Protein Biosynthesis in Mitochondria: Past Simple, Present Perfect, Future Indefinite.

Mitochondria are obligate organelles of most eukaryotic cells that perform many different functions important for cellular homeostasis. The main role of mitochondria is supplying cells with energy in a form of ATP, which is synthesized in a chain of oxidative phosphorylation reactions on the organelle inner membrane. It is commonly believed now that mitochondria have the endosymbiotic origin. In the course of evolution, they have lost most of their genetic material as a result of genome reduction and gene transfer to the nucleus. The majority of mitochondrial proteins are synthesized in the cytosol and then imported to the mitochondria. However, almost all known mitochondria still contain genomes that are maintained and expressed. The processes of protein biosynthesis in the mitochondria - mitochondrial translation - substantially differs from the analogous processes in bacteria and the cytosol of eukaryotic cells. Mitochondrial translation is characterized by a high degree of specialization and specific regulatory mechanisms. In this review, we analyze available information on the common principles of mitochondrial translation with emphasis on the molecular mechanisms of translation initiation in the mitochondria of yeast and mammalian cells.

Mitochondrial Translation Initiation Factor 3: Structure, Functions, Interactions, and Implication in Human Health and Disease.

Mitochondria are essential organelles of eukaryotic cell that provide its respiratory function by means of the electron transfer chain. Expression of mitochondrial genes is organized in a bacterial-like manner; however multiple evolutionary differences are observed between the two systems, including translation initiation machinery. This review is dedicated to the mitochondrial translation initiation factor 3 (IF3mt), which plays a key role in the protein synthesis in mitochondria. Involvement of IF3mt in human health and disease is discussed.

Interaction between Saccharomyces cerevisiae Mitochondrial DNA-Binding Protein Abf2p and Cce1p Resolvase.

Mitochondrial DNA is susceptible to the action of reactive oxygen species generated by the reactions of oxidative phosphorylation. Homologous recombination is one of the mechanisms providing integrity of the mitochondrial genome. Some proteins that take part in this process in budding yeast mitochondria have been identified. These include Abf2p, the major protein of the mt-nucleoid that specifically binds cruciform DNA, and Cce1p - Holliday junction resolvase. Here we show that Abf2p does not significantly affect either binding of Cce1p to branched DNA or rate and specificity of Holliday junction resolution. These data suggest the existence of an alternative homologous recombination pathway in yeast mitochondria.

Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome.

Mitochondrial Matrix Processes.

Mitochondria possess their own genome that, despite its small size, is critically important for their functioning, as it encodes several dozens of RNAs and proteins. All biochemical processes typical for bacterial and nuclear DNA are described in mitochondrial matrix: replication, repair, recombination, and transcription. Commonly, their mechanisms are similar to those found in bacteria, but they are characterized by several unique features. In this review, we provide an overall description of mitochondrial matrix processes paying special attention to the typical features of such mechanisms.

[Effect of heat shock on cells of phytopathogenic mycoplasma Acholeplasma laidlawii PG-8A].

Heat shock caused a more active formation of the "dormant" forms (minibodies), as well as increased production of extracellular membrane vesicles by Acholeplasma laidlawii PG-8A cells. Raise of the amount of the minibodies that have increased resistance to biogenic and abiogenic stress factors and pathogenicity may lead to more successful persistence of mycoplasmas in their hosts. Increased production of the extracellular membrane vesicles containing virulence factors by Acholeplasma laidlawii cells during stress may be an additional burden for the infected organism. It has been recently revealed that the vesicles of A. laidlawii contain appreciable quantities of small heat shock protein IbpA (Hsp20). In this paper, using immune-electron microscopy, have shown that at elevated temperature IbpA is associated with A. laidlawii minibodies. Perhaps, IbpA contributes to increased resistance and pathogenicity of the minibodies, keeping their proteins and polypeptides, including protein virulence factors in the folding-competent state.

Protein biosynthesis in mitochondria.

Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

Complete genome and proteome of Acholeplasma laidlawii.

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

Functional Diversity of Mitochondrial Peptidyl-tRNA Hydrolase ICT1 in Human Cells.

