Early requirement for alpha-SNAP and NSF in the secretory cascade in chromaffin cells.

NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules.     

Transnitrosylating Nitric Oxide Species Directly Activate Type I Protein Kinase A, Providing a Novel Adenylate Cyclase-independent Cross-talk to ��-Adrenergic-like Signaling

The transnitrosylating nitric oxide (NO) donor nitrocysteine (CysNO) induced a disulfide bond between the two regulatory RI subunits of protein kinase A (PKA). The conventional NO donor S-nitroso-N-acetylpenicillamine failed to do this, consistent with our observation that it also did not promote protein S-nitrosylation. This disulfide oxidation event activated PKA and induced vasorelaxation independently of the classical β-adrenergic or NO signaling pathway. Activation of PKA had also been anticipated to exert a positive inotropic effect on the myocardium but did not. The lack of positive inotropy was explained by CysNO concomitantly activating protein kinase G (PKG) Iα. PKG was found to exert a partial negative inotropic influence regardless of whether PKA was activated by classical β-receptor stimulation or by disulfide bond formation. This work demonstrates that NO molecules that can induce S-nitrosylation directly activate type I PKA, providing a novel cross-talk to β-adrenergic-like signaling without receptor or adenylate cyclase stimulation. However, the expected positive inotropic consequences of PKA activation by this novel mechanism are countermanded by the simultaneous dual activation of PKGIα, which is also activated by CysNO.Nitric oxide (NO) initiates cell signaling by binding and activating soluble guanylate cyclase (sGC)² to produce the second messenger cGMP. cGMP primarily allosterically activates protein kinase G (PKG) but can also regulate other proteins. Although this NO-sGC-cGMP-PKG pathway is well defined (1), a second major mechanism of NO-dependent regulation has subsequently emerged. This involves NO covalently adducting to protein thiols, a process known as S-nitrosylation or S-nitrosation (2).Significant evidence continues to accumulate supporting protein S-nitrosylation as a fundamental regulator of protein and thus cell function (3). NO is produced in a regulated way (4), with a defined structural basis for selectivity in the proteins it covalently modifies (5, 6). Additional regulatory control can be achieved by the localization of NO synthase enzymes proximal to target proteins (6) and by reverse denitrosylation being enzymatically controlled (7). Indeed, many proteins appear to be basally S-nitrosylated, offering the potential for attenuation (8) as well as potentiation of signaling.Although stable regulatory S-nitrosylation occurs in some proteins, in others, it serves as an intermediate prior to transition to other redox states, especially disulfides (9). Previously, we searched for proteins that form interprotein disulfides in response to hydrogen peroxide (H₂O₂), identifying the regulatory RI subunit of protein kinase A (PKA) as such a protein (10, 11). This appears to activate the kinase (11), although the mechanism is not yet precisely defined. There is a rational structural basis for interprotein disulfide formation in PKA RI in response to H₂O₂. The RI dimer is held together by an N-terminal amphipathic leucine zipper in which the monomers are aligned antiparallel to each other with both Cys¹⁷ residues directly facing the corresponding Cys³⁸ residues on the opposite chains (12). H₂O₂-mediated RI disulfide formation is likely via protein sulfenic acid formation by one thiol in the Cys¹⁷ and Cys³⁸ disulfide-forming pair, prior to reduction by the other cysteine to yield the covalently conjugated dimer. Intriguingly, this pair of thiol-disulfide switches in RI is located directly on either side of the protein kinase A anchor protein-binding domain (13). This provides a rational structural basis for the PKA RI-protein kinase A anchor protein interaction being redox-modulated, as the interaction is strongly anticipated to change depending on the oxidation state of the cysteine switches, which flank the interaction locus (11).We hypothesized that NO may also be able to drive RI disulfide formation via an S-nitrosylated catalytic redox intermediate in a mechanism analogous to transient sulfenation formation during H₂O₂-induced covalent conjugation. This conceptual link between NO and PKA was investigated by comparing the biochemical and functional responses of cardiovascular tissue to the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and nitrocysteine (CysNO). The authentic NO donor SNAP did not promote RI disulfide formation, whereas CysNO did so efficiently, consistent with its established thiol-oxidizing transnitrosylating ability. We show that disulfide-mediated activation of PKA significantly contributes to vasorelaxation induced by CysNO. However, disulfide activation of PKA failed to exert a positive inotropic influence in isolated hearts exposed to CysNO, which was difficult to reconcile with the kinase being truly activated by oxidation. Further investigations showed that this lack of positive inotropy following CysNO-induced oxidation is explained by the co-activation of PKGIα, which we demonstrated previously can be disulfide-activated (15). PKGIα serves as a master regulator of cardiac inotropy, dominating the system to prevent increases in cardiac contractility. Thus, thiol-oxidizing derivatives of NO can activate PKA and so exert β-adrenergic-like signaling, although dual activation of PKG prevents the anticipated positive inotropy.     

