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Background: Recent findings enlightened the pivotal role of cyclooxygenases-1 and -2 (COX-1 and COX-2) in human diseases with inflammation as the committed earliest stage, such as cancer and neurodegenerative diseases. COXs are the main targets of nonsteroidal anti-inflammatory drugs and catalyze the bis-oxygenation of arachidonic acid into prostaglandin PGH2, then converted into prostaglandins, thromboxane, and prostacyclin by tissue-specific isomerases. A remarkable amount of pure COX-1 is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. Methods: Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine(o)COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. Results and Discussion: An improvement of a previously reported method for pure recombinant oCOX-1 production and isolation has been achieved, leading to a lower starting volume of infected cells for each purification, an increased cell density, an increased number of viral particles per cell, and a shortened infection period. The protocol for the recombinant oCOX-1 expression and purification has been in-depth elaborated to obtain 1 mg/L of protein. Conclusion: The optimized procedure could be suitable for producing other membrane proteins as well, for which an improvement in the solubilization step is necessary to have the availability of high concentration proteins.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649–15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII(2), yielding a small amount of active CI+CIII(2) super-complex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII(2) supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH(2)-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII(2), but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII(2) and CIV(2) and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved “mTERF-motifs”, three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein–protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.

DmTTF is a Drosophila melanogaster mitochondrial DNA-binding protein which binds specifically to two homologous non-coding sequences located at the 30 ends of blocks of genes encoded on opposite strands. In order to test whether this protein acts as transcription termination factor, we assayed the capacity of DmTTF to arrest in vitro the transcription catalyzed by mitochondrial and bacteriophage RNA polymerases. Experiments with human S-100 extracts showed that DmTTF is able to arrest the transcription catalyzed by human mitochondrial RNA polymerase bidirectionally, independently of the orientation of the protein–DNA complex. On the contrary when T3 or T7 RNA polymerases were used, we found that DmTTF prevalently arrests transcription when the DNA-binding site was placed in the reverse orientation with respect to the incoming enzymes. These results demonstrate that DmTTF is a transcription termination factor with a biased polarity and suggest that the DNA-bound protein is structurally asymmetrical, exposing two different faces to RNA polymerases travelling on opposite directions.

The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase. (C) 2011 Elsevier Inc. All rights reserved.

GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.

To study the molecular mechanisms responsible for the regulation of mitochondrial DNA copy number, in vivo and in organello dimethyl sulfate footprinting experiments in human fibroblasts and rat liver mitochondria were carried out. By this approach we identified in both species two specific protein binding sites in the 3′ region of the displacement loop of mitochondrial DNA. One site contains the TAS-D element of human and rat mitochondrial DNA; the other covers TAS-C and TAS-B in human, whereas in rat it comprises part of TAS-B. We suggest that the protected sequences might be the site of action of protein factors involved in the premature termination of mitochondrial DNA heavy-strand synthesis.

A binding protein for single-stranded DNA was purified from Paracentrotus lividus egg mitochondria to near homogeneity by chromatography on DEAE-Sephacel and single-stranded-DNA-cellulose. The protein consists of a single polypeptide of about 15 kDa. Glycerol gradient sedimentation analysis suggested that P. lividus mitochondrial single-stranded-DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein shows a stronger preference for poly(dT) with respect to single-stranded M13, poly(dI) and poly(dC). Binding to poly(dA) takes place with much lower affinity. The binding-site size, determined by gel mobility-shift experiments with oligonucleotides of different length, is approximately 45 nucleotides. The binding to single-stranded DNA occurs with low or no cooperativity and is not influenced by ionic strength. The protein has a very high affinity for the DNA: its apparent macroscopic association constant is 2x10(9) M(-1), a value which is the highest among the mitochondrial single-stranded-DNA-binding proteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein and might be related to the peculiar mechanism of sea urchin mitochondrial DNA replication.

et al., DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila. © 2013 Elsevier B.V., This work was supported by grants from the Center for Biomedical Research on Rare Diseases (CIBERER) [INTRA/08/743.2] to R.G., the Instituto de Salud Carlos III (grants [PI 07/0167] and [PI 10/0703] to R.G.); the Comunidad de Madrid (grants [GEN-0269/2006] and [MITOLAB S2010/BMD-2402] to R.G); the Università di Bari, Progetto di Ricerca di Ateneo (to M.R.); and the National Institutes of Health (grant [GM45295] to L.S.K.).

As a step toward the characterization of the main components of mitochondrial DNA replication apparatus in sea urchin, we report the identification of a DNA-helicase activity inParacentrotus lividusmitochondria. The activity was detected in a protein fraction obtained by fractionating on DEAE-Sephacel a lysate of gradient purified mitochondria fromParacentrotus lividuseggs. The mitochondrial helicase unwound, in the presence of ATP and Mg++, a 39-base oligonucleotide annealed to single-stranded M13mp18 (+) DNA. Its direction of movement is 3′ to 5′ with respect to the single stranded portion of the partial duplex DNA substrate. This polarity is similar to that exhibited by the single stranded portion of the partial duplex DNA substrate. This polarity is similar to that exhibited by theEscherichia coli rephelicase and by the helicase from bovine brain mitochondria. These features suggest that the sea urchin mitochondrial helicase could function in enabling the polymerization of the H-strand during mitochondrial DNA replication.

