Therapeutic effects of TM4SF5-targeting chimeric and humanized monoclonal antibodies in hepatocellular and colon cancer models.

The transmembrane 4 L six family member 5 (TM4SF5) is aberrantly expressed in hepatocellular and colorectal cancers, and has been implicated in tumor progression, suggesting that it could serve as a novel therapeutic target. Previously, we screened a murine antibody phage-display library to generate a novel monoclonal antibody, Ab27, that is specific to the extracellular loop 2 of TM4SF5. In this study, we evaluated the effects of chimeric Ab27 using cancer cells expressing endogenous TM4SF5 or stably overexpressing TM4SF5 in vivo and in vitro. Monotherapy with Ab27 significantly decreased tumor growth in liver and colon cancer xenograft models, including a sorafenib-resistant model, and decreased the phosphorylation of focal adhesion kinase (FAK), p27Kip1, and signal transducer and activator of transcription 3 (STAT3). No general Ab27 toxicity was observed in vivo. Combination treatment with Ab27 and sorafenib or doxorubicin exerted higher antitumor activity than monotherapy. In addition, we humanized the Ab27 sequence by the complementarity-determining region (CDR) grafting method. The humanized antibody Ab27-hz9 had reduced immunogenicity but exhibited target recognition and antitumor activity comparable with those of Ab27. Both Ab27 and Ab27-hz9 efficiently targeted tumor cells expressing TM4SF5 in vivo. These observations strongly support the further development of Ab27-hz9 as a novel therapeutic agent against liver and colorectal cancers.

Efficacy of immune-strengthening functional drinks in top-level athletes: a questionnaire survey-based research.

PURPOSE: Functional beverages are intended to support those who want to maintain optimal physical condition and improve their quality of life through the enhancement of heart health, immunity, and digestion. The purpose of this study was to investigate the performance of top-level athletes consuming immune-strengthening conditioning nutritional drinks. METHODS: A total of 107 top-level athletes (baseball (56 players), pro volleyball (17), athletics (16), cycling (8), golf (6), and fencing (6)) participated in the experiment. They consumed an immune-enhancing functional beverage once a day for 8 weeks and responded to a survey before, during, and after drinking the beverage. RESULTS: Three total aspect-based subfactors were drawn from 24 questions in the factor analysis: physical, satisfaction with mental stability, and activity in performance. The physical, mental stability and performance changes of athletes significantly increased in period 2 (4 weeks after intake) and period 3 (after 8 weeks of intake). CONCLUSION: We evaluated the efficacy of a new conditioned beverage containing Lactobacillus B240 and protein in improving the performance and physiological utility of top athletes. This functional drink may gain popularity among those seeking health benefits and improved exercise performance.

Molecular architecture of mouse and human pancreatic zymogen granules: protein components and their copy numbers.

A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ± 218 and 2039 ± 151 (mean ± SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ± 23 nm (mean ± SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ± 45 and 485 ± 15 (mean ± SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases.

COX7AR is a Stress-inducible Mitochondrial COX Subunit that Promotes Breast Cancer Malignancy.

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in regulating mitochondrial energy production and cell survival. COX subunit VIIa polypeptide 2-like protein (COX7AR) is a novel COX subunit that was recently found to be involved in mitochondrial supercomplex assembly and mitochondrial respiration activity. Here, we report that COX7AR is expressed in high energy-demanding tissues, such as brain, heart, liver, and aggressive forms of human breast cancer cells. Under cellular stress that stimulates energy metabolism, COX7AR is induced and incorporated into the mitochondrial COX complex. Functionally, COX7AR promotes cellular energy production in human mammary epithelial cells. Gain- and loss-of-function analysis demonstrates that COX7AR is required for human breast cancer cells to maintain higher rates of proliferation, clone formation, and invasion. In summary, our study revealed that COX7AR is a stress-inducible mitochondrial COX subunit that facilitates human breast cancer malignancy. These findings have important implications in the understanding and treatment of human breast cancer and the diseases associated with mitochondrial energy metabolism.

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells.

