A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly

Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.     

The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation

Previously, we demonstrated that both 150 mM KCl and alkaline pH inhibit cytochrome c-mediated activation of procaspase-3 in a unique manner. To determine the mechanism of inhibition, we analyzed the effect of KCl and alkaline pH on the formation of apoptosomes (a large complex consisting of cytochrome c, Apaf-1, and procaspase-9/caspase-9) in vitro. Our results suggest that an initial 700-kDa apoptosome matures through a 1.4-MDa intermediate before a 700-kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrate that 150 mM KCl interferes with the conversion of the initial 700-kDa apoptosome to the 1.4-MDa intermediate, while alkaline pH "traps" the apoptosome in the 1.4-MDa intermediate. Analysis of the cleaved state of procaspase-9 and procaspase-3 suggests that the 1.4-MDa intermediate may be required for cleavage of procaspase-9. Consistent with these results, in vivo data suggest that blocking acidification during the induction of apoptosis inhibits activation of procaspase-3. On the basis of these results, we propose a model of apoptosome maturation. caspase; pH; potassium; apoptosis     

A mathematical model for apoptosome assembly: the optimal cytochrome c/Apaf-1 ratio

Apoptosis, a highly conserved form of cell suicide, is regulated by apoptotic signals and their transduction with caspases, a family of cystein proteases. Caspases are constantly expressed in the normal cells as inactive pro-enzymes. The activity of caspase is regulated by the proteolysis. Sequential proteolytic reactions of caspases are needed to execute apoptosis. Mitochondrial pathway is one of these apoptotic signal pathways, in which caspases are oligomerized into characteristic heptamer structure, called apoptosome, with caspase-9 that activate the effector caspases for apoptosis. To investigate the dynamics of signal transduction pathway regulated by oligomerization, we construct a mathematical model for Apaf-1 heptamer assembly process. The model first reveals that intermediate products can remain unconverted even after all assemble reactions are completed. The second result of the model is that the conversion efficiency of Apaf-1 heptamer assembly is maximized when the initial concentration of cytochrome c is equal to that of Apaf-1. When the concentration of cytochrome c is sufficiently larger or smaller than that of Apaf-1, the final Apaf-1 heptamer production is decreased, because intermediate Apaf-1 oligomers (tetramers and bigger oligomers), which themselves are unable to form active heptamer, accumulate too fast in the cells, choking a smooth production of Apaf-1 heptamer. Slow activation of Apaf-1 monomers and small oligomers increase the conversion efficiency. We also study the optimal number of subunits comprising an active oligomer that maximize the conversion efficiency in assembly process, and found that the tetramer is the optimum.     

Splitting the apoptosome

Assembly of the apoptosome in response to mitochondrial permeabilization, the hallmark of the intrinsic apoptotic pathway, involves binding of cytochrome c to Apaf1, recruitment and auto-processing of the apical/signaling pro-caspase-9, and coupled activation of downstream/executioner caspases like caspase 3. Evidence now indicates that certain apoptotic cascades can bypass the apoptosome and activate caspase-9 independent of the mitochondria. Recently, we have demonstrated that caspase-9 can be activated in Apaf1-mutant primary myoblasts, but not fibroblasts, in response to stimuli that are known to act via the mitochondria. Thus, apoptosomal activation of caspase-9 seems to represent only one of the routes for its activation; other pathways, some of which are yet to be discovered, can bypass the requirement for Apaf1 and activate caspase-9 in a tissue and context specific manner.     

Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation

The apoptosome is an Apaf-1 cytochrome c complex that activates procaspase-9. The three-dimensional structure of the apoptosome has been determined at 27 A resolution, to reveal a wheel-like particle with 7-fold symmetry. Molecular modeling was used to identify the caspase recruitment and WD40 domains within the apoptosome and to infer likely positions of the CED4 homology motif and cytochrome c. This analysis suggests a plausible role for cytochrome c in apoptosome assembly. In a subsequent structure, a noncleavable mutant of procaspase-9 was localized to the central region of the apoptosome. This complex promotes the efficient activation of procaspase-3. Therefore, the cleavage of procaspase-9 is not required to form an active cell death complex.     

Theoretical aspects of virus capsid assembly

A virus capsid is constructed from many copies of the same protein(s). Molecular recognition is central to capsid assembly. The capsid protein must polymerize in order to create a three-dimensional protein polymer. More than structure is required to understand this self-assembly reaction: one must understand how the pieces come together in solution.     

