Combination of Artesunate and WNT974 Induces KRAS Protein Degradation by Upregulating E3 Ligase ANACP2 and β-TrCP in the Ubiquitin–Proteasome Pathway

Abstract BackgroundKRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin.MethodsThe synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or β-TrCP. Western blot assay was utilized for the protein levels of KRAS, ANAPC2, β-TrCP, or GSK-3β. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, β-TrCP, or GSK-3β in tumor tissues. Results Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3β and E3 ligase β-TrCP that is known to degrade GSK-3β-phosphorylated KRAS protein. Knockdown of β-TrCP- and inhibition of GSK-3β abolish the combination treatment-induce KRAS ubiquitination and reduction in expression.ConclusionsOur data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin–proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment.

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Combination of Artesunate and WNT974 Induces KRAS Protein Degradation by Upregulating E3 Ligase ANACP2 and β-TrCP in the Ubiquitin–proteasome Pathway

10.21203/rs.3.rs-744967/v1 ◽

2021 ◽

Author(s):

Rui-Hong Gong ◽

Minting Chen ◽

Chunhua Huang ◽

Hoi Leong Xavier Wong ◽

Hiu Yee Kwan ◽

...

Keyword(s):

Mouse Model ◽

Synergistic Effect ◽

Protein Degradation ◽

Combination Treatment ◽

Ubiquitin Proteasome Pathway ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Kras Protein ◽

Gsk 3Β

Abstract BackgroundKRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin.MethodsThe synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or β-TrCP. Western blot assay was utilized for the protein levels of KRAS, ANAPC2, β-TrCP, or GSK-3β. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, β-TrCP, or GSK-3β in tumor tissues.Results Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3β and E3 ligase β-TrCP that is known to degrade GSK-3β-phosphorylated KRAS protein. Knockdown of β-TrCP- and inhibition of GSK-3β abolish the combination treatment-induce KRAS ubiquitination and reduction in expression.ConclusionsOur data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin–proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment.

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Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.37 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Kellie R Machlus ◽

Prakrith Vijey ◽

Thomas Soussou ◽

Joseph E Italiano

Keyword(s):

Protein Degradation ◽

Proteasome Inhibitors ◽

Aurora Kinase ◽

Proteasome Activity ◽

Major Pathway ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

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Mechanisms of Cancer Cachexia

Physiological Reviews ◽

10.1152/physrev.00016.2008 ◽

2009 ◽

Vol 89 (2) ◽

pp. 381-410 ◽

Cited By ~ 672

Author(s):

Michael J. Tisdale

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Adipose Tissue ◽

Protein Degradation ◽

Initiation Factor ◽

Tissue Loss ◽

Α Subunit ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the α-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NFκB. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-α, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment.

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The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.91247.2008 ◽

2009 ◽

Vol 107 (2) ◽

pp. 438-444 ◽

Cited By ~ 41

Author(s):

Heather M. Argadine ◽

Nathan J. Hellyer ◽

Carlos B. Mantilla ◽

Wen-Zhi Zhan ◽

Gary C. Sieck

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Caspase 3 ◽

Diaphragm Muscle ◽

Rat Diaphragm ◽

Ubiquitin Proteasome Pathway ◽

Protein Ubiquitination ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Net Protein

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.

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Oestrogen causes ATBF1 protein degradation through the oestrogen-responsive E3 ubiquitin ligase EFP

Biochemical Journal ◽

10.1042/bj20111890 ◽

2012 ◽

Vol 444 (3) ◽

pp. 581-590 ◽

Cited By ~ 12

Author(s):

Xue-Yuan Dong ◽

Xiaoying Fu ◽

Songqing Fan ◽

Peng Guo ◽

Dan Su ◽

...

Keyword(s):

Cell Proliferation ◽

Protein Degradation ◽

Ubiquitin Ligase ◽

Feedback Loop ◽

E3 Ubiquitin Ligase ◽

Breast Development ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Dependent Cell ◽

Proteasome Pathway

We reported previously that the tumour suppressor ATBF1 (AT motif-binding factor 1) formed an autoregulatory feedback loop with oestrogen–ERα (oestrogen receptor α) signalling to regulate oestrogen-dependent cell proliferation in breast cancer cells. In this loop ATBF1 inhibits the function of oestrogen–ERα signalling, whereas ATBF1 protein levels are fine-tuned by oestrogen-induced transcriptional up-regulation as well as UPP (ubiquitin–proteasome pathway)-mediated protein degradation. In the present study we show that EFP (oestrogen-responsive finger protein) is an E3 ubiquitin ligase mediating oestrogen-induced ATBF1 protein degradation. Knockdown of EFP increases ATBF1 protein levels, whereas overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in oestrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumours the levels of ATBF1 protein are positively correlated with the levels of EFP protein, as both are directly up-regulated ERα target gene products. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for oestrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and oestrogen–ERα signalling and thus implicate ATBF1 in oestrogen-dependent breast development and carcinogenesis.

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Serogroup-Related Escape of Yersinia enterocolitica YopE from Degradation by the Ubiquitin-Proteasome Pathway

Infection and Immunity ◽

10.1128/iai.00528-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4423-4431 ◽

Cited By ~ 16

Author(s):

Moritz Hentschke ◽

Konrad Trülzsch ◽

Jürgen Heesemann ◽

Martin Aepfelbacher ◽

Klaus Ruckdeschel

Keyword(s):

Host Cell ◽

Yersinia Enterocolitica ◽

Virulence Proteins ◽

Ubiquitin Proteasome Pathway ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Protein Secretion System ◽

Proteasome Pathway ◽

Type Iii Protein Secretion ◽

The Stability

ABSTRACT Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis.

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9 Ubiquitin-Proteasome Pathway of Intracellular Protein Degradation

Exercise and Sport Sciences Reviews ◽

10.1249/00003677-199800260-00011 ◽

1998 ◽

Vol 26 ◽

pp. 219???252 ◽

Cited By ~ 17

Author(s):

GEORGE N. DEMARTINO ◽

GEORGE A. ORDWAY

Keyword(s):

Protein Degradation ◽

Intracellular Protein ◽

Ubiquitin Proteasome Pathway ◽

Intracellular Protein Degradation ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

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Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor

Biochemical Journal ◽

10.1042/bj20070688 ◽

2008 ◽

Vol 409 (3) ◽

pp. 751-759 ◽

Cited By ~ 7

Author(s):

Steven T. Russell ◽

Helen L. Eley ◽

Stacey M. Wyke ◽

Michael J. Tisdale

Keyword(s):

Protein Degradation ◽

Kinase Inhibitor ◽

Muscle Protein ◽

Specific Inhibitor ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Phosphoinositide 3 Kinase

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser473 within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin–proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin–proteasome pathway through activation of NF-κB (nuclear factor κB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-κB and that this also allows for a specific synthesis of proteasome subunits.

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