Formation of Amyloid-Like Conformational States of ß-Structured Membrane Proteins on the Example of OMPF Porin from the Yersinia pseudotuberculosis Outer Membrane.

The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with ß-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total ß-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to ß-barrel.

Effect of Non-Specific Porins from the Outer Membrane of Yersinia pseudotuberculosis on Mice Brain Cortex Tissues.

It was found that a single-dose immunization of mice with Yersinia pseudotuberculosis porins OmpF and OmpC causes development of pathological changes in the deep layers of cerebral cortex characterized by dystrophic changes in the cells against the background of the increasing titer of specific antibodies. At the same time, the increased level of caspase-3 expression is observed in the neurons, which indicates induction of proapoptotic signaling pathways. The obtained results indicate potential ability of nonspecific pore-forming proteins of the outer membrane of Gram-negative bacteria to initiate development of degenerative changes in brain cells.

Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

Transposon-mediated telomere destabilization: a driver of genome evolution in the blast fungus.

The fungus Magnaporthe oryzae causes devastating diseases of crops, including rice and wheat, and in various grasses. Strains from ryegrasses have highly unstable chromosome ends that undergo frequent rearrangements, and this has been associated with the presence of retrotransposons (Magnaporthe oryzae Telomeric Retrotransposons-MoTeRs) inserted in the telomeres. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, mapping, and sequencing of parental and novel telomeric restriction fragments (TRFs), along with MinION sequencing of genomic DNA allowed us to document the precise molecular alterations underlying 109 newly-formed TRFs. These included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by 'plain' telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; MoTeR-independent expansion and contraction of subtelomeric tandem repeats; and a variety of rearrangements initiated through breaks in interstitial telomere tracts that are generated during MoTeR integration. Overall, we estimate that alterations occurred in approximately sixty percent of chromosomes (one in three telomeres) analyzed. Most importantly, we describe an entirely new mechanism by which transposons can promote genomic alterations at exceptionally high frequencies, and in a manner that can promote genome evolution while minimizing collateral damage to overall chromosome architecture and function.

Bacterial Group II Intron Genomic Neighborhoods Reflect Survival Strategies: Hiding and Hijacking.

Group II (gII) introns are mobile retroelements that can spread to new DNA sites through retrotransposition, which can be influenced by a variety of host factors. To determine if these host factors bear any relationship to the genomic location of gII introns, we developed a bioinformatic pipeline wherein we focused on the genomic neighborhoods of bacterial gII introns within their native contexts and sought to determine global relationships between introns and their surrounding genes. We found that, although gII introns inhabit diverse regions, these neighborhoods are often functionally enriched for genes that could promote gII intron retention or proliferation. On one hand, we observe that gII introns are frequently found hiding in mobile elements or after transcription terminators. On the other hand, gII introns are enriched in locations in which they could hijack host functions for their movement, potentially timing expression of the intron with genes that produce favorable conditions for retrotransposition. Thus, we propose that gII intron distributions have been shaped by relationships with their surrounding genomic neighbors.

OmpF porin from Yersinia ruckeri as pathogenic factor: Surface antigenic sites and biological properties.

Bacterium Yersinia ruckeri as a pathogen induces causative agent of intestinal fish disease called enteric redmouth disease (ERM) is known. In this study, outer membrane OmpF porin from the Y. ruckeri (YrOmpF) has been identified as a pathogenic factor which affects host macrophage activation and life cycle of eukaryotic cells. Using synthetic peptides corresponding to the sequences of the outer loops of YrOmpF L1 loop of the porin is most involved in the structure of B epitopes on the surface of the microbial cell it was found. T epitopes of the isolated YrOmpF trimer not only by linear, but also by discontinuous determinants, which is due to the secondary structure of the protein are represented. It was shown that YrOmpF was twice more cytotoxic to THP-1 cells (human monocytes, cancer cells) in comparison with CHH-1 cells (Oncorhynchus keta cardiac muscle cell, non-cancer cells). It was found YrOmpF induce cell cycle S-phase arrest in both normal CHH-1 and cancer THP-1 cells. In the cancer cells observed effect was most pronounce. In addition, we have observed an induction of apoptosis in THP-1 cell line treated with YrOmpF for 48 h at IC50 (48.6 µg/ml). Significant cytotoxic effect of YrOmpF on primary mouse peritoneal macrophages been detected as well. Of note, co-incubation of macrophages with anti-YrOmpF antibodies could decrease the amount of lactate dehydrogenase, while the number of living cells significantly increased. YrOmpF stimulates the activity of the phagocytic bactericidal systems especially of the oxygen-independent subsystem it was found. Antibodies against YrOmpF decreased MPO release and CP synthesis by peritoneal macrophages and increased their viability.

