The molecular architecture of galactose mutarotase/UDP-galactose 4-epimerase from Saccharomyces cerevisiae

The metabolic pathway by which beta-D-galactose is converted to glucose 1-phosphate is known as the Leloir pathway and consists of four enzymes. In most organisms, these enzymes appear to exist as soluble entities in the cytoplasm. In yeast such as Saccharomyces cerevisiae, however, the first and last enzymes of the pathway, galactose mutarotase and UDP-galactose 4-epimerase, are contained within a single polypeptide chain referred to as Gal10p. Here we report the three-dimensional structure of Gal10p in complex with NAD(+), UDP-glucose, and beta-D-galactose determined to 1.85-A resolution. The enzyme is dimeric with dimensions of approximately 91 A x 135 A x 108 A and assumes an almost V-shaped appearance. The overall architecture of the individual subunits can be described in terms of two separate N- and C-terminal domains connected by a Type II turn formed by Leu-357 to Val-360. The first 356 residues of Gal10p fold into the classical bilobal topology observed for all other UDP-galactose 4-epimerases studied thus far. This N-terminal domain contains the binding sites for NAD(+) and UDP-glucose. The polypeptide chain extending from Glu-361 to Ser-699 adopts a beta-sandwich motif and harbors the binding site for beta-D-galactose. The two active sites of Gal10p are separated by over 50 A. This investigation represents the first structural analysis of a dual function enzyme in the Leloir pathway.     

Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles

Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.     

Binding dynamics at the quinone reduction (Qi) site influence the equilibrium interactions of the iron sulfur protein and hydroquinone oxidation (Qo) site of the cytochrome bc1 complex

Multiple instances of low-potential electron-transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc(1) complex to varying degrees, ranging from changes in hydroquinone (QH(2)) oxidation and cyt c(1) reduction kinetics to proteolytic accessibility of the hinge region of the iron-sulfur-containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation-dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low-potential electron-transport pathway by Q(i)-site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q(i)-site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster and a second affecting the mobility of this subunit, are revealed. Remarkably, different inhibitors and mutations at or near the Q(i) site induce these two effects differently, indicating that the events occurring at the Q(i) site affect the global structure of the cyt bc(1). Furthermore, occupancy of discrete Q(i)-site subdomains differently impede the location of the Fe/S protein at the Q(o) site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH(2) and Q at the Q(i) site, respectively. Implications of these findings in respect to the Q(o)-Q(i) sites communications and to multiple turnovers of the cyt bc(1) are discussed.     

Molecular structure of human galactose mutarotase

Galactose mutarotase catalyzes the conversion of beta-d-galactose to alpha-d-galactose during normal galactose metabolism. The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells. Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, beta-d-galactose. The polypeptide chain folds into an intricate array of 29 beta-strands, 25 classical reverse turns, and 2 small alpha-helices. There are two cis-peptide bonds at Arg-78 and Pro-103. The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307. Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively. These residues are absolutely conserved among galactose mutarotases. To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans. The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two beta-strands. In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 A. In the C. elegans protein, the first 17 residues are missing, thereby reducing the total number of beta-strands by one.     

Evolution of enzymatic activity in the enolase superfamily: functional studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis

o-Succinylbenzoate synthase (OSBS) from Amycolatopsis, a member of the enolase superfamily, catalyzes the Mn2+-dependent exergonic dehydration of 2-succinyl-6R-hydroxy-2,4-cyclohexadiene-1R-carboxylate (SHCHC) to 4-(2'-carboxylphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB) in the menaquinone biosynthetic pathway. This enzyme first was identified as an N-acylamino acid racemase (NAAAR), with the optimal substrates being the enantiomers of N-acetyl methionine. This laboratory subsequently discovered that this protein is a much better catalyst of the OSBS reaction, with the value of k(cat)/K(M), for dehydration, 2.5 x 10(5) M(-1) s(-1), greatly exceeding that for 1,1-proton transfer using the enantiomers of N-acetylmethionine as substrate, 3.1 x 10(2) M(-1) s(-1) [Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-8]. The efficiency of the promiscuous NAAAR reaction is enhanced with alternate substrates whose structures mimic that of the SHCHC substrate for the OSBS reaction, for example, the value of k(cat)/K(M) for the enantiomers of N-succinyl phenylglycine, 2.0 x 10(5) M(-1) s(-1), is comparable to that for the OSBS reaction. The mechanisms of the NAAAR and OSBS reactions have been explored using mutants of Lys 163 and Lys 263 (K163A/R/S and K263A/R/S), the putative acid/base catalysts identified by sequence alignments with other OSBSs, including the structurally characterized OSBS from Escherichia coli. Although none of the mutants display detectable OSBS or NAAAR activities, K163R and K163S catalyze stereospecific exchange of the alpha-hydrogen of N-succinyl-(S)-phenylglycine with solvent hydrogen, and K263R and K263 catalyze the stereospecific exchange the alpha-hydrogen of N-succinyl-(R)-phenylglycine, consistent with formation of a Mn2+-stabilized enolate anion intermediate. The rates of the exchange reactions catalyzed by the wild-type enzyme exceed those for racemization. That this enzyme can catalyze two different reactions, each involving a stabilized enediolate anion intermediate, supports the hypothesis that evolution of function in the enolase superfamily proceeds by pathways involving functional promiscuity.     

Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone

Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada.     

