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.

Molecular Investigation of Mitochondrial RNA19 Role in the Pathogenesis of MELAS Disease.

In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeu and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases.

Mitochondria Deregulations in Cancer Offer Several Potential Targets of Therapeutic Interventions.

Mitochondria play a key role in cancer and their involvement is not limited to the production of ATP only. Mitochondria also produce reactive oxygen species and building blocks to sustain rapid cell proliferation; thus, the deregulation of mitochondrial function is associated with cancer disease development and progression. In cancer cells, a metabolic reprogramming takes place through a different modulation of the mitochondrial metabolic pathways, including oxidative phosphorylation, fatty acid oxidation, the Krebs cycle, glutamine and heme metabolism.

Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype.

The m.3243A>G mutation within the mitochondrial mt-tRNALeu gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification.

Three-dimensional structure of human cyclooxygenase (hCOX)-1.

The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation.

DNMT1 mutations leading to neurodegeneration paradoxically reflect on mitochondrial metabolism.

ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients.

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.

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.

The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail.

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.

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.

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.

NSUN4 is a dual function mitochondrial protein required for both methylation of 12S rRNA and coordination of mitoribosomal assembly.

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU.

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy.

Characterization of the sea urchin mitochondrial transcription factor A reveals unusual features.

Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail.

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.

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.

D-MTERF5 is a novel factor modulating transcription in Drosophila mitochondria.

The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF.

A modified method for the purification of active large enzymes using the glutathione S-transferase expression system.

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.

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.

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.

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 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.

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.