A Pilot Screen of a Novel Peptide Hormone Library Identified Candidate GPR83 Ligands

2020 ◽  
Vol 25 (9) ◽  
pp. 1047-1063
Author(s):  
Nathan A. Sallee ◽  
Ernestine Lee ◽  
Atossa Leffert ◽  
Silvia Ramirez ◽  
Arthur D. Brace ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Cell Function ◽  
Active Form ◽  
Peptide Hormone ◽  
Biologically Active ◽  
Host Cells ◽  
Functional Screening ◽  
Posttranslational Processing ◽  
Regulated Secretory Pathway ◽  
G Protein Coupled

The identification of novel peptide hormones by functional screening is challenging because posttranslational processing is frequently required to generate biologically active hormones from inactive precursors. We developed an approach for functional screening of novel potential hormones by expressing them in endocrine host cells competent for posttranslational processing. Candidate preprohormones were selected by bioinformatics analysis, and stable endocrine host cell lines were engineered to express the preprohormones. The production of mature hormones was demonstrated by including the preprohormones insulin and glucagon, which require the regulated secretory pathway for production of the active forms. As proof of concept, we screened a set of G-protein-coupled receptors (GPCRs) and identified protein FAM237A as a specific activator of GPR83, a GPCR implicated in central nervous system and regulatory T-cell function. We identified the active form of FAM237A as a C-terminally cleaved, amidated 9 kDa secreted protein. The related protein FAM237B, which is 64% homologous to FAM237A, demonstrated similar posttranslational modification and activation of GPR83, albeit with reduced potency. These results demonstrate that our approach is capable of identifying and characterizing novel hormones that require processing for activity.

Novel salmon cardiac peptide hormone is released from the ventricle by regulated secretory pathway

2000 ◽  
Vol 278 (2) ◽  
pp. E285-E292 ◽  
Author(s):  
Kati Kokkonen ◽  
Heidi Vierimaa ◽  
Sari Bergström ◽  
Virpi Tervonen ◽  
Olli Arjamaa ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Mechanical Load ◽  
Secretory Granules ◽  
Fold Increase ◽  
Peptide Hormone ◽  
Biologically Active ◽  
The Novel ◽  
Endothelin 1 ◽  
Basal Expression ◽  
Regulated Secretory Pathway

We used the secretion of the novel salmon cardiac peptide (sCP) as a model to examine the mechanisms of ventricular hormone release. Mechanical load increased dose dependently the secretion of immunoreactive sCP from isolated perfused salmon ventricle, with 3.3-fold increase when a load of 13 cmH2O was applied. Endothelin-1 (5 nmol/l) was also able to rapidly increase the secretion of sCP. The released peptide corresponded to the biologically active sCP-29, whereas the large ventricular storage consisted of pro-sCP-sized material. With the use of immunoelectron microscopy, a large number of granules containing immunoreactive sCP could be detected in salmon ventricle. As judged by RNA blot analysis, there was very active basal expression of the sCP gene in the ventricle, which was not increased by mechanical load of up to 2-h duration. Our results show that the ventricle actively expresses the gene of sCP, stores the prohormone in secretory granules, and releases the peptide in response to mechanical load and endothelin-1. Thus the salmon ventricle uses the regulated pathway to produce and release a hormone structurally related to the mammalian natriuretic peptides.

scholarly journals Manipulating sorting signals to generate co-expression of somatostatin and eGFP in the regulated secretory pathway from a monocistronic construct

2004 ◽  
Vol 33 (2) ◽  
pp. 523-532 ◽  
Author(s):  
J S Davies ◽  
J L Holter ◽  
D Knight ◽  
S M Beaucourt ◽  
D Murphy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Foreign Protein ◽  
Entry Site ◽  
Fluorescent Markers ◽  
Regulated Secretory Pathway ◽  
Neuroendocrine Systems

Targeted overexpression of biologically active peptides represents a powerful approach to the functional dissection of neuroendocrine systems. However, the requirement to generate separate, biologically active and reporter molecules necessitates the use of internal ribosome entry site (IRES) technology, which often results in preferential translation of the second cistron. We report here a novel approach in which the proteolytic processing machinery of the regulated secretory pathway (RSP) has been exploited to generate multiple mature proteins from a monocistronic construct that encodes a single precursor. This was achieved by duplication of the pre-pro cleavage sites in pre-prosomatostatin cDNA. The duplicated site included 10 flanking amino acids on either side of the Gly-Ala cleavage position. This enabled the incorporation of a foreign protein-coding sequence (in this case, enhanced green fluorescent protein (eGFP)) between these sites. The pre-eGFP-prosomatostatin (PEPS) construct generated co-localized expression of fully processed eGFP and somatostatin to the RSP of transiently transfected AtT20 cells. This approach represents an advance upon bicistronic and other extant approaches to the targeting of multiple, biologically active proteins to neuroendocrine systems, and, importantly, permits the co-expression of fluorescent markers with biologically active neuropeptides. In this study, our demonstration of the fusion of the first 10 amino acids of the prosomatostatin sequence to the N-terminus of eGFP shows that this putative sorting sequence is sufficient to direct expression to the RSP.

