Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

A Sensitive Serodiagnosis of Hepatitis C Virus (HCV) Infection with Two Non-Fused Peptides: Comparison of Antibody Responses Detected with a Newly Developed Assay and a Commercial Second-Generation Test

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of anti-HCV antibody. We assayed for antibodies against either oligopeptide (S29-1) deduced from the nucleocapsid gene or the product of nonstructural region (NS3) synthesized in a recombinant Escherichia coli (S4). To reduce false-positive results induced by non-specific binding of antibodies with a carrier protein and to increase the sensitivity of an immunoassay, non-fused S4 peptide was prepared by the recombinant DNA technique and site-specific proteolysis (by factor Xa). In 71 non-A, non-B hepatitis patients with chronic liver disease, 70 (98.5%) were positive by S29-1/S4 ELISA as well as by a second-generation test (Abbott II). On the other hand, of 40 serum samples from blood donors, in which anti-N14 (core) and C100-3 antibodies were not detected but hepatitis C virus (HCV) RNA was detectable by polymerase chain reaction (PCR), 24 (60%) were positive by S29-1/S4 ELISA, whereas only 18 (45%) were diagnosed by Abbott II. In addition, based on results in a small group of 92 blood donors, detection of anti-S29-1/S4 antibody correlated well with HCV viremia as confirmed by PCR. These results indicated that the preparation of non-fused protein (S4) by recombinant DNA technique and a combination of S29-1 and S4 as immobilized antigens in an ELISA provide a sensitive and specific diagnosis for HCV infection with good correlation with the presence of viral RNA as confirmed by PCR.     

Physiological Activities of Zeanic Acid,a New Plant-growth Promotor from Corn Steep Liquor

Increase in fruit set of grape was observed when vines were sprayed with zeanic acid before blossom, but when sprayed after blossom the effect was not clear.

Promotion of growth of radish cotyledons by zeanic acid was observed in the presence and absence of kinetin. Zeanic acid also promoted growth of dwarf mutant of rice seedlings (cv. Tanginbozu), but inhibited that of normal seedlings (cv. Tonewase). Zeanic acid showed promotive effect on proliferation of rice callus tissue in the absence of kinetin and auxin.

Zeanic acid showed low toxic activity against mice, earthworm and killifish, and no antimicrobial activity.     

Mutation of active site serine residue with cysteine displays change in acyl-acceptor preference of β-peptidyl aminopeptidase from Pseudomonas aeruginosa PAO1

A β-peptidyl aminopeptidase, a peptidase belonging to the P1 family, catalyzes aminolysis in accordance with its hydrolytic activity. We specifically examined β-peptidyl aminopeptidase of Pseudomonas aeruginosa PAO1 (BapF) to assess the effects of mutation of catalytic Ser with Cys or Thr on its catalytic ability. Recombinant BapF and its S237C mutant exhibited p-nitroaniline release activity toward β-homo-Gly-p-nitroanilide (βhGly-pNA), but the products of the enzyme reaction differed completely from one another. Wild-type BapF showed βhGly-βhGly-pNA synthetic activity, but the product vanished in a few minutes and converted to free βhGly. In contrast, the product βhGly-βhGly-pNA was synthesized by S237C BapF efficiently without degradation, indicating that because of the mutation, the enzyme came to recognize only the amine group as an acyl acceptor instead of water. Furthermore, a difference in acyl acceptor preference between that of wild type and S237C BapF was observed. When using cysteamine as an acyl acceptor, βhGly-cysteamine was synthesized only in the reaction using S237C BapF. In contrast, S237C BapF was unable to synthesize βhGly-cystamine when using cystamine as an acyl acceptor, although it was synthesized by wild-type BapF. Such a dynamic change in the acyl acceptor by the mutation of catalytic Ser with Cys is regarded as a unique feature of family P1 peptidases.     

