A cell death assay for assessing the mitochondrial targeting of proteins

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.     

Bacterial proteins predisposed for targeting to mitochondria

Mitochondria evolved from an endosymbiotic proteobacterium in a process that required the transfer of genes from the bacterium to the host cell nucleus, and the translocation of proteins thereby made in the host cell cytosol into the internal compartments of the organelle. According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves. Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria, and propose we that mitochondrial targeting information was preexisting for many proteins of the endosymbiont. We analyzed a family of proteins whose members exist both in bacteria and in mitochondria of eukaryotes and show that the amino-terminal extensions occasionally found in bacterial family members can function as a crude import sequence when the protein is presented to isolated mitochondria. This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.     

Redox proteins in mammalian cell death: an evolutionarily conserved function in mitochondria and prokaryotes

Mammalian cell mitochondria are believed to have prokaryotic ancestry. Mitochondria are not only the powerhouse of energy generation within the eukaryotic cell but they also play a major role in inducing apoptotic cell death through release of redox proteins such as cytochrome c and the apoptosis-inducing factor (AIF), a flavoprotein with NADH oxidase activity. Recent evidence indicates that some present day prokaryotes release redox proteins that induce apoptosis in mammalian cells through stabilization of the tumour suppressor protein p53. p53 interacts with mitochondria either directly or through activation of the genes for pro-apoptotic proteins such as Bax or NOXA or genes that encode redox enzymes responsible for the production of reactive oxygen species (ROS). The analogy between the ancient ancestors of present day bacteria, the mitochondria, and the present day bacteria with regard to their ability to release redox proteins for triggering mammalian cell death is an interesting example of functional conservation during the hundreds of millions of years of evolution. It is possible that the ancestors of the present day prokaryotes released redox proteins to kill the ancestors of the eukaryotes. During evolution of the mitochondria from prokaryotes as obligate endosymbionts, the mitochondria maintained the same functions to programme their own host cell death.     

Viral subversion of immunogenic cell death

While physiological cell death is non-immunogenic, pathogen induced cell death can be immunogenic and hence stimulate an immune response against antigens that derive from dying cells and are presented by dendritic cells (DCs). The obligate immunogenic “eat-me” signal generated by dying cells consists in the exposure of calreticulin (CRT) at the cell surface. This particular “eat-me” signal, which facilitates engulfment by DCs, can only be found on cells that succumb to immunogenic apoptosis, while it is not present on cells dying in an immunologically silent fashion. CRT normally resides in the lumen of the endoplasmic reticulum (ER), yet can translocate to the plasma membrane surface through a complex pathway that involves elements of the ER stress response (e.g., the eIF2α-phosphorylating kinase PERK), the apoptotic machinery (e.g., caspase-8 and its substrate BAP31, Bax, Bak), the anterograde transport from the ER to the Golgi apparatus, and SNARE-dependent exocytosis. A large panoply of viruses encodes proteins that inhibit eIF2α kinases, catalyze the dephosphorylation of eIF2α, bind to caspase-8, Bap31, Bax or Bak, or perturb exocytosis. We therefore postulate that obligate intracellular pathogens have developed a variety of strategies to subvert CRT exposure, thereby avoiding immunogenic cell death.     

Programmed cell death via mitochondria: Different modes of dying

Programmed cell death (PCD)is a major component of normal development, preservation of tissue homeostasis, and elimination of damaged cells. Many studies have subdivided PCD into the three categories of apoptosis, autophagy, and necrosis based on criteria such as morphological alterations, initiating death signal, or the implication of caspases. However, these classifications fail to address the interplay between the three types of PCD. In this review, we will discuss the central role of the mitochondrion in the integration of the cell death pathways. Mitochondrial alterations such as the release of sequestered apoptogenic proteins, loss of transmembrane potential, production of reactive oxygen species (ROS), disruption of the electron transport chain, and decreases in ATP synthesis have been shown to be involved in, and possibly responsible for, the different manifestations of cell death. Thus, the mitochondria can be viewed as a central regulator of the decision between cellular survival and demise.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 284 293.Original Russian Text Copyright ¢ 2005 by Bras, Queenan, Susin.This revised version was published online in April 2005 with corrections to the post codes.     

