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. 2019 Oct 11;10(1):4664.
doi: 10.1038/s41467-019-12710-8.

Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells

Jing Yu  1 , Zaizhou Liu  1 , Yuanyuan Liang  1 , Feng Luo  2 , Jie Zhang  1 , Cuiping Tian  3 , Alex Motzik  4 , Mengmeng Zheng  1 , Jingwu Kang  1 , Guisheng Zhong  3 , Cong Liu  2 , Pengfei Fang  1 , Min Guo  1   5 , Ehud Razin  6   7 , Jing Wang  8
Affiliations

Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells

Jing Yu et al. Nat Commun. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Ap4A integrated two HINT1 dimers into a HINT1-Ap4A-HINT1 structure. a Schematics of adenosine polyphosphates (ApnA). ApnA is composed by two adenosine moieties linked through ribose 5′-carbons to a phosphate group chain of different length (n denotes the number of phosphate groups). b Superimposition of the HINT1-Ap4A complex structure (pink) and HINT1_apo structure (PDB: 1KPB, gray). c Two HINT1 dimers (Dimer I and Dimer II) were integrated into a tetramer in the HINT1-Ap4A complex structure. HINT1 proteins are shown as surface in black or white, and the Ap4A molecule is shown as pink surface. d A new cleft built by two adenosine pockets with a long groove accommodated the Ap4A molecule. The HINT1 proteins are shown as surface and the Ap4A molecule is shown as sticks. e Zoom-in view of the Ap4A-induced tetramer interface between HINT1 Dimer I (black) and Dimer II (white). The interacting residues are shown as sticks
Fig. 2
Fig. 2
Ap4A-promoted HINT1 polymerization. a Electrophoretic mobility shift assay (EMSA) was performed with 25 μM HINT1H114A incubated with 0–700 μM Ap4A or 400–700 μM AMP. The monomer band of HINT1 was detected by Coomassie brilliant blue staining (CBS) and the polymer bands were detected by western blotting against the 6×his-tag fused with HINT1. The experiment was performed three times with similar results. Source data are provided as a Source Data file. b The molecular weights of the experimental polymers correlated with the integral multiples of the theoretical molecular weight of HINT1 with R-squared = 0.996 (error bars represent the SEM of three experimental repeats). Source data are provided as a Source Data file. c–e Negative stain EM images of HINT1WT without Ap4A c, in the presence of 400 μM Ap4A d, or 700 μM Ap4A e
Fig. 3
Fig. 3
HAn formation breaks HINT1-MITF interactions in solution. a A model of (HINT1-Ap4A)n polymer, denoted as HAn, is built based on the HINT1-Ap4A tetramer observed in the crystal structure (circled with a black line). b Interface for MITF (labeled as yellow) overlaps with Ap4A-induced tetramer interface (circled with a black line). Posttranslational modification sites of HINT1 (yellow) is located at the tetramer interface of HINT1. Among them, Lys21 and Tyr109 were reported to directly bind MITF, and modification on these sites disrupted MITF-HINT1 interaction and released MITF transcription activity. c Fluorescence anisotropy assay designed to monitor the interaction between HINT1WT and 5IAF-MITF. Error bars represent the SD of three experimental repeats. Source data are provided as a Source Data file. d Titration of Ap4A and ATP into the 5IAF-MITF-HINT1WT complex at the platform of c. Error bars represent the SD of three experimental repeats. **P < 0.01, from two-tailed Student’s t-test. Source data are provided as a Source Data file. e ATP could not induce HINT1 polymerization and could not release 5IAF-MITF to decrease the fluorescence anisotropy. f Ap4A was able to induce HINT1 polymerization and release 5IAF-MITF to decrease the fluorescence anisotropy
Fig. 4
Fig. 4
HINT1 polymerizes in stimulated RBL cells. a Western blottings of the endogenous HINT1 following by the IgE and antigen stimulation. 0 indicates the unstimulated state. Source data are provided as a Source Data file. b–g Endogenous HINT1 in Rat basophilic leukemia (RBL) cell following the IgE and antigen stimulation (0–4 h) were immunostained and visualized by confocal laser scanning microscope or stimulated emission depletion microscope (STED). Scale bars, 10 μm. Nuclei were labeled with DAPI. One representative experiment out of three is shown. h The transcript level of c-Met and c-Kit following the IgE and antigen stimulation. Error bars represent the SEM of three experimental repeats. Source data are provided as a Source Data file. i Schematic cartoon showing Ap4A induces the formation of HAn polymer to release MITF for transcriptional activity in allergic response
Fig. 5
Fig. 5
The length-dependent regulation of Ap4A on HAn formation. a EMSA was performed with 25 μM HINT1H114A or HINT1WT incubated with 700 μM AMP, Ap3A, Ap4A, Ap5A, and ATP separately. Only Ap4A could induce the HINT1 polymerization. The experiment was performed three times with similar results. Source data are provided as a Source Data file. b FRET assay showing that only Ap4A, but not Ap5A/Ap3A/AMP, could induce the interaction between HINT1V97D-CFP and HINT1V97D-YFP. FRET signal between CFP-YFP tandems was measured and set to 100% (error bars represent the SD of three experimental repeats). *P < 0.05, **P < 0.01, ***P < 0.001, from two-tailed Student’s t-test. Source data are provided as a Source Data file. c–f Negative stain EM of 10 μM HINT1WT incubated with Ap4A, Ap3A, AMP, and Ap5A separately. Only Ap4A could efficiently induce the HINT1 filament formation. Scale bars, 100 nm. g The conformation of ATP in the HINT1H114A-ATP complex structure. Only AMP portion was visible, suggesting the rest part was flexible or disordered. h The conformation of Ap3A in the HINT1H114A-Ap3A complex structure. Only adenosine moiety was visible. i The conformation of Ap5A in the HINT1H114A-Ap5A complex structure. Two adenosines of Ap5A fit in the two adenosine pockets in HINT1 dimer I and HINT1 dimer II separately. The five-phosphate linkage in Ap5A adopted a bent and zigzag conformation, comparing with the straight and tight Ap4A
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