Pancreatic beta cells have well-developed ER to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by 1D SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments and 737 proteins found in all three experiments. GO analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. Further bioinformatics analysis also identified potential beta cell specific ER proteins as well as ER proteins present in the risk genetic loci of type 2 diabetes. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions. All MS data have been deposited in the ProteomeXchange with identifier PXD001081 (http://proteomecentral.proteomexchange.org/dataset/PXD001081).

X-ray solution structure of the native neuronal porosome-synaptic vesicle complex: Implication in neurotransmitter release.

Nanoportals at the cell plasma membrane called porosomes, mediate secretion from cells. In neurons porosomes are 15 nm cup-shaped lipoprotein structure composed of nearly 40 proteins. The size and complexity of the porosome has precluded determination of its atomic structure. Here we report at nanometer resolution the native 3D structure of the neuronal porosome-synaptic vesicle complex within isolated nerve terminals using small-angle X-ray solution scattering. In addition to furthering our understanding of the porosome structure, results from the study suggests the molecular mechanism involved in neurotransmitter release at the nerve terminal.

Down-regulation of microglial activity attenuates axotomized nigral dopaminergic neuronal cell loss.

BACKGROUND: There is growing evidence that inflammatory processes of activated microglia could play an important role in the progression of nerve cell damage in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease which harbor features of chronic microglial activation, though the precise mechanism is unknown. In this study, we presented in vivo and ex vivo experimental evidences indicating that activated microglia could exacerbate the survival of axotomized dopaminergic neurons and that appropriate inactivation of microglia could be neuroprotective. RESULTS: The transection of medial forebrain bundle (MFB) of a rat induced loss of dopaminergic neurons in a time-dependent manner and accompanied with microglial activation. Along with microglial activation, production of reactive oxygen species (ROS) was upregulated and TH/OX6/hydroethidine triple-immunofluorescence showed that the microglia mainly produced ROS. When the activated microglial cells that were isolated from the substantia nigra of the MFB axotomized animal, were transplanted into the substantia nigra of which MFB had been transected at 7 days ago, the survival rate of axotomized dopaminergic neurons was significantly reduced as compared with sham control. Meanwhile, when the microglial activation was attenuated by administration of tuftsin fragment 1-3 (microglia inhibitory factor) into the lateral ventricle using mini-osmotic pump, the survival rate of axotomized dopaminergic neurons was increased. CONCLUSION: The present study suggests that activated microglia could actively produce and secrete unfavorable toxic substances, such as ROS, which could accelerate dopaminergic neuronal cell loss. So, well-controlled blockade of microglial activation might be neuroprotective in some neuropathological conditions.

Aquaporin-assisted and ER-mediated mitochondrial fission: a hypothesis.

It is well established that the status of the endoplasmic reticulum (ER) and mitochondria, and the interactions between them, is critical to numerous cellular functions including apoptosis. Mitochondrial dynamics is greatly influenced by cell stress, and recent studies implicate ER in mitochondrial fission. Although a number of proteins have been identified to participate in ER-induced mitochondrial fission, the molecular mechanism of the process is little understood. In the current study, we confirm the involvement of ER in mitochondrial fission and hypothesize the involvement of water channels or aquaporins (AQP) in the process. Previous studies demonstrate the presence of AQP both in the ER and mitochondrial membranes. Mitochondrial swelling has been observed following mitochondrial calcium overload, and studies report that chelation of cytosolic calcium induces extensive mitochondrial division at ER contact sites. Based on this information, the involvement of ER in mitochondrial division, possibly via water channels, is hypothesized. Utilizing a multi-faceted imaging approach consisting of atomic force microscopy on aldehyde-fixed and semi-dry cells, transmission electron microscopy, and immunofluorescence microscopy on live cells, the physical interactions between the two organelles are demonstrated. Mitochondrial fission following ER stress was abrogated with exposure of cells to the AQP inhibitor mercuric chloride, suggesting the involvement of AQP(s) especially AQP8 and AQP9 known to be present in the mitochondrial membrane, in mitochondrial fission.

Neuronal porosome proteome: Molecular dynamics and architecture.

