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 Decoys provide a scalable platform for the genetic analysis of plant E3 ubiquitin ligases that regulate circadian clock function

2018 ◽  
Author(s):  
Ann Feke ◽  
Wei Liu ◽  
Jing Hong ◽  
Man-Wah Li ◽  
Chin-Mei Lee ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Gene Families ◽  
Dominant Negative ◽  
E3 Ubiquitin Ligases ◽  
Genetic Redundancy ◽  
Clock Period ◽  
Knock Out

ABSTRACTThe circadian clock in all eukaryotes relies on the regulated degradation of clock proteins to maintain 24-hour rhythmicity. Despite this knowledge, we know very few of the components that mediate degradation of proteins to control clock function. This is likely due to high levels of gene duplication and functional redundancy within plant E3 ubiquitin ligase gene families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian clock function, we generated a library of transgenic Arabidopsis lines expressing dominant-negative “decoy” E3 ubiquitin ligases. We determined their effects on the plant circadian clock and identified dozens of new potential regulators of circadian clock function. To demonstrate the potency of the decoy screening methodology to overcome genetic redundancy and identify bona fide clock regulators, we performed follow-up studies on PUB59 and PUB60. Using knock-out studies, we show that they redundantly control circadian clock period by regulating gene splicing. Furthermore, we confirm that they are part of a conserved protein complex that mediates splicing in eukaryotes. This work demonstrates the viability of E3 ubiquitin ligase decoys as a scalable screening platform to overcome traditional genetic challenges and discover E3 ubiquitin ligases that regulate plant developmental processes.

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 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 HaloTag-Targeted Sirtuin Rearranging Ligand (SirReal) for the Development of Proteolysis Targeting Chimeras (PROTACs) Against the Lysine Deacetylase Sirtuin 2 (Sirt2)

2020 ◽  
Author(s):  
Matthias Schiedel ◽  
Attila Lehotzky ◽  
Sándor Szunyogh ◽  
Judit Oláh ◽  
Sören Hammelmann ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Small Molecule ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Selective Inhibitors ◽  
E3 Ligases ◽  
Lysine Deacetylase

We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

scholarly journals E3 Ubiquitin Ligases: Key Regulators of Hormone Signaling in Plants

2018 ◽  
Vol 17 (6) ◽  
pp. 1047-1054 ◽  
Author(s):  
Dior R. Kelley
Keyword(s):  
Plant Hormone ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
Hormone Signaling ◽  
E3 Ubiquitin Ligases ◽  
E3 Ligases ◽  
Experimental Approaches ◽  
Hormone Biosynthesis ◽  
Plant Hormone Signaling ◽  
Insight Into

Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. Although E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis. This review will highlight up-to-date findings that have refined well-known E3 ligase-substrate interactions and defined novel E3 ligase substrates that mediate numerous hormone signaling pathways. Additionally, examples of how particular E3 ligases may mediate hormone crosstalk will be discussed as an emerging theme. Looking forward, promising experimental approaches and methods that will provide deeper mechanistic insight into the roles of E3 ubiquitin ligases in plants will be considered.

scholarly journals HaloTag-Targeted Sirtuin Rearranging Ligand (SirReal) for the Development of Proteolysis Targeting Chimeras (PROTACs) Against the Lysine Deacetylase Sirtuin 2 (Sirt2)

2020 ◽  
Author(s):  
Matthias Schiedel ◽  
Attila Lehotzky ◽  
Sándor Szunyogh ◽  
Judit Oláh ◽  
Sören Hammelmann ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Small Molecule ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Selective Inhibitors ◽  
E3 Ligases ◽  
Lysine Deacetylase

We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

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 Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases

2021 ◽  
Vol 12 (1) ◽  
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

AbstractCryptochromes (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 demonstrate 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, at the CCE domain of 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 Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Gao ◽  
Xianwei Ma ◽  
Ming Yuan ◽  
Yulan Yi ◽  
Guoke Liu ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Type I Interferon ◽  
Zinc Fingers ◽  
E3 Ligase ◽  
Type I ◽  
E3 Ligases ◽  
Protein Posttranslational Modifications ◽  
Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

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