scholarly journals IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

2020 ◽  
Author(s):  
Karen Dunbar ◽  
Thomas J. Macartney ◽  
Gopal P. Sapkota
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Ubiquitin Proteasome System ◽  
Specific Protein ◽  
Protective Role ◽  
Wnt Signalling ◽  
Target Protein ◽  
Genetic Ablation ◽  
Ubiquitin Proteasome ◽  
Canonical Wnt

ABSTRACTImmunomodulatory imide drugs (IMiDs) bind CRBN, a substrate receptor of the Cul4A E3 ligase complex, enabling neo-substrate recruitment and degradation via the ubiquitin-proteasome system. Here, we report FAM83F as such a neo-substrate. We recently showed that the eight FAM83 proteins (A-H) interact with members of the serine/threonine protein kinase CK1 family, to regulate their subcellular distribution and distinct biological roles. CK1α is a well-established IMiD neo-substrate and we demonstrate here that IMiD-induced FAM83F degradation requires its association with CK1α. Despite all FAM83 proteins interacting with CK1α, no other FAM83 protein is degraded by IMiDs. FAM83F is localised to the plasma membrane, and consistent with this, IMiD treatment results in depletion of both FAM83F and CK1α levels from the plasma membrane. We have recently identified FAM83F as a mediator of the canonical Wnt signalling pathway. The IMiD-induced degradation of FAM83F attenuated Wnt signalling in colorectal cancer cells and removed CK1α from the plasma membrane, mirroring the phenotypes observed with genetic ablation of FAM83F. Intriguingly, in many cancer cell lines, IMiD-induced degradation of CK1α is only modest and incomplete. In line with this observation, the expression of FAM83G, which also binds to CK1α, appears to attenuate the IMiD-induced degradation of CK1α, suggesting a protective role for FAM83G on CK1α. Our findings reveal that the efficiency of target protein degradation by IMiDs, and perhaps other degraders such as PROTACs, relies on the nature of the inherent multiprotein complex in which the target protein exists. Our findings unearth opportunities for developing degraders to target specific protein complexes.

scholarly journals IMiDs induce FAM83F degradation via an interaction with CK1α to attenuate Wnt signalling

2020 ◽  
Vol 4 (2) ◽  
pp. e202000804
Author(s):  
Karen Dunbar ◽  
Thomas J Macartney ◽  
Gopal P Sapkota
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Cancer Cells ◽  
Protective Role ◽  
Wnt Signalling ◽  
Target Protein ◽  
Multiprotein Complex ◽  
Genetic Ablation ◽  
Colorectal Cancer Cells ◽  
Canonical Wnt

Immunomodulatory imide drugs (IMiDs) bind CRBN, a substrate receptor of the Cul4A E3 ligase complex, enabling the recruitment of neo-substrates, such as CK1α, and their degradation via the ubiquitinproteasome system. Here, we report FAM83F as such a neo-substrate. The eight FAM83 proteins (A-H) interact with and regulate the subcellular distribution of CK1α. We demonstrate that IMiD-induced FAM83F degradation requires its association with CK1α. However, no other FAM83 protein is degraded by IMiDs. We have recently identified FAM83F as a mediator of the canonical Wnt signalling pathway. The IMiD-induced degradation of FAM83F attenuated Wnt signalling in colorectal cancer cells and removed CK1α from the plasma membrane, mirroring the phenotypes observed with genetic ablation of FAM83F. Intriguingly, the expression of FAM83G, which also binds to CK1α, appears to attenuate the IMiD-induced degradation of CK1α, suggesting a protective role for FAM83G on CK1α. Our findings reveal that the efficiency and extent of target protein degradation by IMiDs depends on the nature of inherent multiprotein complex in which the target protein is part of.

scholarly journals Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1

2020 ◽  
Author(s):  
Katarzyna Szymanska ◽  
Karsten Boldt ◽  
Clare V. Logan ◽  
Matthew Adams ◽  
Philip A. Robinson ◽  
...  
Keyword(s):  
Primary Cilium ◽  
Ubiquitin Proteasome System ◽  
Wnt Signalling ◽  
Abnormal Accumulation ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Proteasome ◽  
Developmental Conditions ◽  
Canonical Wnt ◽  
Conjugating Enzyme

AbstractA functional primary cilium is essential for normal and regulated signalling. Primary ciliary defects cause a group of developmental conditions known as ciliopathies, but the precise mechanisms of signal regulation by the cilium remain unclear. Previous studies have implicated the ubiquitin proteasome system (UPS) in regulation of Wnt signalling at the ciliary basal body. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for a ciliopathy mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitizes cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. To substantiate a direct link between MKS1 and the UPS, we identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalized, particularly during conditions of ciliary resorption, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. Furthermore, we demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1), confirming the fundamental role of UPS defects in the molecular pathogenesis of ciliopathies.

scholarly journals USP7 inhibits Wnt/β-catenin signaling through promoting stabilization of Axin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Lei Ji ◽  
Bo Lu ◽  
Raffaella Zamponi ◽  
Olga Charlat ◽  
Robert Aversa ◽  
...  
Keyword(s):  
Osteoblast Differentiation ◽  
Adipocyte Differentiation ◽  
Ubiquitin Proteasome System ◽  
Cellular Systems ◽  
Negative Regulator ◽  
Scaffolding Protein ◽  
Complex Stability ◽  
Genetic Ablation ◽  
Ubiquitin Proteasome ◽  
Potent Negative Regulator

Abstract Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.

