Mass spectrometric analysis of dimer-disrupting mutations in Plasmodium triosephosphate isomerase

Electrospray ionization mass spectrometry (ESI MS) under nanospray conditions has been used to examine the effects of mutation at two key dimer interface residues, Gln (Q) 64 and Thr (T) 75, in Plasmodium falciparum triosephosphate isomerase. Both residues participate in an intricate network of intra- and intersubunit hydrogen bonds. The gas phase distributions of dimeric and monomeric protein species have been examined for the wild type enzyme (TWT) and three mutants, Q64N, Q64E, and 175S, under a wide range of collision energies (40-160 eV). The results established the order of dimer stability as TWT > T75S > Q64E similar to Q64N. The mutational effects on dimer stability are in good agreement with the previously reported estimates, based on the concentration dependence of enzyme activity. Additional experiments in solution, using inhibition of activity by a synthetic dimer interface peptide, further support the broad agreement between gas phase and solution studies. (C) 2016 Elsevier Inc. All rights reserved.

I. Studies on alkaline phosphatase activity in developing sea urchin embryos. II. Studies on the activation of protein biosynthesis in sea urchin eggs at fertilization

I. Alkaline phosphatase activity in the developing sea urchin Lytechinus pictus has been investigated with respect to intensity at various stages, ionic requirements and intracellular localization. The activity per embryo remains the same in the unfertilized egg, fertilized egg and cleavage stages. At a time just prior to gastrulation (about 10 hours after fertilization) the activity per embryo begins to rise and increases after 300 times over the activity in the cleavage stages during the next 60 hours.

The optimum ionic strength for enzymatic activity shows a wide peak at 0.6 to 1.0. Calcium and magnesium show an additional optimum at a concentration in the range of 0.02 to 0.07 molar. EDTA at concentrations of 0.0001 molar and higher shows a definite inhibition of activity.

The intracellular localization of alkaline phosphatase in homogenates of 72-hour embryos has been studied employing the differential centrifugation method. The major portion of the total activity in these homogenates was found in mitochondrial and microsomal fractions with less than 5% in the nuclear fraction and less than 2% in the final supernatant. The activity could be released from all fractions by treatment with sodium deoxycholate.

II. The activation of protein biosynthesis at fertilization in eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus has been studied in both intact eggs and cell-free homogenates. It is shown that homogenates from both unfertilized and fertilized eggs are dependent on potassium and magnesium ions for optimum amino acid incorporation activity and in the case of the latter the concentration range is quite narrow. Though the optimum magnesium concentrations appear to differ slightly in homogenates of unfertilized and fertilized eggs, in no case was it observed that unfertilized egg homogenates were stimulated to incorporate at a level comparable to that of the fertilized eggs.

An activation of amino acid incorporation into protein has also been shown to occur in parthenogenetically activated non-nucleate sea urchin egg fragments or homogenates thereof. This activation resembles that in the fertilized whole egg or fragment both in amount and pattern of activation. Furthermore, it is shown that polyribosomes form in these non-nucleate fragments upon artificial activation. These findings are discussed along with possible mechanisms for activation of the system at fertilization.

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15,536 Da) and beta (14,873 Da). The mature amino acid sequences of the a (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers Of (alphabeta)(2) and (alphabeta)(4) with molecular weights of 63,680 and 128,518 Da, respectively. The (alphabeta)(2) complex is stabilized by an interchain disulfide bridge between the two alphabeta-heterodimers, whereas the stabilization of the (alphabeta)(4) complex seems to involve non-covalent interactions between the (alphabeta)(2) complexes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Molecular cloning, expression of a peroxiredoxin gene in Chinese shrimp Fenneropenaeus chinensis and the antioxidant activity of its recombinant protein

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions in intracellular signal transduction. A Prx gene was firstly isolated in the crustacean, Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA consists of 942 bp with a 594 bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22041.17 Da with an estimated pI of 5.17. Sequence comparison showed that Prx of F. chinensis shares 76%, 73% and 72% identity with that of Aedes aegypti, Branchiostoma belcheri tsingtaunese and Drosophila melanogaster, respectively. Northern blot analysis revealed the presence of Prx transcripts of F chinensis in all tissues examined. Real-time PCR analysis indicated that the Prx showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with Vibrio anguillarum. In addition, a fusion protein containing Prx was produced in vitro. LC-ESI-MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. And the purified recombinant proteins were shown to reduce H2O2 in the presence of dithiothreitol. (c) 2007 Elsevier Ltd. All rights reserved.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased similar to6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

