scholarly journals A constitutively monomeric UVR8 photoreceptor confers enhanced UV-B photomorphogenesis

2021 ◽  
Vol 118 (6) ◽  
pp. e2017284118
Author(s):  
Roman Podolec ◽  
Kelvin Lau ◽  
Timothée B. Wagnon ◽  
Michael Hothorn ◽  
Roman Ulm
Keyword(s):  
Gene Expression ◽  
Crystal Structure ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ultraviolet B ◽  
Genetic Tool ◽  
Loop Region ◽  
Extreme Uv ◽  
Insight Into

The plant ultraviolet-B (UV-B) photoreceptor UVR8 plays an important role in UV-B acclimation and survival. UV-B absorption by homodimeric UVR8 induces its monomerization and interaction with the E3 ubiquitin ligase COP1, leading ultimately to gene expression changes. UVR8 is inactivated through redimerization, facilitated by RUP1 and RUP2. Here, we describe a semidominant, hyperactive allele, namely uvr8-17D, that harbors a glycine-101 to serine mutation. UVR8G101S overexpression led to weak constitutive photomorphogenesis and extreme UV-B responsiveness. UVR8G101S was observed to be predominantly monomeric in vivo and, once activated by UV-B, was not efficiently inactivated. Analysis of a UVR8 crystal structure containing the G101S mutation revealed the distortion of a loop region normally involved in stabilization of the UVR8 homodimer. Plants expressing a UVR8 variant combining G101S with the previously described W285A mutation exhibited robust constitutive photomorphogenesis. This work provides further insight into UVR8 activation and inactivation mechanisms and describes a genetic tool for the manipulation of photomorphogenic responses.

scholarly journals A constitutively monomeric UVR8 photoreceptor allele confers enhanced UV-B photomorphogenesis

2020 ◽  
Author(s):  
Roman Podolec ◽  
Kelvin Lau ◽  
Timothée B. Wagnon ◽  
Michael Hothorn ◽  
Roman Ulm
Keyword(s):  
Gene Expression ◽  
Crystal Structure ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Genetic Tool ◽  
Loop Region ◽  
Extreme Uv ◽  
Insight Into

AbstractThe plant UV-B photoreceptor UVR8 plays an important role in UV-B acclimation and survival. UV-B absorption by homodimeric UVR8 induces its monomerization and interaction with the E3 ubiquitin ligase COP1, leading ultimately to gene expression changes. UVR8 is inactivated through redimerization, facilitated by RUP1 and RUP2. Here, we describe a novel semi-dominant, hyperactive allele, namely uvr8-17D, that harbors a glycine-101 to serine mutation. UVR8G101S-overexpression led to weak constitutive photomorphogenesis and extreme UV-B responsiveness. UVR8G101S was observed to be predominantly monomeric in vivo and, once activated by UV-B, was not efficiently inactivated. Analysis of a UVR8G101S crystal structure revealed the distortion of a loop region normally involved in stabilization of the UVR8 homodimer. Plants expressing a UVR8 variant combining G101S with the previously described W285A mutation exhibited robust constitutive photomorphogenesis. This work provides further insight into UVR8 activation and inactivation mechanisms, and describes a genetic tool for the manipulation of photomorphogenic responses.

scholarly journals Knockdown of the E3 Ubiquitin ligase UBR5 and its role in skeletal muscle anabolism

2020 ◽  
Author(s):  
Daniel C. Turner ◽  
David C. Hughes ◽  
Leslie M. Baehr ◽  
Robert A. Seaborne ◽  
Mark Viggars ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Muscle Mass ◽  
Ubiquitin Ligase ◽  
Muscle Protein ◽  
E3 Ubiquitin Ligase ◽  
Skeletal Muscle Atrophy ◽  
Human Myotubes ◽  
The Impact

AbstractUBR5 is an E3-ubiquitin-ligase positively associated with anabolism, hypertrophy and recovery from atrophy in skeletal muscle. The precise mechanisms underpinning UBR5’s role in the regulation of skeletal muscle mass remain unknown. The present study aimed to elucidate these mechanisms by silencing the UBR5 gene in-vitro and in-vivo. The siRNA-induced reduction (−77%) in UBR5 gene expression in human myotubes was prevented by mechanical loading, suggesting that UBR5 gene expression may be regulated via mechano-transduction signalling. Therefore, we electroporated a UBR5-RNAi plasmid into mouse tibialis anterior muscle in-vivo to investigate the impact of reduced UBR5 on mechano-transduction signalling MEK/ERK/p90RSK and Akt/p70S6K/4E-BP1/rpS6 pathways. Seven days post UBR5 RNAi electroporation, while reductions in overall muscle mass were not detected, mean CSA of GFP-positive fibers was reduced (−9.5%) and the number of large fibers was lower versus the control. Importantly, UBR5-RNAi significantly reduced total RNA, muscle protein synthesis, ERK1/2 and Akt phosphorylation. Whilst p90RSK phosphorylation significantly increased, total p90RSK protein levels demonstrated a 45% reduction with UBR5-RNAi. Finally, these early signalling events after 7 days of UBR5 knockdown culminated in significant reductions in muscle mass (−4.6%) and larger reductions in fiber CSA (−18.5%) after 30 days. This was associated with increased levels of the phosphatase, PP2Ac, and inappropriate chronic elevation of p70S6K and rpS6 between 7 and 30 days, and corresponding reductions in eIF4e. This study demonstrates UBR5 plays an important role in anabolism/hypertrophy, whereby knockdown of UBR5 culminates in skeletal muscle atrophy.

