scholarly journals Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling

2011 ◽  
Vol 194 (5) ◽  
pp. 737-750 ◽  
Author(s):  
Jason D. Berndt ◽  
Atsushi Aoyagi ◽  
Peitzu Yang ◽  
Jamie N. Anastas ◽  
Lan Tang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Ubiquitin Ligase ◽  
Genetic Interaction ◽  
E3 Ubiquitin Ligase ◽  
Secreted Protein ◽  
Loss Of Function ◽  
Protein Ligands ◽  
Steady State Levels ◽  
Novel Targets

Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steady-state levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/β-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A–mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling.

Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Shirin Doroudgar ◽  
Mirko Völkers ◽  
Donna J Thuerauf ◽  
Ashley Bumbar ◽  
Mohsin Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Misfolding ◽  
Cardiac Myocyte ◽  
Protein Quality ◽  
Heart Function ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Calcium Handling ◽  
Loss Of Function

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P<0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P<0.01), prolonged spark duration (P<0.001), and increased spark width (P<0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P<0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.

scholarly journals Extracellular spreading of Wingless is required for Drosophila oogenesis

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009469
Author(s):  
Xiaoxi Wang ◽  
Kimberly S. LaFever ◽  
Indrayani Waghmare ◽  
Andrea Page-McCaw
Keyword(s):  
Stem Cells ◽  
Wnt Signaling ◽  
Long Range ◽  
Wing Disc ◽  
Secreted Protein ◽  
Loss Of Function ◽  
Drosophila Oogenesis ◽  
Central Source ◽  
Follicle Stem Cells

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 μm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.

scholarly journals AtPPRT1, an E3 Ubiquitin Ligase, Enhances the Thermotolerance in Arabidopsis

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1074
Author(s):  
Yu Liu ◽  
Shuya Xiao ◽  
Haoran Sun ◽  
Linsen Pei ◽  
Yingying Liu ◽  
...  
Keyword(s):  
Heat Stress ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Degradation Pathway ◽  
Vital Role ◽  
Loss Of Function ◽  
Positive Role ◽  
Heat Stress Response ◽  
Acquired Thermotolerance ◽  
Ubiquitin E3 Ligase

E3 ubiquitin ligase plays a vital role in the ubiquitin-mediated heat-related protein degradation pathway. Herein, we report that the expression of AtPPRT1, a C3HC4 zinc-finger ubiquitin E3 ligase gene, was induced by heat stress, and the β-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity after basal and acquired thermotolerance. To further explore the function of AtPPRT1 in heat stress response (HSR), we used the atpprt1 mutant and AtPPRT1-overexpressing lines (OE2 and OE10) to expose in heat shock. In this study, the atpprt1 mutant had a lower germination and survival rate than those of Col-0 when suffered from the heat stress, whereas OEs enhanced basal and acquired thermotolerance in Arabidopsis seedlings. When compared to Col-0 and OEs, loss-of-function in AtPPRT1 resulted in lower chlorophyll retention and higher content of reactive oxygen species (ROS) after heat treatment. Moreover, the transcript levels of AtPPRT1 and several heat-related genes (AtZAT12, AtHSP21 and AtHSFA7a) were upregulated to greater extents in OEs and lower extents in atpprt1 compared to Col-0 after heat treated. Hence, we suggest that AtPPRT1 may act as a positive role in regulating the high temperature by mediating the degradation of unknown target proteins.

scholarly journals Wnt regulation: exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex

2020 ◽  
Vol 31 (10) ◽  
pp. 992-1014 ◽  
Author(s):  
Kristina N. Schaefer ◽  
Mira I. Pronobis ◽  
Clara E. Williams ◽  
Shiping Zhang ◽  
Lauren Bauer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Development ◽  
Wnt Signaling ◽  
Ubiquitin Ligase ◽  
Human Cancer ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Adult Stem Cell ◽  
Cell Homeostasis ◽  
Test Models

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin.

IL-3 and Retinoic Acid-Induced Myeloid Differentiation of Hematopoietic Progenitors Is Negatively Regulated by E3 Ubiquitin Ligase Rnf41 (FLRF).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3160-3160
Author(s):  
Xin Jing ◽  
Luis Dabul ◽  
Ronald G. Nachtman ◽  
Roland Jurecic
Keyword(s):  
Retinoic Acid ◽  
Steady State ◽  
Ligand Binding ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Myeloid Differentiation ◽  
Hematopoietic Progenitors ◽  
Induced Differentiation ◽  
Gm Csf ◽  
Steady State Levels

