Granule Exocytosis Research Articles

Abstract 4485: Enhancing antitumor immune responses with clinical BET bromodomain inhibitor RG6146

Abstract The BET family of proteins bind to acetylated lysine residues on histone proteins and transcription factors to co-activate gene expression. BET proteins regulate the expression of oncogenes and can control the activity of various oncogenic transcription programs and have thereby emerged as therapeutic targets for the treatment of cancer. RG6146 is a novel non-covalent inhibitor of BET proteins that is in early phase clinical trials for the treatment of haematological and solid malignancies. The anti-tumor activity of BET inhibitors has primarily been attributed to tumor cell intrinsic effects, however increasing evidence suggests BET inhibitors modulate anti-tumor immune responses. Here, we examined the anti-solid tumor activity of RG6146 and evaluated the ability of RG6146 to enhance anti-tumor CD8+ T-cell responses. To model anti-tumor CD8+ T-cell responses in vitro, syngeneic colon and breast tumor cells expressing ovalbumin (Ova) antigen were co-cultured with activated CD8+ T-cells derived from OT-1 transgenic mice. RG6146 functionally increased the activity of both wild-type and perforin-deficient OT-1 T-cells, leading to significantly enhanced T cell-mediated tumor cell death in a time- and dose-dependent manner. Mechanistic studies revealed that enhanced tumor cell death induced by RG6146 was dependent upon CD8+ T-cell derived tumor necrosis factor-α (TNF-α), independent of perforin/granzyme-dependent granule exocytosis. As RG6146 did not increase TNF-α production in CD8+ T-cells, we hypothesized RG6146 may sensitize tumors cells to TNF-α. Indeed, screening of cell lines revealed that BET inhibition significantly enhanced TNF-α-induced cell death in solid tumors of diverse origin. Underlying this response, we demonstrate using RNA- and ChIP-sequencing that BET inhibition suppresses transcription of pro-survival NF-kB target genes to elicit a potent pro-apoptotic phenotype. Finally, using syngeneic solid tumor models, we demonstrated that the adaptive immune system promotes the efficacy of RG6146 and evaluated the ability of RG6146 to therapeutically augment cancer immunotherapies in vivo. Taken together, these data demonstrate that RG6146 is a potent BET bromodomain inhibitor with multi-faceted anti-cancer activity against solid tumors. We have identified a novel immunological TNF-α-dependent mechanism of bystander tumor killing by which BET inhibitors promote anti-tumor responses in vivo. Finally, we provide evidence that BET inhibition will augment the activity of cancer immunotherapies, establishing a strong rationale to evaluate these combinations in the clinic. Citation Format: Simon J. Hogg, Lisa Wellinger, Daniel Rohle, Ricky W. Johnstone. Enhancing antitumor immune responses with clinical BET bromodomain inhibitor RG6146 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4485.

SNAP23 is required for constitutive and regulated exocytosis in mouse oocytes†.

Mammalian oocytes are stored in the ovary for prolonged periods, and arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using Trim-away, a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits regulated exocytosis in response to a Ca2+ stimulus (cortical granule exocytosis), as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs.

Acute bilirubin ditaurate exposure attenuates ex vivo platelet reactive oxygen species production, granule exocytosis and activation