Live births and maintenance with levonorgestrel IUD improve disease-free survival after fertility-sparing treatment of atypical hyperplasia and early endometrial cancer.

OBJECTIVE: Our objectives were to (1) compare different regimens of hormonal therapy (HT) in young women with atypical endometrial hyperplasia (AEH) and early endometrial cancer (EC), (2) assess reproductive and oncologic outcomes and (3) explore possible predictors of complete response (CR) and disease free survival (DFS). METHODS: Reproductive age women with AEH and Grade 1-2 endometrioid EC with no or minimal myometrial invasion on MRI treated with different regimens of HT were prospectively analyzed. Treatment protocols included levonorgestrel intrauterine device (LNG IUD), gonadotropin-releasing hormone agonist (aGnRH) or high-dose oral medroxyprogesteron acetate (MPA) separately and in combinations. RESULTS: Total of 418 patients with AEH (n = 228) and EC (n = 190) aged 19-46 years received HT. Overall CR rate was 96% in AEH and 88% in EC patients (р < 0.001). None of the regimens used in AEH (LNG IUD + 2 D&C vs. LNG IUD + aGnRH vs. LNG IUD + 3 D&C) was found inferior to the others (CR of 98%, 95%, 100%, respectively, p > 0.05) except for MPA alone (CR 87%, р = 0.009). Out of four HT regimens used in EC LNG IUD + aGnRH+3 D&C was superior to all others (CR 96%, р = 0.026) where 2 D&Cs were performed or oral MPA was prescribed. The median follow-up for 339 patients was 33 months (range: 3-136), 68% of patients (n = 232) attempted conception, 38% (n = 89) of them used ART. The birth rate was 42% (n = 97). The rate of recurrence was 26% (50/196) in AEH group and 36% (51/143) in EC group (p = 0.05). Birth after treatment (HR = 0.24) or LNG IUD maintenance (HR = 0.18) were associated with superior DFS (p < 0.001 for both). ART use did not influence DFS. CONCLUSION: Hormonal therapy of AEH and early EC with LNG IUD is superior to MPA-containing regimens, however still carries high risk of recurrence. Post-treatment pregnancy rates are satisfactory and can be further improved by broader ART use which was proven safe. Initial diagnosis of AEH, post-treatment child birth and LNG IUD maintenance were associated with decreased rates of recurrence.

Mobile Group II Introns as Ancestral Eukaryotic Elements.

The duality of group II introns, capable of carrying out both self-splicing and retromobility reactions, is hypothesized to have played a profound role in the evolution of eukaryotes. These introns likely provided the framework for the emergence of eukaryotic retroelements, spliceosomal introns and other key components of the spliceosome. Group II introns are found in all three domains of life and are therefore considered to be exceptionally successful mobile genetic elements. Initially identified in organellar genomes, group II introns are found in bacteria, chloroplasts, and mitochondria of plants and fungi, but not in nuclear genomes. Although there is no doubt that prokaryotic and organellar group II introns are evolutionary related, there are remarkable differences in survival strategies between them. Furthermore, an evolutionary relationship of group II introns to eukaryotic retroelements, including telomeres, and spliceosomes is unmistakable.

Porin from Marine Bacterium Marinomonas primoryensis KMM 3633T: Isolation, Physico-Chemical Properties, and Functional Activity.

