Vol 81, No 10 (2016)

Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.

The diverse functions of mitochondria depend on hundreds of different proteins. The vast majority of these proteins is encoded in the nucleus, translated in the cytosol, and must be imported into the organelle. Import was shown to occur after complete synthesis of the protein, with the assistance of cytosolic chaperones that maintain it in an unfolded state and target it to the mitochondrial translocase of the outer membrane (TOM complex). Recent studies, however, identified many mRNAs encoding mitochondrial proteins near the outer membrane of mitochondria. Translation studies suggest that many of these mRNAs are translated locally, presumably allowing cotranslational import into mitochondria. Herein we review these data and discuss its relevance for local protein synthesis. We also suggest alternative roles for mRNA localization to mitochondria. Finally, we suggest future research directions, including revealing the significance of localization to mitochondria physiology and the molecular players that regulate it.

Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed.

Like animal cells, plant cells bear mechanisms for protein synthesis and posttranslational modification (glycosylation and phosphorylation) that allow them to be seriously considered as factories for therapeutic proteins, including antibodies, with the development of biotechnology. The plant platform for monoclonal antibody production is an attractive approach due to its flexibility, speed, scalability, low cost of production, and lack of contamination risk from animal-derived pathogens. Contemporary production approaches for therapeutic proteins rely on transgenic plants that are obtained via the stable transformation of plant cells as well as the transient (temporary) expression of foreign proteins. In this review, we discuss present-day approaches for monoclonal antibody production in plants (MAPP), features of carbohydrate composition, and methods for the humanization of the MAPP carbohydrate profile. MAPPs that have successfully passed preclinical studies and may be promising for use in clinical practice are presented here. Perspectives on using MAPPs are determined by analyzing their economic benefits and production rates, which are especially important in personalized cancer therapy as well as in cases of bioterrorism and pandemics.

Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E. coli in the denatured state. We also discuss the possible mechanistic reasons for this phenomenon.

The core mitochondrial RNA polymerase is a single-subunit enzyme that in yeast Saccharomyces cerevisiae is encoded by the nuclear RPO41 gene. It is an evolutionary descendant of the bacteriophage RNA polymerases, but it includes an additional unconserved N-terminal extension (NTE) domain that is unique to the organellar enzymes. This domain mediates interactions between the polymerase and accessory regulatory factors, such as yeast Sls1p and Nam1p. Previous studies demonstrated that deletion of the entire NTE domain results only in a temperature-dependent respiratory deficiency. Several sequences related to the pentatricopeptide (PPR) motifs were identified in silico in Rpo41p, three of which are located in the NTE domain. PPR repeat proteins are a large family of organellar RNA-binding factors, mostly involved in posttranscriptional gene expression mechanisms. To study their function, we analyzed the phenotype of strains bearing Rpo41p variants where each of these motifs was deleted. We found that deletion of any of the three PPR motifs in the NTE domain does not affect respiratory growth at normal temperature, and it results in a moderate decrease in mtDNA stability. Steady-state levels of COX1 and COX2 mRNAs are also moderately affected. Only the deletion of the second motif results in a partial respiratory deficiency, manifested only at elevated temperature. Our results thus indicate that the PPR motifs do not play an essential role in the function of the NTE domain of the mitochondrial RNA polymerase.

The nuclear isoform of nucleoside diphosphate kinase isoenzyme NDPK-I_n undergoes strong catalytic activation upon its interaction with the active form of phytochrome A (Pfr) in red light. The autophosphorylation or intermolecular transphosphorylation of NDPK-I_n leads to the formation of phosphoester bonds stable in acidic solution. The phosphate residue of the phosphamide bond in the active center of NDPK-I_n can also be transferred to serine and threonine residues localized in other proteins, including phytochrome A. Phytochrome A, similarly to NDPK-I_n, undergoes autophosphorylation on serine and threonine residues and can phosphorylate some potential substrate proteins. The physical interaction between phytochrome A in the Pfr form and NDPK-I_n results in a significant increase in the kinase activity of NDPK-I_n. The results presented in this work indicate that NDPK-I_n may function as a protein kinase regulated by light.

L-asparaginase (EC 3.5.1.1), which catalyzes the deamidation of L-asparagine to L-aspartic acid and ammonia, has been widely used as a key therapeutic tool in the treatment of tumors. The current commercially available L-asparaginases, produced from bacteria, have signs of toxicity and hypersensitivity reactions during the course of tumor therapy. Therefore, searching for L-asparaginases with unique biochemical properties and fewer adverse effects was the objective of this work. In this study, cyanobacterial strain Synechococcus elongatus PCC6803 was found as a novel source of L-asparaginase. The L-asparaginase gene coding sequence (gi:939195038) was cloned and expressed in E. coli BL21(DE3), and the recombinant protein (Se.ASPII) was purified by affinity chromatography. The enzyme has high affinity towards Lasparagine and shows very weak affinity towards L-glutamine. The enzymatic properties of the recombinant enzyme were investigated, and the kinetic parameters (K_m, V_max) were measured. The pH and temperature dependence profiles of the novel enzyme were analyzed. The work was extended to measure the antitumor properties of the novel enzyme against different human tumor cell lines.

Prolonged or excessive increase in the circulatory level of proinflammatory tumor necrosis factor (TNF) leads to abnormal activation and subsequent damage to endothelium. TNF at high concentrations causes apoptosis of endothelial cells. Previously, using mitochondria-targeted antioxidants of SkQ family, we have shown that apoptosis of endothelial cells is dependent on the production of reactive oxygen species (ROS) in mitochondria (mito-ROS). Now we have found that TNF at low concentrations does not cause cell death but activates caspase-3 and caspase-dependent increase in endothelial permeability in vitro. This effect is probably due to the cleavage of β-catenin–an adherent junction protein localized in the cytoplasm. We have also shown that extracellular matrix metalloprotease 9 (MMP9) VE-cadherin shedding plays a major role in the TNF-induced endothelial permeability. The mechanisms of the caspase-3 and MMP9 activation are probably not related to each other since caspase inhibition did not affect VE-cadherin cleavage and MMP9 inhibition had no effect on the caspase-3 activation. Mitochondria-targeted antioxidant SkQR1 inhibited TNF-induced increase in endothelial permeability. SkQR1 also inhibited caspase-3 activation, β-catenin cleavage, and MMP9-dependent VE-cadherin shedding. The data suggest that mito-ROS are involved in the increase in endothelial permeability due to the activation of both caspase-dependent cleavage of intracellular proteins and of MMP9-dependent cleavage of the transmembrane cell-to-cell contact proteins.

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.

The question if mitochondria have some kind of immune system is not trivial. The basis for raising this question is the fact that bacteria, which are progenitors of mitochondria, do have an immune system. The CRISPR system in bacteria based on the principle of RNA interference serves as an organized mechanism for destroying alien nucleic acids, primarily those of viral origin. We have shown that mitochondria are also a target for viral attacks, probably due to a related organization of genomes in these organelles and bacteria. Bioinformatic analysis performed in this study has not given a clear answer if there is a CRISPR-like immune system in mitochondria. However, this does not preclude the possibility of mitochondrial immunity that can be difficult to decipher or that is based on some principles other than those of CRISPR.