A mitochondrial protein, able to specifically bind two double-stranded homologous sequences of seaurchin mitochondrial DNA, has been partially purified from Paracentrotus lividus eggs. This protein, present at a low concentration, is a polypeptide of 40 kDa. One of the binding sequences, located in the main non-coding region, contains the replication origin of the mitochondrial DNA H-strand. By a combination of band-shift, DNase footprinting, and modification interference analyses with homologous and heterologous probes we identified YCYYATCAN(A/T)RC as the minimum sequence required for the binding. The protein also shows a single-stranded DNA-binding activity, as it is able to specifically interact with one of the strands of the binding sites. These features are consistent with a function of the protein in the modulation of sea-urchin mitochondrial DNA replication during the developmental stages.

et al., The DREF [DRE (DNA replication-related element)-binding factor], which regulates the transcription of a group of cell proliferation-related genes in Drosophila, also controls the expression of three genes involved in mtDNA (mitochondrial DNA) replication and maintenance. In the present study, by in silico analysis, we have identified DREs in the promoter region of a gene participating in mtDNA transcription, the DmTTF (Drosophila mitochondrial transcription termination factor). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DREs control DmTTF transcription; moreover, gel-shift and Chip (chromatin immunoprecipitation) assays demonstrated that the analysed DRE sites interact with DREF in vitro and in vivo. Accordingly, DREF knock-down in S2 cells by RNAi (RNA interference) induced it considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the other hand, mtRNApol (mitochondrial RNA polymerase) lacks DREs in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization Studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mRNApol was efficiently transcribed from stages 11-12. Territories where transcription occurred mostly were the gut and Malpighi tubes for DmTTF, and the gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiple roles in the mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.

The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding.

The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA may control the content of this protein in the following stages of embryogenesis. (C) 2000 Academic Press.

The cDNA for the sea urchin mitochondrial D-loop-binding protein (mtDBP), a 40 kDa protein which binds two homologous regions of mitochondrial DNA (the D-loop region and the boundary between the oppositely transcribed ND5 and ND6 genes), has been cloned. Four different 3'-untranslated regions have been detected that are related to each other in pairs and do not contain the canonical polyadenylation signal. The in vitro synthesised mature protein (348 amino acids), deprived of the putative signal sequence, binds specifically to its DNA target sequence and produces a DNase I footprint identical to that given by the natural protein. mtDBP contains two leucine zippers, one of which is bipartite, and two small N- and C-terminal basic domains. A deletion mutation analysis of the recombinant protein has shown that the N-terminal region and the two leucine zippers are necessary for the binding. Furthermore, evidence was provided that mtDBP binds DNA as a monomer. This rules out a dimerization role for the leucine zippers and rather suggests that intramolecular interactions between leucine zippers take place. A database search has revealed as the most significative homology a match with the human mitochondrial transcription termination factor (mTERF), a protein that also binds DNA as a monomer and contains three leucine zippers forming intramolecular interactions. These similarities, and the observation that mtDBP-binding sites contain the 3'-ends of mtRNAs coded by opposite strands and the 3'-end of the D-loop structure, point to a dual function of the protein in modulating sea urchin mitochondrial DNA transcription and replication.

Mitochondrial gene expression has been investigated in synaptic endings from rat cerebral cortex isolated at various stages during the postnatal development and maturation of the animal. The pattern of the mitochondrial translation products labeled in vitro in rat brain synaptosomes revealed some distinctive features when compared with the pattern observed in a rat fibroblast cell line, the most remarkable being the apparent absence of labeling of the ND5 product. This absence contrasted with the presence in synaptosomes of an amount of ND5 mRNA comparable with that found in the rat fibroblast cell line. The rate of mitochondrial protein synthesis per unit amount of mtDNA in brain synaptosomes showed a characteristic reproducible burst at 10-13 days after birth, thereafter declining sharply in the 3rd week to reach a level that remained constant over a 2-year period. The postnatal burst of mitochondrial protein synthesis coincided with a sharp increase in cytochrome c oxidase activity, pointing to a phase of rapid assembly of respiratory complexes. A comparison of the levels of mitochondrial mRNAs with the corresponding rates of protein synthesis during the animal development and maturation showed a lack of correlation. These observations, together with the apparent lack of translation of the ND5 mRNA, indicate that translational control plays a major role in the regulation of gene expression in rat brain synaptic mitochondria.

The unique compactness and economy of gene organization o f the mammalian mitochondrial genome and its very high CCJPY number demand that the regulation of expression of this genome follow unusual rules. Work over the past 10 years in our and other laboratories has identified some of the mechanisms operating in the control of mitochondrial gene expression in mammalian cells in culture and in differentiated mammalian cell systems. As a consequence, a general picture has begun t o emerge concerning the mechanisms by which mammalian cells adapt t o the changing energetic demands in response to functional, developmental and pathological factors.

A new human zinc finger DNA-binding protein was identified by using a yeast one-hybrid selection system. Two versions of the cDNA, encoding the same protein, were detected that differ for a 584 bp extension at the 5′ region. Sequence analysis showed that the longer clone is a full length version containing part of the 5′ untranslated region. The smaller version was fused in frame with the yeast GAL4 activation domain whereas the 5′ region of the longer clone displayed a stop codon interrupting the fusion with the GAL4 domain. Nevertheless, this clone activated the yeast HIS3 reporter gene with the same efficiency as the smaller version. Sequence comparison of the derived protein with the database showed that it belongs to a family of zinc finger DNA-binding proteins which regulate the expression of genes involved in cell proliferation. Expression of the protein in an in vitro system, DNA-binding studies and genetic experiments identify this factor as a new zinc finger DNA-binding protein which binds GC-rich sequences and contains a domain probably functioning as a transcriptional activator. The new human protein identified in this study was therefore named GC-box-binding zinc finger protein).