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PubMed2005

Severe hypercholesterolemia in four British families with the D374Y mutation in the PCSK9 gene: long-term follow-up and treatment response.

OBJECTIVE: Analysis of long-term (30 years) clinical history and response to treatment of 13 patients with the D374Y mutation of PCSK9 (PCSK9 patients) from 4 unrelated white British families compared with 36 white British patients with heterozygous familial hypercholesterolemia attributable to 3 specific mutations in the low-density lipoprotein (LDL) receptor gene (LDLR) known to cause severe phenotype. METHODS AND RESULTS: The PCSK9 patients, when compared with the LDLR patients, were younger at presentation (20.8+/-14.7 versus 30.2+/-15.7 years; P=0.003), had higher pretreatment serum cholesterol levels (13.6+/-2.9 versus 9.6+/-1.6 mmol/L; P=0.004) that remained higher during treatment with simvastatin (10.1+/-3.0 versus 6.5+/-0.9 mmol/L; P=0.006), atorvastatin (9.6+/-2.9 versus 6.4+/-1.0 mmol/L; P=0.006), or current lipid-lowering therapy, including LDL apheresis and partial ileal bypass in 2 PCSK9 patients (7.0+/-1.6 versus 5.4+/-1.0 mmol/L; P=0.001), and were affected >10 years earlier by premature coronary artery disease (35.2+/-4.8 versus 46.8+/-8.9 years; P=0.002). LDL from PCSK9 patients competed significantly less well for binding to fibroblast LDL receptors than LDL from either controls or LDLR patients. CONCLUSIONS: These British PCSK9 patients with the D374Y mutation have an unpredictably severe clinical phenotype, which may be a unique feature for this cohort, and requires early and aggressive lipid-lowering management to prevent cardiovascular complications.

Adolescent↗

PubMedDate not supplied

Solid-phase synthesis of a radiolabeled, biotinylated, and farnesylated Ca(1)a(2)X peptide substrate for Ras- and a-mating factor converting enzyme.

Eukaryotic proteins with carboxyl-terminal Ca(1)a(2) motifs undergo three posttranslational processing reactions--prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding Ca(1)a(2)X endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, localization of Ras2p to the outer membrane is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. A biotinylated, farnesylated Ca(1)a(2)X peptide [(1-N-biotinyl-(13-N-succinimidyl-(S-(E,E-farnesyl)-L-cysteinyl)-L-valinyl-L-isoleucinyl-L-alanine))-4,7,10-trioxatridecanediamine] 1 containing a poly(ethylene glycol) linker was prepared by solid-phase synthesis for use in an assay for Ca(1)a(2)X endoprotease activity that relies on the strong affinity of avidin for biotin. The peptide was radiolabeled in the penultimate step of the synthesis by cleavage of the biotinylated, farnesylated Ca(1)a(2) precursor from Kaiser's oxime resin with [(14)C]-L-alanine methyl ester. [(14)C]1 was a good substrate for yRce1p with K(M) = 1.3 +/- 0.3 microM. Analysis of the carboxyl terminal products by reverse phase HPLC confirmed that VIA was the only radioactive fragment released upon incubation of [(14)C]1 with a yeast membrane preparation of recombinant yRce1p. The solid-phase methodology developed using Kaiser's benzophenone oxime resin to synthesize [(14)C]1 should be generally applicable for peptides containing sensitive side chains. In addition, introduction of the radiolabeled unit at the end of the synthesis mostly circumvents problems associated with handling radioactive materials.

Biotinylation↗

PubMed1998

The pro-protein convertase PC1 is induced in the transected sciatic nerve and is present in cultured Schwann cells: comparison with PC5, furin and PC7, implication in pro-BDNF processing.

Injury of peripheral nerves induces expression of several pro-protein convertases (PCs) involved in processing of precursor proteins into their diverse active end-products. In this study, the focus was on convertase PC1 which, although undetectable in control nerves, is strongly induced in injured nerves. High concentrations of PC1 mRNA of 9.0, 5.5, 3.0, 2.5 and 1.6 kb were observed on day 4 post-lesion in proximal and distal segments. By in situ hybridization PC1 mRNA was detected in most of endoneurial cells, which were further identified by immunocytochemistry as myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase containing Schwann cells. PC1 mRNA and protein were also present in cultured Schwann cells also containing convertases PC5, furin and PC7 as well as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Mostly unprocessed pro-NGF of 35 kDa and pro-BDNF of 35 kDa were found on Western blotting of Schwann cells. Expression of exogenous neurotrophins by infection with vaccinia virus vector showed that mouse pro-NGF and rat pro-BDNF are cleaved intracellularly on smaller forms of 13.5 kDa NGF and 14 kDa BDNF. Infection experiments demonstrated that Schwann cells contain active processing enzymes. In conclusion, this work provides in vivo evidence of the presence of several PCs in the injured rat sciatic nerve and ex vivo in cultured Schwann cells.

Animals↗

PubMed2000

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities.

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

Amino Acid Sequence↗

PubMed1993

Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae.

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.

Base Sequence↗

PubMed2000

Biosynthesis and enzymatic characterization of human SKI-1/S1P and the processing of its inhibitory prosegment.

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca(2+)-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK(137)- and RRLL(186)-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P' residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100-200 nM.

Amino Acid Sequence↗

PubMed1997

Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms.

Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases. To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates having substitutions at only one or two positions. We examined Kex2 sequence recognition for residues at P3, P2, and P1 using two types of fluorogenic peptide substrates, peptidyl-methylcoumarinamides and internally quenched substrates in which cleavage occurs at an actual peptide bond. Kinetic analysis of the two sets of substrates gave comparable data on specificity at these three positions. For the best substrate sequences, high catalytic constants (kCM/KM) of (2-5) x 10(7) M-1 s-1 were seen for cleavage of both peptidyl-methylcoumarinamides and peptide bonds. While no evidence for positive interactions with the P3 residue emerged, Kex2 was found to discriminate against at least one residue Asp. at this position. Specificity at P2 was shown to rely primarily on recognition of a positive charge, although steric constraints on the P2 side chain were also apparent. Kex2 was demonstrated to be exquisitely selective for Arg at P1. Substitutions with similar charge (Lys, ornithine) or similar hydrogen-bonding capability (citrulline) do not confer efficient catalysis. Comparison of otherwise identical substrates having either Arg or citrulline at P1 showed that the positive charge of the Arg guanidinium group stabilizes the transition state by approximately 6.8 kcal/mol.

Arginine↗

PubMed2003

Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans.

Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes.

Amino Acid Motifs↗

PubMed2000

Genomic structure and chromosomal mapping of the human site-1 protease (S1P) gene.

Site-1 protease (S1P) is a subtilisin-related enzyme that cleaves sterol regulatory element-binding proteins (SREBPs) in the lumen of endoplasmic reticulum, thereby initiating the release of transcriptionally active NH2-terminal fragments of SREBPs from membranes. In the experiments reported here, we localized the human S1P gene to chromosome 16q24 by fluorescent in situ hybridization and radiation-hybrid mapping, and determined its genomic structure. This gene is more than 60 kb long and contains 23 exons and 22 introns. Its transcription-initiation site within exon 1 is separate from the initiation codon in exon 2. Analysis of the exon/intron structure revealed that the S1P gene consists of a mosaic of functional units: exon 1 encodes the 5' non-translated region; exon 2 encodes the NH2-terminal signal sequence; and exons 2 and 3 encode the pro-peptide sequence that is released when S1P is self-activated by intramolecular cleavage. Exons 5-10 encode the subtilisin-homology domain that is critical for catalytic activity, and exon 23 encodes the transmembrane region. Analysis of the putative promoter region revealed a highly G/C-rich region containing a binding site for ADD1/SREBP-1, as well as Sp1 and AP2 sites. Therefore, expression of the S1P gene may be under the control of SREBP-1, a key regulator of the expression of genes essential for intracellular lipid metabolism. Our data establish a basis for investigations to detect molecular variants in this gene that may alter levels of plasma lipoproteins and/or otherwise disrupt intracellular lipid metabolism.

Base Sequence↗

PubMed1992

Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.

Relaxin is a polypeptide hormone involved in remodeling of the birth canal during parturition. It is synthesized as a preprohormone precursor, which undergoes specific processing to form the mature two-chain disulfide-linked active species that is secreted by the cell. A major part of this processing requires endoproteolytic cleavage at specific pairs of basic amino acid residues, an event necessary for the maturation of a variety of important biologically active proteins, such as insulin and nerve growth factor. Human type 2 preprorelaxin was coexpressed in human kidney 293 cells with the candidate prohormone convertase-processing enzymes mPC1 or mPC2, both cloned from the mouse pituitary tumor AtT-20 cell line, or with the yeast kex2 alpha-mating factor-converting enzyme from Saccharomyces cerevisiae. Prorelaxin expressed alone in 293 cells was secreted into the culture medium unprocessed. Transient coexpression with mPC1 or kex2, but not with mPC2, resulted in the secretion of a low mol wt species with an electrophoretic mobility very similar, if not identical, to that of authentic mature relaxin purified from human placenta. This species was precipitable by monoclonal antibodies specific for relaxin and had a retention time on reverse phase HPLC comparable to that of relaxin. Its analysis by both electrospray and fast atom bombardment mass spectrometry generated mass data that were consistent only with mature relaxin. The basic residues required for mPC1-dependent cleavage of prorelaxin are defined by site-directed mutagenesis.

Amino Acid Sequence↗

PubMed1998

Role for the ubiquitin-proteasome system in the vacuolar degradation of Ste6p, the a-factor transporter in Saccharomyces cerevisiae.

Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate.

ATP-Binding Cassette Transporters↗

PubMed1995

Role of amino acid sequences flanking dibasic cleavage sites in precursor proteolytic processing. The importance of the first residue C-terminal of the cleavage site.

The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro-proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P'4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P'1, Pro in P6, Gly in P3 and P'2 etc.) whereas some others appeared to be excluded. Most amino acid residues occupying the P'1 position in these precursor cleavage sites were tolerated. But the beta-carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro-ocytocin convertase and Kex2 protease action on a series of pro-ocytocin related synthetic substrates reproducing the Pro7-->Leu15 sequence of the precursor in which the Ala13 residue (P'1 in the LysArg-Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P'1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S'1 convertase subsite. We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro-proteins.

Amino Acid Sequence↗

PubMed1991

Secretion and lipid association of human apolipoprotein E in Saccharomyces cerevisiae requires a host mutation in sterol esterification and uptake.

We have expressed a cDNA to human apolipoprotein E (apoE) in Saccharomyces cerevisiae. Secretion of apoE was achieved only by the use of a mutant (upc2) strain of yeast with the phenotype of enhanced uptake and intracellular esterification of exogenous cholesterol. Approximately 40 ng/ml apoE was secreted by upc2 mutants in the absence of media cholesterol. ApoE secretion was increased 2-3-fold upon the inclusion of cholesterol in the growth media. This response to exogenous cholesterol was not mediated at the transcriptional level, since apoE mRNA levels were constant under all culture conditions. Concomitant with the increase in secretion following cholesterol uptake by upc2 strains, approximately 5% of secreted apoE was associated with lipid; polar and non-polar lipids were detected in this lipoprotein fraction. Intracellular degradation of apoE in non-secreting strains of yeast was minimized by the presence of null mutations in both vacuolar proteases with non-specific activity (pep4) and a Golgi endopeptidase with specificity for paired basic residues (kex2). The approach of expressing human apolipoproteins in yeast may identify factors that mediate lipoprotein biosynthesis in higher cells. One such factor could be the mammalian equivalent of the gene product of UPC2.

