scholarly journals Ubiquitin-Independent Proteasomal Degradation Mediated by Antizyme

2020 ◽  
Author(s):  
Noriyuki Murai
Keyword(s):  
Protein Degradation ◽  
Ornithine Decarboxylase ◽  
Feedback Regulation ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Proteolytic Degradation ◽  
Dependent Manner ◽  
Independent Protein

Most of the proteins in eukaryotic cells are degraded by the proteasome in an ubiquitin-dependent manner. However, ubiquitin-independent protein degradation pathway by the 26S proteasome exists in the cells. Ornithine decarboxylase (ODC) is a well-known protein that is degraded by the 26S proteasome without ubiquitination. Degradation of ODC requires the protein, “antizyme (AZ),” that is induced by polyamine and binds to the ODC monomer to inhibit ODC activity and target it to the 26S proteasome for proteolytic degradation. Namely, AZ contributes the feedback regulation of intracellular polyamine level. ODC has been considered to be the only protein that AZ binds and accelerates its degradation. However, recently AZ-mediated proteasomal protein degradation will gradually increase. Most recently, we found that one of the antizyme families, AZ2, accelerates c-Myc degradation by the proteasome without ubiquitination. In this chapter, we introduce latest several ubiquitin-independent proteasomal degradation mediated by antizyme.

scholarly journals Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

2020 ◽  
Author(s):  
Ganapathi Kandasamy ◽  
Ashis Kumar Pradhan ◽  
R Palanimurugan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Rna Degradation ◽  
26S Proteasome ◽  
Cellular Proteins ◽  
Cellular Homeostasis ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

scholarly journals Ubiquitin-Proteasomal Degradation of COX-2 in TGF-β Stimulated Human Endometrial Cells Is Mediated Through Endoplasmic Reticulum Mannosidase I

Endocrinology ◽  
2012 ◽  
Vol 153 (1) ◽  
pp. 426-437 ◽  
Author(s):  
Mohan Singh ◽  
Parvesh Chaudhry ◽  
Sophie Parent ◽  
Eric Asselin
Keyword(s):  
Endoplasmic Reticulum ◽  
Stromal Cells ◽  
Small Interfering Rna ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Dependent Manner ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Cox 2 ◽  
Interfering Rna

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

scholarly journals COP9 signalosome is an essential and druggable parasite target that regulates protein degradation

2020 ◽  
Author(s):  
Swagata Ghosh ◽  
Laura Farr ◽  
Aditya Singh ◽  
Laura-Ann Leaton ◽  
Jay Padalia ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Therapeutic Potential ◽  
Degradation Pathway ◽  
Dominant Negative ◽  
Cop9 Signalosome ◽  
Dependent Manner ◽  
Parasite Protein ◽  
Upstream Regulator ◽  
Dominant Negative Mutation

ABSTRACTUnderstanding how the protozoan protein degradation pathway is regulated could uncover new parasite biology for drug discovery. We found the COP9 signalosome (CSN) conserved in multiple pathogens such as Leishmania, Trypanosoma, Toxoplasma, and used the severe diarrhea-causing Entamoeba histolytica to study its function in medically significant protozoa. We show that CSN is an essential upstream regulator of parasite protein degradation. Genetic disruption of E. histolytica CSN by two distinct approaches inhibited cell proliferation and viability. Both CSN5 knockdown and dominant negative mutation trapped cullin in a neddylated state, disrupting UPS activity and protein degradation. In addition, zinc ditiocarb (ZnDTC), a main metabolite of the inexpensive FDA-approved alcohol-abuse drug disulfiram, was active against parasites acting in a COP9-dependent manner. ZnDTC, given as disulfiram-zinc, had oral efficacy in clearing parasites in vivo. Our findings provide insights into the regulation of parasite protein degradation, and supports the significant therapeutic potential of COP9 inhibition.Summary sentenceParasite-encoded COP9 signalosome is an essential upstream regulator of ubiquitin-proteasome mediated protein degradation, and shows significant potential as a therapeutic target.

scholarly journals Mode of targeting to the proteasome determines GFP fate

2020 ◽  
Vol 295 (47) ◽  
pp. 15892-15901
Author(s):  
Christopher Eric Bragança ◽  
Daniel Adam Kraut
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Canonical Pathway ◽  
Eukaryotic Cells ◽  
Reporter Protein ◽  
Ubiquitin Proteasome ◽  
Targeting Signal ◽  
Multiple Variants ◽  
Domain Stability

The ubiquitin–proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.

scholarly journals Proteasome pathway operates for the degradation of ornithine decarboxylase in intact cells