Escaping cell death: survival proteins in cancer

Defects in apoptosis signaling pathways are common in cancer cells. Such defects may play an important role in tumor initiation because apoptosis normally eliminates cells with damaged DNA or dysregulated cell cycle, i.e., cells with increased malignant potential. Moreover, impaired apoptosis may enhance tumor progression and promote metastasis by enabling tumor cells to survive the transit in the bloodstream and to grow in ectopic tissue sites lacking the otherwise required survival factors. Finally, raised apoptosis threshold may have deleterious consequences by rendering cancer cells resistant to various forms of therapy. The intensive apoptosis research during the past decade has resulted in the identification of several proteins which may promote tumorigenesis by inhibiting apoptosis. Of special relevance in human cancer are those commonly expressed in primary tumors and functioning at the common part of the signaling pathway leading to apoptosis. Proteins fulfilling these criteria include antiapoptotic members of the Bcl-2 protein family, heat shock proteins, Hsp70 and Hsp27, as well as survivin, the novel cancer-associated member of the inhibitor of apoptosis protein family. Understanding the molecular mechanisms of action of these proteins may offer novel modes of rationally and selectively manipulating the sensitivity of cancer cells to therapy.     

Translation rate underpins specific targeting of N-terminal transmembrane proteins to mitochondria

Protein biogenesis is a complex process, and complexity is greatly increased in eukaryotic cells through specific targeting of proteins to different organelles. To direct targeting, organellar proteins carry an organelle-specific targeting signal for recognition by organelle-specific import machinery. However, the situation is confusing for transmembrane domain (TMD)-containing signal-anchored (SA) proteins of various organelles because TMDs function as an endoplasmic reticulum (ER) targeting signal. Although ER targeting of SA proteins is well understood, how they are targeted to mitochondria and chloroplasts remains elusive. Here, we investigated how the targeting specificity of SA proteins is determined for specific targeting to mitochondria and chloroplasts. Mitochondrial targeting requires multiple motifs around and within TMDs: a basic residue and an arginine-rich region flanking the N- and C-termini of TMDs, respectively, and an aromatic residue in the C-terminal side of the TMD that specify mitochondrial targeting in an additive manner. These motifs play a role in slowing down the elongation speed during translation, thereby ensuring mitochondrial targeting in a co-translational manner. By contrast, the absence of any of these motifs individually or together causes at varying degrees chloroplast targeting that occurs in a post-translational manner.     

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.     

Bcl-2 proteins and mitochondria--specificity in membrane targeting for death

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Plant lectins: targeting programmed cell death pathways as antitumor agents

Lectins, a group of highly diverse, carbohydrate-binding proteins of non-immune origin that are ubiquitously distributed in plants, animals and fungi, are well-characterized to have numerous links a wide range of pathological processes, most notably cancer. In this review, we present a brief outline of the representative plant lectins including Ricin-B family, proteins with legume lectin domains and GNA family that can induce cancer cell death via targeting programmed cell death pathways. Amongst these above-mentioned lectins, we demonstrate that mistletoe lectins (MLs), Ricin, Concanavalin A (ConA) and Polygonatum cyrtonema lectin (PCL) can lead to cancer cell programmed death via targeting apoptotic pathways. In addition, we show that ConA and PCL can also result in cancer cell programmed death by targeting autophagic pathways. Moreover, we summarize the possible anti-cancer therapeutic implications of plant lectins such as ConA, Phaseolus vulgaris lectin (PHA) and MLs that have been utilized at different stages of preclinical and clinical trials. Together, these findings can provide a comprehensive perspective for further elucidating the roles of plant lectins that may target programmed cell death pathways in cancer pathogenesis and therapeutics. And, this research may, in turn, ultimately help cancer biologists and clinicians to exploit lectins as potential novel antitumor drugs in the future.     

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.     

Viral vector targeting.

The field of viral vector targeting is advancing rapidly. Recent advances include the successful use of bifunctional crosslinkers to target adenoviral and retroviral vectors, elucidation of the crystal structures of an adenoviral and a retroviral receptor-binding domain, and definition of strategies for inserting short targeting peptides and larger polypeptide-binding domains into the coat proteins of a number of different viral vectors. Novel targeting strategies based on host range restriction and protease activation have been developed, targeted replication-competent vectors have shown promise as anti-cancer agents and the possibility of selecting targeted vectors from vector display libraries has been established.     

Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

Two ATP-dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM-sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF-dependent pathway is NEM-sensitive and requires the hydrolysis of extra-mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70-dependent pathway is NEM-sensitive and does not require extra-mitochondrial ATP. The NEM-insensitive, hsp70-dependent import became NEM-sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.     

