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As recently demonstrated in patients with factor IX deficiency, adeno-associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and AAV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity., (© 2012 Elsevier B.V. All rights reserved.)

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.

The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K(m) for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.

The hexammineruthenium (HAR) and ferricyanide reductase activities of Complex I (H+-translocating NADH:ubiquinone reductase) from Paracoccus denitrificans and bovine heart mitochondria were studied. The rates of HAR reduction are high, and its steady-state kinetics is similar in both P. denitrificans and bovine Complex I. The deamino-NADH:HAR reductase activity of Complex I from both sources is significantly higher than the respective activity in the presence of NADH. The HAR reductase activity of the bacterial and mitochondrial Complex I is similarly and strongly pH dependent. The pK(a) of this activity could not be determined, however, due to low stability of the enzymes at pH values above 8.0. In contrast to the high similarity between bovine and P. denitrificans Complex I as far as HAR reduction is concerned, the ferricyanide reductase activity of the bacterial enzyme is much lower than in mitochondria. Moreover, ferricyanide reduction in P. denitrificans, but not bovine mitochondria, is partially sensitive to dicyclohexylcarbodiimide (T. Yagi, Biochemistry 26 (1987) 2822-2828). On the other hand, the inhibition of ferricyanide reduction by high concentration of NADH, a typical phenomenon in bovine Complex I, is much weaker in the bacterial enzyme. The functional differences between the two enzymes might be linked to the properties of their binuclear Fe-S clusters.

We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.

We have isolated cDNA clones encoding an iron-sulfur polypeptide subunit of the mitochondrial complex I of Neurospora crassa. The fungal cDNA library was screened by hybridisation with an heterologous probe from Paracoccus denitrificans. The DNA sequence of relevant isolates was determined and revealed an open reading frame encoding a precursor protein of 219 amino acid residues. The gene product is a ferredoxin-like protein that contains two cysteine-rich motives that may each bind a tetranuclear iron-sulfur cluster. The primary structure of the protein is highly homologous to the 23 kDa iron-sulfur subunit of complex I from bovine and P. denitrificans. Interestingly, an alanine residue within the second cluster-binding motif, which is conserved in complex I but replaced by tyrosine in similar chloroplast genes, is substituted for serine in N. crassa.

Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation.

(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.