Prognostic role of vascular endothelial growth factor in hepatocellular carcinoma: systematic review and meta-analysis.

Hepatocellular carcinoma (HCC) is a highly vascular tumour that expresses vascular endothelial growth factor (VEGF). Various studies have evaluated the prognostic value of VEGF levels in HCC. Its overall test performance remains unclear, however. The aim was to perform a systematic review and meta-analysis of prognostic cohort studies evaluating the use of VEGF as a predictor of survival in patients with treated HCC. Eligible studies were identified through multiple search strategies. Studies were assessed for quality using the Newcastle-Ottawa Tool. Data were collected comparing disease-free and overall survival in patients with high VEGF levels as compared to those with low levels. Studies were pooled and summary hazard ratios were calculated. A total of 16 studies were included for meta-analysis (8 for tissue and 8 for serum). Methodological analysis indicated a trend for higher study quality with serum studies as compared to tissue-based investigations. Four distinct groups were pooled for analysis: tissue overall survival (n=251), tissue disease-free survival (n=413), serum overall survival (n=579), and serum disease-free survival (n=439). High tissue VEGF levels predicted poor overall (HR=2.15, 95% CI: 1.26-3.68) and disease-free (HR=1.69, 95% CI: 1.23-2.33) survival. Similarly, high serum VEGF levels predicted poor overall (HR=2.35, 95% CI: 1.80-3.07) and disease-free (HR=2.36, 95% CI 1.76-3.16) survival. A high degree of inter-study consistency was present in three of four groups analysed. Tissue and serum VEGF levels appear to have significant predictive ability for estimating overall survival in HCC and may be useful for defining prognosis in HCC.

A role for rubredoxin in oxidative stress protection in Desulfovibrio vulgaris: catalytic electron transfer to rubrerythrin and two-iron superoxide reductase.

Desulfovibrio vulgaris rubredoxin, which contains a single [Fe(SCys)4] site, is shown to be a catalytically competent electron donor to two enzymes from the same organism, namely, rubrerythrin and two-iron superoxide reductase (a.k.a. rubredoxin oxidoreductase or desulfoferrodoxin). These two enzymes have been implicated in catalytic reduction of hydrogen peroxide and superoxide, respectively, during periods of oxidative stress in D. vulgaris, but their proximal electron donors had not been characterized. We further demonstrate the incorrectness of a previous report that rubredoxin is not an electron donor to the superoxide reductase and describe convenient assays for demonstrating the catalytic competence of all three proteins in their respective functions. Rubrerythrin is shown to be an efficient rubredoxin peroxidase in which the rubedoxin:hydrogen peroxide redox stoichiometry is 2:1 mol:mol. Using spinach ferredoxin-NADP+ oxidoreductase (FNR) as an artificial, but proficient, NADPH:rubredoxin reductase, rubredoxin was further found to catalyze rapid and complete reduction of all Fe3+ to Fe2+ in rubrerythrin by NADPH under anaerobic conditions. The combined system, FNR/rubredoxin/rubrerythrin, was shown to function as a catalytically competent NADPH peroxidase. Another small rubredoxin-like D. vulgaris protein, Rdl, could not substitute for rubredoxin as a peroxidase substrate of rubrerythrin. Similarly, D. vulgaris rubredoxin was demonstrated to efficiently catalyze reduction of D. vulgaris two-iron superoxide reductase and, when combined with FNR, to function as an NADPH:superoxide oxidoreductase. We suggest that, during periods of oxidative stress, rubredoxin could divert electron flow from the electron transport chain of D. vulgaris to rubrerythrin and superoxide reductase, thereby simultaneously protecting autoxidizable redox enzymes and lowering intracellular hydrogen peroxide and superoxide levels.

The crystal structures of Phascolopsis gouldii wild type and L98Y methemerythrins: structural and functional alterations of the O2 binding pocket.

