Caspase-3 Regulatory Mechanisms: Difference between revisions

== Introduction ==

[[Image:Apop.png | thumb| Caspases in the apoptotic pathway]]

Caspases are '''''c'''''ysteine-'''''asp'''''artic acid prote'''''ases''''' and are key protein facilitators for the faithful execution of apoptosis or programmed cell death. Dysregulation in the apoptotic pathway has been implicated in a variety of diseases such as neurodegeneration, cancer, heart disease and some metabolic disorders.  Because of the crucial role of caspases in the the apoptotic pathway, abnormalities  in their functions would cause haywire in the apoptotic cascade and can be deleterious to the cell.  Caspases are thus being considered as therapeutic targets in apoptosis-related diseases.

Any apoptotic signal received by the cell causes the activation of initiator caspases (-8 and -9) by associating with other protein platforms to form a functional holoenzyme.  These initiator caspases then cleave the executioner caspases -3, -6, and -7.  Caspase-3 specifically functions to cleave downstream apoptotic targets as well as both caspase-6 and -7, which in turn cleave their respective targets to induce cell death.  Aside from being able to activate caspase-6 and -7, caspase-3 also regulates caspase-9 activity, operating via a feedback loop.  This dual action of caspase-3 confers its distinct regulatory mechanisms, resulting in a wider extent of its effects in the apoptotic cascade.

== Overview of Caspase-3 Structure ==

===Dimer Formation ===

The active pocket of caspase-3 is defined by <scene name='Sandox_Bay_Serrano/Monomer/2'>Cys-163 and His-121</scene>.  Binding of a <scene name='Sandox_Bay_Serrano/Scene01_substrate/4'>substrate</scene>, such as DEVD-CHO to the active site of the enzyme induces a conformational change that allows the L2 and L2' loops to interlock and stabilize the active site <scene name='Sandox_Bay_Serrano/Scene01_substrate/3'></scene>.  Like caspase-7, caspase-3 recognizes a Asp-X-X-Asp sequence as a cleavage site in its protein substrates.

== Caspase-3 Loop Bundle and Active Site==

=== 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’. <scene name='Caspase-3_Regulatory_Mechanisms/Scene2_nospin_labels/1'>L2'(green spheres) interacts with the opposite half of the dimer by holding up L2 (blue spheres) </scene>. 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.  

In order to be active and cleave 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 <scene name='Caspase-3_Regulatory_Mechanisms/P2/2'>P2</scene> 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 case, valine), helping it stick to the protein. At <scene name='Caspase-3_Regulatory_Mechanisms/P4/2'>P4</scene> 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.  

== Caspase-3 Regulation==

=== Exosite and Allosteric Site===

X-linked inhibitor of apoptosis proteins (XIAP) contains the second baculovirus IAP repeat domain (BIR2) targeting caspase-3 and caspase-7. The <scene name='Caspase-3_Regulatory_Mechanisms/Bir2/2'>BIR2</scene> domain sits directly in the active site of caspase-3 and completely inhibits the protein. The region binding the active site runs in the opposite direction of normal caspase-3 substrates, thus occupying P1 through P4 but avoiding cleavage by the protease. This unique method of inhibition is a critical regulatory mechanism used in cells to control apoptotic caspase activity.

=References=

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.