H. sapiens Caspase-3

Alex Seaver '17 and Coire Gavin-Hanner '18


I. Introduction

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Cysteine Asp-Specific Proteases (CASPASEs) are enzymes that play important roles in apoptosis and inflammation. They all have a cysteine residue used in breaking of a peptide bond[8]. The caspases identified thus far can be organized into three groups: inflammation caspases (1, 4, 5, 11, and 12), initiator caspases (2, 8, 9, 10), and executioner caspases (3, 6, and 7)[1]. Caspase-3 is a key executioner enzyme that, in addition to Caspase-7, is necessary for apoptosis and normal mammalian life[1].
In healthy cells, caspases exist as inactive procaspases composed of a large subunit (p20), a small subunit (p10), and a prodomain of varying length. Once activated, the prodomains form a heterodimer with two subunits. Two heterodimers form a heterotetramer in a mature caspase. Caspases may play a role in many diseases such as Alzheimer's. Their implication in such diseases makes them very attractive targets for drugs.

II. Procaspase and Activation

In healthy cells Caspase-3 exists as inactive Procaspase-3, a homodimer made up of 17 kDa and 12 kDa subunits. The active site components are in a different conformation in Procaspase-3 than in the mature caspase, ensuring inactivity[7]. There are two pathways that activate Procaspase-3, the extrinsic and intrinsic[1]. The extrinsic pathway is activated by a TNFR1-binding substrate, a signal from outside of the cell to begin apoptosis. This pathway activates Caspase-8, an initiator caspase which can cleave the Procaspase-3 homodimer. The intrinsic pathway is activated from within the cell itself as a self-recognition of damage. The intrinsic pathway activates Caspase-9, an initiator capsase similar to Caspase-8 in function. The heterotetramer that makes up Procaspase-3 is held together through internal hydrophobic interactions.

III. General Structure

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The mature Caspase-3 is a homodimer of heterodimers, each formed from the and subunits of the procaspase. At their core, each heterodimer has six surrounded by five [5]. The heterodimers are held together mostly by interactions. Some electrostatic interactions are also present[5].

IV. Active Site

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In the formation of the mature Caspase-3, two Procaspase-3s are cleaved between the p17 and p12 subunits. The subunits come together to form a heterodimer with an active site, which can then self-catalyze the removal of the prodomain through cleavage[6]. Once the prodomain has been removed, the two heterodimers come together in a way such that the heterotetramer formed has two active sites at opposite ends of the molecule[2,4]. The active site itself is formed by four loops. It includes a sulfohydryl group on and an imidazole ring on in the large subunit[2,3].

V. Binding and Catalytic Activity

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The main function of Caspase-3 is to cleave the backbone of other proteins. It needs to be highly specific to avoid performing unwanted cleavage. It gets its specificity from a group of four positions (S1-4) that each recognize a member of a tetrapeptide sequence in the substrate (the P1 through P4 sites). All caspases recognize their substrates in this way, however the specific tetrapeptide sequence varies between caspases. Caspase-3 has a very restrictive pocket in the S1 position that the carboxylate side chain of aspartic acid fits into[4]. The aspartic acid side chain forms H-bonds with and in the p17 subunit and and in the p12 subunit. S2 and S3 are fairly tolerant of substitutions, however the S2 position does prefer a Glutamic acid. The S4 position is almost as stringent as S1 in its requirement of aspartic acid. It has a well-defined pocket formed mostly from and a with a reverse turn over the [4]. With all of the selective characteristics of the Caspase-3 binding site, it only recognizes substrates with the specific amino acid sequence DEXD (S4-S1)[1]. When an appropriate substrate binds to the active site. Caspase-3 cleaves directly after the P1 aspartic acid.

VI. Inhibition

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Most molecules designed to inhibit Caspase-3 rely on the P1-P4 tetrapeptide sequence to reliably bind to caspase-3. Reversible must have an electrophilic component that can attack the Aldehydes, ketones, and nitriles can be used to make reversible inhibitors. Irreversible inhibitors form covalent adducts on the active site. One family of compounds that can perform such a role is the (alcyloxy) methylketone family[4]. Inhibition has been shown to stabilize the mature caspase-3 enzyme[6].

VII. Applications

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The central role that Caspase-3 plays in apoptosis, makes the molecule an attractive target in treatment of many diseases. Synthetic, small-molecule inhibitors my be able to be used to treat certain diseases that involve an excess of apoptosis (Alzheimer's and Huntington's ). Diseases that involve a lack of apoptosis ( Cancer ) are more difficult to treat using therapy designed to affect caspase-3, however it may be possible to selectively activate caspase-3 in cancer cells only[3]

VIII. References

[1] Fu, Guoxing. Structure Based Study of Caspase-3 and D-Arginine Dehydrogenase. Retrieved from UMI 3547084.

[2] Ganesan, Rajkumar, Jelakovic, Peer R.E.M.S., and Grutter, Markus G. 2006. Extended Substrate Recognition in Caspase-3 Revealed by High Resolution X-ray Structure Analysis. Journal of Molecular Biology. 359:1378-1388

[3] Lavrik, Inna N., Golks, Alexander, and Krammer, Peter H. 2005. Caspases: Pharmocological Manipulation of Cell Death. The Journal of Clinical Investigation. 115(10):2665-2672.

[4] Nicholson, DW. 1999. Caspase Structure, Proteolytic Substrates, and Function During Apoptotic Cell Death. Cell Death and Differentiation. 6:1028-1042.

[5] Sulpizi, M., Rothlisberger, U., and Carloni, P. 2003. Molecular Dynamics Studies of Caspase 3. Biophysical Journal. 84:2207-2215.

[6] Tawa, P., Hell, K., Giroux, A., Grimm, E., Han, Y., Nicholson, DW., and Xanthoudaxis, S. 2004. Catalytic Activity of Caspase-3 Is Required for Its Degredation: Stabalization of the Active Complex by Synthetic Inhibitors. Cell Death and Differentiation. 11:439-447.

[7] Thomsen, Nathan D., Koerber, James T., and Wells, James A. 2013. Structural Snapshots Reveal Distinct Mechanisms of Procaspase-3 and -7 Activation. PNAS. 110(21):8477-8482.

[8] Watt, William, Koeplinger, Kenneth A., Mildner, Ana M., Heinrikson, Robert L., Tomaselli, Alfredo G., and Watenpaugh, Keith D. 1999. The Atomic-Resolution Structure of Human Caspase-8, a Key Activator of Apoptosis. Structure. 7(9): 1135-1143.

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