The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium

Bleaching of corals by loss of symbiotic dinoflagellate algae and/or photosynthetic pigments is commonly triggered by elevated temperatures coupled with high irradiance, and is a first-order threat to coral reef communities. In this study, a high-resolution high-performance liquid chromatography method integrated with mass spectrometry was applied to obtain the first definitive identification of chlorophyll and carotenoid pigments of three clades of symbiotic dinoflagellate algae (Symbiodinium) in corals, and their response to experimentally elevated temperature and irradiance. The carotenoids peridinin, dinoxanthin, diadinoxanthin (Dn), diatoxanthin (Dt) and beta-carotene were detected, together with chlorophylls a and c2, and phaeophytin a, in all three algal clades in unstressed corals. On exposure to elevated temperature and irradiance, three coral species (Montastrea franksi and Favia fragum with clade B algae, and Montastrea cavernosa with clade C) bleached by loss of 50-80% of their algal cells, with no significant impact to chlorophyll a or c2, or peridinin in retained algal cells. One species (Agaricia sp. with clade C) showed no significant reduction in algal cells at elevated temperature and irradiance, but lost substantial amounts of chlorophyll a and carotenoid pigments, presumably through photo-oxidative processes. Two coral species (Porites astreoides and Porites porites both bearing clade A algae) did not bleach. The impact of elevated temperature and irradiance on the levels of the photoprotective xanthophylls (Dn + Dt) and beta-carotene varied among the corals, both in pool size and xanthophyll cycling, and was not correlated to coral bleaching resistance.     

Crystal structure of human 3-hydroxy-3-methylglutaryl-CoA Lyase: insights into catalysis and the molecular basis for hydroxymethylglutaric aciduria

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme. We report the experimental determination of a crystal structure at 2.1 A resolution of the recombinant human mitochondrial HMG-CoA lyase containing a bound activator cation and the dicarboxylic acid 3-hydroxyglutarate. The enzyme adopts a (betaalpha)(8) barrel fold, and the N-terminal barrel end is occluded. The structure of a physiologically relevant dimer suggests that substrate access to the active site involves binding across the cavity located at the C-terminal end of the barrel. An alternative hypothesis that involves substrate insertion through a pore proposed to extend through the barrel is not compatible with the observed structure. The activator cation ligands included Asn(275), Asp(42),His(233), and His(235); the latter three residues had been implicated previously as contributing to metal binding or enzyme activity. Arg(41), previously shown to have a major effect on catalytic efficiency, is also located at the active site. In the observed structure, this residue interacts with a carboxyl group of 3-hydroxyglutarate, the hydrolysis product of the competitive inhibitor 3-hydroxyglutaryl-CoA required for crystallization of human enzyme. The structure provides a rationale for the decrease in enzyme activity due to clinical mutations, including H233R, R41Q, D42H, and D204N, that compromise active site function or enzyme stability.     

Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants

In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), we identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((RS)-CIT). Here we described a separate site in hSERT TM3 (Ile-172) that impacts (RS)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (RS)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylenedioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [(125)I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/I172M had a synergistic impact on (RS)-CIT recognition ( approximately 10,000-fold decrease in (RS)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/I172M. An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by stereoisomers of CIT.     

Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.     

Cerulean, Venus, and VenusY67C FRET reference standards

F?rster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.     

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.     

Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis

The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.     

Structures of biologically active oxysterols determine their differential effects on phospholipid membranes

Oxysterols, derivatives of cholesterol that contain a second oxygen moiety, are intermediates in cholesterol catabolism, regulators of lipid metabolism, and toxic sterols with proatherogenic effects. In model membranes, cholesterol and eight selected oxysterols were compared by fluorescence probe techniques that measure changes in bilayer order and phase behavior and by the formation of detergent-resistant membranes (DRM). The oxysterols were modified on the sterol nucleus or on the isooctyl side chain. The model membranes consisted of dipalmitoyl phosphatidylcholine (DPPC) and mixtures of dioleoyl phosphatidylcholine with DPPC and with sphingomyelin. The different oxysterols induced changes in membrane properties according to the differences in their structures. Whereas the effects of some oxysterols on membrane order, fluorescence probe microenvironment, and DRM formation were similar to those of cholesterol, others had little or no effect. An empirical correlation ranking the oxysterols by their ability to modify membrane biophysical properties when compared to cholesterol led to a significant structure/function relationship between the biophysical measurements and an important cellular phenomenon, apoptosis. 7beta-Hydroxycholesterol, which is the most cytotoxic of the eight selected oxysterols, was one of the least cholesterol-like with respect to modification of membrane properties. The results suggest that an underlying mechanism for oxysterol-induced apoptosis in cells, e.g., monocyte/macrophages, should include their biophysical effects on membranes, such as the regulation of the formation and composition of sterol-rich membrane domains.