scholarly journals The proteolytic landscape of cells exposed to non-lethal stresses is shaped by executioner caspases

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
María del Carmen Conde-Rubio ◽  
Roman Mylonas ◽  
Christian Widmann
Keyword(s):  
Caspase 3 ◽  
Environmental Changes ◽  
Proteolytic Processing ◽  
Mass Spectrometry Data ◽  
Apoptotic Cells ◽  
Post Translational Modification ◽  
Gene And Protein Expression ◽  
Caspase 7 ◽  
Executioner Caspases ◽  
In Cells

AbstractCells are in constant adaptation to environmental changes to insure their proper functioning. When exposed to stresses, cells activate specific pathways to elicit adaptive modifications. Those changes can be mediated by selective modulation of gene and protein expression as well as by post-translational modifications, such as phosphorylation and proteolytic processing. Protein cleavage, as a controlled and limited post-translational modification, is involved in diverse physiological processes such as the maintenance of protein homeostasis, activation of repair pathways, apoptosis and the regulation of proliferation. Here we assessed by quantitative proteomics the proteolytic landscape in two cell lines subjected to low cisplatin concentrations used as a mild non-lethal stress paradigm. This landscape was compared to the one obtained in the same cells stimulated with cisplatin concentrations inducing apoptosis. These analyses were performed in wild-type cells and in cells lacking the two main executioner caspases: caspase-3 and caspase-7. Ninety-two proteins were found to be cleaved at one or a few sites (discrete cleavage) in low stress conditions compared to four hundred and fifty-three in apoptotic cells. Many of the cleaved proteins in stressed cells were also found to be cleaved in apoptotic conditions. As expected, ~90% of the cleavage events were dependent on caspase-3/caspase-7 in apoptotic cells. Strikingly, upon exposure to non-lethal stresses, no discrete cleavage was detected in cells lacking caspase-3 and caspase-7. This indicates that the proteolytic landscape in stressed viable cells fully depends on the activity of executioner caspases. These results suggest that the so-called executioner caspases fulfill important stress adaptive responses distinct from their role in apoptosis. Mass spectrometry data are available via ProteomeXchange with identifier PXD023488.

Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications for microfilament reorganization during apoptosis

1999 ◽  
Vol 112 (23) ◽  
pp. 4475-4482 ◽  
Author(s):  
A. Sgorbissa ◽  
R. Benetti ◽  
S. Marzinotto ◽  
C. Schneider ◽  
C. Brancolini
Keyword(s):  
Caspase 3 ◽  
Early Stage ◽  
Proteolytic Processing ◽  
Caspase 7 ◽  
Microfilament System ◽  
Caspase 6 ◽  
Shape Changes ◽  
Mcf 7

Apoptosis is characterized by proteolysis of specific cellular proteins by a family of cystein proteases known as caspases. Gas2, a component of the microfilament system, is cleaved during apoptosis and the cleaved form specifically regulates microfilaments and cell shape changes. We now demonstrate that Gas2 is a substrate of caspase-3 but not of caspase-6. Proteolytic processing both in vitro and in vivo is dependent on aspartic residue 279. Gas2 cleavage was only partially impaired in apoptotic MCF-7 cells which lack caspase-3, thus indicating that different caspases can process Gas2 in vivo. In vitro Gas2 was processed, albeit with low affinity, by caspase-7 thus suggesting that this caspase could be responsible for the incomplete Gas2 processing observed in UV treated MCF-7 cells. In vivo proteolysis of Gas2 was detected at an early stage of the apoptotic process when the cells are still adherent on the substrate and it was coupled to the specific rearrangement of the microfilament characterizing cell death. Finally we also demonstrated that Gas2 in vitro binds to F-actin, but this interaction was unaffected by the caspase-3 dependent proteolytic processing.

scholarly journals Exoenzyme Y induces extracellular active caspase-7 accumulation independent from apoptosis: modulation of transmissible cytotoxicity

2020 ◽  
Vol 319 (2) ◽  
pp. L380-L390
Author(s):  
Phoibe Renema ◽  
Natalya Kozhukhar ◽  
Viktoriya Pastukh ◽  
Domenico Spadafora ◽  
Sunita Subedi Paudel ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Cell Swelling ◽  
Wild Type ◽  
Cell Infection ◽  
Heat Stable ◽  
Caspase 7 ◽  
Executioner Caspases ◽  
Active Caspase

