Cyanidin 3-glucoside Is a Selective Inhibitor of Hepatic Bilirubin Uptake

Background & Aims: One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, as there is no link to the protein/gene entities that carry it out, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology.Methods: We treated isolated, perfused rat livers with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a biofluorometric assay. We tested the ability of nine molecules known to be substrates or inhibitors of sinusoidal membrane transporters to inhibit the hepatic uptake of bilirubin.Results: We found that cyanidin 3-glucoside and malvidin 3-glucoside are the only molecules that inhibit bilirubin uptake. These dietary anthocyanins resemble bromosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cyanidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indomethacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin glucuronide transporter that is inhibited by bilirubin, a fact reported only once in the literature.Conclusions: Data indicate that bilirubin and bilirubin glucuronide are transported into the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver.

Optimization of an in silico protocol to characterize membrane PAINS

Membrane Pan-Assay INterference compoundS (PAINS) are a class of molecules that interact non-specifically with lipid bilayers and alter their physicochemical properties. An early identification of these compounds avoids chasing false leads and the needless waste of time and resources in drug discovery campaigns. In this work, we optimized an in silico protocol based on umbrella sampling (US)/MD simulations to discriminate between compounds with different membrane PAINS behavior. We showed that the method is quite sensitive to membrane thickness fluctuations, which was mitigated by changing the US-reference position to the P-atoms of the closest interacting monolayer. The computational efficiency was improved further by decreasing the number of umbrellas and adjusting their strength and position in our US scheme. The ISDM-calculated membrane permeability coefficients confirmed that resveratrol and curcumin have distinct membrane PAINS characteristics and indicate a misclassification of nothofagin in a previous work. Overall, we have presented here a promising in silico protocol which can be adopted as a future reference method to identify membrane PAINS.

Membrane Transporters Involved in the Antimicrobial Activities of Pyrithione in Escherichia coli

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal–drug compounds.

Insights into the molecular mechanism of amyloid filament formation: Segmental folding of α-synuclein on lipid membranes

Recent advances in the structural biology of disease-relevant α-synuclein fibrils have revealed a variety of structures, yet little is known about the process of fibril aggregate formation. Characterization of intermediate species that form during aggregation is crucial; however, this has proven very challenging because of their transient nature, heterogeneity, and low population. Here, we investigate the aggregation of α-synuclein bound to negatively charged phospholipid small unilamellar vesicles. Through a combination of kinetic and structural studies, we identify key time points in the aggregation process that enable targeted isolation of prefibrillar and early fibrillar intermediates. By using solid-state nuclear magnetic resonance, we show the gradual buildup of structural features in an α-synuclein fibril filament, revealing a segmental folding process. We identify distinct membrane-binding domains in α-synuclein aggregates, and the combined data are used to present a comprehensive mechanism of the folding of α-synuclein on lipid membranes.

Energy and Dynamics of Caveolae Trafficking

Caveolae are 70–100 nm diameter plasma membrane invaginations found in abundance in adipocytes, endothelial cells, myocytes, and fibroblasts. Their bulb-shaped membrane domain is characterized and formed by specific lipid binding proteins including Caveolins, Cavins, Pacsin2, and EHD2. Likewise, an enrichment of cholesterol and other lipids makes caveolae a distinct membrane environment that supports proteins involved in cell-type specific signaling pathways. Their ability to detach from the plasma membrane and move through the cytosol has been shown to be important for lipid trafficking and metabolism. Here, we review recent concepts in caveolae trafficking and dynamics. Second, we discuss how ATP and GTP-regulated proteins including dynamin and EHD2 control caveolae behavior. Throughout, we summarize the potential physiological and cell biological roles of caveolae internalization and trafficking and highlight open questions in the field and future directions for study.

Orthogonal coiled coils enable rapid covalent labelling of two distinct membrane proteins with peptide nucleic acid barcodes

Templated chemistry offers the prospect of addressing specificity challenges occurring in bioconjugation reactions. Here, we show two peptide-templated amide-bond forming reactions that enable the concurrent labelling of two different membrane...

