Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The structure of procaspase 6 is similar to that of active mature caspase 6.

ABSTRACT: To investigate the structural characteristics and activation mechanism of the precursor caspase, genes encoding the inactive pro-form and the active mature form of caspase 6 were expressed in Escherichia coli and the proteins of both forms were purified to homogeneity. The structure of each protein was characterized by chemical cross-linking, size-exclusion chromatography, CD and fluorescence spectroscopies. The pro-form caspase 6 exhibits a dimeric structure and its overall secondary structure was found to be similar to that of the mature caspase 6. Upon the maturation of procaspase 6, the maximum fluorescence wavelength lambda(max) was red-shifted from 330 to 337 nm and the fluorescence intensity of lambda(max) was increased. This fluorescence spectral change indicates that the environment of a tryptophan residue in the substrate-binding site can be changed to a more polar one when the procaspase 6 is processed. Taken together, our results strongly demonstrate that precursor caspase 6 exists as a dimer and its overall structure is similar to that of the active caspase 6. Our results also suggest that the local conformational change at the substrate-binding site, with no drastic change in the overall structure, seems to enable precursor caspase 6 to become the active mature enzyme.

SUBMITTER: Kang BH

PROVIDER: S-EPMC1222610 | biostudies-other | 2002 Jun

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3.

Project description:We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.

| S-EPMC3119717 | biostudies-literature

Crystal structure of procaspase-1 zymogen domain reveals insight into inflammatory caspase autoactivation.

Project description:One key event in inflammatory signaling is the activation of the initiator caspase, procaspase-1. Presented here is the crystal structure of the procaspase-1 zymogen without its caspase recruitment domain solved to 2.05 A. Although the isolated domain is monomeric in solution, the protein appeared dimeric in crystals. The loop arrangements in the dimer provide insight into the first autoproteolytic events that occur during activation by oligomerization. Additionally, in contrast to other caspases, we demonstrate that autoproteolysis at the second cleavage site, Asp316, is necessary for conversion to a stable dimer in solution. Critical elements of secondary structure are revealed in the crystal structure that explain why a dimeric protein is favored after proteolysis at this aspartic acid. Dimer stabilization is concurrent with a 130-fold increase in kcat, the sole contributing kinetic factor to an activated and efficient mediator of inflammation.

| S-EPMC2649088 | biostudies-literature

Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase-1.

Project description:The NLRP1 inflammasome responds to microbial challenges such as Bacillus anthracis infection and is implicated in autoimmune disease such as vitiligo. Human NLRP1 contains both an N-terminal pyrin domain (PYD) and a C-terminal caspase recruitment domain (CARD), with the latter being essential for its association with the downstream effector procaspase-1. Here we report a 2.0 Å crystal structure of the human NLRP1 CARD as a fusion with the maltose-binding protein. The structure reveals the six-helix bundle fold of the NLRP1 CARD, typical of the death domain superfamily. The charge surface of the NLRP1 CARD structure and a procaspase-1 CARD model suggests potential mechanisms for their association through electrostatic attraction.

| S-EPMC3860829 | biostudies-literature

Procaspase-3 activation as an anti-cancer strategy: structure-activity relationship of procaspase-activating compound 1 (PAC-1) and its cellular co-localization with caspase-3.

Project description:A goal of personalized medicine as applied to oncology is to identify compounds that exploit a defined molecular defect in a cancerous cell. A compound called procaspase-activating compound 1 (PAC-1) was reported that enhances the activity of procaspase-3 in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Experimental evidence indicates that PAC-1 activates procaspase-3 in vitro through chelation of inhibitory zinc ions. Described herein is the synthesis and biological activity of a family of PAC-1 derivatives where key functional groups have been systematically altered. Analysis of these compounds reveals a strong correlation between the in vitro procaspase-3 activating effect and their ability to induce death in cancer cells in culture. Importantly, we also show that a fluorescently labeled version of PAC-1 co-localizes with sites of caspase-3 activity in cancer cells. The data presented herein further bolster the hypothesis that PAC-1 induces apoptosis in cancer cells through the direct activation of procaspase-3, has implications for the design and discovery of next-generation procaspase-3 activating compounds, and sheds light on the anti-apoptotic role of cellular zinc.

| S-EPMC2749958 | biostudies-literature

Stress-inducible caspase substrate TRB3 promotes nuclear translocation of procaspase-3.