BackgroundBilirubin, a by-product of haem catabolism, possesses potent endogenous antioxidant and platelet inhibitory properties. These properties may be useful in inhibiting inappropriate platelet activation and ROS production; for example, during storage for transfusion. Given the hydrophobicity of unconjugated bilirubin (UCB), we investigated the acute platelet inhibitory and ROS scavenging ability of a water-soluble bilirubin analogue, bilirubin ditaurate (BRT) on ex vivo platelet function to ascertain its potential suitability for inclusion during platelet storage. MethodsThe inhibitory potential of BRT (10–100 μM) was assessed using agonist induced platelet aggregation, dense granule exocytosis and flow cytometric analysis of P-selectin and GPIIb/IIIa expression. ROS production was investigated by analysis of H2DCFDA fluorescence following agonist simulation while mitochondrial ROS production investigated using MitoSOX™ Red. Platelet mitochondrial membrane potential and viability was assessed using TMRE and Zombie Green™ respectively. ResultsOur data shows ≤35 μM BRT significantly inhibits both dense and alpha granule exocytosis as measured by ATP release and P-selectin surface expression, respectively. Significant inhibition of GPIIb/IIIa expression was also reported upon ≤35 μM BRT exposure. Furthermore, platelet exposure to ≤10 μM BRT significantly reduces platelet mitochondrial ROS production. Despite the inhibitory effect of BRT, platelet viability, mitochondrial membrane potential and agonist induced aggregation were not perturbed. ConclusionsThese data indicate, for the first time, that BRT, a water-soluble bilirubin analogue, inhibits platelet activation and reduces platelet ROS production ex vivo and may, therefore, may be of use in preserving platelet function during storage.

IPLA2 Activation Mediates Granular Exocytosis and Corrects Microbicidal Defects in ROS-Deficient and CGD Human Neutrophils.

The ubiquitous calcium-independent phospholipase A2 enzyme (iPLA2) is inhibited by calmodulin binding and known to be responsible for phospholipid remodeling housekeeping functions including granule exocytosis-associated membrane fusion in normal human neutrophils. We evaluate in human neutrophils the iPLA2 secretagogue effects using normal neutrophils, where reactive oxygen species (ROS) generation has been blocked by diphenyleneiodonium, as well as in neutrophils from chronic granulomatous disease (CGD) patients. Neutrophils were pretreated with W7, a calmodulin inhibitor known to activate iPLA2 and exocytosis of granules, and vesicles as well as intra- and extra-microbicidal activity against Staphylococcus aureus and Aspergillus fumigatus were evaluated. W7 increases exocytosis of primary, secondary, and tertiary granules and vesicles and improves neutrophil microbicidal activity against S. aureus and A. fumigatus. In neutrophils, calmodulin-mediated iPLA2 inhibition controls granule and vesicle exocytosis in the phagosome and in the extracellular microenvironment. Relieving iPLA2 inhibition results in increased exocytosis of primary, secondary, and tertiary granules and secretory vesicles with correction of defective intracellular and extracellular microbicidal activity. In CGD patients presenting ROS defective production, this increase in the non-oxidative killing pathway partially corrects their microbicidal defects.

Recent Advances in the Understanding of Thrombosis.

Recent Advances in the Understanding of Thrombosis.

LPA5 Is an Inhibitory Receptor That Suppresses CD8 T-Cell Cytotoxic Function via Disruption of Early TCR Signaling.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Fusion pore regulation by cAMP/Epac2 controls cargo release during insulin exocytosis.

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

HCV-Induced Beta Cell Dysfunction Contributes to HCV-Associated Type-2 Diabetes

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus (T2DM). Although the mechanisms of insulin resistant in hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of beta cell function in HCV-associated diabetes. Here, we found HCV could infected the beta cell directly and impair pancreatic beta cell function in CHC and mouse model. Both first-phase and second-phase insulin secretion was impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Upregulated p38δ in HCV-infected beta cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of beta cells. These findings provided a new perspective into mechanism of HCV induced type 2 diabetes and a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with glucose intolerance. Funding Statement: This work was supported by grants from the National Natural Sciences Foundation of China [grant number 81770859, 81271828, 81471079, 31271268], the National Basic Research Program of China [grant number 2015CB554304], the Youth Innovation Promotion Association, Open Research Fund Program of the State Key Laboratory of Virology of China (grant number 2018IOV003) Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: Human studies were approved by the Ethics Committee of the First Hospital of Jilin University and the First Affiliated Hospital of Xinjiang Medical University. The written informed consents were received from participants prior to inclusion in the study. All animal experiments were approved by the Institutional Animal Ethical Committee of Wuhan Institute of Virology, Chinese Academy of Sciences (WIV, CAS).

