scholarly journals The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

2020 ◽  
Vol 295 (33) ◽  
pp. 11776-11788
Author(s):  
Molly Hodul ◽  
Rakesh Ganji ◽  
Caroline L. Dahlberg ◽  
Malavika Raman ◽  
Peter Juo
Keyword(s):  
Mammalian Cells ◽  
Deubiquitinating Enzymes ◽  
Post Translational Modification ◽  
Wd40 Repeat ◽  
C Elegans ◽  
Protein Levels ◽  
Specific Protease ◽  
The Stability ◽  
And Function

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in Caenorhabditis elegans. We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identified another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome increased USP-46 abundance, and this effect was nonadditive with increased WDR-48 expression. We found that USP-46 is ubiquitinated and that expression of WDR-48 reduces the levels of ubiquitin–USP-46 conjugates and increases the t1/2 of USP-46. A point-mutated WDR-48 variant that disrupts binding to USP-46 was unable to promote USP-46 abundance in vivo. Finally, siRNA-mediated knockdown of wdr48 destabilizes USP46 in mammalian cells. Together, these results support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism that controls DUB availability and function.

scholarly journals The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

2019 ◽  
Author(s):  
Molly Hodul ◽  
Rakesh Ganji ◽  
Caroline L Dahlberg ◽  
Malavika Raman ◽  
Peter Juo
Keyword(s):  
Mammalian Cells ◽  
Deubiquitinating Enzyme ◽  
Deubiquitinating Enzymes ◽  
Post Translational Modification ◽  
Wd40 Repeat ◽  
C Elegans ◽  
Protein Levels ◽  
The Stability ◽  
And Function

ABSTRACTUbiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, mechanisms that regulate the deubiquitinating enzymes (DUBs) that are responsible for the removal of ubiquitin from target proteins are poorly understood. We previously showed that the DUB USP-46 removes ubiquitin from the glutamate receptor GLR-1 and regulates it trafficking and degradation in C. elegans. We found that WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46. Here, we identify another mechanism by which WDR-48 regulates USP-46. We found that increased expression of WDR-48, but not WDR-20, promotes USP-46 abundance in mammalian cells in culture and in C. elegans neurons in vivo. Inhibition of the proteasome promotes the abundance of USP-46, and this effect is non-additive with increased expression of WDR-48. We found that USP-46 is ubiquitinated, and expression of WDR-48 reduces the levels of ubiquitin-USP-46 conjugates and increases the half-life of USP-46. A point mutant version of WDR-48 that disrupts binding to USP-46 is unable to promote USP-46 abundance in vivo. Together, these data support a model in which WDR-48 binds and stabilizes USP-46 protein levels by preventing the ubiquitination and degradation of USP-46 in the proteasome. Given that a large number of USPs interact with WDR proteins, we propose that stabilization of DUBs by their interacting WDR proteins may be a conserved and widely used mechanism to control DUB availability and function.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

scholarly journals The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

2018 ◽  
Author(s):  
Lewie Zeng ◽  
Rachid El Bejjani ◽  
Marc Hammarlund
Keyword(s):  
Motor Neurons ◽  
Neuronal Development ◽  
Axon Regeneration ◽  
Neuronal Response ◽  
Small Gtpase ◽  
C Elegans ◽  
Protein Levels ◽  
Mature Neurons ◽  
And Function

AbstractMembers of the Amyloid Precursor Protein (APP) family have important functions during neuronal development. However, their physiological functions in the mature nervous system are not fully understood. Here we use the C. elegans GABAergic motor neurons to study the post-developmental function of the APP-like protein APL-1 in vivo. We find that apl-1 has minimum roles in the maintenance of gross neuron morphology and function. However, we show that apl-1 is an inhibitor of axon regeneration, acting on mature neurons to limit regrowth in response to injury. The small GTPase Rab6/RAB-6.2 also inhibits regeneration, and does so in part by maintaining protein levels of APL-1. To inhibit regeneration, APL-1 functions via the E2 domain of its ectodomain; the cytoplasmic tail, transmembrane anchoring, and the E1 domain are not required for this function. Our data defines a novel role for APL-1 in modulating the neuronal response to injury.

scholarly journals USP28 facilitates pancreatic cancer progression through activation of Wnt/β-catenin pathway via stabilising FOXM1

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Leifeng Chen ◽  
Zheng Xu ◽  
Qing Li ◽  
Qian Feng ◽  
Cihua Zheng ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cancer Progression ◽  
Cell Cycle Progression ◽  
Deubiquitinating Enzymes ◽  
Post Translational Modification ◽  
Foxm1 Expression ◽  
Specific Protease