Reported are the X-ray crystal structures of recombinant Phascolopsis gouldii methemerythrin (1.8-A resolution) and the structure of an O2-binding-pocket mutant, L98Y methemerythrin (2.1-A resolution). The L98Y hemerythrin (Hr) has a greatly enhanced O2 affinity, a slower O2 dissociation rate, a larger solvent deuterium isotope effect on this rate, and a greater resistance to autoxidation relative to the wild-type protein. The crystal structures show that the hydrophobic binding pocket of Hr can accommodate substitution of a leucyl by a tyrosyl side chain with relatively minor structural rearrangements. UV/vis and resonance Raman spectra show that in solution L98Y methemerythrin contains a mixture of two diiron site structures differing by the absence or presence of an Fe(III)-coordinated phenolate. However, in the crystal, only one L98Y diiron site structure is seen, in which the Y98 hydroxyl is not a ligand, but instead forms a hydrogen bond to a terminal hydroxo/aqua ligand to the nearest iron. Based on this crystal structure, we propose that in the oxy form of L98Y hemerythrin the non-polar nature of the binding pocket favors localization of the Y98 hydroxyl near the O2 binding site, where it can donate a hydrogen bond to the hydroperoxo ligand. The stabilizing Y98OH-O2H-interaction would account for all of the altered O2 binding properties of L98Y Hr listed above.

Analysis of metal incorporation during overexpression of Clostridium pasteurianum rubredoxin by electrospray FTICR mass spectrometry.

The rate of production of Clostridium pasteurianum rubredoxin overexpressed in Escherichia coli was examined by electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Previous work had shown that this heterologous expression resulted in isolation of both iron-containing (FeRd) and zinc-containing (ZnRd) rubredoxins. In the present work, minimally processed cell lysates of E. coli were analyzed in order to monitor the production of FeRd and ZnRd. The sensitivity of the measurement favored FeRd relative to ZnRd, and this differential sensitivity was quantitated using previously separated and purified rubredoxins. A time course study indicated that ZnRd and FeRd are produced simultaneously during overexpression, but at different rates. The ratio of the concentration of ZnRd to FeRd increased in a linear fashion during 3 h following induction of overexpression. Since only FeRds have been reported from native bacteria and archaea, the data suggest that either Zn2+ is sequestered from rubredoxins during native biosynthesis or that ZnRds may have escaped detection in the native microorganisms. ESI-FTICR mass spectrometry is shown to be a useful tool for monitoring metal insertion during protein biosynthesis.

Five-gene cluster in Clostridium thermoaceticum consisting of two divergent operons encoding rubredoxin oxidoreductase- rubredoxin and rubrerythrin-type A flavoprotein- high-molecular-weight rubredoxin.

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium Clostridium thermoaceticum (Moorella thermoacetica) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as rub (rubredoxin), rbo (rubredoxin oxidoreductase), rbr (rubrerythrin), fprA (type A flavoprotein), and a gene referred to as hrb (high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons, rbr-fprA-hrb and rbo-rub. The rbr, fprA, and rub genes were expressed in Escherichia coli, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against Desulfovibrio vulgaris Rbr reacted with both native and recombinant Rbr from C. thermoaceticum, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of C. thermoaceticum.

Rubrerythrin and rubredoxin oxidoreductase in Desulfovibrio vulgaris: a novel oxidative stress protection system.

Evidence is presented for an alternative to the superoxide dismutase (SOD)-catalase oxidative stress defense system in Desulfovibrio vulgaris (strain Hildenborough). This alternative system consists of the nonheme iron proteins, rubrerythrin (Rbr) and rubredoxin oxidoreductase (Rbo), the product of the rbo gene (also called desulfoferrodoxin). A Deltarbo strain of D. vulgaris was found to be more sensitive to internal superoxide exposure than was the wild type. Unlike Rbo, expression of plasmid-borne Rbr failed to restore the aerobic growth of a SOD-deficient strain of Escherichia coli. Conversely, plasmid-borne expression of two different Rbrs from D. vulgaris increased the viability of a catalase-deficient strain of E. coli that had been exposed to hydrogen peroxide whereas Rbo actually decreased the viability. A previously undescribed D. vulgaris gene was found to encode a protein having 50% sequence identity to that of E. coli Fe-SOD. This gene also encoded an extended N-terminal sequence with high homologies to export signal peptides of periplasmic redox proteins. The SOD activity of D. vulgaris is not affected by the absence of Rbo and is concentrated in the periplasmic fraction of cell extracts. These results are consistent with a superoxide reductase rather than SOD activity of Rbo and with a peroxidase activity of Rbr. A joint role for Rbo and Rbr as a novel cytoplasmic oxidative stress protection system in D. vulgaris and other anaerobic microorganisms is proposed.

