User:Kévin Roger/Sandbox 953

Introduction

Caspases correspond to an abridged version of cysteine-aspartic acid proteases. These proteins are a part of a cysteine protease family which cleaves target proteins after an aspartate residue^[1]. This class-3 enzyme (hydrolase) plays a major role in the execution of programmed cell death also called apoptosis but also in necrosis and inflammation. Some other caspases are crucial in the process of lymphocyte maturation and thus for the well functioning immune system. Dysregulation of caspase activation cascade could induce an apoptosis pathway failure. Because of this, abnormalities in caspase (functional mutation for instance) are often involved in tumorogenesis and auto-immune diseases.

Caspases exist as inactive zymogen form called pro-caspases. They need a proteolytic activation after apoptotic stimuli. It exists two types of caspase: the initiator caspases (caspase 2, 8, 9 and 10) and effector caspases (caspase 3, 6 and 7)^[2]. The activation of initiator caspases is realized in two steps: first by autocatalytic intrachain cleavage and then by the association with a multimeric adaptator complex to finally form a functionnal holoenzyme^[3]. After that, the role of initiator caspases is to activate the effector zymogen caspase (inactive form) according to a cleavage process. Then, active effector caspases will cleave their downstream apoptotic targets.

The caspase-9 is one of the first major actors responsible for killing cells. It will cleave and activate the effector pro-caspase-3 in the mitochondrial apoptotic pathway (intrinsic pathway)^[4]. Nevertheless, several discoveries shall be done to understand the activation of this caspase-9 and the active site interaction. In this purpose, a researcher team engineered a caspase-9: this engineering caspase-9 has been mostly used for a re-evaluation of the induced proximity model for caspase activation.

Engineering Caspase-9

General Structure

Before its activation, the caspase 9 exists primarily as a monomer in solution. Its activation need a multimeric adaptator complex called apoptosome. The apoptosome is composed of seven molecules of Apaf-1 bound together with one molecule of cytochrome c in the presence of ATP/dATP. The Apaf-1 molecules contain a CARD domain (caspase recruitment domain) which interacts with a pro-domain containing an homotypic motif called the CARD domain of pro-caspase-9^[1]. In the engineering caspase-9, there is not CARD domain (it has been removed). This may allow the dimerization and activation of 2 monomers of caspase-9 via the association with the apoptosome. However, it's still uncertain to this day, three different models are still discussed^[2][3].

In this crystal structure, the engineering dimeric caspase-9 has a molecular weight of 60kDa and each monomer is a 278 amino acids length (position 140 to 416) (Δ 139 corresponding to the remove of the pro-domain and the flexible linker). The crystal structure shown here correspond to an homotetramer of caspase-9. The asymmetric unit of the crystal contain (actually this structure does not correspond to the physiological structure)^[2]. The four caspase-9 are represented by the chain A, B, C, D.

This engineering caspase-9 is composed of 2 monomers of caspase-9 (homodimer). A monomer consists of one (position 160 to 304) and one (position 334 to 416)^[4]. Each monomer is oriented in an asymmetric conformation. A molecule of is present in the active site of the chain A. The binding of D-Malate at the engineering caspase-9 is possible according to the creation of an hydrogen bond between the side chain amino group of arginine 355 residue and one of the oxygen of D-Malate acid carboxylic fonction.

Dimer Formation

In each monomer of caspase-9, there are arranged in a β-sheet structure surrounding by α-helices^[2]. ß-sheets from each homodimer interaction resulting in a . Those β strands are located in the hydrophobic core of each monomer of caspase-9. The interaction between 2 monomers of caspase-9 is possible according to the interaction between 2 of the 6 β strand, called respectively . The β6 and β6’ strand are present with a variable constitution of amino acid in each caspase. In the engineering caspase-9, the β6 strand is composed by the in position 402 to 406^[2]. Those amino acids changes do not alter the conformation of the protein. For the wild-type caspase-9, the presence of Phe404 on strand β6 create a steric hindrance for the dimerization which is not the case with the engineering caspase-9. Therefore, it clearly seems that the variation of residues on this strand may likely contribute to the ability to form a dimer structure. The Phe404 is not present in the engineering dimer of caspase-9 so it does not contribute to the formation of the dimeric asymetric structure^[2].

