scholarly journals Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis

2016 ◽  
Author(s):  
Hui Dong ◽  
Jack Dumenil ◽  
Fu-Hao Lu ◽  
Li Na ◽  
Hannes Vanhaeren ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Molecular Mechanisms ◽  
Regulatory Proteins ◽  
Peptidase Activity ◽  
Organ Growth ◽  
E3 Ligases ◽  
Promote Cell Proliferation ◽  
Ring E3 Ligase ◽  
Ring E3 Ligases ◽  
Ring E3

ABSTRACTThe characteristic shapes and sizes of organs are established by cell proliferation patterns and final cell sizes, but the underlying molecular mechanisms coordinating these are poorly understood. Here we characterize a ubiquitin-activated peptidase called DA1 that limits the duration of cell proliferation during organ growth in Arabidopsis thaliana. The peptidase is activated by two RING E3 ligases, BB and DA2, which are subsequently cleaved by the activated peptidase and destabilized. In the case of BB, cleavage leads to destabilization by the RING E3 ligase PRT1 of the N-end rule pathway. DA1 peptidase activity also cleaves the de-ubiquitylase UBP15, which promotes cell proliferation, and the transcription factors TCP15 and TCP22, which promote cell proliferation proliferation and repress endoreduplication. We propose that DA1 peptidase activity regulates the duration of cell proliferation and the transition to endoreduplication and differentiation during organ formation in plants by coordinating the destabilization of regulatory proteins.

scholarly journals Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12

Open Biology ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 200041 ◽  
Author(s):  
Zhuoyao Chen ◽  
Gregory A. Wasney ◽  
Sarah Picaud ◽  
Panagis Filippakopoulos ◽  
Masoud Vedadi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
E3 Ligases ◽  
Kelch Domain ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
New Protein

Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

scholarly journals Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications

2018 ◽  
Vol 475 (9) ◽  
pp. 1569-1582 ◽  
Author(s):  
Adaitya Prasad Behera ◽  
Pritam Naskar ◽  
Shubhangi Agarwal ◽  
Prerana Agarwal Banka ◽  
Asim Poddar ◽  
...  
Keyword(s):  
Dissociation Constants ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
New Gene ◽  
Functional Analyses ◽  
Affinity Interaction ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
Structural Insights

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

scholarly journals Arabidopsis DREB2A-Interacting Proteins Function as RING E3 Ligases and Negatively Regulate Plant Drought Stress–Responsive Gene Expression

2008 ◽  
Vol 20 (6) ◽  
pp. 1693-1707 ◽  
Author(s):  
Feng Qin ◽  
Yoh Sakuma ◽  
Lam-Son Phan Tran ◽  
Kyonoshin Maruyama ◽  
Satoshi Kidokoro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drought Stress ◽  
Interacting Proteins ◽  
E3 Ligases ◽  
Responsive Gene ◽  
Ring E3 Ligases ◽  
Ring E3

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

Designing active RNF4 monomers by introducing a tryptophan: avidity towards E2∼Ub conjugates dictates the activity of ubiquitin RING E3 ligases

2019 ◽  
Vol 476 (10) ◽  
pp. 1465-1482 ◽  
Author(s):  
Sayani Sarkar ◽  
Adaitya Prasad Behera ◽  
Prateeka Borar ◽  
Prerana Agarwal Banka ◽  
Ajit B. Datta
Keyword(s):  
Enzymatic Activity ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Binding Studies ◽  
E3 Ligases ◽  
Ring Domains ◽  
Biochemical Analyses ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
Existing Data