1996 ◽  
Vol 317 (1) ◽  
pp. 77-80 ◽  
Author(s):  
Yasuko MURAKAMI ◽  
Nobuyuki TANAHASHI ◽  
Keiji TANAKA ◽  
Satoshi ŌMURA ◽  
Shin-ichi HAYASHI
Keyword(s):  
Ornithine Decarboxylase ◽  
Proteasome Inhibitor ◽  
Mammalian Cells ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Intact Cells ◽  
Htc Cells ◽  
Proteasome Pathway ◽  
Hypertonic Shock

Ornithine decarboxylase (ODC) is degraded in an ATP-dependent manner in vitro by the 26 S proteasome in the presence of antizyme, an ODC destabilizing protein induced by polyamines. In the present study we examined whether the proteasome catalyses ODC degradation in living mammalian cells. Lactacystin, the most selective proteasome inhibitor, strongly inhibited the degradation of ODC that had been induced in hepatoma tissue-culture (HTC) cells by refeeding with fresh medium. Furthermore the inhibitor inhibited the rapid degradation of ODC that had been induced by hypotonic shock. Interestingly, hypertonic shock was found to increase the proportion of ODC present as a complex with antizyme (the ratio of ODC–antizyme complex to total ODC). Cycloheximide, which partly inhibits rapid ODC degradation caused by hypertonic shock, also partly inhibited the increase in the ratio of ODC–antizyme complex to total ODC. These results suggest that a common ODC degradation pathway, namely the antizyme-dependent and 26 S proteasome-catalysed ODC degradation pathway, is also operating in intact cells for osmoregulated ODC degradation.

scholarly journals Mebendazole Exerts Potent Anti-Leukemic Effects By Downregulating Protein Levels of Hedgehog Transcription Factors GLI1 and GLI2

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5145-5145
Author(s):  
Fabian Freisleben ◽  
Hauke Stamm ◽  
Jana Muschhammer ◽  
Alexander Krispien ◽  
Vanessa Thaden ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Protein Levels

Abstract The relevance of the Hedgehog signaling pathway in the pathophysiology of acute myeloid leukemia (AML) has been demonstrated by us and others. Inhibition of the downstream Hedgehog transcription factors GLI1 and GLI2 results in strong anti-leukemic effects. Therefore, Hedgehog pathway inhibitors represent a promising therapeutic approach in AML. Mebendazole is an anthelmintic drug commonly used for the treatment of various parasitic worm infections. Recently, mebendazole has been shown to exhibit strong anti-tumor effects in different cancer entities including AML. In the work presented here, we investigated the effect of mebendazole on expression and activity of GLI transcription factors and its anti-leukemic activity. To determine the effect of mebendazole on GLI transcription factors, we treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with different concentrations of mebendazole and analyzed its impact on GLI1 and GLI2 protein- and mRNA levels. Furthermore, GLI reporter assays (Cignal GLI Reporter (luc) Kit, Qiagen) were performed to determine the effect of mebendazole on the GLI1 and -2 transcriptional activity. Mebendazole strongly inhibited GLI1 and GLI2 signaling activity in a dose-dependent manner. Exemplarily, treatment with 500 nM mebendazole reduced the GLI1 and -2 transcriptional activity in all cell lines tested by 54.8 % (± 9.6) after 24h and 73.2 % (± 11.6) after 48h. We could demonstrate by Western Blotting that GLI1 and -2 protein levels were clearly reduced 24h and 48h after mebendazole exposure, whereas GLI1 and -2 mRNA levels did not decrease. These data suggest that mebendazole may increase degradation of GLI proteins via the proteasome pathway. Therefore, we evaluated the influence of the 26s proteasome inhibitor bortezomib on GLI levels after mebendazole treatment. Inhibiting the 26s proteasome with 2 nM, 5 nM and 10 nM of bortezomib increased GLI signaling activity by 13.6 % (± 8.0), 84.6 % (± 39.2) and 137.1 % (± 37.9), respectively. Furthermore, 10 nM bortezomib abolished the effect of mebendazole on GLI protein levels. Taken together, mebendazole increased the proteasomal degradation of GLI1 and GLI2. These observations were extended to samples from AML patients. After mebendazole treatment for 24h or 48h all analyzed patients had reductions of GLI1 protein levels as confirmed by Western blotting (n=4), whereas GLI1 and GLI2 mRNA levels were not changed (n=7), indicating that proteasomal degradation was operational in primary blasts as well. Evaluating the anti-leukemic effects of mebendazole, we also investigated its combination with the small molecule GLI inhibitor GANT61. We treated the AML cell lines MV4-11, MOLM-13, THP-1 and OCI-AML3 with combinations of mebendazole and GANT61 and analyzed cell proliferation, apoptosis and colony formation. Mebendazole treatment alone already resulted in decreased proliferation and colony forming capacity as well as increased apoptosis rates in a dose-dependent manner. The combination of mebendazole with the GLI inhibitor GANT61 synergistically increased the anti-proliferative effects of mebendazole on all 4 AML cell lines tested. Additionally, GANT61 further increased the effect of mebendazole on colony formation significantly. Incubation with 100 nM, 200 nM and 500 nM mebendazole inhibited the proliferation of primary blasts from AML patients by 15.1 % (± 7.5), 31.6 % (± 16.8) and 66.0 % (± 17.4), respectively (n=8). Moreover, the combination with GANT61 significantly increased these anti-proliferative effects. This work indicates that mebendazole exerts profound anti-leukemic effects by decreasing GLI1 and GLI2 intracellular levels by promoting its proteasomal degradation. Combining mebendazole with GLI1 and GLI2 inhibitors such as GANT61 enhances this effect considerably. These observations may lead to the introduction of novel treatment strategies in AML. Disclosures Stamm: Amgen Research (Munich) GmbH / Amgen Inc.: Patents & Royalties; Astellas GmbH: Other: Travel Grant. Wellbrock:Amgen Research (Munich) GmbH: Patents & Royalties. Fiedler:GSO: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Teva: Other: support for meeting attendance.

