The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities.

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nß2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

Acyl-CoA thioesterase 9 (ACOT9) in mouse may provide a novel link between fatty acid and amino acid metabolism in mitochondria.

Acyl-CoA thioesterase (ACOT) activities are found in prokaryotes and in several compartments of eukaryotes where they hydrolyze a wide range of acyl-CoA substrates and thereby regulate intracellular acyl-CoA/CoA/fatty acid levels. ACOT9 is a mitochondrial ACOT with homologous genes found from bacteria to humans and in this study we have carried out an in-depth kinetic characterization of ACOT9 to determine its possible physiological function. ACOT9 showed unusual kinetic properties with activity peaks for short-, medium-, and saturated long-chain acyl-CoAs with highest V max with propionyl-CoA and (iso) butyryl-CoA while K cat/K m was highest with saturated long-chain acyl-CoAs. Further characterization of the short-chain acyl-CoA activity revealed that ACOT9 also hydrolyzes a number of short-chain acyl-CoAs and short-chain methyl-branched CoA esters that suggest a role for ACOT9 in regulation also of amino acid metabolism. In spite of markedly different K ms, ACOT9 can hydrolyze both short- and long-chain acyl-CoAs simultaneously, indicating that ACOT9 may provide a novel regulatory link between fatty acid and amino acid metabolism in mitochondria. Based on similar acyl-CoA chain-length specificities of recombinant ACOT9 and ACOT activity in mouse brown adipose tissue and kidney mitochondria, we conclude that ACOT9 is the major mitochondrial ACOT hydrolyzing saturated C2-C20-CoA in these tissues. Finally, ACOT9 activity is strongly regulated by NADH and CoA, suggesting that mitochondrial metabolic state regulates the function of ACOT9.

Repair of UV dimers in skin DNA of patients with basal cell carcinoma.

Epidemiologic studies suggest that exposure to sunlight is the primary etiologic agent for basal cell carcinoma. Formation of UV-induced DNA damage is believed to be a crucial event in the process leading to skin cancer. In this study, repair of photoproducts in DNA was followed in the skin of patients with basal cell carcinoma and control subjects. The subjects were exposed to 800 J/m(2) Commission Internationale de 1'Eclairag of solar-simulating radiation on buttock skin. Biopsies were taken at 0 hour, 24 hours, and 3 weeks after the exposure. Two cyclobutane pyrimidine dimers, TT=C and TT=T, were measured using a sensitive (32)P-postlabeling assay. Initial levels of both TT=C and TT=T differed between individuals in both groups. The levels of TT=T in patients with basal cell carcinoma and controls were similar (9.9 +/- 4.0 and 9.2 +/- 2.9 products per 10(6) normal nucleotides), whereas the level of TT=C was significantly lower in controls than in patients with basal cell carcinoma (6.2 +/- 3.1 versus 10.9 +/- 4.5 products per 10(6) normal nucleotides). The fractions of TT=T remaining after 24 hours and 3 weeks were significantly higher in patients with basal cell carcinoma (72% and 11%) compared with controls (48% and 5%). A slower removal in patients with basal cell carcinoma than in controls was indicated also for TT=C (52% versus 42% remaining at 24 hours); however, the difference between groups was not significant. When including data from our previously reported small-scale study, the fraction of dimers remaining at 24 hours was significantly higher in patients with basal cell carcinoma for both TT=C and TT=T. The data suggest that patients with basal cell carcinoma have a reduced capacity to repair UV-induced DNA lesions.

In vivo gene transfer using cetylated polyethylenimine.

UNLABELLED: This report describes gene transfer in vitro as well as in vivo using cetylated low-molecular mass (600 Da) polyethylenimine (28% of amine groups substituted with cetyl moieties), termed CT-PEI. This compound is hydrophobic and has to be incorporated into liposomes in order to be suitable for gene transfer studies. Serum-induced plasmid DNA degradation assay demonstrated that CT-PEI-containing liposomal carriers could protect complexed DNA (probably via condensation). In vitro luciferase gene expression achieved using medium supplemented with 10% serum was comparable to that achieved in serum-reduced medium and was highest for CT-PEI/cholesterol liposomes, followed by CT-PEI/dioleoylphosphatidylcholine liposomes and PEI 600 Da (uncetylated) carrier. In vivo systemic transfer into mice was most efficient when liposome formulations contained CT-PEI and cholesterol. Higher luciferase expression was then observed in lungs than in liver. IN CONCLUSION: liposomes containing cetylated polyethylenimine and cholesterol are a suitable vehicle for investigating systemic plasmid DNA transfer into lungs.

Effect of the spectral range of a UV lamp on the production of cyclobutane pyrimidine dimers in human skin in situ.

BACKGROUND: Ultraviolet (UV) irradiation has a broad spectrum of biological effects and a capacity to initiate skin carcinogenesis through DNA damage. The effect of different wave bands of UV light on the production of DNA damage in human skin in situ was studied with a broadband UV-B lamp TL-12 and a narrowband UV-B lamp TL-01. METHODS: Eight psoriasis patients participated in the study. Their minimal erythema dose was assessed separately for the two UV-B wave band ranges. Test areas of buttock skin were irradiated with the two spectrally differing lamps using erythemally equivalent UV doses of 40 and 80 mJ/cm2 CIE (Commission International de I'Eclairage). Punch biopsies were taken from the irradiated areas, and UV-induced DNA lesions (cyclobutane pyrimidine dimers, CPDs) in the skin were analyzed with a 32P high-performance liquid chromatography postlabelling method. RESULTS: No UV source-dependent differences in the induced levels of CPDs were detected in this study. CONCLUSION: CPD production with broadband TL-12 and narrowband TL-01 UV-B lamps in situ did not differ when erythemally equivalent UV doses were used. The preliminary result needs to be confirmed in a larger study.