A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA.

The xeroderma pigmentosum group A protein (XPA) plays a central role in nucleotide excision repair (NER). To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system. Here we report a novel cytoplasmic GTP-binding protein, designated XPA binding protein 1 (XAB1). The deduced amino acid sequence of XAB1 consisted of 374 residues with a molecular weight of 41 kDa and an isoelectric point of 4.65. Sequence analysis revealed that XAB1 has four sequence motifs G1-G4 of the GTP-binding protein family in the N-terminal half. XAB1 also contains an acidic region in the C-terminal portion. Northern blot analysis showed that XAB1 mRNA is expressed ubiquitously, and immunofluorescence analysis revealed that XAB1 is localized mainly in the cytoplasm. Consistent with the GTP-binding motif, purified recombinant XAB1 protein has intrinsic GTPase activity. Using the yeast two-hybrid system, we elucidated that XAB1 binds to the N-terminal region of XPA. The deletion of five amino acids, residues 30-34 of XPA, required for nuclear localization of XPA abolished the interaction with XAB1. These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA.

XAB2, a novel tetratricopeptide repeat protein involved in transcription-coupled DNA repair and transcription.

Nucleotide excision repair is a highly versatile DNA repair system responsible for elimination of a wide variety of lesions from the genome. It is comprised of two subpathways: transcription-coupled repair that accomplishes efficient removal of damage blocking transcription and global genome repair. Recently, the basic mechanism of global genome repair has emerged from biochemical studies. However, little is known about transcription-coupled repair in eukaryotes. Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways. The XAB2 protein of 855 amino acids consists mainly of 15 tetratricopeptide repeats. In addition to interacting with XPA, immunoprecipitation experiments demonstrated that a fraction of XAB2 is able to interact with the transcription-coupled repair-specific proteins CSA and CSB as well as RNA polymerase II. Furthermore, antibodies against XAB2 inhibited both transcription-coupled repair and transcription in vivo but not global genome repair when microinjected into living fibroblasts. These results indicate that XAB2 is a novel component involved in transcription-coupled repair and transcription.

Serum carnitine concentrations in patients with idiopathic hypertrophic cardiomyopathy: relationship with impaired myocardial fatty acid metabolism.

We evaluated the clinical significance of serum carnitine concentrations in determining the severity of impaired myocardial fatty acid metabolism in idiopathic hypertrophic cardiomyopathy (HCM). We studied 56 asymptomatic or mildly symptomatic patients with HCM. Serum levels of free carnitine and acylcarnitine were measured by the enzymic cycling method. Myocardial scintigraphy with (123)I-labelled 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) was performed, and the images were analysed quantitatively and semi-quantitatively. Serum free carnitine levels were significantly higher in HCM patients than in normal subjects (52. 5+/-9.5 and 42.3+/-5.5 nmol/ml respectively; P<0.0001). On the other hand, serum acylcarnitine levels and acyl/free carnitine ratios were lower in HCM patients than in normal subjects (10.2+/-4.0 nmol/ml and 0.19+/-0.08, compared with 13.2+/-3.9 nmol/ml and 0.32+/-0.11 respectively; P<0.0001). Clinical characteristics were not significantly different between the patients showing high and normal free carnitine levels, although female patients with high free carnitine levels were few (P=0.02). Both quantitative and semi-quantitative analyses revealed that the severity of decreased myocardial BMIPP uptake was significantly correlated with serum free carnitine levels (quantitative analysis: r=-0.422, P<0.0012; semi-quantitative analysis: r=0.633, P<0.0001). In the presence of reduced carnitine uptake into the myocardium in HCM, there may also be reduced transport of acylcarnitines out of the myocardium into the plasma. Although inborn errors of fatty acid metabolism and carnitine deficiencies are reported to provoke secondary HCM and are associated with low serum carnitine concentrations, this study has revealed that the levels of carnitine are, in contrast, increased in idiopathic HCM. Moreover, serum carnitine concentrations are a sensitive indicator of the severity of impaired myocardial fatty acid metabolism even in asymptomatic patients with HCM.

