Harmaline to Human Mitochondrial Caseinolytic Serine Protease Activation for Pediatric Diffuse Intrinsic Pontine Glioma Treatment.

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4-7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of ß-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline.

Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria.

Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.

POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA.

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA(+) Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid.

Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

Expression and purification of large active GST fusion enzymes.

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.

Drosophila nuclear factor DREF regulates the expression of the mitochondrial DNA helicase and mitochondrial transcription factor B2 but not the mitochondrial translation factor B1.

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.

MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals.

Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.

Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication.

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.

MTERF factors: a multifunction protein family.

The MTERF family is a large protein family, identified in metazoans and plants, which consists of four subfamilies, MTERF1, 2, 3 and 4. Mitochondrial localisation was predicted for the vast majority of MTERF family members and demonstrated for the characterised MTERF proteins. The main structural feature of MTERF proteins is the presence of a modular architecture, based on repetitions of a 30-residue module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes transcription termination factors: human mTERF, sea urchin mtDBP and Drosophila DmTTF. In addition to terminating transcription, they are involved in transcription initiation and in the control of mtDNA replication. This multiplicity of functions seems to flank differences in the gene organisation of mitochondrial genomes. MTERF2 and MTERF3 play antithetical roles in controlling mitochondrial transcription: that is, mammalian and Drosophila MTERF3 act as negative regulators, whereas mammalian MTERF2 functions as a positive regulator. Both proteins contact mtDNA in the promoter region, perhaps establishing interactions, either mutual or with other factors. Regulation of MTERF gene expression in human and Drosophila depends on nuclear transcription factors NRF-2 and DREF, respectively, and proceeds through pathways which appear to discriminate between factors positively or negatively acting in mitochondrial transcription. In this emerging scenario, it appears that MTERF proteins act to coordinate mitochondrial transcription.

Methods for studying mitochondrial transcription termination with isolated components.

Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3'-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP-DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.

The MTERF family proteins: mitochondrial transcription regulators and beyond.

The MTERF family is a wide protein family, identified in Metazoa and plants, which consists of 4 subfamilies named MTERF1-4. Proteins belonging to this family are localized in mitochondria and show a modular architecture based on repetitions of a 30 amino acid module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes the characterized transcription termination factors human mTERF, sea urchin mtDBP and Drosophila DmTTF. In vitro and in vivo studies show that these factors play different roles which are not restricted to transcription termination, but concern also transcription initiation and the control of mtDNA replication. The multiplicity of functions could be related to the differences in the gene organization of the mitochondrial genomes. Studies on the function of human and Drosophila MTERF3 factor showed that the protein acts as negative regulator of mitochondrial transcription, possibly in cooperation with other still unknown factors. The complete elucidation of the role of the MTERF family members will contribute to the unraveling of the molecular mechanisms of mtDNA transcription and replication.

The Drosophila nuclear factor DREF positively regulates the expression of the mitochondrial transcription termination factor DmTTF.

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 a 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 mtRNApol 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.

Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system.

Termination of transcription is a key process in the regulation of mitochondrial gene expression in animal cells. To investigate transcription termination in sea urchin mitochondria, we cloned the mitochondrial RNA polymerase (mtRNAP) of Paracentrotus lividus and used a recombinant form of the enzyme in a reconstituted transcription system, in the presence of the DNA-binding protein mtDBP. Cloning of mtRNAP was performed by a combination of PCR with degenerate primers and library screening. The enzyme contains 10 phage-like conserved motifs, two pentatricopeptide motifs and a serine-rich stretch. The protein expressed in insect cells supports transcription elongation in a promoter-independent assay. Addition of recombinant mtDBP caused arrest of the transcribing mtRNAP when the enzyme approached the mtDBP-binding site in the direction of transcription of mtDNA l-strand. When the polymerase encountered the protein-binding site in the opposite direction, termination occurred in a protein-independent manner, inside the mtDBP-binding site. Pulse-chase experiments show that mtDBP caused true transcription termination rather than pausing. These data indicate that mtDBP acts as polar termination factor and suggest that transcription termination in sea urchin mitochondria could take place by two alternative modes based on protein-mediated or sequence-dependent mechanisms.

The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription.

DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions. To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in D.Mel-2 cells. By a combination of RNase protection and real-time RT-PCR experiments we found that knock-down determines remarkable changes in mitochondrial transcription. In particular, protein depletion increases not only the level of (+) and (-)strand RNAs mapping immediately after of the two protein-binding site, but also that of transcripts located further downstream. Unexpectedly, depletion of the protein also causes the decrease in the content of those transcripts mapping upstream of the protein target sites, including the two rRNAs. The changes in transcript level do not depend on a variation in mitochondrial DNA (mtDNA) content, since mtDNA copy number is unaffected by DmTTF depletion. This work shows conclusively that DmTTF arrests in vivo the progression of the mitochondrial RNA polymerase; this is the first ever-obtained evidence for an in vivo role of an animal mitochondrial transcription termination factor. In addition, the reported data provide interesting insights into the involvement of DmTTF in transcription initiation in Drosophila mitochondria.

Contrahelicase activity of the mitochondrial transcription termination factor mtDBP.

The sea urchin mitochondrial D-loop binding protein (mtDBP) is a transcription termination factor that is able to arrest bidirectionally mitochondrial RNA chain elongation. The observation that the mtDBP binding site in the main non-coding region is located in correspondence of the 3' end of the triplex structure, where the synthesis of heavy strand mitochondrial (mt) DNA is either prematurely terminated or allowed to continue, raised the question whether mtDBP could also regulate mtDNA replication. By using a helicase assay in the presence of the replicative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contrahelicase. The impairing activity of mtDBP is bidirectional as it is independent of the orientation of the protein binding site. The inhibition is increased by the presence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abrogation of mtDBP contrahelicase activity is suggested that is based on the dissociation of mtDBP from DNA caused by the passage of the RNA polymerase through the protein-DNA complex. All these findings favour the view that mtDBP, besides serving as transcription termination factor, could also act as a negative regulator of mtDNA synthesis at the level of D-loop expansion.

In vitro transcription termination activity of the Drosophila mitochondrial DNA-binding protein DmTTF.

DmTTF is a Drosophila melanogaster mitochondrial DNA-binding protein which binds specifically to two homologous non-coding sequences located at the 3' 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.

DmTTF, a novel mitochondrial transcription termination factor that recognises two sequences of Drosophila melanogaster mitochondrial DNA.

Using a combination of bioinformatic and molecular biology approaches a Drosophila melanogaster protein, DmTTF, has been identified, which exhibits sequence and structural similarity with two mitochondrial transcription termination factors, mTERF (human) and mtDBP (sea urchin). Import/processing assays indicate that DmTTF is synthesised as a precursor of 410 amino acids and is imported into mitochondria, giving rise to a mature product of 366 residues. Band-shift and DNase I protection experiments show that DmTTF binds two homologous, short, non-coding sequences of Drosophila mitochondrial DNA, located at the 3' end of blocks of genes transcribed on opposite strands. The location of the target sequences coincides with that of two of the putative transcription termination sites previously hypothesised. These results indicate that DmTTF is the termination factor of mitochondrial transcription in Drosophila. The existence of two DmTTF binding sites might serve not only to stop transcription but also to control the overlapping of a large number of transcripts generated by the peculiar transcription mechanism operating in this organism.