Selective degradation of altered tomato β-fructofuranosidase molecules by neutral protease

Evidence is presented for the selective breakdown of altered tomato β-fructofuranosidase molecules by a neutral protease from Bacillus subtilis.     

Between‐year variations in Little Bustard Tetrax tetrax population densities are influenced by agricultural intensification and rainfall

The Little Bustard is suffering a widespread population decline mainly due to agricultural intensification. This study evaluates the effects of intensification level, habitat availability and rainfall on the population dynamics of this species. The population density of males was monitored for 7 years (2002–2008) at 184 points located within three sites with contrasting levels of agricultural intensification in southern Portugal. Densities decreased along the intensification gradient from 8.2 to 2.3 males / km². Overall, there was an approximately 50% population decline during the period 2002–2008, driven by a decline observed in one of the less intensive sites, whereas in the other two sites densities remained fairly constant. Yearly variations in male densities were influenced by intensification level, amount of grassland habitat and rainfall patterns. Thus, agricultural intensification is having a negative effect on population densities of this threatened species, particularly through the loss of grasslands (fallow fields and pastures) suitable for displaying males. The results also suggest a positive impact of rainfall on male densities, although this is more likely in grasslands within less intensive agricultural regions of poorer soil quality, where higher breeding male densities occur. Grassland habitat quality, driven by both climate and human management, probably plays a major role in the population dynamics of this threatened steppe bird in its strongholds.     

Peroxisomal Degradation of 2-Oxoisocaproate. Evidence for Free Acid Intermediates

Peroxisomes from mung bean hypocotyl (Vigna radiata L.) degrade 2-oxoisocaproate, the transamination product of leucine, via isobutyryl-CoA and propionyl-CoA to acetyl-CoA. The methyl group at the C-3 position forms a barrier to β-oxidation. This barrier is overcome in the peroxisomes by several enzymatic steps. Senecioate (3-methylcrotonate), 2-hydroxyisovalerate, and 2-oxoisovalerate were detected as free acid intermediates. Senecioate, formed from 3-methylcrotonyl-CoA, is transformed by enzymatic hydrolysis to 2-hydroxyisovalerate. 2-Hydroxyisovalerate is then oxidized to 2-oxoisovalerate in an H₂O₂-producing reaction, exhibiting 1:1 stoichiometry of the products, by a 2-hydroxyacid oxidase which is different from the peroxisomal marker enzyme glycollate oxidase. 2-oxoisovalerate is activated by an NAD-dependent oxidative decarboxylation to isobutyryl-CoA. Accumulation of 2-oxoisovalerate in the presence of arsenite, an inhibitor of oxidative decarboxylations, is a feature of this latter pathway of degradation of isovaleryl-CoA or senecioate. It is concluded that the barrier caused by the methyl group of 2-oxoisocaproate is surmounted in higher plant peroxisomes in a manner different to that in mammalian mitochondria.     

Protein Turnover in Brain of the Rat Fetus

Protein synthesis in vivo was studied in whole brain of rat fetuses using continuous intravenous infusion of L-[U-14C]tyrosine into unrestrained pregnant rats at 19 and 21 days gestation. Protein degradation (KD) was calculated by subtracting fractional growth rate of brain protein (KG) from the fractional synthesis rate (KS). KS was high at both gestational ages (0.42 +/- 0.03 days-1 at day 19, 0.47 +/- 0.029 days-1 at 21 days), comparable to values previously reported for newborn rat cerebral hemispheres, and threefold higher than is seen in adult animals. KD was similar at both 19 and 21 days gestation (0.19-0.24) and lower than that reported in neonatal rat brain using similar techniques. Protein accretion during the most rapid phase of brain growth (fetus) is accomplished by similar rates of protein synthesis, but decreased rates of degradation when compared with a slower growth phase (newborn). KD in the brain of the rapidly growing fetus is slightly higher than in adult cerebral hemispheres.     

Enantioselective inclusion complexation of 2,3,4,6-tetra-O-acetyl-D-glycopyranose as a chiral host with (+)-(r)-phenylethylamine: x-ray characterization of the complex

X-ray analysis of the crystalline product obtained by reaction of 2,3,4,6-Tetra-O-acetyl-D-glycopyranose with phenylethylamine revealed the formation of a real one-to-one inclusion complex. This complexaion is highly stereoselective, because only the (+)-R-enantiomeric form of the amine is included. Analogies to the mode of complexation of cellulose triaceate with chiral molecules in ?inclusion chromatography”? are discussed.     

Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains

To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [³²P]-labeled photoreactive partial DNA duplexes containing a 3′-ss/ds-junction (3′-junction) or a 5′-ss/ds-junction (5′-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3′-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5′-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5′-junction. The results show that RPAp70 crosslinked to DNA with a 5′-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.