From endoplasmic reticulum to secretory granules: role of zinc in the secretory pathway of growth hormone

Endocrine and neuroendocrine cells differ from cells which rapidly release all their secreted proteins in that they store some secretory proteins in concentrated forms in secretory granules to be rapidly released when cells are stimulated. Protein aggregation is considered as the first step in the secretory granule biosynthesis and, at least in the case of prolactin and growth hormone, greatly depends on zinc ions that facilitate this process. Hence, regulation of cellular zinc transport especially that within the regulated secretory pathway is of importance to understand. Various zinc transporters of Slc30a/ZnT and Slc39a/Zip families have been reported to fulfil this role and to participate in fine tuning of zinc transport in and out of the endoplasmic reticulum, Golgi complex and secretory granules, the main cellular compartments of the regulated secretory pathway. In this review, we will focus on the role of zinc in the formation of hormone-containing secretory granules with special emphasis on conditions required for growth hormone dimerization/aggregation. In addition, we highlight the role of zinc transporters that govern the process of zinc homeostasis in the regulated hormone secretion.

Direct observation of molecular arrays in the organized smooth endoplasmic reticulum.

BACKGROUND Tubules and sheets of endoplasmic reticulum perform different functions and undergo inter-conversion during different stages of the cell cycle. Tubules are stabilized by curvature inducing resident proteins, but little is known about the mechanisms of endoplasmic reticulum sheet stabilization. Tethering of endoplasmic reticulum membranes to the cytoskeleton or to each other has been proposed as a plausible way of sheet stabilization. RESULTS Here, using fluorescence microscopy we show that the previously proposed mechanisms, such as membrane tethering via GFP-dimerization or coiled coil protein aggregation do not explain the formation of the calnexin-induced organized smooth endoplasmic reticulum membrane stacks. We also show that the LINC complex proteins known to serve a tethering function in the nuclear envelope are excluded from endoplasmic reticulum stacks. Finally, using cryo-electron microscopy of vitreous sections methodology that preserves cellular architecture in a hydrated, native-like state, we show that the sheet stacks are highly regular and may contain ordered arrays of macromolecular complexes. Some of these complexes decorate the cytosolic surface of the membranes, whereas others appear to span the width of the cytosolic or luminal space between the stacked sheets. CONCLUSION Our results provide evidence in favour of the hypothesis of endoplasmic reticulum sheet stabilization by intermembrane tethering.

GPIb is involved in platelet aggregation induced by mucetin, a snake C-type lectin protein from Chinese habu (Trimeresurus mucrosquamatus) venom

Mucetin (Trimeresurus mucrosquamatus venom activator, TMVA) is a potent platelet activator purified from Chinese habu (Trimeresurus mucrosquamatus) venom. It belongs to the snake venom heterodimeric C-type lectin family and exists in several multimeric forms. We now show that binding to platelet glycoprotein (GP) Ib is involved in mucetin-induced platelet aggregation. Antibodies against GPIb as well as the GPIb-blocking C-type lectin echicetin inhibited mucetin-induced platelet aggregation. Binding of GPIb was confirmed by affinity chromatography and Western blotting. Antibodies against GPVI inhibited convulxin- but not mucetin-induced aggregation. Signalling by mucetin involved rapid tyrosine phosphorylation of a number of proteins including Syk, Src, LAT and PLC gamma 2. Mucetin-induced phosphorylation of the Fc gamma chain of platelet was greatly promoted by inhibition of alpha(IIb)beta(3) by the peptidomimetic EMD 132338, suggesting that phosphatases downstream of alpha(IIb)beta(3) activation are involved in dephosphorylation of Fc gamma. Unlike other multimeric snake C-type lectins that act via GPIb and only agglutinate platelets, mucetin activates alpha(IIb)beta(3). Inhibition of alpha(IIb)beta(3) strongly reduced the aggregation response to mucetin, indicating that activation of alpha(IIb)beta(3) and binding of fibrinogen are involved in mucetin-induced platelet aggregation. Apyrase and aspirin also inhibit platelet aggregation induced by mucetin, suggesting that ADP and thromboxane A2 are involved in autocrine feedback. Sequence and structural comparison with closely related members of this protein family point to features that may be responsible for the functional differences.

Inhibition of ongoing allergic reactions using a novel anti-IgE DARPin-Fc fusion protein

Aggregation of the high-affinity IgE receptor (FcεRI) with the low-affinity IgG receptor (FcγRIIb) on basophils or mast cells has been shown to inhibit allergen-induced cell degranulation. Molecules cross-linking these two receptors might therefore be of interest for the treatment of allergic disorders. Here, we demonstrate the generation of a novel bispecific fusion protein efficiently aggregating FcεRI-bound IgE with FcγRIIb on the surface of basophils to prevent pro-inflammatory mediator release.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVI

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (alpha), 20 kDa (beta1) and 22 kDa (beta2), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that beta1 and beta2 subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between beta1 and beta2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the alpha and beta, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against alphaIIbbeta3 inhibited the aggregation response to stejnulxin, indicating that activation of alphaIIbbeta3 and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbalpha or alpha2beta1 as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

The platelet protein kinase C substrate pleckstrin binds directly to SDPR protein

Pleckstrin is a modular platelet protein consisting of N- and C-terminal pleckstrin homology (PH) domains, a central dishevelled egl10 and pleckstrin (DEP) domain and a phosphorylation region. Following agonist-induced platelet stimulation, dimeric pleckstrin translocates to the plasma membrane, is phosphorylated and then monomerizes. A recent study found that pleckstrin null platelets from a knockout mouse have a defect in granule secretion, actin polymerization and aggregation. However, the mechanism of pleckstrin signaling for this function is unknown. Our recent studies have led to the identification of a novel pleckstrin-binding protein, serum deprivation response protein (SDPR), by co-immunoprecipitation, GST-pulldowns and nanospray quadruple time of flight mass spectrometry. We show that this interaction occurs directly through N-terminal sequences of pleckstrin. Both pleckstrin and SDPR are phosphorylated by protein kinase C (PKC), but the interaction between pleckstrin and SDPR was shown to be independent of PKC inhibition or activation. These results suggest that SDPR may facilitate the translocation of nonphosphorylated pleckstrin to the plasma membrane in conjunction with phosphoinositides that bind to the C-terminal PH domain. After binding of pleckstrin to the plasma membrane, its phosphorylation by PKC exerts downstream effects on platelet aggregation/secretion.

Surface charge of polymer coated SPIONs influences the serum protein adsorption, colloidal stability and subsequent cell interaction in vitro

It is known that the nanoparticle-cell interaction strongly depends on the physicochemical properties of the investigated particles. In addition, medium density and viscosity influence the colloidal behaviour of nanoparticles. Here, we show how nanoparticle-protein interactions are related to the particular physicochemical characteristics of the particles, such as their colloidal stability, and how this significantly influences the subsequent nanoparticle-cell interaction in vitro. Therefore, different surface charged superparamagnetic iron oxide nanoparticles were synthesized and characterized. Similar adsorbed protein profiles were identified following incubation in supplemented cell culture media, although cellular uptake varied significantly between the different particles. However, positively charged nanoparticles displayed a significantly lower colloidal stability than neutral and negatively charged particles while showing higher non-sedimentation driven cell-internalization in vitro without any significant cytotoxic effects. The results of this study strongly indicate therefore that an understanding of the aggregation state of NPs in biological fluids is crucial in regards to their biological interaction(s).

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.