5/6/2021 0 Comments Smac Product Key
The IAPs cause the ubiquitination of SMACDiablo thereby targeting it for proteosomal degradation. 25, 26 This mechanism is observed in Drosophila as well.SMACDiablo, HtrA2Omi, AIF, and Endonuclease G have also been identified as proteins with important pro-apoptotic functions.These proteins act on the mitochondria causing it to release several proteins from the intermembrane space into the cytosol or nucleus.
These pro-apoptotic mitochondrial proteins include Cytocrome c, SMACDiablo, HtrA2Omi, AIF and Endo G. Cytochrome c along with APAF-1 form the apoptosome, which functions to activate Caspase-9, which subsequently activates Caspase-3. SMACDiablo and HtrA2Omi function to neutralize the inhibitory effect of IAPs on Caspases thereby allowing Caspase activity. AIF binds chromosomal DNA and causes chromatin condensation and remodeling, which facilitates DNA fragmentation by nucleases such as Endo G. Procaspase-9 is recruited to the apoptosome, via its caspase recruitment domain (CARD), where it is activated and released. Caspases are a family of cysteine proteases expressed as latent zymogens, requiring cleavage for activation. Active initiator Caspases, such as Caspase-9, can amplify the apoptotic cascade through the cleavage and activation of other effector caspases, such as Procaspase-3 and -7, and initiate orderly dismantling of the cell through proteolytic cleavage of other cellular substrates (Figure 1). Once activated, caspase function is further regulated by a family of specific inhibitory proteins. Inhibitor of apoptosis proteins (IAPs) possess one or more BIR (baculoviral IAP repeat) domains and directly bind and inhibit active caspases. XIAP, cIAP, and cIAP-2 each possess 3 BIR domains (BIR1, BIR2, and BIR3) as well as a RING domain (cIAP-1 and -2 also have a CARD domain). XIAP binds and inhibits Caspase-9 via its BIR3 domain and Caspase-3 and -7 via its BIR2 and intervening linker domains. The IAPs, however, may be inhibited themselves, thereby restoring caspase activity. This neutralization of caspase IAP inhibition is accomplished by a family of proteins first identified in Drosophila as Reaper, 10 HID, 11 Grim, 12 and Sickle. These IAP inhibitors possess a common N-terminal, 4-amino acid (aa) sequence called a Reaper or IAP-binding motif (Figure 2). The IAP-binding motif specifically interacts with the BIR domains of IAPs to facilitate IAPIAP inhibitor complex formation, and thereby prevent IAPCaspase complex formation. An IAP-binding motif has also been identified in Caspase-9 (Figure 2). IAP BIR domain binding to caspase and to IAP inhibitor is mutually exclusive suggesting that the two types of complexes, one anti-apoptotic (IAPCaspase) and the other pro-apoptotic (IAPIAP inhibitor), are in equilibrium with each other. While true mammalian homologs of Drosophila IAP inhibitors have yet to be identified, two functional analogs have been described. The consensus sequence of the IAP-binding motif is AVPF (yellow). The search for SMACDiablo began with the observation that detergent-isolated cell extracts possessed a greater ability to activate Caspase-3 than non-detergent extracts. However, this detergent-soluble factor could only activate Caspase-3 in the presence of APAF-1, Cytochrome c, and Procaspase-9. Purification of this factor from solubilized membrane extracts led to the identification of human SMAC (second mitochondria-derived activator of caspases). SMACDiablo is expressed as a 239 (237 in mouse) aa precursor protein with an N-terminal mitochondrial localizing sequence (MLS). Upon translocation to the mitochondria, the N-terminal 55 aa are proteolytically removed to generate mature, 25 kDa SMACDiablo. However, the apparent molecular weight of mature SMACDiablo is 100 kDa upon size exclusion chromatography. Subsequent crystallographic analysis revealed that the mature species forms an elongated, symmetric homodimer through hydrophobic interactions causing it to exhibit a much greater apparent size than a globular protein of similar molecular weight. Immature, but intact 19, 20 SMACDiablo and mature, but N-terminally mutated 21 or deleted 22 SMACDiablo are incapable of caspase activation. SMACDiablo interacts directly with XIAP, cIAP-1, and cIAP-2 BIR domains via its IAP binding motif. While SMACDiablo can interact with both XIAP BIR2 and BIR3 domains, it has strongest affinity for XIAP BIR3. The interaction of SMACDiablo and XIAP BIR3 domain was further investigated by NMR 23 and co-crystallization 24 studies, which indicated that the N-terminal 4 aa of the SMACDiablo IAP-binding motif specifically interacts with a surface groove on XIAP BIR3. SMACDiablo dimer formation is also critical for function as introduction of missense mutations affecting the dimer interface disrupted dimer formation and decreased interaction with XIAP. Since XIAP inhibits Caspase-9 via its BIR3 domain and Caspase-3 via its BIR2 domain and since SMACDiablo inhibits XIAP by binding its BIR2 and BIR3 domains, it is not surprising that SMACDiablo allows Caspase-9 and -3 activity. The IAP RING domain can function as an E3 ubiquitin-protease ligase with specificity for SMACDiablo. ![]() This mechanism is observed in Drosophila as well.
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