T Cell Recognition of Defined Peptides and Autoimmunity

The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, ␤-lactamase, that localizes to parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited ␤-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine ␣-SNAP augmented release. Guanosine 5-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.

Section: Fig 2 Kinetics Of Bla Secretion From Extracellular T Gondiimentioning

confidence: 99%

Constitutive Calcium-independent Release of Toxoplasma gondii Dense Granules Occurs through the NSF/SNAP/SNARE/Rab Machinery

Chaturvedi¹,

Qi²,

Coleman³

et al. 1999

“…Second messengers and protein kinases have also been shown to influence various transport processes. For instance, cAMP and protein kinase A activity have been reported to stimulate apical directed transcytosis and secretion in epithelial cells (3)(4)(5)(6). Protein kinase A seems to exert a differential regulatory role in polarized cells.…”

mentioning

confidence: 99%

A Regulatory Role for cAMP-dependent Protein Kinase in Protein Traffic along the Exocytic Route

Muñiz¹,

Alonso²,

Hidalgo

et al. 1996

The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization.

“…Thus, application of kinase activators or inhibitors (or indeed purified kinases themselves) to permeabilized cells, where receptors and ion channels are bypassed, is a more rigorous demonstration of a role for protein kinases in the direct regulation of the exocytotic machinery. However, a review of the literature reveals only PKA and PKC or their pharmacological effectors produce an almost universal enhancement of Ca 2ϩ -triggered exocytosis in all secretory models studied, for example nerve terminals (11), chromaffin cells (12)(13)(14), PC12 cells (15), , pancreatic acinar cells (17), parotid acinar cells (18), SPOC1 cells (19), neutrophils (20), and mast cells (21). Therefore, identification of PKA or PKC exocytotic substrates will reveal fundamental mechanisms for the direct regulation of exocytosis by phosphorylation.…”

mentioning

confidence: 99%

Phosphorylation of Cysteine String Protein by Protein Kinase A

Evans¹,

Wilkinson

Graham³

et al. 2001

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-␣ (␣-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular ␣-SNAP failed to detect 32 P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser 10 . PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser 10 phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.Exocytosis is the final stage of the secretory pathway and involves the fusion of secretory vesicles with the plasma membrane in a constitutive or regulated manner (1). In regulated exocytosis, vesicles accumulate in the cytoplasm and only fuse with the plasma membrane upon receipt of an appropriate stimulus (usually, but not always, an increase in intracellular free Ca 2ϩ ). As regulated exocytosis is the basis of chemical transmission in the brain, much research has been devoted to uncovering its molecular mechanism. This has revealed the involvement of a large number of proteins (2, 3), which can be classified into three groups. The first group, proteins involved in vesicle fusion events in all eukaryotes, includes the SNAP 1 receptors, SNAPs, RABs, and the Sec1 family. The second group comprises proteins involved in regulated exocytosis in various cell types and diverse organisms but absent in yeast. This group includes the synaptotagmins and cysteine string proteins (CSP). The third class can be defined as proteins whose role in regulated exocytosis is cell type-specific. An example from this group is the synapsins, which are important modulators of the synaptic vesicle cycle in neurons (4). The complex interactions between the numerous proteins of these classes presumably enables sophisticated fine-tuning of exocytosis to suit the particular physiological needs of each cell type.In addition to the cell type-specific repertoire of exocytotic proteins expressed, further control over the exocytotic mechanism can be exerted post-translationally (5). Indeed, a large number of studies have implicated protein kinases in the modulation of regulated exocytosis from many cell types by using cellpermeable inhibitors or activators, including Ca 2ϩ /calmodulindependent protein kinase II (6, 7), mitogen-activated protein kinase (8), cGMP-dependent protein kinase (9), and tyrosine kinases (8). However, one shortfall of t...