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LI-cadherin is a member of the cadherin superfamily. LI-cadherin mediates Ca 2+ -dependent cell-cell adhesion through homodimerization. A previous study reported two single nucleotide polymorphisms (SNPs) in the LI-cadherin-coding gene (CDH17). These SNPs correspond to the amino acid changes of Lys115 to Glu and Glu739 to Ala. Patients with colorectal cancer carrying these SNPs are reported to have a higher risk of lymph node metastasis than patients without the SNPs. Although proteins associated with metastasis have been identified, the molecular mechanisms underlying the functions of these proteins remain unclear, making it difficult to develop effective strategies to prevent metastasis. In this study, we employed biochemical assays and molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which the amino acid changes caused by the SNPs in the LI-cadherin-coding gene increase the risk of metastasis. Cell aggregation assays showed that the amino acid changes weakened the LI-cadherin-dependent cell-cell adhesion. In vitro assays demonstrated a decrease in homodimerization tendency and MD simulations suggested an alteration in the intramolecular hydrogen bond network by the mutation of Lys115. Taken together, our results indicate that the increased risk of lymph node metastasis is due to weakened cell-cell adhesion caused by the decrease in homodimerization tendency., (© 2023. The Author(s).)

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CL pro ) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CL pro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CL pro revealed that the Kcat/Km of the 3CL pro enzyme containing Ser108 was 58% lower than that of Pro108 3CL pro . Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CL pro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CL pro inhibitor., (© 2022. The Author(s).)

Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, FcγRIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with FcγRIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

Phospholipase A 2 (PLA 2 ) is one of the representative toxic components of snake venom. PLA 2 s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA 2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA 2 . The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA 2 remains unknown. In this study, we analyzed a Lys49-PLA 2 homologue from Protobothrops flavoviridis (PflLys49-PLA 2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA 2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.

Severe acute pancreatitis is a lethal inflammatory disease frequently accompanied by pancreatic necrosis. We aimed to identify a key regulator in the development of pancreatic necrosis. A cytokine/chemokine array using sera from patients with acute pancreatitis (AP) revealed that serum CXCL16 levels were elevated according to the severity of pancreatitis. In a mouse model of AP, Cxcl16 expression was induced in pancreatic acini in the late phase with the development of pancreatic necrosis. Cxcl16 -/- mice revealed similar sensitivity as wild-type (WT) mice to the onset of pancreatitis, but better resisted development of acinar cell necrosis with attenuated neutrophil infiltration. A cytokine array and immunohistochemistry revealed lower expression of Ccl9, a neutrophil chemoattractant, in the pancreatic acini of Cxcl16 -/- mice than WT mice. Ccl9 mRNA expression was induced by stimulation with Cxcl16 protein in pancreatic acinar cells in vitro, suggesting a Cxcl16/Ccl9 cascade. Neutralizing antibody against Cxcl16 ameliorated pancreatic injury in the mouse AP model with decreased Ccl9 expression and less neutrophil accumulation. In conclusion, Cxcl16 expressed in pancreatic acini contributes to the development of acinar cell necrosis through the induction of Ccl9 and subsequent neutrophil infiltration. CXCL16 could be a new therapeutic target in AP.

Sweet and umami tastes are perceived by T1r taste receptors in oral cavity. T1rs are class C G-protein coupled receptors (GPCRs), and the extracellular ligand binding domains (LBDs) of T1r1/T1r3 and T1r2/T1r3 heterodimers are responsible for binding of chemical substances eliciting umami or sweet taste. However, molecular analyses of T1r have been hampered due to the difficulties in recombinant expression and protein purification, and thus little is known about mechanisms for taste perception. Here we show the first molecular view of reception of a taste substance by a taste receptor, where the binding of the taste substance elicits a different conformational state of T1r2/T1r3 LBD heterodimer. Electron microscopy has showed a characteristic dimeric structure. Förster resonance energy transfer and X-ray solution scattering have revealed the transition of the dimerization manner of the ligand binding domains, from a widely spread to compactly organized state upon taste substance binding, which may correspond to distinct receptor functional states.

Organotin compounds such as triphenyltin (TPT) and tributyltin (TBT) act as endocrine disruptors through the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway. We recently found that TPT is a particularly strong agonist of PPARγ. To elucidate the mechanism underlying organotin-dependent PPARγ activation, we here analyzed the interactions of PPARγ ligand-binding domain (LBD) with TPT and TBT by using X-ray crystallography and mass spectroscopy in conjunction with cell-based activity assays. Crystal structures of PPARγ-LBD/TBT and PPARγ-LBD/TPT complexes were determined at 1.95 Å and 1.89 Å, respectively. Specific binding of organotins is achieved through non-covalent ionic interactions between the sulfur atom of Cys285 and the tin atom. Comparisons of the determined structures suggest that the strong activity of TPT arises through interactions with helix 12 of LBD primarily via π-π interactions. Our findings elucidate the structural basis of PPARγ activation by TPT.