Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein–protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.

Microphthalmia transcription factor (MITF) is a basic helix-loop-helix leucine zipper (bHLH-Zip) DNA-binding protein. This transcription factor plays a crucial role in the physiological and pathological functions of distinct cell types. MITF transcriptional activity is inhibited by the histidine triad nucleotide-binding protein 1 (HINT1) through direct binding. We previously reported that this association is disrupted by the binding of the second messenger ApA to HINT1. ApA is mainly produced in the mammalian cells by S207-phosphorylated Lysyl-tRNA synthetase. In this study, we found first that HINT1 was subjected to K21 acetylation and Y109 phosphorylation in activated mast cells, together with the ApA-triggered HINT1 dissociation from MITF. Mutational analysis confirmed that these modifications promote MITF transcriptional and oncogenic activity in melanoma cell lines, derived from human melanoma patients. Thus, we provided here an example that manipulation of the LysRS-ApA-HINT1-MITF signalling pathway in melanoma through post-translational modifications of HINT1 can affect the activity of the melanoma oncogene MITF.

The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.

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Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger Ap4A binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by Ap4A remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of Ap4A. These results highlight a direct polymerization signaling mechanism by the second messenger.