Single-molecule FRET investigation of SNARE-mediated exocytosis regulation

In the neuron, neurotransmitter release is mediated by SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) proteins. SNARE-dependent synaptic vesicle membrane and plasma membrane fusion is a multiple-step event and a tightly regulated process. Vesicle-anchored (v-) SNARE from synaptic vesicles associates with target plasma membrane-anchored (t-) SNARE to form a trans-SNAREpin complex. When the triggering signal arrives, v-SNARE and t-SNARE mediate the membrane full fusion and extend on one side of the membrane, forming a cis-conformation. During the whole process, SNARE complex with the help of regulators overcomes the energy barriers to fuse two apposed membranes and ensures that fusion proceeds at the correct time and place. Currently, there are some key questions that remain regarding SNARE-mediated exocytosis regulation. First, among the SNARE regulators, complexin is a small SNARE-binding protein that is thought to inhibit membrane fusion before Ca2+ triggering signal arrives. Although such an inhibitory role of complexin has been reported, its structural basis is very controversially discussed. Second, as the central machinery of neurotransmitters release, all three SNARE proteins are targets of different botulinum neurotoxins (BoNTs). Even though BoNT A and E cleave SNAP-25 at the C-terminus to inhibit SNARE-dependent membrane fusion, the detailed effects of BoNT A and E cleavage on SNARE complex folding pathway, conformation and function remain largely elusive. Third, the cis-SNARE complex contains 16 layers. BoNT E and A cleave SNAP-25 at residue 180 within layer \u27+2\u27 and residue 197 within layer \u27+7\u27, separately. The effect of SNAP-25 layers on SNARE complex formation has not been systematically studied. Also, another knowledge gap is why naturally selected BoNT E and A choose to cleave SNAP-25 at residue 180 and 197. In this thesis, to solve the aforementioned questions, we primarily used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the trans-SNAREpin and cis-SNARE complex formation and structure in the presence of SNARE regulators. Our results demonstrate that complexin splits the SNARE core in the C-terminal region to inhibit further SNARE zippering. We also conclude that the two membranes are necessary for the proper complexin function and are an integral part of the synaptic vesicle fusion regulatory machinery. SNAP-25E, the cleavage product by BoNT E, significantly decreases t- and v-SNARE pairing. The cleavage product by BoNT A SNAP-25A, however, does not affect the t- and v-SNARE pairing but mildly decreases SNARE zippering. In addition, our results unveil a delicate alpha-helix nucleation process at the SNAP-25 C-terminal motif (SC) downstream layers. The results also shed light on why BoNT E but not BoNT A can induce neuron degeneration

Synthesis of heteroatom-doped ZnO nanoparticles as an efficient visible light photocatalyst and its photoelectrochemical performance

Heteroatom-doped ZnO nanoparticles (Zn1-xNixO NPs) have been synthesized by a simple solvothermal approach. The photocatalytic activity of the products has been evaluated by a photoassisted degradation of Rhodamine B in aqueous solution under visible light irradiation. All the heteroatom-doped semiconductors exhibit better photocatalytic activities than pure ZnO, with the 1 mol% Ni2+-doped ZnO showing the best photocatalytic activity. From the transient photocurrent response and electrochemical impedance spectroscopic experiments it is observed that the photogenerated charges of the Ni-doped ZnO show longer lifetime and higher separation than that of pure ZnO, leading to its superior visible light photoactivity. The active species tests indicate that the hydroxyl radical and active holes were primarily responsible for the enhanced photocatalytic performance of Rhodamine B, and the superoxide radical takes part partially in the oxidation process. A possible photocatalytic mechanism is proposed. Good photostability and reusability of the product show that the studied nanoparticles have potential application in dye wastewater treatment

