The crystal structure of augmenter of liver regeneration: A mammalian FAD-dependent sulfhydryl oxidase.
Ontology highlight
ABSTRACT:
SUBMITTER: Wu CK
PROVIDER: S-EPMC2323880 | biostudies-literature | 2003 May
REPOSITORIES: biostudies-literature
Publications
The crystal structure of augmenter of liver regeneration: A mammalian FAD-dependent sulfhydryl oxidase.
Wu Chia-Kuei CK Dailey Tamara A TA Dailey Harry A HA Wang Bi-Cheng BC Rose John P JP
Protein science : a publication of the Protein Society 20030501 5
The crystal structure of recombinant rat augmenter of liver regeneration (ALRp) has been determined to 1.8 A. The protein is a homodimer, stabilized by extensive noncovalent interactions and a network of hydrogen bonds, and possesses a noncovalently bound FAD in a motif previously found only in the related protein ERV2p. ALRp functions in vitro as a disulfide oxidase using dithiothreitol as reductant. Reduction of the flavin by DTT occurs under aerobic conditions resulting in a spectrum characte ...[more]
Similar Datasets
Project description:Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s(-1). However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s(-1) at air saturation) and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While β-mercaptoethanol is a very poor substrate of sfALR (∼0.3 min(-1) at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be physiologically important in preventing the oxidase from catalyzing the potentially harmful oxidation of intracellular glutathione.
| S-EPMC3893697 | biostudies-literature
Project description:The sulfhydryl oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short, cytokine-like, form of ALR (sfALR), and a longer form (lfALR) which resides in the mitochondrial intermembrane space (IMS). lfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia40 during oxidative protein folding within the IMS. Recently, Di Fonzo et al. [Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G. P. (2009) Am. J. Hum. Genet. 84, 594-604] described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human R194H mutant in lf- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR, close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself and from C95 of the intersubunit disulfide of the other protomer) and with the 2'-OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However, the mutation adversely affects the stability of both ALR forms: e.g., by decreasing the melting temperature by about 10 degrees C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NMR spectra of wild-type sfALR and its R194H mutant reveals a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al.
| S-EPMC2914844 | biostudies-literature
Project description:Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive Alr(C131S) mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr(C131S) is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO-mediated knockdown allows the identification of its late developmental role in vertebrate liver organogenesis.
| S-EPMC3266923 | biostudies-literature
Project description:Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases.
| S-EPMC2518652 | biostudies-literature
Project description:Augmenter of liver regeneration (ALR) is both a growth factor and a sulfhydryl oxidase that binds FAD in an unusual helix-rich domain containing a redox-active CxxC disulfide proximal to the flavin ring. In addition to the cytokine form of ALR (sfALR) that circulates in serum, a longer form, lfALR, is believed to participate in oxidative trapping of reduced proteins entering the mitochondrial intermembrane space (IMS). This longer form has an 80-residue N-terminal extension containing an additional, distal, CxxC motif. This work presents the first enzymological characterization of human lfALR. The N-terminal region conveys no catalytic advantage toward the oxidation of the model substrate dithiothreitol (DTT). In addition, a C71A or C74A mutation of the distal disulfide does not increase the turnover number toward DTT. Unlike Erv1p, the yeast homologue of lfALR, static spectrophotometric experiments with the human oxidase provide no evidence of communication between distal and proximal disulfides. An N-terminal His-tagged version of human Mia40, a resident oxidoreductase of the IMS and a putative physiological reductant of lfALR, was subcloned and expressed in Escherichia coli BL21 DE3 cells. Mia40, as isolated, shows a visible spectrum characteristic of an Fe-S center and contains 0.56 +/- 0.02 atom of iron per subunit. Treatment of Mia40 with guanidine hydrochloride and triscarboxyethylphosphine hydrochloride during purification removed this chromophore. The resulting protein, with a reduced CxC motif, was a good substrate of lfALR. However, neither sfALR nor lfALR mutants lacking the distal disulfide could oxidize reduced Mia40 efficiently. Thus, catalysis involves a flow of reducing equivalents from the reduced CxC motif of Mia40 to distal and then proximal CxxC motifs of lfALR to the flavin ring and, finally, to cytochrome c or molecular oxygen.
| S-EPMC2730831 | biostudies-literature
Project description:BACKGROUND: The gene encoding the hepatotrophic factor Augmenter of Liver Regeneration (ALR) has recently been cloned in the rat. The availability of the mouse form of ALR would allow the analysis of the role of this factor in the physiology of liver and other organs, while the identification of the human homolog would allow the transfer of the great wealth of information that has been generated in animal models to clinically oriented pilot trials, and eventually the therapeutic application of this information. MATERIALS AND METHODS: Standard molecular biology approaches have been used to determine the genomic structure of the ALR gene in the mouse, and to characterize the ALR transcript and its protein product. The human ALR cDNA was also isolated and the amino acid sequence of the human gene product deduced. The mapping of mouse and human ALR genes on mouse and human chromosomes was then completed. RESULTS: The protein coding portion of the mouse ALR gene is comprised of three exons, the first containing the 5' untranslated sequence and the initial 18 bases after the ATG translation initiation codon, the second exon encompasses 198 bases, and the third exon contains the remaining portion of the protein coding sequence. Rat, mouse, and human ALR genes (and protein products) were found to be highly conserved and preferentially expressed in the testis and in the liver. The ALR gene maps to the mouse chromosome 17, in a region syntenic with human chromosome 16, where the T/t region has also been mapped. CONCLUSIONS: ALR appears to be a protein with important physiologic properties, not exclusively limited to liver regeneration, with roles that are involved in the synthesis or stability of the nuclear and mitochondrial transcripts that are present in actively regenerating cells, particularly the germ cells of the testes.
