Model of SNARE-Mediated Membrane Adhesion Kinetics

SNARE proteins are conserved components of the core fusion machinery driving diverse membrane adhesion and fusion processes in the cell. In many cases micron-sized membranes adhere over large areas before fusion. Reconstituted in vitro assays have helped isolate SNARE mechanisms in small membrane adhesion-fusion and are emerging as powerful tools to study large membrane systems by use of giant unilamellar vesicles (GUVs). Here we model SNARE-mediated adhesion kinetics in SNARE-reconstituted GUV-GUV or GUV-supported bilayer experiments. Adhesion involves many SNAREs whose complexation pulls apposing membranes into contact. The contact region is a tightly bound rapidly expanding patch whose growth velocity increases with SNARE density . We find three patch expansion regimes: slow, intermediate, fast. Typical experiments belong to the fast regime where depends on SNARE diffusivities and complexation binding constant. The model predicts growth velocities s. The patch may provide a close contact region where SNAREs can trigger fusion. Extending the model to a simple description of fusion, a broad distribution of fusion times is predicted. Increasing SNARE density accelerates fusion by boosting the patch growth velocity, thereby providing more complexes to participate in fusion. This quantifies the notion of SNAREs as dual adhesion-fusion agents

An Ancient Duplication of Exon 5 in the Snap25 Gene Is Required for Complex Neuronal Development/Function

Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Molecular machines governing exocytosis of synaptic vesicles.

Calcium-dependent exocytosis of synaptic vesicles mediates the release of neurotransmitters. Important proteins in this process have been identified such as the SNAREs, synaptotagmins, complexins, Munc18 and Munc13. Structural and functional studies have yielded a wealth of information about the physiological role of these proteins. However, it has been surprisingly difficult to arrive at a unified picture of the molecular sequence of events from vesicle docking to calcium-triggered membrane fusion. Using mainly a biochemical and biophysical perspective, we briefly survey the molecular mechanisms in an attempt to functionally integrate the key proteins into the emerging picture of the neuronal fusion machine

Sex-Differences in the Pattern of Comorbidities, Functional Independence, and Mortality in Elderly Inpatients: Evidence from the RePoSI Register.

BACKGROUND: The RePoSi study has provided data on comorbidities, polypharmacy, and sex dimorphism in hospitalised elderly patients. METHODS: We retrospectively analysed data collected from the 2010, 2012, 2014, and 2016 data sets of the RePoSi register. The aim of this study was to explore the sex-differences and to validate the multivariate model in the entire dataset with an expanded follow-up at 1 year. RESULTS: Among 4714 patients, 51% were women and 49% were men. The disease distribution showed that diabetes, coronary artery disease, chronic obstructive pulmonary disease, chronic kidney disease, and malignancy were more frequent in men but that hypertension, anaemia, osteoarthritis, depression, and diverticulitis disease were more common in women. Severity and comorbidity indexes according to the Cumulative Illness Rating Scale (CIRS-s and CIRS-c) were higher in men, while cognitive impairment, mood disorders, and disability in daily life measured by the Barthel Index (BI) were worse in women. In the multivariate analysis, BI, CIRS, and malignancy significantly increased the risk of death in men at the 1-year follow-up, while age was independently associated with mortality in women. CONCLUSIONS: Our study highlighted the relevance and the validity of our previous predictive model in the identification of sex dimorphism in hospitalised elderly patients underscoring the need of sex-personalised health-care

SNAREing immunity: the role of SNAREs in the immune system

The trafficking of molecules and membranes within cells is a prerequisite for all aspects of cellular immune functions, including the delivery and recycling of cell surface proteins, secretion of immune mediators, ingestion of pathogens and activation of lymphocytes. SNARE (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor)-family members mediate membrane fusion during all steps of trafficking, and function in almost all aspects of innate and adaptive immune responses. Here, we provide an overview of the roles of SNAREs in immune cells, offering insight into one level at which precision and tight regulation are instilled on immune responses

