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K Ahn

Publications and source records attributed to K Ahn.

At least 37 records · Page 2Linked to original sources
PubMed2000

Inhibitor potencies and substrate preference for endothelin-converting enzyme-1 are dramatically affected by pH.

Phosphoramidon has been shown to inhibit endothelin-converting enzyme-1 (ECE-1) in a remarkably pH-dependent manner (Ahn et al. Arch Biochem Biophys 1998;359:258-68). In order to determine whether this dramatic pH-dependence is a general phenomenon of ECE-1, two structurally unrelated ECE-1 inhibitors, PD 069185 and CGS 31447, were tested for ECE-1 inhibition at various pH values. Our data indicate that the potencies of these ECE-1 inhibitors are also highly affected by pH. ECE-1 is known to have a very sharp activity optimum at neutral pH which is in marked contrast to the acidic pH optimum for ECE-2. However, our results show that the pH optimum for ECE-1 activity is highly substrate-dependent. ECE-1 hydrolyzes the small peptide hormones bradykinin and substance P with acidic pH optima of 5.6-5.8, which sharply contrasts the neutral pH optimum with big ET-1 as substrate. These data suggest that the substrate preference for ECE-1 is highly affected by pH and that this pH-dependence for substrate preference might be one way of controlling the specificity of the enzyme in vivo.

Aspartic Acid Endopeptidases↗
PubMed2000

Structural and functional dissection of human cytomegalovirus US3 in binding major histocompatibility complex class I molecules.

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.

Endoplasmic Reticulum↗
PubMed1999

Expanded polyglutamine tract itself induces cell death in cultured cells.

Several neurodegenerative diseases including Huntington disease, Machado-Joseph disease and spinocerebellar ataxias type 1 are caused by expansion of a polyglutamine tract within their respective gene products. In order to assess the role of the tract, 293T cells were transfected with plasmids that contain various lengths of CAG repeat encoding polyglutamine without the repeat disorder proteins: (CAG)27, (CAG)40, (CAG)80, (CAG)130, and (CAG)180. Except for (CAG)27, and (CAG)40, 293T cells showed a common set of morphological alterations such as shrinkage, rounding and surface blebbing when the expanded stretch was expressed. In addition, nuclear staining experiments showed chromatin condensation in COS-7 cells transfected with the vectors containing expanded CAG repeats. These results indicate that expanded polyglutamine itself is able to induce cell death, suggesting existence of a common molecular mechanism in the etiology of neurodegenerative polyglutamine diseases.

Animals↗
PubMed1999

Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli.

A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.

Adenosine Triphosphate↗
PubMed1999

Hydrolysis of peptide hormones by endothelin-converting enzyme-1. A comparison with neprilysin.

Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins.

Amino Acid Sequence↗
PubMed1998

Soluble human endothelin-converting enzyme-1: expression, purification, and demonstration of pronounced pH sensitivity.

Endothelin-converting enzyme-1 (ECE-1) is a type II integral membrane protein that belongs to a family of metalloproteases which includes ECE-2, neprilysin (neutral endopeptidase 24.11, EC 3.4.24. 11), and Kell blood group protein. ECE-1 cleaves its biologically inactive native substrate, big endothelin-1, to generate a powerful vasoactive 21-amino acid peptide, endothelin-1. ECE-1 consists of a short N-terminal cytoplasmic tail, a transmembrane hydrophobic domain, and a large extracellular domain containing the catalytic site with a conserved Zn-binding motif. We have constructed a secreted, soluble form of ECE-1 (solECE-1) by fusing the cleavable N-terminal signal sequence of human alkaline phosphatase in frame with the entire extracellular domain of ECE-1. Stable transfectant CHO cell lines expressing up to 6.1 mg of solECE-1 per liter culture medium were established and solECE-1 was purified to homogeneity using three chromatographic steps with a 24% yield. SolECE-1 behaves as a dimer of 110-kDa subunits. SolECE-1 has a sharp pH optimum, similar to the native form, ECE-1a, but has a slightly more acidic pH optimum of 6.1-6.4 than that of 6.7-6.9 for ECE-1a. At its optimal pH of 6.4, solECE-1 cleaved big ET-1:big ET-2:big ET-3 in a ratio of 8.1:1:1.4, was inhibited by phosphoramidon with an IC50 value of 0.35 +/- 0.05 microM, had a Km value of 4.65 +/- 0.78 microM for big ET-1, and had a kcat value of 5.82 +/- 0.21 min-1, all values comparable to those for ECE-1a at its optimal pH of 6.8. Phosphoramidon inhibition of both ECE-1a and solECE-1 is highly pH-dependent. At pH 5.8, phosphoramidon inhibited ECE-1a and solECE-1 with IC50 values of 14 and 33 nM, respectively, which are 49- and 1224-fold more potent than at pH 7.2. SolECE-1 is highly glycosylated, similar to ECE-1a. Deglycosylation of solECE-1 by peptide N-glycosidase F shifted the apparent molecular weight of solECE-1 to approximately 80 kDa and the deglycosylated form(s) of solECE-1 preserved at least 72% of the activity of the glycosylated form.

Animals↗
PubMed1998

Novel selective quinazoline inhibitors of endothelin converting enzyme-1.

PD 069185 is a highly selective and structurally novel inhibitor of endothelin converting enzyme-1 (ECE-1). PD 069185 is a trisubstituted quinazoline with an IC50 value of 0.9 +/- 0.1 microns for inhibition of human ECE-1 from the solubilized membrane fraction of CHO cells stably transfected with human ECE-1 cDNA. Kinetic analysis revealed that PD 069185 is best fit with a competitive inhibition model with a Ki value of 1.1 +/- 0.1 microns and binds in a reversible manner. The closely related enzyme, ECE-2, is not inhibited at up to 100 microns PD 069185. In addition, PD 069185 at 200-300 microns has little effect on other metalloproteases, such as neutral endopeptidase 24.11, stromelysin, gelatinase A, and collagenase, showing a high ECE-1 specificity. Data are also presented to show that this series of inhibitors are effective in inhibiting ECE-1 in intact cells and in attenuating the increase in perfusion pressure induced by big ET-1 in isolated rat mesentery. These non-peptidic ECE-1 inhibitors should serve as a valuable tool to study the pathophysiological role of endothelin and the therapeutic potential of ECE-1 inhibitors.

Animals↗
PubMed1998

The use of tooth thickness in predicting intermaxillary tooth-size discrepancies.

Intermaxillary tooth-size discrepancies can be assessed using a diagnostic setup or predicted using a mathematical formula, such as the Bolton analysis. However, variations in tooth thickness may produce inaccuracies in the Bolton analysis ratio. To date, no method for incorporating tooth thickness into discrepancy prediction has been proposed. The purpose of this study was to design and test a new method of predicting anterior tooth-size discrepancy that takes into account tooth thickness and width. Forty-four positioner setup models were set to ideal overbite (2.5 mm) and occlusion (Class I canine relationship). Interproximal gaps between the maxillary or mandibular central incisors were allowed in order to optimize tip and torque. The mesiodistal width of all anterior teeth and the labiolingual thickness of the maxillary incisors were measured on these idealized setups to the nearest 0.1 mm. Actual intermaxillary anterior ratios were then calculated. A new method of prediction was developed by assuming a linear relationship between tooth thickness and ideal intermaxillary ratio. Errors in Bolton's method were compared with the new method. The results showed wide variations in mesiodistal tooth widths, tooth thicknesses, and intermaxillary anterior ratios in orthodontically treated patients. The correlation coefficient between the intermaxillary ratio and tooth thickness was r = 0.68 when tooth thickness was < 2.75 mm, and r = 0.28 when tooth thickness was > or = 2.75 mm. The mean absolute errors in predicting the actual intermaxillary ideal ratio was 1.29 +/- 0.81 for Bolton's ratio and 0.84 +/- 0.46 for the new prediction formula. These new formulas were better than Bolton's ratio in predicting tooth-size discrepancies (p = 0.003). Tooth thickness combined with mesiodistal width may be useful in predicting intermaxillary tooth-size discrepancies.

Algorithms↗
PubMed1997

Inhibition of MHC class I antigen presentation by viral proteins.

An essential part of the immune response to viral infections is the recognition and elimination of infected cells by cytotoxic T lymphocytes. For this purpose a display mechanism has evolved which is present in almost all nucleated cells: the major histocompatibility complex class I antigen processing pathway. Both self and foreign antigens are degraded in the cytosol to peptides which are translocated into the endoplasmic reticulum where they are loaded onto MHC class I molecules. Pathogens living inside the cell are evolving under the constant selection pressure of such immune surveillance. As a result such infectious organisms have developed a variety of strategies to prevent their antigens from being presented. Since our understanding of the cell biology of antigen presentation has greatly advanced in recent years, it has now become possible to unravel several of the molecular mechanisms by which viruses interfere with MHC class I antigen presentation. Examples for the interference of viral molecules with components of the MHC class I pathway are presented in this review.

Animals↗
PubMed1997

The ER-luminal domain of the HCMV glycoprotein US6 inhibits peptide translocation by TAP.

Human cytomegalovirus (HCMV) inhibits MHC class I antigen presentation by a sequential multistep process involving a family of unique short (US) region-encoded glycoproteins. US3 retains class I molecules, whereas US2 and US11 mediate the cytosolic degradation of heavy chains by the proteosomes. In US6-transfected cells, however, intracellular transport of class I molecules is impaired because of defective peptide translocation by transporters associated with antigen processing (TAP). Peptide transport is restored in HCMV mutants lacking US6. In contrast to the cytosolic herpes simplex virus protein ICP47, US6 interacts with TAP inside the endoplasmic reticulum lumen, as shown by US6 derivatives lacking the transmembrane and cytoplasmic domains and by the observation that US6 does not prevent peptides from binding to TAP. Thus, HCMV targets TAP for immune escape by a molecular mechanism different from that of herpes simplex virus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Immunohistochemical identification of porcine respiratory coronavirus antigen in the lung of conventional pigs.

Nine 3-week-old conventional pigs were inoculated intranasally with a Korean isolate of porcine respiratory coronavirus (PRCV). Three 3-week-old conventional pigs were kept as noninoculated controls. Three inoculated and one control pig were euthanatized at 5, 10, and 15 days postinoculation (DPI), respectively. All nine inoculated pigs developed respiratory disease. Interstitial pneumonia characterized by mononuclear inflammatory cell infiltration into alveolar septa were seen microscopically in all nine PRCV-infected pigs. Histopathologic changes were severe at DPI, modest at 10 DPI, and almost resolved at 15 DPI. PRCV antigen was not detected in bronchial and bronchiolar epithelium but was detected in interstitial macrophages in the lungs. This Korean isolate of PRCV appeared to replicate primarily within alveolar macrophages in the respiratory system of the pig.

Animals↗
PubMed1996

Phosphohistidyl active sites in polyphosphate kinase of Escherichia coli.

In the synthesis of inorganic polyphosphate (polyP) from ATP by polyphosphate kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N-P-linked phosphoenzyme was previously identified as the intermediate. The phosphate is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N3-phosphohistidine. Tryptic digests of [32P]PPK contain a predominant 32P-labeled peptide that includes His-441. Of the 16 histidine residues in PPK of E. coli, 4 are conserved among several bacterial species. Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, whereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes.

Amino Acid Sequence↗
PubMed1996

Human cytomegalovirus inhibits antigen presentation by a sequential multistep process.

The human cytomegalovirus (HCMV) genomic unique short (US) region encodes a family of homologous genes essential for the inhibition of major histocompatibility complex (MHC) class I-mediated antigen presentation during viral infection. Here we show that US3, the only immediate early (IE) gene within the US region, encodes an endoplasmic reticulum-resident glycoprotein that prevents intracellular transport of MHC class I molecules. In contrast to the rapid degradation of newly synthesized MHC class I heavy chains mediated by the early gene product US11, we found that US3 retains stable MHC class I heterodimers in the endoplasmic reticulum that are loaded with peptides while retained in the ER. Consistent with the expression pattern of US3 and US11, MHC class I molecules are retained but not degraded during the IE period of infection. Our data identify the first nonregulatory role of an IE protein of HCMV and suggest that HCMV uses different T-cell escape strategies at different times during the infectious cycle.

Amino Acid Sequence↗
PubMed1996

In vivo characterization of the proteasome regulator PA28.

A proteasome regulator, termed PA28, has been shown to modulate peptidase activities of the proteasomes in vitro. Two different but homologous PA28 molecules, designated as PA28alpha and PA28beta, have been cloned. Both alpha and beta polypeptides of PA28 are found in PA28 complexes isolated from cells, indicating that both are constituents of functional PA28 complexes. Using antisera specific to PA28alpha, PA28beta, and epitope-tagged PA28 molecules, we show that expression of PA28alpha and PA28beta is coordinately induced by various cytokines in different cell lines and that PA28 subunits and proteasomes have almost identical half-lives. In addition, we show that PA28 complexes are associated with 20 S but not 26 S proteasomes in vivo. Moreover, we demonstrate that PA28 complex is a heterohexamer composed of both alpha and beta subunits with a stoichiometry of alpha3beta3 in an alternating order.

Amino Acid Sequence↗
PubMed1996

Molecular mechanism and species specificity of TAP inhibition by herpes simplex virus ICP47.

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)-mediated translocation of antigen-derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV-infected cells by cytotoxic T-lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species-specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8-azido-ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP-transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100-fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate-binding site of TAP.

Adenosine Triphosphate↗
PubMed1996

Characterization of endothelin converting enzyme from intact cells of a permanent human endothelial cell line, EA.hy926.

Endothelin converting enzyme (ECE) from intact cells of a permanent human endothelial cell line, EA.hy926, was studied by examining the effects of phosphoramidon, an endothelin converting enzyme inhibitor, on the levels of secreted endothelin-1 and big endothelin-1. The specific ECE activity was demonstrated by a phosphoramidon dose-dependent decrease in ET-1 level with a concomitant increase in big ET-1 level. By using a specific neutral endopeptidase 24.11 (NEP 24.11) inhibitor, thiorphan, it was also shown that the phosphoramidon-sensitive ET-1 degrading activity in this cell line is due to the NEP 24.11 activity. Other serine, acid, and cysteine protease inhibitors had no effect on the endogenous synthesis of ET-1 and big ET-1 supporting the evidence that ECE is insensitive to these protease inhibitors as has been demonstrated with the isolated enzyme.

Aspartic Acid Endopeptidases↗