Euthanasia via CO2 inhalation causes premature cortical granule exocytosis in mouse oocytes and influences in vitro fertilization and embryo development.

Generation of high quality mouse metaphase II oocytes is an integral part for efficient in vitro fertilization (IVF), and subsequent embryo production for reproductive studies and genome banking. The main objectives of this study were to investigate the impact of various euthanasia methods on IVF, embryo development, and subcellular structures of MII mouse oocytes. Following superovulation regimen, female mice were euthanized by high flow CO2 (H CO2 ), low flow CO2 (L CO2 ), or cervical dislocation (CD). The MII oocytes obtained from these mice were evaluated for subcellular integrity by assessing their cortical granules and F-actin. Furthermore, fertilization and subsequent embryonic development competence up to blastocyst stage were also evaluated in vitro. The oocytes collected from females euthanized by CD resulted in significantly higher two-cell development rates (p = 0.028) and subsequently lead to in higher embryo development rates (p = 0.027) compared with oocytes from females euthanized by L CO2 . The cortical granule integrity analysis revealed significantly higher rate of premature cortical granules exocytosis (PCGE) for L CO2 group compared with CD and H CO2 groups (p < 0.001). These data collectively suggest that CO2 associated PCGE during euthanasia procedure is the main cause of decreased IVF rates and CD is the optimal euthanasia method for the purpose of obtaining good quality MII oocytes for mouse IVF and other reproductive studies.

PAR1 signaling is involved in TCR-mediated CD8 T cell granule exocytosis

Abstract The Protease Activated Receptor (PAR1) is the receptor of thrombin, the main serine protease of the coagulation cascade. PAR1 is expressed by platelets, endothelial cells and cells of the innate immune system and mediates platelet aggregation and secretion of pro-inflammatory cytokines. PAR1 is expressed by T lymphocytes, however its role in T cell function is not well defined. We have previously shown that human CD4 and CD8 T cells express PAR1. PAR1 expression was higher in CD8 than CD4 T cells and is associated with T cell differentiation. CD8 T cell Effector Memory and Terminal Effector Memory phenotypes have higher PAR1 expression than Naïve T cells. PAR1 activation by thrombin enhanced CD8 T cell function including chemokinesis and cytokine secretion. In the present study, we address the role of PAR1 signaling in CD8 T cell mediated cytotoxicity by the granule exocytosis pathway. We found that expression of PAR1 on CD8 T cells is associated with expression of the transcription factors T-bet and Eomes and effector molecules, granzyme A, granzyme B and perforin. PAR1 activation by thrombin enhanced TcR-induced degranulation in CD8 T cells. In addition, in a redirected killing assay, we show that granule exocytosis and cytotoxicity of target cells was efficiently blocked by PAR1 antagonist (SCH79797). Confocal microscopy of the CD8 T-APC conjugates revealed that PAR1 blockade inhibit actin polymerization and decreased the docking of the Microtubule Organizing Center (MTOC) at the immunological synapse. These findings suggest that PAR1 signaling is involved in several effector functions of CD8 T cells including the cytotoxicity by the granule exocytosis pathway. These data have implications in CD8 T cell function and disease.

Two morphological changes of cancer cells under natural killer cell killing

Abstract Natural killer (NK) cells use granule exocytosis to kill cancer cells, which involves perforin-mediated delivery of granzymes from NK cells to cancer cells. Once inside the cancer cells, granzyme B, a member of serine protease family, activates caspase-3, which activates the serine/threonine kinase Rho-associated coiled coil–containing protein kinase (ROCK) I, an effector of the small GTPase Rho. Activated ROCK I increases the phosphorylation of myosin regulatory light chain and thus stimulates actomyosin contraction, required for apoptotic membrane blebbing. Ultimately, cancer cells under NK cell attack show rounding, shrinkage, and membrane rupture, which are classical morphological changes observed at the onset of apoptosis. Although much has been learned about these morphological changes, a fundamental question of how melanoma cell death is induced by NK cells remains unanswered. Do all melanoma cell lines undergo the classical dying process? If not, is there another dying process? To address this simple issue, we examined the dying of B16 melanoma cell lines under NK cell attack by using time-lapse imaging. Upon contact with NK cells, B16-F10 cells rounded and most of them showed membrane rupture (98 min); however, B16 parent cells showed writhing and delayed membrane rupture (235 min). This morphological difference depended on the expression levels of myosin regulatory light chain 9 (MYL9) but not activating ligands (CD112, CD155, Rae-1, and MULT-1), SPI, FasL, or PD-L1. Taken together, our data show that melanoma cells show two distinct types of morphological changes upon contact with NK cells and suggest that a strategy to decrease MYL9 expression by melanoma cells may improve the efficacy of NK cell–based immunotherapy.

