scholarly journals Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion

2018 ◽  
Author(s):  
Andrew M. Hudson ◽  
Katelynn M. Mannix ◽  
Julianne A. Gerdes ◽  
Molly C. Kottemann ◽  
Lynn Cooley
Keyword(s):  
Actin Cytoskeleton ◽  
Affinity Purification ◽  
Ubiquitin Proteasome System ◽  
Sequence Motif ◽  
Ring Canal ◽  
Cytoskeletal Structure ◽  
Ubiquitin Ligase Complex ◽  
Ring Canals ◽  
Cullin 3 ◽  
Ubiquitin Proteasome

AbstractDuring Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. Here, we use tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3Kelch ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. We present genetic evidence consistent with HtsRC being the CRL3Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. Finally, we identify a short sequence motif in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.

scholarly journals Drosophila Kelch functions with Cullin-3 to organize the ring canal actin cytoskeleton

2010 ◽  
Vol 188 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Repeated Sequence ◽  
Adaptor Proteins ◽  
Sequence Motif ◽  
Filamentous Actin ◽  
E3 Ligases ◽  
Cytoskeletal Structure ◽  
Ring Canals ◽  
Cullin 3 ◽  
New Gene ◽  
Female Germline

Drosophila melanogaster Kelch (KEL) is the founding member of a diverse protein family defined by a repeated sequence motif known as the KEL repeat (KREP). Several KREP proteins, including Drosophila KEL, bind filamentous actin (F-actin) and contribute to its organization. Recently, a subset of KREP proteins has been shown to function as substrate adaptor proteins for cullin-RING (really interesting new gene) ubiquitin E3 ligases. In this study, we demonstrate that association of Drosophila KEL with Cullin-3, likely in a cullin-RING ligase, is essential for the growth of Drosophila female germline ring canals. These results suggest a role for protein ubiquitylation in the remodeling of a complex F-actin cytoskeletal structure.

scholarly journals An UBR-box from a non-N-recognin: Crystal Structure of the UBR domain of UBR6

2014 ◽  
Vol 70 (a1) ◽  
pp. C306-C306
Author(s):  
Juliana Muñoz-Escobar ◽  
Guennadi Kozlov ◽  
Jean-François Trempe ◽  
Kalle Gehring
Keyword(s):  
Crystal Structure ◽  
Ubiquitin Proteasome System ◽  
Zinc Binding ◽  
Binding Motifs ◽  
Clear Explanation ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Proteasome ◽  
Binding Pockets ◽  
F Box Protein

The degradation of many short-lived proteins in eukaryotic cells is carried out by the Ubiquitin Proteasome System. The N-end rule pathway links the half-life of proteins to the identity of its N-terminal residue, also called N-degron. Destabilizing N-degrons, are recognized by E3 ubiquitin ligases termed N-recognins. N-degrons are grouped into type 1, composed of basic residues, and type 2, composed of bulky hydrophobic residues. In mammals, four N-recognins mediate the N-end rule pathway: UBR1, UBR2, UBR4 and UBR5. These proteins share a ~70-residue zinc finger-like motif termed the Ubiquitin Recognin (UBR) box, responsible for their specificity. The mammalian genome encodes at least three more UBR-box proteins: UBR3, UBR6/FBXO11 and UBR7. However, these UBRs cannot recognize any type of N-degrons. Our lab reported the crystal structures of the UBR boxes from the human UBR1 and UBR2, rationalizing the empirical rules for the classification of type 1 N-degrons. Despite the valuable information obtained from those structures there is not a clear explanation for the no recognition of N-degrons by other UBR-box proteins. Here we report the crystal structure of the UBR-box domain from UBR6 also known as FBXO11. UBR6 is a F-box protein of the SKP1-Cullin1-F-box (SCF) ubiquitin ligase complex and does not recognize any type of N-degrons. We crystallized a 77-residue fragment of the UBR-box of UBR6 and determined its structure at 1.7 Å resolution. Unexpectedly, this domain adopts an open conformation compared to UBR1-box, without any N-degron binding pockets. Its zinc-binding residues are conserved as in the N-recognins, but they are arranged in different zinc-binding motifs. Molecules form dimmers stabilized by zinc ions. The crystal had 4 molecules per asymmetric unit and space group P212121. For phasing we used Zn-SAD. With this structure we hope to obtain clues that explain the absence of N-degron recognition in some members of the UBR family.

