scholarly journals alphaII-Spectrin is an in vitro target for caspase-2, and its cleavage is regulated by calmodulin binding

2004 ◽  
Vol 378 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Björn ROTTER ◽  
Yolande KROVIARSKI ◽  
Gaël NICOLAS ◽  
Didier DHERMY ◽  
Marie-Christine LECOMTE
Keyword(s):  
Cell Shape ◽  
Caspase 3 ◽  
Membrane Skeleton ◽  
Calmodulin Binding ◽  
Caspase 7 ◽  
Caspase 6 ◽  
Caspase 2 ◽  
The Stability ◽  
Main Components

The spectrin–actin scaffold underlying the lipid bilayer is considered to participate in cell-shape stabilization and in the organization of specialized membrane subdomains. These structures are dynamic and likely to undergo frequent remodelling during changes in cell shape. Proteolysis of spectrin, which occurs during apoptosis, leads to destabilization of the scaffold. It is also one of the major processes involved in membrane remodelling. Spectrins, the main components of the membrane skeleton, are the targets for two important protease systems: m- and µ-calpains (Ca2+-activated proteases) and caspase-3 (activated during apoptosis). In this paper, we show that caspase-2 also targets spectrin in vitro, and we characterize Ca2+/calmodulin-dependent regulation of spectrin cleavage by caspases. Yeast two-hybrid screening reveals that the large isoform (1/L) of procaspase-2 specifically binds to αII-spectrin, while the short isoform does not. Like caspase-3, caspase-2 cleaves αII-spectrin in vitro at residue Asp-1185. This study emphasizes a role of executioner caspase for caspase-2. We also demonstrated that the executioner caspase-7 but not caspase-6 cleaves spectrin at residue Asp-1185 in vitro. This spectrin cleavage by caspases 2, 3 and 7 is inhibited by the Ca2+-dependent binding of calmodulin to spectrin. In contrast, calmodulin binding enhances spectrin cleavage by calpain at residue Tyr-1176. These results indicate that αII-spectrin cleavage is highly influenced by Ca2+ homoeostasis and calmodulin, which therefore represent potential regulators of the stability and the plasticity of the spectrin-based skeleton.

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 Salmonella-Induced Caspase-2 Activation in Macrophages

2000 ◽  
Vol 192 (7) ◽  
pp. 1035-1046 ◽  
Author(s):  
Veronika Jesenberger ◽  
Katarzyna J. Procyk ◽  
Junying Yuan ◽  
Siegfried Reipert ◽  
Manuela Baccarini
Keyword(s):  
Type Iii Secretion ◽  
Caspase 3 ◽  
Secretion System ◽  
Caspase 1 ◽  
Chemical Inhibition ◽  
Induction Of Apoptosis ◽  
Caspase 2 ◽  
Induced Apoptosis

The enterobacterial pathogen Salmonella induces phagocyte apoptosis in vitro and in vivo. These bacteria use a specialized type III secretion system to export a virulence factor, SipB, which directly activates the host's apoptotic machinery by targeting caspase-1. Caspase-1 is not involved in most apoptotic processes but plays a major role in cytokine maturation. We show that caspase-1–deficient macrophages undergo apoptosis within 4–6 h of infection with invasive bacteria. This process requires SipB, implying that this protein can initiate the apoptotic machinery by regulating components distinct from caspase-1. Invasive Salmonella typhimurium targets caspase-2 simultaneously with, but independently of, caspase-1. Besides caspase-2, the caspase-1–independent pathway involves the activation of caspase-3, -6, and -8 and the release of cytochrome c from mitochondria, none of which occurs during caspase-1–dependent apoptosis. By using caspase-2 knockout macrophages and chemical inhibition, we establish a role for caspase-2 in both caspase-1–dependent and –independent apoptosis. Particularly, activation of caspase-1 during fast Salmonella-induced apoptosis partially relies on caspase-2. The ability of Salmonella to induce caspase-1–independent macrophage apoptosis may play a role in situations in which activation of this protease is either prevented or uncoupled from the induction of apoptosis.

scholarly journals Phytoconstituents from Markhamia Tomentosa Bind To HPV Oncoprotein with Apoptogenic Potential: A Molecular Modeling Approach

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mutiat B. Ibrahim ◽  
Adeola T. Kola-Mustapha ◽  
Niyi S. Adelakun ◽  
Neil A. Koorbanally
Keyword(s):  
Drug Targets ◽  
Active Sites ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Hpv 16 ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Potent Growth ◽  
The Stability