Burrow architecture and digging activity in the Cape dune mole rat

While females are traditionally thought to invest more time and energy into parental care than males, males often invest more resources into searching and displaying for mates, obtaining mates and in male-male conflict. Solitary subterranean mammals perform these activities in a particularly challenging niche, necessitating energetically expensive burrowing to both search for mates and forage for food. This restriction presumably affects males more than females as the former are thought to dig longer tunnels that cover greater distances to search for females. We excavated burrow systems of male and female Cape dune mole rats Bathyergus suillus the, largest truly subterranean mammal, to investigate whether male burrows differ from those of females in ways that reflect mate searching by males. We consider burrow architecture (length, internal dimensions, fractal dimension of tunnel systems, number of nesting chambers and mole mounds on the surface) in relation to mating strategy. Males excavated significantly longer burrow systems with higher fractal dimensions and larger burrow areas than females. Male burrow systems were also significantly farther from one another than females were from other females' burrow systems. However, no sex differences were evident in tunnel cross-sectional area, mass of soil excavated per mound, number of mounds produced per unit burrow length or mass of soil excavated per burrow system. Hence, while males may use their habitat differently from females, they do not appear to differ in the dimensions of the tunnels they create. Thus, exploration and use of the habitat differs between the sexes, which may be a consequence of sex differences in mating behaviour and greater demands for food.

Negative Aspects of Close Relationships as a Predictor of Increased Body Mass Index and Waist Circumference: The Whitehall II Study

Objectives. We investigated whether exposure to negative aspects of close relationships was associated with subsequent increase in body mass index (BMI) and waist circumference.
Methods. Data came from a prospective cohort study (Whitehall II) of 9425 civil servants aged 35 to 55 years at baseline (phase 1: 1985-1988). We assessed negative aspects of close relationships with the Close Persons Questionnaire (range 0-12) at phases 1 and 2 (1989-1990). We measured BMI and waist circumference at phases 3 (1991-1994) and 5 (1997-1999). Covariates at phase 1 included gender, age, marital status, ethnicity, BMI, employment grade, smoking, physical activity, fruit and vegetable consumption, and common mental disorder.
Results. After adjustment for sociodemographic characteristics and health behaviors, participants with higher exposure to negative aspects of close relationships had a higher likelihood of a 10% or greater increase in BMI and waist circumference (odds ratios per 1-unit increase 1.08 [95% confidence interval (CI)=1.02, 1.14; P=.007] and 1.09 [CI=1.04, 1.14; P <= .001], respectively) as well as a transition from the overweight (25 <= BMI <30) to the obese (BMI >= 30) category.
Conclusions. Adverse social relationships may contribute to weight gain.

Subdivision of the bacterioferritin comigratory protein family of bacterial peroxiredoxins based on catalytic activity

Peroxiredoxins are ubiquitous proteins that catalyze the reduction of hydroperoxides, thus conferring resistance to oxidative stress. Using high-resolution mass spectrometry, we recently reclassified one such peroxiredoxin, bacterioferritin comigratory protein (BCP) of Escherichia coli, as an atypical 2-Cys peroxiredoxin that functions through the formation of an intramolecular disulfide bond between the active and resolving cysteine. An engineered E. coli BCP, which lacked the resolving cysteine, retained enzyme activity through a novel catalytic pathway. Unlike the active cysteine, the resolving cysteine of BCP peroxiredoxins is not conserved across all members of the family. To clarify the catalytic mechanism of native BCP enzymes that lack the resolving cysteine, we have investigated the BCP homologue of Burkholderia cenocepacia. We demonstrate that the B. cenocepacia BCP (BcBCP) homologue functions through a 1-Cys catalytic pathway. During catalysis, BcBCP can utilize thioredoxin as a reductant for the sulfenic acid intermediate. However, significantly higher peroxidase activity is observed utilizing glutathione as a resolving cysteine and glutaredoxin as a redox partner. Introduction of a resolving cysteine into BcBCP changes the activity from a 1-Cys pathway to an atypical 2-Cys pathway, analogous to the E. coli enzyme. In contrast to the native B. cenocepacia enzyme, thioredoxin is the preferred redox partner for this atypical 2-Cys variant. BCP-deficient B. cenocepacia exhibit a growth-phase-dependent hypersensitivity to oxidative killing. On the basis of sequence alignments, we believe that BcBCP described herein is representative of the major class of bacterial BCP peroxiredoxins. To our knowledge, this is the first detailed characterization of their catalytic activity. These studies support the subdivision of the BCP family of peroxiredoxins into two classes based on their catalytic activity.

