Hyperthermia impairs liver mitochondrial function in vitro

The effects of temperature on the relationships among the rates of pyruvate carboxylation, O(2) uptake (J(o)), oxidative phosphorylation (J(p)), and the free energy of ATP hydrolysis (G(p)) were studied in liver mitochondria isolated from 250-g female rats. Pyruvate carboxylation was evaluated at 37, 40, and 43 degrees C. In disrupted mitochondria, pyruvate carboxylase maximal reaction velocity increased from 37 to 43 degrees C with an apparent Q(10) of 2.25. A reduction in ATP/ADP ratio decreased enzyme activity at all three temperatures. In contrast, in intact mitochondria, increasing temperature failed to increase pyruvate carboxylation (malate + citrate accumulation) but did result in increased J(o) and decreased extramitochondrial G(p). J(p) was studied in respiring mitochondria at 37 and 43 degrees C at various fractions of state 3 respiration, elicited with a glucose + hexokinase ADP-regenerating system. The relationship between J(o) and G(p) was similar at both temperatures. However, hyperthermia (43 degrees C) reduced the J(p)/J(o) ratio, resulting in lower G(p) for a given J(p). Fluorescent measurements of membrane phospholipid polarization revealed a transition in membrane order between 40 and 43 degrees C, a finding consistent with increased membrane proton conductance. It is concluded that hyperthermia augments nonspecific proton leaking across the inner mitochondrial membrane, and the resultant degraded energy state offsets temperature stimulation of pyruvate carboxylase. As a consequence, at high temperatures approaching 43 degrees C, the pyruvate carboxylation rate of intact liver mitochondria may fail to exhibit a Q(10) effect.     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.     

Structural studies of AntD: an N-Acyltransferase involved in the biosynthesis of D-Anthrose

The unusual dideoxy sugar d-anthrose has been identified as an important component in the endospores of infectious agents such as Bacillus anthracis and Bacillus cereus. Specifically, it is the terminal sugar on the bacterium's exosporium, and it provides a point of interaction between the spore and the host. The biosynthesis of d-anthrose involves numerous steps starting from α-d-glucose 1-phosphate. Here we present a combined structural and functional investigation of AntD from B. cereus. This enzyme plays a key role in d-anthrose biosynthesis by catalyzing the acylation of the C-4″ amino group of dTDP-4-amino-4,6-dideoxyglucose using 3-hydroxy-3-methylbutyryl-CoA as the acyl donor. For this investigation, two ternary complexes of AntD were determined to 1.8 ? resolution: one in which the protein contained bound β-hydroxybutyryl-CoA and dTDP and the second with CoA and dTDP-4-amino-4,6-dideoxyglucose. On the basis of these high-resolution structures, it was shown that the side chain of Asp 94 lies within hydrogen bonding distance of the sugar C-4″ amino group, and the side chain of Ser 84 resides near the carbonyl oxygen of β-hydroxybutyryl-CoA. To test the roles of these residues in the catalytic mechanism of AntD, various site-directed mutant proteins were prepared and subjected to kinetic and structural analyses. The D94A and D94N mutant proteins demonstrated enzymatic activity, albeit with significantly reduced catalytic efficiencies. The S84A mutant protein showed an approximate 10-fold decrease in activity. Interestingly, the S84C and S84T mutant proteins were both active but demonstrated substrate inhibition. The three-dimensional structures of all of the mutant proteins were nearly identical to that of the wild-type enzyme, indicating that the changes in their kinetic parameters were not due to major conformational changes. Taken together, these data suggest that Asp 94 is important for substrate binding, but probably does not function as an enzymatic base, and that Ser 84 most likely plays a role in the formation of an oxyanion hole.     