Lysophosphatidylcholine impairs endothelial barrier function through the G protein-coupled receptor GPR4

Abundant evidence indicates that lysophosphatidylcholine (LPC) is proinflammatory and atherogenic. In the vascular endothelium, LPC increases permeability and expression of proinflammatory molecules such as adhesion molecules and cytokines. Yet, mechanisms by which LPC mediates these activities remain unclear and controversial. Recent evidence implicates involvement of a novel subfamily of G protein-coupled receptors (GPR4, G2A, OGR1, and TDAG8) that are sensitive to lysolipids and protons. We previously reported that one of these receptors, GPR4, is selectively expressed by a variety of endothelial cells and therefore hypothesize that the LPC-stimulated endothelial barrier dysfunction is mediated through GPR4. We developed a peptide Ab against GPR4 that detected GPR4 expression in transfected COS 7 cells and endogenous GPR4 expression in endothelial cells by Western blot. Endothelial cells infected with a retrovirus containing small interference RNA (siRNA) to GPR4 resulted in 40-50% decreased GPR4 expression, which corresponded with partial prevention of the LPC-induced 1) decrease in transendothelial resistance, 2) stress fiber formation, and 3) activation of RhoA. Furthermore, coexpression of the siRNA-GPR4 with a siRNA-resistant mutant GPR4 fully restored the LPC-induced resistance decrease. However, extracellular pH of <7.4 did not alter baseline or LPC-stimulated resistances. The results provide strong evidence that the LPC-mediated endothelial barrier dysfunction is regulated by endogenous GPR4 in endothelial cells and suggest that GPR4 may play a critical role in the inflammatory responses activated by LPC.     

Differential effects of heptanoate and hexanoate on myocardial citric acid cycle intermediates following ischemia-reperfusion.

In the normal heart, there is loss of citric acid cycle (CAC) intermediates that is matched by the entry of intermediates from outside the cycle, a process termed anaplerosis. Previous in vitro studies suggest that supplementation with anaplerotic substrates improves cardiac function during myocardial ischemia and/or reperfusion. The present investigation assessed whether treatment with the anaplerotic medium-chain fatty acid heptanoate improves contractile function during ischemia and reperfusion. The left anterior descending coronary artery of anesthetized pigs was subjected to 60 min of 60% flow reduction and 30 min of reperfusion. Three treatment groups were studied: saline control, heptanoate (0.4 mM), or hexanoate as a negative control (0.4 mM). Treatment was initiated after 30 min of ischemia and continued through reperfusion. Myocardial CAC intermediate content was not affected by ischemia-reperfusion; however, treatment with heptanoate resulted in a more than twofold increase in fumarate and malate, with no change in citrate and succinate, while treatment with hexanoate did not increase fumarate or malate but increased succinate by 1.8-fold. There were no differences among groups in lactate exchange, glucose oxidation, oxygen consumption, and contractile power. In conclusion, despite a significant increase in the content of carbon-4 CAC intermediates, treatment with heptanoate did not result in improved mechanical function of the heart in this model of reversible ischemia-reperfusion. This suggests that reduced anaplerosis and CAC dysfunction do not play a major role in contractile and metabolic derangements observed with a 60% decrease in coronary flow followed by reperfusion.     

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4‐amino‐4,6‐dideoxy‐d‐glucose, also known as d‐viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide‐linked sugar, which in the Mimivirus is thought to be UDP‐d‐glucose. The enzyme required for the installment of the amino group at the C‐4′ position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5′‐phosphate‐dependent enzyme, referred to as L136. For this analysis, three high‐resolution X‐ray structures were determined: the wildtype enzyme/pyridoxamine 5′‐phosphate/dTDP complex and the site‐directed mutant variant K185A in the presence of either UDP‐4‐amino‐4,6‐dideoxy‐d‐glucose or dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP‐d‐glucose or dTDP‐d‐glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three‐dimensional architecture was previously reported by this laboratory. As determined in this investigation,DesI shows a profound preference in its catalytic efficiency for the dTDP‐linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three‐dimensional model for a virally encoded PLP‐dependent enzyme and thus provides new information on sugar aminotransferases in general.     

Toll-like receptor-7 agonist administered subcutaneously in a prolonged dosing schedule in heavily pretreated recurrent breast, ovarian, and cervix cancers

Background

The primary objective was to study the antitumor activity of prolonged subcutaneous dosing of systemic 852A, a Toll-like receptor-7 agonist (TLR-7), in recurrent breast, ovarian and cervix cancer. Secondary objectives included assessment of safety and immune system activation.

Methods

Adults with recurrent breast, ovarian or cervix cancer failing multiple therapies received 0.6 mg/m² of 852A subcutaneously twice weekly for 12 weeks. Doses increased by 0.2 mg/m²/week to a maximum of 1.2 mg/m². Serum was collected to assess immune activation.

Results

Fifteen patients enrolled: 10 ovarian, 2 cervix and 3 breast. Three completed all 24 injections. There were two grade 2 (decreased ejection fractions), nine grade 3 (1 cardiovascular, 1 anorexia, 3 dehydration, 2 infections, 2 renal) and two grade 4 (hepatic and troponin elevation) unanticipated toxicities. Cardiac toxicities included three cardiomyopathies (2 asymptomatic) and one stress-related non-ST elevated myocardial infarction. Five patients discontinued therapy due to possibly associated side effects. One who had stable disease (SD) following 24 doses received 17 additional doses. A cervix patient with SD following 24 doses received chemotherapy after progressing 3 months later, and remains disease free at 18 months. Immune activation, as evidenced by increased IP-10 and IL-1ra, was observed.

Conclusions

In this first human experience of a TLR-7 agonist delivered subcutaneously using a prolonged dosing schedule, 852A demonstrated sustained tolerability in some patients. Clinical benefit was modest, but immune activation was seen suggesting further study of antitumor applications is warranted. Because of cardiac toxicity; 852A should be used cautiously in heavily pretreated patients.