Caspase-3 Regulatory Mechanisms

IntroductionIntroduction

Caspases are cysteine-dependent aspartic acid proteases and are the key facilitators of apoptosis or programmed cell death. Apoptosis is tightly regulated by these caspases, and dysregulation of caspase functions have been implicated in wide variety of diseases such as neurodegeneration, cancer, heart disease and some metabolic disorders. As such, caspases are considered to be attractive drug targets to treat these disorders.

Existing as proenzymes, caspases undergo proteolytic processing at conserved aspartate residues in their intersubunit linker to produce the large and small subunit. These subunits then dimerize to form the active enzyme. Any apoptotic signal received by the cell results in sequential activation of caspases. Upstream or initator caspases (-2,-8, -9 and -10) are first activated by forming a holoenzyme wherein they associate with another protein platform or adaptor protein. Once active, initiator caspases cleave and activate the executioner caspases (-3, -6 and -7) which in turn cleave their respective protein targets initiating cell death.

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Caspase-3 structure

File:Casp-3 with subs.png

Caspase-3 RegulationCaspase-3 Regulation

Regulation of Caspase-3 Exosite and Allosteric Site Caspases have similar structure of active site. Exosite that could be utilized to improve activity has been found in caspase-7 (Boucher, Blais et al. 2012). Caspase-7 also has an inhibitory allosteric site that could bind with small molecule FICA, presenting a zymogen-like conformation (Hardy, Lam et al. 2004). Although there is no evident exosite found in caspase-3, some allosteric sites, (most of which are located on the dimer interface,) has been studied by mutagenesis. Some of mutant residues can modulate the activity of caspase-3 or even procaspase-3. The procaspase-3 was detected only little activity because the orientation of ILA (prematured L2 loop) and ILB loop cannot form an active site pocket (Bose, Pop et al. 2003). V266E is a mutation that improves caspase-3 activity dramatically. Even in the uncleavable procaspase-3 (D5A, D26A, D175A), V266E mutant zymogen is also pseudo-activated (60-fold activity). Interestingly, V266E does not change a lot conformation around active site in the active caspase-3. Based on the crystal structure, L2’ loop is partially disorder at 185’-180’. This active procaspase-3 cannot be inhibited by endogenous XIAP like normal cleaved caspase-3. So it provides us an option for apoptosis stimuli with intrinsic efficiency. It was found recently that many other mutant residues on the dimer interface might play an important role on inhibition of caspase-3 through manipulating the hydrogen bond or remote talking across whole dimer, like V266H, Y197C, E124A. Natural Inhibitors X-linked inhibitor of apoptosis proteins (XIAP) contains the second baculovirus IAP repeat domain (BIR2) targeting caspase-3 and caspase-7. Reference Bose, K., C. Pop, et al. (2003). "An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3." Biochemistry 42(42): 12298-12310. Boucher, D., V. Blais, et al. (2012). "Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysis." Proc Natl Acad Sci U S A 109(15): 5669-5674. Hardy, J. A., J. Lam, et al. (2004). "Discovery of an allosteric site in the caspases." Proc Natl Acad Sci U S A 101(34): 12461-12466.

spinqualitylabelsall models Structure of Caspase-3 with substrate bound (PDB entry 2H5I) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Caspase-3 Active Site and Loop Bundle AnalysisCaspase-3 Active Site and Loop Bundle Analysis

Importance of Loop Orientation Caspases are extremely dependent on the orientation and geometry of their active site loops. If the loops are not ordered properly the enzyme fails to function. Caspase-3 has four active site loops on each half of the dimer constituting the active site bundle. Proteolytic activity is dependent on cleavage of an intersubunit linker, which releases loop 2 (L2) and L2’. . This allows L2 to make critical contacts with L3 and L4, allowing them to organize the active site, bind substrate, and orient the nucleophilic cysteine 163 (bright green) so that it can cleave after aspartate residues. Taking a closer look at L2 and L2’ we can see a critical interaction involving on L2. This residue makes two hydrogen bonds with backbone amides of V189’ and E190’, stabilizing L2 in the proper position. This reinforcement allows L2 to contact L3 so as to twist the active site cysteine into the proper orientation to attack the substrate. In addition, L2 can now contact L4 at K260. This secures L4 and allows it to make contacts in the P4 position, which greatly influence substrate specificity. Caspase-3 Active Site The active site of caspase-3 utilizes a cysteine-histidine dyad, which has an exquisite specificity for cleaving after aspartate residues. Therefore, caspase-3, by definition, will have an aspartate in the pocket. Uncleavable peptide substrates are often used in crystallography to bind to the active site. This will orient the delicate but deadly active site loops in order to facilitate the visualization of the chemistry of cleavage. The nucleophilic Cysteine 163 will work in concert with the second active site residue, Histidine 121, to attack the substrate. This reaction will ultimately cleave the peptide bond following the aspartate. In order to be active and cleave the specific apoptotic targets, Caspase-3 must be able to first bind substrate. There are several essential interactions responsible for securing the substrate before cleavage. The binding pocket at is a hydrophobic patch made up of Y204, W206, and F250 (dark blue residues). This creates a hydrophobic pocket for the P2 residue (in this casse, valine). At there are contacts that contribute to the specificity of caspase-3. Asparagine 208 hydrogen bonds with an aspartate at P4 along with the backbone nitrogen of F250, creating a preference for a carboxylic acid at the P4 site.

spinqualitylabelsall models Structure of Caspase-3 (PDB entry 2H5I) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image