Ca2+ signaling, mitochondria and cell death

In the complex interplay that allows different signals to be decoded into activation of cell death, calcium (Ca2+) plays a significant role. In all eukaryotic cells, the cytosolic concentration of Ca2+ ions ([Ca2+]c) is tightly controlled by interactions among transporters, pumps, channels and binding proteins. Finely tuned changes in [Ca2+]c modulate a variety of intracellular functions ranging from muscular contraction to secretion, and disruption of Ca2+ handling leads to cell death. In this context, Ca2+ signals have been shown to affect important checkpoints of the cell death process, such as mitochondria, thus tuning the sensitivity of cells to various challenges. In this contribution, we will review (i) the evidence supporting the involvement of Ca2+ in the three major process of cell death: apoptosis, necrosis and autophagy (ii) the complex signaling interplay that allows cell death signals to be decoded into mitochondria as messages controlling cell fate.     

Programmed cell death in parasitic protozoans that lack mitochondria

In multicellular organisms and in all protozoans harbouring mitochondria, the pathways leading to programmed cell death (PCD) are localized in the mitochondria. Intriguingly, unicellular parasites devoid of mitochondria such as Trichomonas vaginalis and Giardia intestinalis undergo a form of cell death resembling apoptosis, the most frequent form of PCD. This reinforces the idea that PCD must have evolved before the evolution of multicellularity. Moreover, this leads to the hypothesis of an early emergence of death pathways in eukaryotes preceding mitochondrial endosymbiosis and brings into question the central role of mitochondria in PCD.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Heat shock proteins: endogenous modulators of apoptotic cell death

The highly conserved heat shock proteins (HSPs) accumulate in cells exposed to heat and a variety of other stressful stimuli. HSPs, which function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and HSPs induced in response to stress appear to function at key regulatory points in the control of apoptosis. HSPs include antiapoptotic and proapoptotic proteins that interact with a variety of cellular proteins. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory HSPs, in particular HSP27 and HSP70, may participate in carcinogenesis. This review summarizes apoptosis-regulatory function of HSPs.     

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

It is recognized now that intrinsically disordered proteins (IDPs), which do not have unique 3D structures as a whole or in noticeable parts, constitute a significant fraction of any given proteome. IDPs are characterized by an astonishing structural and functional diversity that defines their ability to be universal regulators of various cellular pathways. Programmed cell death (PCD) is one of the most intricate cellular processes where the cell uses specialized cellular machinery and intracellular programs to kill itself. This cell-suicide mechanism enables metazoans to control cell numbers and to eliminate cells that threaten the animal''s survival. PCD includes several specific modules, such as apoptosis, autophagy, and programmed necrosis (necroptosis). These modules are not only tightly regulated but also intimately interconnected and are jointly controlled via a complex set of protein–protein interactions. To understand the role of the intrinsic disorder in controlling and regulating the PCD, several large sets of PCD-related proteins across 28 species were analyzed using a wide array of modern bioinformatics tools. This study indicates that the intrinsic disorder phenomenon has to be taken into consideration to generate a complete picture of the interconnected processes, pathways, and modules that determine the essence of the PCD. We demonstrate that proteins involved in regulation and execution of PCD possess substantial amount of intrinsic disorder. We annotate functional roles of disorder across and within apoptosis, autophagy, and necroptosis processes. Disordered regions are shown to be implemented in a number of crucial functions, such as protein–protein interactions, interactions with other partners including nucleic acids and other ligands, are enriched in post-translational modification sites, and are characterized by specific evolutionary patterns. We mapped the disorder into an integrated network of PCD pathways and into the interactomes of selected proteins that are involved in the p53-mediated apoptotic signaling pathway.     

Inside an enigma: do mitochondria contribute to cell death in Drosophila?

Mitochondria have been shown to play an important role in cell death in mammalian cells. However, the importance of mitochondria in Drosophila apoptosis is still under investigation. Many proteins involved in the regulation of apoptosis in mammals act at mitochondria or are released from mitochondria, resulting in caspase activation. In addition, these organelles undergo significant ultrastructural changes during apoptosis. This review highlights similarities and differences in the roles of mitochondria and mitochondrial factors in apoptosis between Drosophila and mammals. In Drosophila, many key regulators of apoptosis also appear to localize to this organelle, which also undergoes ultrastructural changes during apoptosis. Although many of the proteins important for the control of apoptosis in mammalian cells are conserved in Drosophila, the role that mitochondria play in apoptosis in this model system remains an area of controversy and active research.