Characterization and evolution of anthranilate 1,2-dioxygenase from Acinetobacter sp. strain ADP1.

The two-component anthranilate 1,2-dioxygenase of the bacterium Acinetobacter sp. strain ADP1 was expressed in Escherichia coli and purified to homogeneity. This enzyme converts anthranilate (2-aminobenzoate) to catechol with insertion of both atoms of O(2) and consumption of one NADH. The terminal oxygenase component formed an alpha(3)beta(3) hexamer of 54- and 19-kDa subunits. Biochemical analyses demonstrated one Rieske-type [2Fe-2S] center and one mononuclear nonheme iron center in each large oxygenase subunit. The reductase component, which transfers electrons from NADH to the oxygenase component, was found to contain approximately one flavin adenine dinucleotide and one ferredoxin-type [2Fe-2S] center per 39-kDa monomer. Activities of the combined components were measured as rates and quantities of NADH oxidation, substrate disappearance, product appearance, and O(2) consumption. Anthranilate conversion to catechol was stoichiometrically coupled to NADH oxidation and O(2) consumption. The substrate analog benzoate was converted to a nonaromatic benzoate 1,2-diol with similarly tight coupling. This latter activity is identical to that of the related benzoate 1, 2-dioxygenase. A variant anthranilate 1,2-dioxygenase, previously found to convey temperature sensitivity in vivo because of a methionine-to-lysine change in the large oxygenase subunit, was purified and characterized. The purified M43K variant, however, did not hydroxylate anthranilate or benzoate at either the permissive (23 degrees C) or nonpermissive (39 degrees C) growth temperatures. The wild-type anthranilate 1,2-dioxygenase did not efficiently hydroxylate methylated or halogenated benzoates, despite its sequence similarity to broad-substrate specific dioxygenases that do. Phylogenetic trees of the alpha and beta subunits of these terminal dioxygenases that act on natural and xenobiotic substrates indicated that the subunits of each terminal oxygenase evolved from a common ancestral two-subunit component.

Transgenic studies of fatty acid oxidation gene expression in nonobese diabetic mice.

Type 1 diabetes mellitus is a devastating disorder affecting both glucose and lipid metabolism. Using the nonobese diabetic (NOD) mouse model, we found that diabetic mice had a liver-specific increase in steady state mRNA levels for enzymes involved in oxidation of fatty acids. Increased mRNA abundance was observed in very long-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase (LCAD), medium-chain acyl-CoA dehydrogenase (MCAD), carnitine palmitoyltransferase I (CPT-1a), and the gluconeogenic enzyme phosphoenolpyruvate carboxykinase, whereas short-chain acyl-CoA dehydrogenase mRNA remained unchanged. In contrast, minimal elevations in LCAD and CPT-1a mRNA were observed in hearts of diabetic mice with no significant differences found for the other enzymes. We developed NOD mice with transgenes containing regulatory elements of human MCAD gene controlling a reporter gene to determine if the increase in MCAD gene expression occurred via the well-characterized nuclear receptor response element (NRRE-1). These results demonstrated that the transgene containing the NRRE-1 and adjacent 5' sequences had elevated liver expression in diabetic mice compared with prediabetic or normal control mice. Surprisingly, the transgene that contains NRRE-1 with adjacent 3' sequences and the transgene with the NRRE-1 deleted showed minimal response to the fulminant diabetic condition.Collectively, these results indicate that in type 1 diabetes there exists an excessive and liver-specific activation of fatty acid oxidation gene expression. Using human MCAD as a prototype gene, we have shown that this increased expression is mediated at the transcriptional level but does not occur via the well-characterized NRRE-1 site responsible for baseline expression in normal mice.

The O(2) binding pocket of myohemerythrin: role of a conserved leucine.

A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.