Microbial modification of host long-distance dispersal capacity

Background  

Dispersal plays a key role in shaping biological and ecological processes such as the distribution of spatially-structured populations or the pace and scale of invasion. Here we have studied the relationship between long-distance dispersal behaviour of a pest-controlling money spider,Erigone atra, and the distribution of maternally acquired endosymbionts within the wider meta-population. This spider persists in heterogeneous environments because of its ability to recolonise areas through active long-distance airborne dispersal using silk as a sail, in a process termed 'ballooning'.     

InsP₃receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2? entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP?Rs (InsP? receptors). The co-localization between Orai1 and IP?Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2? store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2? store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP?Rs and a basolateral pool that interacts with STIM1 following the Ca2? store depletion. Experiments on IP?R knockout animals demonstrated that the apical Orai1 localization does not require IP?Rs and that IP?Rs are not necessary for the activation of SOCE. However, the InsP?-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP?Rs could have an inhibitory effect on this Ca2? entry mechanism.     

A reciprocal autosomal translocation which causes male sterility in the mouse also impairs oogenesis

A quantitative histological analysis of ovaries from 3- and 5-day-old female mice heterozygous for the male-sterile reciprocal autosomal translocation, T(11;19)42H, revealed a marked reduction (by 65%) in the number of oocytes as compared to controls. These findings call into question the widely held view that chromosomal anomalies causing spermatogenic failure have no effect on oogenesis. It is suggested that during meiosis in males and females there is a mechanism operating which tends to eliminate cells which had incomplete chromosome pairing at the pachytene stage.     

Evidence against Stable Protein S-Nitrosylation as a Widespread Mechanism of Post-translational Regulation

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Is NSF a fusion protein?

N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase required for vesicular transport throughout the constitutive secretory and endocytic pathways. Recently, NSF has also been implicated in regulated exocytosis in synapses--based on SNAP-mediated binding in vitro to a complex of neurotoxin substrates (termed 'SNAREs'). This work has generated an hypothesis in which the interaction of SNAREs (SNAP receptors) on the vesicle membrane with those on the target membrane forms a docking complex to which SNAPs bind, thus allowing NSF to bind and elicit membrane fusion. However, current evidence supports an earlier, pre-fusion role for NSF. We speculate that this role may be as a molecular chaperone for the membrane docking/fusion machinery.     

Stimulation of NSF ATPase Activity by α-SNAP Is Required for SNARE Complex Disassembly and Exocytosis

N-ethylmaleimide–sensitive fusion protein (NSF) and α-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH₂- and COOH-terminal truncation mutants of α-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH₂-terminal amino acids had little effect on the ability of α-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH₂-terminal (1–160) and COOH-terminal (160–295) fragments of α-SNAP were able to bind to NSF, suggesting that α-SNAP contains distinct NH₂- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of α-SNAP. Mutation of leucine 294 to alanine (α-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. α-SNAP (1–285) and α-SNAP (L294A) were unable to stimulate Ca²⁺-dependent exocytosis in permeabilized chromaffin cells. In addition, α-SNAP (1–285), and α-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous α-SNAP. α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, α-SNAP (1–285) and α-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that α-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the α-SNAP stimulation of exocytosis.