scholarly journals BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Bo Zhang ◽  
Mattias Holmlund ◽  
Severine Lorrain ◽  
Mikael Norberg ◽  
László Bakó ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Flower Development ◽  
Red Light ◽  
E3 Ubiquitin Ligase ◽  
Plant Responses ◽  
Signaling Mechanism ◽  
Ubiquitin Ligase Complex ◽  
Molecular Determinants ◽  
Subsequent Degradation

Both light and temperature have dramatic effects on plant development. Phytochrome photoreceptors regulate plant responses to the environment in large part by controlling the abundance of PHYTOCHROME INTERACTING FACTOR (PIF) transcription factors. However, the molecular determinants of this essential signaling mechanism still remain largely unknown. Here, we present evidence that the BLADE-ON-PETIOLE (BOP) genes, which have previously been shown to control leaf and flower development in Arabidopsis, are involved in controlling the abundance of PIF4. Genetic analysis shows that BOP2 promotes photo-morphogenesis and modulates thermomorphogenesis by suppressing PIF4 activity, through a reduction in PIF4 protein level. In red-light-grown seedlings PIF4 ubiquitination was reduced in the bop2 mutant. Moreover, we found that BOP proteins physically interact with both PIF4 and CULLIN3A and that a CULLIN3-BOP2 complex ubiquitinates PIF4 in vitro. This shows that BOP proteins act as substrate adaptors in a CUL3BOP1/BOP2 E3 ubiquitin ligase complex, targeting PIF4 proteins for ubiquitination and subsequent degradation.

scholarly journals Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Caitlin M. Miller ◽  
Hisashi Akiyama ◽  
Luis M. Agosto ◽  
Ann Emery ◽  
Chelsea R. Ettinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Culture ◽  
Dendritic Cells ◽  
Ubiquitin Ligase ◽  
Culture System ◽  
E3 Ubiquitin Ligase ◽  
Cell Culture System ◽  
Ubiquitin Ligase Complex ◽  
Hiv 1

ABSTRACT Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4+ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication. IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

scholarly journals SKP1-like protein, CrSKP1-e, interacts with pollen-specific F-box proteins and assembles into SCF-type E3 complex in ‘Wuzishatangju’ (Citrus reticulata Blanco) pollen

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10578
Author(s):  
Yi Ren ◽  
Qingzhu Hua ◽  
Jiayan Pan ◽  
Zhike Zhang ◽  
Jietang Zhao ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Citrus Reticulata ◽  
S Locus ◽  
Binding Assays ◽  
In Vitro Binding ◽  
Genome Wide ◽  
Ubiquitin Ligase Complex ◽  
Vitro Binding

S-ribonuclease (S-RNase)-based self-incompatibility (SI) mechanisms have been extensively studied in Solanaceae, Rosaceae and Plantaginaceae. S-RNase-based SI is controlled by two closely related genes, S-RNase and S-locus F-box (SLF), located at a polymorphic S-locus. In the SI system, the SCF-type (SKP1-CUL1-F-box-RBX1) complex functions as an E3 ubiquitin ligase complex for ubiquitination of non-self S-RNase. Pummelo (Citrus grandis) and several mandarin cultivars are suggested to utilize an S-RNase-based SI system. However, the molecular mechanism of the non-S-factors involved in the SI reaction is not straightforward in Citrus. To investigate the SCF-type E3 complex responsible for the SI reaction in mandarin, SLF, SKP1-like and CUL1 candidates potentially involved in the SI reaction of ‘Wuzishatangju’ (Citrus reticulata Blanco) were identified based on the genome-wide identification and expression analyses. Sixteen pollen-specific F-box genes (CrFBX1-CrFBX16), one pollen-specific SKP1-like gene (CrSKP1-e) and two CUL1 genes (CrCUL1A and CrCUL1B) were identified and cloned from ‘Wuzishatangju’. Yeast two-hybrid (Y2H) and in vitro binding assays showed that five CrFBX proteins could bind to CrSKP1-e, which is an ortholog of SSK1 (SLF-interacting-SKP1-like), a non-S-factor responsible for the SI reaction. Luciferase complementation imaging (LCI) and in vitro binding assays also showed that CrSKP1-e interacts with the N-terminal region of both CrCUL1A and CrCUL1B. These results indicate that CrSKP1-e may serve as a functional member of the SCF-type E3 ubiquitin ligase complex in ‘Wuzishatangju’.

scholarly journals A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62

2010 ◽  
Vol 30 (13) ◽  
pp. 3275-3285 ◽  
Author(s):  
Alexandria Lau ◽  
Xiao-Jun Wang ◽  
Fei Zhao ◽  
Nicole F. Villeneuve ◽  
Tongde Wu ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Defense Mechanism ◽  
Ectopic Expression ◽  
E3 Ubiquitin Ligase ◽  
Direct Interaction ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Ubiquitin Ligase Complex ◽  
Subsequent Degradation ◽  
Kelch Domain

ABSTRACT In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.

scholarly journals Direct phosphorylation of HY5 by SPA1 kinase to regulate photomorphogenesis in Arabidopsis

2020 ◽  
Author(s):  
Wenli Wang ◽  
Inyup Paik ◽  
Junghyun Kim ◽  
Xilin Hou ◽  
Sibum Sung ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Active Form ◽  
E3 Ubiquitin Ligase ◽  
Kinase Domain ◽  
Ubiquitin Ligase Complex ◽  
The Stability ◽  
Target Promoters ◽  
Spa Proteins

