PubMed Health⌕ Search

Biomedical subjects

C H Chung

Publications and source records attributed to C H Chung.

At least 91 records · Page 5Linked to original sources
PubMed1998

Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli.

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

Adenosine Triphosphatases↗
PubMed1998

cDNA cloning and characterization of a human proteasomal modulator subunit, p27 (PSMD9).

We have employed cDNA cloning to deduce the complete primary structure of a new subunit, designated p27, of the modulator trimer complex that stimulates the association of the PA700 regulator with the catalytic 20S proteasome to form the ATP-dependent active 26S proteasome. We found two distinct cDNAs encoding two highly homologous proteins except in the C-terminal region, which are termed tentatively p27-1 and p27-2. The short p27-2 cDNA has a deletion of 65 bp near the 3'-end region of the long p27-1 cDNA, which encodes a large protein with an extended C-terminal region, designated p27-L, whereas the long p27-1 cDNA encodes a small protein named p27-S. The polypeptides of p27-L and p27-S consist of 223 and 209 amino acid residues with calculated molecular masses of 24,852 and 22,764 and isoelectric points of 6.50 and 5.28, respectively. Immunoblot analysis with anti-p27 antibody revealed that p27, together with two other ATPase components, TBP1 and p42, was associated with not only the modulator complex but also significantly with the 26S proteasome complex, suggesting that the three are common/sharing subunits in these two complexes. By the fluorescence in situ hybridization method, the p27 (PSMD9) gene was mapped to the q24.2-q24.3 band of human chromosome 12. Computer-assisted homology analysis revealed the high sequence similarities of p27-L with a possible counterpart in Caenorhabditis elegans and Saccharomyces cerevisiae whose function is yet unknown, the yeast gene that is here termed NAS2 (non-ATPase subunit 2). Disruption of NAS2 had no effect on cell viability, indicating that the subunit is not essential for proliferation of yeast cells.

Adenosine Triphosphatases↗
PubMed1998

Chromosomal localization and immunological analysis of a family of human 26S proteasomal ATPases.

The 26S proteasome is a eukaryotic ATP-dependent protease functioning as a protein death machine. It is a large multisubunit complex, consisting of a catalytic 20S proteasome and two regulatory modules, named PA700. The PA700 complex is composed of multiple subunits of 25-110 kDa, which are classified into two subgroups, a subgroup of at least 6 ATPases that consitute a unique multi-gene family encoding homologous polypeptides conserved during evolution and a subgroup of approximately 15 non-ATPase subunits, most of which are structurally unrelated to each other. In the present study, we report the chromosomal localization and immunological properties of six members of the human 26S proteasomal ATPase family. By use of the fluorescence in situ hybridization method, the S4 (PSMC1), MSS1 (PSMC2), TBP1 (PSMC3), TBP7 (PSMC4), p45 (PSMC5), and p42 (PSMC6) genes were mapped to human chromosomes 19p13.3, 7q22.1-q22.3, 11p11.2, 19q13.11-q13.13, 17q23.1-q23.3, and 12q15, respectively, indicating that the genes for multiple ATPases of the 26S proteasome are located on different chromosomes. Immunoblot analysis revealed that all these ATPases were associated with the purified 26S proteasome and that some of them showed striking heterogeneity in their electrical charges.

Adenosine Triphosphatases↗
PubMed1998

K-ras mutations in the duodenal fluid of patients with pancreatic carcinoma.

