Studies on liver mitochondrial 5′-endonuclease activity in rats fed carcinogenic amines and on their binding to mitochondrial proteins

Mitochondrial 5′-endonuclease activity has been determined at regular time intervals in the livers of rats fed a diet containing 0.09% 2-aminofluorene (AF), 0.09% 2-acetylaminofluorene (AAF) or 0.06% N,N-dimethyl-4-aminoazobenzene (DAB). The results obtained indicate that the 5'-endonuclease activity was not affected significantly.The quantity of AF, AAF or DAB bound to liver homogenate and mitochondrial fraction proteins has also been measured at regular time intervals. The amount of AF and AAF bound to homogenate proteins after 4 weeks of carcinogen feeding is about 60-fold higher than that of DAB. The binding of the AF compounds to the mitochondrial fraction proteins is comparatively more important, reaching a level 300-fold higher than that of DAB. The amount of AF residues bound per mg of mitochondrial fraction proteins is higher than that of the homogenate while that of rats fed DAB is smaller. The present results suggest that no relation can be established between the total amount of these carcinogens bound to liver cellular proteins in vivo and their potential carcinogenic effect.     

Conserved sequence blocks 5′ to start codons of plant mitochondrial genes

Three sequence blocks of 10–12 bp are conserved in sequence and order 5 to putative start codons of several higher-plant mitochondrial genes. At least 25 examples were found, primarily associated with coxII, atp6, and orf25, in monocotyledons and dicotyledons. The proximal block can be 9 bp from start codons, and the three blocks generally occur within 100 bp 5 of start codons. In three examples 5 termini of the blocks represent recombination breakpoints, resulting in conservation of the blocks in resultant configurations. The two proximal blocks can form a secondary structure motif. The occurrence of the blocks near start codons, and conserved sequence and order, is consistent with a possible role in translation initiation or regulation.     

What couples glycolysis to mitochondrial signal generation in glucose-stimulated insulin secretion?

Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis. This is accomplished through the tight coupling of glycolysis to mitochondrial activation. The messenger molecules ATP and glutamate are produced after the metabolism of glycolysis-derived pyruvate in the mitochondria. The entry of monocarboxylates such as pyruvate into the beta cell is limited, explaining why overexpression of monocarboxylate transporters unravels pyruvate-stimulated insulin secretion. NADH generated by glycolysis is efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase. Overexpression of this enzyme does not alter glucose-stimulated insulin secretion, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell. These metabolic features permit the fuel function of glucose to be extended to the generation of signaling molecules, which increases cytosolic Ca2+ and promotes insulin exocytosis.     

Intracellular sterol transport in eukaryotes,a connection to mitochondrial function?

Eukaryotic cells synthesize sterols in the endoplasmatic reticulum (ER) from where it needs to be efficiently transported to the plasma membrane, which harbors approximately 90% of the free sterol pool of the cell. Sterols that are being taken up from the environment, on the other hand, are transported back from the plasma membrane to the ER, where the free sterols are esterified to steryl esters. The molecular mechanisms that govern this bidirectional movement of sterols between the ER and the plasma membrane of eukaryotic cells are only poorly understood. Proper control of this transport is important for normal cell function and development as indicated by fatal human pathologies such as Niemann Pick type C disease and atherosclerosis, which are characterized by an over-accumulation of free sterols within endosomal membranes and the ER, respectively. Recently, a number of complementary approaches using Saccharomyces cerevisiae as a model organism lead to a more precise characterization of the pathways that control the subcellular transport of sterols and led to the identification of components that directly or indirectly affect sterol uptake at the plasma membrane and its transport back to the ER. A genetic approach that is based on the fact that yeast is a facultative anaerobic organism, which becomes auxotrophic for sterols in the absence of oxygen, resulted in the identification of 17 genes that are required for efficient uptake and/or transport of sterols. Unexpectedly, many of these genes are required for mitochondrial functions. A possible connection between mitochondrial biogenesis and sterol biosynthesis and uptake will be discussed in light of the fact that cholesterol transport into the inner membranes of mitochondria is a well established sterol transport route in vertebrates, where it is required to convert cholesterol into pregnenolone, the precursor of steroids.     

How and why does the mitochondrial respiratory chain respond to light?

