Sphingolipid metabolism cooperates with BAK and BAX to promote the mitochondrial pathway of apoptosis.

Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO(4) and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

Digital DNA lifecycle security and privacy: an overview.

DNA sequencing technologies have advanced significantly in the last few years leading to advancements in biomedical research which has improved personalised medicine and the discovery of new treatments for diseases. Sequencing technology advancement has also reduced the cost of DNA sequencing, which has led to the rise of direct-to-consumer (DTC) sequencing, e.g. 23andme.com, ancestry.co.uk, etc. In the meantime, concerns have emerged over privacy and security in collecting, handling, analysing and sharing DNA and genomic data. DNA data are unique and can be used to identify individuals. Moreover, those data provide information on people's current disease status and disposition, e.g. mental health or susceptibility for developing cancer. DNA privacy violation does not only affect the owner but also affects their close consanguinity due to its hereditary nature. This article introduces and defines the term 'digital DNA life cycle' and presents an overview of privacy and security threats and their mitigation techniques for predigital DNA and throughout the digital DNA life cycle. It covers DNA sequencing hardware, software and DNA sequence pipeline in addition to common privacy attacks and their countermeasures when DNA digital data are stored, queried or shared. Likewise, the article examines DTC genomic sequencing privacy and security.

Functions of Cytochrome c oxidase Assembly Factors.

Cytochrome c oxidase is the terminal complex of eukaryotic oxidative phosphorylation in mitochondria. This process couples the reduction of electron carriers during metabolism to the reduction of molecular oxygen to water and translocation of protons from the internal mitochondrial matrix to the inter-membrane space. The electrochemical gradient formed is used to generate chemical energy in the form of adenosine triphosphate to power vital cellular processes. Cytochrome c oxidase and most oxidative phosphorylation complexes are the product of the nuclear and mitochondrial genomes. This poses a series of topological and temporal steps that must be completed to ensure efficient assembly of the functional enzyme. Many assembly factors have evolved to perform these steps for insertion of protein into the inner mitochondrial membrane, maturation of the polypeptide, incorporation of co-factors and prosthetic groups and to regulate this process. Much of the information about each of these assembly factors has been gleaned from use of the single cell eukaryote Saccharomyces cerevisiae and also mutations responsible for human disease. This review will focus on the assembly factors of cytochrome c oxidase to highlight some of the outstanding questions in the assembly of this vital enzyme complex.

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.

Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11-13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450-550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.

Complex formation and turnover of mitochondrial transporters and ion channels.

Mitochondria are responsible for many vital cellular functions in eukaryotic cells, such as ATP production, steroid synthesis and prosthetic group biogenesis. The vital functions of mitochondria are possible due to the compartmental nature of this organelle. Mitochondria form a dynamic network that can exist as a network throughout a cell or as distinct individual structures. Mitochondria are also composed of two membranes, an inner and outer membrane. The inner mitochondrial membrane (IMM) is significantly larger than the outer membrane and must fold upon itself to be contained within the outer mitochondrial membrane (OMM). These folds are known as cristae. Altogether these different membrane compartments specialize in different functions of the mitochondria. The OMM is responsible for passage of small metabolites into and out of the mitochondria while excluding macromolecules. The IMM is a highly selective barrier between the solutes of the cytosol and those within the mitochondrial matrix. Cristae specialize in oxidative phosphorylation. The functions of these membranes are afforded by membrane proteins that are able to transport specific solutes. The appropriate localization, assembly into multi-subunit protein complexes, and wild-type function of these membrane proteins therefore is vital for mitochondria to maintain appropriate function and support cellular survival. This review will address the composition and functions of mitochondrial membrane localized multi-subunit protein complexes along with how these proteins undergo degradation to maintain homeostatic functions of mitochondria in the context of mitochondria specific transporters and ion channels. Due to the large number of known mitochondrial membrane transporters and ion channels this review will focus on the topics presented at the Mitochondrial Ion Channels and Transporters Symposium hosted by the New York University College of Dentistry in September 2015 in honor of Casey Kinnally.

Characterization of cytoplasmic caspase-2 activation by induced proximity.