scholarly journals Angiogenic Effects of Extracellular Human Immunodeficiency Virus Type 1 Tat Protein and Its Role in the Pathogenesis of AIDS-Associated Kaposi's Sarcoma

2002 ◽  
Vol 15 (2) ◽  
pp. 310-326 ◽  
Author(s):  
Giovanni Barillari ◽  
Barbara Ensoli
Keyword(s):  
Human Immunodeficiency Virus ◽  
Kaposi's Sarcoma ◽  
Secretory Pathway ◽  
Active Form ◽  
Kaposi’S Sarcoma ◽  
Biologically Active ◽  
Viral Gene ◽  
Tat Protein ◽  
Immunodeficiency Virus

SUMMARY The Tat protein of human immunodeficiency virus (HIV) type 1 is a transactivator of viral gene expression that is required for virus replication and spread. Moreover, Tat is released by acutely HIV-infected cells via a leaderless secretory pathway and in a biologically active form that exerts effects on both HIV-infected and uninfected cells from different organs and systems. This review focuses on the activities of extracellular Tat protein on endothelial cells, on angiogenesis, and on the pathogenesis of AIDS-associated angioproliferative diseases such as Kaposi's sarcoma. In particular, we discuss results from different groups indicating that Tat mimics the proangiogenic activities of extracellular matrix molecules and that it enhances the effects of angiogenic factors.

scholarly journals Sorting within the regulated secretory pathway occurs in the trans-Golgi network.

1990 ◽  
Vol 110 (1) ◽  
pp. 1-12 ◽  
Author(s):  
W S Sossin ◽  
J M Fisher ◽  
R H Scheller
Keyword(s):  
Protein Trafficking ◽  
Secretory Pathway ◽  
Egg Laying ◽  
Biologically Active ◽  
Dense Core Vesicle ◽  
Trans Golgi Network ◽  
Prohormone Processing ◽  
Regulated Secretory Pathway ◽  
Two Stages ◽  
Golgi Network

Bioactive peptides cleaved from the egg-laying hormone precursor in the bag cell neurons of Aplysia are sorted into distinct dense core vesicle classes (DCVs). Bag cell prohormone processing can be divided into two stages, an initial cleavage occurring in a late Golgi compartment, which is not blocked by monensin, and later cleavages that occur within DCVs and are blocked by monensin. Prohormone intermediates are sorted in the trans-Golgi network. The large soma-specific DCVs turn over, while the small DCVs are transported to processes for regulated release. Thus, protein trafficking differentially regulates the levels and localization of multiple biologically active peptides derived from a common prohormone.

scholarly journals Differential trafficking of soluble and integral membrane secretory granule-associated proteins

1994 ◽  
Vol 124 (1) ◽  
pp. 33-41 ◽  
Author(s):  
SL Milgram ◽  
BA Eipper ◽  
RE Mains
Keyword(s):  
Cell Surface ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Posttranslational Processing ◽  
Endoproteolytic Cleavage ◽  
Associated Proteins ◽  
Storage Granules ◽  
Regulated Secretory Pathway ◽  
And Storage

The posttranslational processing enzyme peptidylglycine alpha-amidating monooxygenase (PAM) occurs naturally in integral membrane and soluble forms. With the goal of understanding the targeting of these proteins to secretory granules, we have compared the maturation, processing, secretion, and storage of PAM proteins in stably transfected AtT-20 cells. Integral membrane and soluble PAM proteins exit the ER and reach the Golgi apparatus with similar kinetics. Biosynthetic labeling experiments demonstrated that soluble PAM proteins were endoproteolytically processed to a greater extent than integral membrane PAM; this processing occurred in the regulated secretory pathway and was blocked by incubation of cells at 20 degrees C. 16 h after a biosynthetic pulse, a larger proportion of soluble PAM proteins remained cell-associated compared with integral membrane PAM, suggesting that soluble PAM proteins were more efficiently targeted to storage granules. The nonstimulated secretion of soluble PAM proteins peaked 1-2 h after a biosynthetic pulse, suggesting that release was from vesicles which bud from immature granules during the maturation process. In contrast, soluble PAM proteins derived through endoproteolytic cleavage of integral membrane PAM were secreted in highest amount during later times of chase. Furthermore, immunoprecipitation of cell surface-associated integral membrane PAM demonstrated that very little integral membrane PAM reached the cell surface during early times of chase. However, when a truncated PAM protein lacking the cytoplasmic tail was expressed in AtT-20 cells, > 50% of the truncated PAM-1 protein reached the cell surface within 3 h. We conclude that the trafficking of integral membrane and soluble secretory granule-associated enzymes differs, and that integral membrane PAM proteins are less efficiently retained in maturing secretory granules.

Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells

1991 ◽  
Vol 260 (5) ◽  
pp. G783-G788 ◽  
Author(s):  
D. F. Daugherty ◽  
C. J. Dickinson ◽  
T. Takeuchi ◽  
D. Bachwich ◽  
T. Yamada
Keyword(s):  
Cell Line ◽  
Peptide Hormone ◽  
Biologically Active ◽  
Molecular Forms ◽  
Posttranslational Processing ◽  
Packaging Cell Line ◽  
Viral Stock ◽  
Peptide Precursors ◽  
Cdna Construct ◽  
Human Gastrin

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.