The plant mitochondrial respiratory system changes in its activity in response to light. This response has been thought to be important for ensuring cooperative function with the photosynthetic system. A recent study addressing light responses of the respiratory chain in Arabidopsis thaliana provided further insight into the role of mitochondria in illuminated leaves. Notably, the nonphosphorylating alternative oxidase is rapidly induced when plants are exposed to the high light stress, and appears to play a key role in keeping cellular redox balance.Key words: alternative oxidase, Arabidopsis thaliana, light acclimation, mitochondria, respiratory chain, organellar crosstalk     

Incorporation of β-sitosterol into mitochondrial membrane enhances mitochondrial function by promoting inner mitochondrial membrane fluidity

Recent findings suggest that mitochondrial membrane fluidity could influence mitochondrial energy metabolism. β-sitosterol (BS) is a common plant sterol that is prevalent in plant oils, nuts, cereals and plant food products. Its chemical structure is very similar to that of cholesterol. As a cholesterol analog, BS is highly lipid soluble and largely resides in the membranes of cells or organelles where it may have an influence on the membrane fluidity. The present study reports that, with the cholesterol chelator 2-hydroxypropyl-β-cyclodextrin (HPβCD) as its carrier, BS is able to increase the fluidity of the inner mitochondrial membrane (IMM) without affecting the fluidity of the outer mitochondrial membrane (OMM), and consequently to increase the mitochondrial membrane potential (?Ψm) and mitochondrial ATP content. It has been previously proposed that a therapeutical boost in adenosine triphosphate (ATP) levels in mitochondria may be beneficial for neurodegenerative diseases such as Alzheimer’s disease (AD). Given that dietary administration of plant sterols could increase brain BS concentrations, these results may provide a better understanding of the beneficial effects of plant sterol-enriched nutrients on neurodegenerative diseases such as AD.     

Mitochondria-specific accumulation of amyloid β induces mitochondrial dysfunction leading to apoptotic cell death

Mitochondria are best known as the essential intracellular organelles that host the homeostasis required for cellular survival, but they also have relevance in diverse disease-related conditions, including Alzheimer's disease (AD). Amyloid β (Aβ) peptide is the key molecule in AD pathogenesis, and has been highlighted in the implication of mitochondrial abnormality during the disease progress. Neuronal exposure to Aβ impairs mitochondrial dynamics and function. Furthermore, mitochondrial Aβ accumulation has been detected in the AD brain. However, the underlying mechanism of how Aβ affects mitochondrial function remains uncertain, and it is questionable whether mitochondrial Aβ accumulation followed by mitochondrial dysfunction leads directly to neuronal toxicity. This study demonstrated that an exogenous Aβ(1-42) treatment, when applied to the hippocampal cell line of mice (specifically HT22 cells), caused a deleterious alteration in mitochondria in both morphology and function. A clathrin-mediated endocytosis blocker rescued the exogenous Aβ(1-42)-mediated mitochondrial dysfunction. Furthermore, the mitochondria-targeted accumulation of Aβ(1-42) in HT22 cells using Aβ(1-42) with a mitochondria-targeting sequence induced the identical morphological alteration of mitochondria as that observed in the APP/PS AD mouse model and exogenous Aβ(1-42)-treated HT22 cells. In addition, subsequent mitochondrial dysfunctions were demonstrated in the mitochondria-specific Aβ(1-42) accumulation model, which proved indistinguishable from the mitochondrial impairment induced by exogenous Aβ(1-42)-treated HT22 cells. Finally, cellular toxicity was directly induced by mitochondria-targeted Aβ(1-42) accumulation, which mimics the apoptosis process in exogenous Aβ(1-42)-treated HT22 cells. Taken together, these results indicate that mitochondria-targeted Aβ(1-42) accumulation is the necessary and sufficient condition for Aβ-mediated mitochondria impairments, and leads directly to cellular death rather than along with other Aβ-mediated signaling alterations.     

Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase?

The interactions between the pyrophosphate (PPi) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PPi per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PPi/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PPi-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PPi. The addition of PPi at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PPi to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PPi binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

ERO1α-dependent endoplasmic reticulum–mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1)

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.     