scholarly journals Old players in new posts: the role of P53, ATM and DNAPK in DNA damage-related ubiquitylation-dependent removal of S2P RNAPII

2021 ◽  
Author(s):  
Barbara N Borsos ◽  
Vasiliki Pantazi ◽  
Zoltán G Páhi ◽  
Hajnalka Majoros ◽  
Zsuzsanna Ujfaludi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase Ii ◽  
E3 Ligase ◽  
Ubiquitin Proteasome System ◽  
Dna Double Strand Breaks ◽  
Damage Site ◽  
Strand Breaks ◽  
Cullin 3 ◽  
Ubiquitin Proteasome ◽  
Dependent Protein

AbstractDNA double-strand breaks are the most deleterious lesions for the cells, therefore understanding the macromolecular interactions in the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) are the two main regulatory kinases of homologous recombination and non-homologous end joining, respectively. Although these kinases are involved in the activation of different repair pathways, they have common target proteins, such as P53. We previously demonstrated that following transcription block, P53 plays a pivotal role in transcription elongation process by interacting with S2P RNAPII. In the current study, we reveal that P53, ATM and DNAPK are involved in the fine-tune regulation of the ubiquitin-proteasome system-related degradation of S2P RNAPII. However, they act differently in this process: P53 delays the removal of S2P RNAPII, while ATM and DNAPK participate in the activation of members of E3 ligase complexes involved in the ubiquitylation of S2P RNAPII. We also demonstrate that WW domain-containing protein 2 (WWP2) and Cullin-3 (CUL3) are interaction partners of S2P RNAPII, thus forming a complex with the transcribing RNAPII complex.Simple SummaryTo ensure the proper repair following DNA double-strand breaks, the eviction of the arrested elongating RNA polymerase II (S2P RNAPII) is required. Here, we report an emerging role of P53, Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) in the ubiquitin-proteasome system-dependent removal of S2P RNAPII. We also identified interactions between S2P RNAPII and WW domain-containing protein 2 (WWP2) or Cullin-3 (CUL3) (members of E3 ligase complexes), which are involved in the ubiquitylation of S2P RNAPII following DNA damage. Furthermore, the RNAPII-E3 ligase complex interactions are mediated by P53, ATM and DNAPK, which suggests potential participation of all three proteins in the effective resolution of transcription block at the damage site. Altogether, our results provide a better comprehension of the molecular background of transcription elongation block-related DNA repair processes and highlight an indispensable function of P53, ATM and DNAPK in these mechanisms.

scholarly journals p97 Negatively Regulates NRF2 by Extracting Ubiquitylated NRF2 from the KEAP1-CUL3 E3 Complex

2017 ◽  
Vol 37 (8) ◽  
Author(s):  
Shasha Tao ◽  
Pengfei Liu ◽  
Gang Luo ◽  
Montserrat Rojo de la Vega ◽  
Heping Chen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Complexes ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Proteasome ◽  
Client Proteins

ABSTRACT Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation (the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system (UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX-containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.

scholarly journals Nek2 Stabilization By Usp7 Leads to Activation of NF-Kb in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4418-4418 ◽  
Author(s):  
Reinaldo Franqui Machin ◽  
Xin Zhan ◽  
Hongwei Xu ◽  
Ivana Frech ◽  
Guido J Tricot ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Affinity Purification ◽  
Ubiquitin Proteasome System ◽  
Negative Regulator ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Ubiquitin Proteasome ◽  
Affinity Purification Mass Spectrometry

