Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.     

Cholesterol-derived bile acids enhance the chaperone activity of α-crystallins

Human lens membranes contain the highest cholesterol concentration of any known biological membranes, but it significantly decreases with age. Oxygenation of cholesterol generates numerous forms of oxysterols (bile acids). We previously showed that two forms of the bile acid components—ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA)—suppressed lens epithelial cell death and alleviated cataract formation in galactosemic rat lenses. We investigated whether these compounds also suppress the thermal aggregation of human lens crystallins. Total water-soluble (WS) proteins were prepared from human lenses, and recombinant human crystallins (αA-, αB-, βB2-, and γC-crystallin) were generated by a prokaryotic expression system and purified by liquid chromatography. The light scattering of proteins in the presence or absence of UDCA or TUDCA was measured using a spectrofluorometer set at Ex/Em = 400/400 nm. Protein blot analysis was conducted for detection of α-crystallins in the human lens WS proteins. High concentrations of UDCA and TUDCA significantly suppressed thermal aggregation of total lens WS proteins, which contained a low level of αA-/αB-crystallin. Spectroscopic analysis with each recombinant human lens crystallin indicated that the bile acids did not suppress the thermal aggregation of γC-, βB2-, αA-, or αB-crystallin. Combination of α-crystallin and bile acid (either UDCA or TUDCA) suppressed thermal aggregation of each individual crystallin as well as a non-crystallin protein, insulin. These results suggest that UDCA or TUDCA protects the chaperone activity of α-crystallin. It is believed that these two naturally occurring intermediate waste products in the lens enhance the chaperone activity of α-crystallin. This finding may lead to the development of UDCA and TUDCA as anticataract agents.     

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core “α-crystallin” domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54–60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54–60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54–60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54–60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.     

Calcium-induced decrease of the thermal stability and chaperone activity of α-crystallin

α-Crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. α-Crystallin aggregation is involved in lens opacification, and high [Ca²⁺] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca²⁺ on the thermal stability of α-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca²⁺-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca²⁺] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca²⁺ concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca²⁺ decreases both secondary and tertiary–quaternary structure stability of α-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca²⁺ alters the structural stability of α-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, α-crystallin aggregation facilitated by Ca²⁺ would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.     

How does the ribosome work? A hypothesis based on the two subunit construction of the ribosome

The following postulates are formulated: 1) the aminoacyl-tRNA binding site and the peptidyl-tRNA binding site are localized mainly on two different subparticles of the ribosome, the 30S and 50S respectively; 2) both sites are localized on the contacting (facing each other) surfaces of the subparticles, so that all the events of peptide bond formation run inside the ribosome, on the border between the subparticles; 3) the complete 70S (or 80S) ribosome can be in two different states: locked, with the particles in a tight association, and unlocked, with the particles loosely associated or slightly drawn apart. On the basis of these postulates the concept is evolved that the periodical unlocking and locking of the subparticles of the ribosome is the driving mechanism providing for the displacements (translocations) of tRNAs, mRNA and peptidyl during translation. A strictly deferred polarity of the displacements results from the unequality of the two subparticles, i.e., from the asymmetry of the ribosome as a whole. It is to the process of unlocking of the subparticles that the energy of GTP cleavage could be primarily applied. A kinematical model of the functioning ribosome is given. A number of observations which were difficult to interprete, are explained on the basis of this hypothesis and some predictions are made which can be subjected to direct experimental testing.     

Role of the specifically targeted lysine residues in the glycation dependent loss of chaperone activity of αA- and αB-crystallins

Earlier studies have shown significant loss of chaperone activity in α-crystallin from diabetic lenses. In vitro glycation studies have suggested that glycation of α-crystallin could be the major cause of chaperone activity loss. The following lysine (K) residues in α-crystallin have been identified as the major glycation sites: K11, K78, and K166 in αA-crystallin and K90, K92, and K166 in αB-crystallin. The present study was aimed to assess the contribution of each of the above glycation site in the overall glycation and loss of chaperone activity by mutating them to threonine followed by in vitro glycation with fructose. Level of glycated protein (GP) was determined by phenylboronate affinity chromatography, advanced glycation end products (AGEs) by direct ELISA using anti-AGE polyclonal antibody, and chaperone activity by using alcohol dehydrogenase as the target protein. K11T, K78, and K166T mutants of αA showed 33, 17, and 27% decrease in GP and 32, 18, and 21% decrease in AGEs, respectively, as compared to αA-wt. Likewise, K90T, K92T, K90T/K92T, and K166T mutants of αB showed 18, 21, 29, and 12% decrease in GP and 22, 24, 32, and 16% decrease in AGEs, respectively. Chaperone activity also showed concomitant increase with decreasing glycation and AGEs formation. αA-K11T and αB-K90T/K92T mutants showed the largest decrease in glycation and increase in chaperone activity.     