A hemerythrin-like domain in a bacterial chemotaxis protein.

Hemerythrin (Hr) is an O(2)-carrying protein found in some marine invertebrates. A conserved sequence motif in all Hrs provides five histidine and two carboxylate ligands to an oxo-/hydroxo-bridged diiron active site, as well as a hydrophobic O(2) binding pocket. Database searches located a previously unrecognized Hr-like sequence motif at the 3' end of the gene, dcrH, from the anaerobic sulfate-reducing bacterium, Desulfovibrio (D.) vulgaris (Hildenborough). This gene encodes a putative methyl-accepting chemotaxis protein, DcrH. We have established by immunoblotting that a full-length DcrH, including the Hr-like domain, is expressed in D. vulgaris (Hildenborough). The C-terminal domain of DcrH, when expressed separately in recombinant form in Escherichia coli, was found to fold into a stable protein, DcrH-Hr. The UV-vis absorption and resonance Raman spectra of DcrH-Hr, and of its azide adduct, provide clear evidence for an oxo-bridged diiron(III) site very similar to that found in Hr. Based on UV-vis absorption spectra, exposure of the reduced (colorless, presumably diferrous) DcrH-Hr to air resulted in formation of an O(2) adduct also very similar to that of Hr. Unlike that of Hr, the O(2) adduct of DcrH-Hr autoxidized within a few minutes at room temperature. The O(2) binding pocket of DcrH-Hr appears to be larger than that of Hr. Given the air-sensitive nature of D. vulgaris and the putative chemotactic function of DcrH, one possible role for the Hr-like domain of DcrH is O(2)-sensing. DcrH-Hr is the first characterized example of a Hr-like protein from any microorganism.

A leucine residue "Gates" solvent but not O2 access to the binding pocket of phascolopsis gouldii hemerythrin.

A leucine residue, Leu-98, lines the O(2)-binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O(2) association rate constants within a factor of 3 of the wild type value. Similarly, the O(2) dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O(2) dissociation rate constant and a large D(2)O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O(2). Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X = Tyr. These increases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O(2), but is evident for solvent.

Thermal stability of Clostridium pasteurianum rubredoxin: deconvoluting the contributions of the metal site and the protein.

To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 degrees C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ << Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 degrees C for Fe2+ Rd and 83 degrees C for Fe3+ Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50-70 degrees C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+ Rd, stoichiometric Fe3+/cysteine-ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.

Modulation of the redox potential of the [Fe(SCys)(4)] site in rubredoxin by the orientation of a peptide dipole.

Rubredoxins (Rds) may be separated into two classes based upon the correlation of their reduction potentials with the identity of residue 44; those with Ala44 have reduction potentials that are approximately 50 mV higher than those with Val44. The smaller side chain volume occupied by Ala44 relative to that occupied by Val44 has been proposed to explain the increase in the reduction potential, based upon changes in the Gly43-Ala44 peptide bond orientation and the distance to the [Fe(SCys)(4)] center in the Pyrococcus furiosus (Pf) Rd crystal structure compared to those of Gly43-Val44 in the Clostridium pasteurianum (Cp) Rd crystal structure. As an experimental test of this hypothesis, single-site Val44 <--> Ala44 exchange mutants, [V44A]Cp and [A44V]Pf Rds, have been cloned and expressed. Reduction potentials of these residue 44 variants and pertinent features of the X-ray crystal structure of [V44A]Cp Rd are reported. Relative to those of wild-type Cp and Pf Rds, the V44A mutation in Cp Rd results in an 86 mV increase in midpoint reduction potential and the [A44V] mutation in Pf Rd results in a 95 mV decrease in midpoint reduction potential, respectively. In the crystal structure of [V44A]Cp Rd, the peptide bond between residues 43 and 44 is approximately 0.3 A closer to the Fe center and the hydrogen bond distance between the residue 44 peptide nitrogen and the Cys42 gamma-sulfur decreases by 0.32 A compared to the analogous distances in the wild-type Cp Rd crystal structure. The results described herein support the prediction that the identity of residue 44 alone determines whether a Rd reduction potential of about -50 or 0 mV is observed.

