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Biomedical subjects

R E Mains

Publications and source records attributed to R E Mains.

At least 37 records · Page 2Linked to original sources
PubMed1999

Activation and routing of membrane-tethered prohormone convertases 1 and 2.

Many peptide hormones and neuropeptides are processed by members of the subtilisin-like family of prohormone convertases (PCs), which are either soluble or integral membrane proteins. PC1 and PC2 are soluble PCs that are primarily localized to large dense core vesicles in neurons and endocrine cells. We examined whether PC1 and PC2 were active when expressed as membrane-tethered proteins, and how tethering to membranes alters the biosynthesis, enzymatic activity, and intracellular routing of these PCs. PC1 and PC2 chimeras were constructed using the transmembrane domain and cytoplasmic domain of the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The membrane-tethered PCs were rerouted from large dense core vesicles to the Golgi region. In addition, the chimeras were transiently expressed at the cell surface and rapidly internalized to the Golgi region in a fashion similar to PAM. Membrane-tethered PC1 and PC2 exhibited changes in pro-domain maturation rates, N-glycosylation, and in the pH and calcium optima required for maximal enzymatic activity against a fluorogenic substrate. In addition, the PC chimeras efficiently cleaved endogenous pro-opiomelanocortin to the correct bioactive peptides. The PAM transmembrane domain/cytoplasmic domain also prevented stimulated secretion of pro-opiomelanocortin products in AtT-20 cells.

Adrenocorticotropic Hormone↗
PubMed1999

Phosphorylation of cytosolic domain Ser(937) affects both biosynthetic and endocytic trafficking of peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine alpha-amidating monooxygenase (PAM), a bifunctional enzyme, catalyzes the COOH-terminal amidation of bioactive peptides. In test tube assays, PAM is phosphorylated by protein kinase C at Ser(937). The roles of phosphorylation and dephosphorylation of Ser(937) in the biosynthetic and endocytic trafficking of integral membrane PAM were examined using an antiserum specific for the phosphorylation of Ser(937) and using AtT-20 cells expressing membrane PAM in which Ser(937) was mutated to Ala or Asp. Although phosphorylation at Ser(937) can occur while PAM is in the endoplasmic reticulum, early steps in the biosynthetic trafficking of membrane PAM were not affected by Ser(937) phosphorylation. The inability to phosphorylate PAM/S937A increased its intracellular degradation and decreased secretion of the soluble monooxygenase portion of PAM. In contrast, the biosynthetic trafficking of PAM/S937D was indistinguishable from wild-type PAM. Despite the fact that Ser(937) is adjacent to the only Tyr-based internalization motif in PAM, internalization and trafficking through early endosomes were unaffected by phosphorylation. However, PAM antibody internalized by wild-type PAM acquired a perinuclear localization, while antibody internalized by PAM/S937A was routed to lysosomes, and antibody bound to PAM/S937D maintained a dispersed, punctate pattern. In cells stimulated with phorbol ester, phosphorylation of Ser(937) increased and phosphorylated PAM accumulated in large vesicular structures. Therefore, phosphorylation of PAM-1 at Ser(937) directs newly synthesized and internalized protein away from lysosomes, while dephosphorylation is needed for a different step in the late endocytic pathway.

Animals↗
PubMed1999

Tonic dopamine inhibition of L-type Ca2+ channel activity reduces alpha1D Ca2+ channel gene expression.

Hormones and neurotransmitters have both short-term and long-term modulatory effects on the activity of voltage-gated Ca2+ channels. Although much is known about the signal transduction underlying short-term modulation, there is far less information on mechanisms that produce long-term effects. Here, the molecular basis of long-lasting suppression of Ca2+ channel current in pituitary melanotropes by chronic dopamine exposure is examined. Experiments involving in vivo and in vitro treatments with the dopaminergic drugs haloperidol, bromocriptine, and quinpirole show that D2 receptors persistently decrease alpha1D L-type Ca2+ channel mRNA and L-type Ca2+ channel current without altering channel gating properties. In contrast, another L-channel (alpha1C) mRNA and P/Q-channel (alpha1A) mRNA are unaffected. The downregulation of alpha1D mRNA does not require decreases in cAMP levels or P/Q-channel activity. However, it is mimicked and occluded by inhibition of L-type channels. Thus, interruption of the positive feedback between L-type Ca2+ channel activity and alpha1D gene expression can account for the long-lasting regulation of L-current produced by chronic activation of D2 dopamine receptors.

