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The core of the membrane fusion machinery that governs neurotransmitter release includes the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin, which form a tight "SNARE complex", and Munc18-1, which binds to the SNARE complex and to syntaxin-1 folded into a closed conformation. Release is also controlled by specialized proteins such as complexins, which also bind to the SNARE complex, and unc13/Munc13s, which are crucial for synaptic vesicle priming and were proposed to open syntaxin-1, promoting SNARE complex assembly. However, the biochemical basis for unc13/Munc13 function and its relationship to other SNARE interactions are unclear. To address this question, we have analyzed interactions of the MUN domain of Muncl3-1, which is key for this priming function, using solution binding assays and cofloatation experiments with SNARE-containing proteoliposomes. Our results indicate that the Muncl3-l MUN domain binds to membrane-anchored SNARE complexes, even though binding is barely detectable in solution. The MUN domain appears to compete with Munc18-1 but not with complexin-1 for SNARE complex binding, although more quantitative assays will be required to verify these conclusions. Moreover, our data also uncover interactions of membrane-anchored syntaxin- 1/SNAP-25 heterodimers with the MUN domain, Munc18-1 and complexin-1. The interaction with complexin-1 is surprising, as it was not observed in previous solution studies. Our results emphasize the importance of studying interactions within the neurotransmitter release machinery in a native membrane environment, and suggest that unc13/Munc13s may provide a template to assemble syntaxin-1/SNAP-25 heterodimers, leading to an acceptor complex for synaptobrevin. [ABSTRACT FROM AUTHOR]

RIM proteins play critical roles in synaptic vesicle priming and diverse forms of presynaptic plasticity. The C-terminal C2B domain is the only module that is common to all RIMs but is only distantly related to well-studied C2 domains, and its three-dimensional structure and interactions have not been characterized in detail. Using NMR spectroscopy, we now show that N- and C-terminal extensions beyond the predicted C2B domain core sequence are necessary to form a folded, stable RIM1α C2B domain. We also find that the isolated RIM1α C2B domain is not sufficient for previously described protein-protein interactions involving the RIM1α C-terminus, suggesting that additional sequences adjacent to the C2B domain might be required for these interactions. However, analytical ultracentrifugation shows that the RIM1α C2B domain forms weak dimers in solution. The crystal structure of the RIM1αq C2B domain dimer at 1.7 Å resolution reveals that it forms a β-sandwich characteristic of C2 domains and that the unique N- and C-terminal extensions form a small subdomain that packs against the β-sandwich and mediates dimerization. Our results provide a structural basis to understand the function of RIM C2B domains and suggest that dimerization may be a crucial aspect of RIM function. [ABSTRACT FROM AUTHOR]

RIMs are large proteins that contain two C2-domains and are localized at presynaptic active zones, where neurotransmitters are released. RIMs play key roles in synaptic vesicle priming and regulation of presynaptic plasticity. A mutation in the RIM 1 C2A-domain has been implicated in autosomal dominant cone–rod dystrophy (CORD7). The RIM C2A-domain does not contain the full complement of aspartate residues that commonly mediate Ca2+ binding at the top loops of C2-domains, and has been reported to interact with SNAP-25 and synaptotagmin 1, two proteins from the Ca2+-dependent membrane fusion machinery. Here we have used NMR spectroscopy and X-ray crystallography to analyze the structure and biochemical properties of the RIM2 C2A-domain, which is closely related to the RIM 1 C2A-domain. We find that the RIM2 C2A-domain does not bind Ca2+. Moreover, little binding of the RIM2 C2A-domain to SNAP-25 and to the C2-domains of synaptotagmin 1 was detected by NMR experiments, suggesting that as yet unidentified interactions of the RIM C2A-domain mediate its function. The crystal structure of the RIM2 C2A-domain using data to 1.4 Å resolution reveals a β-sandwich that resembles those observed for other C2-domains, but exhibits a unique dipolar distribution of electrostatic charges whereby one edge of the β-sandwich is highly positive and the other edge is highly negative. The location of the mutation site implicated in CORD7 at the bottom of the domain and the pattern of sequence conservation suggest that, in contrast to most C2-domains, the RIM C2A-domains may function through Ca2+-independent interactions involving their bottom face. [ABSTRACT FROM AUTHOR]