Energetics and dynamics of SNAREpin folding across lipid bilayers

International audienceMembrane fusion occurs when SNAREpins fold up between lipid bilayers. How much energy is generated during SNAREpin folding and how this energy is coupled to the fusion of apposing membranes is unknown. We have used a surface forces apparatus to determine the energetics and dynamics of SNAREpin formation and characterize the different intermediate structures sampled by cognate SNAREs in the course of their assembly. The interaction energy-versus-distance profiles of assembling SNAREpins reveal that SNARE motifs begin to interact when the membranes are 8 nm apart. Even after very close approach of the bilayers (B2-4 nm), the SNAREpins remain partly unstructured in their membrane-proximal region. The energy stabilizing a single SNAREpin in this configuration (35 k B T) corresponds closely with the energy needed to fuse outer but not inner leaflets (hemifusion) of pure lipid bilayers (40-50 k B T). Intercellular communication and intracellular protein transport rely upon the fusion of cargo-containing vesicles with target membranes. As lipid bilayers are inherently stable, such fusion events are energetically costly and require specialized fusion proteins that harvest the energy made available during their own binding and folding to drive membrane disruption and merging 1-5. In neuronal synapses, the core of the fusion machinery consists of three proteins from the SNARE family: the synaptic vesicle (v)-SNARE protein VAMP-2 and the two target plasma membrane (t)-SNARE proteins syntaxin-1A and SNAP-25 (refs. 6-8). When separately reconstituted into synthetic liposomes or ectopically expressed on the surfaces of cells, neuronal v-and t-SNARE proteins are sufficient to drive membrane fusion through their assembly in the form of SNAREpins 2,9. The interacting domains of SNARE proteins (SNARE motifs) contain 60-70 amino acid residues; they are mostly unstruc-tured as monomers 10-12 and assemble in solution into a highly stable heterotrimer consisting of four a-helices aligned in parallel, with VAMP-2 and syntaxin-1A each contributing one helix and SNAP-25 contributing two helices 13,14. In the context of lipid bilayers, the assembly of SNAREs starts at their membrane-distal N termini and proceeds toward their membrane-proximal C termini (zipper model), a process that also includes passage through a stable intermediate binding state 15-21. This zipper-like assembly progressively brings the membranes into close apposition and creates a tight bridge between them that triggers lipid bilayer fusion. Progressive assembly of SNAREs may culminate in a release of energy sufficient to drive membrane merging. Alternatively, the assembling SNAREs may pass through a series of intermediates, each of which contributes enough energy for advancement through the successive stages of membrane fusion. Characterization of these inter-mediates requires the capacity to measure the interactions between membrane-associated proteins at nanometer distance resolutions. Thermodynamic and atomic force microscopy (AFM) measurements have successfully described the kinetics of SNARE assembly and disassembly in solution 22 and the rupture forces of SNARE complexes affixed to solid supports 23,24. However, none of these studies has been able to offer information about the dynamics and energetics of SNAREpin folding, including conformational changes and distance-energy correlations during SNARE assembly. Furthermore, the previous experiments were not performed in the context of lipid bilayers, preventing any investigation of the interplay of lipids and SNARE proteins in membrane interaction and fusion. Here, we have investigated these questions using the surface forces apparatus (SFA), which directly measures the interaction energy between two facing functionalized membranes as a function of their separation distance and makes it possible to identify molecular rearrangements of interacting species during their association 25. Direct measurements of force versus distance between membrane-embedded neuronal SNARE proteins (derived from mouse and rat) allow us to explore in real time the molecular details of SNAREpin formation across two lipid bilayers, including conformational changes, kinetics of association, binding energy and extent of assembly. RESULTS Interactions between SNAREs in apposing bilayers Force was measured between two mica-supported lipid bilayers reconstituted with the neuronal cognate t-and v-SNARE proteins (Fig. 1). The surfaces were approached toward each other and the