Mechanical aspects of cardiac development

Heart development depends on a dynamic interaction between genetic and epigenetic factors. This paper discusses some of the biomechanical processes that help shape the heart in the embryo. First, an overview is given of some of the critical events that occur during cardiac development. Next, mechanics and modeling strategies are discussed for the morphogenetic processes of cardiac tube formation, cardiac looping, myocardial trabeculation, septation, valve formation, and muscle-fiber alignment. Finally, some considerations for future work in this area are listed.     

Mechanical aspects of chest wall distortion

During passive inflation of the respiratory system, the rib cage (RC) expands because the pressure applied to it [approximately equal to abdominal pressure (Pab)] increases. Similar Pab-tidal volume (VT) relationships between passive and spontaneous inspirations would occur only if 1) Pab acts on RC equally in the two situations (no distortion) or 2) the extradiaphragmatic inspiratory muscles expand RC, compensating for distortion. In anesthetized adult rats and in sleeping human infants the passive relationships between VT and Pab or abdomen motion (AB) were constructed by occluding the airways during expiration. For a given Pab (or AB) in active breathing VT averaged 55% (rats) and 49% (infants) of the passive volume change. With phrenic stimulation in rats VT was only slightly less than during spontaneous breathing, indicating that, in the latter case, the respiratory system was essentially driven only by the diaphragm. In both species occasional breaths with large RC expansion occurred, and VT was then equal to or larger than the passive volume at iso-Pab. We conclude that 1) RC distortion decreases VT to approximately half of the passive value and 2) being on the relaxation curve reflects "compensated" distortion and not absence of it.     

Mechanical aspects of CO2 angiography

The aim of this paper is to clarify some physical–mechanical aspects involved in the carbon dioxide angiography procedure (CO₂ angiography), with a particular attention to a possible damage of the vascular wall.CO₂ angiography is widely used on patients with iodine intolerance. The injection of a gaseous element, in most cases manually performed, requires a long training period. Automatic systems allow better control of the injection and the study of the mechanical behaviour of the gas.CO₂ injections have been studied by using manual and automatic systems. Pressures, flows and jet shapes have been monitored by using a cardiovascular mock. Photographic images of liquid and gaseous jet have been recorded in different conditions, and the vascular pressure rises during injection have been monitored.The shape of the liquid jet during the catheter washing phase is straight in the catheter direction and there is no jet during gas injection. Gas bubbles are suddenly formed at the catheter’s hole and move upwards: buoyancy is the only governing phenomenon and no bubbles fragmentation is detected. The pressure rise in the vessel depends on the injection pressure and volume and in some cases of manual injection it may double the basal vascular pressure values.CO₂ angiography is a powerful and safe procedure which diffusion will certainly increase, although some aspects related to gas injection and chamber filling are not jet well known. The use of an automatic system permits better results, shorter training period and limitation of vascular wall damage risk.     

Mechanical aspects of legged locomotion control

We review the mechanical components of an approach to motion science that enlists recent progress in neurophysiology, biomechanics, control systems engineering, and non-linear dynamical systems to explore the integration of muscular, skeletal, and neural mechanics that creates effective locomotor behavior. We use rapid arthropod terrestrial locomotion as the model system because of the wealth of experimental data available. With this foundation, we list a set of hypotheses for the control of movement, outline their mathematical underpinning and show how they have inspired the design of the hexapedal robot, RHex.     

Mechanical and functional aspects of membrane skeletons

Membrane skeletons can be characterized as cytoskeletal structures lying parallel to the bilayer part of cellular and organelle membranes. Typical examples are spectrin network and actin-myosin cortex. We approach the problem of elucidating the function of membrane skeletons by theoretically analyzing mechanical models of the cellular behavior. Membranes of different physical and chemical properties are considered. In erythrocytes and some organelles membrane bilayers are smooth and simply underlaid or overlaid by membrane skeletons. It is argued that there the role of a membrane skeleton is, either, to keep the membrane composition laterally homogeneous as it is in the case of the erythrocyte, or, that it is involved in the processes of the lateral separation of integral membrane proteins as it is happening in the case of some intermediate steps of the vesicular membrane trafficking. In the second type of membranes the bilayer part is ruffled and folded, and there the membrane skeletons play a role in the determination of the cortical tension. Here we explore in more detail the mechanical behavior of a cell with such properties of its boundary. The shape transformations are described which occur under the influence (i) of different external forces, i.e., when an originally spherical cell is aspirated into the micropipette or when such a cell is adsorbed on a flat surface, and (ii) of different internal forces on the cell boundary exerted by the cytoskeletal elements.     

DARK apoptosome secrets come to light

In this issue of Structure, Yuan et?al. (2011) utilize biochemical approaches to reconstitute an active Drosophila apoptosome, as well as cryo-electron microscopy to generate an improved model for this conserved caspase-activating complex.