AbstractUbiquitination is an important post-translational modification that can be reversed by a family of enzymes called deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 28 (USP28), a member of the DUBs family, functions as a potential tumour promoter in various cancers. However, the biological function and clinical significance of USP28 in pancreatic cancer (PC) are still unclear. Here, we showed that PC tumours had higher USP28 expression compared with that of normal pancreatic tissues, and high USP28 level was significantly correlated with malignant phenotype and shorter survival in patients with PC. Overexpression of USP28 accelerated PC cell growth, whereas USP28 knockdown impaired PC cell growth both in vitro and in vivo. Further, we found that USP28 promoted PC cell growth by facilitating cell cycle progression and inhibiting apoptosis. Mechanistically, USP28 deubiquitinated and stabilised FOXM1, a critical mediator of Wnt/β-catenin signalling. USP28-mediated stabilisation of FOXM1 significantly promoted nucleus β-catenin trans-activation, which in turn led to the activation of the Wnt/β-catenin pathway. Finally, restoration of FOXM1 expression abolished the anti-tumour effects of USP28-silencing. Thus, USP28 contributes to PC pathogenesis through enhancing the FOXM1-mediated Wnt/β-catenin signalling, and could be a potential diagnostic and therapeutic target for PC cases.

USP48 Sustains Chemoresistance and Metastasis in Ovarian Cancer

2020 ◽  
Vol 20 (9) ◽  
pp. 689-699
Author(s):  
Xuemeng Lei ◽  
Xukun Li ◽  
Hongyan Chen ◽  
Zhihua Liu
Keyword(s):  
Ovarian Cancer ◽  
Cancer Patients ◽  
Migration And Invasion ◽  
Clinical Samples ◽  
Deubiquitinating Enzymes ◽  
Potential Therapeutic Target ◽  
Kaplan Meier ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

Background: Ubiquitin specific protease 48 (USP48) is a member of the deubiquitinating enzymes (DUBs) family. However, the function of USP48 in ovarian cancer remains unclear. Objective: The present study reveals that USP48 knockdown could significantly inhibit cell migration and invasion in ES2, 3AO and A2780 cells, without affecting cell proliferation. Methods: After carboplatin (CBP) treatment, the USP48 ablation increases the apoptosis rate, and the cleaved PARP and cleaved caspase 3 expression levels in ES2, 3AO and A2780 cells. The subcutaneous tumor and intraperitoneally injected experiments demonstrated that the USP48 knockdown significantly increases responsiveness to CBP, and alleviates the metastasis in vivo. Meanwhile, USP48 deficiency results in the improved survival of mice. Results: Finally, the analysis of clinical samples and the TCGA and Kaplan-Meier Plot database revealed that the high expression of USP48 in ovarian cancer patients is associated with poor survival and resistance to CBP therapy. Conclusion: In summary, USP48 may be a potential therapeutic target for ovarian cancer patients.

scholarly journals C. elegans germ granules require both assembly and localized regulators for mRNA repression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Scott Takeo Aoki ◽  
Tina R. Lynch ◽  
Sarah L. Crittenden ◽  
Craig A. Bingman ◽  
Marvin Wickens ◽  
...  
Keyword(s):  
Liquid Droplet ◽  
Scaffolding Protein ◽  
P Granules ◽  
C Elegans ◽  
Cytoplasmic Rna ◽  
And Function ◽  
Rnp Granules ◽  
Key Residues ◽  
Reporter Mrna

AbstractCytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

2001 ◽  
Vol 360 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Trevor R. PETTITT ◽  
Mark McDERMOTT ◽  
Khalid M. SAQIB ◽  
Neil SHIMWELL ◽  
Michael J. O. WAKELAM
Keyword(s):  
Liquid Chromatography ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Inducible Promoter ◽  
In Vitro Assays ◽  
Protein Levels ◽  
Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

scholarly journals NLRP7 deubiquitination by USP10 promotes tumor progression and tumor-associated macrophage polarization in colorectal cancer

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Bing Li ◽  
Zhi-Peng Qi ◽  
Dong-Li He ◽  
Zhang-Han Chen ◽  
Jing-Yi Liu ◽  
...  
Keyword(s):  
Tumor Progression ◽  
Acceptor Site ◽  
Protein Levels ◽  
Qrt Pcr ◽  
Chemokine Ligand ◽  
Specific Protease ◽  
Ubiquitin Specific Protease ◽  
Chemokine Ligand 2