Abstract NIMA (Never In Mitosis Gene A)-Related Kinase 2 (Nek2), a centrosomal Serine/Threonine kinase, is a key player in numerous malignancies. Overexpression of Nek2 has been related to many cancers including Multiple Myeloma (MM). In MM, Nek2 is one of the chromosomal instability genes associated with drug resistance and disease relapse. However, very little is known about the mechanisms that lead to these Nek2-driven disparities. Here, we show that the Ubiquitin Specific Peptidase 7 (USP7) stabilizes Nek2 leading to activation of NF-kb pathway. Using gene expression profile (GEP) data from patients and cell lines we discovered that Nek2 overexpression leads to increases of several targets of the NF-kb pathway. We, thus, hypothesize that Nek2 is activating NF-kb. To address this, we overexpressed Nek2 and tested the classic canonical NF-kb hallmarks proteins by western blotting. Nek2 overexpression led to an increase in phosphorylation of IKK, activator of NF-kb, and to decrease levels of IKb-alpha, a negative regulator of the pathway. Nek2 overexpression also increased nuclear and phosphorylated p65 on residue S536, known as active transcriptional site. To further confirm that Nek2 is activating canonical NF-kb luciferase assay was performed. The luciferase reporter is driven by a p65 promoter and in cells overexpressing Nek2 luciferase levels were increased. To characterize Nek2 interacting partners a tandem affinity purification/mass spectrometry (TAP/MS) approach was performed. We found that Nek2 binds to Usp7, a deubiquitinase overexpressed in numerous cancers. This led to hypothesize that that Nek2, a known target of the ubiquitin proteasome system, is being stabilized by the Usp7 contributing to its overexpression and the increased activation of the NF-kb pathway. To test our hypothesis, we treated different cancer cell lines with the commercially available Usp7 inhibitor, P5091, or silenced the protein using shRNA. In both case, we found a reduction in Nek2 protein level. Additionally, we overexpressed Usp7 and Nek2 increased confirming that Usp7 stabilizes Nek2. To further show that Usp7 stabilizes Nek2 by de-ubiquitination, we overexpressed Usp7 and analyzed Nek2 ubiquitination after immunoprecipitation. When Usp7 was overexpressed no ubiquitination of Nek2 was detected. Finally, by using GEP data from MM patients, we found that individuals who overexpressed Nek2 along with an active NF-kb signature have worst event free survival as well as overall survival, indicating Nek2 overexpression leading to increased NF-kb signature has clinical significance. Disclosures No relevant conflicts of interest to declare.

scholarly journals HtsRC-mediated accumulation of F-actin regulates ring canal size during Drosophila melanogaster oogenesis

2020 ◽  
Author(s):  
Julianne A. Gerdes ◽  
Katelynn M. Mannix ◽  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Cytoskeleton ◽  
Fruit Flies ◽  
Follicle Cells ◽  
Filamentous Actin ◽  
Ring Canal ◽  
High Fecundity ◽  
Ring Canals ◽  
Necessary And Sufficient ◽  
Female Germline

AbstractRing canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC, a component specific to female germline ring canals, is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ring-canal-like ectopic actin structures in somatic follicle cells. Finally, we present findings which indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility, a result which is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

scholarly journals Role of the Ubiquitin Proteasome System in the Regulation of Blood Pressure: A Review

2020 ◽  
Vol 21 (15) ◽  
pp. 5358
Author(s):  
Osamu Yamazaki ◽  
Daigoro Hirohama ◽  
Kenichi Ishizawa ◽  
Shigeru Shibata
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Ubiquitin Proteasome System ◽  
Blood Pressure Regulation ◽  
Sodium Reabsorption ◽  
Renal Tubules ◽  
Pressure Regulation ◽  
Cullin 3 ◽  
Ubiquitin Proteasome