Apolipoproteins E↗

PubMed2000

Luv1p/Rki1p/Tcs3p/Vps54p, a yeast protein that localizes to the late Golgi and early endosome, is required for normal vacuolar morphology.

We have characterized LUV1/RKI1/TCS3/VPS54, a novel yeast gene required to maintain normal vacuolar morphology. The luv1 mutant was identified in a genetic screen for mutants requiring the phosphatase calcineurin for vegetative growth. luv1 mutants lack a morphologically intact vacuole and instead accumulate small vesicles that are acidified and contain the vacuolar proteins alkaline phosphatase and carboxypeptidase Y and the vacuolar membrane H(+)-ATPase. Endocytosis appears qualitatively normal in luv1 mutants, but some portion (28%) of carboxypeptidase Y is secreted. luv1 mutants are sensitive to several ions (Zn(2+), Mn(2+), and Cd(2+)) and to pH extremes. These mutants are also sensitive to hygromycin B, caffeine, and FK506, a specific inhibitor of calcineurin. Some vacuolar protein-sorting mutants display similar drug and ion sensitivities, including sensitivity to FK506. Luv1p sediments at 100,000 x g and can be solubilized by salt or carbonate, indicating that it is a peripheral membrane protein. A Green Fluorescent Protein-Luv1 fusion protein colocalizes with the dye FM 4-64 at the endosome, and hemagglutinin-tagged Luv1p colocalizes with the trans-Golgi network/endosomal protease Kex2p. Computer analysis predicts a short coiled-coil domain in Luv1p. We propose that this protein maintains traffic through or the integrity of the early endosome and that this function is required for proper vacuolar morphology.

Amino Acid Sequence↗

PubMed2001

Cloning and rational mutagenesis of kexstatin I, a potent proteinaceous inhibitor of Kex2 proteinase.

Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.

Amino Acid Sequence↗

PubMed1999

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis.

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.

Aspartic Acid Endopeptidases↗

PubMed1999

Identification and chromosomal location of two human genes encoding enzymes potentially involved in proteolytic maturation of farnesylated proteins.

Two human cDNAs encoding proteins similar to yeast enzymes involved in proteolytic processing of farnesylated proteins like a-factor mating pheromone and Ras2p have been cloned from an ovary cDNA library. These proteins have been tentatively called Face-1 and Face-2 (farnesylated protein-converting enzymes 1 and 2), respectively, and are integral membrane proteins, belonging to distinct families of metalloproteinases. Northern blot analysis of poly(A)+ RNAs isolated from a wide variety of human tissues demonstrated that both genes are expressed in all examined tissues, which suggests that these enzymes play housekeeping roles in normal processes. Fluorescence in situ hybridization experiments showed that the human FACE-1 gene maps to 1p34, whereas FACE-2 is located at 11q13, a region frequently amplified in human carcinomas and lymphomas. On the basis of these results, we suggest that inhibition of Face-1 and/or Face-2 could be part of strategies directed to block the functioning of prenylated proteins activated in oncogenic processes, including Ras proteins.

Amino Acid Sequence↗

PubMed1999

A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood.

The integrity of cell membranes is maintained by a balance between the amount of cholesterol and the amounts of unsaturated and saturated fatty acids in phospholipids. This balance is maintained by membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH(2)-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages. The first is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease. This cleavage is unusual because it occurs within a membrane-spanning domain of SREBP. Sterols block SREBP processing by inhibiting S1P. This response is mediated by SREBP cleavage-activating protein (SCAP), a regulatory protein that activates S1P and also serves as a sterol sensor, losing its activity when sterols overaccumulate in cells. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.

Animals↗