Varicella-Zoster Virus infected human neurons are resistant to apoptosis.

Varicella-zoster virus (VZV) is a pathogenic human herpesvirus that causes varicella (chickenpox) as a primary infection following which it becomes latent in ganglionic neurons. Following viral reactivation many years later VZV causes herpes zoster (shingles) as well as a variety of other neurological syndromes. The molecular mechanisms of the conversion of the virus from a lytic to a latent state in ganglia are not well understood. In order to gain insights into the neuron-virus interaction, we studied virus-induced apoptosis in cultures of both highly pure terminally differentiated human neurons and human fetal lung fibroblasts (HFL). It was found that (a) VZV DNA did not accumulate in infected human neurons; (b) VZV transcripts were present at lower levels at all days studied post-infection in neurons; (c) Western blot analysis showed less VZV IE 63 and very little detectable VZV gE proteins in infected neurons compared with HFL; (d) lower levels of the apoptotic marker cleaved Caspase-3 protein were detected in VZV-infected neurons compared with HFL, and higher levels of the known anti-apoptotic proteins Bcl2, Bcl-XL and also the mitochondrial MT-CO2 protein were found in VZV-infected neurons compared with uninfected cells; and (e) both the MT-CO2 protein and VZV IE 63-encoded protein were detected in infected neurons by dual immunofluorescence. These findings showed that neurons are resistant to VZV-induced apoptosis, which may have relevance to the switching of VZV from a lytic to latent ganglionic neuronal infection.

Mitochondria-Targeting, Intracellular Delivery of Native Proteins Using Biodegradable Silica Nanoparticles.

Mitochondria are key organelles in mammalian cells whose dysfunction is linked to various diseases. Drugs targeting mitochondrial proteins provide a highly promising strategy for potential therapeutics. Methods for the delivery of small-molecule drugs to the mitochondria are available, but these are not suitable for macromolecules, such as proteins. Herein, we report the delivery of native proteins and antibodies to the mitochondria using biodegradable silica nanoparticles (BS-NPs). The modification of the nanoparticle surface with triphenylphosphonium (TPP) and cell-penetrating poly(disulfide)s (CPD) facilitated their rapid intracellular uptake with minimal endolysosomal trapping, providing sufficient time for effective mitochondrial localization followed by glutathione-triggered biodegradation and of native, functional proteins into the mitochondria.

Knockdown of human Oxa1l impairs the biogenesis of F1Fo-ATP synthase and NADH:ubiquinone oxidoreductase.

The Oxa1 protein is a founding member of the evolutionarily conserved Oxa1/Alb3/YidC protein family, which is involved in the biogenesis of membrane proteins in mitochondria, chloroplasts and bacteria. The predicted human homologue, Oxa1l, was originally identified by partial functional complementation of the respiratory growth defect of the yeast oxa1 mutant. Here we demonstrate that both the endogenous human Oxa1l, with an apparent molecular mass of 42 kDa, and the Oxa1l-FLAG chimeric protein localize exclusively to mitochondria in HEK293 cells. Furthermore, human Oxa1l was found to be an integral membrane protein, and, using two-dimensional blue native/denaturing PAGE, the majority of the protein was identified as part of a 600-700 kDa complex. The stable short hairpin (sh)RNA-mediated knockdown of Oxa1l in HEK293 cells resulted in markedly decreased steady-state levels and ATP hydrolytic activity of the F(1)F(o)-ATP synthase and moderately reduced levels and activity of NADH:ubiquinone oxidoreductase (complex I). However, no significant accumulation of corresponding sub-complexes could be detected on blue native immunoblots. Intriguingly, the achieved depletion of Oxa1l protein did not adversely affect the assembly or activity of cytochrome c oxidase or the cytochrome bc(1) complex. Taken together, our results indicate that human Oxa1l represents a mitochondrial integral membrane protein required for the correct biogenesis of F(1)F(o)-ATP synthase and NADH:ubiquinone oxidoreductase.

Cloning and sequencing of cDNA encoding for the testis-specific fox (Vulpes vulpes) sperm polypeptide Vb of the cytochrome C oxidase.

Identification of fox (Vulpes vulpes) sperm antigens was carried out to assess their interest as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP8, a fox sperm protein of 14.7 kd. fSP8 was isoantigenic in foxes, as it was recognized by sera of both male and female foxes immunized with fox sperm proteins. No glycosylation was detected, on fSP8, as shown both by deglycosylation assay and lectin labeling. To determine the fSP8 sequence, the NH2-terminal sequence was first obtained, and a piece of cDNA was amplified from testicular RNA by Rapid Amplification of cDNA extremities polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 879 base pairs was obtained, which includes a major open reading frame coding for 128 amino acids. Mass spectrometric analyses have confirmed the position of the open reading frame. Analysis of the predicted amino acids sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated a homology with the Zinc binding site of the subunit Vb of the cytochrome c oxidase. On the C-terminal extremity, fSP8 presents a high homology to the Vb polypeptide of the cytochrome c oxidase from bovine, mouse, and human; however the 34 amino acids on the NH2-extremity were specific to fSP8. Moreover, it was demonstrated that this sequence was testis-specific. This could contribute to the antigenicity of this protein. fSP8 is one of the first fox sperm antigens to be cloned and sequenced.

