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Although exactly how cytochrome c and other mitochondrial intermembrane constituents are released in mammalian cells remains unclear, two major models have been proposed to explain the release. According to one of these models, exposure of a pore in the inner membrane of the mitochondrial wall called the permeability transition pore (PTP). Opening of the PTP allows water to enter and ions to equilibrate, leading to a dissipation of mitochondrial porential. Mitochondrial swelling caused by entry of water leads to the rupturing of the outer membrane and permits release of cytochrome c and other proteins that are located in the intermembrane space. In the second mechanism that has been gaining wide aoceptance, specific proapoptotic members of the Bcl-2 family of proteins (see later), perhaps in association with other proteins, act directly to form pores in the outer mitochondrial membrane through which cytochrome c is released.

More recent work has demonstrated that besides the mitochondria, the endoplasmic reticulum (ER) may be a second compartment that participates in the intrinsic pathway of apoptosis. A major function of the ER is to ensure that only properly folded and modified proteins are passed along to their different destinations. However, prolonged stress to the ER, caused by chemical toxicity or by oxidative stress, leads to an accumulation of unfolded proteins and enhanced calcium release from the ER. The calcium released from the ER is taken up by mitochondria, resulting in a dramatic stimulation of cytochrome c release from the mitochondria, which then activates caspases. Another molecule that plays an important role in ER stress-mediated apoptosis is caspase-12, a member of the caspase family that is normally localized to the ER, Caspase-12 is activated within the ER after exposure to stress ̵causing stimuli and translocates to the cytosol, where it directly cleaves procaspare ̵9 to activate caspase-3.

In addition to the extrinsic and intrinsic pathways, other mechanisms of capase activation have been described in mammalian cells. For example, cytotoxic T cells can trigger apoptosis of their targer cells through a mechanism called the perforin/granzyme B ̵dependent pathway. This pathway, used mainly by the immune system in its surveillance of transformed or virally infected cells, involves a pore ̵ forming proteins, perforin, and a serine protease, granzyme B, which is injected into the cytoplasm of the target cell by the activated cytotoxic T cell. Granzyme B can directly cleave and activate procaspase ̵̵3, thus inducing apoptosis in the target cell.

Caspase Inhibition

The regulation of apoptosis is subject to a complex system of checks and balances.

The activity of caspases can be negatively regulated within cells by endogenously produced proteins. Evidence of the existence of such caspase inhibitory proteins first came from viruses. One of the first viral inhibitors to be identified was CrmA, a protein produced by the cowpox virus. CrmA is structurally similar to serpins, a family of serine proteases inhibitors. But rather than inhibiting serine proteases, CrmA targets caspases. Several forms of herpesviruses inhibit apoptosis by producing a protein called v ̵̵FLIP (viral ̵̵FLICE ̵inhibitory protein), which binds the adaptor protein FADD, preventing it from recruiting caspase ̵8 to the TNF death receptor. Baculoviruses synthesize a potent caspase inhibitor called p35, which prevents the death of insect cells the inhibitor of apoptosis(IAP). Although bearing no structural relationship to p35 or CrmA/serpins, IAPs are found in mammalian cells and are conserved across species including yeast, menatodes, and Drosophia. Mammals express eight different IAP proteins. Stucturally, IAPs are characterized by the presence of one or more conserved amino acid stretches referred to as baculovirus inhibitory repeat domains. Although some IAP proteins have other functions unrelated to the regulation of apoptosis, most IAP family members do inhibit caspase activity. IAPs act by bindingto the cleaved and active form of certain effector caspases, inhibiting their ability to cleave cellular proteins. IAPs may also bind the proform of caspase ̵9, blocking its interaction with Apat ̵1 and the processing of procapase ̵9 into an active enzyme. The activity of IAPs may itself by negatively regulated by other proteins that are released from the mitochondria together with cytochrome c during intrinsic apoptosis signaling. One of these proteins has been called Smac (or Diablo). Smac/Diablo binds to the IAPs, preventing their ability to inhibit caspases.

As described earlier, each caspase recognizes a tetrapeptide sequence within its substrates. Researchers have coupled these tetrapeptide sequences to chemical groups such as fluoro ̵methyl ketones (FMK) and modified the peptides to make them membrane permeable. Such modified but uncleavable tetrapeptides are recognized by caspases and are bound by the enzyme irreversibly. When administered to cells, these pseudo ̵substrate peptides function as potent and highly selective inhibitors of caspases and can inhibit cell death in tissue culture systems and in living animals in response to a variety of different apoptotic stimuli(Fig. 10-10).

Several caspase ̵deficient mice have been generated that exhibit tissue ̵ and cell ̵ specific or stimulus ̵dependent defects in apoptosis. MIce lacking caspase ̵9 have a highly enlarged brain and die in utero. Caspase ̵3 is not active these mice. The brain deformity indicates that caspase ̵9 is required for the developmentally regulated elimination of neurons that occurs during normal brain development. Not unexpectedly, mice lacking Apaf ̵1 also have a larger brain and die prenatally. Mice lacking caspase ̵3 are smaller than normal, display ectopic masses containing excess neurons in several brain regions, and only survive to 3 weeks of age. The milder phenotype of the caspase ̵3 knockout mice as compared with the caspase ̵9 knockout mice is likely to be due to the functioning of other caspase ̵9 ̵activated effector caspases, such as caspase ̵7. Caspase ̵8 and FADD ̵deficient mice have profound cardiac defects and die in utero, indicating that the extrinsic pathway of caspase activation is cretical for embryonic development.