Caspase-1

Caspase specificity profile [1]
The specificity profile for each of 7 recombinant human caspase with 6 different synthetic caspase substrates was analyzed and the results are shown in Supplementary Fig. S1. Each assay involves assaying the activity of a single caspase against different substrates, in the absence of competing caspases. Thus, caspase units were used as a measure of caspase quantity. The same amount of active caspase (1 U) was used in each assay. Ac-YVAD-pNA, Ac-VDVAD-pNA, Ac-VEID-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA are the commercially designated substrates for caspase-1, 2, 6, 8 and 9 respectively. Ac-DEVD-pNA is the substrate for both caspase 3 and 7. The column on the left in Fig. S1 (A, C, E, G, I, K and M) shows the kinetic plots of each caspase with different substrates. The right column (B, D, F, H, J, L and N) shows the relative cleavage efficiency towards different substrates. The activity of each caspase for its designated substrate is defined as 100%. The results show that the caspase-1 substrate (Ac-YVAD-pNA) is the most specific substrate as it is only cleaved by caspase-1. However, the caspase-1 enzyme cleaves both the substrates for caspase-9 (Ac-LEHD-pNA) and caspase-1 (Fig. S1 A and B). Caspase-2 is the most specific caspase as it only cleaves the designated caspase-2 substrate (Ac-VDVAD-pNA) (Fig. S1. C and D). Caspase-3 cleaves 3 different substrates (Ac-VDVAD-pNA, Ac-DEVD-pNA and Ac-VEID-pNA) equally well (Fig S1. E and F). The caspase-6 substrate was cleaved most efficiently by caspase-6 (Fig. S1. G and H). The specificity profile for caspase-7 is similar to that for caspase-3 (Fig. S1. I and J). Interestingly, the highest catalytic activity for caspase-8 was observed for the designated caspase-9 substrate (Ac-LEHD-pNA). This substrate was cleaved approximately 4.5 times more efficiently than the designated substrate for caspase-8 (Ac-IETD-pNA) (Fig. S1. K and L). The caspase-9 substrate (Ac-LEHD-pNA) was the best substrate for caspase-9. However, minimal activity was also seen with substrates for caspase-3, caspase-6 and caspase-8 (Fig. S1. M and N).

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Under our experimental conditions, the designated substrate for caspase-1, Ac-YVAD-pNA, is the most specific substrate as it is only cleaved by caspase-1 (Fig. S1). Caspase-1 is involved in the regulation of inflammatory processes for which the prime substrate is the inflammatory cytokine, pro-IL-1β. Cleavage of pro-IL-β by caspase-1 occurs at the site Tyr-Val-His-Asp116/Ala117 (YVHD/A) which is similar to the commercially available caspase-1 substrate Ac-YVAD-pNA. We show that caspases involved in apoptosis, namely caspase-2, -3, -6, -7, -8 and -9, do not cleave the caspase-1 substrate. This result may imply that a substrate specific for inflammation, such as pro-IL-1β, cannot be cleaved by caspases involved in apoptosis. It has been shown that caspase-2 acts as a specific enzyme due to its strict requirement of a P5 residue for efficient cleavage. Our results confirmed this specificity (Fig. S1 C and D). The designated caspase-2 substrate, Ac-VDVAD-pNA, is efficiently cleaved by caspase-2, -3 and -7 (Fig. S1 E, F, I and J). Therefore, the cleavage of Ac-VDVAD-pNA by apoptotic cells could be due to the combined effects of caspase-2, -3 and -7. Ac-DEVD-pNA, the designated substrate for caspase-3 and -7, is effectively cleaved by these two enzymes (Fig. S1 E, F, I and J). Caspase-3 and -7 also effectively cleaves Ac-VDVAD-pNA and Ac-VEID-pNA, the designated substrates for caspase-2 and -6 respectively (Fig. S1 E, F, I and J). The most surprising result from the specificity study is that caspase-8 cleaves the caspase-9 substrate (Ac-LEHD-pNA) 4.5 times more efficiently than it cleaves its own substrate, Ac-IETD-pNA (Fig. S1 K and L). Since caspase-8 and -9 is involved in the extrinsic and intrinsic death pathway respectively, the significant cleavage of Ac-LEHD-pNA by caspase-8 could lead to difficulties in determining the contributions of each pathway. [1]

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Celastrol inhibited pyroptosis of macrophages [2]
As celastrol prevented the up-regulation of IL-1β and cleavage of caspase-1 induced by LPS and ATP, we hypothesized that celastrol could inhibit pyroptosis in macrophages. As previously mentioned, pyroptosis depends on the activation of caspase-1 and loss of cell membrane integrity, and it causes cell lysis resulting in the leakage of cellular contents. First, we analyzed whether celastrol affected the activation of caspase-1 in macrophages by detecting the cleavage of caspase-1 substrate Ac-YVAD-pNA. LPS and ATP increased the level of activated caspase-1 compared with the control cells (Fig. 3A). When pretreated with celastrol or caspase-1 inhibitor Z-VAD-FMK, the activation of caspase-1 was significantly reduced. Similarly, the results show that the release of LDH induced by LPS and ATP was significantly reduced by celastrol in a dose-dependent manner (Fig. 3B).

Detection of caspase-1 activity [2]
The activity of caspase-1 was assayed using a caspase-1 activity kit, which was based on the ability of caspase-1 to change acetyl-Tyr-Val-Ala-Asp p-nitroanilide (Ac-YVAD-pNA) into the yellow formazan product p-nitroaniline (pNA). The procedure was performed according to the manufacturer's protocol. Cellular extracts (50 μg) were incubated in a 96-well microtiter plate with 20 nmol Ac-YVAD-pNA overnight at 37 °C. The absorbance values of pNA at 405 nm were tested using a microplate reader.

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Caspase specificity profile [1]
The specificity profile for each of 7 recombinant human caspase with 6 different synthetic caspase substrates was analyzed and the results are shown in Supplementary Fig. S1. Each assay involves assaying the activity of a single caspase against different substrates, in the absence of competing caspases. Thus, caspase units were used as a measure of caspase quantity. The same amount of active caspase (1 U) was used in each assay. Ac-YVAD-pNA, Ac-VDVAD-pNA, Ac-VEID-pNA, Ac-IETD-pNA, and Ac-LEHD-pNA are the commercially designated substrates for caspase-1, 2, 6, 8 and 9 respectively. Ac-DEVD-pNA is the substrate for both caspase 3 and 7. The column on the left in Fig. S1 (A, C, E, G, I, K and M) shows the kinetic plots of each caspase with different substrates. The right column (B, D, F, H, J, L and N) shows the relative cleavage efficiency towards different substrates. The activity of each caspase for its designated substrate is defined as 100%. The results show that the caspase-1 substrate (Ac-YVAD-pNA) is the most specific substrate as it is only cleaved by caspase-1. However, the caspase-1 enzyme cleaves both the substrates for caspase-9 (Ac-LEHD-pNA) and caspase-1 (Fig. S1 A and B). Caspase-2 is the most specific caspase as it only cleaves the designated caspase-2 substrate (Ac-VDVAD-pNA) (Fig. S1. C and D). Caspase-3 cleaves 3 different substrates (Ac-VDVAD-pNA, Ac-DEVD-pNA and Ac-VEID-pNA) equally well (Fig S1. E and F). The caspase-6 substrate was cleaved most efficiently by caspase-6 (Fig. S1. G and H). The specificity profile for caspase-7 is similar to that for caspase-3 (Fig. S1. I and J). Interestingly, the highest catalytic activity for caspase-8 was observed for the designated caspase-9 substrate (Ac-LEHD-pNA). This substrate was cleaved approximately 4.5 times more efficiently than the designated substrate for caspase-8 (Ac-IETD-pNA) (Fig. S1. K and L). The caspase-9 substrate (Ac-LEHD-pNA) was the best substrate for caspase-9. However, minimal activity was also seen with substrates for caspase-3, caspase-6 and caspase-8 (Fig. S1. M and N).

[1]. Some commonly used caspase substrates and inhibitors lack the specificity required to monitor individual caspase activity. Biochem Biophys Res Commun. 2008 Dec 19;377(3):873-7.

[2]. A new mechanism of inhibition of IL-1β secretion by celastrol through the NLRP3 inflammasome pathway. Eur J Pharmacol. 2017 Nov 5;814:240-247.

Many designated substrates and inhibitors have been widely used to study the role of caspases in apoptosis during mammalian cell culture. However, the specificity of these substrates and inhibitors has not been systematically evaluated. Therefore, conclusions about the role of specific caspases in apoptotic cells are inaccurate. This study investigated the interactions between seven commercially available human caspases and their designated substrates and inhibitors. The results showed that Ac-YVAD-pNA, the designated substrate for caspase-1, was the most specific substrate. All other tested substrates showed cross-reactivity with multiple caspases. In terms of enzymes, caspase-2 had the highest specificity, followed by caspase-9 and -6. Caspase-3 and -7 were able to efficiently cleave three substrates. The designated substrates for caspase-1 and -8 were not even their optimal substrates. Even at low concentrations, the fluoromethyl ketone (FMK) inhibitor showed no specificity for different caspases. In contrast, the potency of aldehyde inhibitors showed a significant correlation with the cleavage of pNA substrates. These results indicate that some commonly used caspase substrates and inhibitors lack the specificity required to monitor the activity of individual caspases. [1] The NLRP3 (NOD-like receptor protein 3) inflammasome is a multi-protein complex containing caspase-1 that controls the release of IL-1β and is associated with the development of inflammatory diseases. Tripterygium wilfordii is a pharmacologically active ingredient extracted from Tripterygium wilfordii, and its anti-inflammatory activity stems from its inhibitory effect on IL-1β secretion. This study aimed to investigate the possible regulatory role of triptolide on the release of IL-1β and IL-18 mediated by the NLRP3 inflammasome in macrophages. The study showed that triptolide significantly reduced the secretion of IL-1β and IL-18 by inhibiting the expression of NLRP3 and the cleavage of caspase-1 in macrophages induced by lipopolysaccharide (LPS)/ATP. In addition, triptolide inhibited macrophage pyroptosis, which was confirmed by caspase-1 activation, LDH leakage and PI uptake experiments. Moreover, the study found that these inhibitory effects of triptolide are achieved at least in part by reducing the production of reactive oxygen species and the activation of NF-κB. In summary, these findings suggest that triptolide exerts a new anti-inflammatory mechanism by inhibiting the NLRP3 inflammasome. [2]

C29H36N6O10

628.63

628.249

149231-66-3

10484117

Ac-Tyr-Val-Ala-Asp-pNA

YVAD; Ac-YVAD-{pNA}

White to off-white solid powder

1.4±0.1 g/cm3

1108.9±65.0 °C at 760 mmHg

624.5±34.3 °C

0.0±0.3 mmHg at 25°C

1.605

2.61

7

10

14

45

1080

4

C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)NC1=CC=C(C=C1)[N+](=O)[O-])NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)C

YDPNOCSPPGFBPX-XNHCRPTKSA-N

InChI=1S/C29H36N6O10/c1-15(2)25(34-28(42)22(31-17(4)36)13-18-5-11-21(37)12-6-18)29(43)30-16(3)26(40)33-23(14-24(38)39)27(41)32-19-7-9-20(10-8-19)35(44)45/h5-12,15-16,22-23,25,37H,13-14H2,1-4H3,(H,30,43)(H,31,36)(H,32,41)(H,33,40)(H,34,42)(H,38,39)/t16-,22-,23-,25-/m0/s1

(3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-(4-nitroanilino)-4-oxobutanoic acid

Ac-YVAD-pNA; 149231-66-3; Ac-Tyr-Val-Ala-Asp-PNA; (3S)-3-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]-4-(4-nitroanilino)-4-oxobutanoic acid; Caspase-1 Substrate IV, Colorimetric; MFCD00274387; SCHEMBL7694593; DTXSID30440596;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 250 mg/mL (397.69 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)