Cortical mitochondria regulate insulin secretion by local Ca2+ buffering in rodent beta cells.

Mitochondria play an essential role in regulating insulin secretion from beta cells by providing the ATP needed for the membrane depolarization that results in voltage-dependent Ca2+ influx and subsequent insulin granule exocytosis. Ca2+, in turn, is also rapidly taken up by the mitochondria and exerts important feedback regulation of metabolism. The aim of this study was to determine whether the distribution of mitochondria within beta cells is important for the secretory capacity of these cells. We find that cortically localized mitochondria are abundant in rodent beta cells, and that these mitochondria redistribute towards the cell interior following depolarization. The redistribution requires Ca2+-induced remodeling of the cortical F-actin network. Using light-regulated motor proteins, we increased the cortical density of mitochondria twofold and found that this blunted the voltage-dependent increase in cytosolic Ca2+ concentration and suppressed insulin secretion. The activity-dependent changes in mitochondria distribution are likely to be important for the generation of Ca2+ microdomains required for efficient insulin granule release.

OR25-1 Conditional Deletion of Gonadotropic Androgen Receptor Prevents Elevated Androgen Induced Reproductive Dysfunction in Female Mice

Androgen and its receptor (AR) play a critical role in reproductive function, under both normal physiological and clinically pathophysiological conditions. Many women with hyperandrogenemia suffer irregular menses, oligo-anovulation and infertility. Whether high androgen levels directly or secondarily affect reproduction is unknown. Further, most animal models of hyperandrogenemia induced infertility are associated with obesity which adds a confounding variable to analysis. Therefore, we created a lean elevated androgen model by implantation of a low dose dihydrotestosterone (DHT) that separates the effects of obesity from elevated androgen. Reproduction is tightly controlled by the hypothalamus-pituitary-gonadal axis, androgen could theoretically alter neuroendocrine function at the level of brain and/or pituitary. The goal of this study is to understand how high androgen signaling via the androgen receptor (AR) in pituitary gonadotropes affects reproductive function. Mice with disruption of the Ar gene specifically in pituitary gonadotropes (PitARKO) were produced to explore the role of elevated androgen on the development of reproductive dysfunction in female mice. PitARKO-DHT mice showed significantly improved estrous cyclicity and fertility compared to DHT treated control littermates with intact pituitary Ar (Con-DHT). During 90 days of mating, PitARKO-DHT mice had a significantly increased number of litters (2.3±0.6 vs 0.5±0.3) and number of pups (5.3±2.8 vs 17.0±4.5) compared to Con-DHT mice. The improved reproductive function in PitARKO-DHT mice is partially due to improved pituitary responsiveness to GnRH stimulation. Ex vivo pituitary primary culture confirmed that DHT significantly reduced both basal and GnRH stimulated LH secretion. In vivo, LH secretion is preserved in PitARKO-DHT mice after GnRH stimulation compared to CON-DHT mice. GnRH induces the release of gonadotropins via an increase in cytosolic Ca2+ concentration, and reduced cytoplasmic Ca2+ concentrations may interfere with exocytosis of granules of LH. GEM, a calcium binding protein, inhibits voltage-gated Ca2+ channel activity and reduces Ca2+ influx in gonadotropes, which can affect gonadotropin secretion. We observed that Gem mRNA levels were significantly increased in Con-DHT treated mice (6 fold higher) compared to PitARKO-DHT mice. Further, the intracellular calcium influx signals were dramatically attenuated in Con-DHT mice compared to Con-no DHT mice, and were preserved in PitARKO-DHT mice after GnRH stimulation. These findings highlight gonadotrope AR as an extra ovarian regulator playing an important role in reproductive physiology and that high androgens may impact function through AR regulated Gem expression.

