Minor differences in the molecular machinery mediating regulated membrane fusion has major impact on metabolic health.
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ABSTRACT:
SUBMITTER: Valladolid-Acebes I
PROVIDER: S-EPMC5013989 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Paramyxoviruses contain glycoprotein fusion machineries that mediate membrane merger for infection. The molecular framework and mechanistic principles governing receptor-induced triggering of the machinery remain unknown. Using measles virus (MeV) fusion complexes, we demonstrate that receptor binding to only one dimer of the tetrameric attachment protein (H) dimer-of-dimers induces fusion-protein (F) triggering; receptor binding and F triggering can be communicated across the dimer-dimer interface of H; and the physical integrity of the tetramer is maintained during fusion. The central MeV H ectodomain stalk region requires structural flexibility for activation of F, and alanine substitutions in this section, physical stress, or exposure of H to soluble ligands trigger conformational rearrangements in native H tetramers. Binding of soluble receptor to H is sufficient to initiate refolding of F, underscoring the physiological significance of this rearrangement of the H tetramer. These data outline a model of the triggering of the physiological MeV fusion machinery in which unilateral receptor binding to one dimer pair in the H tetramer is sufficient to induce a reorganization of H that affects the conformation of the central stalk section, severing interactions between H and the F trimer and activating refolding of F.
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Project description:Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form a four-helix coiled-coil bundle that juxtaposes two bilayers and drives a basal level of membrane fusion. The Sec1/Munc18 (SM) protein binds to its cognate SNARE bundle and accelerates the basal fusion reaction. The question of how the topological arrangement of the SNARE helices affects the reactivity of the fusion proteins remains unanswered. Here we address the problem for the first time in a reconstituted system containing both SNAREs and SM proteins. We find that to be fusogenic a SNARE topology must support both basal fusion and SM stimulation. Certain topological combinations of exocytic SNAREs result in basal fusion but cannot support SM stimulation, whereas other topologies support SM stimulation without inducing basal fusion. It is striking that of all the possible topological combinations of exocytic SNARE helices, only one induces efficient fusion. Our results suggest that the intracellular membrane fusion complex is designed to fuse bilayers according to one genetically programmed topology.
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Project description:The HIV gp41 protein catalyzes fusion between viral and host cell membranes, and its apolar N-terminal region or "fusion peptide" binds to the host cell membrane and plays a key role in fusion. "HFP" is a construct containing the fusion peptide sequence, induces membrane vesicle fusion, and is an important fusion model system. Earlier solid-state nuclear magnetic resonance (SSNMR) studies showed that when HFP is associated with membranes with ∼30 mol % cholesterol, the first 16 residues have predominant β strand secondary structure and a fraction of the strands form antiparallel β sheet structure with residue 16→1/1→16 or 17→1/1→17 registries for adjacent strands. In some contrast, other SSNMR and infrared studies have been interpreted to support a large fraction of an approximately in-register parallel registry of adjacent strands. However, the samples had extensive isotopic labeling, and other structural models were also consistent with the data. This SSNMR study uses sparse labeling schemes that reduce ambiguity in the determination of the fraction of HFP molecules with parallel β registry. Quantitative analysis of the data shows that the parallel fraction is at most 0.15 with a much greater fraction of antiparallel 16→1/1→16 and 17→1/1→17 registries. These data strongly support a model of HFP-induced vesicle fusion caused by antiparallel rather than parallel registries and provide insight into the arrangement of gp41 molecules during HIV-host cell fusion. This study is an example of quantitative determination of a complex structural distribution by SSNMR, including experimentally validated inclusion of natural abundance contributions to the SSNMR data.
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Project description:Vesicle-associated membrane protein 2 (VAMP2, also known as synaptobrevin-2), encoded by VAMP2 in humans, is a key component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. VAMP2 combined with syntaxin-1A (SYX-1A) and synaptosome-associated protein 25 (SNAP-25) produces a force that induces the formation of fusion pores, thereby mediating the fusion of synaptic vesicles and the release of neurotransmitters. VAMP2 is largely unstructured in the absence of interaction partners. Upon interaction with other SNAREs, the structure of VAMP2 stabilizes, resulting in the formation of four structural domains. In this review, we highlight the current knowledge of the roles of the VAMP2 domains and the interaction between VAMP2 and various fusion-related proteins in the presynaptic cytoplasm during the fusion process. Our summary will contribute to a better understanding of the roles of the VAMP2 protein in membrane fusion.
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Project description:The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved "toolkit" of fusion machinery drives successive NE fusion events during embryogenesis.