BACKGROUND: Many patients with carcinoma of the pancreas die because their disease is not detected until late in its course. Methods that detect these cancers earlier will improve patient outcome. Over 80% of pancreatic carcinomas contain mutations in codon 12 of the K-ras gene. Screening duodenal fluid for these mutations may lead to early detection of these cancers and assist in establishing a diagnosis of pancreatic carcinoma. METHODS: Polymerase chain reaction (PCR), with and without restriction enzyme-mediated mutant enrichment, was performed on DNA isolated from duodenal fluid specimens from 61 patients who underwent pancreaticoduodenectomy (Whipple's operation) for either periampullary cancer or a benign condition of the pancreas. Representative sections of pancreas pathology (primary carcinoma, benign tumor, or chronic pancreatitis) from the patients with duodenal fluid specimens containing amplifiable DNA were also analyzed for K-ras mutations. Wild-type and mutant K-ras were detected by hybridization of the PCR products with K-ras codon 12 mutant and wild-type specific probes. RESULTS: Seven of the 61 duodenal fluid specimens contained DNA that did not amplify. Thirteen (24% of the 54 duodenal fluid specimens with amplifiable DNA and 21% of the total of 61 specimens) contained activating point mutations at codon 12 of the K-ras gene. Mutations were detected in 13 of the 51 duodenal fluid specimens from patients with cancer (sensitivity, 25%), whereas mutations were not detected in any of the 9 amplifiable duodenal fluid specimens from patients with benign conditions of the pancreas (specificity, 100%). One duodenal fluid specimen from a patient with adenocarcinoma of the pancreas had two different K-ras mutations. DNA from three of the primary carcinomas did not amplify or was not available. Twenty-nine (69%) of the 42 primary tumors with amplifiable DNA contained K-ras mutations, whereas 3 (30%) of the 10 pancreata with benign conditions harbored mutations. Twenty-two (65%) of 34 ductal adenocarcinomas of the pancreas with amplifiable DNA had K-ras mutations. It is noteworthy that the same mutation was present in both the duodenal fluid and the primary carcinomas of 11 (92%) of the 12 patients who had primary tumors with amplifiable DNA as well as K-ras mutations in their duodenal fluid specimens. CONCLUSIONS: The identification of genetic alterations in cancer-causing genes in duodenal fluid may form the basis for the development of new approaches to the detection of carcinoma of the pancreas. Some pancreata without cancer, however, may also harbor K-ras mutations, potentially limiting the specificity of K-ras-based tests.

Adenocarcinoma↗
PubMed1998

Induction of apoptosis in human leukemic U937 cells by tetrandrine.

Tetrandrine, a calcium channel antagonist, is a plant alkaloid possessing various pharmacological activities including anti-tumor activity. We studied tetrandrine to determine whether or not this anti-tumor effect occurs through induction of apoptosis. Tetrandrine inhibited both proliferation and clonogenicity of human leukemic U937 cells at an optimal concentration of 2.5 microg/ml. This growth inhibition was dose and time dependent, and accompanied with evidence of apoptotic changes. The characteristic morphological changes of apoptosis were observed in U937 cells under light microscopy and DNA fragmentation was noted by gel electrophoresis. Moreover, flow cytometric detection of surface phosphatidyl serine expression of U937 cells after treatment with tetrandrine confirmed the induction of apoptosis in these cells. The induction of apoptosis by tetrandrine would appear to proceed via non-Ca2+-dependent pathways.

Alkaloids↗
PubMed1998

Calculus-like deposit at apex of tooth with refractory apical periodontitis.

It is generally accepted that bacteria in or outside the root canal are the reason for apical periodontitis and endodontic failures. This case report presents a 60-year-old woman with a periapical lesion and a fistulous tract which did not respond to conventional root canal treatment. During periapical surgery, granulomatous tissue was removed and a calculus-like deposit was observed on the root surface. A radicular cyst was diagnosed. Nine months after this calculus-like deposit had been removed and the cyst enucleated, complete recovery of the bone had occurred. It is suggested that the presence of the calculus-like deposit at the apex of the tooth or its effects may in part have delayed the healing of the periapical inflammation in spite of apparently adequate endodontic treatment.

Chronic Disease↗
PubMed1997

Purification and characterization of UBP6, a new ubiquitin-specific protease in Saccharomyces cerevisiae.

Ubiquitin-specific protease-6 (UBP6) in Saccharomyces cerevisiae was expressed in Escherichia coli and purified from the cells using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a model substrate. The purified UBP6 behaved as a 58-kDa under both nondenaturing and denaturing conditions, indicating that the enzyme comprises a single polypeptide. It was maximally active at pH levels between 8.5 and 9, but showed little or no activity at pH below 7 and above 9.5. As with other UBPs, its activity was strongly inhibited by sulfhydryl-blocking reagents, such as N-ethylmaleimide, and by ubiquitin-aldehyde. In addition to the model substrate, UBP6 hydrolyzed ubiquitin-alphaNH-protein extensions, such as the ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and ubiquitin-alphaNH-dihydrofolate reductase, but not poly-His-tagged diubiquitin. It was also capable of releasing free ubiquitin from branched polyubiquitin chains that are ligated to proteins through epsilonNH-isopeptide bonds, although to a limited extent. These results suggest that UBP6 may play an important role in the generation of free ubiquitins and certain ribosomal proteins from ubiquitin-ribosomal fusion proteins as well as in deubiquitination of certain polyubiquitinated proteins targeted for degradation by the 26S proteasomes.

Amino Acid Sequence↗
PubMed1997

Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle.

A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method. Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs revealed that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and is related to members of the UBP family containing highly conserved Cys and His domains. Chick UBP41 was expressed in E. coli and purified from the cells to apparent homogeneity, using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide. The UBP41 protease cleaved at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes; thus, it is active against ubiquitin-beta-galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids. UBP41 also released free ubiquitin from poly-His-tagged di-ubiquitin. Moreover, it converted poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules were not further degraded to free ubiquitin and lysozyme. These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.

Amino Acid Sequence↗
PubMed1997

ATP binding, but not its hydrolysis, is required for assembly and proteolytic activity of the HslVU protease in Escherichia coli.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. To gain an insight into the role of ATP hydrolysis in protein breakdown, we determined the insulin B-chain-degrading activity and assembly of HslVU in the presence of ATP and its nonhydrolyzable analogs. While beta,gamma-methylene-ATP could not support the proteolytic activity, beta,gamma-imido-ATP supported it to an extent less than 10% of that seen with ATP. Surprisingly, however, HslVU degraded insulin B-chain even more rapidly in the presence of ATPgammaS than with ATP. Furthermore, the ability of ATP and its analogs in supporting the proteolytic activity was closely correlated with their ability in supporting the oligomerization of HslU and the formation of the HslVU complex. However, ADP, which is capable of supporting the HslU oligomerization, could not support the HslVU complex formation or the proteolytic activity, suggesting that the conformation of the ADP-bound HslU oligomer is different from that of ATP-bound form. Thus, it appears that ATP-binding, but not its hydrolysis, is essential for assembly and proteolytic activity of HslVU.

ATP-Dependent Proteases↗
PubMed1997

Opposite effect of intracellular Ca2+ and protein kinase C on the expression of inwardly rectifying K+ channel 1 in mouse skeletal muscle.

The level of inwardly rectifying K+ channel 1 (IRK1) mRNA decreased upon denervation and increased during muscle differentiation in mouse skeletal muscle. To identify the mechanism(s) underlying the regulation of IRK1 mRNA expression, we examined its expression using the well differentiated C2C12 mouse skeletal muscle cell line as a model system. Since nerve-induced muscle activity results in contraction, it was questioned whether the changes in IRK1 expression might be relevant to the increased intracellular calcium that functions as a cytoplasmic messenger in excitation-contraction coupling. Indeed, activation of either L-type calcium channels or ryanodine receptors increased the level of IRK1 mRNA. More directly, ionomycin activated the IRK1 expression in time- and dose-dependent manners, which was abolished by treatment with EGTA. Genistein, a tyrosine kinase inhibitor, also abolished the stimulating effect of ionomycin. Meanwhile, activation of protein kinase C by 12-O-tetradecanoylphorbol acetate (TPA) markedly decreased the level of IRK1 mRNA, which required ongoing protein synthesis. Actinomycin D experiments revealed that ionomycin increased the half-life of IRK1 mRNA from 0.86 to 1.97 h, but TPA decreased it to 0.38 h. However, neither ionomycin nor TPA appreciably altered the rate of IRK1 gene transcription. Based on these observations, we conclude that intracellular calcium and protein kinase C are oppositely involved in the muscle activity-dependent regulation of IRK1 gene expression and that both act at the level of mRNA stability.

Animals↗
PubMed1997

The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-dependent proteinase.

HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome beta-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked. Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.

ATP-Dependent Proteases↗
PubMed1997

Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease.

HslVU in E. coli is a new type of ATP-dependent protease consisting of two heat shock proteins: the HslU ATPase and the HslV peptidase that has two repeated Thr residues at its N terminus, like certain beta-type subunit of the 20S proteasomes. To gain an insight into the catalytic mechanism of HslV, site-directed mutagenesis was performed to replace each of the Thr residues with Ser or Val and to delete the first or both Thr. Also each of the five internal Ser residues in HslV were replaced with Ala. The results obtained by the mutational analysis revealed that the N-terminal Thr acts as the active site nucleophile and that certain Ser residues, particularly Ser124 and Ser172, also contribute to the peptide hydrolysis by the HslVU protease. The mutational studies also revealed that both Thr, Ser103, and Ser172, but not Ser124, are involved in the interaction of HslV with HslU and hence in the activation of HslU ATPase as well as in the HslVU complex formation.

ATP-Dependent Proteases↗
PubMed1997

New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle.

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-alpha-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-alpha-NH-protein extensions, including ubiquitin-alpha-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-alpha-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through epsilon-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.

Amino Acid Sequence↗
PubMed1997

NF-kappaB-dependent expression of nitric oxide synthase is required for membrane fusion of chick embryonic myoblasts.

The activity of nitric oxide synthase (NOS) has recently been shown to increase transiently but dramatically in chick embryonic myoblasts that are competent for fusion and that NO acts as a messenger for the cell fusion. Here we show that the rise in NOS activity is tightly correlated with an increase in NOS protein level, and its synthesis is under transcriptional control. In addition, a partial cDNA sequence of NOS obtained by reverse transcription PCR on total RNA from chick myoblasts was found to be identical with that of the inducible type of NOS (iNOS) from chick macrophage. Thus chick myoblast NOS must belong to the family of iNOS. Consistently, pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor kappaB (NF-kappaB), prevented the expression of myoblast NOS. Furthermore the antioxidant also strongly inhibited cell fusion, and its inhibitory effect was reversed by treatment with sodium nitroprusside, an NO-generating agent. In addition, nuclear extracts obtained from myoblasts that were competent for fusion, but not those from proliferating cells or from fully differentiated myotubes, were capable of binding to the consensus NF-kappaB site in the promoter region of the gene encoding iNOS. These results suggest that NF-kappaB-dependent expression of NOS is an important step in membrane fusion of chick embryonic myoblasts.

Animals↗
PubMed1997

Molecular properties of the proteasome activator PA28 family proteins and gamma-interferon regulation.

BACKGROUND: Recent cDNA cloning of two homologous proteasome activators, PA28 alpha and PA28 beta, indicated the presence of a structurally related third protein, Ki antigen, but a functional relationship between Ki antigen and the two PA28 proteins is unknown. Accumulating evidence has implicated an important role for PA28 in the major histocompatibility complex (MHC) class I-restricted antigen processing pathway. Recently, an immunomodulatory cytokine gamma-interferon (gamma-IFN) was found to increase greatly the messages for PA28 alpha and PA28 beta, but not Ki antigen, in human cells. RESULTS: Ki antigen was co-immunoprecipitated with the 20S proteasome by anti-proteasome antibody, and associated reversibly with the 20S proteasome, as observed for PA28 alpha and PA28 beta. Therefore, Ki antigen was renamed PA28 gamma. Anti-PA28 gamma antibody, however, did not immunoprecipitate PA28 alpha and PA28 beta. gamma-IFN caused an almost complete loss of the PA28 gamma protein in cells without affecting its mRNA level, whereas the levels of both mRNA and protein for PA28 alpha and PA28 beta were coordinately upregulated by gamma-IFN. Finally we showed that the human chromosomal genes of PA28 alpha and PA28 gamma were located on 14q11.2 and 17q21.32-21.33, respectively. CONCLUSION: PA28 gamma (equivalent to Ki antigen) is a new member of the PA28 family proteins. It exists as a unique homopolymer under non-denaturing conditions. gamma-IFN was found to induce the expression of PA28 alpha and PA28 beta, whereas it caused almost complete loss of the PA28 gamma protein in cells. The reciprocal expression of the PA28 family proteins may imply their involvement in distinct biological processes.

Amino Acid Sequence↗
PubMed1997

Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.

Amino Acid Sequence↗