Three-Dimensional Structure of the Apoptosome

Actin-interacting protein 1 (Aip1p) is a 67-kDa WD repeat protein known to regulate the depolymerization of actin filaments by cofilin and is conserved in organisms ranging from yeast to mammals. The crystal structure of Aip1p from Saccharomyces cerevisiae was determined to a 2.3-Å resolution and a final crystallographic R-factor of 0.204. The structure reveals that the overall fold is formed by two connected seven-bladed ␤-propellers and has important implications for the structure of Aip1 from other organisms and WD repeat-containing proteins in general. These results were unexpected because a maximum of 10 WD repeats had been reported in the literature for this protein using sequence data. The surfaces of the ␤-propellers formed by the D-A and B-C loops are positioned adjacent to one another, giving Aip1p a shape that resembles an open "clamshell." The mapping of conserved residues to the structure of Aip1p reveals dense patches of conserved residues on the surface of one ␤-propeller and at the interface of the two ␤-propellers. These two patches of conserved residues suggest a potential binding site for F-actin on Aip1p and that the orientation of the ␤-propellers with respect to one another plays a role in binding an actin-cofilin complex. In addition, the conserved interface between the domains is mediated by a number of interactions that appear to impart rigidity between the two domains of Aip1p and may make a large substrate-induced conformational change difficult.The actin cytoskeleton plays a key role in cell motility, cell morphology, cytokinesis, and the organization of organelles within the cytosol. The organization of actin filaments within the cytoskeleton is tightly regulated by a number of actinbinding proteins. Among these is cofilin, an essential protein conserved in all of the eukaryotes. Cofilin and other members of the actin-depolymerizing factor/cofilin family of proteins are known to aid the rearrangement of the cytoskeleton and increase actin filament turnover by binding and severing F-actin

Section: Structure Of Aip1p As a Model Formentioning

confidence: 87%

The Structure of Aip1p, a WD Repeat Protein That Regulates Cofilin-mediated Actin Depolymerization

Voegtli

Madrona

Wilson

2003

“…Caspases-1, -2, -4, -5, -9, -11, -12, and -13 contain a CARD in each of their prodomains (18). Homotypic interactions between CARDs of caspase-9 and Apaf-1 in the apoptosome contribute to caspase-9 autoactivation (19,30). Similarly, interactions between the CARDs of caspase-2 and CRADD/RAIDD are likely to trigger caspase-2 activation (36,37).…”

Section: Discussionmentioning

confidence: 99%

Activation of Initiator Caspases through a Stable Dimeric Intermediate

Chen

Orozco

Spencer

et al. 2002

Structural and biochemical studies have revealed that procaspases form dimers prior to proteolytic activation. How the two procaspases interact in the dimer is unclear. To study the mechanisms of dimer-dependent caspase activation we used a heterodimeric system so that two caspase molecules can be specifically brought together. Surprisingly, only one caspase partner in the dimer needs to be enzymatically active for caspase processing and activation to occur. Caspase activation is inefficient in the dimer in the absence of intramolecular processing, suggesting that caspase activation is initiated via intramolecular processing. Homodimerization of caspase-8 or caspase-9 leads to the formation of a stable dimeric complex. However, heterodimerization between caspase-8 and caspases-3, -9, or -10 failed to induce stable dimer formation or caspase activation. Our data suggest that the formation of a stable dimeric intermediate initiates caspase activation.

“…These results demonstrate that PKA can inhibit caspase-9 activation by a mechanism that is independent of direct caspase-9 phosphorylation. PKA Inhibits Apoptosome Formation-Cytochrome c induces the ATP/dATP-dependent assembly of Apaf-1 and the recruitment of procaspase-9 into a large oligomeric complex, the apoptosome (7,8). PKA could therefore potentially inhibit caspase-9 activation by regulating the interaction of caspase-9 with Apaf-1.…”

Section: Activation Of Caspase-9 and Caspase-3 Is Inhibited By Campmentioning

confidence: 99%

“…Initiation of an intrinsic apoptotic pathway by a wide variety of stimuli causes the release of cytochrome c from mitochondria (4 -6). In the cytosol, cytochrome c binds the apoptotic factor Apaf-1 and induces Apaf-1 oligomerization, leading to recruitment and activation of procaspase-9 in a large complex termed the apoptosome (7,8). As a result, procaspase-9 autoprocesses and cleaves the effector procaspases 3 and 7, which are activated to cleave key structural and regulatory proteins and thereby bring about the biochemical and morphological changes associated with apoptotic cell death (3).…”

mentioning

confidence: 99%

Protein Kinase A Regulates Caspase-9 Activation by Apaf-1 Downstream of Cytochrome c

Martin

Allan

Lickrish

et al. 2005

The cyclic AMP signal transduction pathway modulates apoptosis in diverse cell types, although the mechanism is poorly understood. A critical component of the intrinsic apoptotic pathway is caspase-9, which is activated by Apaf-1 in the apoptosome, a large complex assembled in response to release of cytochrome c from mitochondria. Caspase-9 cleaves and activates effector caspases, predominantly caspase-3, resulting in the demise of the cell. Here we identified a distinct mechanism by which cyclic AMP regulates this apoptotic pathway through activation of protein kinase A. We show that protein kinase A inhibits activation of caspase-9 and caspase-3 downstream of cytochrome c in Xenopus egg extracts and in a human cell-free system. Protein kinase A directly phosphorylates human caspase-9 at serines 99, 183, and 195. However, mutational analysis demonstrated that phosphorylation at these sites is not required for the inhibitory effect of protein kinase A on caspase-9 activation. Importantly, protein kinase A inhibits cytochrome c-dependent recruitment of procaspase-9 to Apaf-1 but not activation of caspase-9 by a constitutively activated form of Apaf-1. These data indicate that extracellular signals that elevate cyclic AMP and activate protein kinase A may suppress apoptosis by inhibiting apoptosome formation downstream of cytochrome c release from mitochondria.