| S-EPMC2230030 | biostudies-other
Project description:BackgroundAldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents.ResultsThe crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics.ConclusionDifferences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures.SignificanceThe first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.
| S-EPMC3504782 | biostudies-literature
Project description:Background and Aims Hepatocellular carcinoma (HCC) is one of the most common types of cancer, often resulting in death. Augmenter of liver regeneration (ALR), a widely expressed multifunctional protein, has roles in liver disease. In our previous study, we reported that ALR knockdown inhibited cell proliferation and promoted cell death. However, there is no study on the roles of ALR in HCC. Methods We used in vitro and in vivo models to investigate the effects of ALR in HCC as well as its mechanism of action. We produced and characterized a human ALR-specific monoclonal antibody (mAb) and investigated the effects of the mAb in HCC cells. Results The purified ALR-specific mAb matched the predicted molecular weight of IgG heavy and light chains. Thereafter, we used the ALR-specific mAb as a therapeutic strategy to suppress tumor growth in nude mice. Additionally, we assessed the proliferation and viability of three HCC cell lines, Hep G2, Huh-7, and MHC97-H, treated with the ALR-specific mAb. Compared with controls, tumor growth was inhibited in mice treated with the ALR-specific mAb at 5 mg/kg, as shown by hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling. Simultaneous treatment with the ALR-specific mAb and adriamycin promoted apoptosis, whereas treatment with the ALR-specific mAb alone inhibited cell proliferation. Conclusions The ALR-specific mAb might be a novel therapy for HCC by blocking extracellular ALR. Graphical abstract
| S-EPMC10037511 | biostudies-literature
Project description:C-glycosides have a unique structure, in which an anomeric carbon of a sugar is directly bonded to the carbon of an aglycone skeleton. One of the natural C-glycosides, carminic acid, is utilized by the food, cosmetic, and pharmaceutical industries, for a total of more than 200 tons/y worldwide. However, a metabolic pathway of carminic acid has never been identified. In this study, we isolated the previously unknown carminic acid-catabolizing microorganism and discovered a flavoenzyme "C-glycoside 3-oxidase" named CarA that catalyzes oxidation of the sugar moiety of carminic acid. A Basic Local Alignment Search Tool (BLAST) search demonstrated that CarA homologs were distributed in soil microorganisms but not intestinal ones. In addition to CarA, two CarA homologs were cloned and heterologously expressed, and their biochemical properties were determined. Furthermore, a crystal structure of one homolog was determined. Together with the biochemical analysis, the crystal structure and a mutagenesis analysis of CarA revealed the mechanisms underlying their substrate specificity and catalytic reaction. Our study suggests that CarA and its homologs play a crucial role in the metabolism of C-glycosides in nature.
| S-EPMC8501837 | biostudies-literature
Project description:Background and aimsThe augmenter of liver regeneration (ALR) protein is critical for lipid homeostasis and mitochondrial function. We investigated high-fat/high-carbohydrate (HF/HC) diet-induced nonalcoholic fatty liver disease (NAFLD) in wild-type (WT), hepatocyte-specific ALR-knockout (ALR-H-KO), and ALR-heterozygous (ALR-H-HET) mice. ALR was measured in serum of human nonalcoholic steatohepatitis (NASH) and NASH-induced cirrhosis (serum and liver).Approach and resultsHF/HC feeding decreased ALR expression in all groups of mice. The otherwise normal ALR-H-HET mice gained more weight and steatosis than WT mice when challenged metabolically with the HF/HC diet; ALR-H-KO mice gained the least weight and had the least steatosis. These findings were consistent with correspondingly increased triglycerides and cholesterol and altered expression of carnitine palmitoyltransferase 1a, sterol regulatory element-binding protein, acetyl coenzyme A carboxylase, and fatty acid synthase. All HF/HC-fed mice developed insulin resistance, the magnitude being lower in ALR-H-KO mice. HF/HC-fed ALR-H-HET mice were more resistant to glucose challenge than WT or ALR-H-KO mice. The frequency of tumor necrosis factor alpha-producing, interleukin 6 (IL6)-producing, and IL17-producing cells was greater in ALR-H-KO than ALR-H-HET and lowest in WT mice. HF/HC feeding did not increase their number in ALR-H-KO mice, and the increase in ALR-H-HET was greater than that in WT mice except for IL17 cells. Cluster of differentiation 25-positive (CD25+ ) forkhead box P3-positive CD4+ regulatory T-cell frequency was lower in ALR-H-HET than WT mice and further reduced in ALR-H-KO mice; HF/HC reduced regulatory T-cell frequency only in WT mice. HF/HC-fed ALR-H-HET, but not WT, mice developed fibrosis; and ALR-H-KO mice progressed to cirrhosis. White adipose tissue of HF/HC-fed ALR-deficient mice developed strong inflammation, indicating bidirectional interactions with the liver. Hepatic and serum ALR levels were significantly reduced in patients with NASH-cirrhosis. Serum ALR was also significantly lower in patients with NASH.ConclusionsHepatic ALR deficiency may be a critical predisposing factor for aggressive NAFLD progression.
| S-EPMC8025692 | biostudies-literature