Mitochondria are energy producing organelles of the eukaryotic cell, involved in the synthesis of key metabolites, calcium homeostasis and apoptosis. Protein biosynthesis in these organelles is a relic of its endosymbiotic origin. While mitochondrial translational factors have homologues among prokaryotes, they possess a number of unique traits. Remarkably as many as four mammalian mitochondrial proteins possess a clear similarity with translation termination factors. The review focuses on the ICT1, which combines several functions. It is a non-canonical termination factor for protein biosynthesis, a rescue factor for stalled mitochondrial ribosomes, a structural protein and a regulator of proliferation, cell cycle, and apoptosis. Such a diversity of roles demonstrates the high functionality of mitochondrial translation associated proteins and their relationship with numerous processes occurring in a living cell.

[Oligomeric forms, functions and cellular localization of alpha-crystallin type protein from Acholeplasma laidlawii].

Alpha-Crystallin type heat shock protein (alpha-HSP) IbpA from Acholeplasma laidlawii was expressed in Escherichia coil and isolated from cell extract on Ni-sepharose column. Recombinant IbpA, like other alpha-HSPs, spontaneously formed oligomeres in vitro. High resolution electron microscopy revealed regular structures with 15 nm in diameter. Evaluation of molecular mass of IbpA oligomers was performed by gel filtration. Most of oligomers consist of 24 subunits. Recombinant IbpA prevents heat denaturation of soluble proteins in cell extract of E. coli and displays a mild positive effect on thermotolerance of E. coli cells during severe heat shock. We investigated a localization of IbpA in A. laidlawii cell by immunocytochemistry. We suppose that IbpA may protect various intracellular structures from damage during heat shock.

[Localization of the division protein FtsZ in mycoplasma cells Mycoplasma hominis].

Localization of the protein FtsZ in Mycoplasma hominis cells was determined. Ultra thin sections were treated by rabbit polyclonal antibodies against FtsZ M. hominis: a conjugate of protein A with colloidal gold particles was used instead of secondary antibodies. Considerable polymorphism of cells was seen on electron microscopy pictures of M. hominis cells, which is typical for mycoplasmas. Among a wide variety of cell shapes we distinguished dumbbell-shaped dividing cells, and the cells connected with each other with the aid of thin membrane tubules (former constrictions). Dominants distribution of the label in the constriction area of dividing M. hominis cells and in the area of the thin membrane tubules was observed. We revealed the cross septum in the mycoplasma cells for the first time, as well as the gold labeling of this structure. Furthermore, in some rounded and oval cells colloidal gold particles labeled the whole plasma membrane in ring-shaped manner. Probably, the label in these cases marks a submembrane contractile ring (Z-ring). The facts mentioned above confirm that FtsZ of M. hominis plays an active role in the mycoplasma cytokinesis. In a series of cases spiral-like distribution of gold particles was observed. Probably, FtsZ protofilaments in M. hominis cells can form spiral structures similar to Z-spirals of Bacillus subtilis and Escherichia coli. Its presence in mycoplasma cells may be considered as an important argument in favour of model of Z-ring assembling through reorganization of Z-spirals. FtsZ also may participate in maintenance of mycoplasma cell shape (membrane localization).

[Molecular genetic methods of typing of the Bartonellae].

The primer systems for the PCR detection of four house-keeping genes of bartonellae in clinical material were developed and tested. The tactics of the species RFLP typing was also developed and tested. The scheme of the species RFLP typing of bartonellae was tested using as an example two strains for the first time isolated in Russia from patients with endocarditis and fever of uncertain origin. The results of the typing were supported by sequencing of the amplicons obtained. According to the sequencing the isolates were attributed to the sub species Bartonella vinsonii, subsp. arupensis. The necessity of molecular epidemiological analysis of bartonelloses in Russia was substantiated.

[Wild small mammals are the reservoir hosts of the Bartonella genus bacteria in the south of Moscow region].

