Misframed proteins and neurodegeneration: a novel view on Alzheimer's and Parkinson's diseases.

Sporadic forms of Alzheimer's and Parkinson's diseases are the most frequent forms of their kind. Together with Huntington's disease, they belong to the so called 'conformational diseases' as they share a common feature in the accumulation of insoluble protein deposits. In this review, we focus on the significance of the ubiquitin-proteasome system in conformational diseases and the possible consequences due to the accumulation of aberrant proteins. In all forms of Alzheimer's and Huntington's diseases, but not in Parkinson's disease, we have shown the presence of misframed proteins such as misframed ubiquitin (UBB(+1)) of which we have determined the functional relevance in vitro and in vivo.Misframed proteins are the result of the inaccurate transcription of monotonic sequences in the genome and their subsequent translation. This process has been called 'molecular misreading'. In the present review, we will discuss the present state of the art with regard to UBB(+1) and amyloid precursor protein APP(+1).

A methodology for calculating transport emissions in cities with limited traffic data: Case study of diesel particulates and black carbon emissions in Murmansk.

This paper presents a methodology for calculating exhaust emissions from on-road transport in cities with low-quality traffic data and outdated vehicle registries. The methodology consists of data collection approaches and emission calculation methods. For data collection, the paper suggests using video survey and parking lot survey methods developed for the International Vehicular Emissions model. Additional sources of information include data from the largest transportation companies, vehicle inspection stations, and official vehicle registries. The paper suggests using the European Computer Programme to Calculate Emissions from Road Transport (COPERT) 4 model to calculate emissions, especially in countries that implemented European emissions standards. If available, the local emission factors should be used instead of the default COPERT emission factors. The paper also suggests additional steps in the methodology to calculate emissions only from diesel vehicles. We applied this methodology to calculate black carbon emissions from diesel on-road vehicles in Murmansk, Russia. The results from Murmansk show that diesel vehicles emitted 11.7 tons of black carbon in 2014. The main factors determining the level of emissions are the structure of the vehicle fleet and the level of vehicle emission controls. Vehicles without controls emit about 55% of black carbon emissions.

Aminoacylation of tRNA gene transcripts is strongly affected by 3'-extended and dimeric substrate RNAs.

Kinetic parameters of aminoacylation by E. coli phenylalanyl-tRNA synthetase vary for phage T5 tRNA(Phe) gene transcript from 0.950 to 2.545 microM for Km and from 550 to 400 min(-1) for kcat. To reveal the source of this variability for various RNA preparations, homogeneity of the transcripts has been examined. Presence of 3' extensions and dimer formation in transcript preparations reduced the catalytic efficiency kcat/Km several-fold. We have shown that the proportion of dimers and 3'-extended transcripts in tRNA preparations is sensitive to single-base substitutions in tRNA. While wild-type phage T5 tRNA(Phe) gene transcript contains about half of dimeric molecules, for some mutants this value increases up to 90% or drops to 0%. Phage T5 tRNA(Phe) gene with anticodon stem nucleotide substitutions used as a template in run-off transcription produces 5 times less 3'-extended molecules than the wild-type gene. In view of all these results kinetic parameters of aminoacylation reaction for many wild-type and mutant tRNA gene transcripts should be reevaluated.

Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration.

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

Protein tyrosine phosphatases.

The molecular mechanisms of signal transduction have been at the focus of increasingly intense scientific research. As a result, our understanding of protein tyrosine kinase-mediated signaling has advanced at an unprecedented pace during the past decade. In contrast, the study of protein tyrosine phosphatases has lagged behind, but is now gathering momentum and is predicted to become a "hot topic" in the field within the next few years. This review summarizes the current state-of-the art in our understanding of the structure, regulation and role of protein tyrosine phosphatases with emphasis on the lymphocyte system.

Novel vectors for co-expression of two proteins in E. coli.