NADH peroxidase activity of rubrerythrin.

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.

Oxygen-carrying proteins: three solutions to a common problem.

Nature has used transition-metal ions with unpaired d-electrons to overcome the kinetic inertness of O2 and to control its thermodynamic tendency towards reduction. High-resolution X-ray crystal structures of O2-carrying proteins show that Nature has devised three distinct solutions to the problem of reversible O2 binding. The three types can be classified according to their active sites: Hb (haem iron); Hr (non-haem di-iron); and Hcy (dicopper). The reversible O2 binding to the three types of active site are formally oxidative additions: Fe(II) to Fe(III)-O2- for Hb; [Fe(II),Fe(II)] to [Fe(III),Fe(III)O(2)2-] for Hr; and [Cu(I),Cu(I)] to [Cu(II)(mu-O(2)2-) Cu(II)] for Hcy. In all cases the O-O bond is weakened, but not cleaved, upon binding. The 'textbook' explanation for discrimination against CO and O2 binding to Hb has been revised: steric constraints to the preferred linear Fe-C-O geometry imposed by the 'distal' histidine are no longer thought to play a major role. Instead, recent experimental evidence indicates that the polarity of the binding pocket favours the polar Fe-O-O unit over the relatively non-polar Fe-C-O unit, and that a C-O-binding pocket near the haem also inhibits the preferred linear Fe-C-O geometry. Reversible O2 binding to the di-iron site of Hr involves an internal proton transfer as well as electron transfer to O2, but the elementary steps governing the rates of O2 binding and release, especially the effects of the surrounding protein, remain to be delineated. An unusual side-on-bonded O2 that bridges the two copper ions explains both the unusually low O-O stretching frequency and the diamagnetism of oxyHcy. O2-activating-enzyme counterparts exist for each of the three known types of O2-carrying protein. Detailed comparisons of these protein/enzyme pairs are likely to clarify the factors that tune the delicate balance between reversible O2 binding and controlled O-O bond cleavage.

Targeted disruption of mouse long-chain acyl-CoA dehydrogenase gene reveals crucial roles for fatty acid oxidation.

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/- mice yielded an abnormally low number of LCAD +/- and -/- offspring, indicating frequent gestational loss. LCAD -/- mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD -/- males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD -/- mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

Abnormal nonshivering thermogenesis in mice with inherited defects of fatty acid oxidation.

When placed in the cold (4 degreesC), BALB/cByJ mice of both genders rapidly lose body temperature as compared with the control strain, C57BL/6J. This sensitivity to cold resembles that previously described for mice with a defect in nonshivering thermogenesis due to the targeted inactivation of the brown adipocyte-specific mitochondrial uncoupling protein gene, Ucp1. Genetic mapping of the trait placed the gene on chromosome 5 near Acads, a gene encoding the short chain acyl CoA dehydrogenase, which is mutated in BALB/cByJ mice. The analysis of candidate genes in the region indicated a defect only in the expression of Acads. Confirmation of the importance of fatty acid oxidation to thermogenesis came from our finding that mice carrying the targeted inactivation of the long chain acyl CoA dehydrogenase gene (Acadl) are also sensitive to the cold. Both of these mutations attenuate the induction of genes normally responsive to adrenergic signaling in brown adipocytes. These results suggest that the action of fatty acids as regulators of gene expression has been perturbed in the mutant mice. From a clinical perspective, it is important to determine whether defects in thermogenesis may be a phenotype in human neonates with inherited deficiencies in fatty acid beta-oxidation.

Structural characterization of the mouse long-chain acyl-CoA dehydrogenase gene and 5' regulatory region.

