Tertiary structural rearrangements upon oxidation of Methionine145 in calmodulin promotes targeted proteasomal degradation

The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.     

Structure and reactivity of the chromophore of a GFP-like chromoprotein from Condylactis gigantea

Here we present the study of the chromophore structure of the purple chromoprotein from Condylactis gigantea. Tandem mass spectrometry and 1H and 13C NMR of the chromopeptide reveal that the protein contains a chromophore with a chemical structure identical to that of the red fluorescent protein from Discosoma sp. A single A63G substitution demonstrates that the nature of the first amino acid of the XYG chromophore-forming sequence is dispensable for the chromoprotein red shift development. It has been recently proposed that post-translational reactions at the acylimine, a chemical group that accounts for the red fluorescence, might be an additional source of spectral diversity of proteins homologous to the Aequorea victoria green fluorescent protein (GFP). We have examined the reactivity of the chromophore acylimine group within the C. gigantea purple chromoprotein. Like other proteins with the acylimine-modified chromophore, the purple chromoprotein suffers a hypsochromic spectral shift to the GFP-like absorbance (386 nm) upon mild denaturation. NMR analysis of the chromopeptide suggests this hypsochromic spectral shift is due to H2O addition across the C=N bond of the acylimine. However, unlike the red fluorescent protein from Discosoma sp., denatured under harsh conditions, the wild-type chromoprotein exhibits only slight fragmentation, which is induced by complete hydrolysis of the acylimine. A model suggesting the influence of the amino acid X side chain on protein fragmentation is presented.     

Evolution of blue-flowered species of genus <Emphasis Type="Italic">Linum</Emphasis> based on high-throughput sequencing of ribosomal RNA genes

Background

The species relationships within the genus Linum have already been studied several times by means of different molecular and phylogenetic approaches. Nevertheless, a number of ambiguities in phylogeny of Linum still remain unresolved. In particular, the species relationships within the sections Stellerolinum and Dasylinum need further clarification. Also, the question of independence of the species of the section Adenolinum still remains unanswered. Moreover, the relationships of L. narbonense and other species of the section Linum require further clarification. Additionally, the origin of tetraploid species of the section Linum (2n?=?30) including the cultivated species L. usitatissimum has not been explored. The present study examines the phylogeny of blue-flowered species of Linum by comparisons of 5S rRNA gene sequences as well as ITS1 and ITS2 sequences of 35S rRNA genes.

Results

High-throughput sequencing has been used for analysis of multicopy rRNA gene families. In addition to the molecular phylogenetic analysis, the number and chromosomal localization of 5S and 35S rDNA sites has been determined by FISH.Our findings confirm that L. stelleroides forms a basal branch from the clade of blue-flowered flaxes which is independent of the branch formed by species of the sect. Dasylinum. The current molecular phylogenetic approaches, the cytogenetic analysis as well as different genomic DNA fingerprinting methods applied previously did not discriminate certain species within the sect. Adenolinum. The allotetraploid cultivated species L. usitatissimum and its wild ancestor L. angustifolium (2n?=?30) could originate either as the result of hybridization of two diploid species (2n?=?16) related to the modern L. gandiflorum and L. decumbens, or hybridization of a diploid species (2n?=?16) and a diploid ancestor of modern L. narbonense (2n?=?14).

Conclusions

High-throughput sequencing of multicopy rRNA gene families allowed us to make several adjustments to the phylogeny of blue-flowered flax species and also reveal intra- and interspecific divergence of the rRNA gene sequences.

     

Transpiration of Pinus rotundata on a wooded peat bog in central Europe

Transpiration of a central European endemic tree species, Pinus rotundata Link, growing on a wooded peat bog in the Třeboň Basin, Czech Republic, was studied in 1999–2000. Transpiration was measured by sap flow techniques (heat field deformation method) on individual trees and scaled up to stand level. The radial patterns of sap flow density showed narrow peaks in the outer part of the xylem, sapwood accounted for 47–60% of the xylem radius and 72–84% of the xylem basal area. Adult trees tolerated well both short-term flooding during the growing season and drawdown of the water table to a depth of 60 cm below ground level. The maximum and mean daily transpiration rates were 3.0 and 1.8 mm per day, and were thus similar to published data for Scots pine. The seasonal total transpiration (25 April–20 October 2000, 180 days) amounted to 322 mm, or 62% of the potential evapotranspiration over this period. This canopy transpiration was compensated by 319 mm of precipitation. The difference between the accumulated precipitation and the accumulated transpiration (derived from seasonal sap flow measurements) closely mimicked the seasonal course of the water table.     

sumSTAAR: A flexible framework for gene-based association studies using GWAS summary statistics

Gene-based association analysis is an effective gene-mapping tool. Many gene-based methods have been proposed recently. However, their power depends on the underlying genetic architecture, which is rarely known in complex traits, and so it is likely that a combination of such methods could serve as a universal approach. Several frameworks combining different gene-based methods have been developed. However, they all imply a fixed set of methods, weights and functional annotations. Moreover, most of them use individual phenotypes and genotypes as input data. Here, we introduce sumSTAAR, a framework for gene-based association analysis using summary statistics obtained from genome-wide association studies (GWAS). It is an extended and modified version of STAAR framework proposed by Li and colleagues in 2020. The sumSTAAR framework offers a wider range of gene-based methods to combine. It allows the user to arbitrarily define a set of these methods, weighting functions and probabilities of genetic variants being causal. The methods used in the framework were adapted to analyse genes with large number of SNPs to decrease the running time. The framework includes the polygene pruning procedure to guard against the influence of the strong GWAS signals outside the gene. We also present new improved matrices of correlations between the genotypes of variants within genes. These matrices estimated on a sample of 265,000 individuals are a state-of-the-art replacement of widely used matrices based on the 1000 Genomes Project data.     

Presence of oxygen-consuming ribonucleotide reductase in corrinoid-deficientPropionibacterium freudenreichii

Corrinoid-deficient Propionibacterium freuden- reichii subsp. shermanii showed adenosylcobalamin-(AdoCbl)-independent ribonucleotide reductase (RNR) activity in the presence of air. Increasing the incubation time with free access of O₂ led to an increase in RNR activity. As polarographic estimations of O₂ uptake demonstrated, AdoCbl-independent RNR activity (with ADP as substrate) in a cell-free system of corrinoid-deficient P. freudenreichii was accompanied by specific molecular oxygen consumption. The activity was not inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCCP). The activity was present in the cytoplasmic membrane-free soluble fraction of the cell extract, and it was inhibited by hydroxyurea. Manganese ions were important for the cell division of corrinoid-deficient P. freudenreichii and stimulated RNR activity after 8-hydroxyquinoline or EDTA treatment of the cell extract. We therefore concluded that P. freudenreichii is able to form DNA (deoxyribosylic precursors) using AdoCbl-dependent ribonucleotide reductase and also with an alternative AdoCbl-independent molecular-oxygen-consuming RNR system. Received: 29 May 1995 / Accepted: 14 August 1995     

Free Fatty Acids of Surface Film of Water in the Sydinsky Bay of the Krasnoyarsk Reservoir

During the summer of 1990 composition and concentrations of free fatty acids (FFA) in the surface film of water were investigated. 23 FFA species were identified. Using cluster analysis in conjunction with ANOVAR correspondence of FFA contents in the surface film with seasonal patterns of pelagic plankton was proved. Three different groups of FFA were revealed, acids of every group had similar seasonal dynamics and presumably were of similar origination. Data obtained gave a chance to suppose that main part of FFA were originated by exudation of living cells of phytoplankton as well as postmortal exudation of bacterioplankton. On the basis of FFA contents in a single sample from the surface film identification of sign of derivatives of phytoplankton biomass was suggested. This express-assay may be useful for monitoring.     

The early effects of uninephrectomy on rat kidney metabolic state using optical imaging

Uninephrectomy (UNX) is known to result in structural and metabolic changes to the remaining kidney, although it is uncertain if this alters the mitochondrial redox state and how soon such changes may occur. A custom‐designed fluorescence cryo‐imaging technique was used to quantitatively assess the effect of UNX by measuring the levels of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the remaining kidney. Kidneys were snap‐frozen 3 days following UNX, and the intrinsic fluorescence of NADH and FAD were optically acquired. The 3D images were created to characterize the NADH/FAD redox ratios (RR) of the right kidneys, which underwent UNX and the remaining kidneys 3 days following UNX. Both the NADPH‐oxidases (Nox2 and Nox4) and the mitochondria are the main sources of reactive oxygen species (ROS) production in tubular epithelial cells. Responses to the UNX were obtained in kidneys of normal Sprague Dawley (SD) rats, Dahl salt‐sensitive (SS) rats and SS rats in which NADPH‐oxidase isoform 4 (Nox4) was knocked out (SS^Nox4?/?). The results found that each of the strains exhibited similar increase in kidney weights averaging 17% after 3 days of UNX. SD and SS^Nox4?/?rats both exhibited global reductions of the RR (P < .05) with a similar tendency observed in SS rats (P < .08), indicating increased ROS production. The unexpected reduction of the RR in the remnant kidneys of SS^Nox4?/? rats indicates that mechanisms independent of H₂O₂ produced from Nox4 may be responsible for this global increase of ROS. We propose that the reduced RR was largely a consequence of enhanced mitochondrial bioenergetics due to increased tubular workload of the remaining kidney. The data indicate that mitochondria become the dominant source of increased ROS following UNX and could represent an important hypertrophic signaling mechanism.