In vivo phosphorylation sites of barley tonoplast proteins identified by a phosphoproteomic approach

In plants the vacuolar functions are the cellular storage of soluble carbohydrates, organic acids, inorganic ions and toxic compounds. Transporters and channels located in the vacuolar membrane, the tonoplast, are modulated by PTMs to facilitate the optimal functioning of a large number of metabolic pathways. Here we present a phosphoproteomic approach for the identification of in vivo phosphorylation sites of tonoplast (vacuolar membrane) proteins. Highly purified tonoplast and tonoplast‐enriched microsomes were isolated from photosynthetically induced barley (Hordeum vulgare) mesophyll protoplasts. Phosphopeptides were enriched by strong cation exchange (SCX) chromatography followed either by IMAC or titanium dioxide (TiO₂) affinity chromatography and were subsequently analysed using LC‐ESI‐MS/MS. In total, 65 phosphopeptides of 27 known vacuolar membrane proteins were identified, including the two vacuolar proton pumps, aquaporins, CAX transporters, Na⁺/H⁺ antiporters as well as other known vacuolar transporters mediating the transfer of potassium, sugars, sulphate and malate. The present study provides a novel source to further analyse the regulation of tonoplast proteins by protein phosphorylations, especially as most of the identified phosphorylation sites are highly conserved between Hordeum vulgare (Hv) and Arabidopsis thaliana.     

Adaptive basis of geographic variation: genetic,phenotypic and environmental differences among beach mouse populations

A major goal in evolutionary biology is to understand how and why populations differentiate, both genetically and phenotypically, as they invade a novel habitat. A classical example of adaptation is the pale colour of beach mice, relative to their dark mainland ancestors, which colonized the isolated sandy dunes and barrier islands on Florida''s Gulf Coast. However, much less is known about differentiation among the Gulf Coast beach mice, which comprise five subspecies linearly arrayed on Florida''s shoreline. Here, we test the role of selection in maintaining variation among these beach mouse subspecies at multiple levels—phenotype, genotype and the environments they inhabit. While all beach subspecies have light pelage, they differ significantly in colour pattern. These subspecies are also genetically distinct: pair-wise F_st-values range from 0.23 to 0.63 and levels of gene flow are low. However, we did not find a correlation between phenotypic and genetic distance. Instead, we find a significant association between the average ‘lightness’ of each subspecies and the brightness of the substrate it inhabits: the two most genetically divergent subspecies occupy the most similar habitats and have converged on phenotype, whereas the most genetically similar subspecies occupy the most different environments and have divergent phenotypes. Moreover, allelic variation at the pigmentation gene, Mc1r, is statistically correlated with these colour differences but not with variation at other genetic loci. Together, these results suggest that natural selection for camouflage—via changes in Mc1r allele frequency—contributes to pigment differentiation among beach mouse subspecies.     

A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.     

Studies on the metabolic fate of n-3 polyunsaturated fatty acids

Several different processes involved in the metabolic fate of docosahexaenoic acid (DHA, C22:6n-3) and its precursor in the biosynthesis route, C24:6n-3, were studied. In cultured skin fibroblasts, the oxidation rate of [1-14C] 24:6n-3 was 2.7 times higher than for [1-14C]22:6n-3, whereas [1-14C]22:6n-3 was incorporated 7 times faster into different lipid classes than was [1-14C]24:6n-3. When determining the peroxisomal acyl-CoA oxidase activity, similar specific activities for C22:6(n-3)-CoA and C24:6(n-3)-CoA were found in mouse kidney peroxisomes. Thioesterase activity was measured for both substrates in mouse kidney peroxisomes as well as mitochondria, and C22:6(n-3)-CoA was hydrolyzed 1.7 times faster than C24:6(n-3)-CoA. These results imply that the preferred metabolic fate of C24:6(n-3)-CoA, after its synthesis in the endoplasmic reticulum (ER), is to move to the peroxisome, where it is beta-oxidized, producing C22:6(n-3)-CoA. This DHA-CoA then preferentially moves back, probably as free fatty acid, to the ER, where it is incorporated into membrane lipids.     

UniFrag and GenomePrimer: selection of primers for genome-wide production of unique amplicons

The complementary programs UniFrag and GenomePrimer were developed to provide a reliable high-throughput method to select the most unique regions within genomic DNA sequence(s) and design primers therein, involving minimal user intervention and maximum flexibility.     

