scholarly journals Cryptochrome 2 competes with COP1 substrates to repress COP1 ubiquitin ligase activity duringArabidopsisphotomorphogenesis

2019 ◽  
Vol 116 (52) ◽  
pp. 27133-27141 ◽  
Author(s):  
Jathish Ponnu ◽  
Tabea Riedel ◽  
Eva Penner ◽  
Andrea Schrader ◽  
Ute Hoecker
Keyword(s):  
Blue Light ◽  
Ubiquitin Ligase ◽  
Dependent Manner ◽  
In Planta ◽  
Yeast Two Hybrid ◽  
Ubiquitin Ligase Activity ◽  
Binding Pockets ◽  
Cryptochrome 2 ◽  
Two Hybrid

In plants, the cryptochrome photoreceptors suppress the activity of the COP1/SPA ubiquitin ligase to initiate photomorphogenesis in blue light. Both CRY1 and CRY2 interact with the COP1/SPA complex in a blue light-dependent manner. The mechanisms underlying the inhibition of COP1 activity through direct interactions with photoactivated CRYs are not fully understood. Here we tested the hypothesis that CRY2 inhibits COP1 by displacing the degradation substrates from COP1. To this end, we analyzed the role of a conserved valine-proline (VP) motif in the C-terminal domain of CRY2 (CCT2), which resembles the core COP1-WD40–binding sequences present in the substrates of COP1. We show that the VP motif in CRY2 is essential for the interaction of CRY2 with COP1 in yeast two-hybrid assays andin planta. Mutations in the VP motif of CRY2 abolished the CRY2 activity in photomorphogenesis, indicating the importance of VP. The interaction between COP1 and its VP-containing substrate PAP2 was prevented in the presence of coexpressed CRY2, but not in the presence of CRY2 carrying a VP mutation. Thus, since both PAP2 and CRY2 engage VP motifs to bind to COP1, these results demonstrate that CRY2 outcompetes PAP2 for binding to COP1. We further found that the previously unknown interaction between SPA1-WD and CCT2 occurs via the VP motif in CRY2, suggesting structural similarities in the VP-binding pockets of COP1-WD40 and SPA1-WD40 domains. A VP motif present in CRY1 is also essential for binding to COP1. Thus, CRY1 and CRY2 might share this mechanism of COP1 inactivation.

scholarly journals Proteasomal degradation of JAZ9 by SALT- AND DROUGHT-INDUCED RING FINGER1 during pathogen infection

2021 ◽  
Author(s):  
Vemanna Ramu ◽  
Garima Pal ◽  
Sunhee Oh ◽  
Kirankumar S Mysore
Keyword(s):  
Ubiquitin Ligase ◽  
Disease Susceptibility ◽  
Plant Immunity ◽  
E3 Ubiquitin Ligase ◽  
In Planta ◽  
Yeast Two Hybrid ◽  
Pathogen Infection ◽  
Jaz Proteins ◽  
Molecular Fluorescence ◽  
Two Hybrid

E3 ubiquitin ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) plays a novel role in modulating plant immunity against pathogens. The molecular interactors of SDIR1 during pathogen infection are not known. SDIR1 interacting Jasmonate ZIM-domain (JAZ) proteins were identified through a yeast two-hybrid (Y2H) screen. Full length JAZ9 interacts with SDIR1 only in the presence of coronatine, a bacteria secreted toxin, or jasmonic acid (JA) in Y2H assay. The bi-molecular fluorescence complementation and pull-down assays confirm the in planta interaction of these proteins. JAZ9 proteins, negative regulators of JA-mediated plant defense, were degraded during the pathogen infection by SDIR1 through a proteasomal pathway causing disease susceptibility against hemibiotrophic pathogens.

scholarly journals E3 Ligase ITCH Interacts with the Z Matrix Protein of Lassa and Mopeia Viruses and Is Required for the Release of Infectious Particles

Viruses ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 49 ◽  
Author(s):  
Nicolas Baillet ◽  
Sophie Krieger ◽  
Xavier Carnec ◽  
Mathieu Mateo ◽  
Alexandra Journeaux ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Hemorrhagic Fever ◽  
Lassa Fever ◽  
Lymphocytic Choriomeningitis ◽  
Lassa Virus ◽  
Yeast Two Hybrid ◽  
Ubiquitin Ligase Activity ◽  
Two Hybrid

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release.

scholarly journals Liganded ERα Stimulates the E3 Ubiquitin Ligase Activity of UBE3C to Facilitate Cell Proliferation

2015 ◽  
Vol 29 (11) ◽  
pp. 1646-1657 ◽  
Author(s):  
Maiko Okada ◽  
Fumiaki Ohtake ◽  
Hiroyuki Nishikawa ◽  
Wenwen Wu ◽  
Yasushi Saeki ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Cyclin B1 ◽  
Dependent Manner ◽  
Ubiquitin Ligase Activity ◽  
Dependent Cell ◽  
Estrogen Antagonist