Effect of sports activity on carnitine metabolism. Measurement of free carnitine, gamma-butyrobetaine and acylcarnitines by tandem mass spectrometry.

The effects of sports activity on carnitine metabolism were studied using mass spectrometry. Serum levels of free carnitine, acylcarnitines (acetylcarnitine, propionylcarnitine, C4-, C5- and C8-acylcarnitine) and gamma-butyrobetaine, a carnitine precursor, were determined by tandem mass spectrometry in liquid secondary ion mass ionization mode. The coefficients of variation at three different concentrations were 2.8-7.9% for gamma-butyrobetaine, and 1.2 to approximately 6.7% for free carnitine. The recoveries added to serum were 109.1% for gamma-butyrobetaine, 89.3% for free carnitine. Sports activity caused increased serum levels of gamma-butyrobetaine, acetylcarnitine, C4- and C8-acylcarnitines and decreased serum levels of free carnitine. This method requires a small amount of sample volume (20 microl of serum) and short total instrumental time for the analysis (1 h for preparation, 2 min per sample for mass spectrometric analysis). Therefore, this method can be applied to study carnitine metabolism under various conditions that affect fatty acid oxidation.

Resonance assignments, solution structure, and backbone dynamics of the DNA- and RPA-binding domain of human repair factor XPA.

XPA is involved in the damage recognition step of nucleotide excision repair (NER). XPA binds to other repair factors, and acts as a key element in NER complex formation. The central domain of human repair factor XPA (residues Met98 to Phe219) is responsible for the preferential binding to damaged DNA and to replication protein A (RPA). The domain consists of a zinc-containing subdomain with a compact globular structure and a C-terminal subdomain with a positively charged cleft in a novel alpha/beta structure. The resonance assignments and backbone dynamics of the central domain of human XPA were studied by multidimensional heteronuclear NMR methods. 15N relaxation data were obtained at two static magnetic fields, and analyzed by means of the model-free formalism under the assumption of isotropic or anisotropic rotational diffusion. In addition, exchange contributions were estimated by analysis of the spectral density function at zero frequency. The results show that the domain exhibits a rotational diffusion anisotropy (Dparallel/Dperpendicular) of 1.38, and that most of the flexible regions exist on the DNA binding surface in the cleft in the C-terminal subdomain. This flexibility may be involved in the interactions of XPA with various kinds of damaged DNA.

Solution structure of the DNA- and RPA-binding domain of the human repair factor XPA.

The solution structure of the central domain of the human nucleotide excision repair protein XPA, which binds to damaged DNA and replication protein A (RPA), was determined by nuclear magnetic resonance (NMR) spectroscopy. The central domain consists of a zinc-containing subdomain and a C-terminal subdomain. The zinc-containing subdomain has a compact globular structure and is distinct from the zinc-fingers found in transcription factors. The C-terminal subdomain folds into a novel alpha/beta structure with a positively charged superficial cleft. From the NMR spectra of the complexes, DNA and RPA binding surfaces are suggested.

The effect of carnitine on ketogenesis in perfused livers from juvenile visceral steatosis mice with systemic carnitine deficiency.

