In Vitro Analysis of E3 Ubiquitin Ligase Function

10.3791/62393 ◽  
2021 ◽  
Author(s):  
Leonie Müller ◽  
Carl Elias Kutzner ◽  
Vishnu Balaji ◽  
Thorsten Hoppe
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ligase Function

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

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.

scholarly journals Axotrophin/MARCH7 acts as an E3 ubiquitin ligase and ubiquitinates tau protein in vitro impairing microtubule binding

2014 ◽  
Vol 1842 (9) ◽  
pp. 1527-1538 ◽  
Author(s):  
Katharina Flach ◽  
Ellen Ramminger ◽  
Isabel Hilbrich ◽  
Annika Arsalan-Werner ◽  
Franziska Albrecht ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Tau Protein ◽  
E3 Ubiquitin Ligase ◽  
Microtubule Binding

scholarly journals Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

2007 ◽  
Vol 179 (5) ◽  
pp. 935-950 ◽  
Author(s):  
K.G. Suresh Kumar ◽  
Hervé Barriere ◽  
Christopher J. Carbone ◽  
Jianghuai Liu ◽  
Gayathri Swaminathan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Interferon Α ◽  
Type I ◽  
Lysosomal Degradation ◽  
Linear Motif ◽  
Site Specific ◽  
Receptor Endocytosis ◽  
Β Receptor

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Shirin Doroudgar ◽  
Mirko Völkers ◽  
Donna J Thuerauf ◽  
Ashley Bumbar ◽  
Mohsin Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Misfolding ◽  
Cardiac Myocyte ◽  
Protein Quality ◽  
Heart Function ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Calcium Handling ◽  
Loss Of Function

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P<0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P<0.01), prolonged spark duration (P<0.001), and increased spark width (P<0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P<0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.

scholarly journals SLAP2 adaptor binding disrupts c-CBL autoinhibition to activate ubiquitin ligase function

2020 ◽  
Author(s):  
Leanne E. Wybenga-Groot ◽  
Andrea J. Tench ◽  
Craig D. Simpson ◽  
Jonathan St. Germain ◽  
Brian Raught ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
Adaptor Proteins ◽  
Negative Regulator ◽  
Alternative Mechanism ◽  
Kinase Signaling ◽  
Tyrosine Kinase Signaling ◽  
Ligase Function

AbstractCBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Tat-binding protein-1, a component of the 26S proteasome, contributes to the E3 ubiquitin ligase function of the von Hippel–Lindau protein

2003 ◽  
Vol 35 (3) ◽  
pp. 229-237 ◽  
Author(s):  
Paul G Corn ◽  
E Robert McDonald ◽  
James G Herman ◽  
Wafik S El-Deiry
Keyword(s):  
Ubiquitin Ligase ◽  
Binding Protein ◽  
E3 Ubiquitin Ligase ◽  
26S Proteasome ◽  
Von Hippel Lindau ◽  
Ligase Function

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

Blood ◽  
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.

Identification of Ubiquitin Ligase From Grapevine Ring C3H2C3E3 and Characterization of Drought Resistance Function of VyRCHC114

2020 ◽  
Author(s):  
Yihe Yu ◽  
Shengdi Yang ◽  
Lu Bian ◽  
Keke Yu ◽  
Xiangxuan Meng ◽  
...  
Keyword(s):  
Drought Stress ◽  
Ubiquitin Ligase ◽  
Stress Responses ◽  
Phylogenetic Trees ◽  
Expression Profiles ◽  
E3 Ubiquitin Ligase ◽  
Pcr Analysis ◽  
Biotic And Abiotic Stress ◽  
Element Analysis

Abstract Background: RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, playing an important role in plants’ development and growth and their biotic and abiotic stress responses. Results: A total of 143 RING C3H2C3-type genes (RCHCs) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, with these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VyRCHCs family together. Structural analysis showed that most VyRCHCs(67.13%) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis-acting element analysis showed the diversity of VyRCHCs regulation. The expression profiles of eight DEGs in RNA-Seq after drought stress were similar to those in qRT-PCR analysis. The in vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance, moreover, the transgenic plant survival rate increased by 30%, accompanied by changing of electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. AtCOR15a, AtRD29A, AtERD15 and AtP5CS1 were expressed quantitatively, the results showed that they participated in the drought stress response may be regulated by the expression of VyRCHC114.Conclusions: Valuable new information on the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress emerged from this research.

scholarly journals COP1 destabilizes DELLA proteins inArabidopsis

2020 ◽  
Vol 117 (24) ◽  
pp. 13792-13799 ◽  
Author(s):  
Noel Blanco-Touriñán ◽  
Martina Legris ◽  
Eugenio G. Minguet ◽  
Cecilia Costigliolo-Rojas ◽  
María A. Nohales ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Plant Cells ◽  
Transcriptional Regulators ◽  
Growth Responses ◽  
Warm Temperature ◽  
Della Proteins ◽  
The Stability ◽  
Dependent Pathway

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele inArabidopsis thaliana. Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed inNicotiana benthamiana.DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OFphyA-1051 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

scholarly journals Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

2015 ◽  
Vol 36 (5) ◽  
pp. 794-808 ◽  
Author(s):  
Sarah M. Pedersen ◽  
Waipan Chan ◽  
Rakhi P. Jattani ◽  
deMauri S. Mackie ◽  
Joel L. Pomerantz
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
E3 Ubiquitin Ligase ◽  
Antigen Receptor ◽  
Resonance Energy ◽  
B Cell Lymphoma ◽  
Receptor Signaling ◽  
Antigen Receptor Signaling

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activityin vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.

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