Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.

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Autophagy as a Regulated Pathway of Cellular Degradation

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

In many instances, permeabilization of mitochondrial membranes is a rate-limiting step of apoptotic or necrotic cell demise. This has important consequences for the pathophysiology of cell death, as well as for its pharmacological control.

Mitochondrial control of cell death

Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca2+, etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox stress may involve mitochondrial channels such as mPTP and inner membrane anion channel (IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to ROS release. This regenerative cycle of mitochondrial ROS formation and release was named ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of accumulated potentially toxic levels of ROS (and Ca2+). At higher ROS levels, longer mPTP openings may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites (shown in vitro) and their relevance to the mitochondrial ROS production in vivo.

Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release

In the endoplasmic reticulum (ER), secretory and transmembrane proteins fold into their native conformation and undergo posttranslational modifications important for their activity and structure. When protein folding in the ER is inhibited, signal transduction pathways, which increase the biosynthetic capacity and decrease the biosynthetic burden of the ER to maintain the homeostasis of this organelle, are activated. These pathways are called the unfolded protein response (UPR). In this review, we briefly summarize principles of protein folding and molecular chaperone function important for a mechanistic understanding of UPR-signaling events. We then discuss mechanisms of signal transduction employed by the UPR in mammals and our current understanding of the remodeling of cellular processes by the UPR. Finally, we summarize data that demonstrate that UPR signaling feeds into decision making in other processes previously thought to be unrelated to ER function, e.g., eukaryotic starvation responses and differentiation programs.

The mammalian unfolded protein response

This article reviews the involvement of the mitochondrial permeability transition pore in necrotic and apoptotic cell death. The pore is formed from a complex of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and cyclophilin-D (CyP-D) at contact sites between the mitochondrial outer and inner membranes. In vitro, under pseudopathological conditions of oxidative stress, relatively high Ca2+ and low ATP, the complex flickers into an open-pore state allowing free diffusion of low-Mr solutes across the inner membrane. These conditions correspond to those that unfold during tissue ischaemia and reperfusion, suggesting that pore opening may be an important factor in the pathogenesis of necrotic cell death following ischaemia/reperfusion. Evidence that the pore does open during ischaemia/reperfusion is discussed. There are also strong indications that the VDAC-adenine nucleotide translocase-CyP-D complex can recruit a number of other proteins, including Bax, and that the complex is utilized in some capacity during apoptosis. The apoptotic pathway is amplified by the release of apoptogenic proteins from the mitochondrial intermembrane space, including cytochrome c, apoptosis-inducing factor and some procaspases. Current evidence that the pore complex is involved in outer-membrane rupture and release of these proteins during programmed cell death is reviewed, along with indications that transient pore opening may provoke 'accidental' apoptosis.

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The mitochondrial permeability transition pore and its role in cell death.

The capacity of cyclosporin A to inhibit opening of a Ca2+-dependent pore in the inner membrane of heart mitochondria was investigated. Whereas in the presence of 25 nmol of Ca2+/mg of mitochondrial protein and 5 mM-Pi mitochondria were unable to maintain accumulated Ca2+, inner-membrane potential and sucrose impermeability, all three parameters were preserved when cyclosporin was included. Pore opening was assayed directly by [14C]sucrose entry and entrapment in the matrix space. [14C]Sucrose entry induced by both Ca2+ plus Pi and Ca2+ plus t-butyl hydroperoxide was almost completely inhibited by 60 pmol of cyclosporin/mg of mitochondrial protein. It is concluded that cyclosporin A is a potent inhibitor of the pore.

Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.

This chapter reviews recent advances in the identification of the structural elements of the permeability transition pore. The discovery that cyclosporin A (CsA) inhibits the pore proved instrumental. Various approaches indicate that CsA blocks the pore by binding to cyclophilin (CyP)-D. In particular, covalent labelling of CyP-D in situ by a photoactive CsA derivative has shown that pore ligands have the same effects on the degree to which CsA both blocks the pore and binds to CyP-D. The recognition that CyP-D is a key component has enabled the other constituents to be resolved. Use of a CyP-D fusion protein as affinity matrix has revealed that CyP-D binds very strongly to 1:1 complexes of the voltage-dependent anion channel (from the outer membrane) and adenine nucleotide translocase (inner membrane). Our current model envisages that the pore arises as a complex between these three components at contact sites between the mitochondrial inner and outer membranes. This is in line with recent reconstitutions of pore activity from protein fractions containing these proteins. The strength of interaction between these proteins suggests that it may be a permanent feature rather than assembled only under pathological conditions. Calcium, the key activator of the pore, does not appear to affect pore assembly; rather, an allosteric action allowing pore flicker into an open state is indicated. CsA inhibits pore flicker and lowers the binding affinity for calcium. Whether adenine nucleotide translocase or the voltage-dependent anion channel (via inner membrane insertion) provides the inner membrane pore has not been settled, and data relevant to this issue are also documented.

The mitochondrial permeability transition pore.

A cyclophilin-D affinity matrix was employed to isolate components of the mitochondrial permeability transition pore. A cDNA encoding cyclophilin-D was cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S-transferase/cyclophilin-D fusion protein in Escherichia coli XL1 cells. The cyclophilin-D in the fusion was functionally normal as judged by its peptidylprolyl cis-trans-isomerase activity and its inhibition by cyclosporin A. The fusion protein was bound to glutathione-agarose to form the cyclophilin-D affinity matrix. The matrix selectively bound 32-kDa proteins of mitochondrial membrane extracts, but no H2O-soluble proteins were bound. The 32-kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage-dependent anion channel (porin) and the adenine nucleotide translocase. These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A. The thus-purified voltage-dependent anion channel, adenine nucleotide translocase and the fusion protein were incorporated into phosphatidylcholine liposomes containing fluorescein sulphonate. The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A. These properties are identical to those of the permeability transition pore in mitochondria. It is concluded that the basic permeability transition pore structure comprises the voltage-dependent anion channel (outer membrane), adenine nucleotide translocase (inner membrane) and cyclophilin-D, and forms at contact sites between the two membranes.

Cyclophilin-d binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2880%2980806-4

Martin Crompton

Papers

The mitochondrial permeability transition pore and its role in cell death.

Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.

The mitochondrial permeability transition pore.

Cyclophilin-d binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore

Mitochondrial calcium transport