Caspase-2 is an initiator caspase activated in response to heat shock and other stressors that induce apoptosis. Activation of caspase-2 requires induced proximity resulting after recruitment to caspase-2 activation complexes such as the PIDDosome. We have adapted bimolecular fluorescence complementation (BiFC) to measure caspase-2 induced proximity in real time in single cells. Nonfluorescent fragments of the fluorescent protein Venus that can associate to reform the fluorescent complex were fused to caspase-2, allowing visualization and kinetic measurements of caspase-2 induced proximity after heat shock and other stresses. This revealed that the caspase-2 activation platform occurred in the cytosol and not in the nucleus in response to heat shock, DNA damage, cytoskeletal disruption, and other treatments. Activation, as measured by this approach, in response to heat shock was RAIDD dependent and upstream of mitochondrial outer-membrane permeabilization. Furthermore, we identify Hsp90alpha as a key negative regulator of heat shock-induced caspase-2 activation.

Assembly of the rotor component of yeast mitochondrial ATP synthase is enhanced when Atp9p is supplied by Atp9p-Cox6p complexes.

The Atp9p ring is one of several assembly modules of yeast mitochondrial ATP synthase. The ring, composed of 10 copies of Atp9p, is part of the rotor that couples proton translocation to synthesis or hydrolysis of ATP. We present evidence that before its assembly with other ATP synthase modules, most of Atp9p is present in at least three complexes with masses of 200-400 kDa that co-immunopurify with Cox6p. Pulse-labeling analysis disclosed a time-dependent reduction of radiolabeled Atp9p in the complexes and an increase of Atp9p in the ring form of wild type yeast and of mss51, pet111, and pet494 mutants lacking Cox1p, Cox2p, and Cox3p, respectively. Ring formation was not significantly different from wild type in an mss51 or atp10 mutant. The atp10 mutation blocks the interaction of the Atp9p ring with other modules of the ATP synthase. In contrast, ring formation was reduced in a cox6 mutant, consistent with a role of Cox6p in oligomerization of Atp9p. Cox6p involvement in ATP synthase assembly is also supported by studies showing that ring formation in cells adapting from fermentative to aerobic growth was less efficient in mitochondria of the cox6 mutant than the parental respiratory-competent strain or a cox4 mutant. We speculate that the constitutive and Cox6p-independent rate of Atp9p oligomerization may be sufficient to produce the level of ATP synthase needed for maintaining a membrane potential but limiting for optimal oxidative phosphorylation.

Mitochondrial pathway of apoptosis is ancestral in metazoans.

The mitochondrial pathway of apoptosis is the major mechanism of physiological cell death in vertebrates. In this pathway, proapoptotic members of the Bcl-2 family cause mitochondrial outer membrane permeabilization (MOMP), allowing the release of cytochrome c, which interacts with Apaf-1 to trigger caspase activation and apoptosis. Despite conservation of Bcl-2, Apaf-1, and caspases in invertebrate phyla, the existence of the mitochondrial pathway in any invertebrate is, at best, controversial. Here we show that apoptosis in a lophotrochozoan, planaria (phylum Platyhelminthes), is associated with MOMP and that cytochrome c triggers caspase activation in cytosolic extracts from these animals. Further, planarian Bcl-2 family proteins can induce and/or regulate cell death in yeast and can replace Bcl-2 proteins in mammalian cells to regulate MOMP. These results suggest that the mitochondrial pathway of apoptosis in animals predates the emergence of the vertebrates but was lost in some lineages (e.g., nematodes). In further support of this hypothesis, we surveyed the ability of cytochrome c to trigger caspase activation in cytosolic extracts from a variety of organisms and found this effect in cytosolic extracts from invertebrate deuterostomes (phylum Echinodermata).

Characterization of assembly intermediates containing subunit 1 of yeast cytochrome oxidase.

Mitochondrial-encoded Cox1p, one of the three core subunits of yeast cytochrome oxidase (COX), was previously shown to associate with regulatory proteins and nuclear-encoded subunits into five high molecular weight complexes that were proposed to constitute the pathway for biogenesis of the Cox1p assembly module. One of the intermediates (D5) was inferred, but not directly shown to exist. In the present study mitochondria of strains expressing C-terminal-tagged subunits of COX that had not been looked at previously were pulse-labeled and analyzed for the presence of newly translated Cox1p in the immunoprecipitates. These studies revealed that of the eight nuclear-encoded COX subunits, only Cox5ap, Cox6p, and Cox8p are present in the Cox1p module. Both Cox5ap and Cox8p share interfaces with Cox1p in the holoenzyme, whereas Cox6p interacts indirectly through Cox5ap. These results suggest that the subunit contacts in the holoenzyme are probably established during biogenesis of the Cox1p module. To confirm the existence of the largest Cox1p intermediates (D5), which was only inferred previously, radiolabeled Cox1p with a C-terminal tag was expressed in COX-deficient pet111 and pet494 mutants. Pulldown assays confirmed the presence of newly translated Cox1p in D5, which in wild type cannot be demonstrated directly because of its co-migration with COX in the native electrophoresis system used to separate the intermediates. Jointly, the results of these analyses substantiate our previous proposal that COX is assembled from separate assembly modules, each containing one of the mitochondrial-translated core subunits in association with a unique set of nuclear-encoded subunits.

