Nele Festjens

Toxic proteins released from mitochondria in cell death

A plethora of apoptotic stimuli converge on the mitochondria and affect their membrane integrity. As a consequence, multiple death-promoting factors residing in the mitochondrial intermembrane space are liberated in the cytosol. Pro- and antiapoptotic Bcl-2 family proteins control the release of these mitochondrial proteins by inducing or preventing permeabilization of the outer mitochondrial membrane. Once released into the cytosol, these mitochondrial proteins activate both caspase-dependent and -independent cell death pathways. Cytochrome c was the first protein shown to be released from the mitochondria into the cytosol, where it induces apoptosome formation. Other released mitochondrial proteins include apoptosis-inducing factor (AIF) and endonuclease G, both of which contribute to apoptotic nuclear DNA damage in a caspase-independent way. Other examples are Smac/DIABLO (second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI) and the serine protease HtrA2/OMI (high-temperature requirement protein A2), which both promote caspase activation and instigate caspase-independent cytotoxicity. The precise mode of action and importance of cytochrome c in apoptosis in mammalian cells has become clear through biochemical, structural and genetic studies. More recently identified factors, for example HtrA2/OMI and Smac/DIABLO, are still being studied intensively in order to delineate their functions in apoptosis. A better understanding of these functions may help to develop new strategies to treat cancer.

Caspases in cell survival, proliferation and differentiation

Caspases, a family of evolutionarily, conserved cysteinyl proteases, mediate both apoptosis and inflammation through aspartate-specific cleavage of a wide number of cellular substrates. Most substrates of apoptotic caspases have been conotated with cellular dismantling, while inflammatory caspases mediate the proteolytic activation of inflammatory cytokines. Through detailed functional analysis of conditional caspase-deficient mice or derived cells, caspase biology has been extended to cellular responses such as cell differentiation, proliferation and NF-κB activation. Here, we discuss recent data indicating that non-apoptotic functions of caspases involve proteolysis exerted by their catalytic domains as well as non-proteolytic functions exerted by their prodomains. Homotypic oligomerization motifs in the latter mediate the recruitment of adaptors and effectors that modulate NF-κB activation. The non-apoptotic functions of caspases suggest that they may become activated independently of – or without – inducing an apoptotic cascade. Moreover, the existence of non-catalytic caspase-like molecules such as human caspase-12, c-FLIP and CARD-only proteins further supports the non-proteolytic functions of caspases in the regulation of cell survival, proliferation, differentiation and inflammation.

Mitochondrial intermembrane proteins in cell death.

Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Mitochondria have, next to their function in respiration, an important role in the apoptotic-signaling pathway. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. Some of these factors, such as AIF and endonuclease G, appear to be highly conserved during evolution. Other factors, like cytochrome c, have gained their apoptogenic function later during evolution. In this review, we focus on the role of cytochrome c, AIF, endonuclease G, Smac/DIABLO, Omi/HtrA2, Acyl-CoA-binding protein, and polypyrimidine tract-binding protein in the initiation and modulation of cell death in different model organisms. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death.

RIP1, a kinase on the crossroads of a cell's decision to live or die.

Binding of inflammatory cytokines to their receptors, stimulation of pathogen recognition receptors by pathogen-associated molecular patterns, and DNA damage induce specific signalling events. A cell that is exposed to these signals can respond by activation of NF-κB, mitogen-activated protein kinases and interferon regulatory factors, resulting in the upregulation of antiapoptotic proteins and of several cytokines. The consequent survival may or may not be accompanied by an inflammatory response. Alternatively, a cell can also activate death-signalling pathways, resulting in apoptosis or alternative cell death such as necrosis or autophagic cell death. Interplay between survival and death-promoting complexes continues as they compete with each other until one eventually dominates and determines the cells fate. RIP1 is a crucial adaptor kinase on the crossroad of these stress-induced signalling pathways and a cells decision to live or die. Following different upstream signals, particular RIP1-containing complexes are formed; these initiate only a limited number of cellular responses. In this review, we describe how RIP1 acts as a key integrator of signalling pathways initiated by stimulation of death receptors, bacterial or viral infection, genotoxic stress and T-cell homeostasis.

Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features

Necroptosis, necrosis and secondary necrosis following apoptosis represent different modes of cell death that eventually result in similar cellular morphology including rounding of the cell, cytoplasmic swelling, rupture of the plasma membrane and spilling of the intracellular content. Subcellular events during tumor necrosis factor (TNF)-induced necroptosis, H2O2-induced necrosis and anti-Fas-induced secondary necrosis were studied using high-resolution time-lapse microscopy. The cellular disintegration phase of the three types of necrosis is characterized by an identical sequence of subcellular events, including oxidative burst, mitochondrial membrane hyperpolarization, lysosomal membrane permeabilization and plasma membrane permeabilization, although with different kinetics. H2O2-induced necrosis starts immediately by lysosomal permeabilization. In contrast, during TNF-mediated necroptosis and anti-Fas-induced secondary necrosis, this is a late event preceded by a defined signaling phase. TNF-induced necroptosis depends on receptor-interacting protein-1 kinase, mitochondrial complex I and cytosolic phospholipase A2 activities, whereas H2O2-induced necrosis requires iron-dependent Fenton reactions.

