Found at high concentrations in the brain and bordering tissues, Caspase-6 has been implicated in several neurological diseases including Alzheimer's and dementia. It's primarily involved in apoptosis through a largely ambiguous mechanism. It is classified as an [1]endopeptidase as it cleaves an internal peptide bond of its substrate. It has relatively low specificity in the binding site which allows for a variety of substrates, including other caspase enzymes to bind. Furthermore, it is a part of the cysteine aspartate family, which have these critical amino acid residues in the active site of the enzyme. Caspase-6 has both an inactive zinc-bound conformation and an active ligand-bound conformation, which are largely regulated by variations in zinc concentration.
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Structure
Active Site
Caspase 6 is a part of the cystine aspartic protease family that cleaves proteins at the TEVD sequence. in its monomeric form with protein ligand bound, its catalytic residues are , , and .
Cystine Aspartase mechanism
Together, these residues form a that cleaves a .
Zinc Exoside
Activation of Caspase-6
Caspase-6 has a small pro-domain. It shares 41% sequence identity with Caspase-3 and 37% sequence identity with Caspase-7. Both of these caspases are classified as effectors and because of it's similarites to these other Caspases, Caspase-6 is also classified as an effector. Caspase-6, does however, have many unique features compared to the other effectors, it has similar substrate specificity to that of initiator Caspases-8 and -9. Inhibitors of Apoptosis or IAPs, which are known to inhibit Caspase-3, -7, and -9, do not inhibit Caspase-6. Caspase-6 is known to undergo self-processing and activation in vitro and in vivo. High activity of Caspase-6 protein do not induce apoptosis of in HEK293 cells. Caspase-6 also has a relatively low zymogenicty, which is the ratio of activity for cleaved protein to the activity of uncleaved protein, of about 200, which is comparable to the zymogenicities of Caspase-8 and Caspase-9, which are both classified as initiator caspases. Caspase-6’s zymogenicity is also much lower than Caspase-3, which is another effector. This is interesting because the Caspase-6 protein shows low activity when it is not cleaved, similar to the initiator caspases, but Caspase-3, another effector, has basically no zymogen activity. Caspase-6 is classified as an effector, but it can also act as an initiator and cleave Caspases-2 and -8. It can also induce the mitochondrial membrane to become permeable, which leads to cytochrome c release and activation of other effector caspases.
Activation of Caspase-6
It is expressed as a dimeric zymogen, it contains a short prodomain, a large subunuit, known as p20, an intersubunit linker, and a small subunit, known as p10. Caspase-6 contains three cleavage sites, the first following the residues TETD23 that follows the prodomain, the next sit follows the residue sequence DVVD179, and the third cleavage site falls within the intersubunit linker following the sequence TEVD193. To activate effector caspases there must be a cleavage at the intersubunit linker, which releases the N terminus of p10, the N terminus then rotates about 180⁰ to form a loop bundle with the four other loops of an adjacent catalytic unit, this stabilizes the substrate binding pockets. It is sufficient to cleave either or both of the intersubunit linkers to activate Caspase-6. It is also important to point out that the prodomain of Caspase-6 inhibits in vivo. Caspase-6 can either undergo autoactivation or it can be activated by Capase-3, but the patterns for these two modes of activation are different. When Caspase-6 is self-activating it loses the prodomain first by cleavage at TETD23. Then it self-cleaves at TEVD23, which results in the formation of the loop bundle. The final cleavage of autoactivation is at DVVD179. In comparison, when Caspase-3 is activating Caspase-6 the first cleavage is at DVVD179, then the next cleavage is at TETD23, and the final cleavage occurs at TEVD193.
Autoactivation Mechanism of Caspase-6
It has been found that Caspase-6 can undergo activation without any other caspases, in vivo and in vitro, so there is a proposed intramolecular self-cleavage mechanism for Caspase-6. The intramolecular cleavage of TEVD193 is essential for the initiation caspase-6 activation without Caspase-3 present. The prodomain somehow inhibits the intramolecular cleavage of TEVD193, but currently the mechanism for this is unknown. The TETD23 and TEVD193 cleavage sites are similar, but the TETD23 cleavage site is always cleaved before TEVD193. This indicates that the TETD23 cleavage site is always more readily available for cleavage. The result of the TETD23 cleavage site priority is that the prodomain acts as a “suicide protector”, which protects the TEVD193 cleavage site from intermolecular self-cleavage. This protection is useful when there are low levels of protein, such as when it is in vivo, it also helps explain why the prodomain inhibits self-activation in vivo, but not in vitro.
Subunits involved in activation
Self cleavage
Inhibition
- Zinc Inhibition
Primary inhibition of Caspase-6 occurs when a zinc ion binds to the exosite containing Lys-36, Glu-244, and His-287 of the active dimer. In addition to these residues, the zinc interacts with one water molecule from the cytoplasm. It has been proposed that helices of the active dimer must rotate or move in some other way to provide these ideal interactions with zinc. This subtle shift is most likely the cause for allosteric inhibition. As the helices move to bind zinc, the amino acids of the active site become misaligned. The altered positions of the amino acids no longer provide ideal interactions for incoming substrates. After zinc binds, no new substrates enter the active site. Thus, Caspase-6 is effectively inhibited.
- Phosphorylation
The function of Caspase-6 can be inhibited by phosphorylation of Ser-257. The exact mechanism of this reaction remains unidentified at the time of publication, but proceeds when ARK5 kinase is present. This modification can occur before and after zymogen activation or auto-processing. The phosphoryl group inhibits Caspase-6 through steric interference. When Ser-257 is phosphorylated, the amino acid residue interacts with Pro-201, causing a shift in the helices of Caspase-6. The shift misaligns and disrupts residues found in the active site. This conformational difference prevents the inter-subunit loop from entering during zymogen activation and the self-cleaved active dimer cannot be formed. Additionally, no new substrate is able to enter the active site.
- Zymogen Activation
Zinc Inhibition
Phosphoylation
Function
Caspase-6 involvement in Alzheimer's Disease
Caspase-6 activity is associated with the formation of lesions within the Alzheimer's Disease (AD) and they can become present very early on during the disease's progression. Proapoptotic protein p53 is present at increased levels within AD brains, which seems to directly increase the transcription of Caspase-6. Treatments of Alzheimer's include targeting active Caspase-6 proteins because staining has found active Caspase-6 within the hippocampus and cortex of the Brain within in mild, moderate, and severe cases of AD, which indicates that Caspase-6 plays a predominate role in the pathophysiology of Alzheimer's. There has been research conducted that shows activation of Caspase-6 in AD could cause disruption of the cytoskeleton network of neurons, which then causes handicapped synaptic plasticity.
Luke's free space
If is , the activity of this protein is inhibited.
If binds to the protein, the activity of the active site is inhibited.
Inactive state of caspase 6:
Relevance