Porosomes are the universal secretory portals at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the outside during cell secretion. In the past decade, the neuronal porosome complex, a 10-15nm cup-shaped lipoprotein structure has been isolated, its partial composition and 3D contour map determined, and it has been functionally reconstituted into artificial lipid membrane. Here we further determine the composition of the neuronal porosome proteome using immunoisolation and gel filtration chromatography, followed by tandem mass spectrometry. Results from the study demonstrate nearly 40 proteins to constitute the neuronal porosome proteome. Furthermore, interaction of proteins within the porosome and their resulting arrangement is predicted. The association and dissociation of proteins at the porosome following stimulation of cell secretion demonstrate the dynamic nature of the organelle.

Endoplasmic reticulum factor ERLIN2 regulates cytosolic lipid content in cancer cells.

Increased de novo lipogenesis is a hallmark of aggressive cancers. Lipid droplets, the major form of cytosolic lipid storage, have been implicated in cancer cell proliferation and tumorigenesis. Recently, we identified the ERLIN2 [ER (endoplasmic reticulum) lipid raft-associated 2) gene that is amplified and overexpressed in aggressive human breast cancer. Previous studies demonstrated that ERLIN2 plays a supporting oncogenic role by facilitating the transformation of human breast cancer cells. In the present study, we found that ERLIN2 supports cancer cell growth by regulating cytosolic lipid droplet production. ERLIN2 is preferably expressed in human breast cancer cells or hepatoma cells and is inducible by insulin signalling or when cells are cultured in lipoprotein-deficient medium. Increased expression of ERLIN2 promotes the accumulation of cytosolic lipid droplets in breast cancer cells or hepatoma cells in response to insulin or overload of unsaturated fatty acids. ERLIN2 regulates activation of SREBP (sterol regulatory element-binding protein) 1c, the key regulator of de novo lipogenesis, in cancer cells. ERLIN2 was found to bind to INSIG1 (insulin-induced gene 1), a key ER membrane protein that blocks SREBP activation. Consistent with the role of ERLIN2 in regulating cytosolic lipid content, down-regulation of ERLIN2 in breast cancer or hepatoma cells led to lower cell proliferation rates. The present study revealed a novel role for ERLIN2 in supporting cancer cell growth by promoting the activation of the key lipogenic regulator SREBP1c and the production of cytosolic lipid droplets. The identification of ERLIN2 as a regulator of cytosolic lipid content in cancer cells has important implications for understanding the molecular basis of tumorigenesis and the treatment of cancer.

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex.

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.

Pharmacologic ER stress induces non-alcoholic steatohepatitis in an animal model.

Endoplasmic reticulum (ER) stress refers to a condition of accumulation of unfolded or misfolded proteins in the ER lumen, which is known to activate an intracellular stress signaling termed Unfolded Protein Response (UPR). A number of pharmacologic reagents or pathophysiologic stimuli can induce ER stress and activation of the UPR signaling, leading to alteration of cell physiology that is associated with the initiation and progression of a variety of diseases. Non-alcoholic steatohepatitis (NASH), characterized by hepatic steatosis and inflammation, has been considered the precursor or the hepatic manifestation of metabolic disease. In this study, we delineated the toxic effect and molecular basis by which pharmacologic ER stress, induced by a bacterial nucleoside antibiotic tunicamycin (TM), promotes NASH in an animal model. Mice of C57BL/6J strain background were challenged with pharmacologic ER stress by intraperitoneal injection of TM. Upon TM injection, mice exhibited a quick NASH state characterized by hepatic steatosis and inflammation. An increase in hepatic triglycerides (TG) and a decrease in plasma lipids, including plasma TG, plasma cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL), were observed in the TM-treated mice. In response to TM challenge, cleavage of sterol responsive binding protein (SREBP)-1a and SREBP-1c, the key trans-activators for lipid and sterol biosynthesis, was dramatically increased in the liver. Consistent with the hepatic steatosis phenotype, expression of some key regulators and enzymes in de novo lipogenesis and lipid droplet formation was up-regulated, while expression of those involved in lipolysis and fatty acid oxidation was down-regulated in the liver of mice challenged with TM. Moreover, TM treatment significantly increased phosphorylation of NF-κB inhibitors (IκB), leading to the activation of NF-κB-mediated inflammatory pathway in the liver. Our study not only confirmed that pharmacologic ER stress is a strong "hit" that triggers NASH, but also demonstrated crucial molecular links between ER stress, lipid metabolism, and inflammation in the liver in vivo.