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 Bifunctional Small Molecules That Mediate the Degradation of Extracellular Proteins

2020 ◽  
Author(s):  
David Caianiello ◽  
Mengwen Zhang ◽  
Jason Ray ◽  
Jake Swartzel ◽  
Emily Branham ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Extracellular Proteins ◽  
Target Protein ◽  
Disease Treatment ◽  
Bifunctional Molecules ◽  
Synthetic Molecules ◽  
Ubiquitin Proteasome ◽  
Mode A

<p>Targeted protein degradation (TPD) has emerged as a promising and exciting therapeutic strategy. The majority of existing TPD technologies rely on the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modularly designed, bifunctional synthetic molecules called <b>MoDE-A</b>s (<b>Mo</b>lecular <b>D</b>egraders of <b>E</b>xtracellular proteins through the <b>A</b>sialoglycoprotein receptor (ASGPR)), which are capable of mediating the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and the ASGPR, which is expressed primarily on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing bifunctional molecules that induce the degradation of both antibody and pro-inflammatory cytokine proteins. To our knowledge, these data represent the first experimental evidence that non-proteinogenic, synthetic molecules can be employed for the TPD of extracellular proteins both <i>in vitro</i> and <i>in vivo</i>. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.</p>

scholarly journals Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Daichao Xu ◽  
Bing Shan ◽  
Byung-Hoon Lee ◽  
Kezhou Zhu ◽  
Tao Zhang ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Signaling ◽  
Ubiquitin Proteasome System ◽  
Specific Protein ◽  
Negative Control ◽  
Common Mechanism ◽  
Wide Range ◽  
Specific Protease ◽  
Ubiquitin Proteasome

Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells.

scholarly journals Protein repertoire impact of Ubiquitin–Proteasome System impairment: Insight into the protective role of beta-estradiol

2012 ◽  
Vol 75 (4) ◽  
pp. 1440-1453 ◽  
Author(s):  
Annamaria D'Alessandro ◽  
Simona D'Aguanno ◽  
Maria Teresa Cencioni ◽  
Luisa Pieroni ◽  
Adamo Diamantini ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Protective Role ◽  
Ubiquitin Proteasome ◽  
Insight Into

scholarly journals Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

2008 ◽  
Vol 181 (7) ◽  
pp. 1095-1105 ◽  
Author(s):  
Daniel C. Scott ◽  
Randy Schekman
Keyword(s):  
Degradation Process ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Specific Protein ◽  
Cysteine Residue ◽  
Misfolded Proteins ◽  
Er Quality Control ◽  
Substrate Protein ◽  
Ubiquitin Proteasome ◽  
Protein Components

Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin–proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p.

scholarly journals Clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins and preimplantation development

Development ◽  
2021 ◽  
Vol 148 (14) ◽  
Author(s):  
Akihito Morita ◽  
Yuhkoh Satouh ◽  
Hidetaka Kosako ◽  
Hisae Kobayashi ◽  
Akira Iwase ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Ubiquitin Proteasome System ◽  
Maternal Plasma ◽  
Early Embryogenesis ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Selective Degradation ◽  
Kinase C ◽  
Ubiquitin Proteasome

ABSTRACT Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.

Much to know about proteolysis: intricate proteolytic machineries compromise essential cellular functions

2008 ◽  
Vol 36 (5) ◽  
pp. 781-785 ◽  
Author(s):  
Gemma Marfany ◽  
Rosa Farràs ◽  
Eduardo Salido ◽  
Dimitris P. Xirodimas ◽  
Manuel S. Rodríguez
Keyword(s):  
Protein Complexes ◽  
Ubiquitin Proteasome System ◽  
Cellular Functions ◽  
Cell Functions ◽  
Starting Point ◽  
Wide Range ◽  
Cellular Factors ◽  
Ubiquitin Proteasome ◽  
The University ◽  
Intracellular Proteolysis

Proteolysis has traditionally been considered as a radical way to terminate the function of a protein. However, protein destruction also is the starting point for many processes as they can only occur when the way has been cleared for the action of other proteins. Protein destruction can occur virtually in all compartments and organelles of the cell, associated with cell membranes or large protein complexes, it determines subcellular partitioning, association with positive or negative regulators which conditions the action of many critical cellular factors. The third intracellular proteolysis meeting held by the University La Laguna, Canary Islands, Spain, included speakers working with some of the most important proteolytic systems present in higher eukaryotes, such as the UPS (ubiquitin–proteasome system) and autophagy. Owing to the fact that these pathways directly or indirectly regulate many cell functions, this meeting brought together an audience with a wide range of research interests, including genetic, cell biological, biochemical and structural aspects of protein degradation. Some of these topics inspired interesting discussions and a significant number of these are developed in the issues reviewed herein.

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