Met-HGF/SF signal transduction induces mimp, a novel mitochondrial carrier homologue, which leads to mitochondrial depolarization.

Met-hepatocyte growth factor/scatter factor (HGF/SF) signaling plays an important role in epithelial tissue morphogenesis, lumen formation, and tumorigenicity. We have recently demonstrated that HGF/SF also alters the metabolic activity of cells by enhancing both the glycolytic and oxidative phosphorylation pathways of energy production. Using differential display polymerase chain reaction, we cloned a novel gene, designated mimp (Met-Induced Mitochondrial Protein), which is upregulated in NIH-3T3 cells cotransfected with both HGF/SF and Met (HMH cells). Northern and Western blot analyses showed that mimp is induced in several Met-expressing cell lines following treatment with HGF/SF. Mimp encodes a 33-kDa protein that shows sequence homology to the family of mitochondrial carrier proteins (MCPs). Murine Mimp (mMimp) is expressed in a wide variety of tissues, exhibiting an expression pattern similar to Met. Predominant expression is seen in liver, kidney, heart, skeletal muscle, and testis. Using immunostaining for HA-tagged mMimp and a GFP-mMimp chimeric protein as well as subcellular fractionation, we determined that Mimp is primarily localized to the mitochondria. Ectopic expression of mMimp in the Met-responsive adenocarcinoma cell line, DA3, reduced the mitochondrial membrane potential (uncoupling activity). The extent of the mitochondrial depolarization positively correlated with the level of Mimp expression. Our results demonstrate that Mimp is a novel mitochondrial carrier homologue upregulated by Met-HGF/SF signal transduction, which leads to mitochondrial depolarization, and suggest novel links among tyrosine kinase signaling, mitochondrial function, and cellular bioenergetics.

Identification of immunogenic antigens using a phage-displayed cDNA library from an invasive ductal breast carcinoma tumour.

The identification of immunogenic antigens for serological testing and vaccine development is a major challenge facing cancer immunology research. To study the humoral immune response in patients with breast cancer, a T7 phage display cDNA library from an invasive ductal breast carcinoma was panned on patient serum IgG antibodies. By monitoring the selection with an immunoscreening technique, positive phage-displayed cDNA products reacting with breast cancer patient IgG antibodies were selected. Sequence analysis identified immunogenic antigens such as the cytochrome oxidase I, sp100 and Ran GTPase activating protein. Additionally, immunogenic uncharacterized gene products were also identified. Both the known and unknown immunoselected gene products should offer an additional source for cancer gene discovery for diagnostic testing and vaccine development.

Localization of glucocorticoid hormone receptors in mitochondria of human cells.

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their cognate receptors. In addition, these hormones also modulate mitochondrial gene transcription by mechanisms which are as yet poorly understood. Using immunofluorescence labeling and confocal laser scanning microscopy we show that the glucocorticoid receptor of HeLa and Hep-2 cells is specifically enriched at the sites of the mitochondria which were visualized by labeling with the vital dye CMX and antibodies against cytochrome oxidase subunit I. Immunogold electron microscopy demonstrated that the receptor was located within the inner space of the mitochondria. Immunoblotting experiments also revealed the presence of glucocorticoid receptor in mitochondria isolated from HeLa and Hep-2 cells. Finally, living HeLa cells expressing green fluorescent-glucocorticoid receptor fusion protein revealed a distinct mitochondrial GFP fluorescence. Our results support the concept of a receptor-mediated direct action of steroid hormones on mitochondrial gene transcription.

Identification of differentially expressed genes in Con A-activated carp (Cyprinus carpio L.) leucocytes.

A cDNA library was constructed from the message RNA (mRNA) obtained from Con A-induced head kidney (HK) leucocytes of carp (Cyprinus carpio L.). Differential screening of the cDNA was carried out by hybridization against the total cDNA probes from normal, Con A-uninduced HK leucocytes or Con A-induced HK leucocytes of carp. The differential expression patterns of certain cDNA clones were confirmed by Southern-blot and Northern-blot analysis. Single-pass of the sequencing analysis and homology search in Genbank (EMBL) revealed those differentially expressed cDNA clones encode for cytochrome c oxidase sub-unit II and III (COII and COIII), elongation factor-1 beta (EF-1 beta), bleomycin hydrolase (BH), heat shock cognate protein 70 (HSC70) and 16S ribosomal RNA (16S rRNA).