Evaluating the Role of Piccolo Splice Variants in Regulated Secretion of Insulin from Rat Pancreatic Cells

Secretion of insulin from pancreatic beta‐cells is required for proper glucose homeostasis; however, the molecular mechanism of insulin granule exocytosis has not been fully elucidated. In pancreatic beta‐cells, glucose‐induced insulin secretion is synergistically potentiated through an Epac2‐Rim2‐Piccolo signaling response mechanism. Both Piccolo and Rim2 were originally identified as active zone proteins in neurons and have been shown to regulate synaptic vesicle cycling and neurotransmitter signaling. A recent study showed that a number of active zone proteins, including Rim2 and Piccolo, are downregulated in human pancreatic islet cells isolated from patients with type 2 diabetes (T2D). Understanding the role of Piccolo in regulated secretion of insulin may provide valuable insights into the causes of T2D. The C‐terminal region of Piccolo contains a Ca2+‐binding C2A domain. It is likely that Piccolo may regulate secretion from insulin granule vesicles by serving as a Ca2+‐sensor. Alternative splicing of exons within the coding region of the C2A domain results in an exon‐skipping event generating either a splice variant that lacks the 27‐nucleotide exon 16 (short C2A) or a variant that retains this exon (long C2A). Exclusion of the 9 amino acid residue sequence in the short C2A domain results in enhanced Ca2+‐binding affinity. In this study, we sought to examine the differential roles of the short C2A and long C2A variants in Ca2+‐mediated insulin secretion from rat INS‐832/13 pancreatic beta‐cells. We demonstrate that INS‐832/13 cells express both long and short variants of the C2A domain and real‐time qPCR demonstrated differential expression of the variants. In addition, we examined the effects of overexpression of the short and long C2A domains on glucose‐stimulated insulin secretion from INS‐832/13 cells.Support or Funding InformationMaximizing Access to Research Careers Program at Fort Lewis College (NIH‐T34GM092711)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A truncation mutation in human SLC12A2 leads to abnormal goblet cell mucus secretion

The basolateral Na+‐K+‐2Cl− cotransporter (NKCC1) plays a critical role in maintaining the intracellular concentrations of Na+, K+, and Cl− ions, and cell volume. The cotransporter also plays a key role in mediating epithelial fluid secretion. In previous studies, we have described the first known human SLC12A2 (NKCC1) mutation (Cold Spring Harbor Mol. Case Stud. 2(6): a001289, 2016). The now 16‐year‐old patient carries a carboxyl‐terminal truncation mutation (NKCC1‐DFX) in the transporter that leads to complex clinical features including complete gastrointestinal and bladder failure, endocrine insufficiencies, small intestinal dysmotility, seizure‐like episodes, and multi‐organ failure. The 11 bp deletion leads to truncation of the cytosolic COOH‐terminal domain of the cotransporter, making it non‐functional. In polarized epithelial MDCK cells and a mouse model that recapitulates the patient mutation we demonstrated that expression of the truncated Na‐K‐2Cl cotransporter causes mistargeting of the mutant and wild‐type cotransporter to the apical membrane of Cl− secreting epithelia and affects proper lumen formation in MDCK cysts (Am. J. Physiol. Cell Physiol. 315: C258–C276, 2018). Recently due to chronic infections, the patient's colon was surgically resected and lately, the patient has been battling chronic yeast infections in the small intestine. To investigate whether NKCC1‐DFX affect the integrity and function of the gut epithelia, we used MDCK cells expressing the mutant transporter and a mouse model recapitulating the patient mutation. We show that expression of the NKCC1‐DFX mutant impairs tight junction integrity, as indicated by a decrease in trans‐epithelial electrical resistance and inability of MDCK‐I cells expressing NKCC1‐DFX to re‐establish barrier function after a calcium‐switch challenge. Compared to wild‐type mice, NKCC1‐DFX have increased intestinal transit time and displayed an increase in unhealthy mitochondria characterize by dense inclusion bodies. Relative to wild‐type mice, NKCC1‐DFX mice showed decreased expression of Claudin‐2, a tight junction protein involved in paracellular Na+ and water transport. NKCC1‐DFX expression causes defective goblet cells mucus granules exocytosis leading to secretion of intact granules in the lumen of the large intestine. In addition, NKCC1‐DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Taken together, these data indicate that NKCC1‐DFX likely impairs gut barrier function by affecting epithelia tight junction integrity and mucus secretion.Support or Funding InformationSupported by NIH grant GM118944.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular Docking and Dynamics Simulation Studies Predict Munc18b as a Target of Mycolactone: A Plausible Mechanism for Granule Exocytosis Impairment in Buruli Ulcer Pathogenesis.

Ulcers due to infections with Mycobacterium ulcerans are characterized by complete lack of wound healing processes, painless, an underlying bed of host dead cells and undermined edges due to necrosis. Mycolactone, a macrolide produced by the mycobacterium, is believed to be the toxin responsible. Of interest and relevance is the knowledge that Buruli ulcer (BU) patients remember experiencing trauma previously at the site of the ulcers, suggesting an impairment of wound healing processes, the plausible effect due to the toxin. Wound healing processes involve activation of the blood platelets to release the contents of the dense granules mainly serotonin, calcium ions, and ADP/ATP by exocytosis into the bloodstream. The serotonin release results in attracting more platelets and mast cells to the wound site, with the mast cells also undergoing degranulation, releasing compounds into the bloodstream by exocytosis. Recent work has identified interference in the co-translational translocation of many secreted proteins via the endoplasmic reticulum and cell death involving Wiskott-Aldrich syndrome protein (WASP), Sec61, and angiotensin II receptors (AT2R). We hypothesized that mycolactone by being lipophilic, passively crosses cell membranes and binds to key proteins that are involved in exocytosis by platelets and mast cells, thus inhibiting the initiation of wound healing processes. Based on this, molecular docking studies were performed with mycolactone against key soluble n-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and regulators, namely Vesicle-associated membrane protein (VAMP8), Synaptosomal-associated protein (SNAP23, syntaxin 11, Munc13-4 (its isoform Munc13-1 was used), and Munc18b; and also against known mycolactone targets (Sec61, AT2R, and WASP). Munc18b was shown to be a plausible mycolactone target after the molecular docking studies with binding affinity of −8.5 kcal/mol. Structural studies and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding energy calculations of the mycolactone and Munc18b complex was done with 100 ns molecular dynamics simulations using GROMACS. Mycolactone binds strongly to Munc18b with an average binding energy of −247.571 ± 37.471 kJ/mol, and its presence elicits changes in the structural conformation of the protein. Analysis of the binding interactions also shows that mycolactone interacts with Arg405, which is an important residue of Munc18b, whose mutation could result in impaired granule exocytosis. These findings consolidate the possibility that Munc18b could be a target of mycolactone. The implication of the interaction can be experimentally evaluated to further understand its role in granule exocytosis impairment in Buruli ulcer.

Munc18-2, but not Munc18-1 or Munc18-3, regulates platelet exocytosis, hemostasis, and thrombosis

Platelet degranulation, a form of regulated exocytosis, is crucial for hemostasis and thrombosis. Exocytosis in platelets is mediated by SNARE proteins, and in most mammalian cells this process is controlled by Munc18 (mammalian homolog of Caenorhabditis elegans uncoordinated gene 18) proteins. Platelets express all Munc18 paralogs (Munc18-1, -2, and -3), but their roles in platelet secretion and function have not been fully characterized. Using Munc18-1, -2, and -3 conditional knockout mice, here we deleted expression of these proteins in platelets and assessed granule exocytosis. We measured products secreted by each type of platelet granule and analyzed EM platelet profiles by design-based stereology. We observed that the removal of Munc18-2 ablates the release of alpha, dense, and lysosomal granules from platelets, but we found no exocytic role for Munc18-1 or -3 in platelets. In vitro, Munc18-2-deficient platelets exhibited defective aggregation at low doses of collagen and impaired thrombus formation under shear stress. In vivo, megakaryocyte-specific Munc18-2 conditional knockout mice had a severe hemostatic defect and prolonged arterial and venous bleeding times. They were also protected against arterial thrombosis in a chemically induced model of arterial injury. Taken together, our results indicate that Munc18-2, but not Munc18-1 or Munc18-3, is essential for regulated exocytosis in platelets and platelet participation in thrombosis and hemostasis.

Senescent cells: Living or dying is a matter of NK cells.

NK cells are lymphocytes of the innate immune system, which are able to deal promptly with stressed cells. Cellular senescence is a cell stress response leading to cell cycle arrest that plays a key role during tissue homeostasis and carcinogenesis. In this review, how senescent cells trigger an immune response and, in particular, the ability of NK cells to recognize and clear senescent cells are discussed. Special attention is given to the NK cell-mediated clearance of senescent tumor cells. NK cells kill senescent cells through a mechanism involving perforin- and granzyme-containing granule exocytosis, and produce IFN-γ following senescent cell interaction, leading to hypothesize that NK cell-mediated immune clearance of senescent cells not only relies on direct killing but also on cytokine production, that in turn can promote macrophage activation. These aspects, as well as the ability of the senescence-associated secretory phenotype and senescent cell-produced extracellular vesicles to modulate NK cell effector functions, are described.

Glucose-dependent phosphorylation signaling pathways and crosstalk to mitochondrial respiration in insulin secreting cells

BackgroundGlucose is the main secretagogue of pancreatic beta-cells. Uptake and metabolism of the nutrient stimulates the beta-cell to release the blood glucose lowering hormone insulin. This metabolic activation is associated with a pronounced increase in mitochondrial respiration. Glucose stimulation also initiates a number of signal transduction pathways for the coordinated regulation of multiple biological processes required for insulin secretion.MethodsShotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on lysates from glucose-stimulated INS-1E cells was used to identify glucose regulated phosphorylated proteins and signal transduction pathways. Kinase substrate enrichment analysis (KSEA) was applied to identify key regulated kinases and phosphatases. Glucose-induced oxygen consumption was measured using a XF96 Seahorse instrument to reveal cross talk between glucose-regulated kinases and mitochondrial activation.ResultsOur kinetic analysis of substrate phosphorylation reveal the molecular mechanism leading to rapid activation of insulin biogenesis, vesicle trafficking, insulin granule exocytosis and cytoskeleton remodeling. Kinase-substrate enrichment identified upstream kinases and phosphatases and time-dependent activity changes during glucose stimulation. Activity trajectories of well-known glucose-regulated kinases and phosphatases are described. In addition, we predict activity changes in a number of kinases including NUAK1, not or only poorly studied in the context of the pancreatic beta-cell. Furthermore, we pharmacologically tested whether signaling pathways predicted by kinase-substrate enrichment analysis affected glucose-dependent acceleration of mitochondrial respiration. We find that phosphoinositide 3-kinase, Ca2+/calmodulin dependent protein kinase and protein kinase C contribute to short-term regulation of energy metabolism.ConclusionsOur results provide a global view into the regulation of kinases and phosphatases in insulin secreting cells and suggest cross talk between glucose-induced signal transduction and mitochondrial activation.

Calcium negatively regulates secretion from dense granules in Toxoplasma gondii.

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+, and we show that this Ca2+‐mediated response is contingent on the function of calcium‐dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.