scholarly journals Small molecules that inhibit the late stage of Munc13-4–dependent secretory granule exocytosis in mast cells

2018 ◽  
Vol 293 (21) ◽  
pp. 8217-8229 ◽  
Author(s):  
Stephen Bruinsma ◽  
Declan J. James ◽  
Melanie Quintana Serrano ◽  
Joseph Esquibel ◽  
Sang Su Woo ◽  
...  
Keyword(s):  
Mast Cells ◽  
Secretory Granule ◽  
Small Molecule ◽  
Secretory Pathway ◽  
Small Molecule Inhibitors ◽  
Protein Isoforms ◽  
Secretory Cells ◽  
Granule Exocytosis ◽  
Regulated Secretory Pathway ◽  
Liposome Fusion

Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain–containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4–membrane interactions and thereby inhibit Munc13-4–dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.

scholarly journals A biosynthetic regulated secretory pathway in constitutive secretory cells.

1996 ◽  
Vol 133 (6) ◽  
pp. 1177-1191 ◽  
Author(s):  
R A Chavez ◽  
S G Miller ◽  
H P Moore
Keyword(s):  
Secretory Pathway ◽  
Phorbol Esters ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Secretory Cells ◽  
Ionophore A23187 ◽  
Intact Cells ◽  
Rab Protein ◽  
Regulated Secretory Pathway ◽  
Dense Core Granules

It has frequently been proposed that while the constitutive secretory pathway is present in all cells, the regulated secretory pathway is found only in specialized cells such as neuronal, endocrine, or exocrine types. In this study we provide evidence that suggests that this distinction is not as restrictive as proposed. We have identified a population of post-Golgi storage vesicles in several constitutive secretory cells using [35S]SO4-labeled glycosaminoglycan (GAG) chains as a marker. A fraction of this pool of vesicles can undergo exocytosis in response to stimuli such as cytoplasmic Ca2+ and phorbol esters. The effect of Ca2+ was demonstrated both in intact cells in the presence of the ionophore A23187 and in streptolysin-O-permeabilized semi-intact cells. N-ethylmaleiimide, under conditions known to block regulated and constitutive secretion, inhibited the stimulated secretion from these cells, suggesting that the observed release of labeled GAG chains was not due to a leakage artefact. Subcellular fractionation revealed that the stored GAG chains were in low-density membrane granules (d approximately 1.12 g/ml), whose size was greater than that of synaptic-like vesicles found in PC12 cells. In addition, in CHO cells that express epitope-tagged rab 3D, the labeled GAG chains were found to cofractionate with the exogenous rab protein. When expressed in the regulated cell line AtT-20, this tagged rab protein was found to colocalize with ACTH-containing dense-core granules by indirect immunofluorescence. Taken together, these results provide evidence for the presence of a cryptic regulated secretory pathway in "constitutive" cells and suggest that the regulated secretory pathway is more widespread amongst different cell types than previously believed.

scholarly journals Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

2021 ◽  
Vol 22 (21) ◽  
pp. 12007
Author(s):  
Eva Pauwels ◽  
Ralf Schülein ◽  
Kurt Vermeire
Keyword(s):  
Small Molecule ◽  
Cell Biology ◽  
High Throughput Screening ◽  
Secretory Pathway ◽  
Small Molecule Inhibitors ◽  
Protein Translocation ◽  
Central Component ◽  
Antibiotic Drugs ◽  
Screening Strategies ◽  
Translocation Pathway

Proteins targeted to the secretory pathway start their intracellular journey by being transported across biological membranes such as the endoplasmic reticulum (ER). A central component in this protein translocation process across the ER is the Sec61 translocon complex, which is only intracellularly expressed and does not have any enzymatic activity. In addition, Sec61 translocon complexes are difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its function has thus been notoriously difficult. However, such translocation inhibitors may not only be valuable tools for cell biology, but may also represent novel anticancer drugs, given that cancer cells heavily depend on efficient protein translocation into the ER to support their fast growth. In this review, different inhibitors of protein translocation will be discussed, and their specific mode of action will be compared. In addition, recently published screening strategies for small molecule inhibitors targeting the whole SRP-Sec61 targeting/translocation pathway will be summarized. Of note, slightly modified assays may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex, in order to identify novel antibiotic drugs.

Two proteins associated with secretory granule membranes identified in chicken regulated secretory cells

1994 ◽  
Vol 107 (5) ◽  
pp. 1297-1308
Author(s):  
J.L. Thomas ◽  
A. Stieber ◽  
N. Gonatas
Keyword(s):  
Secretory Granule ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Cell Types ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Electrophoretic Patterns ◽  
Granule Membrane ◽  
The Central Nervous System ◽  
Ultrathin Frozen Sections