Project description:Pseudokinase TRB3 is a stress-inducible nuclear protein, which has recently been shown to be involved in ER stress-induced apoptosis. However, it remains unclear how TRB3 contributes to the process. We recently demonstrated that TRB3 was cleaved by caspase-3 (CASP3) in vitro and also in apoptosis-induced cells. Thus, we investigate the role of TRB3 cleavage in the apoptotic process to address the above question. Overexpression studies revealed that the cleavage of TRB3 promoted CASP3/7 activation and apoptosis. In contrast, the anti-apoptotic effects were found under TRB3 non-cleavable conditions, such as ER stress, and also when the CASP3/7 activation was enhanced by knockdown of endogenous TRB3 expression. Interestingly, nuclear translocation of procaspase-3 (proCASP3) was observed in cells either overexpressing TRB3 or under tunicamycin-induced ER stress. Although forced cytoplasmic expression of proCASP3 enhanced apoptosis significantly, its nuclear expression did not produce any pro-apoptotic effect, suggesting that nuclear distribution of proCASP3 is not critical for the execution of apoptosis. Thus, TRB3 might prevent cytoplasmic activation of CASP3 by promoting proCASP3 entry into the nucleus, and thereby inhibit apoptosis. Taken together, our results suggest that TRB3, through its own cleavage, functions as a molecular switch between the cell survival and apoptotic pathways under stressful conditions.

| S-EPMC3415431 | biostudies-literature

Apoptosome structure, assembly, and procaspase activation.

Project description:Apaf-1-like molecules assemble into a ring-like platform known as the apoptosome. This cell death platform then activates procaspases in the intrinsic cell death pathway. In this review, crystal structures of Apaf-1 monomers and CED-4 dimers have been combined with apoptosome structures to provide insights into the assembly of cell death platforms in humans, nematodes, and flies. In humans, the caspase recognition domains (CARDs) of procaspase-9 and Apaf-1 interact with each other to form a CARD-CARD disk, which interacts with the platform to create an asymmetric proteolysis machine. The disk tethers multiple pc-9 catalytic domains to the platform to raise their local concentration, and this leads to zymogen activation. These findings have now set the stage for further studies of this critical activation process on the apoptosome.

| S-EPMC3644875 | biostudies-literature

Caspase-9 holoenzyme is a specific and optimal procaspase-3 processing machine.

Project description:Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome, and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper-linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC but is much less active than C9Holo for the physiological substrate procaspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for procaspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for procaspase-3, which is important for procaspase-3 activation at the physiological concentration.

| S-EPMC2904439 | biostudies-literature

The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3.

Project description:Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.

| S-EPMC3155825 | biostudies-literature

Caspase-2 primes cancer cells for TRAIL-mediated apoptosis by processing procaspase-8.

Project description:Although caspase-2 is believed to be involved in death receptor-mediated apoptosis, the exact function, mode of activation, and regulation of caspase-2 remain unknown. Here we show that protein kinase (PK) CK2 phosphorylates procaspase-2 directly at serine-157. When intracellular PKCK2 activity is low or downregulated by specific inhibitors, procaspase-2 is dephosphorylated, dimerized, and activated in a PIDDosome-independent manner. The activated caspase-2 then processes procaspase-8 monomers between the large and small subunits, thereby priming cancer cells for TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The processed procaspase-8 that is recruited to death-inducing signaling complex by TRAIL engagement becomes fully activated, and cancer cells undergo apoptosis. PKCK2 activity is low in TRAIL-sensitive cancer cell lines but high in TRAIL-resistant cancer cell lines. Thus, downregulating PKCK2 activity is required for TRAIL-mediated apoptosis to occur in TRAIL-resistant cancer cells. Our data provide novel insights into the regulation, mode of activation, and function of caspase-2 in TRAIL-mediated apoptosis.

| S-EPMC1276710 | biostudies-literature

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence.

Project description:Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.

| S-EPMC8489496 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data