细胞生物学

Alpha-protein kinase 1 (ALPK1) is normally activated by bacterial ADP-heptose as part of the innate immune response, leading to the initiation of downstream signalling events that culminate in the activation of transcription factors such as NF-κB and AP-1. In contrast, disease-causing mutations in ALPK1 that cause ROSAH syndrome or spiradenoma allow ALPK1 to be activated in cells in the absence of bacterial infection (i.e., without ADP-heptose). This protocol describes a semi-quantitative reporter assay based on ALPK1 knockout HEK-Blue cells that measures the activity of transfected wildtype and disease-causing forms of ALPK1 by virtue of their ability to activate the transcription factors NF-κB and AP-1. These cells express a synthetic gene encoding alkaline phosphatase under the control of an NF-κB/AP-1-dependent promoter, and consequently, the activation of ALPK1 leads to the production of alkaline phosphatase, which is secreted into the culture media and can be measured colorimetrically at 645 nm after the addition of a detection reagent.

Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as ‘click chemistry’ serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in Drosophila melanogaster.

Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows quantitative analysis of modulation of protein or mRNA stability under different treatments or culturing conditions.