Abstract P117: Does Mammalian Target of Rapamycin Complex-2 Regulate Protein Degradation Pathways in the Heart?

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Pankaj S Shende ◽  
Christian Morandi ◽  
Marijke Brink
Keyword(s):  
Gene Expression ◽  
Protein Degradation ◽  
Mammalian Target Of Rapamycin ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Target Of Rapamycin ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Hypertrophic Response ◽  
Akt Phosphorylation

Background: Mammalian target of rapamycin (mTOR) occurs in the cell in two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2, which contain raptor and rictor, respectively. We have recently demonstrated that mTORC1 activity is required for the hypertrophic response to aortic constriction and for the normal cardiac homeostasis. Moreover, we showed that raptor deletion causes Akt hyperphosphorylation and lower gene expression of Atrogin-1 and MuRF1, two muscle specific E3 enzymes part of the proteasomal degradation pathway. These results suggested that, as a counter-regulatory response to mTORC1 inactivation, mTORC2 reduces protein degradation via phosphorylation of Akt at Ser473. It has previously been shown that the phosphorylation state of Akt regulates Atrogin-1 and MuRF1 gene expression at the transcriptional level via FoxO. In the present study, we have tested whether mTORC2 inactivation induces the ubiquitin-proteasomal degradation pathway. Methods and results: In 10 week-old male mice, transgenic for MerCreMer driven by the α-MHC promoter and homozygous for floxed rictor, deletion of the rictor gene was induced by tamoxifen. Protein and RNA extracts were analyzed at three weeks after tamoxifen by Western blotting and qPCR, respectively. The rictor gene was efficiently ablated from the heart as its protein levels were reduced. Phosphorylation of Akt and PKC-α, direct targets of mTORC2, was abolished, identifying these signaling molecules as downstream targets of mTORC2 in the heart. However, the reduced Akt phosphorylation was not associated with any changes in the mRNA levels of Atrogin-1, MuRF1, and MuRF3. Conclusion: Our study suggests that mTORC2-induced phosphorylation of Akt is not required for the maintenance of low expression levels of these genes. Further studies are ongoing to identify the factors that modulate Atrogin-1 and MuRF1 gene transcription in the heart.

scholarly journals 2-Methoxy-7-Acetonyljuglone Isolated from Reynoutria japonica Increases the Activity of Nuclear Factor Erythroid 2-Related Factor-2 through Inhibition of Ubiquitin Degradation in HeLa Cells

Antioxidants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 398 ◽  
Author(s):  
Jung-Hwan Kim ◽  
Atif Ali Khan Khalil ◽  
Hye-Jin Kim ◽  
Sung-Eun Kim ◽  
Mi-Jeong Ahn
Keyword(s):  
Hela Cells ◽  
Target Genes ◽  
Proteasomal Degradation ◽  
Degradation Pathway ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Signaling Mechanism ◽  
Reynoutria Japonica

