Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes 1 , but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives < 1 second) precluding their detection with many commonly-used high throughput methods 2 .Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t 1/2 ≤ 0.1 sec) with a low false positive rate 3 . The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.The recombinant protein libraries are produced using a convenient and high-level mammalian expression system 4
ScopeFlavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti‐inflammatory effects of flavonoid metabolites relative to their precursor structures.Methods and resultsSix flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1–10 μM) were screened for their ability to reduce LPS‐induced tumor necrosis factor‐α (TNF‐α) secretion in THP‐1 monocytes. One micromolar peonidin‐3‐glucoside, cyanidin‐3‐glucoside, and the metabolites isovanillic acid (IVA), IVA‐glucuronide, vanillic acid‐glucuronide, protocatechuic acid‐3‐sulfate, and benzoic acid‐sulfate significantly reduced TNF‐α secretion when in isolation, while there was no effect on TNF‐α mRNA expression. Four combinations of metabolites that included 4‐hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF‐α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS‐induced IL‐1β and IL‐10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL‐1β secretion but none of the flavonoids or metabolites significantly modified IL‐10 secretion.ConclusionThis study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites.
Pseudokinases are a class of kinases which are structurally designated as lacking kinase activity. Despite the lack of kinase domain sequence conservation, there is increasing evidence that a number of pseudokinases retain kinase activity and/or have critical cellular functions, casting aside previous notions that pseudokinases simply exist as redundant kinases. Moreover, a number of recent studies have implicated pseudokinases as critical components in cancer formation and progression. The present review discusses the interactions and potential functions that nuclear receptor-binding protein 1, a pseudokinase recently described to have a tumour-suppressive role in cancer, may play in cellular homoeostasis and protein regulation. The recent findings highlighted in the present review emphasize the requirement to fully determine the function of pseudokinases in vitro and in vivo, the understanding of which may ultimately uncover new directions for drug discovery.
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