scholarly journals Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells

1999 ◽  
Vol 340 (1) ◽  
pp. 127-133 ◽  
Author(s):  
Xue-min ZHANG ◽  
Hong LIN ◽  
Catheryne CHEN ◽  
Ben D.-M. CHEN
Keyword(s):  
Cell Death ◽  
Proteasome Inhibitor ◽  
Caspase 3 ◽  
Specific Inhibitor ◽  
Apoptotic Pathway ◽  
Principal Mechanism ◽  
Ubiquitin Proteasome Pathway ◽  
Gm Csf ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Here we examine the possibility that ubiquitin-proteasome is involved in regulating the levels of Bcl-2, which is abundantly expressed in M-07e cells, a granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent human leukaemic cell line. Apoptosis in M-07e cells, induced by GM-CSF withdrawal, was associated with a gradual cleavage of Bcl-2 into a 22 kDa fragment. Treatment of M-07e cells with benzyloxycarbonyl-Leu-Leu-L-leucinal (Z-LLL-CHO; MG-132), a reversible ubiquitin-proteasome inhibitor, markedly accelerated the cleavage of Bcl-2 and promoted cell death through the apoptotic pathway. The cleavage of Bcl-2 was inhibited by a caspase-3 (CPP32)-specific inhibitor [acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO)] but not caspase 1 inhibitor (acetyl-Tyr-Val-Ala-Asp-CHO), suggesting that Bcl-2 is a proteolytic substrate of a caspase-3-like protease activated during apoptosis. The simultaneous addition of recombinant human GM-CSF (rhGM-CSF) to M-07e cultures delayed the activation of caspase 3 and Bcl-2 cleavage triggered by Z-LLL-CHO, suggesting that the activation of the GM-CSF signalling pathway can partly overcome the apoptotic effect induced by Z-LLL-CHO. Apoptosis induced by inhibition of the proteasome pathway was verified in studies with lactacystin, a highly specific and irreversible proteasome inhibitor. Lactacystin-induced apoptosis in M-07e cells was remarkably similar to that induced by Z-LLL-CHO, which included caspase 3 activation, cleavage of Bcl-2 into a 22 kDa fragment and, ultimately, cell death. These results showed that inhibition of the ubiquitin-proteasome pathways can lead to the activation of a DEVD-CHO-sensitive caspase and induces Bcl-2 cleavage, which might have a role in mediating apoptosis in M-07e cells.

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 44: TAK1 Regulates Caspase 8 Activation and Necroptotic Signaling via Multiple Cell Death Checkpoints

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Xaioyun Guo ◽  
Haifeng Yin ◽  
Yi Chen ◽  
Lei Li ◽  
Jing Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Adaptor Protein ◽  
Regulatory Mechanisms ◽  
Caspase 8 ◽  
Ubiquitin Proteasome Pathway ◽  
Pathological Conditions ◽  
Ubiquitin Proteasome ◽  
Multiple Cell ◽  
Proteasome Pathway

Necroptosis has emerged as a new form of programmed cell death implicated in a number of pathological conditions such as ischemic injury, neurodegenerative disease, and viral infection. Recent studies indicate that TGFβ-activated kinase 1 (TAK1) is nodal regulator of necroptotic cell death, but the underlying molecular regulatory mechanisms remain elusive. Here we reported that TAK1 regulates necroptotic signaling as well as caspase 8 activation through both NFκB-dependent and -independent mechanisms. Inhibition of TAK1 promoted TNFα-induced necroptosis through the induction of RIP1 phosphorylation/activation and necrosome formation, in the presence of ongoing caspase activation. Further, inhibition of TAK1 triggered two caspase 8 activation pathways through the induction of RIP1-FADD-caspase 8 complex as well as FLIP cleavage/degradation. Mechanistically, our data uncovered an essential role of the adaptor protein TRADD in caspase 8 activation and necrosome formation triggered by TAK1 inhibition. Moreover, ablation of the deubiqutinase CYLD prevented both apoptotic and necroptotic signaling induced by TAK1 inhibition, whereas deletion of the E3 ubiquitin ligase TRAF2 had the opposite effect. Finally, blocking the ubiquitin-proteasome pathway prevented the degradation of key necroptotic signaling proteins and necrosome formation. Thus we identified novel regulatory mechanisms underling the critical role of TAK1 in necroptotic signaling through regulation of multiple cell death checkpoints. Targeting key components of the necroptotic pathway (e.g., TRADD and CYLD) and the ubiquitin-proteasome pathway may represent novel therapeutic strategies for pathological conditions driven by necroptosis.

scholarly journals The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN DegradationIn VivoandIn Vitro

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Wei Huang ◽  
Chen Yang ◽  
Qinling Nan ◽  
Chenlin Gao ◽  
Hong Feng ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
High Glucose ◽  
Proteasome Inhibitor ◽  
Kidney Damage ◽  
Control Group ◽  
Glomerular Mesangial Cells ◽  
Proteasome Inhibitor Mg132 ◽  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Transforming growth factor-β(TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-βsignaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-βwere observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-βwere significantly increased (P<0.05), whereas SnoN was significantly decreased in the DC group (P<0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P<0.05), but Arkadia expression gradually increased due to high glucose stimulation (P<0.05), which could be almost completely reversed by MG132 (P<0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-βactivation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.

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.

scholarly journals Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma

Blood ◽  
2007 ◽  
Vol 110 (9) ◽  
pp. 3281-3290 ◽  
Author(s):  
Deborah J. Kuhn ◽  
Qing Chen ◽  
Peter M. Voorhees ◽  
John S. Strader ◽  
Kevin D. Shenk ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Proteasome Inhibitor ◽  
Hematologic Malignancies ◽  
Irreversible Inhibitor ◽  
Inhibition Of Proliferation ◽  
Mitochondrial Membrane Depolarization ◽  
Dependent Inhibition ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

AbstractThe proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.

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.

The ubiquitin-proteasome pathway regulates lysosomal degradation of the growth hormone receptor and its ligand

2001 ◽  
Vol 29 (4) ◽  
pp. 488-493 ◽  
Author(s):  
P. van Kerkhof ◽  
G. J. Strous
Keyword(s):  
Growth Hormone ◽  
Proteasome Inhibitor ◽  
Growth Hormone Receptor ◽  
Proteasome Inhibitors ◽  
Plasma Membrane Protein ◽  
Lysosomal Degradation ◽  
Ubiquitin Proteasome Pathway ◽  
Gh Receptor ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

The growth hormone (GH) receptor (GHR) is a mammalian plasma membrane protein whose internalization is mediated by the ubiquitin-proteasome pathway. GH internalization and degradation are inhibited when cells are treated with proteasome inhibitors. Here we show that a GHR truncated at residue 369 can enter the cells in the presence of a proteasome inhibitor, but that the subsequent lysosomal degradation of GH is blocked. Lysosomal inhibitors prolong the half-life of both receptor and ligand. Experiments with antibodies against different receptor tail sections show that degradation of the GHR cytosolic domain precedes degradation of the extracellular GH-binding domain. A possible role for the ubiquitin-proteasome pathway in the degradation of the receptor and ligand is discussed.

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