Expression and processing of prohormones in nonendocrine cells

1993 ◽  
Vol 264 (3) ◽  
pp. G553-G560
Author(s):  
C. J. Dickinson ◽  
T. Takeuchi ◽  
Y. J. Guo ◽  
B. T. Stadler ◽  
T. Yamada
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Secretory Granules ◽  
Fibroblast Cell ◽  
Peptide Hormone ◽  
Biologically Active ◽  
Cdna Clones ◽  
Posttranslational Processing ◽  
Pancreatic Cell ◽  
Carboxy Terminal

Pancreatic polypeptide (PP) and gastrin are initially synthesized as larger precursors that require posttranslational processing to produce the biologically active peptides. These steps include tryptic cleavage at paired basic residues, their subsequent removal by carboxypeptidase H, and formation of a carboxy-terminal amide moiety via the action of peptidyl-glycine alpha-amidating monooxygenase (PAM). Although these posttranslational processing reactions are presumed to occur primarily in the secretory granule of endocrine cells, nonendocrine cells that do not possess these structures nevertheless are able to posttranslationally process a wide variety of proteins destined for export. In these studies we sought to determine whether the mechanisms for prohormone processing are present in nonendocrine cell lines. We examined two fibroblast cell lines (psi-2, BHK) a hepatocyte cell line (Hepa), and an exocrine pancreatic cell line (AR42J). We used the pZIPneo(SVX) retroviral vector to express cDNA clones encoding human PP and gastrin in the nonendocrine cells. Transfected psi-2, BHK, and Hepa cells produced a precursor of PP that appeared to be secreted constitutively, with little remaining in intracellular stores. Almost no posttranslational processing of the PP precursor was evident in these cells. By contrast, AR42J cells were capable of expressing and storing fully processed and carboxy-terminally amidated PP and gastrin. These data support the notion that the sorting mechanisms in endocrine and exocrine cells are similar and that the posttranslational processing of peptide hormone precursors requires storage in secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Minireview: How Peptide Hormone Vesicles Are Transported to the Secretion Site for Exocytosis

2008 ◽  
Vol 22 (12) ◽  
pp. 2583-2595 ◽  
Author(s):  
Joshua J. Park ◽  
Y. Peng Loh
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Hormone Secretion ◽  
Release Site ◽  
Peptide Hormone ◽  
Adaptor Proteins ◽  
Transport Systems ◽  
Cytosolic Proteins ◽  
Readily Releasable Pool ◽  
Regulated Secretory Pathway

Abstract Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.

scholarly journals Developmental Changes in the Pattern of Ghrelin’s Acyl Modification and the Levels of Acyl-Modified Ghrelins in Murine Stomach

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2709-2715 ◽  
Author(s):  
Yoshihiro Nishi ◽  
Hiroshi Hiejima ◽  
Hiroharu Mifune ◽  
Takahiro Sato ◽  
Kenji Kangawa ◽  
...  
Keyword(s):  
Active Form ◽  
Peptide Hormone ◽  
Decanoic Acid ◽  
Biologically Active ◽  
Acylated Ghrelin ◽  
Amino Acid Peptide ◽  
Total Ghrelin ◽  
Wet Weight ◽  
Milk Lipids ◽  
Acyl Ghrelin

Abstract Ghrelin is an acylated peptide hormone secreted primarily from endocrine cells in the stomach. The major active form of ghrelin is a 28-amino acid peptide with an n-octanoyl modification at Ser3 (n-octanoyl ghrelin), which is essential for its activity. In addition to n-octanoyl ghrelin, other forms of ghrelin peptide exist, including des-acyl ghrelin, which lacks an acyl modification, and other minor acylated ghrelin species, such as n-decanoyl ghrelin, whose Ser3 residue is modified by n-decanoic acid. Multiple reports have identified various physiological functions of ghrelin. However, until now, there have been no reports that explore the process of ghrelin acyl modification, and only a few studies have compared the levels of des-acyl, n-octanoyl, and/or other minor populations of acylated ghrelin peptides. In this study we report that the amount of n-octanoyl ghrelin in murine stomachs increases gradually during the suckling period to a maximal level at 3 wk of age and falls sharply after the initiation of weaning. However, the concentration (picomoles per milligram of wet weight tissue) of total ghrelin, which includes des-acyl and all acylated forms of ghrelin peptides with intact C termini in murine stomach, remains unchanged across this suckling-weaning transition. Prematurely weaned mice exhibited a significant decrease in the amount of n-octanoyl or n-decanoyl ghrelin in the stomach. Orally ingested glyceryl trioctanoate, a medium-chain triacylglyceride rich in milk lipids, significantly increased the level of n-octanoyl-modified ghrelin in murine stomach. Fluctuations in the proportion of this biologically active, acyl-modified ghrelin could contribute to or be influenced by the change in energy metabolism during the suckling-weaning transition.

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