Abstract The proliferation and differentiation of hematopoietic cells is regulated by interaction of diverse group of cytokines with corresponding receptors. Regulation of cytokine signaling occurs at multiple levels, including the regulation of the amount of receptors present before and after ligand binding. However, the mechanisms that govern pre-determined steady-state amount of cytokine receptors are poorly understood. Previously we have reported that new E3 ubiquitin ligase Rnf41 (FLRF/Nrdp1) regulates cytokine-induced differentiation of hematopoietic progenitors through negative regulation of steady-state receptor levels. Increased levels of Rnf41 protein significantly attenuate differentiation of multipotent progenitors in response to IL-3, Epo and GM-CSF, due to a significant and constitutive decrease in the steady-state amount of IL-3, Epo and GM-CSF receptors. Immunoprecipitation and Western analysis of proteins from several types of hematopoietic progenitors has confirmed that Rnf41 protein associates with IL-3, Epo and GM-CSF receptors, and has shown that Rnf41-mediated down-regulation of receptors is independent of the ligand binding. Thus, by regulating steady-state amounts of IL-3, Epo and GM-CSF receptors Rnf41 could be maintaining optimal levels of signaling necessary for proper lineage commitment and differentiation of HSC and progenitors. Retinoic acid (RA) is known to augment IL-3 and GM-CSF induced differentiation of progenitors into myeloid lineages. Surprisingly, instead of improving the RA treatment further suppressed IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors over-expressing Rnf41. The RA functions through RARα and RXR receptors, whihc act as transcription factors and interact with specific DNA targets as hetero- (RAR-RXR) or homodimers (RXR-RXR). Interestingly, Western analysis has shown that hematopoietic progenitors over-expressing Rnf41 exhibit a reduction in the steady-state levels of RARα receptors, whereas levels of RXR receptors remain unchanged. Moreover, the treatment of hematopoietic progenitors over-expressing Rnf41 with RA leads to even further decrease of RARα receptor levels. The transient over-expression of Rnf41 in BaF3 cell line also decreases steady-state levels of RARα receptors, while RXR receptor levels remain unchanged. Protein IP with α-Rnf41, α-RARα or α-RXR antibodies has shown that endogenous Rnf41 protein associates with RARα receptors in hematopoietic progenitors. Taken together, these results suggest that Rnf41 influences RA-mediated myeloid differentiation of hematopoietic progenitors by negatively regulating the levels of RARα receptors. Several studies have reported that IL-3 and GM-CSF-induced myeloid differentiation of hematopoietic progenitors leads to activation of RARa through induction of the Jak2/Stat5 pathway. These studies have defined a previously unknown cytokine-RAR interaction during myelopoiesis and suggested that RAR activation might be a critical downstream event following IL-3 and GM-CSF signaling during myeloid differentiation. Combined together the existing data suggest a model in which Rnf41 negatively regulates myeloid differentiation of hematopoietic progenitors by independently regulating steady state levels of IL-3, GM-CSF and RAR receptors, and thus impacting cytokine and RA signaling. Ongoing studies with RA agonists and antagonists are aimed at unraveling further the role of Rnf41 in regulation of RA signaling during progenitor cell differentiation.

scholarly journals Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

2016 ◽  
Vol 27 (5) ◽  
pp. 744-756 ◽  
Author(s):  
Yuki Niwa ◽  
Takehiro Suzuki ◽  
Naoshi Dohmae ◽  
Siro Simizu
Keyword(s):  
Mass Spectrometry ◽  
Enzymatic Activity ◽  
Wnt Signaling ◽  
Protein Interactions ◽  
Secreted Protein ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Rare Type ◽  
Synthesis Activity ◽  
Canonical Wnt

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W153 and W156. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W153 and W156 by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W156 but not W153 based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W156 and found that DPY19L3-mediated C-mannosylation of Rspo1 at W156 is required for its secretion.

scholarly journals PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection

2021 ◽  
Author(s):  
Salik Miskat Borbora ◽  
R.S. Rajmani ◽  
Kithiganahalli N Balaji
Keyword(s):  
Ubiquitin Ligase ◽  
Lipid Accumulation ◽  
Ex Vivo ◽  
E3 Ubiquitin Ligase ◽  
Mycobacterial Infection ◽  
Drug Regimen ◽  
Loss Of Function ◽  
Methyl Transferase ◽  
Accumulation Of Lipids

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux genes. Here, we define an Mtb-orchestrated modulation of the ubiquitination mechanism of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In our study, perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in primary murine macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights on the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies.

scholarly journals Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination

2018 ◽  
Vol 115 (8) ◽  
pp. E1906-E1915 ◽  
Author(s):  
Jinggeng Zhou ◽  
Derui Liu ◽  
Ping Wang ◽  
Xiyu Ma ◽  
Wenwei Lin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Differentiation ◽  
Residence Time ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Endocytic Pathway ◽  
Protein Abundance ◽  
Unique Mechanism

Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase–substrate association regulated by phosphorylation.

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