Caspase-3 and -7 are executioner caspases whose enzymatic activity is necessary to complete apoptotic cell death. Here, we questioned whether endothelial cell infection leads to caspase-3/7-mediated cell death. Pulmonary microvascular endothelial cells (PMVECs) were infected with Pseudomonas aeruginosa (PA103). PA103 caused cell swelling with a granular appearance, paralleled by intracellular caspase-3/7 activation and cell death. In contrast, PMVEC infection with ExoY+ (PA103 Δ exoUexoT::Tc pUCP exoY) caused cell rounding, but it did not activate intracellular caspase-3/7 and it did not cause cell death. However, ExoY+ led to a time-dependent accumulation of active caspase-7, but not caspase-3, in the supernatant, independent of apoptosis. To study the function of extracellular caspase-7, caspase-7- and caspase-3-deficient PMVECs were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Caspase-7 activity was significantly reduced in supernatants from infected caspase-7-deficient cells but was unchanged in supernatants from infected caspase-3 deficient cells, indicating an uncoupling in the mechanism of activation of these two enzymes. Because ExoY+ leads to the release of heat stable amyloid cytotoxins that are responsible for transmissible cytotoxicity, we next questioned whether caspase-7 contributes to the severity of this process. Supernatants obtained from infected caspase-7-deficient cells displayed significantly reduced transmissible cytotoxicity when compared with supernatants from infected wild-type controls, illustrating an essential role for caspase-7 in promoting the potency of transmissible cytotoxicity. Thus, we report a mechanism whereby ExoY+ infection induces active caspase-7 accumulation in the extracellular space, independent of both caspase-3 and cell death, where it modulates ExoY+-induced transmissible cytotoxicity.

scholarly journals Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)–mediated inhibition of caspase 9

2007 ◽  
Vol 405 (1) ◽  
pp. 11-19 ◽  
Author(s):  
Jean-Bernard Denault ◽  
Brendan P. Eckelman ◽  
Hwain Shin ◽  
Cristina Pop ◽  
Guy S. Salvesen
Keyword(s):  
Caspase 3 ◽  
Alternative Mechanism ◽  
Caspase 9 ◽  
Inhibitory Interaction ◽  
Iap Antagonist ◽  
Apoptosis Protein ◽  
Caspase 7 ◽  
Amplification Process ◽  
Amplification Loop ◽  
Executioner Caspases

During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp330, and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.

scholarly journals Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Matthew Mannarino ◽  
Hosni Cherif ◽  
Li Li ◽  
Kai Sheng ◽  
Oded Rabau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Back Pain ◽  
Protein Expression ◽  
Disc Degeneration ◽  
Cell Senescence ◽  
Enzyme Linked Immunosorbent Assay ◽  
Matrix Synthesis ◽  
Gene And Protein Expression ◽  
Pain Patients ◽  
In Cells

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16INK4a, SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

Antiapoptotic function of Bcl-2 in mast cells is dependent on its association with heat shock protein 90β

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1413-1420 ◽  
Author(s):  
Cellina Cohen-Saidon ◽  
Irit Carmi ◽  
Avishai Keren ◽  
Ehud Razin
Keyword(s):  
Mast Cell ◽  
Rna Interference ◽  
Mast Cells ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Caspase 3 ◽  
Caspase 7 ◽  
Antiapoptotic Activity ◽  
Novel Strategy ◽  
First Time

In the present study, we demonstrated that the antiapoptotic function of Bcl-2 in mast cells is significantly dependent on its association with the heat shock protein 90β (Hsp90β). Dissociation of these 2 proteins inhibits the antiapoptotic activity of Bcl-2 by initiating the release of cytochrome c from mitochondria into cytosol and increasing the activity of caspase 3 and caspase 7, resulting in mast-cell apoptosis. The antiapoptotic activity of Bcl-2 was greatly affected by knocking-out specifically Hsp90β using the RNA interference approach. Thus, for the first time, it has been shown that Hsp90β might modulate the antiapoptotic activity of Bcl-2 at least in mast cells. These findings could have implications for a novel strategy of regulating apoptosis in patients with mastocytosis and other mast cell–associated diseases.

scholarly journals Cleavage and Inactivation of ATM during Apoptosis

1999 ◽  
Vol 19 (9) ◽  
pp. 6076-6084 ◽  
Author(s):  
Graeme C. M. Smith ◽  
Fabrizio d’adda di Fagagna ◽  
Nicholas D. Lakin ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Caspase 3 ◽  
Cysteine Proteases ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Dna Damage Signalling ◽  
In Cells

ABSTRACT The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

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