Previous research has widely overlooked the respiratory risks associated with cosmetic powder, a type of mixed particulate matter with intricate chemical compositions, especially in the context of wearing masks. This study investigated the inhalation risks posed by five face powders, focusing on both particulate matter (minerals and primary microplastics) and soluble components (preservatives and organic UV filters). Wearing masks significantly increased the inhalation risk of face powders, with exposure levels influenced by factors such as particle size, density, and composition. Additionally, different samples demonstrated irregular behavioral patterns when exposed to various human tissue environments. Soluble components analysis revealed that multiple additives dissolved in six body fluids, with a higher degree of release observed in the respiratory tract fluid compared to the digestive tract fluid. The alveoli may serve as a specific target for exposure to organic UV filters due to the solubilization effect of pulmonary surfactants. These findings revealed the importance of considering both particulate matter and soluble components when assessing respiratory and digestive exposure risks from cosmetic powders. Furthermore, understanding the interactions between cosmetic particles and body fluids, as well as potential synergistic toxic effects, is crucial for ensuring the safety of cosmetic products and safeguarding public health.

A magnetic solid-phase extraction method using ionic liquid (IL)-micelle-functionalized mesoporous Fe3O4 microspheres (MFMs) was proposed for the preconcentration of anthraquinones in dietary supplements. The analytes were then determined by ultraperformance liquid chromatography combined with an ultraviolet detector. The extraction parameters, such as the choice of ILs, the concentrations of ILs and MFMs, the pH of diluent, and the concentration of acetic acid in the eluent, were presented. Under the optimized conditions, the limits of detection and limits of quantitation were 0.4-2.8 ng mL(-1) and 1.4-9.4 ng mL(-1), respectively. The accuracy of the proposed method was investigated by recovery in herb and granules of Radix et Rhizoma Rhei, yielding values between 89.25% and 96.48%. The use of the proposed method in the sample pretreatment of complex dietary supplements is feasible due to the high surface area and excellent adsorption capacity of MFMs after modification with IL.

An ionic liquid (IL)-based one-step micellar extraction procedure was developed for the extraction of multiclass polar analytes (protocatechuic acid, chlorogenic acid, epicatechin, hyperoside, isoquercitrin, quercetin) from hawthorn fruits and their determination using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF/MS). Compared to conventional organic solvent extractions, this newly proposed method was much easier, more sensitive, environmentally friendly, and effective as well. Several important parameters influencing the micellar extraction efficiency are discussed, such as selection of ILs, surfactant concentration, and extraction time. Under the optimal conditions, good linearity was achieved for each analyte with correlation coefficients (r(2)) ranging from 0.9934 to 0.9999, and the recovery values ranged from 89.3 to 106% with relative standard deviations lower than 5.5%. Results suggest that the IL-based one-step micellar extraction could be an alternative and promising means in future food analysis.

Carba-NAD is a synthetic compound identical to NAD except for one substitution, where an oxygen atom adjacent to the anomeric linkage bearing nicotinamide is replaced with a methylene group. Because it is inert in nicotinamide displacement reactions, carba-NAD is an unreactive substrate analogue for NAD-consuming enzymes. SIRT3 and SIRT5 are NAD-consuming enzymes that are potential therapeutic targets for the treatment of metabolic diseases and cancers. We report an improved carba-NAD synthesis, including a pyrophosphate coupling method that proceeds in approximately 60% yield. We also disclose the X-ray crystal structures of the ternary complexes of SIRT3 and SIRT5 bound to a peptide substrate and carba-NAD. These X-ray crystal structures provide critical snapshots of the mechanism by which human sirtuins function as protein deacylation catalysts.