Animals↗
PubMed1999

Inducible genetic suppression of neuronal excitability.

Graded, reversible suppression of neuronal excitability represents a logical goal of therapy for epilepsy and intractable pain. To achieve such suppression, we have developed the means to transfer "electrical silencing" genes into neurons with sensitive control of transgene expression. An ecdysone-inducible promoter drives the expression of inwardly rectifying potassium channels in polycistronic adenoviral vectors. Infection of superior cervical ganglion neurons did not affect normal electrical activity but suppressed excitability after the induction of gene expression. These experiments demonstrate the feasibility of controlled ion channel expression after somatic gene transfer into neurons and serve as the prototype for a novel generalizable approach to modulate excitability.

Action Potentials↗
PubMed1999

Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.

Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.

Actins↗
PubMed1999

Kalirin, a multifunctional PAM COOH-terminal domain interactor protein, affects cytoskeletal organization and ACTH secretion from AtT-20 cells.

The production and regulated secretion of bioactive peptides require a series of lumenal enzymes to convert inactive precursors into bioactive peptides plus several cytosolic proteins to govern granule formation, maturation, translocation, and exocytosis. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for biosynthesis of many peptides, is an integral membrane protein with trafficking information in both its lumenal and cytosolic domains. Kalirin, a PAM cytosolic domain interactor protein with spectrin-like repeats and GDP/GTP exchange factor activity for Rac1, is expressed with PAM in neurons but is not expressed in the anterior pituitary or AtT-20 corticotrope cells. Expression of Kalirin alters the cytoskeletal organization of Chinese hamster ovary and AtT-20 cells expressing membrane PAM. Expression of membrane PAM also alters cytoskeletal organization, demonstrating the presence of endogenous proteins that can mediate this effect. Significant amounts of both PAM and Kalirin fractionate with cytoskeletal elements. Since cytoskeletal organization is critical for exocytosis, constitutive-like and regulated secretions were evaluated. Whereas the constitutive-like secretion of adrenocorticotropic hormone (ACTH) is increased by expression of membrane PAM, regulated secretion is eliminated. Expression of Kalirin in AtT-20 cells expressing membrane PAM restores stimulated secretion of ACTH. Thus, Kalirin or its homologue may be essential for regulated secretion, and the PAM-Kalirin interaction may coordinate intragranular with cytosolic events.

Actins↗
PubMed1999

Kalirin inhibition of inducible nitric-oxide synthase.

Nitric oxide (NO) acts as a neurotransmitter. However, excess NO produced from neuronal NO synthase (nNOS) or inducible NOS (iNOS) during inflammation of the central nervous system can be neurotoxic, disrupting neurotransmitter and hormone production and killing neurons. A screen of a hippocampal cDNA library showed that a unique region of the iNOS protein interacts with Kalirin, previously identified as an interactor with a secretory granule peptide biosynthetic enzyme. Kalirin associates with iNOS in vitro and in vivo and inhibits iNOS activity by preventing the formation of iNOS homodimers. Expression of exogenous Kalirin in pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Thus Kalirin may play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity.

Adrenocorticotropic Hormone↗
PubMed1999

Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.

Animals↗
PubMed1999

A molluscan peptide alpha-amidating enzyme precursor that generates five distinct enzymes.

Mechanisms underlying the specificity and efficiency of enzymes, which modify peptide messengers, especially with the variable requirements of synthesis in the neuronal secretory pathway, are poorly understood. Here, we examine the process of peptide alpha-amidation in individually identifiable Lymnaea neurons that synthesize multiple proproteins, yielding complex mixtures of structurally diverse peptide substrates. The alpha-amidation of these peptide substrates is efficiently controlled by a multifunctional Lymnaea peptidyl glycine alpha-amidating monooxygenase (LPAM), which contains four different copies of the rate-limiting Lymnaea peptidyl glycine alpha-hydroxylating monooxygenase (LPHM) and a single Lymnaea peptidyl alpha-hydroxyglycine alpha-amidating lyase. Endogenously, this zymogen is converted to yield a mixture of monofunctional isoenzymes. In vitro, each LPHM displays a unique combination of substrate affinity and reaction velocity, depending on the penultimate residue of the substrate. This suggests that the different isoenzymes are generated in order to efficiently amidate the many peptide substrates that are present in molluscan neurons. The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide alpha-amidation.