Lately, we have identified two polypeptides of 92–94 kDa (GRL1) and 45–60 kDa (GRL2), expressed in cytoplasmic granules of chicken granulocytes and thrombocytes. Here, we report that GRL1 and GRL2 are widely distributed in all exocrine and several endocrine cell types, but not in neurons of the central nervous system, during late stages of embryonic development, as well as in newly hatched and two-month-old chickens. Immunogold studies in ultrathin frozen sections of pancreatic acinar cells show that GRL1 and GRL2 are co-localized at the periphery of zymogen granules, in granules fused with apical acinar membranes and on apical membranes of acini, while the pregranular compartments of the secretory pathway are weakly or not labeled. Semiquantitative morphometric studies indicate that GRL1 and GRL2 are equally distributed in secretory granules. A variety of physical and metabolic studies reveal that GRL2, a highly N-glycosylated polypeptide, is an intrinsic membrane protein, while GRL1 is a peripheral membrane polypeptide released by Na2CO3 treatment of granulocyte membranes. In all hematopoietic, exocrine or endocrine cells examinated, GRL1 shows identical electrophoretic patterns, while GRL2 is identified as a diffuse band, at 40–65 kDa, in hematopoietic and pancreatic cells. Taken together, the morphological and biochemical studies indicate that GRL1 and GRL2 are components of the secretory granule membrane in chicken exocrine, endocrine and hemopoietic cell types.

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.

scholarly journals Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts

2003 ◽  
Vol 369 (3) ◽  
pp. 453-460 ◽  
Author(s):  
Chun-Fa ZHANG ◽  
Savita DHANVANTARI ◽  
Hong LOU ◽  
Y. Peng LOH
Keyword(s):  
Fusion Protein ◽  
Lipid Rafts ◽  
Secretory Granule ◽  
Secretory Pathway ◽  
Full Length ◽  
Density Gradients ◽  
Carboxypeptidase E ◽  
Neuro2a Cells ◽  
Sorting Receptor ◽  
Regulated Secretory Pathway

Carboxypeptidase E (CPE) functions as a regulated secretory pathway sorting receptor for several prohormones, including pro-opiomelanocortin (POMC), proenkephalin and proinsulin. The association of CPE with lipid rafts in the trans-Golgi network and secretory granule membranes is necessary for its sorting receptor function. We now provide evidence that a domain within the C-terminal 25 residues of CPE functions as a signal for both raft association and the sorting of CPE to the regulated secretory pathway. A fusion protein containing the extracellular domain of the human interleukin-2 receptor Tac (N-Tac) and the C-terminal 25 amino acids of CPE was transfected into Neuro2A cells. This fusion protein floated in sucrose density gradients, indicating raft association, and co-localized with chromogranin A (CGA), a secretory granule marker. To define further a minimum sequence required for raft association and sorting, deletion mutants of CPE that lacked the C-terminal four or 15 residues (CPE-Δ4 and CPE-Δ15 respectively) were transfected into a clone of CPE-deficient Neuro2A cells. In contrast with full-length CPE, neither CPE-Δ4 nor CPE-Δ15 floated in sucrose density gradients. The sorting of both CPE-Δ4 and CPE-Δ15 to the regulated secretory pathway was impaired, as indicated by significantly increased basal secretion and a lack of response to stimulation. Additionally, there was a significant decrease in the co-localization of mutant CPE immunofluorescence with CGA when compared with full-length CPE. Finally, the sorting of the prohormone POMC to the regulated pathway was impaired in cells transfected with either CPE-Δ4 or CPE-Δ15. We conclude that the sorting of CPE to the regulated secretory pathway in endocrine cells is mediated by lipid rafts, and that the C-terminal four residues of CPE, i.e. Thr431-Leu-Asn-Phe434, are required for raft association and sorting.

scholarly journals Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles

1998 ◽  
Vol 141 (2) ◽  
pp. 359-371 ◽  
Author(s):  
Judith Klumperman ◽  
Regina Kuliawat ◽  
Janice M. Griffith ◽  
Hans J. Geuze ◽  
Peter Arvan
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Coated Vesicle ◽  
Secretory Cells ◽  
Β Cells ◽  
Mannose 6 Phosphate ◽  
Regulated Secretory Pathway ◽  
Granule Maturation

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ∼90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.

scholarly journals Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2

1993 ◽  
Vol 291 (1) ◽  
pp. 289-296 ◽  
Author(s):  
F A Leblond ◽  
G Viau ◽  
J Lainé ◽  
D Lebel
Keyword(s):  
Secretory Granule ◽  
Secretory Pathway ◽  
Relative Proportion ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Granule Membrane ◽  
Regulated Secretory Pathway ◽  
Golgi Network

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. To elucidate the biogenesis of the secretory granule in the exocrine pancreas, we reconstituted in vitro the conditions of pH and ions believed to exist in the TGN using the end product of this sorting process, the zymogen granule contents. Protein aggregation was dependent on pH (acidic) and on the presence of cations (10 mM Ca2+, 150 mM K+) to reproduce the pattern of proteins found in the granule. The constitutive secretory protein IgG was excluded from these aggregates. Zymogen aggregation correlated with the relative proportion of the major granule membrane protein GP-2 in the assay. These results show that the glycosylphosphatidylinositol-anchored protein GP-2 co-aggregates with zymogens in the acidic environment believed to exist in the pancreatic TGN, and thus suggest that GP-2 would function as a membrane anchor for zymogen aggregates, facilitating their entrapment in budding vesicles directed towards the regulated secretory pathway.

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