Structural and functional studies with membrane-associated proteins in synaptic exocytosis and endocytosis

In the cell, there are about 60% of the total proteins are membrane and membrane-associated proteins. Exocytosis, or its reverse phase endocytosis, contains a variety of membrane proteins such as synaptobrevin2, syntaxin-1A etc and membrane-associated proteins, for example SNAP25 and epsin1 etc. Synaptobrein2 is also called vesicle-associated protein or v-SANRE which interacts with t-SNAREs syntaxin-1A and SNAP25 binary complex to form a four helical-bundle complex, which was the minimal fusion machinery for synaptic vesicle fused with plasma membrane in neuron. In addition, many other proteins such as Munc18, synaptotagmin, complexin etc are essential regulators to control neuronal transmitter release spatially and temporally. Epsin1, a membrane-associated protein binding with the plasma membrane by interaction with lipid phosphatidylinositol 4, 5-biphosphate (PIP2), on the contrary, plays a role in the clathrin-mediated endocytosis process. The purpose of this work is to investigate the structures of TMD (transmembrane domain) of synaptobrevin2 and N-termini (residues 1-14) of ENTH domain of epsin1 by EPR (electron paramagnetic resonance) technique. In addition, lipid mixing assay with fluorescence dyes was also applied to mimic the kinetics of membrane fusion with recombinant SNARE proteins on the liposome in vitro. EPR studies with TMD of synaptobrevin2 revealed that TMD was characterized as a loose dimmer with interaction mostly at the N-half of TMD, while C-half interacted with each other to form a dimmer only on the vesicle with 40% cholesterol. Thus, a scissors mechanism exists for TMD of synaptobrevin2 with the stimulation of cholesterol molecule. Moreover, disulfide cross-linked lipid mixing assay confirmed different functions for these two confirmations of TMD. Meanwhile, the EPR work of N-termini of ENTH domain provided evidence that the N-termini of ENTH domain forms an amphiphilic helix when it inserts into the vesicle contained 3% of lipid PIP2. Our power saturation study unraveled the topology of this helix with hydrophobic residues L6 and M10 deep insertion into the membrane. Most interestingly, the work helps us to identify the anti-parallel dimerization of N-termini of ENTH domain on the surface of membrane. Functional studies both in vitro and in vivo confirmed that such a self-association of ENTH domains on the membrane surface plays an important role to tubulate the membrane during endocytosis process

Network motif comparison rationalizes Sec1/Munc18-SNARE regulation mechanism in exocytosis

BackgroundNetwork motifs, recurring subnetwork patterns, provide significant insight into the biological networks which are believed to govern cellular processes. MethodsWe present a comparative network motif experimental approach, which helps to explain complex biological phenomena and increases the understanding of biological functions at the molecular level by exploring evolutionary design principles of network motifs. ResultsUsing this framework to analyze the SM (Sec1/Munc18)-SNARE (N-ethylmaleimide-sensitive factor activating protein receptor) system in exocytic membrane fusion in yeast and neurons, we find that the SM-SNARE network motifs of yeast and neurons show distinct dynamical behaviors. We identify the closed binding mode of neuronal SM (Munc18-1) and SNARE (syntaxin-1) as the key factor leading to mechanistic divergence of membrane fusion systems in yeast and neurons. We also predict that it underlies the conflicting observations in SM overexpression experiments. Furthermore, hypothesis-driven lipid mixing assays validated the prediction. ConclusionTherefore this study provides a new method to solve the discrepancies and to generalize the functional role of SM proteins

The LDBC Financial Benchmark

The Linked Data Benchmark Council's Financial Benchmark (LDBC FinBench) is a new effort that defines a graph database benchmark targeting financial scenarios such as anti-fraud and risk control. The benchmark has one workload, the Transaction Workload, currently. It captures OLTP scenario with complex, simple read queries and write queries that continuously insert or delete data in the graph. Compared to the LDBC SNB, the LDBC FinBench differs in application scenarios, data patterns, and query patterns. This document contains a detailed explanation of the data used in the LDBC FinBench, the definition of transaction workload, a detailed description for all queries, and instructions on how to use the benchmark suite.Comment: For the source code of this specification, see the ldbc_finbench_docs repository on Githu

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049

Structural and functional studies with membrane-associated proteins in synaptic exocytosis and endocytosis