Abstract Markhamia tomentosa crude extract and fractions exhibited potent growth inhibitory effects capable to induce apoptosis in cervical (HeLa) cancer cell line via in vitro model. Presently, interaction of M. tomentosa phytoconstituents with molecular drug targets to exert its anticancer property is evaluated via in silico study. Identified phytoconstituents from M. tomentosa were retrieved from PubChem database and docked in active sites of HPV 16 E6, caspase -3 and caspase -8 targets using AutoDockVina from PyRx software. Screening for druglikeness; and absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions was carried out with the use of SwissADME and pkCSM web servers. Standard melphalan and co-crystallized ligands of caspases -3 and -8 enzymes were used to validate protein-ligand interactions. Molecular dynamic simulation was used to validate the stability of the hit molecules complexed with caspases -3 and -8. All identified phytoconstituents from M. tomentosa showed binding affinity for HPV with docking scores range of - 5.4 to -2.6 kcal/mol. Ajugol, carnosol, luteolin and phytol showed good docking energy range of -6.8 to -3.6 kcal/mol; and -4.8 to -1.9 kcal/mol for the active sites of caspases -3 and -8 targets respectively. Based on docking scores; drug-likeliness; and ADMET predictions; luteolin and carnosol were selected as hit compounds. These molecules were found to be stable within the binding site of caspase -3 target throughout the 40ns simulation time. These findings identified hit ligands from M. tomentosa phytoconstituents that inhibit HPV 16 E6 oncogene expression with stimulation of caspases -3 and -8 targets.

scholarly journals Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

2016 ◽  
Vol 113 (14) ◽  
pp. E2001-E2010 ◽  
Author(s):  
Olivier Julien ◽  
Min Zhuang ◽  
Arun P. Wiita ◽  
Anthony J. O’Donoghue ◽  
Giselle M. Knudsen ◽  
...  
Keyword(s):  
Cleavage Sites ◽  
Protein Cleavage ◽  
Peptide Substrates ◽  
Rate Analysis ◽  
Substrate Preferences ◽  
Sequence Of Events ◽  
Caspase 7 ◽  
Caspase 6 ◽  
Caspase 2 ◽  
Few Data

Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

scholarly journals Caspase-Mediated Cleavage of Human Cortactin during Influenza A Virus Infection Occurs in Its Actin-Binding Domains and Is Associated with Released Virus Titres

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 87 ◽  
Author(s):  
Da-Yuan Chen ◽  
Matloob Husain
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Caspase 3 ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Apoptotic Pathway ◽  
Binding Domains ◽  
Caspase 7 ◽  
Caspase 6 ◽  
Late Stages

Influenza A virus (IAV) exploits host factors to multiply and cause disease. An in-depth knowledge of this interaction of IAV with the host will aid the development of anti-IAV intervention strategies. Previously, we demonstrated that host cortactin, an actin filament-binding protein promotes IAV infection, but undergoes degradation via a lysosome-associated apoptotic pathway during the late stages of IAV infection. Next, we wanted to further understand the mechanisms and significance of this phenomenon. By using the RNA interference screens and site-directed mutagenesis followed by western blotting, we found that lysosome protease, cathepsin C is involved in cortactin degradation in human cells infected with IAV. Furthermore, executioner apoptotic caspase, caspase-3 not caspase-6 or caspase-7 is involved in cortactin degradation during IAV infection, and caspase-3 cleavage site is located in the first actin-binding repeat of cortactin polypeptide. Finally, when expressed ectopically, the cleavage-resistant cortactin mutants decreased the amount of IAV progeny released from infected cells that was enhanced by the cleavage-sensitive cortactin wild type. These data strengthen the hypothesis proposed earlier that host cortactin plays an inhibitory role during the late stages of IAV infection, and IAV is facilitating its degradation to undermine such function.

scholarly journals Hexarelin Modulation of MAPK and PI3K/Akt Pathways in Neuro-2A Cells Inhibits Hydrogen Peroxide—Induced Apoptotic Toxicity

2021 ◽  
Vol 14 (5) ◽  
pp. 444
Author(s):  
Ramona Meanti ◽  
Laura Rizzi ◽  
Elena Bresciani ◽  
Laura Molteni ◽  
Vittorio Locatelli ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Viability ◽  
Caspase 3 ◽  
Mrna Levels ◽  
Protective Effects ◽  
Neuroprotective Effects ◽  
Caspase 7 ◽  
Induced Apoptosis

