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Aim: To elucidate the pathogenic mechanism of amyloid formation in corned amyloidosis with trichiasis. Methods: Ophthalmological examination was performed in nine patients to determine secondary corned amyloidosis with trichiasis. Congo red staining and immunohistochemistry using anti-human lactoferrin antibody were used for biopsied corneal samples. For genetic analyses, single strand conformation polymorphism (SSCP), direct DNA sequence analysis, and polymerase chain reaction (KR) induced mutation restriction analysis (IMRA) were employed to detect lactoferrin gene polymorphism. Results: All patients had had trichiasis at least for 1 year, and all amyloid-like deposits were found in one eye with trichiasis. Ophthalmological examination revealed that eight patients showed gelatinous type of amyloid deposition and one showed lattice type of amyloid deposition. Studies of biopsied corned samples with Congo red stain revealed positive staining just under the corned epithelial cells. Immunoreactivity of anti-human lactoferrin antibodies was recognised in all tissues with positive Congo red staining. lactoferrin gene analysis revealed that seven patients were heterozygotic and two were homozygotic for lactoferrin Glu561 Asp. The frequency of the polymorphism in the patients was significantly different from that in 56 healthy control subjects. Conclusion: lactoferrin G1u561 Asp is a key polymorphism related to facilitating amyloid formation in corneal amyloidosis with trichiasis. [ABSTRACT FROM AUTHOR]

Nuclear factor κB (NF-κB) is activated by various inflammatory and infectious molecules and is involved in immune responses. It has been elucidated that ADP-β-D-manno-heptose (ADP-Hep), a metabolite in gram-negative bacteria, activates NF-κB through alpha-kinase 1 (ALPK1)-TIFA-TRAF6 signaling. ADP-Hep stimulates the kinase activity of ALPK1 for TIFA phosphorylation. Complex formation between phosphorylation-dependent TIFA oligomer and TRAF6 promotes the polyubiquitination of TRAF6 for NF-κB activation. TIFAB, a TIFA homolog lacking a phosphorylation site and a TRAF6 binding motif, is a negative regulator of TIFA-TRAF6 signaling and is implicated in myeloid diseases. TIFAB is indicated to regulate TIFA-TRAF6 signaling through interactions with TIFA and TRAF6; however, little is known about its biological function. We demonstrated that TIFAB forms a complex not with the TIFA dimer, an intrinsic form of TIFA involved in NF-κB activation, but with monomeric TIFA. The structural analysis of the TIFA/TIFAB complex and the biochemical and cell-based analyses showed that TIFAB forms a stable heterodimer with TIFA, inhibits TIFA dimer formation, and suppresses TIFA-TRAF6 signaling. The resultant TIFA/TIFAB complex is a "pseudo-TIFA dimer" lacking the phosphorylation site and TRAF6 binding motif in TIFAB and cannot form the orderly structure as proposed for the phosphorylated TIFA oligomer involved in NF-κB activation. This study elucidated the molecular and structural basis for the regulation of TIFA-TRAF6 signaling by TIFAB., Competing Interests: Competing interests statement:The authors declare no competing interest.

African swine fever virus is a complex DNA virus that causes high fatality in pigs and wild boar and has a great socio-economic impact. An attenuated genotype II strain was constructed by replacing the gene for wildtype CD2v protein with versions in which single or double amino acid substitutions were introduced to reduce or abrogate the binding to red blood cells and reduce virus persistence in blood. The mutant CD2v proteins were expressed at similar levels to the wildtype protein on the surface of infected cells. Three recombinant viruses also had K145R, EP153R, and in one virus DP148R genes deleted. Following immunization of pigs, the virus with a single amino acid substitution in CD2v, Q96R, induced moderate levels of replication, and 100% protection against virulent ASFV. Two additional recombinant viruses had two amino acid substitutions in CD2v, Q96R, and K108D, and induced no binding to red blood cells in vitro . In immunized pigs, reduced levels of virus in blood and strong early ASFV-specific antibody and cellular responses were detected. After challenge low to moderate replication of challenge virus was observed. Reduced clinical signs post-challenge were observed in pigs immunized with the virus from which DP148R gene was deleted. Protection levels of 83-100% were maintained across a range of doses. Further experiments with virus GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D showed low levels of virus dissemination in tissue and transient clinical signs at high doses. The results support further evaluation of GeorgiaΔDP148RΔK145RΔEP153R-CD2v_mutantQ96R/K108D as a vaccine candidate.

Human MutT homolog 1 (MTH1), also known as Nudix-type motif 1 (NUDT1), hydrolyzes 8-oxo-dGTP and 2-oxo-dATP with broad substrate recognition and has attracted attention in anticancer therapeutics. Previous studies on MTH1 have proposed that the exchange of the protonation state between Asp119 and Asp120 is essential for the broad substrate recognition of MTH1. To understand the relationship between protonation states and substrate binding, we determined the crystal structures of MTH1 at pH 7.7-9.7. With increasing pH, MTH1 gradually loses its substrate-binding ability, indicating that Asp119 is deprotonated at pH 8.0-9.1 in 8-oxo-dGTP recognition and Asp120 is deprotonated at pH 8.6-9.7 in 2-oxo-dATP recognition. These results confirm that MTH1 recognizes 8-oxo-dGTP and 2-oxo-dATP by exchanging the protonation state between Asp119 and Asp120 with higher pK a ., (© 2023 Federation of European Biochemical Societies.)