Strain transfer through the aortic valve

The complex structural organization of the aortic valve (AV) extracellular matrix (ECM) enables large and highly nonlinear tissue level deformations. The collagen and elastin (elastic) fibers within the ECM form an interconnected fibrous network (FN) and are known to be the main load-bearing elements of the AV matrix. The role of the FN in enabling deformation has been investigated and documented. However, there is little data on the correlation between tissue level and FN-level strains. Investigating this correlation will help establish the mode of strain transfer (affine or nonaffine) through the AV tissue as a key feature in microstructural modeling and will also help characterize the local FN deformation across the AV sample in response to applied tissue level strains. In this study, the correlation between applied strains at tissue level, macrostrains across the tissue surface, and local FN strains were investigated. Results showed that the FN strain distribution across AV samples was inhomogeneous and nonuniform, as well as anisotropic. There was no direct transfer of the deformation applied at tissue level to the fibrous network. Loading modes induced in the FN are different than those applied at the tissue as a result of different local strains in the valve layers. This nonuniformity of local strains induced internal shearing within the FN of the AV, possibly exposing the aortic valve interstitial cells (AVICs) to shear strains and stresses.     

Genome-Wide Expression Profiling Identifies Type 1 Interferon Response Pathways in Active Tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains the leading cause of mortality from a single infectious agent. Each year around 9 million individuals newly develop active TB disease, and over 2 billion individuals are latently infected with M.tb worldwide, thus being at risk of developing TB reactivation disease later in life. The underlying mechanisms and pathways of protection against TB in humans, as well as the dynamics of the host response to M.tb infection, are incompletely understood. We carried out whole-genome expression profiling on a cohort of TB patients longitudinally sampled along 3 time-points: during active infection, during treatment, and after completion of curative treatment. We identified molecular signatures involving the upregulation of type-1 interferon (α/β) mediated signaling and chronic inflammation during active TB disease in an Indonesian population, in line with results from two recent studies in ethnically and epidemiologically different populations in Europe and South Africa. Expression profiles were captured in neutrophil-depleted blood samples, indicating a major contribution of lymphocytes and myeloid cells. Expression of type-1 interferon (α/β) genes mediated was also upregulated in the lungs of M.tb infected mice and in infected human macrophages. In patients, the regulated gene expression-signature normalized during treatment, including the type-1 interferon mediated signaling and a concurrent opposite regulation of interferon-gamma. Further analysis revealed IL15RA, UBE2L6 and GBP4 as molecules involved in the type-I interferon response in all three experimental models. Our data is highly suggestive that the innate immune type-I interferon signaling cascade could be used as a quantitative tool for monitoring active TB disease, and provide evidence that components of the patient’s blood gene expression signature bear similarities to the pulmonary and macrophage response to mycobacterial infection.     

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 2: α-Branched quaternary amines

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 μg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC. As such, pyrazinoyl quaternary amines represent a promising new class of ENaC blockers for the treatment of cystic fibrosis that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride.     

A structural study of GDP-4-keto-6-deoxy-D-mannose-3-dehydratase: caught in the act of geminal diamine formation

Di- and trideoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction.     

The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380

Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.     

Cytochrome P450--redox partner fusion enzymes

The cytochromes P450 (P450s) are a broad class of heme b-containing mono-oxygenase enzymes. The vast majority of P450s catalyse reductive scission of molecular oxygen using electrons usually derived from coenzymes (NADH and NADPH) and delivered from redox partner proteins. Evolutionary advantages may be gained by fusion of one or more redox partners to the P450 enzyme in terms of e.g. catalytic efficiency. This route was taken by the well characterized flavocytochrome P450(BM3) system (CYP102A1) from Bacillus megaterium, in which soluble P450 and cytochrome P450 reductase enzymes are covalently linked to produce a highly efficient electron transport system for oxygenation of fatty acids and related molecules. However, genome analysis and ongoing enzyme characterization has revealed that there are a number of other novel classes of P450-redox partner fusion enzymes distributed widely in prokaryotes and eukaryotes. This review examines our current state of knowledge of the diversity of these fusion proteins and explores their structural composition and evolutionary origins.