Abstract Ubiquitin RING E3 ligases (E3s) catalyze ubiquitin (Ub) transfer to their substrates by engaging E2∼Ub intermediates with the help of their RING domains. Different E3s have been found to contain a conserved tryptophan residue in their RING that plays an essential role in E2 binding and, hence, enzymatic activity. Many active E3s, however, lack this specific residue. We mined through the existing data to observe that the conservation of the tryptophan and quaternary organization of the RING domains are remarkably correlated. Monomeric RINGs possess the tryptophan while all well-characterized dimeric RINGs, except RNF8, contain other amino acid residues. Biochemical analyses on representative E3s and their mutants reveal that the tryptophan is essential for optimal enzymatic activity of monomeric RINGs whereas dimeric E3s with tryptophan display hyperactivity. Most critically, the introduction of the tryptophan restores the activity of inactive monomeric RNF4 mutants, an obligatory dimeric E3. Binding studies indicate that monomeric RINGs retained the tryptophan for their optimal functionality to compensate for weak Ub binding. On the other hand, tryptophan was omitted from dimeric RINGs during the course of evolution to prevent unwanted modifications and allow regulation of their activity through oligomerization.

scholarly journals Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

2018 ◽  
Author(s):  
Viduth K Chaugule ◽  
Connor Arkinson ◽  
Rachel Toth ◽  
Helen Walden
Keyword(s):  
Dna Damage ◽  
Repair Pathway ◽  
Core Complex ◽  
Site Specific ◽  
E3 Ligases ◽  
Interstrand Crosslink ◽  
Damage Repair ◽  
Molecular Signals ◽  
Ring E3 Ligases ◽  
Ring E3

AbstractIn eukaryotes, DNA damage repair is implemented by a host of proteins that are coordinated by defined molecular signals. One such signal that transpires during the Fanconi Anemia (FA) - interstrand crosslink (ICL) repair pathway is the site-specific monoubiquitination of FANCD2 and FANCI proteins by a large, multi-protein FA core complex. The mechanics for this exquisitely specific monoubiquitin signal has been elusive. Here we show FANCL, the RING E3 module of the FA core complex, allosterically activates its cognate E2 Ube2T for monoubiquitination by a mechanism distinct from the typical RING-based catalysis. FANCL triggers intricate re-wiring of Ube2T’s intra-residue network thus activating the E2 for precision targeting. This network is intrinsically regulated by conserved gates and loops which can be engineered to yield Ube2T variants that enhance FANCD2 ubiquitination by ~30-fold without compromising on target specificity. Finally, we also uncover allosteric networks in other ubiquitin E2s that can be leveraged by RING E3 ligases to drive specific ubiquitination.

scholarly journals Keratin 6a reorganization for ubiquitin–proteasomal processing is a direct antimicrobial response

2017 ◽  
Vol 217 (2) ◽  
pp. 731-744 ◽  
Author(s):  
Jonathan K.L. Chan ◽  
Don Yuen ◽  
Priscilla Hiu-Mei Too ◽  
Yan Sun ◽  
Belinda Willard ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Immune Defense ◽  
Antigenic Peptides ◽  
Corneal Epithelial ◽  
E3 Ligases ◽  
Appreciable Increase ◽  
Host Defense Response ◽  
Ubiquitin Proteasome ◽  
Ring E3 Ligases ◽  
Ring E3

Skin and mucosal epithelia deploy antimicrobial peptides (AMPs) to eliminate harmful microbes. We reported that the intermediate filament keratin 6a (K6a) is constitutively processed into antimicrobial fragments in corneal epithelial cells. In this study, we show that K6a network remodeling is a host defense response that directly up-regulates production of keratin-derived AMPs (KAMPs) by the ubiquitin–proteasome system (UPS). Bacterial ligands trigger K6a phosphorylation at S19, S22, S37, and S60, leading to network disassembly. Mutagenic analysis of K6a confirmed that the site-specific phosphorylation augmented its solubility. K6a in the cytosol is ubiquitinated by cullin-RING E3 ligases for subsequent proteasomal processing. Without an appreciable increase in K6a gene expression and proteasome activity, a higher level of cytosolic K6a results in enhanced KAMP production. Although proteasome-mediated proteolysis is known to produce antigenic peptides in adaptive immunity, our findings demonstrate its new role in producing AMPs for innate immune defense. Manipulating K6a phosphorylation or UPS activity may provide opportunities to harness the innate immunity of epithelia against infection.

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