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity., (© 2022. The Author(s).)

Mammalian MutY homologue (MUTYH) is an adenine DNA glycosylase that excises adenine inserted opposite 8-oxoguanine (8-oxoG). The inherited variations in human MUTYH gene are known to cause MUTYH-associated polyposis (MAP), which is associated with colorectal cancer. MUTYH is involved in base excision repair (BER) with proliferating cell nuclear antigen (PCNA) in DNA replication, which is unique and critical for effective mutation-avoidance. It is also reported that MUTYH has a Zn-binding motif in a unique interdomain connector (IDC) region, which interacts with Rad9-Rad1-Hus1 complex (9-1-1) in DNA damage response, and with apurinic/apyrimidinic endonuclease 1 (APE1) in BER. However, the structural basis for the BER pathway by MUTYH and its interacting proteins is unclear. Here, we determined the crystal structures of complexes between mouse MUTYH and DNA, and between the C-terminal domain of mouse MUTYH and human PCNA. The structures elucidated the repair mechanism for the A:8-oxoG mispair including DNA replication-coupled repair process involving MUTYH and PCNA. The Zn-binding motif was revealed to comprise one histidine and three cysteine residues. The IDC, including the Zn-binding motif, is exposed on the MUTYH surface, suggesting its interaction modes with 9-1-1 and APE1, respectively. The structure of MUTYH explains how MAP mutations perturb MUTYH function., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-β-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA-TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-κB activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA-TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants-Thr9 mutated to either Asp or Glu to mimic the phosphorylation state-to obtain the structural information for oligomer formation of the TIFA-TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA-TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA-TRAF6 signalling complex.

T-cell checkpoint inhibition has a profound impact on cancer care and the programmed cell death protein 1 (PD-1)-targeted antibodies nivolumab and pembrolizumab have been two of the lead molecules of this therapeutic revolution. Their clinical comparability is a highly relevant topic of discussion, but to a significant degree is a consequence of their molecular properties. Here we provide a molecular, preclinical, and early clinical comparison of the two antibodies, based on the available data and recent literature. We acknowledge the limitations of such comparisons, but suggest that based on the available data, differences in clinical trial outcomes between nivolumab and pembrolizumab are more likely drug-independent than drug-dependent., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif ( i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

CD200R is an inhibitory receptor expressed on myeloid cells and some lymphoid cells, and plays important roles in negatively regulating immune responses. CD200 is the only known ligand of CD200R and broadly distributed in a variety of cell types. Here we identified novel CD200 homologues, designated iSEC1 and iSEC2, that are expressed exclusively by secretory cell lineages in the gastrointestinal epithelium while authentic CD200 is expressed by none of epithelial cells including secretory cells. Both iSEC1 and iSEC2 could bind to CD200R but not other members of the CD200R family. Notably, CD200R expression was confined to intraepithelial lymphocytes (IELs) among cells in the gastrointestinal epithelium. Binding of iSEC1 to CD200R on IELs resulted in the suppression of cytokine production and cytolytic activity by activated IELs. Thus, iSEC1 is a previously unappreciated CD200R ligand with restricted expression in gastrointestinal secretory cells and may negatively regulate mucosal immune responses.

The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

PD-1, a receptor expressed by T cells, B cells, and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology but differs at the edge of the GFCC' sheet, which is flexible and completely lacks a C" strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whereas binding is centered on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1 · ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The 3-4-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the 3-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T cells with antigen-presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1 and reveal that potent inhibitory signaling can be initiated by weakly interacting receptors.

Background: This study investigated whether a relationship exists between the presence of de novo antibodies and the clinical manifestations of familial amyloidotic polyneuropathy (FAP)., Methods: Serum samples were collected from 25 Japanese and 6 Swedish FAP amyloidogenic transthyretin (ATTR) Valine30Methionine (V30M) patients, 4 asymptomatic Japanese ATTR V30M gene carriers, and 24 Japanese healthy volunteers. Study methods included enzyme-linked immunosorbent assay (ELISA) and mass spectrometry., Results: Three Japanese and 5 Swedish patients had significantly higher levels of antibodies against ATTR than did healthy volunteers and asymptomatic gene carriers (P<0.05). All 8 patients with higher antibody levels were late-onset cases. The ratio of wild-type TTR to ATTR V30M in serum from the high-antibody group was higher than that of the low-antibody group. ELISA results revealed two epitopes at positions 24-35 and 105-115 of ATTR V30M. We found a significant positive correlation between levels of the antibody at positions 24-35 and the age at FAP onset (r=0.751, P<0.05). An age-dependent increase in the occurrence of antibodies was observed in these patients with an epitope at positions 24-35., Conclusions: These findings may help explain the differences in early- and late-onset FAP and/or the progression of FAP., (Copyright © 2013 Elsevier B.V. All rights reserved.)