Abstract Background NOD-like receptors affect multiple stages of cancer progression in many malignancies. NACHT, LRR, and PYD domain-containing protein 7 (NLRP7) is a member of the NOD-like receptor family, although its role in tumorigenesis remains unclear. By analyzing clinical samples, we found that NLRP7 protein levels were upregulated in colorectal cancer (CRC). We proposed the hypothesis that a high level of NLRP7 in CRC may promote tumor progression. Here, we further investigated the role of NLRP7 in CRC and the underlying mechanism. Methods NLRP7 expression in human CRC and adjacent non-tumorous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry. The effect of NLRP7 in CRC progression was investigated in vitro and in vivo. Proteins interacting with NLRP7 were identified by immunoprecipitation and mass spectrometry analysis while immunofluorescence staining revealed the cellular location of the proteins. Cellular ubiquitination and protein stability assays were applied to demonstrate the ubiquitination effect on NLRP7. Cloning and mutagenesis were used to identify a lysine acceptor site that mediates NLRP7 ubiquitination. Cytokines/chemokines affected by NLRP7 were identified by RNA sequencing, qRT-PCR, and enzyme-linked immunosorbent assay. Macrophage phenotypes were determined using qRT-PCR, flow cytometry, and immunohistochemistry. Results NLRP7 protein levels, but not mRNA levels, were upregulated in CRC, and increased NLRP7 protein expression was associated with poor survival. NLRP7 promoted tumor cell proliferation and metastasis in vivo and in vitro and interacted with ubiquitin-specific protease 10, which catalyzed its deubiquitination in CRC cells. NLRP7 stability and protein levels in CRC cells were modulated by ubiquitination and deubiquitination, and NLRP7 was involved in the ubiquitin-specific protease 10 promotion of tumor progression and metastasis in CRC. K379 was an important lysine acceptor site that mediates NLRP7 ubiquitination in CRC cells. In CRC, NLRP7 promoted the polarization of pro-tumor M2-like macrophages by inducing the secretion of C-C motif chemokine ligand 2. Furthermore, NLRP7 promoted NF-κB nuclear translocation and activation of C-C motif chemokine ligand 2 transcription. Conclusions We showed that NLRP7 promotes CRC progression and revealed an as-yet-unidentified mechanism by which NLRP7 induces the polarization of pro-tumor M2-like macrophages. These results suggest that NLRP7 could serve as a biomarker and novel therapeutic target for the treatment of CRC.

scholarly journals Prep1 Deficiency Induces Protection from Diabetes and Increased Insulin Sensitivity through a p160-Mediated Mechanism

2008 ◽  
Vol 28 (18) ◽  
pp. 5634-5645 ◽  
Author(s):  
Francesco Oriente ◽  
Luis Cesar Fernandez Diaz ◽  
Claudia Miele ◽  
Salvatore Iovino ◽  
Silvia Mori ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Glucose Uptake ◽  
Normal Glucose Tolerance ◽  
Glucose Disposal ◽  
Protein Levels ◽  
Normal Glucose ◽  
Enhanced Expression ◽  
The Stability

ABSTRACT We have examined glucose homeostasis in mice hypomorphic for the homeotic transcription factor gene Prep1. Prep1-hypomorphic (Prep1 i / i ) mice exhibit an absolute reduction in circulating insulin levels but normal glucose tolerance. In addition, these mice exhibit protection from streptozotocin-induced diabetes and enhanced insulin sensitivity with improved glucose uptake and insulin-dependent glucose disposal by skeletal muscle. This muscle phenotype does not depend on reduced expression of the known Prep1 transcription partner, Pbx1. Instead, in Prep1 i / i muscle, we find normal Pbx1 but reduced levels of the recently identified novel Prep1 interactor p160. Consistent with this reduction, we find a muscle-selective increase in mRNA and protein levels of PGC-1α, accompanied by enhanced expression of the GLUT4 transporter, responsible for insulin-stimulated glucose uptake in muscle. Indeed, using L6 skeletal muscle cells, we induced the opposite effects by overexpressing Prep1 or p160, but not Pbx1. In vivo skeletal muscle delivery of p160 cDNA in Prep1 i / i mice also reverses the molecular phenotype. Finally, we show that Prep1 controls the stability of the p160 protein. We conclude that Prep1 controls insulin sensitivity through the p160-GLUT4 pathway.

FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

2021 ◽  
Author(s):  
Bin Qiu ◽  
Zhaohui Zhong ◽  
Shawn Righter ◽  
Yuxue Xu ◽  
Jun Wang ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Translational Regulation ◽  
Disease Onset ◽  
Fold Increase ◽  
Knock Out ◽  
Fk506 Binding Protein ◽  
Protein Levels ◽  
And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

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