Protein Modeling/Apoptosis

The 2012 topic of Protein Modeling deals with cell apoptosis. The pre-build model is of caspase-3.

See the CBM webpage for more details.

Caspase-3
Caspase-3 is the pre-build model for 2012, based on file 1I3O.pdb.

Caspase-3 is a cysteine-aspartic acid protease, known as an "executioner" protein because of the direct role it plays in dismantling other cell proteins during apoptosis. It is inhibited by XIAP, and it acts upon PARP, among other proteins. In particular, its substrates include structural proteins such as spectrin, actin, and gelsolin, as well as endonuclease inhibitors (inhibitors of proteins that cleave DNA) such as ICAD/DFF45.

The active form of caspase-3 is created when caspase-8 or caspase-9 cleaves procaspase-3, an inactive precursor, into a 17 kDa and a 12 kDa subunit (chains A and B in 1I3O.pdb, respectively). The active form of caspase-3 is a heterotetramer (two copies each of the 17 kDa and 12 kDa subunits), attached via the large beta sheet in the middle of the protein.

Its active site contains a cysteine which directly attacks the substrate, cleaving the target protein at an aspartic acid residue; hence the name "cysteine-aspartic acid protease". Caspase-3 targets the specific substrate sequence DXXD, meaning that it requires an aspartic acid at the P1 and P4 positions relative to the cleavage site (where D-X-X-D is P4-P3-P2-P1, because the caspase cleaves its substrate after the second D; the residue after P1 is known as P1'). The P2 and P3 positions (the two amino acids between the two aspartic acids) are less specific, but a polar residue capable of hydrogen bonding is preferred at the P3 position, and a hydrophobic residue is preferred at the P2 position.

The catalytic cysteine (CYS285 under the numbering system used in 1I3O) and a nearby histidine (HIS237 in 1I3O) participate in the proteolytic reaction together: HIS237 accepts a proton from CYS285, making CYS285 highly nucleophilic (wanting to donate an electron pair). The nucleophilic cysteine then forms a covalent bond with the carboxyl carbon (part of the backbone) of the substrate aspartic acid, creating a new amino terminus for one of the substrate fragments. The bond between the caspase cysteine and the substrate backbone is then hydrolyzed, creating the carboxy terminus of the other substrate fragment, and the fragments are released from the active site.

When functional caspase-3 is not present, cells do not exhibit the traditional hallmarks of apoptosis – DNA fragmentation, chromatin condensation, and membrane blebbing.

Diablo
Diablo is the onsite model for Invitationals.

The Diablo homolog is an inhibitor of XIAP.

XIAP
XIAP is the onsite model for regionals, also based on file 1I3O.pdb.

XIAP is the X-linked Inhibitor of Apoptosis Protein, which inhibits apoptosis by binding to Caspase-3 (and other caspases) to prevent them from cleaving their substrates. It is called "X-linked" because the gene responsible for it is found on the X-chromosome.

XIAP is a competitive inhibitor (for caspase-3, at least; it has other inhibition mechanisms for other proteins), in that it binds directly to the active site of caspase-3, blocking the binding of its usual substrates.

Its BIR2 domain (the part that binds directly to caspase-3) contains a zinc finger in which one histidine and three cysteines coordinate a zinc ion.

PARP
PARP, or poly(ADP-ribose) polymerase, is the onsite model for state tournaments. PARP is part of a DNA repair pathway: it flags single-strand breakages in DNA by attaching poly(ADP-ribose) (PAR) to nearby proteins, bringing the DNA repair proteins to the site.

PARP consists of a DNA binding domain, an "automodification domain" (an area in which PARP can attach PAR to itself, which allows it to detach from DNA when its function is completed), and a catalytic domain, which does the actual PAR synthesis and attachment. The file on which this year's state tournament on-site is based (3OD8.pdb) contains only the DNA binding domain of PARP.

During the apoptosis cascade, caspase-3 cleaves PARP into two fragments – a 24 kDa piece that contains the DNA binding domain, and a much larger, 89 kDa piece that contains the catalytic and automodification domains. By detaching the DNA binding domain from the catalytic domain, this cleavage effectively inactivates PARP.

Inactivation of PARP prevents DNA repair, which facilitates the nuclear disassembly (DNA fragmentation, chromatin condensation, etc) that accompanies apoptosis. It is hypothesized that PARP inactivation may allow endonucleases, the proteins responsible for dismantling the DNA in an apoptotic cell, to better access the chromatin; alternately, or possibly at the same time, the now-cleaved DNA binding domain of PARP has been shown to bind irreversibly to single-strand breakages, preventing DNA repair enzymes from accessing the damage.

Because PARP requires NAD+ as a substrate, overactivation (or lack of full inactivation) of PARP in apoptotic cells can lead to ATP depletion. Even a normal amount of PARP remaining in the cell (such as in experiments where cells were made to express a mutant PARP that could not be cleaved by caspase-3 but was otherwise normally functional) consumes a massive amount of NAD+ in a cell undergoing apoptosis, because despite the efforts of the DNA repair proteins, DNA breakages accrue at an accelerating rate. ATP depletion leads to cell death as well, but via necrotic pathways (which can cause tissue damage) rather than apoptosis.

MHC
MHC is the onsite model for nationals.

Apoptosis
Apoptosis is the programmed death of a cell. It is triggered by signals either from mitochondria (i.e., the release of cytochrome c) or extrinsic signals caused by exposure to radiation, etc. In the process of apoptosis, structural and DNA-repair proteins are broken down. It contrasts with necrosis, in which cells die because of an injury.

Apoptosis can occur in response to external conditions (toxins, radiation, etc), as part of a disease (such as cerebral ischemia, in which neurons undergo apoptosis inappropriately), or as part of normal development and differentiation (such as the death of skin cells from the "webbing" between the fingers in developing fetuses).

Mechanism
During apoptosis, mitochondria release SMACs, which are small mitochondria-derived activators of caspases. These bind to inhibitors of apoptotic proteins (IAPs) and deactivate them. Once this inhibition is removed, the mitochondrion can release cytochrome c, which forms a complex with other substances. This complex is called an apoptosome, which binds and cleaves procaspase-9 to form active caspase-9, beginning the caspase cascade. Caspase-9 and similar caspases early in the cascade are known as "initiator caspases" for this reason.

Other caspases, such as caspase-3 and caspase-7, are called "executioner caspases" because of their direct role in dismantling cell machinery. They cleave a variety of substrates, including structural and DNA repair proteins, as well as endonuclease inhibitors, allowing endonucleases (DNA enzymes) to break down chromatin into fragments. With its DNA breaking down, the nucleus condenses. Cleavage of structural proteins causes the cell membrane to form "blebs", or protrusions, which eventually break off from the cell as apoptotic bodies containing the remains of the dismantled organelles.

These apoptotic bodies are then consumed by macrophages, cells which break down cellular debris. In this way, apoptosis allows a cell to be dismantled without releasing potentially damaging contents into the environment of other cells.

Characteristics
Apoptosis is characterized by the shrinking of the cytoplasm and a dense appearance in the organelles. The cell membrane forms irregular protrusions called blebs, which become more pronounced until they break apart into smaller bodies called apoptotic bodies, which are consumed by other cells.