Two new vectors, pAC28 and pEGST, for the co-expression of recombinant genes in E. coli were developed. This two-plasmid system allows for an efficient expression and purification of large amounts of protein-protein complexes formed in bacterial cells. We have utilized this new system to express and isolate a stable complex of two human proteins, hematopoietic cell tyrosine phosphatase (HePTP) and mitogen-activated proteins kinase Erk2. This approach is useful for biochemical and structural studies of protein-protein interactions.

[A new method to measure the functional activity of class-1 translation termination factor eRF1]. / Novyi metod opredeleniia funktsional'noi aktivnosti factora terminatsii transliatsii pervogo klassa eRF1.

Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.

[Suppression of nonsense mutations in the Dystrophin gene by a suppressor tRNA gene]. / Ispol'zovanie gena supressornoi tRNK dlia ispravleniia nonsens-mutatsii v gene distrofina.

Nonsense mutations in the dystrophin gene are the cause of Duchenne muscular dystrophy (DMD) in 10-15% of patients. In such an event, one approach to gene therapy for DMD is the use of suppressor tRNAs to overcome the premature termination of translation of the mutant mRNA. We have carried out cotransfection of the HeLa cell culture with constructs containing a suptRNA gene (pcDNA3suptRNA) and a marker LacZ gene (pNTLacZhis) using their polymer VSST-525 complexes. It was found that the number of cells producing beta-galactosidase depends inversely on the dose of the suptRNA gene. A single in vivo injection of the construct providing for expression of the suptRNAochre gene into mdx mouse muscle resulted in the production of dystrophin in 2.5% of fibers. This suggests that suppressor tRNAs are applicable in gene therapy for hereditary diseases caused by nonsense mutations.

The ubiquitin proteasome system in neurodegenerative diseases: culprit, accomplice or victim?

A shared hallmark for many neurodegenerative disorders is the accumulation of toxic protein species which is assumed to be the cause for these diseases. Since the ubiquitin proteasome system (UPS) is the most important pathway for selective protein degradation it is likely that it is involved in the aetiology neurodegenerative disorders. Indeed, impairment of the UPS has been reported to occur during neurodegeneration. Although accumulation of toxic protein species (amyloid ß) are in turn known to impair the UPS the relationship is not necessarily causal. We provide an overview of the most recent insights in the roles the UPS plays in protein degradation and other processes. Additionally, we discuss the role of the UPS in clearance of the toxic proteins known to accumulate in the hallmarks of neurodegenerative diseases. The present paper will focus on critically reviewing the involvement of the UPS in specific neurodegenerative diseases and will discuss if UPS impairment is a cause, a consequence or both of the disease.

[Molecular changes in inbred mice with individual vulnerability vs resilience to stress-induced depressive-like state].

The C57BL/6 mice were subjected to a chronic combined stress which resulted in the induction of a depressive-like state. The occurrence of a depressive-like state was defined by a decrease in sensitivity to the reward determined by the diminished preference of sweetened solutions over regular drinking water. Such decrease is generally considered as a sign of an unhedonic-like state: one of the key features of clinical depression. Applied here, the paradigm in mice allows unhedonia induction in a subpopulation of stressed animals (54% in the current study); remaining mice are regarded as resilient to stress-induced hedonic deficit. The resilient subgroup is taken, therefore, as a "functional control" for those effects of stress that are not accompanied by development of the stress-induced depressive-like state in mice. The analysis of the mRNA extracted from the hippocampi of stress-subjected and home-cage control mice enabled the assessment of gene expression level of over 13 000 genes. This study showed that unhedonic mice are characterized by an up-regulation of 278 and down-regulation of 174 genes related mostly to the CNS development and functions, inter-cellular interactions and signalling, neurological disorders, apoptosis and behavioural regulation. Resilient animals demonstrated up-regulation of 924 and down-regulation of only 29 genes that control formation of cell assemblies, molecular transport, CNS functioning, neurological disorders and various biochemical reactions. Thus, gene expression profiles in the hippocampus of susceptible vs resilient to stress-induced unhedonia inbred subgroups of animals are strictly distinct in both quantity and quality.

