Muscle atrophy phenotype gene expression during spaceflight is linked to a metabolic crosstalk in both the liver and the muscle in mice.

Human expansion in space is hampered by the physiological risks of spaceflight. The muscle and the liver are among the most affected tissues during spaceflight and their relationships in response to space exposure have never been studied. We compared the transcriptome response of liver and quadriceps from mice on NASA RR1 mission, after 37 days of exposure to spaceflight using GSEA, ORA, and sparse partial least square-differential analysis. We found that lipid metabolism is the most affected biological process between the two organs. A specific gene cluster expression pattern in the liver strongly correlated with glucose sparing and an energy-saving response affecting high energy demand process gene expression such as DNA repair, autophagy, and translation in the muscle. Our results show that impaired lipid metabolism gene expression in the liver and muscle atrophy gene expression are two paired events during spaceflight, for which dietary changes represent a possible countermeasure.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Detection of caspase activity using antibody-based techniques.

A number of antibodies have been generated that recognize caspases from mammalian model organisms. These include antibodies that recognize specific caspase pro-forms and others that bind caspase cleavage fragments. These antibodies are excellent reagents for identifying which executioner caspases have been activated following application or induction of a specific apoptotic stimulus. This approach is more difficult to use with initiator caspases, however, because cleavage does not necessarily correlate with caspase activation. In this protocol, cultured cells are treated with a proapoptotic stimulus, and then protein lysates are prepared from the treated cells. The proteins are then separated by gel electrophoresis and transferred to a suitable membrane. The fragment-specific antibodies that recognize executioner caspases are used in a western analysis to determine the extent of activation and to aid in identifying which caspases have been activated.

The Cox3p assembly module of yeast cytochrome oxidase.

Yeast cytochrome oxidase (COX) was previously inferred to assemble from three modules, each containing one of the three mitochondrially encoded subunits and a different subset of the eight nuclear gene products that make up this respiratory complex. Pull-down assays of pulse-labeled mitochondria enabled us to characterize Cox3p subassemblies that behave as COX precursors and contain Cox4p, Cox7p, and Cox13p. Surprisingly, Cox4p is a constituent of two other complexes, one of which was previously proposed to be an intermediate of Cox1p biogenesis. This suggests that Cox4p, which contacts Cox1p and Cox3p in the holoenzyme, can be incorporated into COX by two alternative pathways. In addition to subunits of COX, some Cox3p intermediates contain Rcf1p, a protein associated with the supercomplex that stabilizes the interaction of COX with the bc1 (ubiquinol-cytochrome c reductase) complex. Finally, our results indicate that although assembly of the Cox1p module is not contingent on the presence of Cox3p, the converse is not true, as none of the Cox3p subassemblies were detected in a mutant blocked in translation of Cox1p. These studies support our proposal that Cox3p and Cox1p are separate assembly modules with unique compositions of ancillary factors and subunits derived from the nuclear genome.

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.

Stabilization of Cox1p intermediates by the Cox14p-Coa3p complex.

Cox14p and Coa3p have been shown to regulate translation of the mitochondrial COX1 mRNA and to be required for assembly of cytochrome oxidase. We present evidence that Cox14p and Coa3p stabilize previously identified Cox1p intermediates and that in the absence of either protein, Cox1p aggregates with itself and other mitochondrial gene products, including cytochrome b, Var1p and Cox2p. Our evidence suggests that Cox1p assembly intermediates are in close proximity to other mitochondrially translated proteins and that an important function of Cox14p and Coa3p is to prevent Cox1 from entering into unproductive aggregation pathways.

Modular assembly of yeast cytochrome oxidase.

Previous studies of yeast cytochrome oxidase (COX) biogenesis identified Cox1p, one of the three mitochondrially encoded core subunits, in two high-molecular weight complexes combined with regulatory/assembly factors essential for expression of this subunit. In the present study we use pulse-chase labeling experiments in conjunction with isolated mitochondria to identify new Cox1p intermediates and place them in an ordered pathway. Our results indicate that before its assimilation into COX, Cox1p transitions through five intermediates that are differentiated by their compositions of accessory factors and of two of the eight imported subunits. We propose a model of COX biogenesis in which Cox1p and the two other mitochondrial gene products, Cox2p and Cox3p, constitute independent assembly modules, each with its own complement of subunits. Unlike their bacterial counterparts, which are composed only of the individual core subunits, the final sequence in which the mitochondrial modules associate to form the holoenzyme may have been conserved during evolution.