Acetylation of αA-crystallin in the human lens: Effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N^ε-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

The chaperone activity of α-synuclein: Utilizing deletion mutants to map its interaction with target proteins

α-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, α-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. α-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. α-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61-140 [α-syn(61-140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments α-syn(1-60) and α-syn(96-140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar α-synuclein had greater chaperone activity than nonfibrillar α-synuclein. It is concluded that the amyloidogenic NAC region (residues 61-95) contains the chaperone-binding site which is optimized for target protein binding as a result of its β-sheet formation and/or ordered aggregation by α-synuclein. On the other hand, the first 60 residues of α-synuclein modulate the protein's chaperone-active site, while at the same time protecting α-synuclein from fibrillation. On its own, however, this fragment [α-syn(1-60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of α-synuclein in its chaperone activity have been delineated.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Annual cycle of activity of the neurocytes of the ventral lateral septum in the brainof Rana esculenta L.∗

Abstract

Sexually mature male and femaleRana esculenta L. were captured in their natural habitat in six phases of the annual cycle. Nuclear volumes of neurocytes in the ventral lateral septum (VLS) were found to fluctuate distinctly in the course of the year. In both sexes nuclear volumes of neurocytes were maximal in the phases preceding the breeding season (3rd decade of January, and 1st decadeof April), and minimal in the phaseat the beginning of hibernation (3rd decade of October). The results lead to the assumption that the VLS ofRana esculenta may have an active role in controlling gonadotropin release.     

Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for Δ12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the Δ12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the Δ12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.     

Difference spectroscopic studies on binding of Cibacron blue F3GA to ribosome inactivating proteins: Effect of β-mercaptoethanol on the interaction with ricin

The interaction of Cibacron blue F3GA with ribosome inactivating proteins, ricin, ricin A-chain and momordin has been investigated using difference absorption spectroscopy. Ricin was found to bind the dye with a 20- and 2-fold lower affinity than ricin A-chain and momordin, respectively. A time dependent increase in the amplitude of Cibacron blue difference spectrum in the presence of ricin was observed on addition of β-mercaptoethanol. Analysis of the kinetic profile of this increase showed a biphasic phenomenon and the observed rates were found to be independent of the concentration of β-mercaptoethanol. Kinetics of reduction of the intersubunit disulphide bond in ricin by β-mercaptoethanol showed that reductionper se is a second order reaction. Therefore, the observed changes in the difference spectra of Cibacron blue probably indicate a slow change in the conformation of ricin, triggered by reduction of the intersubunit disulphide bond.     

Polyadenylation site-specific differences in the activity of the neuronal βCstF-64 protein in PC-12 cells

Recent genome-wide analyses have implicated alternative polyadenylation — the process of regulated mRNA 3′ end formation — as a critical mechanism that influences multiple steps of mRNA metabolism in addition to increasing the protein-coding capacity of the genome. Although the functional consequences of alternative polyadenylation are well known, protein factors that regulate this process are poorly characterized. Previously, we described an evolutionarily conserved family of neuronal splice variants of the CstF-64 mRNA, βCstF-64, that we hypothesized to function in alternative polyadenylation in the nervous system. In the present study, we show that βCstF-64 mRNA and protein expression increase in response to nerve growth factor (NGF), concomitant with differentiation of adrenal PC-12 cells into a neuronal phenotype, suggesting a role for βCstF-64 in neuronal gene expression. Using PC-12 cells as model, we show that βCstF-64 is a bona fide polyadenylation protein, as evidenced by its association with the CstF complex, and by its ability to stimulate polyadenylation of luciferase reporter mRNA. Using luciferase assays, we show that βCstF-64 stimulates polyadenylation equivalently at the two weak poly(A) sites of the β-adducin mRNA. Notably, we demonstrate that the activity of βCstF-64 is less than CstF-64 on a strong polyadenylation signal, suggesting polyadenylation site-specific differences in the activity of the βCstF-64 protein. Our data address the polyadenylation functions of βCstF-64 for the first time, and provide initial insights into the mechanism of alternative poly(A) site selection in the nervous system.     