Long-chain acyl-CoA dehydrogenase (LCAD) is one of four enzymes involved in the initial step of mitochondrial beta-oxidation of straight-chain fatty acids. It is a member of the acyl-CoA dehydrogenase (Acad or ACAD) gene family of enzymes, which also includes very-long-chain (VLCAD), medium-chain (MCAD), and short-chain (SCAD) acyl-CoA dehydrogenases. These enzymes all have similar activity but differ only in the chain length specificity for their substrate. Mitochondrial beta-oxidation provides an important source of energy especially during times of fasting. In order to understand the role of LCAD in this pathway, we have cloned and characterized the entire mouse (Mus musculus) gene encoding LCAD (Acadl). Acadl is a single-copy, nuclear encoded gene approximately 35 kb in size. We have sequenced the entire coding region, all intron/exon boundaries, 1.7 kb of its 5' regulatory region, and mapped the transcription start site. The gene contains 11 coding exons ranging in size from 67 bp to 275 bp, interrupted by 10 introns ranging in size from 1.0 kb to 6.6 kb in size. The Acadl 5' regulatory region, like other members of the Acad family, lacks a TATA or CAAT box and is GC rich. This region does contain multiple, putative cis-acting DNA elements recognized by either SP1 or members of the steroid-thyroid family of nuclear receptors, which has been shown with other members of the ACAD gene family to be important in regulated expression. The characterization of the mouse Acadl gene will allow further study of LCAD in an in vivo model, and how its expression may be coordinated with other members of the Acad gene family.

Electrospray mass spectrometry studies of non-heme iron-containing proteins.

The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer. The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli. ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms. Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms. Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr). ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein. Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer. The masses of the monomer ions produced in this manner are assigned to the aposubunit, [subunit + Fe - 3H]+, and [subunit + 2Fe - 6 H]+. Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit. Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer. Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3). In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da. Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.

EXAFS comparison of the dimanganese core structures of manganese catalase, arginase, and manganese-substituted ribonucleotide reductase and hemerythrin.

The solution structures of the binuclear Mn centers in arginase, Mn catalase, and the Mn-substituted forms of the Fe enzymes ribonucleotide reductase and hemerythrin have been determined using X-ray absorption spectroscopy (XAS). X-ray absorption near edge structure (XANES) spectra for these proteins were compared to those obtained for Mn(II) models. The Mn model spectra show an inverse correlation between the XANES peak maximum and the root-mean-square (RMS) deviation in metal-ligand bond lengths. For these complexes, the XANES maxima appear to be more effective than the 1s --> 3d areas as an indicator of metal-site symmetry. Arginase and Mn-substituted ribonucleotide reductase have symmetric nearest neighbor environments with low RMS deviation in bond length, while Mn catalase and Mn-substituted hemerythrin appear to have a larger RMS bond length deviation. The 1s --> 3d areas for arginase and Mn-substituted ribonucleotide reductase are consistent with six coordinate Mn, while the 1s --> 3d areas for Mn catalase and Mn-substituted hemerythrin are larger, suggesting that one or both of the Mn ions are five-coordinate in these proteins. Extended x-ray absorption fine structure (EXAFS) spectra were used to determine the Mn2 core structure for the four proteins. In order to quantitate the number of histidine residues bound to the Mn2 centers, EXAFS data for the crystallographically characterized model hexakis-imidazole Mn(II) dichloride tetrahydrate were used to calibrate the Mn-imidazole multiple scattering interactions. These calibrated parameters allowed the outer shell EXAFS to be fit to give a lower limit on the number of bound histidine residues. The EXAFS spectra for Mn-substituted ribonucleotide reductase and arginase are nearly identical, with symmetric Mn-nearest neighbor environments and outer shell scattering consistent with a lower limit of one histidine per Mn2 core. In contrast, the EXAFS data for Mn catalase and Mn-substituted hemerythrin show two distinct Mn-nearest neighbor shells, modeled as Mn-O at ca. 2.1 A and Mn-N at ca. 2.3 A, and outer shell carbon scattering consistent with a lower limit of ca. 2-3 His residues per Mn2 core. Only Mn catalase shows clear evidence for Mn...Mn scattering. The observed Mn...Mn distance is 3.53 A, which is significantly longer than the approximately 3.3 A distances that are typically observed for Mn(II)2 cores with two single atom bridges, but which is typical of the distances seen in Mn(II)2 cores having one single atom bridge (e.g., aqua or hydroxo) together with one or two carboxylate bridges. The absence of EXAFS-detectable Mn...Mn interactions for the other three proteins suggests either that there are no single atom bridges in these cases or that the Mn...Mn interactions are more disordered.