User:Kévin Roger/Sandbox 953
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Caspases correspond to an abridged version of '''c'''ysteine-'''asp'''artic acid prote'''ases'''. These proteins are a part of a cysteine protease family which cleaves target proteins after an '''aspartate residue'''<ref name="apoptosome">PMID: 16977332</ref>. 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 correspond to an abridged version of '''c'''ysteine-'''asp'''artic acid prote'''ases'''. These proteins are a part of a cysteine protease family which cleaves target proteins after an '''aspartate residue'''<ref name="apoptosome">PMID: 16977332</ref>. 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 | + | 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)<ref name="EngCasp9">PMID: 15941357</ref>. 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<ref name="MDCasp9">PMID: 25313070</ref>. 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 | + | 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)<ref name="HoloApop">PMID: 21827945</ref>. 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. |
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=== General Structure === | === 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 cytochrome c in the presence of ATP/dATP. The Apaf-1 molecules contain a CARD domain (caspase recruitment domain) which | + | 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<ref name=" apoptosome" />. 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<ref name=" EngCasp9" /><ref name=" MDCasp9" />. |
| - | 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''' | + | 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 <scene name='67/676986/Homotetramer/1'>2 molecules of the dimeric caspase-9 </scene>(actually this structure does not correspond to the physiological structure)<ref name=" EngCasp9" />. 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 <scene name='67/676986/P10/1'>p10 subunit</scene> (position 160 to 304) and one <scene name='67/676986/P20/2'>p20 subunit</scene> (position 334 to 416)<ref name=" HoloApop" />. Each monomer is oriented in an asymmetric conformation. A molecule of <scene name='67/676986/D-malate/4'>D-Malate</scene> is present in the active site of the chain A. The binding of D-Malate at the engineering caspase-9 is | + | This engineering caspase-9 is composed of 2 monomers of caspase-9 '''(homodimer)'''. A monomer consists of one <scene name='67/676986/P10/1'>p10 subunit</scene> (position 160 to 304) and one <scene name='67/676986/P20/2'>p20 subunit</scene> (position 334 to 416)<ref name=" HoloApop" />. Each monomer is oriented in an asymmetric conformation. A molecule of <scene name='67/676986/D-malate/4'>D-Malate</scene> 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 === | === Dimer Formation === | ||
| - | In each monomer of caspase-9, there | + | In each monomer of caspase-9, there are <scene name='67/676986/Beta-strands/1'>6 β-strands</scene> arranged in a '''β-sheet structure''' surrounding by α-helices<ref name=" EngCasp9" />. ß-sheets from each homodimer interaction resulting in a <scene name='67/676986/12_beta-strands/1'>large ß-sheet structure composed of 12 ß strains</scene>. 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 <scene name='67/676986/Strand_6/1'>β6 and β6’</scene>. The β6 and β6’ strands are present with a '''variable constitution''' of amino acid in each caspase. In the engineering caspase-9, the β6 strand is composed by the <scene name='67/676986/Civsm/1'>CIVSM amino acids</scene> in position '''402 to 406'''<ref name=" EngCasp9" />. 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'''<ref name=" EngCasp9" />. |
=== Active site === | === Active site === | ||
| - | The active site is located on the '''surface''' of each monomer of caspase-9 because it needs an <scene name='67/676986/Hydrophilic_solvent/1'>hydrophilic environment</scene> (hydrophilic solvent) due to its function, '''hydrolysing''' the effector pro-caspase 3. There is therefore H20 molecules | + | The active site is located on the '''surface''' of each monomer of caspase-9 because it needs an <scene name='67/676986/Hydrophilic_solvent/1'>hydrophilic environment</scene> (hydrophilic solvent) due to its function, '''hydrolysing''' the effector pro-caspase 3. There is therefore H20 molecules in the actif site, however it is not represented in the crystal structure. Moreover, the actif site is composed of 4 loops, '''labelled L1 to L4'''<ref name=" EngCasp9" /> which form the active site of all monomer of caspase including caspase-9. The catalytic cysteine is located at the '''beginning of the L2 loops''' at the <scene name='67/676986/Catalytic_amino_acid/1'> position 287 remplaced in the crystal structure of dimeric caspase by a serine </scene> (Cys287->Ser287 because of a crystallization issue)<ref name=" EngCasp9" />. 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). The support of the L2' critical loop in the other monomer is crucial to have a catalytic activity<ref name=" EngCasp9" />. |
==Function of caspase-9== | ==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 | + | 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, it will cleave pro-caspase 3 in a specific sequence. This sequence is characterize by a '''high conserved motif''' : it corresponds to a sequence of '''Leu-Gly-His-Asp-|-Xaa''' where Xaa represents any amino acid. This sequence had absolutely an '''asparagine''' at '''position P1''' (caspase definition) and a preference for '''histidine''' at '''position P2''' <ref name="inhibitor">PMID: 23516580</ref>. The hydrolysis is carry out between the asparagine residue and the X residue. After all is said and done, the activated caspase 3 '''amplify the cell disassembly signal''', cleaving crucial cellular proteins leading to '''apoptosis'''. |
== References == | == References == | ||
<references/> | <references/> | ||
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