Juvenile visceral steatosis (JVS) mice have been reported to have systemic carnitine deficiency, and the carnitine concentration in the liver of JVS mice was markedly lower than that of controls (11.6 +/- 2.6 versus 393.5 +/- 56.4 nmol/g of wet liver). To evaluate the role of carnitine in mitochondrial beta-oxidation in liver, we examined the effects of carnitine on ketogenesis in perfused liver from control and JVS mice. In control mice, ketogenesis was increased by the infusion of 0.3 mM oleate, but not by L-carnitine. In contrast, although ketogenesis in JVS mice was not increased by the infusion of oleate, it was increased 2.5-fold by the addition of 1000 microM L-carnitine. Addition of 50, 100, and 200 microM L-carnitine increased ketogenesis in a dose-dependent manner. The infusion of 0.3 mM octanoate or butyrate increased ketogenesis in a carnitine-independent fashion in both control and JVS mice. These findings suggest that endogenous long chain fatty acids from accumulated triglycerides may be used as substrates in the presence of carnitine in JVS mice. The relationship between ketogenesis and free carnitine concentration was examined in livers from JVS mice. Ketogenesis increased as free carnitine levels increased until concentrations exceeded about 100 nmol/g of wet liver (340 microM). The free carnitine concentration required for half-maximal ketone body production in liver of JVS mice was 45 microM (13 nmol/g of wet liver), which corresponds to a K(m) value of carnitine palmitoyltransferase I. We conclude that carnitine is a rate-limiting factor for beta-oxidation in liver only when the carnitine level in liver is very low.

Pivalate affects carnitine status but causes no severe metabolic changes in rat liver.

To investigate the carnitine deficiency induced by pivalate, rats had free access to drinking water with or without pivalate. Consumption of 20 mmol/L pivalate for 1 wk decreased the levels of both free and total carnitine in plasma to approximately 20% of levels before treatment. After 4 wk, the concentrations of free carnitine in the liver, heart and muscle of pivalate-treated rats were approximately 60-80% of the control, and in the kidney, 26% of the control. Fractional excretion of free carnitine (FEFC) in pivalate-treated rats was measured; however, the treatment for 3 or 8 d did not affect the values relative to those obtained before treatment. Treatment with pivalate for 4 wk did not affect plasma concentrations of glucose, ammonia and free fatty acids (FFA) in the rats; however, the concentration of 3-hydroxybutyrate (3-OHB) was higher, and the FFA/3-OHB ratio was lower than those of controls. In a liver perfusion study, ketogenesis from oleate and gluconeogenesis from lactate and pyruvate in rats treated with pivalate for 4 wk were not different from controls. These results suggest that administration of pivalate did not induce the excessive excretion of free carnitine in urine, and secondary carnitine deficiency induced by intake of 20 mmol/L pivalate for 4 wk did not cause severe metabolic changes in rat liver.

Enhancement of damage-specific DNA binding of XPA by interaction with the ERCC1 DNA repair protein.

The human XPA and ERCC1 proteins, which are involved in early steps of nucleotide excision repair of DNA, specifically interacted in an in vitro binding assay and a yeast two-hybrid assay. A stretch of consecutive glutamic acid residues in XPA was needed for binding to ERCC1. Binding of XPA to damaged DNA was markedly increased by the interaction of the XPA and ERCC1 proteins. ERCC1 did not enhance binding to DNA when a truncated XPA protein, MF122, was used in place of the XPA protein. MF122 retains damaged DNA binding activity but lacks the region for protein-protein interaction including the E-cluster region. These results suggest that the XPA/ERCC1 interaction may participate in damage-recognition as well as in incision at the 5' site of damage during nucleotide excision repair.

Biomedical applications of high-performance liquid chromatography-mass spectrometry with continuous-flow fast atom bombardment.

This report describes the application of high-performance liquid chromatography combined with continuous-flow fast atom bombardment mass spectrometry to analytical problems in the biomedical laboratory. Applications include the compound-specific detection of diagnostic acylcarnitines in human urine, the separation and analysis of acyl-coenzyme A thioesters, and qualitative studies on complex mixtures of modified peptides (dansyl and dinitrophenyl derivatives). For each of these applications standard analytical columns (3.9 mm I.D.) and 1 ml/min flow-rates were employed with post-column stream splitting (1:100) before mass spectrometry. Various mobile phase compositions and solvent gradients were employed. The addition of 1-5% glycerol to the mobile phase was shown to have little effect on the chromatography. For all compounds studied (acylcarnitines, acyl-coenzyme A thioesters, and derivatized peptides) molecular weight information was obtained and sufficient sensitivity was achieved to allow unambiguous identification of trace components in complex mixtures.