Hexarelin, a synthetic hexapeptide, exerts cyto-protective effects at the mitochondrial level in cardiac and skeletal muscles, both in vitro and in vivo, may also have important neuroprotective bioactivities. This study examined the inhibitory effects of hexarelin on hydrogen peroxide (H2O2)-induced apoptosis in Neuro-2A cells. Neuro-2A cells were treated for 24 h with various concentrations of H2O2 or with the combination of H2O2 and hexarelin following which cell viability and nitrite (NO2−) release were measured. Cell morphology was also documented throughout and changes arising were quantified using Image J skeleton and fractal analysis procedures. Apoptotic responses were evaluated by Real-Time PCR (caspase-3, caspase-7, Bax, and Bcl-2 mRNA levels) and Western Blot (cleaved caspase-3, cleaved caspase-7, MAPK, and Akt). Our results indicate that hexarelin effectively antagonized H2O2-induced damage to Neuro-2A cells thereby (i) improving cell viability, (ii) reducing NO2− release and (iii) restoring normal morphologies. Hexarelin treatment also reduced mRNA levels of caspase-3 and its activation, and modulated mRNA levels of the BCL-2 family. Moreover, hexarelin inhibited MAPKs phosphorylation and increased p-Akt protein expression. In conclusion, our results demonstrate neuroprotective and anti-apoptotic effects of hexarelin, suggesting that new analogues could be developed for their neuroprotective effects.

scholarly journals In vitro and In silico Evaluation of Structurally Diverse Benzyl-pyrrolidine-3-ol Analogues as Apoptotic Agents via Caspase Activation

2021 ◽  
Vol 68 (3) ◽  
pp. 667-682
Author(s):  
Tahira Naqvi ◽  
Asif Amin ◽  
Shujat Ali ◽  
Mohsin Y. Lone ◽  
Nadeem Bashir ◽  
...  
Keyword(s):  
Cell Lines ◽  
In Silico ◽  
Caspase 3 ◽  
Human Cancer ◽  
Md Simulations ◽  
Human Cancer Cell Lines ◽  
Lead Compounds ◽  
Model Protein ◽  
The Stability

The activation of caspases is central to apoptotic process in living systems. Defects in apoptosis have been implicated with carcinogenesis. Need to develop smart agents capable of inducing apoptosis in tumor cells is obvious. With this motive, diversity oriented synthesis of 1-benzylpyrrolidin-3-ol analogues was envisaged. The multi component Ugi reaction synthesized library of electronically diverse analogues was explored for cytotoxic propensity towards a panel of human cancer cell lines at 10 μM. The lead compounds exhibit a selective cytotoxicity towards HL-60 cells as compared to cell lines derived from solid tumors. Besides, their milder cytotoxic effect on non-cancerous cell lines reaffirm their selective action towards cancer cells only.The lead molecules were tested for their ability to target caspase-3, as a vital protease triggering apoptosis. The lead compounds were observed to induce apoptosis in HL-60 cells around 10 μM concentration. The lead compounds exhibited various non-covalent supra type interactions with caspase-3 key residues around the active site. The binding ability of lead compounds with caspase-3 was studied via molecular docking and molecular dynamic (MD) simulations. MD simulations indicated the stability of compound-caspase-3 complex throughout the 50 ns simulation run. The stability and bio-availability of the lead compounds under physiological conditions was assessed by their interaction with Bovine Serum Albumin (BSA) as model protein. BSA interactions of lead compounds were studied by various bio-physical methods and further substantiated with in silico MD simulations.

In Vitro Proteolysis of Myofibrillar Proteins from Beef Skeletal Muscle by Caspase-3 and Caspase-6

2011 ◽  
Vol 59 (17) ◽  
pp. 9658-9663 ◽  
Author(s):  
Feng Huang ◽  
Ming Huang ◽  
Guanghong Zhou ◽  
Xinglian Xu ◽  
Mei Xue
Keyword(s):  
Skeletal Muscle ◽  
Caspase 3 ◽  
Myofibrillar Proteins ◽  
Caspase 6

scholarly journals Selective Cleavage of BLM, the Bloom Syndrome Protein, during Apoptotic Cell Death

2001 ◽  
Vol 276 (15) ◽  
pp. 12068-12075 ◽  
Author(s):  
Oliver Bischof ◽  
Sanjeev Galande ◽  
Farzin Farzaneh ◽  
Terumi Kohwi-Shigematsu ◽  
Judith Campisi
Keyword(s):  
Caspase 3 ◽  
Apoptotic Cell ◽  
Autosomal Recessive Disorder ◽  
Bloom Syndrome ◽  
Recognition Sequence ◽  
Blm Helicase ◽  
Caspase 6 ◽  
Syndrome Protein ◽  
Condensed Dna

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6,in vitro. The caspase 3 recognition sequence412TEVD415was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.

Sign in / Sign up

Export Citation Format

Share Document