In situ trapping of activated initiator caspases reveals a role for caspase-2 in heat shock-induced apoptosis.

Activation of 'initiator' (or 'apical') caspases-2, -8 or -9 (refs 1-3) is crucial for induction of apoptosis. These caspases function to activate executioner caspapses that, in turn, orchestrate apoptotic cell death. Here, we show that a cell-permeable, biotinylated pan-caspase inhibitor (bVAD-fmk) both inhibited and 'trapped' the apical caspase activated when apoptosis was triggered. As expected, only caspase-8 was trapped in response to ligation of death receptors, whereas only caspase-9 was trapped in response to a variety of other apoptosis-inducing agents. Caspase-2 was exclusively activated in heat shock-induced apoptosis. This activation of caspase-2 was also observed in cells protected from heat-shock-induced apoptosis by Bcl-2 or Bcl-xL. Reduced sensitivity to heat-shock-induced death was observed in caspase-2(-/-) cells. Furthermore, cells lacking the adapter molecule RAIDD failed to activate caspase-2 after heat shock treatment and showed resistance to apoptosis in this setting. This approach unambiguously identifies the apical caspase activated in response to apoptotic stimuli, and establishes caspase-2 as a proximal mediator of heat shock-induced apoptosis.

Modular biogenesis of mitochondrial respiratory complexes.

Mitochondrial function relies on the activity of oxidative phosphorylation to synthesise ATP and generate an electrochemical gradient across the inner mitochondrial membrane. These coupled processes are mediated by five multi-subunit complexes that reside in this inner membrane. These complexes are the product of both nuclear and mitochondrial gene products. Defects in the function or assembly of these complexes can lead to mitochondrial diseases due to deficits in energy production and mitochondrial functions. Appropriate biogenesis and function are mediated by a complex number of assembly factors that promote maturation of specific complex subunits to form the active oxidative phosphorylation complex. The understanding of the biogenesis of each complex has been informed by studies in both simple eukaryotes such as Saccharomyces cerevisiae and human patients with mitochondrial diseases. These studies reveal each complex assembles through a pathway using specific subunits and assembly factors to form kinetically distinct but related assembly modules. The current understanding of these complexes has embraced the revolutions in genomics and proteomics to further our knowledge on the impact of mitochondrial biology in genetics, medicine, and evolution.

Regulation of Mitochondrial Dynamics by Proteolytic Processing and Protein Turnover.

The mitochondrial network is a dynamic organization within eukaryotic cells that participates in a variety of essential cellular processes, such as adenosine triphosphate (ATP) synthesis, central metabolism, apoptosis and inflammation. The mitochondrial network is balanced between rates of fusion and fission that respond to pathophysiologic signals to coordinate appropriate mitochondrial processes. Mitochondrial fusion and fission are regulated by proteins that either reside in or translocate to the inner or outer mitochondrial membranes or are soluble in the inter-membrane space. Mitochondrial fission and fusion are performed by guanosine triphosphatases (GTPases) on the outer and inner mitochondrial membranes with the assistance of other mitochondrial proteins. Due to the essential nature of mitochondrial function for cellular homeostasis, regulation of mitochondrial dynamics is under strict control. Some of the mechanisms used to regulate the function of these proteins are post-translational proteolysis and/or turnover, and this review will discuss these mechanisms required for correct mitochondrial network organization.

In Vitro Use of Peptide Based Substrates and Inhibitors of Apoptotic Caspases.

Caspases are proteases that are essential components of apoptotic cell death pathways. There are approximately one dozen apoptotic caspases found in organisms where cells die via apoptosis. These caspases are responsible for initiation or execution of apoptosis through the proteolytic cleavage of specific substrates. These substrates contain specific motifs that are recognized and cleaved by caspases that result in alterations of substrate function that promotes the apoptotic phenotype. Analysis of caspase involvement, much like any other protease, can be followed using peptides corresponding to cleavage motifs of these substrates, which can be used as substrates, inhibitors, or affinity-based probes.Different caspases have different substrates and therefore different motifs are recognized by each different caspase. However, these different caspases have a common amino acid recognition pattern containing an aspartic acid residue at the amino-side of the cleavage site. Therefore, caspase substrates have a certain overlap in the cleavage motif as this aspartic acid is found in almost every one. This means that certain peptide motifs are not exclusively cleaved by one single caspase. This lack of exclusive cleavage has brought the use of these motif-based probes into question and spurred the development of truly caspase-specific motifs. This chapter describes the use of peptide-based probes to measure caspase activity while highlighting the limitations of these reagents.

