H. sapiens Caspase-3
Alex Seaver '17 and Coire Gavin-Hanner '18
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. 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). Caspase-3 is a key executioner enzyme that, in
addition to Caspase-7, is necessary for apoptosis and normal mammalian
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
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. There are two
pathways that activate Procaspase-3, the extrinsic and intrinsic.
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
The mature Caspase-3 is a
homodimer of heterodimers, each formed from the
subunits of the procaspase. At their core, each heterodimer has
surrounded by five
. The heterodimers are held together mostly by
interactions. Some electrostatic interactions are also present.
IV. Active Site
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. 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
and an imidazole ring on
in the large subunit[2,3].
V. Binding and Catalytic Activity
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. The aspartic acid side
chain forms H-bonds with
in the p17 subunit
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
with a reverse turn over the
. 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). When an appropriate
substrate binds to the active site. Caspase-3 cleaves directly
after the P1 aspartic acid.
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
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. Inhibition has been shown to stabilize the mature caspase-3 enzyme.
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
). Diseases that involve a lack of apoptosis (
) 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.
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