VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways

AbstractInflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow–derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8–deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8–deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3–positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways

Preparation and In Vitro

For preventing premature drug release in neutral environment and avoiding them being trapped into the endosomal/lysosomal system, we developed a novel iron silicate@liposome hybrid (ILH) formulation, which can be used as a carrier to transport doxorubicin (DOX) in a pH-sensitive manner and to escape from endosomal/lysosomal trapping through “proton-sponge” effect. The high intensity of photoacoustic signal from in vitro photoacoustic imaging (PAI) experiments suggests that it is a promising candidate for PAI agent, providing the potential for simultaneously bioimaging and cancer-targeting drug delivery. Cytotoxicity of our formulation toward tumor cells was remarkably higher than free DOX (48.4±7.7% and 26.2±8.4%, P<0.001). Confocal laser scanning microscopy experiments showed the enhanced transportation and enrichment process of DOX in QSG-7703 cells. Taking together, we developed an easy approach to construct a multifunctional anticancer drug delivery/imaging system with a potency as a PAI agent. The strategy of combining drug carrier and imaging agent is an emerging platform for further construction of nanoparticle and may play a significant role in cancer therapy and diagnosis

Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT

Severe acute respiratory syndrome coronavirus (SARS-CoV) 7a is an accessory protein with no known homologues. In this study, we report the interaction of a SARS-CoV 7a and small glutamine-rich tetratricopeptide repeat-containing protein (SGT). SARS-CoV 7a and human SGT interaction was identified using a two-hybrid system screen and confirmed with interaction screens in cell culture and cellular co-localization studies. The SGT domain of interaction was mapped by deletion mutant analysis and results indicated that tetratricopeptide repeat 2 (aa 125-158) was essential for interaction. We also showed that 7a interacted with SARS-CoV structural proteins M (membrane) and E (envelope), which have been shown to be essential for virus-like particle formation. Taken together, our results coupled with data from studies of the interaction between SGT and HIV-1 vpu indicated that SGT could be involved in the life-cycle, possibly assembly of SARS-CoV.IS

A role for oxysterol-binding protein–related protein 5 in endosomal cholesterol trafficking

ORP5 works together with Niemann Pick C-1 to facilitate exit of cholesterol from endosomes and lysosomes

SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane

G protein–coupled receptors rely on the PDZ domain of SNX27 for endosomal recycling

The Seoul National University AGN Monitoring Project. IV. Hα Reverberation Mapping of Six AGNs and the Hα Size–Luminosity Relation

The broad-line region (BLR) size–luminosity relation has paramount importance for estimating the mass of black holes in active galactic nuclei (AGNs). Traditionally, the size of the Hβ BLR is often estimated from the optical continuum luminosity at 5100 Å, while the size of the Hα BLR and its correlation with the luminosity is much less constrained. As a part of the Seoul National University AGN Monitoring Project, which provides 6 yr photometric and spectroscopic monitoring data, we present our measurements of the Hα lags of high-luminosity AGNs. Combined with the measurements for 42 AGNs from the literature, we derive the size–luminosity relations of the Hα BLR against the broad Hα and 5100 Å continuum luminosities. We find the slope of the relations to be 0.61 ± 0.04 and 0.59 ± 0.04, respectively, which are consistent with the Hβ size–luminosity relation. Moreover, we find a linear relation between the 5100 Å continuum luminosity and the broad Hα luminosity across 7 orders of magnitude. Using these results, we propose a new virial mass estimator based on the Hα broad emission line, finding that the previous mass estimates based on scaling relations in the literature are overestimated by up to 0.7 dex at masses lower than 107M⊙

The Seoul National University AGN Monitoring Project III: Hβ\beta lag measurements of 32 luminous AGNs and the high-luminosity end of the size--luminosity relation

We present the main results from a long-term reverberation mapping campaign carried out for the Seoul National University Active Galactic Nuclei (AGN) Monitoring Project. High-quality data were obtained during 2015-2021 for 32 luminous AGNs (i.e., continuum luminosity in the range of $10^{44-46}$ erg s$^{-1}$) at a regular cadence, of 20-30 days for spectroscopy and 3-5 days for photometry. We obtain time lag measurements between the variability in the H$\beta$ emission and the continuum for 32 AGNs; twenty-five of those have the best lag measurements based on our quality assessment, examining correlation strength, and the posterior lag distribution. Our study significantly increases the current sample of reverberation-mapped AGNs, particularly at the moderate to high luminosity end. Combining our results with literature measurements, we derive a H$\beta$ broad line region size--luminosity relation with a shallower slope than reported in the literature. For a given luminosity, most of our measured lags are shorter than the expectation, implying that single-epoch black hole mass estimators based on previous calibrations could suffer large systematic uncertainties.Comment: Accepted by ApJ; 39 pages, 22 figure

Trafficking-Deficient G572R-hERG and E637K-hERG Activate Stress and Clearance Pathways in Endoplasmic Reticulum

Background: Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins. Methods: pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined. Conclusion: Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin wa