impacts on circadian clock period and seasonal flowering. We identify a group of circadian clock transcriptional regulators that interact with ZTL, LKP2, and FKF1 in vivo providing a host of potential substrates that have not been seen previously. We then validate one substrate of ZTL, the plant circadian clock transcription factor CHE, and show that ZTL mediates CHE ubiquitylation and that the levels of the CHE protein cycle in daily timecourses. This work further untangles the complicated genetic roles of this family of E3 ligases and suggests that ZTL is a master regulator of a diverse set of critical clock transcription factors. Furthermore, the method that is validated here can be tool employed widely to overcome traditional challenges in studying redundant plant E3 ubiquitin ligases.

scholarly journals Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation

2016 ◽  
Vol 48 (7) ◽  
pp. 502-512 ◽  
Author(s):  
Barbara Medvar ◽  
Viswanathan Raghuram ◽  
Trairak Pisitkun ◽  
Abhijit Sarkar ◽  
Mark A. Knepper
Keyword(s):  
Human Genome ◽  
Large Scale ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
Bayes Rule ◽  
E3 Ubiquitin Ligases ◽  
E3 Ligases ◽  
Aquaporin 2 ◽  
Large Scale Data ◽  
Level Data

Aquaporin-2 (AQP2) is regulated in part via vasopressin-mediated changes in protein half-life that are in turn dependent on AQP2 ubiquitination. Here we addressed the question, “What E3 ubiquitin ligase is most likely to be responsible for AQP2 ubiquitination?” using large-scale data integration based on Bayes' rule. The first step was to bioinformatically identify all E3 ligase genes coded by the human genome. The 377 E3 ubiquitin ligases identified in the human genome, consisting predominant of HECT, RING, and U-box proteins, have been used to create a publically accessible and downloadable online database ( https://hpcwebapps.cit.nih.gov/ESBL/Database/E3-ligases/ ). We also curated a second database of E3 ligase accessory proteins that included BTB domain proteins, cullins, SOCS-box proteins, and F-box proteins. Using Bayes' theorem to integrate information from multiple large-scale proteomic and transcriptomic datasets, we ranked these 377 E3 ligases with respect to their probability of interaction with AQP2. Application of Bayes' rule identified the E3 ligases most likely to interact with AQP2 as (in order of probability): NEDD4 and NEDD4L (tied for first), AMFR, STUB1, ITCH, ZFPL1. Significantly, the two E3 ligases tied for top rank have also been studied extensively in the reductionist literature as regulatory proteins in renal tubule epithelia. The concordance of conclusions from reductionist and systems-level data provides strong motivation for further studies of the roles of NEDD4 and NEDD4L in the regulation of AQP2 protein turnover.

scholarly journals DDB2 regulates DNA replication through PCNA-independent degradation of CDT2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaojun Wu ◽  
Min Yu ◽  
Zhuxia Zhang ◽  
Feng Leng ◽  
Yue Ma ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cancer Therapy ◽  
Target Genes ◽  
Ubiquitin Ligases ◽  
Ovarian Teratoma ◽  
Control Group ◽  
E3 Ubiquitin Ligases ◽  
Small Interfering Rnas ◽  
Interacting Protein

Abstract Background Targeting ubiquitin-dependent proteolysis is one of the strategies in cancer therapy. CRLCDT2 and CRLDDB2 are two key E3 ubiquitin ligases involved in DNA replication and DNA damage repair. But CDT2 and DDB2 are opposite prognostic factors in kinds of cancers, and the underlining mechanism needs to be elucidated. Methods Small interfering RNAs were used to determine the function of target genes. Co-immunoprecipitation (Co-IP) was performed to detect the interaction between DDB2 and CDT2. Immunofluorescence assays and fluorescence activating cell sorting (FACS) were used to measure the change of DNA content. In vivo ubiquitination assay was carried out to clarify the ubiquitination of CDT2 mediated by DDB2. Cell synchronization was performed to arrest cells at G1/S and S phase. The mechanism involved in DDB2-mediated CDT2 degradation was investigated by constructing plasmids with mutant variants and measured by Western blot. Immunohistochemistry was performed to determine the relationship between DDB2 and CDT2. Paired two-side Student’s t-test was used to measure the significance of the difference between control group and experimental group. Results Knockdown of DDB2 stabilized CDT2, while over-expression of DDB2 enhanced ubiquitination of CDT2, and subsequentially degradation of CDT2. Although both DDB2 and CDT2 contain PIP (PCNA-interacting protein) box, PIP box is dispensable for DDB2-mediated CDT2 degradation. Knockdown of PCNA had negligible effects on the stability of CDT2, but promoted accumulation of CDT1, p21 and SET8. Silencing of DDB2 arrested cell cycle in G1 phase, destabilized CDT1 and reduced the chromatin loading of MCMs, thereby blocked the formation of polyploidy induced by ablation of CDT2. In breast cancer and ovarian teratoma tissues, high level of DDB2 was along with lower level of CDT2. Conclusions We found that CRL4DDB2 is the novel E3 ubiquitin ligases of CDT2, and DDB2 regulates DNA replication through indirectly regulates CDT1 protein stability by degrading CDT2 and promotes the assembly of pre-replication complex. Our results broaden the horizon for understanding the opposite function of CDT2 and DDB2 in tumorigenesis, and may provide clues for drug discovery in cancer therapy.

scholarly journals Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase

2020 ◽  
Vol 319 (4) ◽  
pp. C700-C719 ◽  
Author(s):  
David C. Hughes ◽  
Leslie M. Baehr ◽  
Julia R. Driscoll ◽  
Sarah A. Lynch ◽  
David S. Waddell ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Atrophy ◽  
Splice Variant ◽  
Splice Variants ◽  
Ring Finger ◽  
Ubiquitin Ligases ◽  
Skeletal Muscle Atrophy ◽  
E3 Ubiquitin Ligases ◽  
Muscle Mass Loss