A total of 103 blood samples collected from wild small mammals captured in the Prioksko-Terrasny Reserve on the south of Moscow region were studied to determine the bartonellae prevalence. The examined species were the yellow-necked mice Apodemus flavicollis (35 samples), the European wood mouse Apodemus uralensis (10 samples), the bank vole Clethrionomys glareolus (51 samples), the house mouse Mus musculus (3 samples), the common vole Microtus arvalis (2 samples), and the shrew Sorex araneus (2 samples). Initially, we obtained 76 bacterial Bartonella-like isolates after plating onto the surface of the solid nutrient media. 66 of them were PCR-positive at least for three of four targets, gltA, ftsZ, ribC and 16S RNA. Thus, the percentage of the infection in the studied community was 64%. Subsequent RFLP assay showed that obtained isolates belonged to the Bartonella grahamii and/or B. taylorii species. In 7 cases we found both bartonellae species in one animal. These data were confirmed by direct sequencing of four ftsZ, four ribC and two gltA amplicons. According to our data, there is no any marked host specificity for these bartonellae species. Now we have laid the bartonellae strain collection consisting of 31 isolates. To our knowledge, this is the first investigation of the bartonellae prevalence in wild small mammals performed in Russia. The comparison of our data with those obtained by European researchers and issues of coinfection by different bartonellae species and host specificity are discussed.

[Identification of signal sequences determining the specific nucleolar localization of fibrillarin in HeLa cells]. / Vyiavlenie signal'nykh posledovatel'nostei v molekule fibrillarina, opredeliaiushchikh ego spetsificheskuiu lokalizatsiiu v iadryshkakh kletok HeLa.

Fibrillarin is one of the major nucleolar proteins and is involved in pre-rRNA maturation. Its three main regions are a glycine and arginine-rich (GAR) domain, an RNA-binding domain, and an alpha-helical region, which presumably has a methyltransferase activity. Yet the roles of these regions in nucleolus-specific localization of fibrillarin are still unclear. To elucidate this issue, a series of plasmids was constructed to express human fibrillarin mutants fused with the green fluorescent protein. Localization of the chimeric proteins was studied in interphase and mitotic HeLa cells after single transfection with the plasmids. Deletion or a mutation of any domain proved to alter the specific fibrillarin location coinciding with sites of pre-rRNA synthesis. The GAR domain and the first spacer together were sufficient for fibrillarin migration into the nucleolus. Fibrillarin mutants located within the interphase nucleolus did not differ in mitotic location from the wild-type fibrillarin.

Localization of C. trachomatis Inc proteins in expression of their genes in HeLa cell culture.

Co-localization of Chlamydia trachomatis incorporation membrane proteins with cell organelles was studied in HeLa cell culture after transfection by expressing vectors carrying incA, incB, incC, incD, incE, incF, incG genes, respectively, fused with the marker green fluorescent protein (EGFP) gene. The prokaryotic proteins were co-located with compartments of the secretory pathway of the eukaryotic cell in the course of biogenesis.

Recombinant plasmid constructs expressing gene for antimicrobial peptide melittin for the therapy of Mycoplasma and chlamydia infections.

In view of growing number of pathogenic microbial strain resistant to routine antibiotics, antimicrobial peptides become promising agents for the therapy of infectious diseases. We studied in vivo effects of melittin, an antimicrobial peptide expressed in a recombinant plasmid vector, on infection with urogenital pathogens Chlamydia trachomatis, Mycoplasma hominis, and Mycoplasma gallisepticum. We obtained recombinant plasmid constructs, where melittin gene is under the control of tetracycline-dependent human cytomegalovirus promoter. Inhibition of experimental C. trachomatis, M. hominis, and M. gallisepticum infection after administration of recombinant plasmid vectors expressing melittin gene to BALB/c mice was demonstrated.

Expression of Chlamydia trachomatis inclusion membrane protein genes IncB and IncC in Escherichia coli.

In this study, we have cloned the Chlamydia trachomatis genes incB and incC into the expression plasmid vectors from pET series for the subsequent isolation of recombinant proteins. As a result, we have obtained the first full-length recombinant C. trachomatis proteins IncB and IncC, which can be used for following antibody production and for study of their protein-protein interaction.