scholarly journals The Cullin3 Ubiquitin Ligase Functions as a Nedd8-bound Heterodimer

2007 ◽  
Vol 18 (3) ◽  
pp. 899-909 ◽  
Author(s):  
Wananit Wimuttisuk ◽  
Jeffrey D. Singer
Keyword(s):  
Ubiquitin Ligase ◽  
Covalent Attachment ◽  
Winged Helix ◽  
Btb Domain ◽  
C Terminus ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitination Pathway ◽  
Insight Into ◽  
Ligase Function

Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cell-expressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases.

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 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 The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

2000 ◽  
Vol 11 (7) ◽  
pp. 2315-2325 ◽  
Author(s):  
Joel D. Leverson ◽  
Claudio A.P. Joazeiro ◽  
Andrew M. Page ◽  
Han-kuei Huang ◽  
Philip Hieter ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Protein Substrates ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

Abstract 241: MDM2 E3 Ligase-mediated Ubiquitination and Degradation of HDAC1 in Vascular Calcification

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Hyun Kook ◽  
Duk-Hwa Kwon ◽  
Gwang Hyeon Eom ◽  
Hyun-Ki Min ◽  
Somy Yoon ◽  
...  
Keyword(s):  
Vascular Calcification ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Metabolic Diseases ◽  
Genetic Ablation ◽  
Histone Deacetylase 1 ◽  
Ubiquitination Pathway ◽  
Mdm2 Inhibitor

Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC.

scholarly journals Fat-regulated adaptor protein Dlish binds the growth suppressor Expanded and controls its stability and ubiquitination

2019 ◽  
Vol 116 (4) ◽  
pp. 1319-1324 ◽  
Author(s):  
Xing Wang ◽  
Yifei Zhang ◽  
Seth S. Blair
Keyword(s):  
Ubiquitin Ligase ◽  
Hippo Pathway ◽  
E3 Ubiquitin Ligase ◽  
Growth Control ◽  
Adaptor Protein ◽  
Domain Growth ◽  
C Terminus ◽  
The Hippo Pathway

The Drosophila protocadherin Fat controls organ size through the Hippo pathway, but the biochemical links to the Hippo pathway components are still poorly defined. We previously identified Dlish, an SH3 domain protein that physically interacts with Fat and the type XX myosin Dachs, and showed that Fat’s regulation of Dlish levels and activity helps limit Dachs-mediated inhibition of Hippo pathway activity. We here characterize a parallel growth control pathway downstream of Fat and Dlish. Using immunoprecipitation and mass spectrometry to search for Dlish partners, we find that Dlish binds the FERM domain growth repressor Expanded (Ex); Dlish SH3 domains directly bind sites in the Ex C terminus. We further show that, in vivo, Dlish reduces the subapical accumulation of Ex, and that loss of Dlish blocks the destabilization of Ex caused by loss of Fat. Moreover, Dlish can bind the F-box E3 ubiquitin ligase Slimb and promote Slimb-mediated ubiquitination of Expanded in vitro. Both the in vitro and in vivo effects of Dlish on Ex require Slimb, strongly suggesting that Dlish destabilizes Ex by helping recruit Slimb-containing E3 ubiquitin ligase complexes to Ex.

Enzymes of ubiquitination and deubiquitination

2012 ◽  
Vol 52 ◽  
pp. 37-50 ◽  
Author(s):  
Melanie Neutzner ◽  
Albert Neutzner
Keyword(s):  
Protein Trafficking ◽  
Current Knowledge ◽  
Covalent Attachment ◽  
Cellular Processes ◽  
Regulatory Processes ◽  
Substrate Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
The Many ◽  
Enzymatic Machinery ◽  
Insight Into

Ubiquitination, the covalent attachment of the small protein modifier ubiquitin to a substrate protein is involved in virtually all cellular processes by mediating the regulated degradation of proteins. Aside from proteasomal degradation, ubiquitination plays important roles in transcriptional regulation, protein trafficking, including endocytosis and lysosomal targeting, and activation of kinases involved in signalling processes. A three-tiered enzymatic cascade consisting of E1 or ubiquitin-activating enzyme, E2 or ubiquitin-conjugating enzyme, and E3, or ubiquitin ligases, is necessary to achieve the many forms of ubiquitination known to date. In this chapter, we summarize the current knowledge on the enzymatic machinery necessary for ubiquitin activation and ligation, as well as its removal, and provide some insight into the complexity of regulatory processes governed by ubiquitination.

scholarly journals Mechanistic insight into the nucleus–vacuole junction based on the Vac8p–Nvj1p crystal structure

2017 ◽  
Vol 114 (23) ◽  
pp. E4539-E4548 ◽  
Author(s):  
Hanbin Jeong ◽  
Jumi Park ◽  
Hye-In Kim ◽  
Miriam Lee ◽  
Young-Joon Ko ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Direct Interaction ◽  
Endoplasmic Reticulum Membrane ◽  
Cellular Functions ◽  
C Terminus ◽  
Self Association ◽  
Vacuolar Protein ◽  
Superhelical Structure ◽  
Insight Into

Formation of the nucleus–vacuole junction (NVJ) is mediated by direct interaction between the vacuolar protein Vac8p and the outer nuclear endoplasmic reticulum membrane protein Nvj1p. Herein we report the crystal structure of Vac8p bound to Nvj1p at 2.4-Å resolution. Vac8p comprises a flexibly connected N-terminal H1 helix followed by 12 armadillo repeats (ARMs) that form a right-handed superhelical structure. The extended 80-Å-long loop of Nvj1p specifically binds the highly conserved inner groove formed from ARM1−12 of Vac8p. Disruption of the Nvj1p–Vac8p interaction results in the loss of tight NVJs, which impairs piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae. Vac8p cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1p are also critical to the recognition of Atg13p, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8p. Indeed, mutation of the cationic triad abolishes CVT of Ape1p in vivo. Combined with biochemical data, the crystal structure reveals a Vac8p homodimer formed from ARM1, and this self-association, likely regulated by the flexible H1 helix and the C terminus of Nvj1p, is critical for Vac8p cellular functions.

scholarly journals The conserved RNA recognition motif and C3H1 domain of the Not4 ubiquitin ligase regulate in vivo ligase function

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Hongfeng Chen ◽  
Tirupataiah Sirupangi ◽  
Zhao-Hui Wu ◽  
Daniel L. Johnson ◽  
R. Nicholas Laribee
Keyword(s):  
Ubiquitin Ligase ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Ligase Function

scholarly journals The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function

2019 ◽  
Vol 294 (46) ◽  
pp. 17249-17261 ◽  
Author(s):  
Liang Qin ◽  
Arda Mizrak ◽  
Dimitrius Santiago P. S. F. Guimarães ◽  
Hana M. Tambrin ◽  
David O. Morgan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Biochemical Analysis ◽  
Anaphase Promoting Complex ◽  
Amino Acid Motif ◽  
Conserved Sequence ◽  
High Affinity ◽  
Substrate Degradation ◽  
C Terminus ◽  
Necessary And Sufficient

The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif–dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.

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.

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