Single-molecule FRET investigation of SNARE-mediated exocytosis regulation

In the neuron, neurotransmitter release is mediated by SNARE (soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) proteins. SNARE-dependent synaptic vesicle membrane and plasma membrane fusion is a multiple-step event and a tightly regulated process. Vesicle-anchored (v-) SNARE from synaptic vesicles associates with target plasma membrane-anchored (t-) SNARE to form a trans-SNAREpin complex. When the triggering signal arrives, v-SNARE and t-SNARE mediate the membrane full fusion and extend on one side of the membrane, forming a cis-conformation. During the whole process, SNARE complex with the help of regulators overcomes the energy barriers to fuse two apposed membranes and ensures that fusion proceeds at the correct time and place. Currently, there are some key questions that remain regarding SNARE-mediated exocytosis regulation. First, among the SNARE regulators, complexin is a small SNARE-binding protein that is thought to inhibit membrane fusion before Ca2+ triggering signal arrives. Although such an inhibitory role of complexin has been reported, its structural basis is very controversially discussed. Second, as the central machinery of neurotransmitters release, all three SNARE proteins are targets of different botulinum neurotoxins (BoNTs). Even though BoNT A and E cleave SNAP-25 at the C-terminus to inhibit SNARE-dependent membrane fusion, the detailed effects of BoNT A and E cleavage on SNARE complex folding pathway, conformation and function remain largely elusive. Third, the cis-SNARE complex contains 16 layers. BoNT E and A cleave SNAP-25 at residue 180 within layer '+2' and residue 197 within layer '+7', separately. The effect of SNAP-25 layers on SNARE complex formation has not been systematically studied. Also, another knowledge gap is why naturally selected BoNT E and A choose to cleave SNAP-25 at residue 180 and 197. In this thesis, to solve the aforementioned questions, we primarily used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the trans-SNAREpin and cis-SNARE complex formation and structure in the presence of SNARE regulators. Our results demonstrate that complexin splits the SNARE core in the C-terminal region to inhibit further SNARE zippering. We also conclude that the two membranes are necessary for the proper complexin function and are an integral part of the synaptic vesicle fusion regulatory machinery. SNAP-25E, the cleavage product by BoNT E, significantly decreases t- and v-SNARE pairing. The cleavage product by BoNT A SNAP-25A, however, does not affect the t- and v-SNARE pairing but mildly decreases SNARE zippering. In addition, our results unveil a delicate alpha-helix nucleation process at the SNAP-25 C-terminal motif (SC) downstream layers. The results also shed light on why BoNT E but not BoNT A can induce neuron degeneration.</p

Synthesis of heteroatom-doped ZnO nanoparticles as an efficient visible light photocatalyst and its photoelectrochemical performance

566-570<span style="font-size:9.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" "times="" roman";mso-ansi-language:en-us;mso-fareast-language:en-us;="" mso-bidi-language:ar-sa"="" lang="EN-US">Heteroatom-doped ZnO nanoparticles (Zn1-xNixO NPs) have been synthesized by a simple solvothermal approach. The photocatalytic activity of the products has been evaluated by a photoassisted degradation of Rhodamine B in aqueous solution under visible light irradiation. All the heteroatom-doped semiconductors exhibit better photocatalytic activities than pure ZnO, with the 1 mol% Ni2+-doped ZnO showing the best photocatalytic activity. From the transient photocurrent response and electrochemical impedance spectroscopic experiments it is observed that the photogenerated charges of the Ni-doped ZnO show longer lifetime and higher separation than that of pure ZnO, leading to its superior visible light photoactivity. The active species tests indicate that the hydroxyl radical and active holes were primarily responsible for the enhanced photocatalytic performance of Rhodamine B, and the superoxide radical takes part partially in the oxidation process. A possible photocatalytic mechanism is proposed. Good photostability and reusability of the product show that the studied nanoparticles have potential application in dye wastewater treatment.</span

Synthesis of Paclitaxel Derivatives for Remote Loading into Liposomes and Improved Therapeutic Effect

A series of novel paclitaxel derivatives modified by boronic acid according to the characteristics of the interaction between RB(OH)2 and different strapping agents of intraliposomal aqueous phase were designed and synthesized, which were then used to develop remote poorly water-soluble drugs loading into liposomes. Meanwhile, we screened nineteen paclitaxel boronic acid derivatives for their cytotoxic activities against three cancer cell lines (A549, HCT-116 and 4T1) and one normal cell line (LO2), and performed liposome formulation screening of active compounds. Among all the compounds, the liposome of 4d, with excellent drug-encapsulated efficiency (&gt;95% for drug-to-lipid ratio of 0.1 w/w), was the most stable. Furthermore, the liposomes of compound 4d (8 mg/kg, 4 times) and higher dose of compound 4d (24 mg/kg, 4 times) showed better therapeutic effect than paclitaxel (8 mg/kg, 4 times) in the 4T1 tumor model in vivo, and the rates of tumor inhibition were 74.3%, 81.9% and 58.5%, respectively. This study provided a reasonable design strategy for the insoluble drugs to improve their drug loading into liposomes and anti-tumor effect in vivo

Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy

Discovery of anti-inflammatory dihydroxylated phenolic acids in patients with severe cardiac symptoms and conditions associated with inflammation and hypoxia

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe cardiac symptoms when compared with healthy subjects. In view of the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on a multi-centre investigation to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients with severe cardiac symptoms and those being treated for conditions associated with inflammation (WBC ≥ 10×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled, their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. Proteus vulgaris and P. mirabilis were significantly more abundant in DHPLA-positive patients’ fecal samples than healthy volunteers. In culture, these bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest anti-inflammatory DPAs are generated de novo as part of a human’s defense mechanism against disease. Given that DHPLA isolated from Radix Salvia miltiorrhizae has multi-dimensional pharmacological activities, these data demonstrate not only scientific basis of the ethnopharmacological uses of this medicinal plant but also highlight the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans