assay protocol


Brief discription:

Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation.

Caspases are essential in cells for apoptosis, or programmed cell death, in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis is one of the main contributions to tumour development and autoimmune diseases; this, coupled with the unwanted apoptosis that occurs with ischemia or Alzheimer's disease, has stimulated interest in caspases as potential therapeutic targets.

Inactive protease of caspase family is in state of pro-enzyme which amino acid end has a sequence called "pro-domain". When zymogen is being activated, the pro-domain is cleaved and the rest part is cut into two subunits called P20 and P10. Active zymogens consist of these two subunits in forms of (P20/P10)2. This activation reaction is also Asp-specific for reason that the cleavage occurs between Asp of conserved sequences in pro-enzyme and the amino acids sequence after Asp. In the cleavage the small subunit at the carboxyl end is cleaved first and pro-domain is then cut off the amino side of the big subunit. The cleavage can be self-catalyzing of pro-enzyme and mediated enzyme or functioned by other proteases of ICE family.

More information of caspases are listed in the below table:

Protase Alias Recognized sequence Substrate
caspase 1 ICE YVAD pro-IL-1b , pro-caspase 3, pro-caspase 4
caspase 4 TX, ICH-2, ICErel-II   pro-caspase 1
caspase 5 ICErel-III, TY    
caspase 2 ICH-1   PARP
caspase 9 ICE-LAP6, Mch6   PARP
caspase 3 CPP32, Yama, apopain DEVD PARP, DNA-PK, SRE/BP, rho-GDI, KCq
caspase 6 Mch2 VEID lamin A
caspase 7 Mch3, ICE-LAP3, CMH-1 DEVD PARP, pro-caspase 6
caspase 8 MACH, FLICE, Mch5 DEVD pro-caspase3,4,7,9,10
caspase 10 Mch4 YVAD  
caspase 11 ICH3, FLICE2    

The homology of caspases is as below:


In 1994 Fernandez-Alnemri with his coworkers found a sequence that is homologous to active center of ICE/CED-3 in EST (expression sequence tag) database of GenBank. Probing with this sequence they cloned a new gene filtering cDNA library of Jurkat T lymphocyte. This gene encodes cysteine protease protein (32kD). In 1996 this protease was named as caspase-3. Now caspase-3 is considered the major terminal shear enzyme and an important participant of mechanism of CTL cell damage.

  • Caspase-3: structure

Pro-caspase-3 has 277 amino acids, molecular weight of 32kD, 30% homology with ICE and 35% homology with CED-3. In caspase family pro-caspase-3 is the most homologous to CED-3 both in structure and substrate specificity. The pro-domain of caspase-3 is shorter than that of ICE which has 28 amino acids, but its activity center and conserved amino acids that are related with substrate binding are the same with ICE. In activation, pro-caspase-3 is cleaved at two sites: Asp28~Ser29 and Asp175~Ser176, giving rise to two fragments: P17 (29~175) and P10 (182~277) which are close to P20 and P10 of ICE. The two subunits combine and form active caspase-3. When being activated, pro-caspase-3 is not active of catalyzing before being cleaved by granzyme B (GrzB) or caspase-10 at D175. Other caspases e.g. ICE might participate in cleaving pro-domain of caspase-3.

  • Caspase-3: activation and participation in apoptosis

Caspase-3 is indispensible in apoptosis. It triggers apoptosis when being transfected into insect Sf9 cells. This process can be blocked by BCL-2. Exclusion of caspase-3 in extractions of apoptotic cells leads to loss of capability of inducing apoptosis. Adding of caspase-3 let it regain the capability of inducing apoptosis.

Caspase-3 can be activated by various factors. In CTL-mediated killing, caspase-3 can be activated both by Fas/FasL pathway and by granzyme B pathway. Granzyme B is a kind of serine esterase in cells, and is the only protease that cleaves after Asp except caspases in mammals. Granzyme B can specificly cleave IxxD sequence at the C terminal of catalyzing subunit of ICE family and activate caspase-2, 3,6,7,8,9,10. ICE can be cleaved by granzyme B, too, but it can't be activated after cleavage.

  • Caspase-3: mechanism of inducing apoptosis

The major substrate of caspase-3 is Poly ADP-ribose polymerase (PARP). It correlates with DNA repairment and monitoring of gene intergration. In initiation of apoptosis, PARP 116kD is cleaved by caspase-3 into two fragments, 31kD and 85kD at Asp216-Gly217, separating its two zinc finger domain that binds with DNA from its catalyzing domain of carboxyl end, and loses its normal function. Then the activity of endonuclease which is down regulated by PARP and dependent on Ca2+/Mg2 increases, and DNA in nucleosomes is lysed which triggers apoptosis. This lysis process can be inhibited by Ac-DEVD-CHO, a specific inhibitor of caspase-3, but can't be inhibited by CrmA. Caspase-3 can also cleave U1-70K、DNA-PK、PKCd and PKCq. Both PKCd and PKCq belong to novel PKC (nPKC). After being cleaved by caspase-3 which cuts off the regulation domain, they become active PKC. Moreover, over expression of PKCd and PKCq can trigger apoptosis, which illustrates they participate in inducing of apoptosis.