In the cell, there are about 60% of the total proteins are membrane and membrane-associated proteins. Exocytosis, or its reverse phase endocytosis, contains a variety of membrane proteins such as synaptobrevin2, syntaxin-1A etc and membrane-associated proteins, for example SNAP25 and epsin1 etc. Synaptobrein2 is also called vesicle-associated protein or v-SANRE which interacts with t-SNAREs syntaxin-1A and SNAP25 binary complex to form a four helical-bundle complex, which was the minimal fusion machinery for synaptic vesicle fused with plasma membrane in neuron. In addition, many other proteins such as Munc18, synaptotagmin, complexin etc are essential regulators to control neuronal transmitter release spatially and temporally. Epsin1, a membrane-associated protein binding with the plasma membrane by interaction with lipid phosphatidylinositol 4, 5-biphosphate (PIP2), on the contrary, plays a role in the clathrin-mediated endocytosis process. The purpose of this work is to investigate the structures of TMD (transmembrane domain) of synaptobrevin2 and N-termini (residues 1-14) of ENTH domain of epsin1 by EPR (electron paramagnetic resonance) technique. In addition, lipid mixing assay with fluorescence dyes was also applied to mimic the kinetics of membrane fusion with recombinant SNARE proteins on the liposome in vitro. EPR studies with TMD of synaptobrevin2 revealed that TMD was characterized as a loose dimmer with interaction mostly at the N-half of TMD, while C-half interacted with each other to form a dimmer only on the vesicle with 40% cholesterol. Thus, a scissors mechanism exists for TMD of synaptobrevin2 with the stimulation of cholesterol molecule. Moreover, disulfide cross-linked lipid mixing assay confirmed different functions for these two confirmations of TMD. Meanwhile, the EPR work of N-termini of ENTH domain provided evidence that the N-termini of ENTH domain forms an amphiphilic helix when it inserts into the vesicle contained 3% of lipid PIP2. Our power saturation study unraveled the topology of this helix with hydrophobic residues L6 and M10 deep insertion into the membrane. Most interestingly, the work helps us to identify the anti-parallel dimerization of N-termini of ENTH domain on the surface of membrane. Functional studies both in vitro and in vivo confirmed that such a self-association of ENTH domains on the membrane surface plays an important role to tubulate the membrane during endocytosis process.</p

Network motif comparison rationalizes Sec1/Munc18-SNARE regulation mechanism in exocytosis

BackgroundNetwork motifs, recurring subnetwork patterns, provide significant insight into the biological networks which are believed to govern cellular processes. MethodsWe present a comparative network motif experimental approach, which helps to explain complex biological phenomena and increases the understanding of biological functions at the molecular level by exploring evolutionary design principles of network motifs. ResultsUsing this framework to analyze the SM (Sec1/Munc18)-SNARE (N-ethylmaleimide-sensitive factor activating protein receptor) system in exocytic membrane fusion in yeast and neurons, we find that the SM-SNARE network motifs of yeast and neurons show distinct dynamical behaviors. We identify the closed binding mode of neuronal SM (Munc18-1) and SNARE (syntaxin-1) as the key factor leading to mechanistic divergence of membrane fusion systems in yeast and neurons. We also predict that it underlies the conflicting observations in SM overexpression experiments. Furthermore, hypothesis-driven lipid mixing assays validated the prediction. ConclusionTherefore this study provides a new method to solve the discrepancies and to generalize the functional role of SM proteins.This article is from BMC Systems Biology 6 (2012): 19, doi:10.1186/1752-0509-6-19, Posted with permission.</p

Synaptotagmin 1 and Ca2+ drive trans SNARE zippering

Synaptotagmin 1 (Syt1) is a major Ca(2+)-sensor that evokes neurotransmitter release. Here we used site-specific fluorescence resonance energy transfer (FRET) assay to investigate the effects of Syt1 on SNAREpin assembly. C2AB, a soluble version of Syt1, had virtually no stimulatory effect on the rate of the FRET at N-terminus of SNARE complex both with and without Ca(2+), indicating C2AB does not interfere with the initial nucleation of SNARE assembly. However, C2AB-Ca(2+) accelerated the FRET rate significantly at membrane proximal region, indicating C2AB-Ca(2+) promotes the transition from a partially assembled SNARE complex to the fusion-competent SNAREpin. Similar enhancement was also observed at the end of the transmembrane domain of SNARE proteins. The stimulatory effect disappeared if there was no membrane or only neutral membrane present