Amino Acid Sequence↗
PubMed1998

Post-translational N-glycosylation of a truncated form of a peptide processing enzyme.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the carboxyl-terminal amidation of bioactive peptides through a two-step reaction involving the monooxygenase and lyase domains. PAM undergoes endoproteolytic cleavage in neuroendocrine cells in the lyase domain. To determine which of the two possible paired basic sites is utilized, truncated PAM proteins ending at these sites were stably expressed in Chinese hamster ovary cells. While characterizing the truncation mutants, it became apparent that N-glycosylation occurred post-translationally at the single site localized near the carboxyl terminus of the lyase domain. The post-translational N-glycosylation of this site does not require the newly synthesized protein to remain tightly bound to membranes. Both malfolded, secretion incompetent proteins and fully active, secreted proteins were subject to post-translational N-glycosylation.

Animals↗
PubMed1998

Structural elements of PC2 required for interaction with its helper protein 7B2.

The structures of the eukaryotic subtilisin protease family members can be divided into four distinct domains as follows: the proregion, the catalytic domain, the P domain, and the carboxyl-terminal region. Although these enzymes are evolutionarily related, only prohormone convertase 2 (PC2) requires 7B2 for activation. To examine the potential contribution of each domain of PC2 to PC2-7B2 interactions, we performed sequential deletions, site-directed mutagenesis, and domain swapping to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments/amino acids of pro-PC1. These chimeras and mutant enzyme molecules were then expressed in AtT-20 cells and analyzed for 7B2 binding, maturation ability, and enzymatic activity. The results revealed that 1) the PC2 proregion is required but is not sufficient to confer 7B2 binding; 2) the P domain is required for the stabilization of PC2 structure and is not exchangeable with the P domain of PC1; and 3) the carboxyl-terminal domain is not involved in 7B2 binding. Site-directed mutagenesis of pro-PC2 further showed that a single residue replacement in the catalytic domain, Tyr-194 --> Asp, prevented pro-PC2 from binding 7B2 and blocked activation. This residue is present within a loop rich in aromatic amino acids which appears to be on the surface of the molecule as extrapolated from the crystal structure of subtilisin. This loop may represent the primary recognition site for 7B2 within the catalytic domain.

Amino Acid Sequence↗
PubMed1997

Expression of PACE4 in chemically induced carcinomas is associated with spindle cell tumor conversion and increased invasive ability.

Gene expression changes associated with the conversion of squamous cell carcinoma (SCC) to a more advanced malignant spindle cell carcinoma (SPCC) were determined by differential display. Using an animal model of human SCC progression, we provide evidence of increased PACE4 expression in SPCC cell lines and primary tumors induced by chemical carcinogenesis protocols, thus implicating this proprotein convertase in the process of tumor progression. Exogenous overexpression of PACE4 cDNA in mouse SCC cells of low invasive ability resulted in enhanced tumor cell invasiveness that was absent in parental or mock-transfected SCC cells. In addition, the PACE4-transfected cells acquired the ability to process prostromelysin 3 into its active enzyme form. Taken together, these results show that up-regulation of PACE4 expression is associated with SCC conversion to SPCC and suggests that activation of essential PACE4 substrates, such as the metalloproteinase stromelysin 3, is required for tumor cell invasion.

Animals↗
PubMed1997

Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase.

Many neuropeptides and peptide hormones require amidation at the carboxyl terminus for activity. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of these diverse physiological regulators. The amino-terminal domain of the bifunctional PAM protein is a peptidylglycine alpha-hydroxylating monooxygenase (PHM) with two coppers that cycle through cupric and cuprous oxidation states. The anomalous signal of the endogenous coppers was used to determine the structure of the catalytic core of oxidized rat PHM with and without bound peptide substrate. These structures strongly suggest that the PHM reaction proceeds via activation of substrate by a copper-bound oxygen species. The mechanistic and structural insight gained from the PHM structures can be directly extended to dopamine beta-monooxygenase.

Animals↗
PubMed1997

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.

Animals↗
PubMed1997

Kalirin, a cytosolic protein with spectrin-like and GDP/GTP exchange factor-like domains that interacts with peptidylglycine alpha-amidating monooxygenase, an integral membrane peptide-processing enzyme.