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22–193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12–16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

scholarly journals Coupled monoubiquitylation of the co-E3 ligase DCNL1 by Ariadne-RBR E3 ubiquitin ligases promotes cullin-RING ligase complex remodeling

2018 ◽  
Vol 294 (8) ◽  
pp. 2651-2664 ◽  
Author(s):  
Ian R. Kelsall ◽  
Yosua A. Kristariyanto ◽  
Axel Knebel ◽  
Nicola T. Wood ◽  
Yogesh Kulathu ◽  
...  
Keyword(s):  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases

scholarly journals Regulation of Cullin RING E3 Ubiquitin Ligases by CAND1 In Vivo

PLoS ONE ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. e16071 ◽  
Author(s):  
Yee Shin Chua ◽  
Boon Kim Boh ◽  
Wanpen Ponyeam ◽  
Thilo Hagen
Keyword(s):  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Ring E3

scholarly journals The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel–Lindau tumor suppressor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Antonella Falconieri ◽  
Giovanni Minervini ◽  
Raissa Bortolotto ◽  
Damiano Piovesan ◽  
Raffaele Lopreiato ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Protein Interactions ◽  
Specific Interaction ◽  
Hypoxia Inducible Factor ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Von Hippel Lindau ◽  
Intrinsically Disordered

Abstract Mutations of the von Hippel–Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1 α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein–protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). We found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already shown for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell metabolic activity and necrosis, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data show an isoform-specific interaction of pVHL with MDM2, suggesting an interplay between these two E3 ubiquitin ligases.

scholarly journals Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases

2020 ◽  
Author(s):  
Yadi Chen ◽  
Xiaohua Hu ◽  
Siyuan Liu ◽  
Tiantian Su ◽  
Hsiaochi Huang ◽  
...  
Keyword(s):  
Blue Light ◽  
E3 Ligase ◽  
Light Regulation ◽  
Light Response ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Light Conditions ◽  
E3 Ligases ◽  
Dependent Activity ◽  
Evolutionary Lineages

Abstract Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1-SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrated the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2 to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1-SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

scholarly journals RIPs Interact with OsFH5 and Control the Rice Morphology by Affecting Stability of OsFH5

2021 ◽  
Author(s):  
Shuwei Chang ◽  
Litao Yang ◽  
Wanqi Liang ◽  
Qi Xie ◽  
Guoqiang Huang ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Degradation Rate ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Wild Type ◽  
Knock Out ◽  
Target Proteins ◽  
Transgenic Lines ◽  
And Control

Abstract E3 ubiquitin ligases have been more widely reported to mediate the degradation of target proteins. In our research, one SIAH type E3 ligase was identified, named RIP1, and there are five genes were found to be highly homologous to it, named RIP2 to RIP6 dividedly. Y2H and BiFC were carried out to prove the interaction between RIPs and OsFH5, moreover, homo- and hetero- oligomers can be formed among RIPs and they all can affect the localization of OsFH5. Degradation experiments showed that RIP1 is able to slow down the degradation rate of OsFH5, and also RIP5, RIP6. There was no obvious phenotype in both RIP1 overexpression and mutant transgenic lines, whereas in rips multi-knock out lines showed up similar defects to osfh5, such as, dwarf height; less smooth and rounder seeds; leaves width became wilder. Otherwise, rips also exhibit some special phenotypes, for instance, the angles between leaves and stems were appreciable enlarged; spiral crimped roots. In addition, microfilaments organization in rips appeared disordered than wild type. All above illustrate that RIPs influence morphology by interacting with OsFH5 and affecting its subcellular localization, stability or maybe involved in other pathways in rice.

scholarly journals CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells

2019 ◽  
Vol 30 (10) ◽  
pp. 1230-1244 ◽  
Author(s):  
Divya Ganapathi Sankaran ◽  
Alexander J. Stemm-Wolf ◽  
Chad G. Pearson
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Alternative Polyadenylation ◽  
Centrosome Amplification ◽  
Breast Cancer Cell Lines ◽  
Microtubule Organizing Center ◽  
Relative Level ◽  
Centriole Duplication

The centrosome, composed of two centrioles surrounded by pericentriolar material, is the cell’s central microtubule-organizing center. Centrosome duplication is coupled with the cell cycle such that centrosomes duplicate once in S phase. Loss of such coupling produces supernumerary centrosomes, a condition called centrosome amplification (CA). CA promotes cell invasion and chromosome instability, two hallmarks of cancer. We examined the contribution of centriole overduplication to CA and the consequences for genomic stability in breast cancer cells. CEP135, a centriole assembly protein, is dysregulated in some breast cancers. We previously identified a short isoform of CEP135, CEP135mini, that represses centriole duplication. Here, we show that the relative level of full-length CEP135 (CEP135full) to CEP135mini (the CEP135full:mini ratio) is increased in breast cancer cell lines with high CA. Inducing expression of CEP135full in breast cancer cells increases the frequency of CA, multipolar spindles, anaphase-lagging chromosomes, and micronuclei. Conversely, inducing expression of CEP135mini reduces centrosome number. The differential expression of the CEP135 isoforms in vivo is generated by alternative polyadenylation. Directed genetic mutations near the CEP135mini alternative polyadenylation signal reduces the CEP135full:mini ratio and decreases CA. We conclude that dysregulation of CEP135 isoforms promotes centriole overduplication and contributes to chromosome segregation errors in breast cancer cells.

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