Dae-Hyuk Kweon

Dynamic Ca2+-Dependent Stimulation of Vesicle Fusion by Membrane-Anchored Synaptotagmin 1

A Trick of the Tail The synaptic vesicle protein, synaptotagmin 1 (Syt1), acts as the main Ca2+-dependent switch for neurotransmitter release. In vitro studies of the truncated Syt1, which lacks the transmembrane domain, have unveiled the fusion-triggering mechanism of Syt1. However, in vitro approaches using the full-length, membrane-anchored Syt1 have not only failed to recapitulate Ca2+-triggered membrane fusion, but could even inhibit vesicle fusion. In contrast, the membrane anchor is conserved across the Syt family, suggesting a critical functional role for the membrane anchor. Now, using a single vesicle fusion assay, H.-K. Lee et al. (p. 760) show that the membrane anchor is indeed essential for Syt1 to induce physiological rates of Ca2+-induced vesicle fusion on a 100-millisecond time scale. A synaptic vesicle protein must be membrane-anchored to stimulate fusion in vitro at physiological Ca2+ concentrations. In neurons, synaptotagmin 1 (Syt1) is thought to mediate the fusion of synaptic vesicles with the plasma membrane when presynaptic Ca2+ levels rise. However, in vitro reconstitution experiments have failed to recapitulate key characteristics of Ca2+-triggered membrane fusion. Using an in vitro single-vesicle fusion assay, we found that membrane-anchored Syt1 enhanced Ca2+ sensitivity and fusion speed. This stimulatory activity of membrane-anchored Syt1 dropped as the Ca2+ level rose beyond physiological levels. Thus, Syt1 requires the membrane anchor to stimulate vesicle fusion at physiological Ca2+ levels and may function as a dynamic presynaptic Ca2+ sensor to control the probability of neurotransmitter release.

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking.

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.

Selective Killing of Bacterial Persisters by a Single Chemical Compound without Affecting Normal Antibiotic-Sensitive Cells

ABSTRACT We show that 3-[4-(4-methoxyphenyl)piperazin-1-yl]piperidin-4-yl biphenyl-4-carboxylate (C10), screened out of a chemical library, selectively kills bacterial persisters that tolerate antibiotic treatment but does not affect normal antibiotic-sensitive cells. C10 led persisters to antibiotic-induced cell death by causing reversion of persisters to antibiotic-sensitive cells. This work is the first demonstration in which the eradication of bacterial persisters is based on single-chemical supplementation. The chemical should be versatile in elucidating the mechanism of persistence.

Inhibitory Effect of Agrimonia pilosa Ledeb. on Inflammation by Suppression of iNOS and ROS Production

Herbal medicines including Agrimonia pilosa Ledeb. (APL) have been traditionally used to treat inflammations including allergic disease as valuable medicinal properties. To investigate the attenuating ability of APL on inflammation, the NO release and ROS production, which play a key role in inflammatory and immune responses, was first tested using in vitro assay. The 80% ethanol extract of APL showed a significant activity to inhibit NO release and ROS production. In additional extracts from 80% ethanol extract of APL, n-butanol (BuOH) extract displayed the most potent anti-inflammatory effects based on in vitro assay. The extract also significantly reduced nitric oxide in lipopolysaccharide-activated RAW 264.7 macrophage cells (p < 0.05), and suppressed the nitric oxide synthase (iNOS) expression, whereas the extract showed no inhibitory effect on cyclooxygenase-2 (COX-2) expression, suggesting that the BuOH extract of APL could reduce the NO production through suppression of iNOS, but not COX-2. The BuOH extract also showed a significant effect in a carrageenan-induced rat paw edema in vivo model, consistent with our in vitro results. Our findings suggest that the BuOH extract of APL shows a potential anti-inflammatory activity, substantiating its traditional use in medicine.

Butein is a novel anti-adipogenic compound

Rhus verniciflua Stokes (RVS) has been used as a traditional herbal medicine for its various biological activities including anti-adipogenic effects. Activity-guided separation led to the identification of the anti-adipogenic functions of butein. Butein, a novel anti-adipogenic compound, robustly suppressed lipid accumulation and inhibited expression of adipogenic markers. Molecular studies showed that activated transforming growth factor-β (TGF-β) and suppressed signal transducer and activator of transcription 3 (STAT3) signaling pathways were mediated by butein. Analysis of the temporal expression profiles suggests that TGF-β signaling precedes the STAT3 in the butein-mediated anti-adipogenic cascade. Small interfering RNA-mediated silencing of STAT3 or SMAD2/3 blunted the inhibitory effects of butein on adipogenesis indicating that an interaction between two signaling pathways is required for the action of butein. Upon butein treatments, stimulation of TGF-β signaling was still preserved in STAT3 silenced cells, whereas regulation of STAT3 signaling by butein was significantly impaired in SMAD2/3 silenced cells, further showing that TGF-β acts upstream of STAT3 in the butein-mediated anti-adipogenesis. Taken together, the present study shows that butein, a novel anti-adipogenic compound from RVS, inhibits adipocyte differentiation through the TGF-β pathway followed by STAT3 and peroxisome proliferator-activated receptor γ signaling, further implicating potential roles of butein in TGF-β- and STAT3-dysregulated diseases.

Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin.

Expression with a fusion partner is now a popular scheme to produce a protein of interest because it provides a generic tool for expression and purification. In our previous study, a strong polycationic tail has been harnessed for an efficient purification scheme. Here, the same polycation tail attached to a protein of interest is shown to hold versatility for a solid‐phase refolding method that utilizes a charged adsorbent as a supporting material. Cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at the C‐terminus (CGTK10ase) retains the ability to bind to a cation exchanger even in a urea‐denatured state. When the denatured and adsorbed CGTK10ase is induced to refold, the bound CGTK10ase aggregates little even at a g/L range. The renatured CGTK10ase can also be simply recovered from the solid support by adding high concentration of NaCl. The CGTK10ase refolded on a solid support retains specific enzyme activity virtually identical to that of the native CGTK10ase. Several factors that are important in improving the refolding efficiency are explored. Experimental results indicate that nonspecific electrostatic interactions between the charge of the ion exchanger and the local charge of CGTase other than the polycationic tag should be reduced to obtain higher refolding yield. The solid‐phase refolding method utilizing a strong polycationic tag resulted in a remarkable increase in the refolding performance. Taken together with the previous report in which a series of polycations were explored for efficient purification, expression of a target protein fused with a strong polycation provides a straightforward protein preparation scheme.

Dissection of SNARE-driven membrane fusion and neuroexocytosis by wedging small hydrophobic molecules into the SNARE zipper

Neuronal SNARE proteins mediate neurotransmitter release at the synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from the vesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the C-terminal ends. Here, we demonstrate that SNARE zippering can be modulated in the midways by wedging with small hydrophobic molecules. Myricetin, which intercalated into the hydrophobic inner core near the middle of the SNARE complex, stopped SNARE zippering in motion and accumulated the trans-complex, where the N-terminal region of v-SNARE VAMP2 is in the coiled coil with the frayed C-terminal region. Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering. Neuronal SNARE complex in PC12 cells showed the same pattern of vulnerability to small hydrophobic molecules. We propose that the half-zipped trans-SNARE complex is a crucial intermediate waiting for a calcium trigger that leads to fusion pore opening.

Overproduction of Phytolacca insularis protein in batch and fed-batch culture of recombinant Escherichia coli

Escherichia coli TG1 transformed with an expression plasmid pMS carrying the gene encoding Phytolacca insularis protein (PIP) under the control of the tac promoter was cultivated in various conditions to improve the productivity of PIP. Production of PIP was dramatically increased by the addition of yeast extract as a nitrogen source in both batch and fed-batch cultures. High levels of PIP were obtained by adopting a fed-batch process and an optimized induction strategy. The optimized fed-batch culture resulted in 59.0 g of cell mass and 466.5 mg of soluble PIP per litre of culture, corresponding to a 9.1 fold increase in cell mass and a 17.3 fold enhancement in PIP production compared with a simple batch culture in Luria broth.

Multiple conformations of a single SNAREpin between two nanodisc membranes reveal diverse pre-fusion states

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized, pre-fusion conformations have been elusive. We used single-molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. Although the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure, the membrane-proximal C-terminal half shows high FRET, representing a helical bundle (45%), low FRET, reflecting a frayed conformation (39%) or mid FRET revealing an as-yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1.

A Biosynthetic Pathway for Hexanoic Acid Production in Kluyveromyces marxianus

Hexanoic acid can be used for diverse industrial applications and is a precursor for fine chemistry. Although some natural microorganisms have been screened and evolved to produce hexanoic acid, the construction of an engineered biosynthetic pathway for producing hexanoic acid in yeast has not been reported. Here we constructed hexanoic acid pathways in Kluyveromyces marxianus by integrating 5 combinations of seven genes (AtoB, BktB, Crt, Hbd, MCT1, Ter, and TES1), by which random chromosomal sites of the strain are overwritten by the new genes from bacteria and yeast. One recombinant strain, H4A, which contained AtoB, BktB, Crt, Hbd, and Ter, produced 154mg/L of hexanoic acid from galactose as the sole substrate. However, the hexanoic acid produced by the H4A strain was re-assimilated during the fermentation due to the reverse activity of AtoB, which condenses two acetyl-CoAs into a single acetoacetyl-CoA. This product instability could be overcome by the replacement of AtoB with a malonyl CoA-acyl carrier protein transacylase (MCT1) from Saccharomyces cerevisiae. Our results suggest that Mct1 provides a slow but stable acetyl-CoA chain elongation pathway, whereas the AtoB-mediated route is fast but unstable. In conclusion, hexanoic acid was produced for the first time in yeast by the construction of chain elongation pathways comprising 5-7 genes in K. marxianus.