Marinomonas primoryensis KMM 3633T, extreme living marine bacterium was isolated from a sample of coastal sea ice in the Amursky Bay near Vladivostok, Russia. The goal of our investigation is to study outer membrane channels determining cell permeability. Porin from M. primoryensis KMM 3633T (MpOmp) has been isolated and characterized. Amino acid analysis and whole genome sequencing were the sources of amino acid data of porin, identified as Porin_4 according to the conservative domain searching. The amino acid composition of MpOmp distinguished by high content of acidic amino acids and low content of sulfur-containing amino acids, but there are no tryptophan residues in its molecule. The native MpOmp existed as a trimer. The reconstitution of MpOmp into black lipid membranes demonstrated its ability to form ion channels whose conductivity depends on the electrolyte concentration. The spatial structure of MpOmp had features typical for the classical gram-negative porins. However, the oligomeric structure of isolated MpOmp was distinguished by very low stability: heat-modified monomer was already observed at 30 °C. The data obtained suggest the stabilizing role of lipids in the natural membrane of marine bacteria in the formation of the oligomeric structure of porin.

The roles of mechanotransduction, vascular wall cells, and blood cells in atheroma induction.

BACKGROUND: This paper describes and analyzes the cellular and molecular mechanisms underlying atherosclerosis development. In particular, the roles of monocytes/macrophages, smooth muscle cells, and vascular endothelium in the formation of stable/unstable atheromatous plaques, and the contributions of some processes to atheroma formation. METHODS AND RESULTS: In this study we analyzed endothelium: function, dysfunction, and involvement into atherogenesis; cell proteins mediating mechanotransduction; proatherogenic role of monocytes; the role of macrophages in the development of unstable atheromatous plaques and smooth muscle cell origin in atherosclerosis. Smooth muscle cell phenotypic switching; their functioning; the ability to retain cholesterol and lipoproteins as well as secretion of pro- and anti-inflammatory molecules and extracellular matrix proteins, their response to extracellular stimuli secreted by other cells, and the effect of smooth muscle cells on the cells surrounding atheromatous plaques are fundamentally important for the insight into atherosclerosis molecular basis. CONCLUSION: Atheromatous plaque transcriptome studies will be helpful in the identification of the key genes involved in atheroma transformation and development as well as discovery of the new targets for diagnosis and therapy.

Carotid Endarterectomy with Autoarterial Remodeling of Bifurcation of the Common Carotid Artery and Carotid Endarterectomy with Patch Closure: Comparison of Methods.

BACKGROUND: The objectives of our research were to identify whether the new method of carotid endarterectomy (CEA) with autoarterial remodeling of bifurcation of the common carotid artery (ARBCCA) influences daily parameters of blood pressure and heart rate (HR) while monitoring them on a daily basis and to assess the efficacy of the suggested method. MATERIALS AND METHODS: It is a prospective randomized comparative study. The first group (n = 100) included patients that underwent ARBCCA, the second group (n = 100) included patients that underwent "classic" CEA with xenopericardial patch closure. Diurnal Holter recording of blood pressure and (HR) was performed before and after the surgical treatment in both groups. RESULTS: Surgical treatment in both groups leads to an increase of HR, arterial hypertension time index by systolic blood pressure, and arterial hypertension time index by diastolic arterial blood pressure. The damage of carotid artery bulb increases sympathetic innervation and causes dysregulation of the baroreceptor mechanism. CONCLUSIONS: In our study, we did not reveal a significant difference in the incidence of postoperative hypertension and the dependence of HR on the choice of surgical technique. Thus, the proposed ARBCCA method does not lead to an increased risk of pre-existing arterial hypertension development. A significant difference is found out on the parameter of the clamping time of carotid arteries in favor to ARBCCA group. Another advantage of the suggested technique is the number of restenosis greater than 50% during the 2-year follow-up (4 [4%] cases (ARBCCA group) versus 12 [12%] cases ["classic" CEA], respectively, P = .037).

Mechanisms Underlying Atheroma Induction: The Roles of Mechanotransduction, Vascular Wall Cells, and Blood Cells.

