Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy.

Intrinsic Disorder of the Neuronal SNARE Protein SNAP25a in its Pre-fusion Conformation.

The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments.

Modulation of K4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current () is generated by the activation of K4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by K4.3/KChIP2, K4.

Forces, Kinetics, and Fusion Efficiency Altered by the Full-Length Synaptotagmin-1 -PI(4,5)P Interaction in Constrained Geometries.

A mechanism for full-length synaptotagmin-1 (syt-1) to interact with anionic bilayers and to promote fusion in the presence of SNAREs is proposed. Colloidal probe force spectroscopy in conjunction with tethered particle motion monitoring showed that in the absence of Ca the binding of syt-1 to membranes depends on the presence and content of PI(4,5)P. Addition of Ca switches the interaction forces from weak to strong, eventually exceeding the cohesion of the C2A domain of syt-1 leading to partial unfolding of the protein.

Dynamic Excimer (DYNEX) Imaging of Lipid Droplets.

Unraveling cellular physiological processes via luminescent probes that target specific cellular microenvironments is quite challenging due to the uneven distribution of probes. Herein, we designed a new dynamic excimer (DYNEX) imaging method that involves the sensitive detection of nanosecond-scale dynamic molecular contacts of a fluorescent acridone derivative and reveals the cell microenvironment polarity. Using our method, we specifically tracked cell lipid droplets in fibroblast colon carcinoma cells.

An overview of the synaptic vesicle lipid composition.

Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steady-state levels.

Characterization of PROPPIN-Phosphoinositide Binding by Stopped-Flow Fluorescence Spectroscopy.

PROPPINs (β-propellers that bind polyphosphoinositides) are a protein family that binds preferentially phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) via its FRRG motif. PROPPINs are involved in autophagic functions, but their molecular mechanism is still elusive. To unravel the molecular mechanism of PROPPINs, it is essential to understand the PROPPIN-phosphoinositide binding.

Structural dynamics and transient lipid binding of synaptobrevin-2 tune SNARE assembly and membrane fusion.

Intrinsically disordered proteins (IDPs) and their conformational transitions play an important role in neurotransmitter release at the neuronal synapse. Here, the SNARE proteins are essential by forming the SNARE complex that drives vesicular membrane fusion. While it is widely accepted that the SNARE proteins are intrinsically disordered in their monomeric prefusion form, important mechanistic aspects of this prefusion conformation and its lipid interactions, before forming the SNARE complex, are not fully understood at the molecular level and remain controversial.

Structure and pro-toxic mechanism of the human Hsp90/PPIase/Tau complex.

The molecular chaperone Hsp90 is critical for the maintenance of cellular homeostasis and represents a promising drug target. Despite increasing knowledge on the structure of Hsp90, the molecular basis of substrate recognition and pro-folding by Hsp90/co-chaperone complexes remains unknown. Here, we report the solution structures of human full-length Hsp90 in complex with the PPIase FKBP51, as well as the 280 kDa Hsp90/FKBP51 complex bound to the Alzheimer's disease-related protein Tau.

Plekhg5-regulated autophagy of synaptic vesicles reveals a pathogenic mechanism in motoneuron disease.

Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures.

Structure based biophysical characterization of the PROPPIN Atg18 shows Atg18 oligomerization upon membrane binding.

PROPPINs (β-propellers that bind polyphosphoinositides) are PtdIns3P and PtdIns(3,5)P binding autophagy related proteins. They contain two phosphatidylinositolphosphate (PIP) binding sites and a conserved FRRG motif is essential for PIP binding. Here we present the 2.

Synaptotagmin-1 binds to PIP(2)-containing membrane but not to SNAREs at physiological ionic strength.

The Ca(2+) sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored.

Characterization of PROPPIN-Phosphoinositide Binding and Role of Loop 6CD in PROPPIN-Membrane Binding.

PROPPINs (β-propellers that bind polyphosphoinositides) are a family of PtdIns3P- and PtdIns(3,5)P2-binding proteins that play an important role in autophagy. We analyzed PROPPIN-membrane binding through isothermal titration calorimetry (ITC), stopped-flow measurements, mutagenesis studies, and molecular dynamics (MD) simulations. ITC measurements showed that the yeast PROPPIN family members Atg18, Atg21, and Hsv2 bind PtdIns3P and PtdIns(3,5)P2 with high affinities in the nanomolar to low-micromolar range and have two phosphoinositide (PIP)-binding sites.

Control of membrane gaps by synaptotagmin-Ca2+ measured with a novel membrane distance ruler.

Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1 (syt-1), resulting in fusion of synaptic vesicles with the presynaptic membrane. Syt-1 possesses two Ca(2+)-binding C2 domains that tether membranes via interactions with anionic phospholipids. It is capable of crosslinking membranes and has recently been speculated to trigger fusion by decreasing the gap between them.

Phosphatidylinositol-4,5-bisphosphate enhances anionic lipid demixing by the C2 domain of PKCα.

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca2+, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP2) was present in the membrane. When PIP2 was the labeled phospholipid, in the form of TopFluor-PIP2, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine.

Phosphatidylinositol 4,5-bisphosphate decreases the concentration of Ca2+, phosphatidylserine and diacylglycerol required for protein kinase C α to reach maximum activity.

The C2 domain of PKCα possesses two different binding sites, one for Ca(2+) and phosphatidylserine and a second one that binds PIP2 with very high affinity. The enzymatic activity of PKCα was studied by activating it with large unilamellar lipid vesicles, varying the concentration of Ca(2+) and the contents of dioleylglycerol (DOG), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphadidylserine (POPS) in these model membranes. The results showed that PIP2 increased the Vmax of PKCα and, when the PIP2 concentration was 5 mol% of the total lipid in the membrane, the addition of 2 mol% of DOG did not increase the activity.

The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition.

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2).

Curcumin modulates PKCα activity by a membrane-dependent effect.

Curcumin modulates the activity of protein kinase Cα (PKCα) when assayed in the presence of vesicles including phosphatidylcholine, phosphatidylserine and diacylglycerol. Increasing concentrations of curcumin progressively increased PKCα activity at concentrations lower than 20μM, but at higher concentrations of curcumin the activity decreased although, at concentrations of curcumin of up to 100μM the activity was always higher than the basal one (in the absence of curcumin). The maximum activity was reached at 3μM curcumin, at 20 and 30mol% of phosphatidylserine, 10μM Ca(2+) and 2mol% diacylglycerol.

Curcumin disorders 1,2-dipalmitoyl-sn-glycero-3-phosphocholine membranes and favors the formation of nonlamellar structures by 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine.

Curcumin is a polyphenol present in turmeric, a spice widely used in Asian traditional medicine and cooking. It has many and diverse biological effects and is incorporated in cell membranes. This paper describes the mode in which curcumin modulates the physical properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dielaidyl-sn-glycero-3-phosphoetnanolamine (DEPE) multilamellar membranes.

Antithrombin Murcia (K241E) causing antithrombin deficiency: a possible role for altered glycosylation.

Background: Identification of mutations in the SERPINC1 gene has revealed different mechanisms responsible for antithrombin deficiency. Deletions and nonsense mutations associate with type I deficiency. Certain missense mutations cause type II deficiency by affecting the heparin binding site or the reactive center loop, while others result in type I deficiency by intracellular retention or RNA instability.

A comparison of the membrane binding properties of C1B domains of PKCgamma, PKCdelta, and PKCepsilon.

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical gamma, and the novel delta and epsilon), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols.