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J LaMendola

Publications and source records attributed to J LaMendola.

11 recordsLinked to original sources
PubMed1998

Regulatory roles of the P domain of the subtilisin-like prohormone convertases.

A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and alpha-TC1-6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592-594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in alpha-TC1-6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases.

Animals↗
PubMed1997

Expression of PC3, carboxypeptidase E and enkephalin in human monocyte-derived macrophages as a tool for genetic studies.

Circulating monocytes in human peripheral blood are readily available, easily obtained, and can be cultured in vitro. Once plated, the monocytes spontaneously differentiate into macrophages. Undifferentiated human monocytes do not express carboxypeptidase E (CPE), prohormone convertase 3 (PC3/PC1) or proenkephalin (ENK), suggesting that gene induction during differentiation results in the expression of these genes. RT-PCR of human monocyte-derived macrophage (HMDM) mRNA showed detectable levels of ENK mRNA at 48 h after plating, followed by PC3 and CPE mRNAs at 72 h. PC3 expression was confirmed by Western blotting in THP-1 cells. Similarities in expression of enzymes involved in the conversion of neuroendocrine precursors, such as proenkephalin, into functionally mature peptides underscores the close association HMDMs may share with the neuroendocrine system. Because of this, HMDMs may prove to be a valuable, non-surgical source of human tissue for clinical genetic diagnosis of some convertase disorders.

Aspartic Acid Endopeptidases↗
PubMed1994

Nucleotide sequence and analysis of the mouse SPC3 promoter region.

Insulin is converted from the higher molecular weight proprotein, proinsulin by highly specific proteolytic cleavage at two dibasic amino acid sites. SPC3 and SPC2, two recently identified prohormone convertase that are specifically expressed in beta cells and other neuroendocrine cells, appear to be responsible for those cleavages. We have sequenced the 5'-upstream region of the SPC3 gene and examined its promotor/enhancer activity and most of several deletion mutants in several cell lines. This region contains no CAAT box but has several non-functional TATA-like sequences and several putative transcriptional regulatory elements, including AP-1, Sp1 and cAMP response elements. These features are not unlike those of the human SPC2 upstream region. In beta TC3 insulinoma cells, the sequence between the EcoRI (620 bp) and NsiI (702 bp) sites seems to be important for gene expression, while the sequence between the NsiI and DraI (775 bp) sites may contain strong enhancer element(s).

Amino Acid Sequence↗
PubMed1994

Expression of cone transducin, Gz alpha, and other G-protein alpha-subunit messenger ribonucleic acids in pancreatic islets.

The G-proteins are a family of heterotrimeric guanine nucleotide-binding proteins that play important roles in signal transduction and whose expression is regulated in a tissue-specific manner. Here we have surveyed the expression of G-protein alpha-subunits in mouse pancreatic islets. Degenerate oligonucleotide primers corresponding to conserved primary sequences in known G alpha-subunits were used in a reverse transcriptase-polymerase chain reaction, and the amplified complementary DNA (cDNA) fragments were subcloned and sequenced. Over 100 clones were analyzed, from which we determined that islet cells express at least seven G alpha-subunits: G8 alpha, Gi1 alpha or Gi3 alpha, Gi2 alpha, G11 alpha, G14 alpha, Gz alpha, and Gt2 alpha (cone transducin). In particular, the identification of Gz alpha and Gt2 alpha was of interest in that previous studies had indicated that the expression of Gz alpha was restricted mainly to the brain, retina, and adrenal gland, whereas Gt2 alpha was expressed predominantly in retinal cone photoreceptors. By Western blot analysis, we estimated that the amount of Gz alpha protein present in mouse islets was about 40% of that in retina. To further investigate the expression of Gt2 alpha, mouse Gt2 alpha cDNA was cloned from a retinal library and sequenced. The cDNA was used as a probe for Northern blot analysis, and the results confirmed that mouse islets contained a substantial level of Gt2 alpha messenger RNA (mRNA), albeit less than that found in retina (approximately 5-fold lower). Gt2 alpha mRNA was also shown to be present in a clonal mouse pancreatic alpha-cell line (alpha TC1-6) as well as in adrenal gland, pituitary, and a clonal mouse anterior pituitary cell line (AtT20). In situ hybridization revealed that Gt2 alpha mRNA was expressed essentially throughout the islet, suggesting that it is normally expressed in the abundant islet beta-cells and possibly others. In situ analysis also showed that Gt2 alpha mRNA expressed in the pituitary was limited to the intermediate and anterior lobes. We conclude that islet cells express multiple G-proteins, including several that are normally expressed at high levels in certain neuronal cells.

Amino Acid Sequence↗
PubMed1993

Human G(olf) alpha: complementary deoxyribonucleic acid structure and expression in pancreatic islets and other tissues outside the olfactory neuroepithelium and central nervous system.

G(olf) alpha is a G-protein originally believed to mediate signal transduction exclusively within the olfactory neuroepithelium and subsequently found to be a major stimulatory G-protein in the basal ganglia. Here we present evidence that G(olf) alpha is expressed in several other tissues. The human isoform of G(olf) alpha was isolated from two human insulinoma cDNA libraries. Comparison of the human sequence with rat G(olf) alpha shows 91% nucleotide identity (within the coding region) and 99% identity at the amino acid level. Northern and reverse transcriptase-polymerase chain reaction analyses indicated that G(olf) alpha is expressed in all human insulinomas examined thus far as well as in normal pancreatic islets. G(olf) alpha mRNA was also detected in testis, retina, brain, and liver. Western blot analysis of various mouse tissues demonstrated that the level of G(olf) alpha protein in islets is lower than that in the olfactory neuroepithelium and other parts of the brain; its expression in retina, lung, and spleen was moderately higher than that in islets, and its expression in testis approached that in olfactory neuroepithelium. G(olf) alpha was also detected by immunohistochemistry in mouse islets, human insulinomas, the epithelial lining of mouse epididymis, photoreceptor cells of mouse retina, and mouse lung alveoli. These findings suggest a role for G(olf) alpha in a diverse population of cells located outside the olfactory neuroepithelium and central nervous system.

Amino Acid Sequence↗
PubMed1992

Conservation of the prohormone convertase gene family in metazoa: analysis of cDNAs encoding a PC3-like protein from hydra.

A subclass of proteolytic enzymes that correctly cleave precursor proteins at paired basic residues and are structurally related to the bacterial subtilisins has recently been identified. In yeast, a single membrane-bound proteolytic processing enzyme encoded by the kex2 gene has been found, whereas in higher vertebrates cDNAs encoding four distinct enzymes (PC2, PC3, furin, and PACE 4) have been identified. Like kex2, furin (also known as PACE) contains a hydrophobic transmembrane domain, but PC2, PC3, and PACE 4 lack this feature. All five enzymes exhibit striking similarities in their catalytic domains, and this suggests that they have arisen from a common ancestral subtilisin-like gene. We report here the identification of cDNAs encoding a protein that is similar in structure to PC3 from a simple metazoan, Hydra vulgaris (formerly Hydra attenuata). cDNAs encoding two isoforms of this PC3-like enzyme were obtained that differ only in their carboxyl-terminal sequences, probably due to alternative splicing of a common pre-mRNA. Neither form contains a transmembrane domain. Predicted amino acid sequence comparisons revealed that the hydra PC3-like enzyme is 55.4% and 56.7% identical in the catalytic domain to mouse PC3 and human furin, respectively. RNA blot analyses revealed that the PC3-like RNA is expressed predominantly in the hydra body column and not in the head region, although the hydra head contains a high density of nerve cells, which synthesize a variety of neuropeptides. For this reason, we suspect that another proprotein cleavage enzyme isoform may be expressed in head nerve cells. The isolation of a PC3-like cDNA from hydra is consistent with the presence of neuroendocrine cells and indicates that the PC/furin gene family has been well conserved in all metazoa. A simplified nomenclature for the group of mammalian processing proteases is proposed.

Amino Acid Sequence↗
PubMed1992

Identification and analysis of the gene encoding human PC2, a prohormone convertase expressed in neuroendocrine tissues.