The kidney and the vasculature play crucial roles in regulating blood pressure. The ubiquitin proteasome system (UPS), a multienzyme process mediating covalent conjugation of the 76-amino acid polypeptide ubiquitin to a substrate protein followed by proteasomal degradation, is involved in multiple cellular processes by regulating protein turnover in various tissues. Increasing evidence demonstrates the roles of UPS in blood pressure regulation. In the kidney, filtered sodium is reabsorbed through diverse sodium transporters and channels along renal tubules, and studies conducted till date have provided insights into the complex molecular network through which ubiquitin ligases modulate sodium transport in different segments. Components of these pathways include ubiquitin ligase neuronal precursor cell-expressed developmentally downregulated 4-2, Cullin-3, and Kelch-like 3. Moreover, accumulating data indicate the roles of UPS in blood vessels, where it modulates nitric oxide bioavailability and vasoconstriction. Cullin-3 not only regulates renal salt reabsorption but also controls vascular tone using different adaptor proteins that target distinct substrates in vascular smooth muscle cells. In endothelial cells, UPS can also contribute to blood pressure regulation by modulating endothelial nitric oxide synthase. In this review, we summarize current knowledge regarding the role of UPS in blood pressure regulation, focusing on renal sodium reabsorption and vascular function.

scholarly journals HtsRC-Mediated Accumulation of F-Actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis

Genetics ◽  
2020 ◽  
Vol 216 (3) ◽  
pp. 717-734
Author(s):  
Julianne A. Gerdes ◽  
Katelynn M. Mannix ◽  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Drosophila Melanogaster ◽  
Actin Cytoskeleton ◽  
Fruit Flies ◽  
Follicle Cells ◽  
Filamentous Actin ◽  
Ring Canal ◽  
High Fecundity ◽  
Ring Canals ◽  
Necessary And Sufficient ◽  
Female Germline

Ring canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton, but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, we show that HtsRC—a component specific to female germline ring canals—is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ectopic actin structures in somatic follicle cells. Finally, we present findings that indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility—a result that is consistent with our understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

scholarly journals Cullin 1 (CUL1) Promotes Primary Ciliogenesis through the Induction of Ubiquitin-Proteasome-Dependent Dvl2 Degradation

2021 ◽  
Vol 22 (14) ◽  
pp. 7572
Author(s):  
Sun-Ok Kim ◽  
Kyoung Sang Cho ◽  
Bo Yeon Kim ◽  
Kyung Ho Lee
Keyword(s):  
Primary Cilia ◽  
Ubiquitin Proteasome System ◽  
Human Diseases ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Ubiquitin Ligase Complex ◽  
Cellular Signal ◽  
Ubiquitin Proteasome ◽  
Novel Target ◽  
And Function

Primary cilia are nonmotile cellular signal-sensing antenna-like structures composed of microtubule-based structures that distinguish them from motile cilia in structure and function. Primary ciliogenesis is regulated by various cellular signals, such as Wnt, hedgehog (Hh), and platelet-derived growth factor (PDGF). The abnormal regulation of ciliogenesis is closely related to developing various human diseases, including ciliopathies and cancer. This study identified a novel primary ciliogenesis factor Cullin 1 (CUL1), a core component of Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, which regulates the proteolysis of dishevelled 2 (Dvl2) through the ubiquitin-proteasome system. Through immunoprecipitation-tandem mass spectrometry analysis, 176 Dvl2 interacting candidates were identified, of which CUL1 is a novel Dvl2 modulator that induces Dvl2 ubiquitination-dependent degradation. Neddylation-dependent CUL1 activity at the centrosomes was essential for centrosomal Dvl2 degradation and primary ciliogenesis. Therefore, this study provides a new mechanism of Dvl2 degradation by CUL1, which ultimately leads to primary ciliogenesis, and suggest a novel target for primary cilia-related human diseases.

scholarly journals FBXO25-associated Nuclear Domains: A Novel Subnuclear Structure

2008 ◽  
Vol 19 (5) ◽  
pp. 1848-1861 ◽  
Author(s):  
Adriana O. Manfiolli ◽  
Ana Leticia G.C. Maragno ◽  
Munira M.A. Baqui ◽  
Sami Yokoo ◽  
Felipe R. Teixeira ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Striated Muscle ◽  
Ubiquitin Proteasome System ◽  
Immunoblot Analysis ◽  
High Concentration ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Ligase Complex ◽  
Mouse Tissues ◽  
Ubiquitin Proteasome ◽  
Nuclear Domains

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

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