Caspase-9 is anenzyme that in humans is encoded by theCASP9gene. It is an initiatorcaspase,[5] critical to the apoptotic pathway found in many tissues.[6] Caspase-9 homologs have been identified in all mammals for which they are known to exist, such asMus musculus andPan troglodytes.[7]
Similar to other caspases, caspase-9 has three domains: N-terminal pro-domain, large subunit, and a small subunit.[8] The N-terminal pro-domain is also called the long pro-domain and this contains the caspase activation domain (CARD)motif.[11] The pro-domain is linked to the catalytic domain by a linker loop.[12]
The caspase-9monomer consists of one large and one small subunit, both comprising thecatalytic domain.[13] Differing from the normally conserved active site motif QACRG in other caspases, caspase-9 has the motif QACGG.[14][12]
When dimerized, caspase-9 has two different active site conformations within eachdimer.[13] One site closely resembles the catalytic site of other caspases, whereas the second has no 'activation loop', disrupting the catalytic machinery in that particular active site.[13] Surface loops around the active site are short, giving rise to broad substrate specificity as the substrate-binding cleft is more open.[15] Within caspase-9's active site, in order for catalytic activity to occur there has to be specific amino acids in the right position. Amino acid Asp at position P1 is essential, with a preference for amino acid His at position P2.[16]
Caspase-9 in humans is expressed in fetus and adult tissues.[14][12] Tissue expression of caspase-9 is ubiquitous with the highest expression in the brain and heart, specifically at the developmental stage of an adult in the heart's muscle cells.[18] The liver, pancreas, and skeletal muscle express this enzyme at a moderate level, and all other tissues express caspase-9 at low levels.[18]
Active caspase-9 works as an initiating caspase by cleaving, thus activating downstream executioner caspases, initiating apoptosis.[19] Once activated, caspase-9 goes on to cleave caspase-3, -6, and -7, initiating the caspase cascade as they cleave several other cellular targets.[8]
When caspase-9 is inactive, it exists in the cytosol as azymogen, in its monomer form.[13][20] It is then recruited and activated by the CARDs in apaf-1, recognizing the CARDs in caspase-9.[21]
Before activation can occur, caspase-9 has to be processed.[22] Initially, caspase-9 is made as an inactive single-chain zymogen.[22] Processing occurs when the apoptosome binds to pro-caspase-9 as apaf-1 assists in the autoproteolytic processing of the zymogen.[22] The processed caspase-9 stays bound to the apoptosome complex, forming a holoenzyme.[23]
Negative regulation of caspase-9 occurs throughphosphorylation.[8] This is done by a serine-threoninekinase, Akt, on serine-196 which inhibits the activation and protease activity of caspase-9, suppressing caspase-9 and further activation of apoptosis.[25] Akt acts as anallosteric inhibitor of caspase-9 because the site of phosphorylation of serine-196 is far from the catalytic site.[25] The inhibitor affects the dimerization of caspase-9 and causes a conformational change that affects the substrate-binding cleft of caspase-9.[25]
Akt can act on both processed and unprocessed caspase-9 in-vitro, where phosphorylation on processed caspase-9 occurs on the large subunit.[26]
A deficiency in caspase-9 largely affects the brain and its development.[27] The effects of having a mutation or deficiency in this caspase compared to others is detrimental.[27] The initiating role caspase-9 plays in apoptosis is the cause for the severe effects seen in those with an atypical caspase-9.
Mice with insufficient caspase-9 have a mainphenotype of an affected or abnormal brain.[8] Larger brains due to a decrease in apoptosis, resulting in an increase of extra neurons is an example of a phenotype seen in caspase-9 deficient mice.[28] Thosehomozygous for no caspase-9 die perinatally as a result of an abnormally developedcerebrum.[8]
The effects of abnormal caspase-9 levels or function impacts the clinical world. The impact caspase-9 has on the brain can lead to future work in inhibition through targeted therapy, specifically with diseases associated with the brain as this enzyme may take part in the developmental pathways of neuronal disorders.[8]
The introduction of caspases may also have medical benefits.[19] In the context ofgraft versus host disease, caspase-9 can be introduced as an inducible switch.[31] In the presence of a small molecule, it will dimerize and trigger apoptosis, eliminatinglymphocytes.[31]
iCasp9 (inducible caspase-9) is a type of control system forchimeric antigen receptor T cells (CAR T cells). CAR T cells are genetically modifiedT cells that exhibitcytotoxicity totumor cells. Evidence shows that CAR T cells are effective in treatingB-cell malignancies. However, as CAR T cells introduce toxicity, user control of the cells and their targets is critical.[32] One of the various ways to exert control over CAR T cell is through drug-controlled synthetic systems. iCasp9 was created by modifying caspase-9 and fusing it with theFK506 binding protein.[32] iCasp9 can be added to the CAR T cells as an inducible suicide gene.[33]
If therapy with CAR T cells results in severe side effects, iCasp9 can be used to halt treatment. Administering a small-molecule drug such asrapamycin causes the drug to bind to the FK506 domain.[33] This, in turn, induces expression of caspase-9, which triggers cell death of the CAR T cells.[33]
Isoform 2 doesn't include exons 3, 4, 5, and 6; it is missing amino acids 140-289.[11][34] Caspase-9S doesn't have central catalytic domain, therefore it functions as an inhibitor of caspase-9α by attaching to the apoptosome, suppressing the caspase enzyme cascade and apoptosis.[11][35] Caspase-9β is referred to as theendogenous dominant-negative isoform.
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