1H, 13C and 15N resonance assignments for a chemokine receptor-binding domain of FROUNT, a cytoplasmic regulator of chemotaxis

Yoshinaga, Sosuke; Ishida, Norihito; Tsuji, Tatsuichiro; Sonoda, Akinaga; Yunoki, Kaori; Takeda, Mitsuhiro; Toda, Etsuko; Terashima, Yuya; Matsushima, Kouji; Terasawa, Hiroaki

doi:10.1007/s12104-018-9819-2

Cited by 3 publications

(5 citation statements)

References 9 publications

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“…The titration was performed in 20 mM Tris-HCl buffer (pH 7.5, 50 mM NaCl, prepared in 95% H 2 O and 5% D 2 O at a final concentration of 12.5-50 µM DSF or DDC) and 50 µM CRBD of FROUNT (a.a. 532-656) bearing the L538E/P612S mutations or its mutant (C603S). The assignments of the backbone 1 H N and 15 N NMR signals of CRBD were performed, based on the previous paper 30 . The NMR signal assignments of CRBD after the titration of DSF were separately performed (manuscript in preparation).…”

Section: Methodsmentioning

confidence: 99%

“…5a) 29 . For this analysis, L538E/ P612S mutations were introduced to improve the protein solubility and spectral quality, without affecting the chemokine receptor-binding activity 30 . Recently, we determined the three-dimensional structure of CRBD by NMR, in which the region of a.a. 534-648 is well structured (PDB ID: 6L5C).…”

Section: Frount Expression Is Associated With Poor Cancer Prognosismentioning

confidence: 99%

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Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties

et al. 2020

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Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumorpromoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

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Section: Methodsmentioning

confidence: 99%

Section: Frount Expression Is Associated With Poor Cancer Prognosismentioning

confidence: 99%

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties

et al. 2020

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“…Taken together, we concluded that CRBD_LEPS exists in a monodispersed state in solution, which is quite suitable for NMR experiments. Indeed, in a separate paper, we reported that almost all of the backbone and side-chain signals of CRBD_LEPS could be assigned by applying conventional triple resonance experiments …”

Section: Resultsmentioning

confidence: 99%

“…Indeed, in a separate paper, we reported that almost all of the backbone and side-chain signals of CRBD_LEPS could be assigned by applying conventional triple resonance experiments. 28 CCR2 Pro-C-Binding Assay of Wild-Type CRBD and Its Mutants. To examine whether the mutants of CRBD retain CCR2 Pro-C-binding activities comparable to that of the wildtype CRBD protein, we performed an HTRF assay, as previously described (Figures 1I and 8).…”

Section: ■ Resultsmentioning

confidence: 99%

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents

et al. 2020

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Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.

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“…C-C chemokine receptor types 2 (CCR2) and C-C chemokine receptor types 5 (CCR5) and their respective ligands play important roles in the recruitment of monocytes and macrophages to inflammation sites [1][2][3][4][5][6][7][8][9][10][11]. A protein called FROUNT (NUP85, Nucle-oporin85) was shown to promote the phosphatidylinositol-3-OH kinase (PI3K) cascade and cell migration by binding directly to the intracellular C-terminal region of CCR2 and CCR5 [12][13][14][15][16]. Disulfiram (DSF) was identified in a drug screen as a FROUNT inhibitor; DSF binds directly to a specific site on FROUNT and inhibits FROUNT-CCR2 and -CCR5 interactions [17].…”

Section: Introductionmentioning

confidence: 99%

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model

Ikebukuro

Arima

Kasamatsu

et al. 2023

IJMS

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FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

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1H, 13C and 15N resonance assignments for a chemokine receptor-binding domain of FROUNT, a cytoplasmic regulator of chemotaxis

Cited by 3 publications

References 9 publications

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model

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