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 TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhou Yu ◽  
Xuelian Li ◽  
Mingjin Yang ◽  
Jiaying Huang ◽  
Qian Fang ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
B Cell Lymphoma ◽  
Antiviral Response ◽  
Type I Interferons ◽  
Type I ◽  
Innate Response ◽  
Innate Antiviral Response

AbstractSensing of pathogenic nucleic acids by pattern recognition receptors (PRR) not only initiates anti-microbe defense but causes inflammatory and autoimmune diseases. E3 ubiquitin ligase(s) critical in innate response need to be further identified. Here we report that the tripartite motif-containing E3 ubiquitin ligase TRIM41 is required to innate antiviral response through facilitating pathogenic nucleic acids-triggered signaling pathway. TRIM41 deficiency impairs the production of inflammatory cytokines and type I interferons in macrophages after transfection with nucleic acid-mimics and infection with both DNA and RNA viruses. In vivo, TRIM41 deficiency leads to impaired innate response against viruses. Mechanistically, TRIM41 directly interacts with BCL10 (B cell lymphoma 10), a core component of CARD proteins−BCL10 − MALT1 (CBM) complex, and modifies the Lys63-linked polyubiquitylation of BCL10, which, in turn, hubs NEMO for activation of NF-κB and TANK-binding kinase 1 (TBK1) − interferon regulatory factor 3 (IRF3) pathways. Our study suggests that TRIM41 is the potential universal E3 ubiquitin ligase responsible for Lys63 linkage of BCL10 during innate antiviral response, adding new insight into the molecular mechanism for the control of innate antiviral response.

scholarly journals Effects of diethylcarbamazine and ivermectin treatment onBrugia malayigene expression in infected gerbils (Meriones unguiculatus)

2019 ◽  
Vol 5 ◽  
Author(s):  
Mary J. Maclean ◽  
W. Walter Lorenz ◽  
Michael T. Dzimianski ◽  
Christopher Anna ◽  
Andrew R. Moorhead ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Action ◽  
Meriones Unguiculatus ◽  
Current Control ◽  
Direct Result ◽  
Ivermectin Treatment ◽  
Treatment Groups ◽  
Parasite Survival ◽  
Insight Into

AbstractLymphatic filariasis (LF) threatens nearly 20% of the world's population and has handicapped one-third of the 120 million people currently infected. Current control and elimination programs for LF rely on mass drug administration of albendazole plus diethylcarbamazine (DEC) or ivermectin. Only the mechanism of action of albendazole is well understood. To gain a better insight into antifilarial drug actionin vivo, we treated gerbils harbouring patentBrugia malayiinfections with 6 mg kg−1DEC, 0.15 mg kg−1ivermectin or 1 mg kg−1albendazole. Treatments had no effect on the numbers of worms present in the peritoneal cavity of treated animals, so effects on gene expression were a direct result of the drug and not complicated by dying parasites. Adults and microfilariae were collected 1 and 7 days post-treatment and RNA isolated for transcriptomic analysis. The experiment was repeated three times. Ivermectin treatment produced the most differentially expressed genes (DEGs), 113. DEC treatment yielded 61 DEGs. Albendazole treatment resulted in little change in gene expression, with only 6 genes affected. In total, nearly 200 DEGs were identified with little overlap between treatment groups, suggesting that these drugs may interfere in different ways with processes important for parasite survival, development, and reproduction.

scholarly journals p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLprovia E3 ubiquitin ligase RCHY1

2016 ◽  
Vol 113 (35) ◽  
pp. E5192-E5201 ◽  
Author(s):  
Yue Ma-Lauer ◽  
Javier Carbajo-Lozoya ◽  
Marco Y. Hein ◽  
Marcel A. Müller ◽  
Wen Deng ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Nonstructural Protein ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Immune Recognition ◽  
Major Player ◽  
Nonstructural Protein 3 ◽  
Human Coronaviruses ◽  
Unique Domain

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLprofusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLproalone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

scholarly journals Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

Open Biology ◽  
2014 ◽  
Vol 4 (3) ◽  
pp. 130172 ◽  
Author(s):  
Barbara Franke ◽  
Alexander Gasch ◽  
Dayté Rodriguez ◽  
Mohamed Chami ◽  
Muzamil M. Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Quaternary Structure ◽  
E3 Ubiquitin Ligase ◽  
Coiled Coil ◽  
Trim Protein ◽  
Muscle Catabolism ◽  
And Function ◽  
Helical Region ◽  
Structural Significance

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.

scholarly journals Photocontrollable PROTAC molecules: Structure and mechanism of action

2021 ◽  
Vol 71 (3) ◽  
pp. 161-176
Author(s):  
Mladen Koravović ◽  
Gordana Tasić ◽  
Milena Rmandić ◽  
Bojan Marković
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Binding Sites ◽  
E3 Ubiquitin Ligase ◽  
Drug Binding ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Target Drug ◽  
Traditional Drug

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.

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