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From CellBiology

--Mark Hill 18:56, 12 May 2009 (EST) Please read my comments on your discussion page.

Caspase 3

Introduction; General Caspase Overview

Apoptosis is the preferred mechanism of degradation compared to necrosis, as it provides a safe route of disposal of potentially toxic components contained within the desired cell of removal. Caspases are essential for apoptosis, evidence provided is that their zymogens are processed during apoptosis, they cut proteins whose cleavage is associated with apoptotic cell death, and specific inhibitors prevent the development of apoptosis [El-Diery et al., 2005].

Cysteine-aspartic acid proteases or caspases are a family of proteins belonging to a group of enzymes known as cysteine proteases. The study of caspases began with the discovery that the C.elegans ced-3 gene encodes a homologue of the interleukin-1B processing enzyme (ICE) [Yuan et al., 1993]. 14 different caspases have been identified in mammals, 2 existing in mice only, numbered according to the order of identification. They are synthesized as inactive zymogens which can be cleaved to form active enzymes. Caspases share a similar structure consisting of a prodomain followed by large and small subunits.

Caspases are divided into three categories; [El-Deiry et al., 2005]

  • Inflammatory group
    • Consists of caspases 1, 4, 5, 11, 12, 13 and 14
    • Involved in inflammation not apoptosis, concerned mainly with the maturation of proinflammatory cytokines [Gewies 2003]
  • Apoptotic initiator group
    • Comprised of caspases 2, 8, 9 and 10
    • Primarily focused on the activation of the effector caspases
    • Long prodomains contain either a death effector domain (DED) (Casp 8 and 10) or a caspase activation and recruitment domain (CARD) (Casp 2 and 9)
      • These domains moderate the interaction with upstream adaptor molecules
  • Apoptotic effector group
    • Consist of caspases 3, 6 and 7
    • Nicknamed the executioner proteins
    • Short prodomains
    • Processed and activated by their up-stream counterparts (initiator caspases)

This study will be focusing on caspase-3.

Caspase-3; The executioner

Introduction; Structure, Location and Function

Structure

Procaspase-3 structure consists of a noncatalytic prodomain at the N-terminus and two catalytic subunits; a large subunit of 17kDa (p17) and a small subunit of 12kDa (p12).

The procaspase is processed to form the active caspase-3 structure. This involves the removal of the prodomain and the formation of the new structure [Gewies 2003]. Caspase-3 exists as a heterotetramer that consists of two anti-parallel arranged heterodimers, each one formed by a p17 and a p12 subunit.

Location

Procaspase-3 is released from the mitochondria into the cell cytoplasm where it is processed into caspase-3. Caspase-3 is largely found in cells in the lung, spleen, heart, liver and kidney, but can also be found in moderate levels in the brain and skeletal muscle. Very low levels are found in the testis.

Function

Activated through the caspase cascade, it acts as an apoptotic executioner. It proteolytically cleaves poly(ADP-ribose) polymerase (PARP). It cleaves and activates sterol regulatory element binding proteins (SREBP). It also cleaves and activates caspases -6, -7 and -9.

Pathway; Activation, Targets, Actions and Inhibition

Fig.1 Schematic drawing of Caspase-3 activation and pathway inducing apoptosis.

Activation

Intrinsic pathway

This is a mitochondria-mediated pathway which involves procaspase-9 activation by Apaf-1 complex with cytochrome c and ATP (apoptosome) forming caspase-9. Once the initiator caspases have been activated, they in turn activate the executioner caspases, such as caspase-3.

Extrinsic pathway

The recruitment of procaspase-8 to the DISC complex formed by the ligation of a member of the tumor necrosis factor receptor (TNFR) family. Many procaspase-8 proteins are in close proximity and autoactivate, forming caspase-8. Caspase-8 is then responsible for activating caspase-3 as well as others [Gewies 2003].

Granzyme B is another extrinsic factor responsible for caspase-3 activation. It acts both indirectly by promoting the apoptosome formation and procaspase-9 activation and procaspase-8 activation, and it works directly promoting procapase-3 activation.

Targets

Caspase targets are divided into four categories; mediators and regulators of apoptosis, structural proteins, cellular DNA repair proteins and cell cycle-related proteins. Caspase-3 is a mediator and regulator of apoptosis [El-Diery et al., 2005].

Caspase-3 cleaves and inactivates proteins crucial for the maintenance of the cellular cytoskeleton, DNA repair, signal transduction and cell-cycle control [Hengatner et al., 2000]

Acts at receptors, PARP, U170K and DFF, to initiate actions within the nucleus [Stennicke at al., 1998]

Actions

Initiates DNA fragmentation

Inactivates DNA repair

Inactivates mRNA splicing

Inhibition

Inhibitors of caspase-3 have been described as being promising cardioprotectants, neuroprotectants and hepatoprotectants [Sharma et al., 2008].

IAP – inhibitors of apoptosis proteins. They are the most important negative regulator of apoptosis. So far 8 human IAPs have been identified [El-Diery et al., 2005], all containing a common feature, the Baculovirus IAP repeat (BIR) [Riedl et al., 2001]. XIAP is the most powerful caspase inhibitor, containing three BIR domains, it completely blocks the activity of caspase-3 via the region close to BIR2 [Chai et al., 2001]. XIAP binds to the substrate groove of caspase-3 blocking it from having any contact with its substrates [Fadeel et al., 2005].

Smac/Diablo is an IAP antagonist which acts as a competitive inhibitor. Released from the mitochondria, it inhibits XIAP blocking the inhibition of caspase-3 allowing the cell to undergo apoptosis [El-Diery et al., 2005].