Although the integral membrane proteins that catalyze steps in the biosynthesis of neuroendocrine peptides are known to contain routing information in their cytosolic domains, the proteins recognizing this routing information are not known. Using the yeast two-hybrid system, we previously identified P-CIP10 as a protein interacting with the cytosolic routing determinants of peptidylglycine alpha-amidating monooxygenase (PAM). P-CIP10 is a 217-kDa cytosolic protein with nine spectrin-like repeats and adjacent Dbl homology and pleckstrin homology domains typical of GDP/GTP exchange factors. In the adult rat, expression of P-CIP10 is most prevalent in the brain. Corticotrope tumor cells stably expressing P-CIP10 and PAM produce longer and more highly branched neuritic processes than nontransfected cells or cells expressing only PAM. The turnover of newly synthesized PAM is accelerated in cells co-expressing P-CIP10. P-CIP10 binds to selected members of the Rho subfamily of small GTP binding proteins (Rac1, but not RhoA or Cdc42). P-CIP10 (kalirin), a member of the Dbl family of proteins, may serve as part of a signal transduction system linking the catalytic domains of PAM in the lumen of the secretory pathway to cytosolic factors regulating the cytoskeleton and signal transduction pathways.

Amino Acid Sequence↗
PubMed1997

Neuropeptide amidation in Drosophila: separate genes encode the two enzymes catalyzing amidation.

In vertebrates, the two-step peptide alpha-amidation reaction is catalyzed sequentially by two enzymatic activities contained within one bifunctional enzyme called PAM (peptidylglycine alpha-amidating mono-oxygenase). Drosophila head extracts contained both of these PAM-related enzyme activities: a mono-oxygenase (PHM) and a lyase (PAL). However, no bifunctional PAM protein was detected. We identified cDNAs encoding an active mono-oxygenase that is highly homologous to mammalian PHM. PHM-like immunoreactivity was found within diverse larval tissues, including the CNS, endocrine glands, and gut epithelium. Northern and Western blot analyses demonstrate RNA and protein species corresponding to the cloned PHM, but not to a bifunctional PAM, leading us to predict the existence of separate PHM and PAL genes in Drosophila. The Drosophila PHM gene displays an organization of exons that is highly similar to the PHM-encoding portion of the rat PAM gene. Genetic analysis was consistent with the prediction of separate PHM and PAL gene functions in Drosophila: a P element insertion line containing a transposon within the PHM transcription unit displayed strikingly lower PHM enzyme levels, whereas PAL levels were increased slightly. The lethal phenotype displayed by the dPHM P element insertion indicates a widespread essential function. Reversion analysis indicated that the lethality associated with the insertion chromosome likely is attributable to the P element insertion. These combined data indicate a fundamental evolutionary divergence in the genes coding for critical neurotransmitter biosynthetic enzymes: in Drosophila, the two enzyme activities of PAM are encoded by separate genes.

Amides↗
PubMed1997

PACE4: a subtilisin-like endoprotease with unique properties.

PACE4 is one of the neuroendocrine-specific mammalian subtilisin-related endoproteases believed to function in the secretory pathway. The biosynthesis and secretion of PACE4 have been studied using transfected neuroendocrine and fibroblast cell lines. as well as primary pituitary cultures. ProPACE4 (approx. 106 kDa) is cleaved intracellularly before secretion of PACE4 (approx. 97 kDa); the N-terminal propeptide cleavage is accelerated in a truncated form of PACE4 lacking the Cys-rich C-terminal region (PACE4s). Neither PACE4 nor PACE4s is stored in regulated neuroendocrine secretory granules, whereas pro-opiomelanocortin-derived peptides and prohormone convertase I enter the regulated secretory pathway efficiently. The relatively slow cleavage of the proregion of proPACE4 in primary anterior pituitary cells, followed by rapid secretion of PACE4, is similar to the results for proPACE4 in transfected cell lines. The enzyme activity of PACE4 is distinct from furin and prohormone convertases, both in the marked sensitivity of PACE4 to inhibition by leupeptin and the relative insensitivity of PACE4 to inhibition by Ca2+ chelators and dithiothreitol; PACE4 is not inhibited by the alpha1-antitrypsin Portland variant that is very potent at inhibiting furin. The unique biosynthetic and enzymic patterns seen for PACE4 suggest a role for this neuroendocrine-specific subtilisin-like endoprotease outside the pathway for peptide biosynthesis.

Blotting, Western↗