Rational design of tropoelastin peptide-based inhibitors of metalloproteinases

Abnormal production of matrix metalloproteinases (MMPs) has been observed in a variety of diseases, such as emphysema, atherosclerosis, and cancer metastasis. Destruction of connective tissue ensues and elastin is often a key target. Three of the main elastolytic MMPs are the gelatinases MMP-2 and MMP-9 and the metalloelastase MMP-12. To investigate the possibility of using peptides to inhibit the elastolytic activity of these enzymes, we mapped the sites within tropoelastin recognized by MMP-9 and MMP-12. Peptides that correspond to regions overlapping these sites were then tested for their ability to inhibit these MMPs. These included an unmodified peptide directed against MMP-9 (peptide PP), cysteine-containing peptides that mimicked either the MMP-9 (peptide NCP) or the MMP-12 (peptide lin24) cleavage sites in tropoelastin and their cyclized forms (CP and cyc24, respectively), and a peptide containing a zinc-chelating hydroxamate group directed against MMP-9 (HP). The presence of a free sulfhydryl or hydroxamate group capable of chelating the zinc ion in the active site of the MMPs was generally found to increase the inhibitory activity of the peptides. The specificity of the inhibitors varied, with some of the inhibitors showing activity against all of the MMPs examined. None of the inhibitors had any significant effect on the activity of the unrelated serine protease, plasmin. K(i) values for the inhibitors were in the micromolar range. Our results suggest ways of developing other MMP inhibitors based on substrate recognition sites that may provide greater levels of inhibition.     

Structural changes and facilitated association of tropoelastin

Circular dichroism studies of tropoelastin secondary structure show 4+/-1% alpha-helix in aqueous solutions. This is in contrast to the substantially higher amounts (up to 23+/-7%) of alpha-helix predicted by computer algorithms, which propose that regions of alpha-helix are limited to the alanine-rich cross-linking domains. Through the addition of trifluoroethanol, the amount of alpha-helix increased to 17+/-1%, equivalent to that expected on the basis of primary structure. The physiological ability of the protein to coacervate and the critical concentration of monomer required for coacervation were unaffected by levels of alpha-helix. However, the temperature required for coacervation decreased linearly with increasing alpha-helical structure, which correlates with the participation of alpha-helices in association. We propose that the alanine-rich cross-linking domains exist as nascent helices in tropoelastin in aqueous solution. We further suggest a novel mechanism for coacervation whereby formation of alpha-helices and subsequent helical side chain interactions limit the conformational flexibility of the polypeptide, to facilitate associations between hydrophobic domains during elastogenesis.     

Correlation of repolarization of ventricular monophasic action potential with ECG in the murine heart

Transgenic mice have become important experimental models in the investigation of mechanisms causing cardiac arrhythmias because of the ability to create strains with alterations in repolarizing membrane currents. It is important to relate alterations in membrane currents in cells to their phenotypic expression on the electrocardiogram (ECG). The murine ECG, however, has unusual characteristics that make interpretation of the phenotypic expression of changes in ventricular repolarization uncertain. The major deflection representing the QRS (referred to as "a") is often followed by a secondary slower deflection ("b") and sometimes a subtle third deflection ("c"). To determine whether the second or third deflections or both represent ventricular repolarization, we recorded the ventricular monophasic action potential (MAP) in open-chest mice and correlated repolarization with the ECG. There was no significant correlation by linear regression, between action potential duration to 50% or 90% repolarization (APD(50) or APD(90)), respectively, of the MAP and either the interval from onset of Q to onset of b (Qb interval) or onset of c (Qc interval). Administration of 4-aminopyridine (4-AP) significantly prolonged APD(50) and APD(90) and the Qb interval, indicating that this deflection on the ECG represents part of ventricular repolarization. After 4-AP, the c wave disappeared, also suggesting that it represents a component of ventricular repolarization. Although it appears that both the b and c waves that follow the Q wave on the ECG represent ventricular repolarization, neither correlates exactly with APD(90) of the MAP. Therefore, an accurate measurement of complete repolarization of the murine ventricle cannot be obtained from the surface ECG.     

Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency

We identified a new peroxisomal disorder caused by a deficiency of the enzyme alpha-methylacyl-coenzyme A (CoA) racemase. Patients with this disorder show elevated plasma levels of pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid (DHCA and THCA), which are all substrates for the peroxisomal beta-oxidation system. alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (2S)-isomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. In this study we used liquid chromatography/tandem mass spectrometry (LC-MS/MS) to analyze the bile acid intermediates that accumulate in plasma from patients with a deficiency of alpha-methylacyl-CoA racemase and, for comparison, in plasma from patients with Zellweger syndrome and patients with cholestatic liver disease.We found that racemase-deficient patients accumulate exclusively the (R)-isomer of free and taurine-conjugated DHCA and THCA, whereas in plasma of patients with Zellweger syndrome and patients with cholestatic liver disease both isomers were present. On the basis of these results we describe an easy and reliable method for the diagnosis of alpha-methylacyl-CoA racemase-deficient patients by plasma analysis. Our results also show that alpha-methylacyl-CoA racemase plays a unique role in bile acid formation. - Ferdinandusse, S., H. Overmars, S. Denis, H. R. Waterham, R. J. A. Wanders, and P. Vreken. Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency. J. Lipid Res. 2001. 42: 137;-141.