Gene-environment interaction research and transgenic mouse models of Alzheimer's disease.

The etiology of the sporadic form of Alzheimer's disease (AD) remains largely unknown. Recent evidence has suggested that gene-environment interactions (GxE) may play a crucial role in its development and progression. Whereas various susceptibility loci have been identified, like the apolipoprotein E4 allele, these cannot fully explain the increasing prevalence of AD observed with aging. In addition to such genetic risk factors, various environmental factors have been proposed to alter the risk of developing AD as well as to affect the rate of cognitive decline in AD patients. Nevertheless, aside from the independent effects of genetic and environmental risk factors, their synergistic participation in increasing the risk of developing AD has been sparsely investigated, even though evidence points towards such a direction. Advances in the genetic manipulation of mice, modeling various aspects of the AD pathology, have provided an excellent tool to dissect the effects of genes, environment, and their interactions. In this paper we present several environmental factors implicated in the etiology of AD that have been tested in transgenic animal models of the disease. The focus lies on the concept of GxE and its importance in a multifactorial disease like AD. Additionally, possible mediating mechanisms and future challenges are discussed.

Dimer formation by tRNAs.

Available data on formation of dimers and other oligomers by "mature" tRNAs and the transcripts of their genes show that the dimers may be formed in vitro during the purification of individual tRNAs as well as on heating and subsequent cooling of tRNA preparations. The functional activity of dimers varies for different tRNAs. Structural analysis of tRNAs that are able or unable to form dimers suggests the importance of the hairpin melting temperature for dimer formation. Pathways of dimer formation and specific structural features of tRNA dimers are discussed.

Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers.

Significant amounts (10-30%) of 3'-extended products with one or two extra nucleotides are synthesized in the course of run-off tRNA gene transcription with T7 RNA polymerase. Denaturing polyacrylamide gel electrophoresis appeared to be insufficient to provide preparative amounts of pure correct-size transcripts. Formation of dimers by tRNA gene transcripts as side products in the course of their activation is also another obstacle in preparation of biologically active transcripts. Here, we have shown that EF-Tu affinity chromatography and/or non-denaturing electrophoresis are simple and efficient tools for isolation of highly active correct-size transcripts. Conditions for transcript activation in vitro should be carefully controlled to prevent dimer formation and obtain reliable data on tRNA transcript structure and function.

Mg2+ binding and structural stability of mature and in vitro synthesized unmodified Escherichia coli tRNAPhe.

Mature tRNAPhe from Escherichia coli and the transcript of its gene lacking modified nucleotides were compared by a variety of physical techniques. Melting experiments revealed that at a low Mg2+level the transcript was partially denatured, while the mature tRNA possessed intact tertiary interactions. Mg2+binding to both tRNAs was studied by CD and UV techniques as well as by using the Mg2+-sensitive fluorescence indicator, 8-hydroxyquinoline 5-sulfonic acid. Both tRNA forms exhibited a single strong Mg2+-binding site, its dissociation constant was 10-fold higher for the transcript. Conformational changes in response to Mg2+ addition measured by CD and UV spectrometry revealed no difference for the estimated binding cooperativity and strong differences for affinities of Mg2+-binding sites for the two tRNA forms. Conformational transitions in mature and in in vitro synthesized tRNA required the binding of two Mg2+ ions per molecule and therefore should be associated not only with a single strong binding site. The Mg2+ dependence of Stokes radii measured by gel-filtration revealed insignificant differences between the overall sizes of the two tRNA forms at physiological Mg2+ levels (>1 mM). Taken together, these results suggest that modified nucleotides stabilize tertiary interactions and increase the structure stability without affecting the mechanism of Mg2+binding and overall folding of the tRNA molecule. This conclusion is supported by the known biological activity of the E. coli tRNAPhe gene transcript.