Caspase-3/Sandbox: Difference between revisions

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{{STRUCTURE_1qx3 | PDB=1qx3 | SCENE=Caspase-3/Sandbox/Unliganded_human_caspase-3/4 | CAPTION= Crystal Structure of Unliganded Human Caspase-3}}

Caspases are proteases that function via a cysteine residue in the active site to cleave substrates after aspartic acid residues. Caspases are crucial for the initiation (e.g. caspase-8, -9, -10) and execution (e.g. caspase-3, -6, -7) of apoptosis, or programmed cell death either via the intrinsic or extrinsic pathway (Degterev, Boyce et al. 2003). In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, ~~p18~~/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998).

Caspases are proteases that function via a cysteine residue in the active site to cleave substrates after aspartic acid residues. Caspases are crucial for the initiation (e.g. caspase-8, -9, -10) and execution (e.g. caspase-3, -6, -7) of apoptosis, or programmed cell death either via the intrinsic or extrinsic pathway (Degterev, Boyce et al. 2003). In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, p17/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998).

==Structure & Function==

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====Salt Bridge====

[[Image:Casp3Salt bridge.png|500px|left]]

Lys242 is located at the C-terminus of helix 5 and Glu246 is located at the base of loop L4 of caspase-3. It has been suggested that the salt bridge between Lys242-Glu246 (NH3+ of Lys with COO- of Glu) is not found in the zymogen form of caspase-3 but forms only upon caspase-3 maturation. Rather, these residues are involved in other interactions in procaspase-3. Lys242 maintains the native procaspase dimer, whereas glutamate is involved in stabilizing loop L3, the substrate binding loop. In procaspase-3, Glu246 participates in hydrogen bonding with Trp214. In the mature, active caspase-3, the stability of the active site of caspase-3 depends on the salt bridge between Lys242 and Glu246 to maintain the correct conformation of loop L4. In caspase-3, Glu246 neutralizes the positive charge of Lys242 by forming a salt bridge within the same hydrophobic cluster of residues between helices 4 and 5 that buries the aliphatic portion of Lys242. Mutations of either K242A or E246A resulted in the complete loss of procaspase-3 activity as measured by fluorescence emission following addition of substrate (Ac-DEVD-AFC) and excitation at 400nm. These mutants were active in the mature, fully cleaved caspase-3, although to a much lower extent than wild type mature caspase-3, perhaps due to altered accessibility of its active site. The measurements of kcat/Km for these mutants at the optimal pH (7.5) for caspase-3 activity are 14 to 60-fold lower than that of wild type, with an overall increase in Km and a decrease in kcat. This may be due to alterations caused by the mutation(s) in the loop bundle interaction between loops L2, L4 and L2’. Loss of the Lys242-Glu246 salt bridge may alter the position of helix 4, which consequently alter the position of loop L3, leading to altered hydrogen-bonding in the P4 site. This may explain the decrease in activity seen in the mutants. Furthermore, because Phe250 in loop L4 participates in hydrogen bonding to P4, mutations in loop L4 could result in disruptions of these interactions (Feeney, 2004; Walters, 2011).

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====Caspase-3====

[[Image:ICAD.png|300px|right]]

In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, ~~p18~~/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave itself as well as other caspase. It also cleaves substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998). Cleavage of poly(ADP-ribose) polymerase (PARP) between Asp216-Gly217 also contributes to apoptosis. (http://www.phosphosite.org/proteinAction.do?id=4672&showAllSites=true)

In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, p17/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave itself as well as other caspase. It also cleaves substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998). Cleavage of poly(ADP-ribose) polymerase (PARP) between Asp216-Gly217 also contributes to apoptosis. (http://www.phosphosite.org/proteinAction.do?id=4672&showAllSites=true)