A minor catalase/peroxidase from Burkholderia cenocepacia is required for normal aconitase activity

The opportunistic bacterium Burkholderia cenocepacia C5424 contains two catalase/peroxidase genes, katA and katB. To investigate the functions of these genes, katA and katB mutants were generated by targeted integration of suicide plasmids into the katA and katB genes. The catalase/peroxidase activity of the katA mutant was not affected as compared with that of the parental strain, while no catalase/peroxidase activity was detected in the katB mutant. However, the katA mutant displayed reduced resistance to hydrogen peroxide under iron limitation, while the katB mutant showed hypersensitivity to hydrogen peroxide, and reduced growth under all conditions tested. The katA mutant displayed reduced growth only in the presence of carbon sources that are metabolized through the tricarboxylic acid (TCA) cycle, as the growth defect was abrogated in cultures supplemented with glucose or glycerol. This phenotype was also correlated with a marked reduction in aconitase activity. In contrast, aconitase activity was not reduced in the katB mutant and parental strains. The authors conclude that the KatA protein is a specialized catalase/peroxidase that has a novel function by contributing to maintain the normal activity of the TCA cycle, while KatB is a classical catalase/peroxidase that plays a global role in cellular protection against oxidative stress.

Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein

We have developed a new technique for quantifying methionine sulfoxide (MetSO) in protein to assess levels of oxidative stress in physiological systems. In this procedure, samples are hydrolyzed with methanesulfonic acid (MSA) in order to avoid the conversion of MetSO to methionine (Met) that occurs during hydrolysis of protein in HCl. The hydrolysate is fractionated on a cation exchange column to remove the nonvolatile MSA from amino acids, and the amino acids are then derivatized as their trimethylsilyl esters for analysis by selected ion monitoring-gas chromatography/mass spectrometry. The limit of detection of the assay is 200 pmol of MetSO per analysis, and the interassay coefficient of variation is 5.8%. Compared to current methods, the SIM-GC/MS assay avoids the potential for conversion of Met to MetSO during sample preparation, requires less sample preparation time, has lower variability, and uses mass spectrometry for sensitive and specific analyte detection.

Rethinking the capsid proteins of enveloped viruses: multifunctionality from genome packaging to genome transfection

© 2015 FEBS

Automatic internal calibration in liquid chromatography/Fourier transform ion cyclotron resonance mass spectrometry of protein digests

Accurately measured peptide masses can be used for large-scale protein identification from bacterial whole-cell digests as an alternative to tandem mass spectrometry (MS/MS) provided mass measurement errors of a few parts-per-million (ppm) are obtained. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) routinely achieves such mass accuracy either with internal calibration or by regulating the charge in the analyzer cell. We have developed a novel and automated method for internal calibration of liquid chromatography (LC)/FTICR data from whole-cell digests using peptides in the sample identified by concurrent MS/MS together with ambient polydimethyl-cyclosiloxanes as internal calibrants in the mass spectra. The method reduced mass measurement error from 4.3 +/- 3.7 ppm to 0.3 +/- 2.3 ppm in an E. coli LC/FTICR dataset of 1000 MS and MS/MS spectra and is applicable to all analyses of complex protein digests by FTICRMS. Copyright (c) 2006 John Wiley & Sons, Ltd.

Enzyme Activity of Catalase Immobilized in Langmuir-Blodgett Films of Phospholipids

A major challenge for producing low cost biosensors based on nanostructured films with control of molecular architectures is to preserve the catalytic activity of the immobilized biomolecules. In this study, we show that catalase (HRP) keeps its activity if immobilized in Langmuir-Blodgett (LB) films of dipalmitoyl phosphatidylglycerol (DPPG). The incorporation of catalase into a DPPG monolayer at the at interface was demonstrated with surface pressure and surface potential isotherms, in addition to polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). According to the PM-IRRAS data. catalase was not denatured upon adsorption on a preformed DPPG monolayer and could be transferred onto a solid substrate. The catalytic activity of catalase in a mixed LB film with DPPG was ca. 13% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allows catalase-containing LB films to be used in sensing hydrogen peroxide.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Population biology of Uca thayeri Rathbun, 1900 (Brachyura, Ocypodidae) in a subtropical South American mangrove area: Results from transect and catch-per-unit-effort techniques

The population biology of Uca thayeri was studied in a subtropical mangrove in Ubatuba, State of São Paulo, Brazil. Two sampling techniques were used: transect sampling and catch-per-unit-effort. Size frequency distribution, sex-ratio, and reproductive period were analysed. For juvenile crabs (CW < 4.6 mm), the transect procedure was most efficient, while ovigerous females were most collected during the capture effort. Males were most numerous in the transect technique, while in the catch-per-unit-effort there was no difference between sexes. The species showed a different size frequency distribution for each sampling procedure. The reproduction of U. thayeri is seasonal, being more pronounced in the warmer months of the year. However, juveniles occurred all over the year, although more numerous in the colder months. The success of the species in reproductive activity and the constant colonization of the area can be attributed to the availability of food resources and differential occupation of the habitat by ovigerous females.