Abnormalities

Overactivation of apoptosis is associated with neurodegenerative disorders such as; Alzheimer’s disease, Huntington’s disease and Parkinson’s disease. The abnormal rate is also associated with brain ischemia, myocardial infarction and liver disease [Sharma et al., 2008]. Current treatments are aimed at improving the affected cells rather than preventing the apoptosis pathway.

Genetic evidence shows that the loss of caspase-3 results in gross brain malformation and premature death [Ranger et al., 2001]. Caspase-3 knockout mice display an apoptotic defect in response to both intrinsic and extrinsic pathway stimuli. These results further demonstrate the significance of caspase-3 as a crucial executioner caspase [El-Diery et al., 2005].

Alzheimer's disease (AD) is the extensve loss of neurons in the brain. It is speculated to be caused by a dysregulation in appoptosis. The defining feature of AD is the accumulation of amyloid plaques formed by aggregates of Aβ peptide fragments generated by proteolytic processing of amyloid precursor protein (APP) [Fadeel et al., 2005]. APP is a substrate for caspase-3, suggesting that caspase-3 is one of the the prime causes of AD.

Therapeutical Opportunities

Targeted caspase inhibition could provide protection against stroke, myocardial infarction, hepatic injury, neurodegenerative diseases, spinal cord injury, sepsis and others [Fadeel et al., 2005].

By the same concept, but in reverse, activating the caspase cascade to initiate apoptosis could prove beneficial for conditions such as cancer and AIDS. Such a concept could be acheived by enhancing the release or activation of Smac/Diablo from the mitochondria to inhibit the inhibitors of the caspases.

Examples of compounds that have shown promise experimentally

M-826; A reversible caspase-3 inhibitor

Experimental effects; protects mice against neonatal hypoxia, and has been found to save neurons in a Huntungton's disease model [Schulze-Osthoff et al., 2005]

M-791; specific caspase-3 inhibitor

Experimental effects; decreased lymphocyte apoptosis in thymus and spleen of mice subjected to sepsis [Schulze-Osthoff et al., 2005]

Concluding Remarks

Caspase-3 is an important protein in the caspase cascade. It has been nicknamed the 'henchman' as it is a pivotal player in the execution of cells. More research is needed regarding the structure and activities of this important player. Caspase-3 shows a lot of potential for future therapeutical targets as it is a major common link between varying stimuli and apoptosis. With scientifically specific inhibition or excitation of this protein, many diseases/disorders would be controllable.

References

Chai, J., Shiozaki, E., Srinivasula, S. M., Wu, Q., Datta, P., Alnemri, E. S., Shi, Y., Dataa, P. (2001). Structural basis of caspases-7 inhibition by XIAP. Cell. 104:769-780

El-Deiry, W. S., Jin, Z. (2005). Overview of cell death signalling pathways. Cancer biology and therapy. 4(2):139-163

Fadeel, B., Orrenius, S. (2005). Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. Journal of internal medicine. 258:479-517

Fischer, U., Schulze-Osthoff, K. (2005). Apoptosis-based therapies and drug targets. Cell death and differentiation. 12:942-961

Gewies, A. (2003). Introduction to apoptosis. Aporeview. 1-26

Mazumder, S., Plesca, D., Almasan, A. (2005). Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis. Methods in molecular biology. 414:13-21

Ranger, A. M., Malynn, B. A., Korsmeyer, S. J. (2001). Mouse models of cell death. Nat genet. 28:113-118

Riedl, S. J., Renatus, M., Schwarzenbacher, R., Zhou, Q., Sun, C., Fesik, S. W., Liddington, R. C., Salvesen, G. S. (2001). Structural basis for the inhibition of caspase-3 by XIAP. Cell. 104:791-800

Sharma, S., Sahu, K., Jain, P., Mourya, V. K., Agrawal, R. K., (2008). QSAR study of 1,3-dioxo-4-methyl-2,3-dihydro-1h-pyrrolo[3,4-c]quinolines as caspase-3 inhibitors. Med chem res. 17:399-411

Stennicke, H. R., Salvesen, G. S. (1998). Properties of caspases. Biochemica et biophysica acta. 1387:17-31

Stennicke, H. R., Salvesen, G. S. (2000). Caspases - controlling intracellular signals by protease zymogen activation. Biochem biophys acta. 1477:299-306

Lecture Feedback

Lecture 15 - Cell Cycle

S = synthesis

Lecture 14 - Confocal Microscopy

Two methods are spinning and laser

Lecture 10 - Cytoskeleton - Intermediate Filaments

Stratum spinosum in located between stratum basale and stratum granulosum. It synthesises cytokeratins which are a form of intermediate filament composed of keratin

Lecture 8 - Cell Junctions

L-CAM = Liver Cell Adhesion Molecule I-CAM = Intercellular Adhesion Molecule N-CAM = Neuronal Cell Adhesion Molecule Ng-CAM = Neuron-glial Cell Adehesion Molecule

Lecture 7 - Cell Mitochondria

Needed in cells with high energy such as muscle, sperm tail and flagella. It is also needed for cellular respiration and the electron transport chain

Lecture 5 - Exocytosis

The concept that I'm struggling to wrap my head around would be the way in which proteins are transported around the cell. What pathways they are following and how it's achieved with perfection. From the lecture I understand that different motility proteins are added to propel these transport vesicles along, but the mechanisms by which it does this and how the vesicle knows where to go is beyond me.

Lecture 4 - Nucleus

I find it fascinating that an entire strand of DNA is kept in every cell nucleus. If unwound would stretch out to 2 metres long. From this strand every protein can be made, depending on the cell, will determine which proteins will actually be synthesised, but this then decides the functions and the impact that this cell will have on itself or on surrounding cells and that will ultimately affect the entire organism.