Structural basis for the binding of the membrane‐proximal C‐terminal region of chemokine receptor <scp>CCR</scp>2 with the cytosolic regulator <scp>FROUNT</scp>

Esaki, Kaori; Yoshinaga, Sosuke; Tsuji, Tatsuichiro; Toda, Etsuko; Terashima, Yuuya; Saitoh, Takashi; Kohda, Daisuke; Kohno, Toshiyuki; Osawa, Masatoshi; Ueda, Takumi; Shimada, Ichio; Matsushima, Kouji; Terasawa, Hiroaki

doi:10.1111/febs.13096

Cited by 12 publications

(6 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, these findings have not yet been translated into effective clinical therapies, possibly due to species-specific differences in chemokine receptors or due to redundancy in chemokine signaling. FROUNT promotes chemotactic signaling via CCR2 and CCR5 by binding to a conserved intracellular region of these receptors 14,15,25,40 . Since FROUNT is a molecule involved in signal amplification, its inhibition is expected to have effects different from that of the inhibition of chemokine or chemokine-receptor blockade, as evidenced by the minimal effect on steady-state monocyte population in Frount-deficient mice ( Supplementary Fig.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…FROUNT interacts with the membrane‐proximal C‐terminal regions of chemokine receptors, CCR2 and CCR5, through FNT‐C (Terashima et al., ; Toda et al., ). The intracellular interaction promotes leukocyte migration, and thus, FROUNT represents a promising target for drug development (Esaki et al., , ; Terashima et al., ; Toda et al., , ).…”

Section: Introductionmentioning

confidence: 99%

Efficient identification of compounds suppressing protein precipitation via solvent screening using serial deletion mutants of the target protein

et al. 2018

Self Cite

View full text Add to dashboard Cite

The control of protein solubility is a subject of broad interest. Although several solvent screening methods are available to search for compounds that enhance protein solubilization, their performance is influenced by the intrinsic solubility of the tested protein. We now present a method for screening solubilizing compounds, using an array of N- or C-terminal deletion mutants of the protein. A key behind this approach is that such terminal deletions of the protein affect its aggregation propensity. The solubilization activities of trial solvents are individually assessed, based on the number of solubilized mutants. The solubilizing compounds are then identified from the screened solvents. In this study, the C-terminal chemokine receptor-binding region of the cytoplasmic protein, FROUNT (FNT-C), which mediates intracellular signals leading to leukocyte migration, was subjected to the multicomponent screening. In total, 192 solution conditions were tested, using eight terminal deletion mutants of FNT-C. We identified five solvent conditions that solubilized four or five mutants of FNT-C, and the compounds in the screened solvents were then, respectively, assessed in terms of their solubilization ability. The best compound for solubilizing FNT-C was 1,6-hexanediol. Indeed, 1,6-hexanediol bound to FNT-C and suppressed its precipitation, as showed by NMR and dynamic light scattering analyses.

show abstract

“…We have been studying the cytoplasmic protein FROUNT (UniProtKB Q9BW27), − a 656-amino acid protein that binds to the membrane-proximal C-terminal intracellular region (Pro-C) of the chemokine receptor CCR2 via its C-terminal 493–656 region, as revealed by yeast two-hybrid assays . FROUNT also binds to Pro-C of CCR5, thereby participating in CCR5-related cell migration .…”

mentioning

confidence: 99%

“…In our structural and functional studies of the interactions between FROUNT and chemokine receptors, we identified a 16-amino acid region of CCR2 Pro-C as the minimal segment responsible for binding to FROUNT . CCR2 Pro-C adopted an amphiphilic helical conformation upon binding to FROUNT and a typical amphiphilic α-helix upon binding to the membrane . The binding sites of FROUNT and the membrane overlap on the same side of the CCR2 Pro-C structure, and Pro-C dynamically switches between the two binding partners.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Structural basis for the binding of the membrane‐proximal C‐terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT

Cited by 12 publications

References 56 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

Efficient identification of compounds suppressing protein precipitation via solvent screening using serial deletion mutants of the target protein

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

Contact Info

Product

Resources

About