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 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 Ubiquitination, Biotech Startups, and the Future of TRIM Family Proteins: A TRIM-Endous Opportunity

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1015
Author(s):  
Utsa Bhaduri ◽  
Giuseppe Merla
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Genetic Disorders ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Post Translational Modification ◽  
Disease Biology ◽  
The Future

Ubiquitination is a post-translational modification that has pivotal roles in protein degradation and diversified cellular processes, and for more than two decades it has been a subject of interest in the biotech or biopharmaceutical industry. Tripartite motif (TRIM) family proteins are known to have proven E3 ubiquitin ligase activities and are involved in a multitude of cellular and physiological events and pathophysiological conditions ranging from cancers to rare genetic disorders. Although in recent years many kinds of E3 ubiquitin ligases have emerged as the preferred choices of big pharma and biotech startups in the context of protein degradation and disease biology, from a surface overview it appears that TRIM E3 ubiquitin ligases are not very well recognized yet in the realm of drug discovery. This article will review some of the blockbuster scientific discoveries and technological innovations from the world of ubiquitination and E3 ubiquitin ligases that have impacted the biopharma community, from biotech colossuses to startups, and will attempt to evaluate the future of TRIM family proteins in the province of E3 ubiquitin ligase-based drug discovery.

scholarly journals Genome-wide analysis of HECT E3 ubiquitin ligase gene family in Solanum lycopersicum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bhaskar Sharma ◽  
Harshita Saxena ◽  
Harshita Negi
Keyword(s):  
Gene Family ◽  
Ubiquitin Ligase ◽  
Plant Development ◽  
Stress Responses ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
E3 Ligases ◽  
Genome Wide Analysis ◽  
Genome Wide

AbstractThe E3 ubiquitin ligases have been known to intrigue many researchers to date, due to their heterogenicity and substrate mediation for ubiquitin transfer to the protein. HECT (Homologous to the E6-AP Carboxyl Terminus) E3 ligases are spatially and temporally regulated for substrate specificity, E2 ubiquitin-conjugating enzyme interaction, and chain specificity during ubiquitylation. However, the role of the HECT E3 ubiquitin ligase in plant development and stress responses was rarely explored. We have conducted an in-silico genome-wide analysis to identify and predict the structural and functional aspects of HECT E3 ligase members in tomato. Fourteen members of HECT E3 ligases were identified and analyzed for the physicochemical parameters, phylogenetic relations, structural organizations, tissue-specific gene expression patterns, and protein interaction networks. Our comprehensive analysis revealed the HECT domain conservation throughout the gene family, close evolutionary relationship with different plant species, and active involvement of HECT E3 ubiquitin ligases in tomato plant development and stress responses. We speculate an indispensable biological significance of the HECT gene family through extensive participation in several plant cellular and molecular pathways.

scholarly journals The Ubiquitin E3 Ligase MaLUL2 Is Involved in High Temperature-Induced Green Ripening in Banana Fruit

2020 ◽  
Vol 21 (24) ◽  
pp. 9386
Author(s):  
Wei Wei ◽  
Jian-ye Chen ◽  
Ze-xiang Zeng ◽  
Jian-fei Kuang ◽  
Wang-jin Lu ◽  
...  
Keyword(s):  
High Temperature ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Negative Regulator ◽  
Banana Fruit ◽  
E3 Ubiquitin Ligases ◽  
Stay Green ◽  
E3 Ligases ◽  
Ring Type

Harvested banana fruit ripened under warm temperatures above 24 °C remain green peel, leading to severe economic loss. E3 ubiquitin-ligases, as the major components in the ubiquitination pathway, have been implicated to play important roles in temperature-stress responses. However, the molecular mechanism underlying high temperature-triggered stay-green ripening bananas in association with E3 ubiquitin-ligases, remains largely unknown. In this study, a RING-type E3 ubiquitin ligase termed MaLUL2, was isolated and characterized from banana fruit. The MaLUL2 gene contains 1095 nucleotides and encodes a protein with 365 amino acids. The MaLUL2 protein contains a domain associated with RING2 (DAR2) and a RING domain, which are the typical characteristics of RING-type E3 ligases. MaLUL2 expression was up-regulated during high temperature-induced green ripening. Subcellular localization showed that MaLUL2 localized in the nucleus, cytoplasm, and plasma membrane. MaLUL2 displayed E3 ubiquitin ligase activity in vitro. More importantly, transient overexpression of MaLUL2 in banana fruit peel increased the level of ubiquitination in vivo and led to a stay-green phenotype, accompanying with decreased expression of chlorophyll catabolic genes. Collectively, these findings suggest that MaLUL2 might act as a negative regulator of chlorophyll degradation and provide novel insights into the regulatory mechanism of high temperature-induced green ripening bananas.