BACKGROUND: The objective of this article is to review cellular mechanism of atherosclerosis (AS) development. The pathogenesis of AS comprises a sequence of biological events leading to build up of a dense or loose atheromatous plaque (AP). METHODS: In this review, we tried to attempt to analyze the cellular mechanisms underlying AS development, including the roles of monocytes/macrophages and smooth muscle cells in the formation of stable/unstable APs. RESULTS: As a rule, APs are formed in the regions with irregular blood flow; both mechanical perturbations of the vascular wall and several biological events contribute to plaque formation. Blood lipid/lipoprotein deposition, recruitment of monocytes/macrophages, foam cell formation, migration and proliferation of smooth muscle cells, secretion of extracellular matrix, and formation of the connective tissue in plaques are among the latter events. CONCLUSIONS: The review briefs the contributions of different processes to atheroma formation and describes the molecular mechanisms involved in AS development. AP transcriptome studies will be helpful in the identification of the key genes involved in atheroma transformation and development as well as discovery of the new targets for diagnosis and therapy.

Single channel activity of OmpF-like porin from Yersinia pseudotuberculosis.

To gain a mechanistic insight in the functioning of the OmpF-like porin from Yersinia pseudotuberculosis (YOmpF), we compared the effect of pH variation on the ion channel activity of the protein in planar lipid bilayers and its binding to lipid membranes. The behavior of YOmpF channels upon acidification was similar to that previously described for Escherichia coli OmpF. In particular, a decrease in pH of the bathing solution resulted in a substantial reduction of YOmpF single channel conductance, accompanied by the emergence of subconductance states. Similar subconductance substates were elicited by the addition of lysophosphatidylcholine. This observation, made with porin channels for the first time, pointed to the relevance of lipid-protein interactions, in particular, the lipid curvature stress, to the appearance of subconductance states at acidic pH. Binding of YOmpF to membranes displayed rather modest dependence on pH, whereas the channel-forming potency of the protein tremendously decreased upon acidification.

Intein Clustering Suggests Functional Importance in Different Domains of Life.

Inteins, also called protein introns, are self-splicing mobile elements found in all domains of life. A bioinformatic survey of genomic data highlights a biased distribution of inteins among functional categories of proteins in both bacteria and archaea, with a strong preference for a single network of functions containing replisome proteins. Many nonorthologous, functionally equivalent replicative proteins in bacteria and archaea carry inteins, suggesting a selective retention of inteins in proteins of particular functions across domains of life. Inteins cluster not only in proteins with related roles but also in specific functional units of those proteins, like ATPase domains. This peculiar bias does not fully fit the models describing inteins exclusively as parasitic elements. In such models, evolutionary dynamics of inteins is viewed primarily through their mobility with the intein homing endonuclease (HEN) as the major factor of intein acquisition and loss. Although the HEN is essential for intein invasion and spread in populations, HEN dynamics does not explain the observed biased distribution of inteins among proteins in specific functional categories. We propose that the protein splicing domain of the intein can act as an environmental sensor that adapts to a particular niche and could increase the chance of the intein becoming fixed in a population. We argue that selective retention of some inteins might be beneficial under certain environmental stresses, to act as panic buttons that reversibly inhibit specific networks, consistent with the observed intein distribution.

Production of recombinant porin from Y. pseudotuberculosis in a water-soluble form for pseudotuberculosis diagnostics.

OmpF porin from the outer membrane of Yersinia pseudotuberculosis was cloned into pET-40b(+) plasmid. Using E. coli Rosetta (DE3) strain, MX medium, IPTG concentration of 0.2 mm and post-induction cultivation at 14°C overnight allowed us to obtain a water-soluble form of the recombinant protein (rs-OmpF). Rs-OmpF was shown to have the ordered spatial structure at the levels of secondary and tertiary structure. Rs-OmpF was found to be effective as diagnostic antigen in ELISA for pseudotuberculosis diagnostics.

Post-translational environmental switch of RadA activity by extein-intein interactions in protein splicing.

Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.

Interaction between conjugative and retrotransposable elements in horizontal gene transfer.