In recent studies we have identified PC2 and PC3, members of a family of serine proteases that are related structurally to subtilisin, and have provided evidence that these are involved in the tissue-specific processing of prohormones and neuropeptides. PC2 is expressed at high levels in the islets of Langerhans, where it participates in the processing of proinsulin to insulin (S.P.S. and D.F.S., unpublished data). To evaluate the regulated expression of the human PC2 (hPC2) gene we have analyzed its structure and characterized its promoter. A map of the gene was constructed by using 11 clones isolated from two human genomic DNA libraries. The gene spans greater than 130 kilobase pairs and consists of 12 exons. Comparison with the structure of the gene encoding human furin, another member of this superfamily, revealed a high degree of conservation of exon-intron junctions. The hPC2 gene was localized to chromosome 20, band p11.2. The 5' flanking region of the hPC2 gene is very G+C-rich and contains six potential Sp1 binding sites but no TATA or CAAT box. Expression of chloramphenicol acetyltransferase reporter fusions containing the putative promoter region was observed to occur in beta TC-3 mouse insulinoma cells but not in HepG2 human hepatoma cells, consistent with the known tissue-specific pattern of expression of the hPC2 gene. Analysis of the level of chloramphenicol acetyltransferase activity with several deletion mutants identified the region from -1100 to -539 from the translation start site as essential for hPC2 promoter activity.

Amino Acid Sequence↗
PubMed1991

Identification of a cDNA encoding a second putative prohormone convertase related to PC2 in AtT20 cells and islets of Langerhans.

PC2 and furin are two recently identified members of a class of mammalian proteins homologous to the yeast precursor processing protease kex2 and the bacterial subtillisins. We have used the polymerase chain reaction to identify and clone a cDNA (PC3) from the mouse AtT20 anterior pituitary cell line that represents an additional member of this growing family of mammalian proteases. PC3 encodes a 753-residue protein that begins with a signal peptide and contains a 292-residue domain closely related to the catalytic modules of PC2, furin, and kex2. Within this region 58%, 65%, and 50% of the amino acids of PC3 are identical to those of the aligned PC2, furin, and kex2 sequences, respectively, and the catalytically important Asp, His, and Ser residues are all conserved. On Northern blots, PC3 hybridizes to two transcripts of 3 and 5 kilobases. Tissue distribution studies indicate that both PC2 and PC3 are expressed in a variety of neuroendocrine tissues, including pancreatic islets and brain, but are not expressed in liver, kidney, skeletal muscle, and spleen. The high degree of similarity of PC3, PC2, and furin suggests that they are all members of a superfamily of mammalian proteases that are involved in the processing of prohormones and/or other protein precursors. In contrast to furin, PC3, like PC2, lacks a hydrophobic transmembrane anchor, but it has a potential C-terminal amphipathic helical segment similar to the putative membrane anchor of carboxypeptidase H. These and other differences suggest that these proteins carry out compartmentalized proteolysis within cells, such as processing within regulated versus constitutive secretory pathways.

Adrenal Gland Neoplasms↗
PubMed1991

Sequence and functional expression in Xenopus oocytes of a human insulinoma and islet potassium channel.

Regulation of insulin secretion involves the coordinated control of ion channels in the beta-cell membrane. We have isolated and characterized cDNA and genomic clones encoding a voltage-dependent K+ channel isoform expressed in human islets and in a human insulinoma. This K+ channel isoform, designated hPCN1, with a deduced amino acid sequence of 613 residues (Mr = 67,097), is related to the Shaker family of Drosophila K+ channels. hPCN1 is homologous to two other human K+ channel isoforms we have isolated, hPCN2 and hPCN3, with 55% and 65% amino acid sequence identity, respectively. The electrophysiological characteristics of hPCN1 were determined after microinjection of synthetic RNA into Xenopus oocytes. Two-microelectrode voltage-clamp recordings of oocytes injected with hPCN1 RNA revealed a voltage-dependent outward K+ current that inactivated slowly with time. Outward currents were inhibited by 4-aminopyridine with a Ki less than 0.10 mM and were relatively insensitive to tetraethylammonium ion or Ba2+. A delayed rectifier K+ channel such as hPCN1 could restore the resting membrane potential of beta cells after depolarization and thereby contribute to the regulation of insulin secretion.

Amino Acid Sequence↗
PubMed1989

Cloning and sequence determination of human placental aldose reductase gene.

The human aldose reductase gene has been cloned by screening a human placental cDNA library with antibodies against bovine lens aldose reductase. The nucleotide sequence of the entire coding region has been determined. The deduced amino acid sequence indicates that the human enzyme is 84% identical to the bovine lens aldose reductase and 85% identical to the rat lens aldose reductase. It is also very similar to the human aldehyde reductase, the bovine prostaglandin F synthase, and to the European common frog rho-crystallin. The deduced amino acid sequence also indicates that maturation of aldose reductase involves removal of the N-terminal methionine.

Alcohol Dehydrogenase↗