The nuclear factor erythroid-derived 2-related factor 2 (NRF2) is a key transcription factor for the activation of genes responsible for oxidative stress and drug detoxification. Thus, it is important to identify NRF2 activators, which can be used to protect the cells from oxidative damage. Here, we investigated the effect of juglone derivatives isolated from Reynoutria japonica on the activity of NRF2 in HeLa cells. We demonstrated that among the juglone derivatives, 2-methoxy-7-acetonyljuglone (MA) strongly stimulated the antioxidant response element (ARE)-luciferase activity in a dose-dependent manner. In addition, MA significantly increased the nuclear localization of NRF2 and, consequently, increased the expression of NRF2 target genes, including heme oxygenase-1(HO-1), NAD(P)H: quinine oxidoreductase-1 (NQO-1), and glutamate-cysteine ligase catalytic (GCLC). To gain insights into the NRF2 signaling mechanism by MA, we measured the activities of RAC-alpha serine/threonine-protein kinase (AKT) and mitogen-activated protein (MAP) kinase family proteins, including extracellular signal-regulated kinase (ERK) and p38. Our results showed that MA induced NRF2 activity through p38 and AKT signaling. Subsequently, we found that MA significantly enhanced NRF2 stability by inhibiting ubiquitin-dependent proteasomal degradation. Thus, MA might protect cells by enhancing the activity and stability of NRF2 through inhibition of the proteasomal degradation pathway.

scholarly journals Multi-Step Ubiquitin Decoding Mechanism for Proteasomal Degradation

2020 ◽  
Vol 13 (6) ◽  
pp. 128
Author(s):  
Hikaru Tsuchiya ◽  
Akinori Endo ◽  
Yasushi Saeki
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Diversity ◽  
Proteasomal Degradation ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Present Evidence ◽  
Major Function ◽  
Chain Lengths ◽  
Liquid Liquid Phase Separation

The 26S proteasome is a 2.5-MDa protease complex responsible for the selective and ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Proteasome-mediated protein degradation accounts for ~70% of all cellular proteolysis under basal conditions, and thereby any dysfunction can lead to drastic changes in cell homeostasis. A major function of ubiquitylation is to target proteins for proteasomal degradation. Accompanied by deciphering the structural diversity of ubiquitin chains with eight linkages and chain lengths, the ubiquitin code for proteasomal degradation has been expanding beyond the best-characterized Lys48-linked ubiquitin chains. Whereas polyubiquitylated proteins can be directly recognized by the proteasome, in several cases, these proteins need to be extracted or segregated by the conserved ATPases associated with diverse cellular activities (AAA)-family ATPase p97/valosin-containing protein (VCP) complex and escorted to the proteasome by ubiquitin-like (UBL)–ubiquitin associated (UBA) proteins; these are called substrate-shuttling factors. Furthermore, proteasomes are highly mobile and are appropriately spatiotemporally regulated in response to different cellular environments and stresses. In this review, we highlight an emerging key link between p97, shuttling factors, and proteasome for efficient proteasomal degradation. We also present evidence that proteasome-containing nuclear foci form by liquid–liquid phase separation under acute hyperosmotic stress.

Diacylglycerol acyltransferase-2 and monoacylglycerol acyltransferase-2 are ubiquitinated proteins that are degraded by the 26S proteasome

2016 ◽  
Vol 473 (20) ◽  
pp. 3621-3637 ◽  
Author(s):  
Curtis Brandt ◽  
Pamela J. McFie ◽  
Scot J. Stone
Keyword(s):  
Degradation Pathway ◽  
Diacylglycerol Acyltransferase ◽  
26S Proteasome ◽  
Dependent Manner ◽  
Limited Information ◽  
Stored Energy ◽  
Ubiquitinated Proteins ◽  
Triacylglycerol Synthesis ◽  
Eukaryotic Organisms ◽  
Important Form

Acyl-CoA:1,2-diacylglycerol acyltransferase (DGAT)-2 is one of the two DGAT enzymes that catalyzes the synthesis of triacylglycerol, which is an important form of stored energy for eukaryotic organisms. There is currently limited information available regarding how DGAT2 and triacylglycerol synthesis are regulated. Recent studies have indicated that DGAT2 can be regulated by changes in gene expression. How DGAT2 is regulated post-transcriptionally remains less clear. In this study, we demonstrated that DGAT2 is a very unstable protein and is rapidly degraded in an ubiquitin-dependent manner via the proteasome. Many of the 25 lysines present in DGAT2 appeared to be involved in promoting its degradation. However, the six C-terminal lysines were the most important in regulating stability. We also demonstrated that acyl-CoA:monoacylglycerol acyltransferase (MGAT)-2, an enzyme with extensive sequence homology to DGAT2 that catalyzes the synthesis of diacylglycerol, was also ubiquitinated. However, MGAT2 was found to be much more stable than DGAT2. Interestingly, when co-expressed, MGAT2 appeared to stabilize DGAT2. Finally, we found that both DGAT2 and MGAT2 are substrates of the endoplasmic reticulum-associated degradation pathway.

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