Quantitative enzyme- and immunohistochemistry of hexokinase and cytochrome c oxidase of spinal neurons in the zebrafish.

A combined quantitative enzyme- and immunohistochemical procedure to demonstrate hexokinase (HK) was developed and tested on sections of spinal cord tissue of the zebrafish. In both procedures, the amount of final reaction product was linearly related with section thickness. When applied to serial sections of fish spinal neurons, the enzyme- and immunohistochemical activities appeared to correlate significantly (r = 0.61; p < 0.001). As HK and cytochrome c oxidase (COX) histochemistry have been used regularly to screen the average level of chronic activity of neurons, we subsequently analysed the relationship between HK and COX in fish spinal neurons, using previously published methods of quantitative enzyme- and immunohistochemistry for COX. The enzyme- as well as the immunohistochemical localisation patterns of HK showed a weak correlation with the enzyme- and immunohistochemical COX localisation respectively. Therefore, it is concluded that both enzyme- and immunohistochemical localisation of COX provide a poor estimate for the relative level of glucose utilisation in fish spinal neurons.

A cytochrome ba3 functions as a quinol oxidase in Paracoccus denitrificans. Purification, cloning, and sequence comparison.

A quinol oxidase has been purified from the cytoplasmic membrane of Paracoccus denitrificans; its heme composition and CO binding properties identify it as a cytochrome ba3. On SDS gels, the purified enzyme complex is separated into five polypeptides. Using partial peptide sequence information for subunit II, the gene locus has been cloned and sequenced. In a typical operon pattern, four genes were identified: qoxA, -B, -C, and -D, coding for subunits II, I, III, and IV. DNA-derived amino acid sequence comparisons reveal extensive similarities to other members of the terminal oxidase superfamily.

Tissue-specific regulation of bovine heart cytochrome-c oxidase activity by ADP via interaction with subunit VIa.

The activity of reconstituted cytochrome-c oxidase (EC 1.9.3.1) from bovine heart is stimulated by intraliposomal ADP but not by NaCl of the same ionic strength. A monoclonal antibody which reacts with subunits VIa-H (heart-type) and VIc, due to the evolutionary relationship between these subunits, also stimulates the activity of the enzyme from bovine heart but not from bovine liver. The antibody induces a conformational change in the heart enzyme but not in the liver enzyme, as shown by the visible difference spectrum. Preincubation of heart cytochrome-c oxidase with the antibody prevents stimulation of activity by intraliposomal ADP after reconstitution in liposomes. Reconstituted liver cytochrome c oxidase is not stimulated by intraliposomal ADP. The data suggest tissue-specific regulation of the activity of cytochrome-c oxidase by ADP via interaction with the matrix domain of subunit VIa-H.

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.

The molecular pathology of respiratory-chain dysfunction in human mitochondrial myopathies.

Some of the different molecular pathologies of respiratory-chain dysfunction in human mitochondrial myopathies will be reviewed in relation to the findings in 58 cases. Deletions of mitochondrial DNA were identified in 21 cases [36%]. There was some correlation between the sites of the deletion and the mitochondrial biochemistry in patients with defects of Complex I but not in cases with more extensive loss of respiratory chain activity. Complex I and Complex IV polypeptides were usually normal in deleted cases. Non-deleted cases, however, often showed specific subunit deficiencies which involved the products of both nuclear and mitochondrial genes. Immunoblots of respiratory-chain polypeptides in one case pointed to defective translocation of the Rieske precursor from the cytosol into the mitochondria. The pathogenic role of circulating autoantibodies to specific matrix proteins and the nature of the target antigens in two patients with mitochondrial encephalomyopathies and respiratory-chain dysfunction will also be discussed.

Brain cytochrome oxidase: purification, antibody production, and immunohistochemical/histochemical correlations in the CNS.