Mobile genetic elements either encode their own mobilization machineries or hijack them from other mobile elements. Multiple classes of mobile elements often coexist within genomes and it is unclear whether they have the capacity to functionally interact and even collaborate. We investigate the possibility that molecular machineries of disparate mobile elements may functionally interact, using the example of a retrotransposon, in the form of a mobile group II intron, found on a conjugative plasmid pRS01 in Lactococcus lactis. This intron resides within the pRS01 ltrB gene encoding relaxase, the enzyme required for nicking the transfer origin (oriT) for conjugal transmission of the plasmid into a recipient cell. Here, we show that relaxase stimulates both the frequency and diversity of retrotransposition events using a retromobility indicator gene (RIG), and by developing a high-throughput genomic retrotransposition detection system called RIG-Seq. We demonstrate that LtrB relaxase not only nicks ssDNA of its cognate oriT in a sequence- and strand-specific manner, but also possesses weak off-target activity. Together, the data support a model in which the two different mobile elements, one using an RNA-based mechanism, the other using DNA-based transfer, do functionally interact. Intron splicing facilitates relaxase expression required for conjugation, whereas relaxase introduces spurious nicks in recipient DNA that stimulate both the frequency of intron mobility and the density of events. We hypothesize that this functional interaction between the mobile elements would promote horizontal conjugal gene transfer while stimulating intron dissemination in the donor and recipient cells.

Convergent evolution of ribonuclease h in LTR retrotransposons and retroviruses.

Ty3/Gypsy long terminals repeat (LTR) retrotransposons are structurally and phylogenetically close to retroviruses. Two notable structural differences between these groups of genetic elements are 1) the presence in retroviruses of an additional envelope gene, env, which mediates infection, and 2) a specific dual ribonuclease H (RNH) domain encoded by the retroviral pol gene. However, similar to retroviruses, many Ty3/Gypsy LTR retrotransposons harbor additional env-like genes, promoting concepts of the infective mode of these retrotransposons. Here, we provide a further line of evidence of similarity between retroviruses and some Ty3/Gypsy LTR retrotransposons. We identify that, together with their additional genes, plant Ty3/Gypsy LTR retrotransposons of the Tat group have a second RNH, as do retroviruses. Most importantly, we show that the resulting dual RNHs of Tat LTR retrotransposons and retroviruses emerged independently, providing strong evidence for their convergent evolution. The convergent resemblance of Tat LTR retrotransposons and retroviruses may indicate similar selection pressures acting on these diverse groups of elements and reveal potential evolutionary constraints on their structure. We speculate that dual RNH is required to accelerate retrotransposon evolution through increased rates of strand transfer events and subsequent recombination events.

Acquisition of an Archaea-like ribonuclease H domain by plant L1 retrotransposons supports modular evolution.

Although a variety of non-LTR retrotransposons of the L1 superfamily have been found in plant genomes over recent decades, their diversity, distribution, and evolution have yet to be analyzed in depth. Here, we perform comprehensive comparative and evolutionary analyses of L1 retrotransposons from 29 genomes of land plants covering a wide range of taxa. We identify numerous L1 elements in these genomes and detect a striking diversity of their domain composition. We show that all known land plant L1 retrotransposons can be grouped into five major families based on their phylogenetic relationships and domain composition. Moreover, we trace the putative evolution timeline that created the current variants and reveal that evolutionary events included losses and acquisitions of diverse putative RNA-binding domains and the acquisition of an Archaea-like ribonuclease H (RNH) domain. We also show that the latter RNH domain is autonomously active in vitro and speculate that retrotransposons may play a role in the horizontal transfer of RNH between plants, Archaea, and bacteria. The acquisition of an Archaea-like RNH domain by plant L1 retrotransposons negates the hypothesis that RNH domains in non-LTR retrotransposons have a single origin and provides evidence that acquisition happened at least twice. Together, our data indicate that the evolution of the investigated retrotransposons can be mainly characterized by repeated events of domain rearrangements and identify modular evolution as a major trend in the evolution of plant L1 retrotransposons.