Abstract Estrogen receptor (ER)α is a well-characterized ligand-dependent transcription factor. However, the global picture of its nongenomic functions remains to be illustrated. Here, we demonstrate a novel function of ERα during mitosis that facilitates estrogen-dependent cell proliferation. An E3 ubiquitin ligase, UBE3C, was identified in an ERα complex from estrogen-treated MCF-7 breast cancer cells arrested at mitosis. UBE3C interacts with ERα during mitosis in an estrogen-dependent manner. In vitro, estrogen dramatically stimulates the E3 activity of UBE3C in the presence of ERα. This effect was inhibited by the estrogen antagonist tamoxifen. Importantly, estrogen enhances the ubiquitination of cyclin B1 (CCNB1) and destabilizes CCNB1 during mitosis in a manner dependent on endogenous UBE3C. ERα, UBE3C, and CCNB1 colocalize in prophase nuclei and at metaphase spindles before CCNB1 is degraded in anaphase. Depletion of UBE3C attenuates estrogen-dependent cell proliferation without affecting the transactivation function of ERα. Collectively, these results demonstrate a novel ligand-dependent action of ERα that stimulates the activity of an E3 ligase. The mitotic role of estrogen may contribute to its effects on proliferation in addition to its roles in target gene expression.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals The role of Clt1-regulated-Xylan metabolism in the melanin and toxin formation for the pathogenicity of Curvularia lunata in Maize

2021 ◽  
Author(s):  
Jinxin Gao ◽  
Jie Chen
Keyword(s):  
Toxin Production ◽  
Bimolecular Fluorescence Complementation ◽  
Curvularia Lunata ◽  
Yeast Two Hybrid ◽  
Acetyl Xylan Esterase ◽  
Btb Domain ◽  
Intermediate Metabolites ◽  
Regulative Mechanism ◽  
Two Hybrid

We previously reported that the BTB domain-containing protein Clt1 regulates melanin and toxin synthesis, conidiation, and pathogenicity in Curvularia lunata, but the interacting proteins and regulative mechanism of Clt1 are unclear. In this research, we identified two proteins, which respectively correspond to xylanase (Clxyn24) and acetyl xylan esterase (Claxe43) from C. lunata were regulated by Clt1. Yeast two-hybrid (Y2H), and bimolecular fluorescence complementation assays were conducted to verify the interaction of Clt1 with full-length Clxyn24 and Claxe43. Furthermore, the Y2H assay revealed that Clt1 physically interacted with Clxyn24 and Claxe43 through its BTB domain to degrade xylan which was used as a carbon source for C. lunata growth. The utilization of xylan provides acetyl-CoA for the synthesis of melanin and toxin, as well as energy and other intermediate metabolites for conidiation. Furthermore, transcriptome analysis revealed that PKS18 and its 13 flanking genes are found clustered in a region spanning 57.89 kb on scaffold 9 of the C. lunata CX-3 genome were down-regulated in toxin production deficient mutant T806, and this cluster is possibly responsible for toxin biosynthesis of C. lunata.

Cryptochrome 2 is involved in betacyanin decomposition induced by blue light in Suaeda salsa

2006 ◽  
Vol 33 (7) ◽  
pp. 697 ◽  
Author(s):  
Wang Chang-Quan ◽  
Liu Tao
Keyword(s):  
Western Blot ◽  
Blue Light ◽  
Hypocotyl Elongation ◽  
Suaeda Salsa ◽  
Biosynthesis Pathway ◽  
Tyrosine Hydroxylation ◽  
Key Enzyme ◽  
Cryptochrome 2 ◽  
Hydroxylation Activity

Seeds of the halophyte Suaeda salsa (L.) Pall. were cultured in 24 h dark and 14 h blue light / 10 h dark to examine the role of blue light and the blue-light-absorbing photoreceptor cryptochrome 2 (CRY2) in betacyanin accumulation, hypocotyl elongation and cotyledon opening in S. salsa seedlings. Darkness significantly promoted betacyanin accumulation and hypocotyl elongation but inhibited cotyledon opening. Blue light suppressed betacyanin accumulation and hypocotyl elongation but stimulated cotyledon opening. Betacyanin in S. salsa seedlings decomposed with time in blue light. Western blot analysis showed that CRY2 protein accumulated both in hypocotyls and cotyledons of S. salsa seedlings grown in dark, but degraded with time in blue light, which was paralleled by a decrease of tyrosine hydroxylation activity of tyrosinase, a key enzyme involved in the betalain biosynthesis pathway. These results suggest that CRY2 protein mediates betacyanin decomposition via inactivation of tyrosinase in S. salsa seedlings, and the blue-light-dependent degradation of CRY2 protein is crucial to its function.

scholarly journals Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

2003 ◽  
Vol 23 (6) ◽  
pp. 1968-1982 ◽  
Author(s):  
Irfan Saadi ◽  
Adisa Kuburas ◽  
Jamison J. Engle ◽  
Andrew F. Russo
Keyword(s):  
Dominant Negative ◽  
Hill Coefficient ◽  
Homeodomain Protein ◽  
Yeast Two Hybrid ◽  
Autosomal Dominant Disorder ◽  
Rieger Syndrome ◽  
A Charge ◽  
Two Hybrid

ABSTRACT Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

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