Isolation of a New Quinoline, 2,8-Dihydroxycinchoninic Acid from Corn Steep Liquor

Androgen receptor (AR) signaling is the master regulator of prostate cell growth. Here, to better understand AR signaling, we searched for AR-interacting proteins by yeast two-hybrid screening and identified protein arginine methyltransferase 10 (PRMT10) as one of the interacting proteins. PRMT10 was highly expressed in reproductive tissues, such as prostate. Immunostaining showed that PRMT10 was expressed in the nucleus of both epithelia and stroma of rat prostate. In human prostate cancer LNCaP cells, PRMT10 co-immunoprecipitated with AR in both the presence and absence of dihydrotestosterone (DHT). Knockdown of PRMT10 by siRNA decreased DHT-dependent LNCaP cell growth and induction of prostate-specific antigen, an AR-target gene, without apparent loss of AR. DHT decreased PRMT10 at both the mRNA and protein levels. The decrease in PRMT10 was canceled by knockdown of AR or an AR antagonist. These results indicate that PRMT10 plays an important role in androgen-dependent proliferation of prostate cancer cells.     

Effects of dimethylsulfoxide on sphingomyelinase in cultured human fibroblasts and correction of sphingomyelinase deficiency in fibroblasts from Niemann-Pick patients

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity were studied using human skin fibroblasts from normal individuals and Niemann-Pick patients. Sphingomyelinase activity in normal fibroblasts increased up to 230% of controls by 2% DMSO while the cell growth was inhibited. Other lysosomal hydrolases showed a rather smaller extent of increment in activity. There was no direct effect of DMSO in cell-free system and no evidence of any activating factor of sphingomyelinase in homogenates. Sphingomyelinase deficiency in fibroblasts from a Niemann-Pick patient (type C) was corrected by 2% DMSO with regaining its enzyme activity.     

Reduction of acid sphingomyelinase activity in human fibroblasts induced by AY-9944 and other cationic amphiphilic drugs

AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride), a cationic amphiphilic drug, caused a rapid, irreversible and dose-dependent reduction of acid sphingomyelinase activity in normal human fibroblasts without changing the activities of other lysosomal hydrolases tested. Examinations of activities against synthetic substrates and of the pH-dependency of sphingomyelinase in the drug-treated cells also suggested that the reduction of activity was specific to acid sphingomyelinase. Such a specific reduction was also found with 12 other cationic amphiphilic drugs, most of which have been shown to be inducers of experimental phospholipidosis in animals and/or cultured cells. These results strongly suggest that acid sphingomyelinase is involved in the process of drug-induced lipidosis. The reduction of acid sphingomyelinase seemed not to be due to direct inhibition by these drugs, a specific loss of the enzyme into the culture medium, the presence of inhibitor in the drug-treated cells, or impaired synthesis of the enzyme. There was no indication that changes in the catalytic properties of the enzyme, or changes in the requirement of detergents for its activity occurred in the cell. These results suggest that AY-9944 and other cationic amphiphilic drugs may cause the reduction of acid sphingomyelinase activity by inducing an increased rate of degradation of the enzyme or by causing an irreversible inactivation via some undetected factor.     

Three-dimensional visualization of basal body structures and some cytoskeletal components in the apical zone of tracheal ciliated cells

The ciliated cells of tracheal epithelium were mechanically fragmented to remove the cytoplasmic soluble contents, and the apical zone was examined to clarify the three-dimensional structures of basal body and cytoskeletal filaments using freeze-fracture-etch approaches. The basal body was connected to the apical plasma membrane by definite laminae, formerly called alar sheets. The distal one-half of the basal foot was composed of several smooth-surfaced 12-nm fibrils. Intermediate filament networks extended to the lower half plane of the basal body, and enmeshed the basal body tightly by tiny 5- to 8-nm fibrils. Actin core bundles of microvilli also had tiny crosslinking fibrils. Some actin filaments were seen to run horizontally at the upper half plane of the basal body. Tracheal cilated cells also had circular actin filament bundles just inside the zonula adherens as many other epithelial cells. These cytoskeletal networks which enmeshed both basal bodies and core filaments of microvilli may function as a coordinator of ciliary beating.     

Derivation of Human Trophoblast Stem Cells

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