SUMMARYELONGATED HYPOCOTYL5 (HY5) is a key transcription factor which promotes photomorphogenesis by regulating complex downstream growth programs. Previous studies suggest that the regulation of HY5 mainly depends on the CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) - SUPPRESSOR OF PHYTOCHROME A-105 (SPA) E3 ubiquitin ligase complex, which degrades positively acting transcription factors of light signaling to repress photomorphogenesis in the dark. SPA proteins function not only as a component of the E3 ubiquitin ligase complex but also as a kinase of PHYTOCHROME INTERACTING FACTOR1 (PIF1) through its N-terminal kinase domain. Here, we show that HY5 is a new substrate of SPA1 kinase. SPA1 can directly phosphorylate HY5 in vitro and in vivo. We also demonstrate that unphosphorylated HY5 strongly interacts with both COP1 and SPA1 than phosphorylated HY5, is the preferred substrate for degradation, whereas phosphorylated HY5 is more stable in the dark. In addition, unphosphorylated HY5 actively binds to the target promoters, and is physiologically more active form. Consistently, the transgenic plants expressing unphosphorylated mutant of HY5 displays enhanced photomorphogenesis. Collectively, our study revealed that SPA1 fine-tunes the stability and the activity of HY5 to regulate photomorphogenesis.

scholarly journals CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1

2006 ◽  
Vol 26 (4) ◽  
pp. 1235-1244 ◽  
Author(s):  
Shih-Ching Lo ◽  
Mark Hannink
Keyword(s):  
Ubiquitin Ligase ◽  
Redox Homeostasis ◽  
Ectopic Expression ◽  
E3 Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Bzip Transcription Factor ◽  
Ubiquitin Ligase Complex ◽  
Steady State Levels

ABSTRACT The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2. Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to repress steady-state levels of Nrf2 and Nrf2-dependent transcription. Cullin-dependent ubiquitin ligase complexes have been proposed to undergo dynamic cycles of assembly and disassembly that enable substrate adaptor exchange or recycling. In this report, we have characterized the importance of substrate adaptor recycling for regulation of Keap1-mediated repression of Nrf2. Association of Keap1 with Cul3 was decreased by ectopic expression of CAND1 and was increased by small interfering RNA (siRNA)-mediated knockdown of CAND1. However, both ectopic overexpression and siRNA-mediated knockdown of CAND1 decreased the ability of Keap1 to target Nrf2 for ubiquitin-dependent degradation, resulting in stabilization of Nrf2 and activation of Nrf2-dependent gene expression. Neddylation of Cul3 on Lys 712 is required for Keap1-dependent ubiquitination of Nrf2 in vivo. However, the K712R mutant Cul3 molecule, which is not neddylated, can still assemble with Keap1 into a functional ubiquitin ligase complex in vitro. These results provide support for a model in which substrate adaptor recycling is required for efficient substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes.

scholarly journals Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex

2004 ◽  
Vol 24 (24) ◽  
pp. 10941-10953 ◽  
Author(s):  
Donna D. Zhang ◽  
Shih-Ching Lo ◽  
Janet V. Cross ◽  
Dennis J. Templeton ◽  
Mark Hannink
Keyword(s):  
Oxidative Stress ◽  
Steady State ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Bzip Transcription Factor ◽  
Critical Determinant ◽  
Ubiquitin Ligase Complex ◽  
Steady State Levels

ABSTRACT The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

scholarly journals Multisite Phosphorylation of the Saccharomyces cerevisiae Filamentous Growth Regulator Tec1 Is Required for its Recognition by the E3 Ubiquitin Ligase Adaptor Cdc4 and Its Subsequent Destruction In Vivo

2010 ◽  
Vol 9 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Marie Z. Bao ◽  
Teresa R. Shock ◽  
Hiten D. Madhani
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Filamentous Growth ◽  
Mitogen Activated Protein Kinase ◽  
Ubiquitin Ligase Complex ◽  
Mitogen Activated Protein ◽  
Substrate Phosphorylation

ABSTRACT In Saccharomyces cerevisiae, the pheromone-induced ubiquitylation and degradation of the filamentation pathway-specific activator, Tec1, suppresses cross talk between the mating and filamentous growth mitogen-activated protein kinase (MAPK) pathways. The mating pathway MAPK, Fus3, phosphorylates Tec1, resulting in its recognition by the SCF (for Skp1, Cullin, F-box containing) E3 ubiquitin ligase complex, leading to its proteolysis. Previously, it was found that Tec1 destruction requires phosphorylation on threonine 273 (T273). T273 is embedded in the sequence LLpTP, which is identical to the canonical binding site for Cdc4, a conserved F-box substrate adaptor for the SCF complex. However, recent work on both Cdc4 and the human Cdc4 ortholog Fbw7 has shown that a second substrate phosphorylation can be required for optimal Cdc4 binding in vitro. We report here that high-affinity binding of recombinant Cdc4 to Tec1 phosphopeptides requires phosphorylation of not only T273 but also a second site, T276. Significantly, both phospho-sites on Tec1 and a conserved basic pocket on Cdc4 are critical for Tec1 proteolysis in response to pheromone treatment of cells, establishing a role for two-phosphate recognition by yeast Cdc4 in substrate targeting in vivo.

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.

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