==Disease==

Caspase-3/Sandbox: Difference between revisions

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@@ Line 1: / Line 1: @@
 {{STRUCTURE_1qx3 |  PDB=1qx3  |  SCENE=Caspase-3/Sandbox/Unliganded_human_caspase-3/4 | CAPTION= Crystal Structure of Unliganded Human Caspase-3}}
-Caspases are proteases that function via a cysteine residue in the active site to cleave substrates after aspartic acid residues. Caspases are crucial for the initiation (e.g. caspase-8, -9, -10) and execution (e.g. caspase-3, -6, -7) of apoptosis, or programmed cell death either via the intrinsic or extrinsic pathway (Degterev, Boyce et al. 2003). In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, p18/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998).
+Caspases are proteases that function via a cysteine residue in the active site to cleave substrates after aspartic acid residues. Caspases are crucial for the initiation (e.g. caspase-8, -9, -10) and execution (e.g. caspase-3, -6, -7) of apoptosis, or programmed cell death either via the intrinsic or extrinsic pathway (Degterev, Boyce et al. 2003). In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, p17/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998).
 ==Structure & Function==
@@ Line 9: / Line 9: @@
 ====Salt Bridge====
+[[Image:Casp3Salt bridge.png|500px|left]]
 Lys242 is located at the C-terminus of helix 5 and Glu246 is located at the base of loop L4 of caspase-3. It has been suggested that the salt bridge between Lys242-Glu246 (NH3+ of Lys with COO- of Glu) is not found in the zymogen form of caspase-3 but forms only upon caspase-3 maturation. Rather, these residues are involved in other interactions in procaspase-3. Lys242 maintains the native procaspase dimer, whereas glutamate is involved in stabilizing loop L3, the substrate binding loop. In procaspase-3, Glu246 participates in hydrogen bonding with Trp214. In the mature, active caspase-3, the stability of the active site of caspase-3 depends on the salt bridge between Lys242 and Glu246 to maintain the correct conformation of loop L4. In caspase-3, Glu246 neutralizes the positive charge of Lys242 by forming a salt bridge within the same hydrophobic cluster of residues between helices 4 and 5 that buries the aliphatic portion of Lys242. Mutations of either K242A or E246A resulted in the complete loss of procaspase-3 activity as measured by fluorescence emission following addition of substrate (Ac-DEVD-AFC) and excitation at 400nm. These mutants were active in the mature, fully cleaved caspase-3, although to a much lower extent than wild type mature caspase-3, perhaps due to altered accessibility of its active site. The measurements of kcat/Km for these mutants at the optimal pH (7.5) for caspase-3 activity are 14 to 60-fold lower than that of wild type, with an overall increase in Km and a decrease in kcat. This may be due to alterations caused by the mutation(s) in the loop bundle interaction between loops L2, L4 and L2’. Loss of the Lys242-Glu246 salt bridge may alter the position of helix 4, which consequently alter the position of loop L3, leading to altered hydrogen-bonding in the P4 site. This may explain the decrease in activity seen in the mutants. Furthermore, because Phe250 in loop L4 participates in hydrogen bonding to P4, mutations in loop L4 could result in disruptions of these interactions (Feeney, 2004; Walters, 2011).
@@ Line 47: / Line 48: @@
 ====Caspase-3====
 [[Image:ICAD.png|300px|right]]
-In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, p18/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave itself as well as other caspase. It also cleaves substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998). Cleavage of poly(ADP-ribose) polymerase (PARP) between Asp216-Gly217 also contributes to apoptosis. (http://www.phosphosite.org/proteinAction.do?id=4672&showAllSites=true)
+In its inactive form, procaspase-3 consist of a large subunit and small subunit, interjected by an aspartic acid residue. This aspartate is the site recognized by activated initiator caspases, such as caspase-8 and caspase-9. Upon cleavage, procaspase-3 is separated into two subunits, p17/20 and p12/10 respectively, which heterodimerize to yield the active form of caspase-3. In its active form, caspase-3 is able to cleave itself as well as other caspase. It also cleaves substrates such as ICAD (inhibitor of caspase-activated deoxyribonuclease). Cleavage of ICAD leads to abrogation of its inhibitory effect on CAD, allowing CAD to migrate into the nucleus and cause double-strand breaks in DNA, thus contributing to apoptosis(Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998). Cleavage of poly(ADP-ribose) polymerase (PARP) between Asp216-Gly217 also contributes to apoptosis. (http://www.phosphosite.org/proteinAction.do?id=4672&showAllSites=true)
 ==Disease==