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

2009 ◽  
Vol 107 (2) ◽  
pp. 438-444 ◽  
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.

Mechanisms of Cancer Cachexia

2009 ◽  
Vol 89 (2) ◽  
pp. 381-410 ◽  
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.

scholarly journals Inhibition of the Ubiquitin–Proteasome Pathway Does Not Protect against Ventilator-induced Accelerated Proteolysis or Atrophy in the Diaphragm

2014 ◽  
Vol 121 (1) ◽  
pp. 115-126 ◽  
Author(s):  
Ashley J. Smuder ◽  
W. Bradley Nelson ◽  
Matthew B. Hudson ◽  
Andreas N. Kavazis ◽  
Scott K. Powers
Keyword(s):  
Caspase 3 ◽  
Ring Finger ◽  
Diaphragm Muscle ◽  
Contractile Dysfunction ◽  
E3 Ligases ◽  
Ubiquitin Proteasome Pathway ◽  
Life Saving ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Fiber Atrophy

Abstract Background: Mechanical ventilation (MV) is a life-saving intervention in patients with acute respiratory failure. However, prolonged MV results in ventilator-induced diaphragm dysfunction (VIDD), a condition characterized by both diaphragm fiber atrophy and contractile dysfunction. Previous work has shown that calpain, caspase-3, and the ubiquitin–proteasome pathway (UPP) are all activated in the diaphragm during prolonged MV. However, although it is established that both calpain and caspase-3 are important contributors to VIDD, the role that the UPP plays in the development of VIDD remains unknown. These experiments tested the hypothesis that inhibition of the UPP will protect the diaphragm against VIDD. Methods: The authors tested this prediction in an established animal model of MV using a highly specific UPP inhibitor, epoxomicin, to prevent MV-induced activation of the proteasome in the diaphragm (n = 8 per group). Results: The results of this study reveal that inhibition of the UPP did not prevent ventilator-induced diaphragm muscle fiber atrophy and contractile dysfunction during 12 h of MV. Also, inhibition of the UPP does not affect MV-induced increases in calpain and caspase-3 activity in the diaphragm. Finally, administration of the proteasome inhibitor did not protect against the MV-induced increases in the expression of the E3 ligases, muscle ring finger-1 (MuRF1), and atrogin-1/MaFbx. Conclusion: Collectively, these results indicate that proteasome activation does not play a required role in VIDD development during the first 12 h of MV.

scholarly journals Toll-like Receptor 4 Signaling in Ventilator-induced Diaphragm Atrophy

2012 ◽  
Vol 117 (2) ◽  
pp. 329-338 ◽  
Author(s):  
Willem-Jan M. Schellekens ◽  
Hieronymus W. H. van Hees ◽  
Michiel Vaneker ◽  
Marianne Linkels ◽  
P. N. Richard Dekhuijzen ◽  
...  
Keyword(s):  
Mechanical Ventilation ◽  
Caspase 3 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Toll Like Receptor 4 ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Diaphragm Atrophy ◽  
Deficient Mice

Background Mechanical ventilation induces diaphragm muscle atrophy, which plays a key role in difficult weaning from mechanical ventilation. The signaling pathways involved in ventilator-induced diaphragm atrophy are poorly understood. The current study investigated the role of Toll-like receptor 4 signaling in the development of ventilator-induced diaphragm atrophy. Methods Unventilated animals were selected for control: wild-type (n = 6) and Toll-like receptor 4 deficient mice (n = 6). Mechanical ventilation (8 h): wild-type (n = 8) and Toll-like receptor 4 deficient (n = 7) mice.Myosin heavy chain content, proinflammatory cytokines, proteolytic activity of the ubiquitin-proteasome pathway, caspase-3 activity, and autophagy were measured in the diaphragm. Results Mechanical ventilation reduced myosin content by approximately 50% in diaphragms of wild-type mice (P less than 0.05). In contrast, ventilation of Toll-like receptor 4 deficient mice did not significantly affect diaphragm myosin content. Likewise, mechanical ventilation significantly increased interleukin-6 and keratinocyte-derived chemokine in the diaphragm of wild-type mice, but not in ventilated Toll-like receptor 4 deficient mice. Mechanical ventilation increased diaphragmatic muscle atrophy factor box transcription in both wild-type and Toll-like receptor 4 deficient mice. Other components of the ubiquitin-proteasome pathway and caspase-3 activity were not affected by ventilation of either wild-type mice or Toll-like receptor 4 deficient mice. Mechanical ventilation induced autophagy in diaphragms of ventilated wild-type mice, but not Toll-like receptor 4 deficient mice. Conclusion Toll-like receptor 4 signaling plays an important role in the development of ventilator-induced diaphragm atrophy, most likely through increased expression of cytokines and activation of lysosomal autophagy.

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

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.

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

2021 ◽  
Author(s):  
RUIHONG GONG ◽  
Minting Chen ◽  
Chunhua Huang 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.

scholarly journals mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

2015 ◽  
Vol 112 (52) ◽  
pp. 15790-15797 ◽  
Author(s):  
Jinghui Zhao ◽  
Bo Zhai ◽  
Steven P. Gygi ◽  
Alfred Lewis Goldberg
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Protein Degradation ◽  
Mammalian Cells ◽  
Cholesterol Biosynthesis ◽  
Mammalian Target Of Rapamycin ◽  
Ubiquitin Proteasome System ◽  
Mtor Inhibition ◽  
Protein Ubiquitination ◽  
Ubiquitin Proteasome

Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440–443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth.

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

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.

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

2008 ◽  
Vol 409 (3) ◽  
pp. 751-759 ◽  
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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