Interactions between proteins implicated in exocytosis and voltage-gated calcium channels.
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Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel. (+info)
Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance.
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The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface. (+info)
The intracellular parasite Theileria parva protects infected T cells from apoptosis.
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Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells. (+info)
Effects of calcium channel antagonists on LPS-induced hepatic iNOS expression.
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The onset of liver injury is a pivotal event during endotoxemia. Lipopolysaccharide (LPS) activates the Kupffer cells (KC), the resident macrophages of the liver, to generate an abundance of inflammatory substances, including nitric oxide (NO). Elevated levels of NO are thought to contribute to the propagation of liver injury during sepsis. Calcium, a major second messenger in several cellular signaling events, is required by the KC for the generation of inducible nitric oxide synthase (iNOS). The purpose of this study was to determine whether calcium channel antagonists limit hepatic injury and iNOS expression in vivo following LPS exposure and to evaluate their effects on the regulation of iNOS expression in cultured KC. In rats subjected to LPS for 6 h, the serum alanine aminotransferase (ALT) level was elevated significantly; this response was accompanied by an increase in iNOS mRNA formation in the intact liver. Pretreatment of rats with calcium channel antagonists (i.e., diltiazem, nifedipine, or verapamil) before LPS exposure attenuated the serum ALT level and iNOS mRNA expression in the liver. Pretreatment of cultured KC with calcium channel antagonists for 1 h followed by the addition of LPS markedly repressed iNOS protein and mRNA expression. Time-course studies revealed that calcium channel antagonists were most effective at inhibiting LPS-induced iNOS mRNA formation by KC when added before LPS. Treatment of KC with calcium channel antagonists prior to the addition of LPS decreased nuclear levels of the p65 subunit of nuclear factor-kappaB and prevented the LPS-dependent degradation of the inhibitory protein IkappaBalpha. Thus our findings indicate that under endotoxemic conditions calcium channel antagonists limit hepatocellular injury that is accompanied by an inhibition of LPS-mediated iNOS expression in rat liver KC. (+info)
Nuclear factor-kappaB and cell death after experimental intracerebral hemorrhage in rats.
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BACKGROUND AND PURPOSE: Nuclear factor-kappaB (NF-kappaB) is a ubiquitous transcription factor that, when activated, translocates to the nucleus, binds to DNA, and promotes transcription of many target genes. Its activation has been demonstrated in chronic inflammatory conditions, cerebral ischemia, and apoptotic cell death. The present study evaluated the presence and activation of NF-kappaB in relation to cell death surrounding intracerebral hemorrhage (ICH). METHODS: Striatal ICH was induced in rats by the double blood injection method. Animals were killed 2, 8, and 24 hours and 4 days after ICH. To examine changes in NF-kappaB protein, Western blot was performed on brain extract. We determined NF-kappaB activity using electrophoretic mobility shift assay (EMSA) and immunohistochemistry, using an antibody that only recognizes active NF-kappaB. DNA fragmentation was detected with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining. RESULTS: Western blot analysis of the NF-kappaB p65 subunit showed that there was no difference in p65 protein levels in the control, 2-hour, 8-hour, or 24-hour groups. However, ipsilateral perilesional samples from the 4-day group revealed a 1.8- to 2.5-fold increase compared with the contralateral hemisphere. Western blotting showed no differences in the inhibitor of NF-kappaB, IkappaBalpha, in any group. EMSA showed 1.3-, 2.1-, and 3.6-fold increased NF-kappaB activation in the ipsilateral striatum from the 8-hour, 24-hour, and 4-day groups, respectively, compared with the contralateral hemisphere. Immunohistochemistry, in which an activation-dependent anti-NF-kappaB antibody was used, demonstrated perivascular NF-kappaB activation as early as 2 hours after ICH with more generalized activation at 8 hours, in agreement with the EMSA results. NF-kappaB activation colocalized to cells containing fragmented DNA measured by TUNEL. CONCLUSIONS: The present study suggests a relationship between NF-kappaB and the pathobiology of perilesional cell death after ICH. (+info)
Di-leucine signals mediate targeting of tyrosinase and synaptotagmin to synaptic-like microvesicles within PC12 cells.
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One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents. (+info)
Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.
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The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex. (+info)
Direct interaction between synaptotagmin and the intracellular loop I-II of neuronal voltage-sensitive sodium channels.
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Synaptotagmin, a synaptic vesicle protein involved in Ca(2+)-regulated exocytosis, displayed direct high affinity interaction with neuronal sodium channels. Monoclonal antibodies directed against synaptotagmins I and II adsorbed in a concentration-dependent and -specific manner [(3)H]saxitoxin prelabeled sodium channels extracted with detergent from nerve endings. Conversely, co-immunoprecipitation of synaptotagmin was achieved by antibodies against sodium channel subunits. Consistent with the co-immunoprecipitation assays, solubilized [(3)H]saxitoxin-prelabeled sodium channels were trapped on immobilized maltose binding protein (MBP)-synaptotagmin I. In vitro recombinant protein assays were employed to identify the interaction site of synaptotagmin I, which was located on the cytoplasmic loop between domains I and II of the sodium channel alphaIIA subunit. The co-immunoprecipitated synaptotagmin-sodium channel complexes were found to be Ca(2+)-dependent; this effect was mimicked by Ba(2+) and Sr(2+) but not Mg(2+). Finally the complex was shown to be distinct from the synaptotagmin-SNARE protein complex that can selectively interact with presynaptic calcium channels (N and P/Q types). Thus, our findings demonstrate an unexpected and direct interaction between sodium channels and synaptotagmin. The Ca(2+)-regulated association between sodium channels and a protein implicated in vesicular fusion may have intriguing consequences for the establishment and regulation of neuronal excitability. (+info)