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Project description:Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and pathogenic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our data suggest a model for how HOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part of the membrane fusion machinery.
| S-EPMC9592082 | biostudies-literature
Project description:PurposeLiterature shows that orthogeriatric co-management improves the outcomes of patients with hip fractures. Corresponding research with more diverse fragility fracture groups is lacking. Therefore, an examination was performed prospectively as a 2 year-follow-up on an orthogeriatric co-managed ward, comparing relevant outcome parameters for major and minor fragility fractures.MethodsAll patients treated on an orthogeriatric co-managed ward from February 2014 to January 2015 were included and their injuries, orthogeriatric parameters such as the Barthel Index (BI), Parker Mobility Score (PMS) and place of residence (POR). Patients were separated into two groups of either immobilizing major (MaF) or non-immobilizing minor (MiF) fractures. 2 years later, a follow-up was conducted via telephone calls and questionnaires mailed to patients and/or their relatives.Results740 (574 major vs. 166 minor injuries) patients were initially assessed, with a follow-up rate of 78.9%. The in-house, 1-year, and 2-year-mortality rates were 2.7, 27.4, and 39.2%, respectively. Mortality was significantly higher for MaF in the short term, but not after 2 years. On average, during the observation period, patients regained their BI by 36.7 points (95% CI: 33.80-39.63) and PMS was reduced by 1.4 points (95% CI: 1.16-1.68). No significant differences were found in the readmission rate, change in BI, PMS or POR between the MaF and MiF groups.ConclusionThe relevance of orthogeriatric treatment to improving functional and socioeconomic outcomes was confirmed. The similarity of the results from both fracture groups emphasizes the need for a multidisciplinary approach also for minor fractures.
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Project description:The flavivirus envelope glycoproteins prM and E drive the assembly of icosahedral, spiky immature particles that bud across the membrane of the endoplasmic reticulum. Maturation into infectious virions in the trans-Golgi network involves an acid-pH-driven rearrangement into smooth particles made of (prM/E)2 dimers exposing a furin site for prM cleavage into "pr" and "M". Here we show that the prM "pr" moiety derives from an HSP40 cellular chaperonin. Furthermore, the X-ray structure of the tick-borne encephalitis virus (pr/E)2 dimer at acidic pH reveals the E 150-loop as a hinged-lid that opens at low pH to expose a positively-charged pr-binding pocket at the E dimer interface, inducing (prM/E)2 dimer formation to generate smooth particles in the Golgi. Furin cleavage is followed by lid-closure upon deprotonation in the neutral-pH extracellular environment, expelling pr while the 150-loop takes the relay in fusion loop protection, thus revealing the elusive flavivirus mechanism of fusion activation.
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Project description:A key step during the entry of enveloped viruses into cells is the merger of viral and cell lipid bilayers. This process is driven by a dedicated membrane fusion protein (MFP) present at the virion surface, which undergoes a membrane-fusogenic conformational change triggered by interactions with the target cell. Viral MFPs have been extensively studied structurally, and are divided into three classes depending on their three-dimensional fold. Because MFPs of the same class are found in otherwise unrelated viruses, their intra-class structural homology indicates horizontal gene exchange. We focus this review on the class II fusion machinery, which is composed of two glycoproteins that associate as heterodimers. They fold together in the ER of infected cells such that the MFP adopts a conformation primed to react to specific clues only upon contact with a target cell, avoiding premature fusion in the producer cell. We show that, despite having diverged in their 3D fold during evolution much more than the actual MFP, the class II accompanying proteins (AP) also derive from a distant common ancestor, displaying an invariant core formed by a β-ribbon and a C-terminal immunoglobulin-like domain playing different functional roles-heterotypic interactions with the MFP, and homotypic AP/AP contacts to form spikes, respectively. Our analysis shows that class II APs are easily identifiable with modern structural prediction algorithms, providing useful information in devising immunogens for vaccine design.
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Project description:Activation of the phosphoinositide (PI) cycle generates the second messengers that control various aspects of cellular signaling. We have previously shown that two PI cycle enzymes, type II phosphatidylinositol 5-phosphate 4-kinase (PIPK IIα) and phosphoinositide 3-kinase (PI3K), are activated through light stimulation. In our earlier studies, we measured enzyme activities, instead of directly measuring the products, due to lack of sensitive analytical techniques. Cells have very low levels of PIs, compared to other lipids, so special techniques and sensitive analytical instruments are necessary for their identification and quantification. There are also other considerations, such as different responses in different cell types, which may complicate quantification of PIs. For example, although light activated PIPK IIα, there was no increase in PI-4,5-P2 measured by liquid chromatography-mass spectrometry (LC/MS) This discrepancy is due to the heterogeneous nature of the retina, which is composed of various cell types. In this study, we examined PI generation in situ using immunohistochemistry with specific PI antibodies. PIs were generated in specific retinal cell layers, suggesting that analyzing PIs from the total retina by LC/MS underscores the significance. This suggests that PI-specific antibodies are useful tools to study the cell-specific regulation of PIs in the retina.
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