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美国论文代写生物硕士代写dissertation留学生论文代写范例

美国论文代写生物硕士代写dissertation留学生论文代写范例
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TABLE OF CONTENTS

ABSTRACT iii

CHAPTER 1: GENERAL INTRODUCTION 1

Introduction 1

Thesis Organization 4

References 4

Figures and Captions 8

CHAPTER 2: α-SYN’S INHIBITION EFFECT ON SNARE-MEDIATED MEMBRANE FUSION 13

Abstract 13

Introduction 14

Results and Discussion 15

Materials and Methods 17

References 19

Figures and Captions 22

CHAPTER 3: C2AB AND Ca2+ STIMULATE SNARE-MEDIATED LIPID MIXING WHILE α-SYN CAN INHIBIT THIS STIMULATORY FUNCTION 30

Abstract 30

Introduction 31

Results and Discussion 32

Materials and Methods 33

References 35

Figures and Captions 39

CHAPTER 4: CONCLUDING REMARKS 44

Conclusions 44 ii

 

References 45

ACKNOWLEDGEMENTS 47 iii

ABSTRACT

Neurotransmitter release is a precisely orchestrated process in terms of time and space in a neuron. SNAREs have been identified to function as the basic machinery mediating membrane fusion during neutotransmitter release. Many forms of neurodegeneration initiate presynaptically, however, only a few of their molecular mechanisms have been revealed clearly. α-Synuclein (α-Syn) is a highly conserved synaptic vesicle-associated protein. Aggregated α-Syn is a major component of the Lewy bodies, which is characteristic of Parkinson’s disease (PD). We studied the effect of α-Syn on SNARE-mediated membrane fusion using fluorescent methods. Bulk lipid mixing assay shows that α-Syn has a role of inhibition of? fusion and this effect requires phosphatidylserine (PS) on the vesicles. Disease related α-Syn mutants, A30P and E46K, show higher inhibition effect in the lipid mixing assay than wild type. Synaptotagmin-1 (Syt-1) is a Ca2+ sensor localized to synaptic vesicles and regulates neuronal exocytosis. Here we also show that C2AB, a soluble version of Syt-1 that lacks the transmembrane region, enhances the FRET signal in lipid mixing significantly. This effect also needs PS on the vesicles. However, C2AB’s stimulatory effect can be inhibited by α-Syn to a large extent. Thus, α-Syn can inhibit both SNARE only mediated membrane fusion event and C2AB enhanced fusion. 1

 

CHAPTER 1: GENERAL INTRODUCTION

Introduction

Membrane fusion and SNARE proteins

A series of membrane fusion happens in cell to maintain its basic function. Membrane fusion, a process of two separate lipid bilayers merging to become one, is a universal reaction that varies vastly in space and time. One of the most studied membrane fusions is exocytosis. At the synapse, exocytosis is very important to ensure the efficient delivery of chemical signals. Synaptic vesicle fusion is mediated by a central fusion machinery called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), while also controlled by various regulators.

SNAREs vary widely in size and structure1. They are recognized by sharing a SNARE motif, which contains eight heptad repeats of 60-70 amino acids (Fig 1). The SNARE core complex is basically a parallel four-helix bundle made up of these SNARE motifs intertwined with each other (Fig 2). SNAREs can be divided into two broad categories, t-SNAREs in target plasma membranes and v-SNAREs in transport vesicles. These synaptic SNARE proteins are one of the best characterized and studied paradigms. In this system, syntaxin-1 and SNAP-25 on the plasma membrane are t-SNAREs; Synaptobrevin/VAMP2 on the vesicle is v-SNARE. Most SNAREs contain a single, C-terminal transmembrane domain adjacent to the SNARE motif. SNARE motifs spontaneously assemble into a four-helix bundle between membranes to drive fusion2.

Vesicle docking and fusion process is mediated via SNARE assembly, during which SNARE motifs of t-SNARE and v-SNARE zipper from their membrane-distal N-terminus to membrane-proximal C-terminus. According to the predominant fusion model, there are three concerted steps involved3, 4. Firstly, by forming a tight ternary complex, the SNARE motifs bring two opposing membranes together. Secondly, the outer leaflets of membranes contact with each other and merge into a hemifusion state5, 6, 7, in which the outer leaflets, but not the inner leaflets, merge together. Thirdly, fusion pore is formed after hemifusion and expands to enable content mixing. The ternary complex which resides on two membranes is called trans-SNARE and it transits into 2

 

cis-SNARE after membranes merge together (Fig 3). This ternary core complex was found to be resistant to denaturation by SDS8, 9.

Multiple studies have suggested that additional regulatory proteins are essential for the fast neurotransmitter release process10. Several proteins have been identified to play important roles in SNARE assembly, such as Munc-18, Synaptotagmin (Syt), complexin, etc. For fusion events to occur in vitro, the presence of some of these regulatory proteins is not required at high SNARE concentrations.

α-Syn

α-Syn is a cytosolic protein that is highly conserved and enriched in mature nerve terminals11. More evidence has emerged that implicates its involvement in neurodegenerative diseases12, 13. Aggregation of α-Syn into amyloid fibrils is a major component of the Lewy Body deposits, which is the pathological hallmark of Pakinson’s Disease (PD) (Fig 4). Duplication and triplication of the wild-type α-Syn gene, and several missense mutations have been proposed to be linked with rare familial forms of early-onset PD. It has been shown that excess accumulation of α-Syn leads to cellular toxicity when α-Syn, or PD-related α-Syn mutants, is overexpressed in mouse, rat and even yeast. Despite intense studies, the exact function of α-Syn is still unclear.

A small protein of 140-143 amino acids as it is, α-Syn is natively unfolded in solution. According to circular dichroism measurements, the conformation of α-Syn in solution is quite random. However, in the presence of lipid vesicles, α-Syn adopts a highly helical structure in its N-terminal region and remains unstructured at C-terminus14.

The extremely well conserved α-Syn sequence in evolution implies functional constraints on its three-dimensional structure. The presence of seven imperfect 11-mer repeats in the sequence has a high resemblance to 11-mer repeats of apolipoproteins, suggesting a lipid interaction role of α-Syn. The recurring 11-residue periodicity enables α-Syn to have a capacity to fold into an amphipathic α-helix. A helical wheel model has been proposed to represent α-Syn. In this model, several of the α-Syn 11-mers display a distinctive distribution of polar and nonpolar residues on the opposite faces of the helix11. The structural observation of α-Syn led us to hypothesize that α-

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