Pharmacological ER stress promotes hepatic lipogenesis and lipid droplet formation.

Endoplasmic Reticulum (ER) stress refers to a condition of accumulation of unfolded or misfolded proteins in the ER lumen. A variety of biochemical stimuli or pathophysiologic conditions can directly or indirectly induce ER stress, leading to activation of an ER-originated adaptive signaling response called Unfolded Protein Response (UPR). Recent studies demonstrated that ER stress and UPR signaling are critically involved in the initiation and progression of many diseases, such as metabolic disease, cardiovascular disease, neurodegenerative disease, and cancer. In this study, we show that ER stress induced by pharmacologic reagents, including tunicamycin (TM) and thapsigargin (Tg), promotes hepatic lipogenesis and lipid droplet formation. Using quantitative gene expression analysis, we identified 3 groups of key lipogenic regulators or enzymes that are inducible by pharmacological ER stress in a human hepatoma cell line Huh-7. These ER stress-inducible lipogenic factors include: 1) lipogenic trans-activators including CCAAT/ enhancer binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coacti-vator 1-alpha (PGC1α), and Liver X receptor alpha (LXRα); 2) components of lipid droplets including fat-specific protein 27 (FSP27), adipose differentiation related protein (ADRP), fat-inducing transcript 2 (FIT2), and adipocyte lipid-binding protein (AP2); 3) key enzymes involved in de novo lipogenesis including acetyl-CoA carboxylase 1 (ACC1) and stearoyl-CoA desaturase-1 (SCD1). Supporting the role of pharmacologic ER stress in up-regulating de novo lipogenesis, TM or Tg treatment significantly increased accumulation of cytosolic lipid droplet formation in the hepatocytes. Moreover, we showed that forced expression of an activated form of X-box binding protein 1 (XBP1), a potent UPR trans-activator, can dramatically increase expression of PPARγ and C/EBPα in Huh-7 cells. The identification of ER stress-inducible lipogenic regulators provides important insights into the molecular basis by which acute ER stress promotes de novo lipogenesis. In summary, the findings from this study have important implication in understanding the link between ER stress and metabolic disease.

Water channels in platelet volume regulation.

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein-mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl(2)-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G(αo), G(αi-1) and G(αi-3) proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein-mediated pathway.

Lysophosphatidylcholine inhibits membrane-associated SNARE complex disassembly.

In cells, N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors called SNAREs are involved in membrane fusion. In neurons, for example, target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the pre-synaptic membrane, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, is part of the conserved protein complex involved in neurotransmission. Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes. In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly. Exposure of cholesterol-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP results in dissociated vesicles. In contrast, exposure of LPC-associated t-SNARE and v-SNARE liposome mixtures to NSF-ATP, results in inhibition of t-/v-SNARE disassembly and the consequent accumulation of clustered vesicles. Similarly, exposure of isolated rat brain slices and pancreas to cholesterol or LPC, also demonstrates LPC-induced inhibition of SNARE complex disassembly. Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer. The altered plasma LPC levels observed in various cancers may in part contribute to defects in SNARE assembly-disassembly and membrane fusion, consequently affecting protein maturation and secretion in cancer cells.

Membrane-directed molecular assembly of the neuronal SNARE complex.

Since the discovery and implication of N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full-length recombinant t-SNAREs and v-SNARE present in opposing liposomes, leading to the assembly of a t-/v-SNARE ring complex. Using high-resolution electron microscopy, the electron density maps and 3D topography of the membrane-directed SNARE ring complex was determined at nanometre resolution. Similar to the t-/v-SNARE ring complex formed when 50 nm v-SNARE liposomes meet a t-SNARE-reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t-SNARE-reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane-directed t-/v-SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome-reconstituted recombinant t-/v-SNARE proteins, and native v-SNARE present in isolated SV membrane, the membrane-directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.

Involvement of ß-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release.

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The G(αo) -mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates G(o) protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous ß-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using ß-adrenergic receptor antibody, and vesicle swelling experiments using ß-adrenergic agonists and antagonists, demonstrate the presence of functional ß-adrenergic receptors at the SV membrane. Since a recent study shows vH(+) -ATPase to be upstream of AQP-6 in the pathway leading from G(αo) -mediated swelling of SV, participation of an endogenous ß-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.

Conformation states of the neuronal porosome complex.

Porosomes are the universal secretory machinery of the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents to the environment during cell secretion. In neurons, 12- to 17-nm cup-shaped lipoprotein structures possessing a central plug are present at the presynaptic membrane, where 40-50 nm in diameter synaptic vesicles transiently dock and fuse to release neurotransmitters. The neuronal porosome complex has been isolated, its composition determined and it has been both structurally and functionally reconstituted in artificial lipid membranes. Earlier studies using AFM (atomic force microscopy), EM (electron microscopy), electron density and 3D contour mapping provide the structure and assembly of proteins within the neuronal porosome complex at the nanoscale level. A set of eight protein units lining the neuronal porosome cup is present, each connected via spoke-like elements to a central plug, hypothesized for the rapid opening and closing of the structure to the outside. In the present study, ultrahigh-resolution imaging of the presynaptic membrane of isolated synaptosome preparations demonstrate, for the first time, the presence of neuronal porosomes in both their open and close conformations. The results suggests that the central plug is retracted into the porosome cup in its open conformation and pushed outward to seal the porosome opening, supporting the hypothesis that it operates as the opening-closing device of the complex.

Involvement of cholesterol in synaptic vesicle swelling.

Studies demonstrate that cholesterol plays a critical role in the regulation of neurotransmitter release and that secretory vesicle swelling is a requirement for the regulated expulsion of intravesicular contents during cell secretion. In view of this, the involvement of cholesterol in synaptic vesicle swelling was hypothesized and tested in the present study, using isolated synaptic vesicles from rat brain and the determination of their swelling competency in the presence and absence of cholesterol. The involvement of the water channel aquaporin-6 (AQP-6) and proton pump vH(+)-ATPase in GTP-G(alpha o)-mediated synaptic vesicle swelling has been reported previously. Mastoparan, the amphiphilic tetradecapeptide from wasp venom, known to activate the GTPase activity of G(alpha o/i) proteins, stimulates synaptic vesicle swelling in the presence of GTP. In the current study, using nanometer-scale precision measurements of isolated synaptic vesicles, we report for the first time that depletion of cholesterol from synaptic vesicle membrane results in a significant loss of GTP-mastoparan-stimulable synaptic vesicle swelling. In contrast, incorporation of cholesterol into the synaptic vesicle membrane potentiates GTP-mastoparan-stimulable vesicle swelling. Our study further demonstrates that this effect of cholesterol is due, in part, to its involvement in the interactions between AQP-6, vH(+)-ATPase and the GTP-binding G(alpha o) protein at the synaptic vesicle membrane.

Involvement of vH(+)-ATPase in synaptic vesicle swelling.

Secretory vesicle swelling is central to cell secretion, but the underlying mechanism of vesicle swelling, particularly synaptic vesicles, is not completely understood. The G(alphai3)-PLA2-mediated involvement of water channel AQP-1 in the regulation of secretory vesicle swelling in exocrine pancreas and the G(alphao)-mediated AQP-6 involvement in synaptic vesicle swelling in neurons have previously been reported. Furthermore, the role of vH(+)-ATPase in neurotransmitter transport into synaptic vesicles has also been shown. Using nanometer-scale precision measurements of isolated synaptic vesicles, the present study reports for the first time the involvement of vH(+)-ATPase in GTP-G(alphao)-mediated synaptic vesicle swelling. Results from this study demonstrate that the GTP-G(alphao)-mediated vesicle swelling is vH(+)-ATPase dependent and pH sensitive. Zeta potential measurements of isolated synaptic vesicles further demonstrate a bafilomycin-sensitive vesicle acidification, following the GTP-G(alphao)-induced swelling stimulus. Water channels are bidirectional and the vH(+)-ATPase inhibitor bafilomycin decreases both the volume of isolated synaptic vesicles and GTP-mastoparan stimulated swelling, suggesting that vH(+)-ATPase is upstream of AQP-6, in the pathway leading from G(alphao)-stimulated swelling of synaptic vesicles. Vesicle acidification is therefore a prerequisite for AQP-6-mediated gating of water into synaptic vesicles.