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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

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.     

Perturbations in the impact of mutational activity on Vλ genes in systemic lupus erythematosus

To assess the impact of somatic hypermutation and selective influences on the Vλ light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of mutations were analyzed in individual CD19⁺ B cells from a patient with previously undiagnosed SLE. The mutational frequency of nonproductive and productive rearrangements in the SLE patient was greater (3.1 × 10^-2 vs 3.4 × 10^-2, respectively) than that in normal B cells (1.2 × 10^-2 vs 2.0 × 10^-2, both P < 0.001). The frequencies of mutated rearrangements in both the nonproductive and productive repertoires were significantly higher in the patient with SLE than in normal subjects. Notably, there were no differences in the ratio of replacement to silent (R/S) mutations in the productive and nonproductive repertoires of the SLE patient, whereas the R/S ratio in the framework regions of productive rearrangements of normal subjects was reduced, consistent with active elimination of replacement mutations in this region. The pattern of mutations was abnormal in the SLE patient, with a significant increase in the frequency of G mutations in both the productive and nonproductive repertoires. As in normal subjects, however, mutations were found frequently in specific nucleotide motifs, the RGYW/WRCY sequences, accounting for 34% (nonproductive) and 46% (productive) of all mutations. These data are most consistent with the conclusion that in this SLE patient, the mutational activity was markedly greater than in normal subjects and exhibited some abnormal features. In addition, there was decreased subsequent positive or negative selection of mutations. The enhanced and abnormal mutational activity along with disturbances in selection may play a role in the emergence of autoreactivity in this patient with SLE.     

Impact of epistasis and QTL×environment interaction on the developmental behavior of plant height in rice (Oryza sativa L.)

QTLs with epistatic effects and environmental interaction effects for the developmental behavior of plant height in rice were studied by conventional and conditional methods for quantitative trait loci (QTLs) by mapping with a doubled-haploid population of 123 lines from IR64/Azucena in three environments. The results showed that epistatic effects were important and most epistasis could be detected only by conditional QTL mapping, while most non–epistatic QTLs could be detected by both conventional and conditional methods. Many modificative QTLs showed only epistatic effects without their own additive effects at some stages. QTL×environment (QE) interaction effects were detected more often than QTL main effects for plant-height behavior, which might indicate that gene expression could be greatly affected by the environment. No QTLs had effects during the whole of ontogeny. Conditional QTL mapping might be a valid way to reveal dynamic gene expression for the development of quantitative traits, especially for epistatic effects. Received: 19 May 2000 / Accepted: 27 October 2000     

Localization of the human c-kit protooncogene on the q11–q12 region of chromosome 4

Summary Using a 166-nucleotide-long DNA synthetic probe corresponding to the v-kit sequence (1458-1623), we have mapped the human c-kit gene to chromosome 4 at the q11–q12 band by in situ hybridization on chromosomes from human lymphocyte preparations.     

Regulation of α-galactosidase activity and the hydrolysis of galactomannan in the endosperm of the fenugreek (Trigonella foenum-graecum L.) seed

When endosperms were isolated from fenugreek seeds 5 h after sowing and incubated in a small volume of water, the development of α-galactosidase activity and the breakdown of the galactomannan storage polysaccharide were both inhibited relative to control endosperms incubated in larger volumes. The inhibition could be relieved by pre-washing the endosperms, and reimposed by the wash-liquors. If the endosperms were isolated 24 h after sowing, no inhibition was observed. Removal of the embryonic axis from germinating fenugreek seeds and from germinated seedlings also inhibited the development of α-galactosidase activity and galactomannan breakdown in the endosperms; the inhibition was more pronounced the earlier the axis was removed. Axis excision 5 h after sowing caused a delay in the onset of galactomannan breakdown and of the appearance of α-galactosidase activity in the endosperms. It also led to a decrease in the rates of galactomannan breakdown and α-galactosidase production. Axis excision 24 h after sowing caused only a slowing of the rates of galactomannan breakdown and α-galactosidase increase. The inhibition caused by axis removal at 5 h could be relieved partially by gibberellin (10^-4 M), benzyladenine (10^-5 M), mixtures of these and by the herbicide SAN 9789 [4-chloro-5-(methylamine)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone]. These substances had no effect on the inhibition caused by axis-removal at 24 h. Excision of the cotyledons at 5 h-leaving the separated axis and the endosperm-also caused inhibition of galactomannan breakdown and α-galactosidase development. The results are consistent with the presence in the fenugreek seed endosperm of diffusible inhibitors of galactomannan mobilisation which are removed or inactivated during normal germination and early seedling development. They are also consistent with a role for the seedling axis in the control of galactomannan breakdown in the endosperm. Initially the axis appears to have a regulatory function (via gibberellins and/or cytokinins?) in determining the onset of α-galactosidase production in the endosperm. Thereafter its continued presence is necessary to ensure maximal rates of α-galactosidase production and galactomannan hydrolysis. The role of the axis may be initially to counteract the endogenous inhibitors in the endosperm and then to act as a sink for the galactomannan breakdown products released in the endosperm and taken up by the cotyledons.     

Initial data on biological activity of taiga–steppe soils in the lower reaches of the Kolyma River

Microbiological and enzyme activities of extrazonal taiga-steppe soils in the lower reaches of the Kolyma River have been studied for the first time. Contrary to north-taiga cryometamorphic soils, predominating in the area, microbial cenoses under herb–sedge petrophytic and grass–sagebrush–herb thermophytic steppes are characterized by features typical for arid soils. The saturation of the soil profile with microorganisms is greater, and the development of actinomycetes is more intensive. The enzyme complex is characterized by high activity of dehydrogenases.