Caspase 9

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Introduction

There are two best-described apoptotic pathways in mammals, the receptor-induced pathway, and the mitochondrial stress-induced pathway, which are also called the extrinsic and the intrinsic pathways, respectively. These two apoptotic pathways use the caspase cascade. The apoptotic pathways realize the dissolution of cell structure by executioner proteases known as caspases, which are usually divided into initiator and effector caspases according to their position in the apoptotic signal transduction. Procaspases are initially synthesized as a single-chain inactive zymogen, which promotes cell death when activated. Procaspase-9 exists as a monomer and serves as an initiator caspase of the intrinsic apoptotic pathway. It has a long prodomain that contains a caspase activation domain (CARD) motif through which caspase-9 is activated and then breaks up cells into apoptotic bodies. Due to its vital function, it can convert the death signal into the first proteolytic event and directly mediate the activation of the lethal executioner protease. Its manipulation of upstream events, especially the control of caspase-9, makes it a promising therapeutic target for proliferative and degenerative diseases.  

Structure and activation of caspase-9

The CARD motif at the N-terminus of the long prodomain in caspase-9 can selectively bind to the CARD in Apaf-1. Following this is a linker loop that connects the prodomain and the catalytic domain composed of large and small subunits (Figure 1B). The long linker loop between subunits is supposed to be movable and gain access to the active site without being cleaved due to its length and function of connecting large and small subunits. The dimerization of caspase-9 leads to rapid autocatalytic cleavage, producing caspase-9 (p35/p12).

Two hypotheses have been proposed for the activation of caspase-9. One is the "induced conformation model", which means that Apaf-1 apoptosome altersposi the conformation of caspase-9 by binding, which is necessary for the caspase-9 activation. The crystal structure of 1:1 complex between the CARD domains of caspase-9 and Apaf-1 demonstrates the complementary interface essential for caspase-9 activation. The other hypothesis is the "induced proximity model", which assumes that apoptosome provides a platform for the dimerization of caspase-9. Caspase 9 must maintain its binding to apoptosome to retain its catalytic activity. Recent studies have shown that procaspase-9 is more affinitive with the apoptosome in contrast to its cleaved form. In addition, the purpose of procaspase-9 autoprocessing is not to activate caspase-9, but to start a molecular timer to stimulate the duration of apoptosome activity. Although these new findings are contradictory and controversial to a certain extent, they promote the development of our cognition in the mechanism that controls the activation and the activity of caspase-9.

Figure 1. The predicted amino acid structure of human caspase-9 (Li, P.; et al. 2000)

Inducible caspase-9 suicide gene and its application

The inducible caspase 9 (iCasp9) provides a mechanism to eliminate CAR T cells without proper activation, and has been proven by clinical trials to be a novel suicide gene. The optimized iCasp9 molecule is based on the drug binding domain FK506-F36V, which is linked to ΔCaspase9 (a caspase-9 without its physiological CARD) through a short Ser-Gly-Gly-Gly-Ser linker. AP1903 or AP20187 acts as a chemical inducer of dimerization (CID) binds to iCasp9 and activates the dimerization of ΔCaspase9, so that the downstream caspase cascade is activated, leading to apoptosis. The iCasp9 safety switch has been clinically validated in haploidentical hematopoietic stem cell transplantation (haplo-HSCT). In the preclinical study of this system, the severe combined immunodeficiency (SCID) mouse-human xenograft model was studied, in which the dose of CID killed more than 99% of circulating human GFP + T cells within three days. A study provides strong support that this iCasp9-based conditional safety switch can quickly remove T cells in a murine model and prevent ongoing autoimmune attacks. In a clinical trial study, five recipients with relapsed acute leukemia who had undergone stem cell transplantation were treated with infusions of iCasp9-expressing donor T cells. In four patients with graft-versus-host disease (GVHD), the iCasp9 suicide switch cleared more than 90% of engineered T cells and did not reoccurrence half an hour after the administration of a single dose of CID. Long-term follow-up (3.5 years) showed that iC9-T cell infusion and the administration of CID effectively helped the patient rebuild the immune system after the haplo-HSCT and prevented the patient from pathogenic infections. In general, the iCasp9 suicide gene technology has shown significant advantages in improving the safety of CAR T cell technology and expanding its clinical application.    

Figure 2. The apoptosis of transduced cells incurred by activated iCasp9 (Li, P.; et al. 2000)