Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

It has been proposed that plasma low-density lipoprotein (LDL) undergoes oxidative modification before it can give rise to foam cells in atherosclerosis. Oxidation of LDL generates a variety of reactive aldehyde products including 4-hydroxy-2-nonenal (HNE), which may covalently attach to the LDL apolipoproteins. We here present direct evidence that HNE derivatization of LDL forms Michael addition-type adducts of HNE with histidine and lysine residues of apolipoprotein B-100 (apoB) and also demonstrate the utility of an antibody specific to the HNE adducts generated in the LDL treated with HNE or oxidatively modified by Cu2+ or cultured endothelial cells. HNE adducts present in the LDL that had been treated with HNE were attested to be Michael addition-type adducts on the basis of the fact that incubation of LDL with 1 mM HNE (2 h, 37 degrees C) resulted primarily in the formation of Michael addition-type HNE-histidine (39.9 mol/mol of LDL) and HNE-lysine (19.3 mol/mol of LDL) adducts. An enzyme-linked immunosorbent assay (ELISA) and an SDS-polyacrylamide gel electrophoresis (SDS-PAGE)/immunoblot analysis of HNE-modified LDL demonstrated that these HNE adducts were detectable with the HNE-specific antibody affinity-purified with the Michael adduct (HNE-histidine) as a ligand. The following lines of evidence indicated the presence of Michael addition-type HNE adducts in the oxidatively modified LDL in vitro: (i) Amino acid analysis of LDL that had been treated with Cu2+ (24 h, 37 degrees C) demonstrated the presence of a Michael addition-type HNE-histidine adduct (7-9 mol/mol of LDL).(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear.

The RecQ helicase superfamily has been implicated in DNA repair and recombination. At least five human RecQ-related genes exist: RecQ1, BLM, WRN, RecQ4 and RecQ5. Mutations in BLM, WRN and RecQ4 are associated with Bloom, Werner and Rothmund-Thomson syndromes, respectively, involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability. RecQ5 is small, containing only a core part of the RecQ helicase, but three isomer transcripts code for small RecQ5alpha (corresponding to the original RecQ5 with 410 amino acids), new large RecQ5beta (991 amino acids) and small RecQ5gamma (435 amino acids) proteins that contain the core helicase motifs. By determining the genomic structure, we found that the three isoforms are generated by differential splicing from the RecQ5 gene that contains at least 19 exons. Northern blot analysis using a RecQ5beta-specific probe indicates that RecQ5beta mRNA is expressed strongly in the testis. Immunocytochemical staining of three N-terminally tagged RecQ5 isomers expressed in 293EBNA cells showed that RecQ5beta migrates to the nucleus and exists exclusively in the nucleoplasm, while the small RecQ5alpha and RecQ5gamma proteins stay in the cytoplasm. Immunoprecipitation and an extended cytochemical experiment suggested that the nucleoplasmic RecQ5beta, like yeast Sgs1 DNA helicase, binds to topoisomerases 3alpha and 3beta, but not to topoisomerase 1. These results predict that RecQ5beta may have an important role in DNA metabolism and may also be related to a distinct genetic disease.

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