The effects of salicylate on ketogenesis, gluconeogenesis and urea production in rat liver perfusion.

In order to elucidate the relation between the hepatotoxicity of salicylate (SA) and the pathogenesis of Reye's syndrome (RS), urea production, gluconeogenesis and ketogenesis were investigated in isolated perfused rat livers in the presence of salicylate (SA) and oleate. Although urea formation from 0.5 mM NH4Cl, 2 mM ornithine and 0.3 mM oleate was not inhibited by infusion of SA, 3 mM SA caused a 26% decrease of ketogenesis, 85% decrease of 3-hydroxybutyrate/acetoacetate ratio (30HB/AcAc) and 45% increase of oxygen consumption. Glucose production from 2 mM pyruvate in the presence of 0.3 mM oleate decreased by 33% after administration of 3 mM SA, and 30HB/AcAc ratio also decreased by 33%. The decrement of gluconeogenesis and that of the 30HB/AcAc ratio were very close. These results suggested that ATP production was maintained but that the intra-mitochondrial redox state was changed to a more oxidized state after SA administration in perfused rat livers. This change in redox state could be responsible for the decrease of gluconeogenesis. Metabolic characteristics found in RS were not obtained by infusion of 3 mM SA and 0.3 mM oleate in rat livers. Therefore, some other factors in addition to SA seem necessary to establish an animal model of RS.

2,4-Dienoyl-coenzyme A reductase deficiency: a possible new disorder of fatty acid oxidation.

Several inherited disorders of fatty acid beta-oxidation have been described that relate mainly to saturated precursors. This study is the first report of an enzyme defect related only to unsaturated fatty acid oxidation and provides the first in vivo evidence that fat oxidation in humans proceeds by the reductase-dependent pathway. The patient was a black female, presenting in the neonatal period with persistent hypotonia. Biochemical studies revealed hyperlysinemia, hypocarnitinemia, normal organic acid profile, and an unusual acylcarnitine species in both urine and blood. The new metabolite was positively identified by mass spectrometry as 2-trans,4-cis-decadienoylcarnitine, derived from incomplete oxidation of linoleic acid. In spite of dietary therapy, the patient died of respiratory acidosis at four months of age. Samples of liver and muscle from the autopsy were assayed for 2,4-dienoyl-coenzyme A reductase activity. Using the substrate 2-trans,4-cis-decadienoylcoenzyme A, the reductase activity was 40% of the control value in liver and only 17% of that found in normal muscle. It is suggested that unsaturated substrates should be used for in vitro testing to cover the full range of potential beta-oxidation defects and that acylcarnitine species identification be used for in vivo detection of this disorder.

Quantitative assay of free and total carnitine using tandem mass spectrometry.

A new, specific method for isotope dilution assay of total and free carnitine in urine has been developed. The method utilizes fast atom bombardment ionization with tandem mass spectrometry and requires minimal sample preparation. It compared well with radioenzymatic assay in terms of specificity, precision and accuracy, but was much more convenient in terms of analysis time and sample throughput. The new method is also applicable to the determination of free and short-chain total carnitine in plasma.

Carnitine homeostasis in the organic acidurias.

The detection of acylcarnitines by FAB-MS in human physiological fluids has added another valuable diagnostic tool for the recognition of specific metabolic diseases. The newly developed technique of FAB-MS/MS, which embodies the principles of tandem mass spectrometry, affords a quantum leap in specificity, enabling the detection and quantification of acylcarnitines in concentrations well within the physiological ranges in urine, blood and tissue. The therapeutic use of L-carnitine appears to offer protection against the harmful consequences of catabolism. A reduction in the number of clinical episodes requiring hospitalization has been observed in most cases. When such occurrences have necessitated intravenous fluids, the addition of L-carnitine to the IV infusate has led to dramatic reduction in recovery times. It has also been shown that there is no discernable toxicity associated with carnitine therapy, even under high-dose IV conditions.