Cytochrome oxidase (CO) is a mitochondrial energy-generating enzyme used in brain studies as a marker of neural functional activity. The activity of CO in different brain regions, revealed histochemically, is distributed nonhomogeneously but in distinct patterns. Localized differences in CO activity could arise from localized differences in enzyme amount or from localized regulation of enzyme turnover number (molecular activity). To distinguish between these alternatives, we used antibodies against purified calf brain CO to assess the immunohistochemical distribution of CO amount (protein immunoreactivity) in several brain regions. Calf brain mitochondria (synaptic and nonsynaptic populations) were isolated from gray matter homogenates by differential centrifugation. CO was purified from detergent extracts of the mitochondria by cytochrome c-Sepharose 4B affinity chromatography. Antisera against the purified CO were raised in rabbits. The antibodies reacted specifically with CO, predominantly subunit IV, in SDS immunoblots. The antibodies did not react in SDS immunoblots with any other proteins solubilized from mitochondria or caudate nucleus but did cross-react with brain CO from other mammalian species and with bovine heart CO. The immunohistochemical distribution of CO amount matched the histochemical distribution of CO activity in all regions tested, including the monkey hippocampus and the mouse olfactory bulb, somatosensory (barrel) cortex, and cerebellum. Thus, the amount of CO in neural tissue is distributed in the same nonhomogeneous pattern as the histochemical activity of CO. The results suggest that mechanisms exist by which CO molecules are selectively distributed within neurons to meet local metabolic demands posed by neural functional activity.

Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.

Two monoclonal antibody lines directed against subunit IV of cytochrome oxidase: a study of opposite effects.

Two monoclonal lines of antibodies were isolated with specificities against the amino half of Subunit IV of beef heart cytochrome oxidase. The lines had nonoverlapping epitopes. Both bound to the matrix face of membranous oxidase, neither bound to the cytoplasmic face. One line (QA4/C4) stimulated electron transfer in soluble or membranous oxidase, while the other (QA4) inhibited that activity by both oxidase preparations. These effects on electron transfer activity were not altered by the inclusion or omission of detergent. ATP depressed the binding of either antibody to either soluble or membranous oxidase. In the absence of ATP, QA4/C4 stimulated electron transfer only in the high affinity phase of cytochrome c oxidation (with decreased KM and increased Vmax), causing slight inhibition in the low affinity phase (with decreased KM). In the presence of ATP, QA4/C4 abolished the high affinity phase, but did not alter the ATP influence on the low affinity phase. In the absence of ATP, antibodies of line QA4 abolished the low affinity phase, leaving a high affinity phase similar to that induced by ATP. In the presence of ATP, QA4 abolished the high affinity phase, leaving a low affinity phase similar to that seen with ATP alone. This behavior is consistent with the dissection of two catalytic sites for cytochrome c and more than one ATP affector site.

Renal oncocytoma. I. Cytochrome c oxidase in normal and neoplastic renal tissue as detected by immunohistochemistry--a valuable aid to distinguish oncocytomas from renal cell carcinomas.

Using a polyclonal antibody raised against bovine heart cytochrome c oxidase, the occurrence of this mitochondrial marker enzyme has been investigated in 63 kidney tumors (ten renal oncocytomas, 43 renal cell carcinomas and ten tubulopapillary adenomas) as well as in normal renal tissue by an immunoperoxidase method (PAP-technique). The differentiation between renal oncocytomas and mitochondria-rich carcinomas represents a problem of histopathology since these tumors have a different prognosis and require different patient managements. The strong immunoreactivity in renal oncocytomas contrasted with the much weaker reactivity in renal carcinomas and adenomas. Even mitochondria-rich (granular cell type) carcinomas exhibited only moderate staining intensity. Furthermore, single strongly stained oncocytes or small complexes were sometimes detected in normal renal tissue. The demonstration of marked differences in enzyme content between renal oncocytomas and granular cell carcinomas renders this method suitable for unequivocal distinction between these renal neoplasms. The antibody proved to be a valuable marker for detecting "true" oncocytic transformation in renal tumors and was useful in defining even single oncocytes or small oncocytic lesions.

Separation, amino-terminal sequence and cell-free synthesis of the smallest subunit of sweet potato cytochrome c oxidase.

The smallest subunit (V) of sweet potato cytochrome c oxidase was separated into three polypeptides, Va, Vb and Vc with different molecular masses (7.4 kDa, 6.8 kDa and 6.2 kDa respectively) by highly resolving sodium dodecylsulfate polyacrylamide gel electrophoresis. Antibody against subunit V reacted specifically with the polypeptide Vc. When polyadenylated mRNA from sweet potato root tissue was translated in a wheat germ cell-free system, the smallest subunit (Vc) of the polypeptides was synthesized to the same size as the mature form, which suggests that the mature subunit retains the signal for import into mitochondria. Within the N-terminal first 25 amino acids there is a stretch of 16 non-polar residues, periodically linked by basic residues, which might form an amphiphilic helix as the targeting signal.

Isolation and characterization of human heart cytochrome c oxidase.

Cytochrome c oxidase was isolated from human hearts and separated by SDS gel electrophoresis. The identity of polypeptide bands with known subunits was demonstrated by immunoblotting with monospecific antisera to rat liver cytochrome c oxidase subunits. The polarographically determined kinetics of cytochrome c oxidation were similar to those reported for the bovine heart enzyme.

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera.

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.