Active-site directed probes are powerful in studies of
enzymatic
function. We report an active-site directed probe based on a warhead
so far considered unreactive. By replacing the C-terminal carboxylate
of ubiquitin (Ub) with an alkyne functionality, a selective reaction
with the active-site cysteine residue of de-ubiquitinating enzymes
was observed. The resulting product was shown to be a quaternary vinyl
thioether, as determined by X-ray crystallography. Proteomic
analysis of proteins bound to an immobilized Ub alkyne probe confirmed
the selectivity toward de-ubiquitinating enzymes. The observed reactivity
is not just restricted to propargylated Ub, as highlighted by
the selective reaction between caspase-1 (interleukin converting enzyme)
and a propargylated peptide derived from IL-1β, a caspase-1
substrate.
Owing to the low abundance of signaling proteins and transcription factors, their protein complexes are not easily identified by classical proteomics. The isolation of these protein complexes from endogenous plant tissues (rather than plant cell cultures) is therefore an important technical challenge. Here, we describe a sensitive, quantitative proteomics-based procedure to determine the composition of plant protein complexes. The method makes use of fluorophore-tagged protein immunoprecipitation (IP) and label-free mass spectrometry (MS)-based quantification to correct for nonspecifically precipitated proteins. We provide procedures for the isolation of membrane-bound receptor complexes and transcriptional regulators from nuclei. The protocol consists of an IP step (~6 h) and sample preparation for liquid chromatography-tandem MS (LC-MS/MS; 2 d). We also provide a guide for data analysis. Our single-step affinity purification protocol is a good alternative to two-step tandem affinity purification (TAP), as it is shorter and relatively easy to perform. The data analysis by label-free quantification (LFQ) requires a cheaper and less challenging experimental setup compared with known labeling techniques in plants.
Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue-type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9 EV, degranulation was accompanied by the release of CD63 EV, which correlated with release of the soluble mediator β-hexosaminidase. This CD63 EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9 EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63 EV from connective tissue-type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.
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