scholarly journals SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

2013 ◽  
Vol 201 (1) ◽  
pp. 49-63 ◽  
Author(s):  
Daniel W. Buster ◽  
Scott G. Daniel ◽  
Huy Q. Nguyen ◽  
Sarah L. Windler ◽  
Lara C. Skwarek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ubiquitin Ligase ◽  
Interphase Nucleus ◽  
Chromosome Organization ◽  
Interphase Chromatin ◽  
Nuclear Reorganization ◽  
Ubiquitin Ligase Complex ◽  
Drosophila Cells ◽  
Mitosis And Meiosis

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCFSlimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCFSlimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCFSlimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.

scholarly journals In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation

2001 ◽  
Vol 21 (13) ◽  
pp. 4276-4291 ◽  
Author(s):  
Richard G. Gardner ◽  
Alexander G. Shearer ◽  
Randolph Y. Hampton
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Sequence Similarity ◽  
High Specificity ◽  
Specific Sequence ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

ABSTRACT Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRDcomplex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.

scholarly journals Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

2020 ◽  
Author(s):  
Damien Garrido ◽  
Mohammed Bourouh ◽  
Éric Bonneil ◽  
Pierre Thibault ◽  
Andrew Swan ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
Anaphase Promoting Complex ◽  
Cells In Culture ◽  
Cyclin B3 ◽  
Mitosis And Meiosis ◽  
In Cells

ABSTRACTIn mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its Cdc20 co-activator. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 is required for meiotic anaphase in flies, worms and vertebrates, but whether it activates the APC/C is unclear. We found that Drosophila Cyclin B3 (CycB3) collaborates with PP2A-B55/Tws in embryonic development, indicating that CycB3 also promotes anaphase in mitosis. Moreover, CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its co-activators. We propose that CycB3-Cdk1 directly phosphorylates the APC/C to activate it in both meiosis and mitosis.

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 Regulation of photoprotection gene expression in Chlamydomonas by a putative E3 ubiquitin ligase complex and a homolog of CONSTANS

2019 ◽  
Vol 116 (35) ◽  
pp. 17556-17562 ◽  
Author(s):  
Stéphane T. Gabilly ◽  
Christopher R. Baker ◽  
Setsuko Wakao ◽  
Thien Crisanto ◽  
Katharine Guan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
High Light ◽  
Forward Genetics ◽  
Regulatory Function ◽  
Regulatory Sequences ◽  
Low Light ◽  
Green Algal ◽  
Ubiquitin Ligase Complex

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii, the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii. The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis-regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis-regulatory CrCO-binding sites at key photoprotection genes.

scholarly journals miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential

2018 ◽  
Vol 215 (8) ◽  
pp. 2115-2136 ◽  
Author(s):  
Sara E. Meyer ◽  
David E. Muench ◽  
Andrew M. Rogers ◽  
Tess J. Newkold ◽  
Emily Orr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ubiquitin Ligase ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Leukemia Stem Cells ◽  
Monocytic Differentiation ◽  
Self Renewal ◽  
Ubiquitin Ligase Complex ◽  
Pharmacologic Inhibition

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell–like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2–containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

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 Condensin II inactivation in interphase does not affect chromatin folding or gene expression

2018 ◽  
Author(s):  
Nezar Abdennur ◽  
Wibke Schwarzer ◽  
Aleksandra Pekowska ◽  
Indra Alon Shaltiel ◽  
Wolfgang Huber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromosome Organization ◽  
Domain Formation ◽  
Interphase Chromatin ◽  
Control Of Gene Expression ◽  
Chromosomal Architecture ◽  
Chromatin Folding ◽  
Mouse Hepatocytes ◽  
Or Gene ◽  
And Control

SummaryCondensin complexes have been proposed to play a prominent role in interphase chromatin organization and control of gene expression. Here, we report that the deletion of the central condensin II kleisin subunit Ncaph2 in differentiated mouse hepatocytes does not lead to significant changes in chromosome organization or in gene expression. Both observations challenge current views that implicate condensin in interphase chromosomal domain formation and in enhancer-promoter interactions. Instead, we suggest that the previously reported effects of condensin perturbation may result from their structural role during mitosis, which might indirectly impact the re-establishment of interphase chromosomal architecture after cell division.

scholarly journals Obtusaquinone is a cysteine modifying compound that targets Keap1 for degradation

2020 ◽  
Author(s):  
Christian E. Badr ◽  
Cintia Carla da Hora ◽  
Aleksandar B. Kirov ◽  
Elie Tabet ◽  
Romain Amante ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Multidisciplinary Approach ◽  
Antineoplastic Agent ◽  
Pharmacokinetic Analysis ◽  
Quantitative Mass Spectrometry ◽  
Functional Studies ◽  
Ubiquitin Ligase Complex ◽  
Molecular Mechanism Of Action ◽  
Xenograft Models

ABSTRACTWe have previously identified the natural product Obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and charaterize the molecular mechanism of action of OBT. We here show that OBT and analogs, which have improved pharmacological properties, bind to cysteine residues with particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway.

scholarly journals The CRL4VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258683
Author(s):  
N. Max Schabla ◽  
Patrick C. Swanson
Keyword(s):  
B Cells ◽  
Cell Line ◽  
Ubiquitin Ligase ◽  
Receptor Gene ◽  
E3 Ubiquitin Ligase ◽  
Proteasome Inhibition ◽  
Fibroblast Cell ◽  
Ubiquitin Ligase Complex ◽  
Evolutionarily Conserved

The development of B and T lymphocytes critically depends on RAG1/2 endonuclease activity to mediate antigen receptor gene assembly by V(D)J recombination. Although control of RAG1/2 activity through cell cycle- and ubiquitin-dependent degradation of RAG2 has been studied in detail, relatively little is known about mechanisms regulating RAG1 stability. We recently demonstrated that VprBP/DCAF1, a substrate adaptor for the CRL4 E3 ubiquitin ligase complex, is required to maintain physiological levels of RAG1 protein in murine B cells by facilitating RAG1 turnover. Loss of VprBP/DCAF1 in vivo results in elevated RAG1 expression, excessive V(D)J recombination, and immunoglobulin light chain repertoire skewing. Here we show that RAG1 is constitutively degraded when ectopically expressed in a human fibroblast cell line. Consistent with our findings in murine B cells, RAG1 turnover under these conditions is sensitive to loss of VprBP, as well as CRL4 or proteasome inhibition. Further evidence indicates that RAG1 degradation is ubiquitin-dependent and that RAG1 association with the CRL4VPRBP/DCAF1 complex is independent of CUL4 activation status. Taken together, these findings suggest V(D)J recombination co-opts an evolutionarily conserved and constitutively active mechanism to ensure rapid RAG1 turnover to restrain excessive RAG activity.

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