Membrane Catalysed Formation of Nucleotide Clusters and Its Role in the Origins of Life

<div>Non-enzymatic polymerisation explained how life could assemble spontaneously on the early Earth. Prior research has discovered that the non-enzymatic polymerisation can be mediated by various environmental settings, most widely accepted are the reactions driven by the cycles of hydration and dehydration and organising matrices such as clay, salt, membranes and mineral surface. Membranes have a unique feature which prepares it as a sensitive organising matrix, as compared to mineral surface and clay, which transforms its phase with small changes in temperature, hydration and composition of lipids. Multi-component membranes can show phase separation where two or more distinct phases can co-exist. To the best of our knowledge, this is for the fi?rst time the influence of different phases of the membrane in the nucleotide organisation and polymerisation in a simulated prebiotic setting in this work have been explored. This study encompasses the energetics of inserting a mononucleotide, UMP, in distinct membrane settings which is probed utilising the PMF. The preferential partitioning of UMP is also investigated; which is coupled to the location of the insertion and the composition of the PSM/DOPC/Chol membranes. Combined with umbrella-sampling calculations, AA-MD simulations were performed to estimate the role of the</div><div>monophasic and diphasic membrane in modifying the behaviour of UMPs and their clustering mechanism. A mathematical model was also developed based on lattice model and random walk and some of the parameters were trained from the AA-MD simulations. The results indicated that the membranes were capable of concentrating nucleotides and organised into anisotropic clusters, and the thermodynamic studies along with mathematical model showed that it had a sharp preference for the Lo/Ld domain interface for clustering.</div>

Membrane Catalysed Formation of Nucleotide Clusters and Its Role in the Origins of Life

<div>Non-enzymatic polymerisation explained how life could assemble spontaneously on the early Earth. Prior research has discovered that the non-enzymatic polymerisation can be mediated by various environmental settings, most widely accepted are the reactions driven by the cycles of hydration and dehydration and organising matrices such as clay, salt, membranes and mineral surface. Membranes have a unique feature which prepares it as a sensitive organising matrix, as compared to mineral surface and clay, which transforms its phase with small changes in temperature, hydration and composition of lipids. Multi-component membranes can show phase separation where two or more distinct phases can co-exist. To the best of our knowledge, this is for the fi?rst time the influence of different phases of the membrane in the nucleotide organisation and polymerisation in a simulated prebiotic setting in this work have been explored. This study encompasses the energetics of inserting a mononucleotide, UMP, in distinct membrane settings which is probed utilising the PMF. The preferential partitioning of UMP is also investigated; which is coupled to the location of the insertion and the composition of the PSM/DOPC/Chol membranes. Combined with umbrella-sampling calculations, AA-MD simulations were performed to estimate the role of the</div><div>monophasic and diphasic membrane in modifying the behaviour of UMPs and their clustering mechanism. A mathematical model was also developed based on lattice model and random walk and some of the parameters were trained from the AA-MD simulations. The results indicated that the membranes were capable of concentrating nucleotides and organised into anisotropic clusters, and the thermodynamic studies along with mathematical model showed that it had a sharp preference for the Lo/Ld domain interface for clustering.</div>

Masters of asymmetry – lessons and perspectives from 50 years of septins

Septins are a unique family of GTPases, which were discovered 50 years ago as essential genes for the asymmetric cell shape and division of budding yeast. Septins assemble into filamentous nonpolar polymers, which associate with distinct membrane macrodomains and subpopulations of actin filaments and microtubules. While structurally a cytoskeleton-like element, septins function predominantly as spatial regulators of protein localization and interactions. Septin scaffolds and barriers have provided a long-standing paradigm for the generation and maintenance of asymmetry in cell membranes. Septins also promote asymmetry by regulating the spatial organization of the actin and microtubule cytoskeleton, and biasing the directionality of membrane traffic. In this 50th anniversary perspective, we highlight how septins have conserved and adapted their roles as effectors of membrane and cytoplasmic asymmetry across fungi and animals. We conclude by outlining principles of septin function as a module of symmetry breaking, which alongside the monomeric small GTPases provides a core mechanism for the biogenesis of molecular asymmetry and cell polarity.