Stability against temperature of Sulfolobus solfataricus elongation factor 1 alpha, a multi-domain protein

The elongation factors (EF-Tu/EF-1 alpha) are universal proteins, involved in protein biosynthesis. A detailed characterization of the stability against temperature of SsEF-1 alpha, a three-domain protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus is presented. Thermal denaturation of both the GDP-bound (SsEF-1 alpha*.GDP) and the ligand-free (nfSsEF-1 alpha) forms was investigated by means of circular dichroism and fluorescence measurements, over the 4.0-7.5 pH interval. Data indicate that the unfolding process is cooperative with no intermediate species and that the few inter-domain contacts identified in the crystal structure of SsEF-1 alpha play a role also at high temperatures. Finally, it is shown that the enzyme exhibits two different interchangeable thermally denatured states, depending on pH.     

Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation

Background

Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free α-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal α-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s).

Methodology/Principal Findings

Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H₂O₂; 0.1–0.5 mM range). Results show that A) Cells overexpressing “wild type” human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H₂O₂ concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H₂O₂ treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase.

Conclusion/Significance

Apoptosis can be modulated by “on/off” switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.     

Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers

Oligomeric procyanidins were isolated from the leaves and flowers of hawthorn (Crataegus laevigata). A trimer, epicatechin-(4β→8)-epicatechin-(4β→6)-epicatechin, and a pentamer consisting of (−)-epicatechin units linked through C-4β/C-8 bonds have been isolated from hawthorn for the first time, in addition to known procyanidins including dimers B-2, B-4 and B-5, trimers C-1 and epicatechin-(4β→6)-epicatechin-(4β→8)-epicatechin, and tetramer D-1. A fraction containing a hexamer was also found.     

Elongation factor Ts from the Antarctic eubacterium Pseudoalteromonas haloplanktis TAC 125: biochemical characterization and cloning of the encoding gene

The elongation factor Ts was isolated from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis TAC 125 strain (PhEF-Ts), and its functional properties were studied. At 0 degrees C PhEF-Ts enhanced the [(3)H]GDP/GDP exchange rate on the preformed PhEF-Tu.[(3)H]GDP complex by 2 orders of magnitude even at very low Tu:Ts ratio, by lowering the energy of activation of the exchange reaction. PhEF-Ts is a monomeric protein, and in solution it forms a stable dimeric complex with PhEF-Tu. The PhEF-Ts encoding gene was cloned and sequenced. Its structural organization was similar to that of Escherichia coli because it showed at its 5' end the gene encoding the ribosomal protein S2. The translated amino acid sequence had a calculated molecular weight of 30762, and showed a high sequence identity with E. coli (68%) and Thermus thermophilus (44%) EF-Ts. The PhEF-Ts primary structure contains well-preserved almost all the amino acid residues interacting at the interfaces of the E. coli EF-Ts.EF-Tu complex. Finally, the high concentration of PhEF-Ts in this psychrophilic eubacterium might represent an adaptive tool to ensure an efficient nucleotide exchange even at low temperature.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strains isolated from moisture-damaged buildings produced surfactin and a substance toxic to mammalian cells

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 g dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (_m) and the plasma membrane (_p) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K⁺:Na⁺:Ca²⁺ of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.Abbreviations RP-HPLC Reversed-phase high-performance liquid chromatography - BLM Black lipid membrane - DAD Diode-array detector - _m Mitochondrial membrane potential - _p Plasma membrane potential - JC-1 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenz-imidazolo carbocyanine iodide - AM Calcein acetoxymethyl ester - PI Propidium iodide - MALDI-TOF-MS Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - ESI-IT-MS Electrospray ionization ion trap mass spectrometry - EC₅₀ Endpoint concentration which caused 50% change in the viability parameters - FCCP Carbonyl cyanide 4-trifluoromethoxyphenylhydrazone     

Systematic evaluation of medium-throughput mRNA abundance platforms

Profiling of mRNA abundances with high-throughput platforms such as microarrays and RNA-seq has become an important tool in both basic and biomedical research. However, these platforms remain prone to systematic errors and have challenges in clinical and industrial applications. As a result, it is standard practice to validate a subset of key results using alternate technologies. Similarly, clinical and industrial applications typically involve transitions from a high-throughput discovery platform to medium-throughput validation ones. These medium-throughput validation platforms have high technical reproducibility and reduced sample input needs, and low sensitivity to sample quality (e.g., for processing FFPE specimens). Unfortunately, while medium-throughput platforms have proliferated, there are no comprehensive comparisons of them. Here we fill that gap by comparing two key medium-throughput platforms—NanoString''s nCounter Analysis System and ABI''s OpenArray System—to gold-standard quantitative real-time RT-PCR. We quantified 38 genes and positive and negative controls in 165 samples. Signal:noise ratios, correlations, dynamic range, and detection accuracy were compared across platforms. All three measurement technologies showed good concordance, but with divergent price/time/sensitivity trade-offs. This study provides the first detailed comparison of medium-throughput RNA quantification platforms and provides a template and a standard data set for the evaluation of additional technologies.     

Patterns of climate‐induced density shifts of species: poleward shifts faster in northern boreal birds than in southern birds

Climate change has been shown to cause poleward range shifts of species. These shifts are typically demonstrated using presence–absence data, which can mask the potential changes in the abundance of species. Moreover, changes in the mean centre of weighted density of species are seldom examined, and comparisons between these two methods are even rarer. Here, we studied the change in the mean weighted latitude of density (MWLD) of 94 bird species in Finland, northern Europe, using data covering a north–south gradient of over 1000 km from the 1970s to the 2010s. The MWLD shifted northward on average 1.26 km yr^?1, and this shift was significantly stronger in northern species compared to southern species. These shifts can be related to climate warming during the study period, because the annual temperature had increased more in northern Finland (by 1.7 °C) than in southern Finland (by 1.4 °C), although direct causal links cannot be shown. Density shifts of species distributed over the whole country did not differ from shifts in species situated on the edge of the species range in southern and northern species. This means that density shifts occur both in the core and on the edge of species distribution. The species‐specific comparison of MWLD values with corresponding changes in the mean weighted latitude using presence–absence atlas data (MWL) revealed that the MWLD moved more slowly than the MWL in the atlas data in the southern species examined, but more rapidly in the northern species. Our findings highlight that population densities are also moving rapidly towards the poles and the use of presence–absence data can mask the shift of population densities. We encourage use of abundance data in studies considering the effects of climate change on biodiversity.     

The A26G replacement in the consensus sequence A-X-X-X-X-G-K-[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus.

A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A-X-X-X-X-G-K-[T,S] of the elongation factors EF-G or EF-2 into the corresponding G-X-X-X-X-G-K-[T,S] motif which is present in all the other GTP-binding proteins. The rate of poly(U)-directed poly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsEF-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide-binding pocket of EF-2 or EF-G is discussed.     

The interaction between the archaeal elongation factor 1alpha and its nucleotide exchange factor 1beta.

In Sulfolobus solfataricus the binding of the exchange factor 1beta (SsEF-1beta) to SsEF-1alpha-GDP displaces the nucleotide and the SsEF-1alpha-SsEF-1beta complex is formed. The complex itself is stable, but it dissociates upon the addition of GDP or Gpp(NH)p but not ATP. Since the rate of the formation of the SsEF-1alpha-SsEF-1beta complex is significatively slower than the rate of the nucleotide exchange catalyzed by SsEF-1beta it can be inferred that in vivo the GDP/GTP exchange reaction proceeds via an SsEF-1alpha-SsEF-1beta interaction without involving the formation of a stable binary complex as an intermediate.