Loss of Ssq1 leads to mitochondrial dysfunction,activation of autophagy and cell cycle arrest due to iron overload triggered by mitochondrial iron–sulfur cluster assembly defects in Candida albicans

Iron–sulfur clusters perform essential functions in enzymatic catalysis and homeostatic regulation. Here we for the first time identified Ssq1 as an essential component for iron–sulfur cluster assembly in Candida albicans. Ssq1 played an important role in cell growth. Shutting off SSQ1 led to accumulation of intracellular iron, especially in mitochondria, and disorder of intracellular iron regulation. In tetO-SSQ1, iron overloading triggered the oxidative damage of mitochondrial function. Surprisingly, disruption of SSQ1 activated autophagic pathway. The mitochondrial dysfunction was further aggravated when CCZ1 (which is essential for autophagy) and SSQ1 was simultaneously deleted, suggesting that autophagy played a critical role in maintenance of mitochondrial function in tetO-SSQ1. In addition, double deletion of SSQ1 and CCZ1 further elevated cellular iron levels in comparison with tetO-SSQ1, indicating that autophagy participated in maintenance of iron homeostasis. Furthermore, we found that loss of SSQ1 led to increasing protein expression of Rnr1 and redistribution of Rnr2 from the nucleus to cytoplasm, and further resulted in cell cycle arrest. The results implied that cell cycle arrest was caused by activating the checkpoint pathway because of impairing the iron–sulfur cluster assembly in tetO-SSQ1. Shutting off SSQ1 led to a significant defect in filamentous development. Interestingly, the tetO-SSQ1ccz1Δ/Δ growth was inhibited on hyphae-inducing solid media. Both tetO-SSQ1 and tetO-SSQ1ccz1Δ/Δ exhibited extremely attenuated virulence, indicating that Ssq1 might provide a promising target for antifungal drugs development. In summary, our findings provide new insights into the understanding of iron–sulfur cluster assembly-related gene in C. albicans.     

D-loop mutations in mitochondrial DNA: link with mitochondrial DNA depletion?

Clinical presentation of the patients with mitochondrial DNA depletion is quite diverse and is suggestive of genetic heterogeneity. Autosomal recessive inheritance of the disease appears likely, thus implying the nuclear origin of the disease. This has been demonstrated recently in large families with neonatal presentation of the disease. Here, we report upon a family with one child having a late-onset disease associated with severe mitochondrial DNA depletion. The presence of mitochondrial alterations in the muscle of the patient's mother prompted us to extensively analyse the mitochondrial DNA in the family. We found mitochondrial DNA multiple deletions, but also three heteroplasmic point mutations of the D-loop region, two of which (T119C and T408A) affect conserved regions involved in the mtDNA replication process. These mutations were non-randomly distributed in the maternal lineage and, for one of them, among single muscle fibres. Involvement of the mitochondrial DNA in its own depletion appears therefore possible. It may act in close relationship with a hypothetical modified nuclear factor.     

Does cholesterol use the mitochondrial contact site as a conduit to the steroidogenic pathway?

The first and rate-limiting step of steroidogenesis is the transfer of cholesterol from the outer mitochondrial membrane to the inner membrane where it is converted to pregnenolone by cytochrome P450 side-chain cleavage (P450scc). This reaction is modulated in the gonads and adrenals by the steroidogenic acute regulatory protein (StAR), however, the mechanism used by StAR is not understood. The outer and inner mitochondrial membranes are joined at contact sites that are thought to be held in place by protein complexes that bridge the two membranes. While it is generally accepted that proteins are imported into the mitochondrion via contact sites, it is not clear whether cholesterol takes the same conduit to the inner membrane. Strategies to combat diseases caused by interrupted cholesterol transfer will rely on a full understanding of the steroidogenic mechanism. The challenge for the future is to determine whether StAR relies on the molecular architecture that spans the mitochondrial intermembrane space to deliver its cargo.     

IL-15Rα deficiency leads to mitochondrial and myofiber differences in fast mouse muscles

The purpose of this study was to determine mitochondrial changes in fast muscles from interleukin-15 receptor alpha knockout (IL-15RαKO) mice. We tested the hypothesis that fast muscles from IL-15RαKO mice would have a greater mitochondrial density and altered internal structure compared to muscles from control mice. In fast muscles from IL-15RαKO mice, mitochondrial density was 48% greater with a corresponding increase in mitochondrial DNA content. Although there were no differences in the relative size of isolated mitochondria, internal complexity was lower in mitochondria from IL-15RαKO mice. These data support an increase in mitochondrial biogenesis and provide direct evidence for a greater density and altered internal structure of mitochondria in EDL muscles deficient in IL-15Rα.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes.