Search PubMed⌕ Search

Biomedical subjects

Y P Loh

Publications and source records attributed to Y P Loh.

At least 19 recordsLinked to original sources
PubMed2001

Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis.

We present evidence that regulation of dense-core secretory granule biogenesis and hormone secretion in endocrine cells is dependent on chromogranin A (CGA). Downregulation of CGA expression in a neuroendocrine cell line, PC12, by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected prohormone, and reduction of secretory granule proteins. Transfection of bovine CGA into a CGA-deficient PC12 clone rescued the regulated secretory phenotype. Stable transfection of CGA into a CGA-deficient pituitary cell line, 6T3, lacking a regulated secretory pathway, restored regulated secretion. Overexpression of CGA induced dense-core granules, immunoreactive for CGA, in nonendocrine fibroblast CV-1 cells. We conclude that CGA is an "on/off" switch that alone is sufficient to drive dense-core secretory granule biogenesis and hormone sequestration in endocrine cells.

Animals↗
PubMed2000

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor.

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.

Animals↗
PubMed2000

Oligomerization of pro-opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway.

Studies indicate that pro-opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane-bound carboxypeptidase E (CPE) [Cool et al. (1997) Cell 88, 73-83]. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self-associate to form oligomers, prior or subsequent to binding to CPE. Using cross-linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. delta N-POMC, which lacks the N-terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.

Animals↗
PubMed2000

In vivo processing of nonanchored Yapsin 1 (Yap3p).

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3) was overexpressed in yeast and its processing through the secretory pathway was followed by pulse-labeling and immunoprecipitation studies. In the soluble cell extract, three forms of yapsin 1-87, 74, and 18 kDa-were found. Identification of these forms of yapsin 1 using different antisera suggests that the 87-kDa form is pro-yapsin 1, which is processed into two subunits, alpha (18 kDa) and beta (74 kDa), by cleavage at a loop region not found in traditional aspartic proteases. By use of a temperature-sensitive mutant strain, sec18, the generation of the two subunits was found to occur in the endoplasmic reticulum. An active site-mutated yapsin 1 was not processed into the two subunits, suggesting that this process occurs in an autocatalytic manner.

Adenosine Triphosphatases↗
PubMed1999

Identification and characterization of Saccharomyces cerevisiae yapsin 3, a new member of the yapsin family of aspartic proteases encoded by the YPS3 gene.

A new aspartic protease from Saccharomyces cerevisiae, with a high degree of similarity with yapsin 1 and yapsin 2 and a specificity for basic residue cleavage sites of prohormones, has been cloned. This enzyme was named yapsin 3. Expression of a C-terminally truncated non-membrane anchored yapsin 3 in yeast yielded a heterogeneous protein between 135-200 kDa which, upon treatment with endoglycosidase H, migrated as a 60 kDa form. Amino-acid analysis of the N-terminus of expressed yapsin 3 revealed two different N-terminal residues, serine-48 and phenylalanine-54, which followed a dibasic and a monobasic residue respectively. Cleavage of several prohormones by non-anchored yapsin 3 revealed a specificity distinct from that of yapsin 1.

Amino Acid Sequence↗
PubMed1999

Altered biosynthesis and secretion of pro-opiomelanocortin in the intermediate and anterior pituitary of carboxypeptidase E-deficient, Cpe(fat)/ Cpe(fat)mice.

The biosynthesis and secretion of pro-opiomelanocortin (POMC) was examined in the pituitary of Cpe(fat)/ Cpe(fat)mice, which are deficient in carboxypeptidase E, a sorting receptor for the regulated secretory pathway (Cool D R, Normant E, Shen F S, et al. Cell 1997; 83: 73-83). Dopamine inhibited forskolin-stimulated accumulation of cAMP in the intermediate lobe of Cpe(fat)/ Cpe(fat)mice, showing that their dopamine receptors were fully functional. This result indicates that the elevated, dopamine-insensitive POMC secretion previously observed in the intermediate pituitary of Cpe(fat)/ Cpe(fat)mice was constitutive, rather than due to defective dopamine receptors. Concomitant with the increase in POMC secretion was a twofold increase in POMC mRNA levels and [(35)S]-methionine incorporation into POMC. In the anterior pituitary of Cpe(fat)/ Cpe(fat)mice, a 1.6-fold increase in basal release of POMC was accompanied by a similar increase in [(35)S]-methionine incorporation into POMC, although POMC mRNA levels were unchanged. Thus, the intermediate and anterior pituitary of Cpe(fat)/ Cpe(fat)mice compensate for the constitutive secretion of POMC by upregulating biosynthesis.

Animals↗
PubMed1999

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor.

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.

Animals↗
PubMed1998

Carboxypeptidase E is a sorting receptor for prohormones: binding and kinetic studies.

The binding of pro-opiomelanocortin,(POMC), pro-insulin, pro-enkephalin and chromogranin A (CGA) to the regulated secretory pathway sorting receptor, carboxypeptidase E (CPE), in bovine pituitary secretory granule (SG) membranes was investigated. N-POMC1-26, which contains the POMC sorting signal, bound to CPE in the SG membranes with low affinity and the binding was ion independent. Pro-insulin bound CPE with similar kinetics. Pro-enkephalin, but not CGA bound to CPE with similar IC50 as pro-insulin and N-POMC1-26. Crosslinking studies showed that pro-insulin and pro-enkephalin bound specifically to SG membrane CPE, similar to N-POMC1-26 reported previously. CPE was extracted from the SG membranes with NaHCO3 or KSCN, but not Triton X-100/1 M NaCl. The results show that CPE is tightly associated with SG membranes and binds several prohormones, but not CGA, with similar kinetics, providing further evidence that membrane CPE has the characteristics to function as a common sorting receptor for targeting prohormones to the regulated secretory pathway.

Animals↗
PubMed1998

Cleavage efficiency of the novel aspartic protease yapsin 1 (Yap3p) enhanced for substrates with arginine residues flanking the P1 site: correlation with electronegative active-site pockets predicted by molecular modeling.

Yapsin 1, a novel aspartic protease with unique specificity for basic residues, was shown to cleave CCK13-33 at Lys23. Molecular modeling of yapsin 1 identified the active-site cleft to have negative residues close to or within the S6, S3, S2, S1, S1', S2', and S3' pockets and is more electronegative than rhizopuspepsin or endothiapepsin. In particular, the S2' subsite has three negative charges in and close to this pocket that can provide strong electrostatic interactions with a basic residue. The model, therefore, predicts that substrates with a basic residue in the P1 position would be favored with additional basic residues binding to the other electronegative pockets. A deletion of six residues close to the S1 pocket in yapsin 1, relative to rhizopuspepsin and other aspartic proteases of known 3D structure, is likely to affect its specificity. The model was tested using CCK13-33 analogues. We report that yapsin 1 preferentially cleaves a CCK13-33 substrate with a basic residue in the P1 position since the substrates with Ala in P1 were not cleaved. Furthermore, the cleavage efficiency of yapsin 1 was enhanced for CCK13-33 analogues with arginine residues flanking the P1 position. An alanine residue, substituting for the arginine residue in the P6 position in CCK13-33, resulted in a 50% reduction in the cleavage efficiency. Substitution with arginine residues downstream of the cleavage site at the P2', P3', or P6' position increased the cleavage efficiency by 21-, 3- and 7-fold, respectively. Substitution of Lys23 in CCK13-33 with arginine resulted not only in cleavage after the substituted arginine residue, but also forced a cleavage after Met25, suggesting that an arginine residue in the S2' pocket is so favorable that it can affect the primary specificity of yapsin 1. These results are consistent with the predictions from the molecular model of yapsin 1.

Alanine↗
PubMed1998

Activation and processing of non-anchored yapsin 1 (Yap3p).

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel sub-class of aspartic proteases with specificity for basic residues (designated the Yapsins), was overexpressed and purified to apparent homogeneity, yielding approximately 1 microg of yapsin 1/g of wet yeast. N-terminal amino acid analysis of the purified protein confirmed that the propeptide was absent and that the mature enzyme began at Ala68. The mature enzyme was shown to be composed of approximately equimolar amounts of two subunits, designated alpha and beta, that were associated to each other by a disulfide bond. C-terminally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at approximately 4.0. When expressed without its pro-region, it was localized intracellularly and lacked activity, indicating that the pro-region was required for the correct folding of the enzyme. The activation of proyapsin 1 in vitro exhibited linear kinetics and generated an intermediate form of yapsin 1 or pseudo-yapsin 1.

Amino Acid Sequence↗
PubMed1998

Vasopressin gene expression in rat choroid plexus.

Vasopressin (VP) levels in cerebrospinal fluid (CSF) change in response to physiological stimuli and under various pathological conditions. The sources of CSF VP have yet to be clarified, however. In the present study, we provide evidence indicating that VP is synthesized in the choroid plexus, the primary site of CSF formation. All experiments were performed on adult male Sprague-Dawley rats. The presence of VP mRNA in choroid plexus epithelium was demonstrated by in situ hybridization histochemistry using the 35S-labeled riboprobe that was complementary to cDNA fragment of rat VP encoding the C-terminus part of proVP. In situ hybridization findings were confirmed by reverse transcriptase-polymerase chain reaction analysis. Immunohistochemistry for VP-associated neurophysin (VP-NP), a polypeptide component of proVP, revealed subapical accumulation of VP-NP-immunopositive product in choroidal epithelial cells. Immunoprecipitation and immunoblotting of choroidal protein extracts with anti-VP-NP antibody demonstrated the presence of a approximately 10-kD polypeptide that was also detected in hypothalamus. We hypothesize that the choroid plexus-derived VP exerts autocrine and/or paracrine effects on tissues near the CSF system.

Animals↗
PubMed1998

Depletion of carboxypeptidase E, a regulated secretory pathway sorting receptor, causes misrouting and constitutive secretion of proinsulin and proenkephalin, but not chromogranin A.

Previous studies have shown that the prohormone POMC is sorted to the regulated secretory pathway (RSP), at the trans-Golgi network, by binding of a conformation-dependent sorting signal to a sorting receptor, identified as membrane-bound carboxypeptidase E (CPE) (Cool et al., 1997, Cell, 88:73-83). In this study, the role of CPE as a sorting receptor for other RSP proteins that contain sorting signals (proinsulin, proenkephalin, and chromogranin A) was investigated in neuroendocrine cells (Neuro-2a) stably expressing CPE antisense RNA. Whereas these cells were depleted of CPE by greater than 85%, electron microscopy showed that they contain dense core secretory granules identical to wild-type Neuro-2a cells, indicating that CPE is not essential for granulogenesis. Secretion and immunocytochemical studies showed that, in wild-type Neuro-2a cells, endogenous proenkephalin and transfected proinsulin/insulin were localized to punctate secretory granules and were released via the RSP. However, in CPE-depleted cells, these two prohormones were released constitutively and had a Golgi-like distribution but were not localized to punctate secretory granules. In contrast, chromogranin A was present in punctate secretory granules and released via the RSP, in wild-type and CPE-depleted Neuro-2a cells. Thus, the sorting of proinsulin and proenkephalin to the RSP, like POMC, necessitates binding to CPE, and hence, CPE acts as a common sorting receptor for targeting these prohormones to the RSP. In contrast, the sorting signal of chromogranin A does not use CPE as a sorting receptor, suggesting the existence of other sorting receptors for the RSP.

Carboxypeptidase H↗
PubMed1997

High-performance liquid chromatographic-colorimetric assay for glycine carboxypeptidase activity.

A rapid and sensitive assay method for the determination of glycine carboxypeptidase activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe, enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a TSK gel ODS-80TM reversed-phase column by isocratic elution. This method is sensitive enough to measure 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 7.5 min per sample for separation and quantitation. The pH optimum for glycine carboxypeptidase activity was 4.8 to 5.4. The Km and Vmax values were respectively 21.1 micromol and 3.73 pmol/microg/h with the use of enzyme extract obtained from bovine pituitary. Glycine carboxypeptidase activity was strongly inhibited by Ag+, Cu2+ and p-chloromercuriphenylsulfonic acid. Among the organs examined in a mouse, the highest specific activity of the enzyme was found in testis. The sensitivity and selectivity of this method will aid in efforts to examine the physiological role of this peptidase.

Animals↗
PubMed1997

Intracellular misrouting and abnormal secretion of adrenocorticotropin and growth hormone in cpefat mice associated with a carboxypeptidase E mutation.

Cpefat mice carry a mutation in the carboxypeptidase E/H gene which encodes an exopeptidase that removes C-terminal basic residues from endoproteolytically cleaved hormone intermediates. These mice have endocrine disorders including obesity, infertility, and hyperproinsulinemia-diabetes syndrome, but the etiology remains an enigma. Because studies have identified membrane carboxypeptidase E as a sorting receptor for targeting prohormones to the regulated secretory pathway for processing and secretion, the intracellular routing and secretion of pro-opiomelanocortin/adrenocorticotropin and growth hormone from anterior pituitary cells were investigated in Cpefat mice. In Cpefat mice, pro-opiomelanocortin was accumulated 24-fold above normal animals in the pituitary and it was poorly processed to adrenocorticotropin. Furthermore, pro-opiomelanocortin was secreted constitutively at high levels, showing no response to stimulation by corticotropin-releasing hormone. Similarly, growth hormone release was constitutive and did not respond to high K+ stimulation. Both pro-opiomelanocortin and growth hormone levels were elevated in the circulation of Cpefat mice versus normal mice. These data provide evidence that the lack of carboxypeptidase E, the sorting receptor, results in the intracellular misrouting and secretion of pro-opiomelanocortin and growth hormone via the constitutive pathway in the pituitary of Cpefat mice.

Adrenocorticotropic Hormone↗
PubMed1997

The structure of synenkephalin (pro-enkephalin 1-73) is dictated by three disulfide bridges.

Mass spectrometry of fragments produced by limited proteolytic digestion of pro-enkephalin was used to locate the disulfide bridges in synenkephalin (pro-enkephalin 1-73), a domain which contains sorting information for targeting the pro-neuropeptide to the granules of the regulated secretory pathway in neuroendocrine cells. Mass spectrometric analysis was optimized by using chemicals that gave low interference with the ionization and desorption processes, and computer software which simplified the identification of all possible disulfide-linked peptide fragments. Three disulfide bridges between Cys2-Cys24, Cys6-Cys28, and Cys9-Cys41 were identified. Protein conformational prediction of synenkephalin1-42 shows beta-turns which facilitate the formation of these disulfide bonds.

Amino Acid Sequence↗
PubMed1997

Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice.

A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.

Amino Acid Sequence↗