MOLECULAR MECHANISMS AND PATHOPHYSIOLOGY OF NECROTIC CELL DEATH

Necrotic cell death has long been considered an accidental and uncontrolled mode of cell death. But recently it has become clear that necrosis is a molecularly regulated event that is associated with pathologies such as ischemia-reperfusion (IR) injury, neurodegeneration and pathogen infection. The serine/threonine kinase receptor-interacting protein 1 (RIP1) plays a crucial role during the initiation of necrosis induced by ligand-receptor interactions. On the other hand, ATP depletion is an initiating factor in ischemia-induced necrotic cell death. Common players in necrotic cell death irrespective of the stimulus are calcium and reactive oxygen species (ROS). During necrosis, elevated cytosolic calcium levels typically lead to mitochondrial calcium overload, bioenergetics effects, and activation of proteases and phospholipases. ROS initiates damage to lipids, proteins and DNA and consequently results in mitochondrial dysfunction, ion balance deregulation and loss of membrane integrity. Membrane destabilization during necrosis is also mediated by other factors, such as acid-sphingomyelinase (ASM), phospholipase A(2) (PLA(2)) and calpains. Furthermore, necrotic cells release immunomodulatory factors that lead to recognition and engulfment by phagocytes and the subsequent immunological response. The knowledge of the molecular mechanisms involved in necrosis has contributed to our under-standing of necrosis-associated pathologies. In this review we will focus on the intracellular and intercellular signaling events in necrosis induced by different stimuli, such as oxidative stress, cytokines and pathogen-associated molecular patterns (PAMPs), which can be linked to several pathologies such as stroke, cardiac failure, neurodegenerative diseases, and infections.

Caspase Inhibitors Promote Alternative Cell Death Pathways

The use of caspase inhibitors has revealed the existence of alternative backup cell death programs for apoptosis. The broad-spectrum caspase inhibitor zVAD-fmk modulates the three major types of cell death. Addition of zVAD-fmk blocks apoptotic cell death, sensitizes cells to necrotic cell death, and induces autophagic cell death. Several studies have shown a crucial role for the kinase RIP1 and the adenosine nucleotide translocator (ANT)–cyclophilin D (CypD) complex in necrotic cell death. The underlying mechanism of zVAD-fmk–mediated sensitization to necrotic cell death involves the inhibition of caspase-8–mediated proteolysis of RIP1 and disturbance of the ANT-CypD interaction. RIP1 is also involved in autophagic cell death. Caspase inhibitors and knockdown studies have revealed negative roles for catalase and caspase-8 in autophagic cell death. The positive role of RIP1 and the negative role of caspase-8 in both necrotic and autophagic cell death suggest that the pathways of these two types of cell death are interconnected. Necrotic cell death represents a rapid cellular response involving mitochondrial reactive oxygen species (ROS) production, decreased adenosine triphosphate concentration, and other cellular insults, whereas autophagic cell death first starts as a survival attempt by cleaning up ROS-damaged mitochondria. However, when this process occurs in excess, autophagy itself becomes cytotoxic and eventually leads to autophagic cell death. A better understanding of the molecular mechanisms of these alternative cell death pathways may provide therapeutic tools to combat cell death associated with neurodegenerative diseases, ischemia-reperfusion pathologies, and infectious diseases, and may also facilitate the development of alternative cytotoxic strategies in cancer treatment.

Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells

The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the ‘zipper’-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3′-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.

Tumor necrosis factor-mediated cell death: to break or to burst, that's the question.

In this review, we discuss the signal-transduction pathways of three major cellular responses induced by tumor necrosis factor (TNF): cell survival through NF-κB activation, apoptosis, and necrosis. Recruitment and activation of caspases plays a crucial role in the initiation and execution of TNF-induced apoptosis. However, experimental inhibition of caspases reveals an alternative cell death pathway, namely necrosis, also called necroptosis, suggesting that caspases actively suppress the latter outcome. TNF-induced necrotic cell death crucially depends on the kinase activity of receptor interacting protein serine-threonine kinase 1 (RIP1) and RIP3. It was recently demonstrated that ubiquitination of RIP1 determines whether it will function as a pro-survival or pro-cell death molecule. Deeper insight into the mechanisms that control the molecular switches between cell survival and cell death will help us to understand why TNF can exert so many different biological functions in the etiology and pathogenesis of human diseases.

Bcl-2 Family Members as Sentinels of Cellular Integrity and Role of Mitochondrial Intermembrane Space Proteins in Apoptotic Cell Death

In addition to their function as major energy-providing organelles of the cell, mitochondria accomplish a crucial role in apoptosis. The pro-apoptotic BH3-only members of the Bcl-2 family continuously sense the cellular integrity and well-being at various subcellular levels. If these sentinels are induced, released or activated, they converge on the release of mitochondrial intermembrane space proteins such as cytochrome c, the oxidoreductase AIF, endonuclease G, Smac/DIABLO and the serine protease Omi/HtrA2. We discuss how Bcl-2 family members integrate diverse survival and death signals and act as central regulators of apoptosis. Furthermore, we describe the current knowledge on the role of mitochondrial proteins in apoptotic cell death, discuss the molecular mechanisms of their release and the apoptotic role of mitochondria from a phylogenetic and immunological point of view.