Effects of phospholipids on the antioxidant activity of α-tocopherol in the singlet oxygen oxidation of canola oil

This study evaluated the effects of phosphatidylcholine (PC) or phosphatidylethanolamine (PE) on the antioxidative activity of α-tocopherol during oxidation of canola oil by singlet oxygen at 10°C for seven hours. Singlet oxygen was produced by chlorophyll b (4 ppm) under 1,700 lux. The oxidation of oil was evaluated by headspace oxygen consumption by gas chromatography and peroxide values (POVs). Concentrations of PC, PE, chlorophyll, and α-tocopherol were determined by HPLC. PC and PE protected chlorophyll from degradation, but they accelerated the degradation of α-tocopherol under singlet oxygen. Contents of PC and PE did not change for seven hours under singlet oxygen. α-Tocopherol significantly lowered POV and headspace oxygen consumption of canola oil under singlet oxygen, and its antioxidant activity was increased by the co-presence of PC and PE. PC and PE increased chemical quenching of singlet oxygen by tocopherol in decreasing the oil oxidation.     

Migration of fallout-radionuclides in the soil: effect of non-uniformity of the sorption properties on the activity–depth profiles

Experimentally observed activity–depth profiles of fallout radionuclides in the soil frequently exhibit a comparatively fast moving tail in soil layers below the peak concentration (tailing). Monte Carlo calculations on the basis of the convection-dispersion model show that this phenomenon can be explained by assuming that either the hydraulic properties of the soil (characterised by the diffusion/dispersion coefficient and pore water velocity) or the sorption properties of the soil (characterised by the distribution coefficient K _d ), or both, exhibit a horizontal variability according to a log-normal distribution. Modifications of the activity–depth profile due to a K _d value which decreases linearly with depth were examined by using a random walk approach, based also on the convection-dispersion model. In this case, however, a pronounced tailing effect of the activity–depth profile did not result. Interpretation and realistic modelling of an experimentally observed activity–depth profile which exhibits a tailing effect is thus not unambiguously possible without any additional information on the spatial variability of the hydraulic parameters and, independently, also for the sorption properties. Received: 5 January 2001 / Accepted: 1 May 2001     

RuBisCo activase—a catalytic chaperone involved in modulating the RuBisCo activity and heat stress-tolerance in wheat

Journal of Plant Biochemistry and Biotechnology - RuBisCo activase modulate the activity of RuBisCo and protect the nascent proteins from aggregation under heat stress (HS). Here, we have...     

Influence of truncation of avian β-defensin-4 on biological activity and peptide-membrane interaction

Defensins are important components in host defense systems. The therapeutic use of β-defensins is limited by their innate toxicity and high cost due to the size and complex disulfide pairing. In this study, we used linear avian β- defensin-4 (RL38) without disulfide bonds as model peptide to derive two peptides by the truncation. GL23 is the C-terminal truncated sequence of RL38, and GLI23 is the derivative of GL23 by the replacement of cysteines with isoleucines. Results showed that these peptides exhibited strong antibacterial activity against gram-negative and gram-positive bacteria. An exception was that GL23 showed weak antimicrobial activity against gallinaceous pathogenic bacteria Salmonella Pullorum C79-13. Two truncated peptides GL23 and GLI23 displayed no or weak hemolysis, which was in accordance with little blue shifts of the peptides in the presence of synthetic eukaryotic membranes. CD spectroscopy demonstrated that these peptides appeared to be unfolded in aqueous solution but acquire structure in the presence of membrane- mimicking phospholipids. GLI23 kept the antibacterial activity at high concentrations of NaCl or low concentration of divalent cations (Mg2+ and Ca2+). The peptides preferentially bound to negatively charged phospholipids over zwitterionic phospholipids, which led to greater cell selectivity. The outer and inner membranes assay displayed that GLI23 killed bacteria by targeting the cell membrane. These results suggest the peptides derived by truncation of linear β-defensins may be a promising candidate for future antibacterial agent.