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 The ARRE RING-Type E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis thaliana by Controlling ECERIFERUM1 and ECERIFERUM3 Protein Levels

2021 ◽  
Vol 12 ◽  
Author(s):  
Shuang Liu ◽  
Meixuezi Tong ◽  
Lifang Zhao ◽  
Xin Li ◽  
Ljerka Kunst
Keyword(s):  
Ubiquitin Ligase ◽  
Uv Light ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Cuticular Wax ◽  
26S Proteasome ◽  
Covalent Attachment ◽  
Wax Deposition ◽  
E3 Ligases ◽  
Ring Type

The outer epidermal cell walls of plant shoots are covered with a cuticle, a continuous lipid structure that provides protection from desiccation, UV light, pathogens, and insects. The cuticle is mostly composed of cutin and cuticular wax. Cuticular wax synthesis is synchronized with surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. Cuticular wax deposition is tightly regulated by well-established transcriptional and post-transcriptional regulatory mechanisms, as well as post-translationally via the ubiquitin-26S proteasome system (UPS). The UPS is highly conserved in eukaryotes and involves the covalent attachment of polyubiquitin chains to the target protein by an E3 ligase, followed by the degradation of the modified protein by the 26S proteasome. A large number of E3 ligases are encoded in the Arabidopsis genome, but only a few have been implicated in the regulation of cuticular wax deposition. In this study, we have conducted an E3 ligase reverse genetic screen and identified a novel RING-type E3 ubiquitin ligase, AtARRE, which negatively regulates wax biosynthesis in Arabidopsis. Arabidopsis plants overexpressing AtARRE exhibit glossy stems and siliques, reduced fertility and fusion between aerial organs. Wax load and wax compositional analyses of AtARRE overexpressors showed that the alkane-forming branch of the wax biosynthetic pathway is affected. Co-expression of AtARRE and candidate target proteins involved in alkane formation in both Nicotiana benthamiana and stable Arabidopsis transgenic lines demonstrated that AtARRE controls the levels of wax biosynthetic enzymes ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3). CER1 has also been confirmed to be a ubiquitination substrate of the AtARRE E3 ligase by an in vivo ubiquitination assay using a reconstituted Escherichia coli system. The AtARRE gene is expressed throughout the plant, with the highest expression detected in fully expanded rosette leaves and oldest stem internodes. AtARRE gene expression can also be induced by exposure to pathogens. These findings reveal that wax biosynthesis in mature plant tissues and in response to pathogen infection is controlled post-translationally.

scholarly journals Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16

2018 ◽  
Author(s):  
Xiaoyu Zhang ◽  
Vincent M. Crowley ◽  
Thomas G. Wucherpfennig ◽  
Melissa M. Dix ◽  
Benjamin F. Cravatt
Keyword(s):  
Protein Degradation ◽  
Protein Content ◽  
Substrate Recognition ◽  
Ubiquitin Ligases ◽  
Nuclear Proteins ◽  
E3 Ubiquitin Ligases ◽  
E3 Ligases ◽  
Dependent Protein

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy to discover that DCAF16 – a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases – promotes nuclear-restricted protein degradation upon modification by cysteine-directed heterobifunctional electrophilic compounds.

scholarly journals Expression Patterns and Functional Analysis of E3 Ubiquitin Ligase Genes in Rice

2021 ◽  
Author(s):  
huijuan zhang ◽  
Dewei Zheng ◽  
Fengming Song ◽  
Ming Jiang
Keyword(s):  
Ubiquitin Ligase ◽  
Abiotic Stresses ◽  
Magnaporthe Grisea ◽  
Function Analysis ◽  
Expression Patterns ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
E3 Ubiquitin Ligases ◽  
Biotic And Abiotic Stresses ◽  
Genes Expression

Abstract Background: E3 ubiquitin ligases involve in many processes, containing the response to biotic and abiotic stresses. However, the functions of E3 ubiquitin ligases in rice were rarely studied.Results: In this research, 11 E3 ubiquitin ligase genes were selected and the function analysis was done in rice. These 11 E3 ubiquitin ligase genes showed different expression patterns under different treatments. The BMV:Os06g13870- infiltrated seedlings showed decreased resistance to Magnaporthe grisea (M. grisea) when compared with BMV:00-infiltrated seedlings, while BMV:Os04g34030- and BMV:Os02g33590-infiltrated seedlings showed increased resistance. They involved in the resistance against M. grisea maybe by regulating the accumulation of reactive oxygen species (ROS) and expression levels of defense-related genes. The BMV:Os06g34390-infiltrated seedlings showed decreased tolerance to drought stress while BMV:Os02g33590-infiltraed seedlings showed increased tolerance, maybe through regulating proline content, sugar content and drought-responsive genes’ expression. BMV:Os05g01940-infiltrated seedlings showed decreased tolerance to cold stress by regulating malondial dehyde (MDA) content and cold-responsive genes’ expression.Conclusion: These results showed that E3 ubiquitin ligases involved in the resistance to biotic and abiotic stresses in rice.

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 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.

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