The permeability transition pore complex: another view.

Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca(2+)], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl-prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca(2+) or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys(56) and Cys(159)) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys(159) modification whilst oxidation of Cys(56) increases CyP-D binding to the ANT, probably at Pro(61).

Identification of active caspases using affinity-based probes.

Small-molecule inhibitors of caspases can be modified with moieties such as biotin or fluorescent molecules. After the inhibitor molecule has bound to an active caspase, the caspase itself becomes labeled and can be isolated using affinity purification. This protocol describes the use of the biotinylated pan-caspase inhibitor VAD-FMK and streptavidin beads to isolate active caspases. These caspases are then separated by gel electrophoresis and identified with caspase-specific antibodies using western blotting techniques. Other caspase inhibitors bound with biotin or other labels can be substituted in this assay; labeled inhibitors are available commercially as either pan-caspase or caspase-specific probes.

Verification of a putative caspase substrate.

Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. These approaches provide a comprehensive list of substrates and give clues to which pathways are altered during apoptosis by activated caspases. However, substrates identified by large-scale proteomic screening need to be validated as bona fide caspase targets. This ensures that conclusions derived from the screen are based on real substrates and not on artifacts of the proteomic screen. The validation method described in this protocol uses radiolabeled versions of the putative substrates synthesized using in vitro transcription/translation methods. These are incubated with purified caspases to determine whether they are genuine caspase substrates.

Measuring apoptosis: caspase inhibitors and activity assays.

Caspases are proteases that initiate and execute apoptotic cell death. These caspase-dependent events are caused by cleavage of specific substrates that propagate the proapoptotic signal. A number of techniques have been developed to follow caspase activity in vitro and from apoptotic cellular extracts. Many of these techniques use molecules that are based on optimal peptide motifs for each caspase and on our understanding of caspase cleavage events that occur during apoptosis. Although these approaches are useful, there are several drawbacks associated with them. The optimal peptide motifs are not unique recognition sites for each caspase, so techniques that use them may yield information about more than one caspase. Furthermore, caspase cleavage does not take into account the different caspase activation mechanisms. Recently, probes having greater specificity for individual caspases have been developed and are being used successfully. This introduction provides background on the various caspases and introduces a set of complementary techniques to examine the activity, substrate specificity, and activation status of caspases from in vitro or cell culture experiments.

Preparation of cytosolic extracts and activation of caspases by cytochrome c.

It can be useful to explore the caspase activation process in an in vitro setting. In this protocol, cytosolic extracts prepared from cell culture are incubated with cytochrome c and adenosine triphosphate (dATP), leading to the oligomerization of apoptotic protease activating factor-1 (APAF-1) and the formation of the apoptosome. The apoptosome serves as an activation platform for caspase-9, which binds to the apoptosome through heterodimeric caspase recruitment domain (CARD) interactions and then dimerizes. This leads to cleavage of the executioner, caspase-3. These extracts contain highly active caspases that can be analyzed using a variety of biochemical assays.

Assaying caspase activity in vitro.

Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g., a cytosolic extract), can be achieved by measuring substrate cleavage. Chromogenic or fluorogenic substrates are available for many caspases. These substrates usually consist of the four-amino-acid motif that is optimal for each caspase and a moiety that, when cleaved, generates either a chromophore or a fluorophore that can be detected using spectrophotometric or fluorimetric means. In this protocol, we describe how to use these substrates to monitor caspase activity in samples containing active caspases (e.g., apoptotic cells). Caspase inhibitors, which contain a moiety that covalently attaches to the active site of the caspase, can be used in these assays. These assays will ascertain whether caspases are involved in a specific process (e.g., whether caspases are activated after an apoptotic stimulus) and are particularly informative if a purified caspase is used. However, the substrates and inhibitors are not specific for a particular caspase in an environment containing multiple caspases. So, if cytosolic or apoptotic cell extracts are used in these assays, additional experiments must be performed to identify exactly which caspases are involved.