Active site

The active site is located on the surface of each monomer of caspase-9 because it needs an (hydrophilic solvent) due to its function, hydrolysing the effector pro-caspase 3. There is therefore H20 molecules within the actif site, however it is not represent in the crystal structure. Moreover, the actif site is composed of 4 loops, labelled L1 to L4^[2] which form the active site of all monomer caspases including caspase-9. The catalytic cysteine is located at the beginning of the L2 loops at the (Cys287->Ser287 because of a crystallization issue)^[2]. In the dimeric form, caspase-9 has a better catalytic activity because of the presence of two active sites (one in each monomer) in an opposite position (asymmetry of the dimer). Indeed, the support of the L2' critical loop in the other monomer is crucial to have a catalytic activity^[2].

Function of caspase-9

Consequently to the caspase-9 activation, this initiator protein will associate with its substrate and activate it by cleavage. The substrate of caspase-9 is equivalent to an effector protein: it is the protein caspase-3. This protein is present downstream of caspase-9 in the mitochondrial cell death pathway and it is also present as a pro-enzyme before its activation. Once caspase-9 is activated (as we explained previously), it will cleave pro-caspase 3 in a specific sequence. This sequence is characterize by a high conserved motif : it correspond to a sequence of Leu-Gly-His-Asp-|-Xaa which Xaa represent any amino acid. This sequence had absolutely an asparagine at position P1 (caspase definition) and with a preference for His at position P2 ^[5]. The hydrolysis is carry out between the asparagine residue and the X residue. After all is said and done, caspase 3 activated amplify the cell disassembly signal, cleaving crucial cellular proteins leading to apoptosis.

References

1. ↑ ^{1.0 1.1} Bao Q, Shi Y. Apoptosome: a platform for the activation of initiator caspases. Cell Death Differ. 2007 Jan;14(1):56-65. Epub 2006 Sep 15. PMID:16977332 doi:http://dx.doi.org/10.1038/sj.cdd.4402028
2. ↑ ^{2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8} Chao Y, Shiozaki EN, Srinivasula SM, Rigotti DJ, Fairman R, Shi Y. Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation. PLoS Biol. 2005 Jun;3(6):e183. Epub 2005 May 10. PMID:15941357 doi:10.1371/journal.pbio.0030183
3. ↑ ^{3.0 3.1} Hu Q, Wu D, Chen W, Yan Z, Yan C, He T, Liang Q, Shi Y. Molecular determinants of caspase-9 activation by the Apaf-1 apoptosome. Proc Natl Acad Sci U S A. 2014 Nov 18;111(46):16254-61. doi:, 10.1073/pnas.1418000111. Epub 2014 Oct 13. PMID:25313070 doi:http://dx.doi.org/10.1073/pnas.1418000111
4. ↑ ^{4.0 4.1} Yuan S, Yu X, Asara JM, Heuser JE, Ludtke SJ, Akey CW. The holo-apoptosome: activation of procaspase-9 and interactions with caspase-3. Structure. 2011 Aug 10;19(8):1084-96. doi: 10.1016/j.str.2011.07.001. PMID:21827945 doi:http://dx.doi.org/10.1016/j.str.2011.07.001
5. ↑ Blasche S, Mortl M, Steuber H, Siszler G, Nisa S, Schwarz F, Lavrik I, Gronewold TM, Maskos K, Donnenberg MS, Ullmann D, Uetz P, Kogl M. The E. coli effector protein NleF is a caspase inhibitor. PLoS One. 2013;8(3):e58937. doi: 10.1371/journal.pone.0058937. Epub 2013 Mar 14. PMID:23516580 doi:http://dx.doi.org/10.1371/journal.pone.0058937