Enzymatic activity of cGAS in the presence of three types of DNAs: limited cGAS stimulation by single-stranded HIV-1 SL2 DNA.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is activated by binding to DNA. Activated cGAS produces 2'3'-cGAMP, which subsequently binds to the adaptor protein STING (stimulator of interferon genes). This interaction triggers the cGAS/STING signaling pathway, leading to the production of type I interferons. Three types of DNA, namely double-stranded DNA longer than 40 base pairs, a 70-nucleotide single-stranded HIV-1 DNA known as SL2, and Y-form DNA with unpaired guanosine trimers (G3 Y-form DNA), induce interferon production by activating cGAS/STING signaling. However, the extent of cGAS activation by each specific DNA type remains unclear. The comparison of cGAS stimulation by various DNAs is crucial for understanding the mechanisms underlying cGAS-mediated type I interferon production in the innate immune response. Here, we revealed that cGAS produces 2'3'-cGAMP at a significantly lower rate in the presence of single-stranded SL2 DNA than in the presence of double-stranded DNA or G3 Y-form DNA. Furthermore, the guanine-to-cytosine mutations and the deletion of unpaired guanosine trimers significantly reduced the 2'3'-cGAMP production rate and the binding of cGAS to Y-form DNA. These studies will provide new insights into the cGAS-mediated DNA-sensing in immune response.

Trypsin-induced aggregation of transthyretin Valine 30 variants associated with hereditary amyloidosis.

Amyloid fibrils of transthyretin (TTR) consist of full-length TTR and C-terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C-terminal fragment remains unclear. Here, we investigated trypsin-induced aggregation and urea-induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less-bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin-induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin-induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion.

Resveratrol Derivatives Inhibit Transthyretin Fibrillization: Structural Insights into the Interactions between Resveratrol Derivatives and Transthyretin.

Hereditary ATTR amyloidosis is a disease caused by the deposition of amyloid fibrils formed by mutated transthyretin (TTR), a protein that binds to thyroid hormone in the serum, in the organs. The development of a small molecule that binds to and stabilizes TTR is a promising strategy for the treatment of ATTR amyloidosis. In the present study, we demonstrated that the resveratrol derivatives including pterostilbene available as a dietary supplement inhibit the fibrillization of V30M-TTR to the same extent as the approved drug tafamidis. Furthermore, based on a thermodynamic and X-ray crystallographic analysis, the binding of the resveratrol derivative to TTR was shown to be enthalpy-driven, with the binding enthalpy being acquired by hydrogen bonding to S117. Moreover, direct observation of hydrogen atoms by neutron crystallography provided details of the hydrogen bond network by S117 and emphasized the importance of the CH···π interaction by L110 in the ligand binding.

Design and structural optimization of thiadiazole derivatives with potent GLS1 inhibitory activity.

GLS1 is an attractive target not only as anticancer agents but also as candidates for various potential pharmaceutical applications such as anti-aging and anti-obesity treatments. We performed docking simulations based on the complex crystal structure of GLS1 and its inhibitor CB-839 and found that compound A bearing a thiadiazole skeleton exhibits GLS1 inhibition. Furthermore, we synthesized 27 thiadiazole derivatives in an effort to obtain a more potent GLS1 inhibitor. Among the synthesized derivatives, 4d showed more potent GLS1 inhibitory activity (IC50 of 46.7 µM) than known GLS1 inhibitor DON and A. Therefore, 4d is a very promising novel GLS1 inhibitor.

Rafoxanide, a salicylanilide anthelmintic, interacts with human plasma protein transthyretin.

Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4 ) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti-ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU-approved anthelmintic drug for domesticated animals, binds to the T4 -binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone-related proteins, including serum albumin and thyroid hormone receptor ß. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone-related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid-disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors.

Benziodarone and 6-hydroxybenziodarone are potent and selective inhibitors of transthyretin amyloidogenesis.

Transthyretin amyloidosis is a progressive systemic disorder that is caused by the amyloid deposition of transthyretin in various organs. Stabilization of the native transthyretin is an effective strategy for the treatment of transthyretin amyloidosis. In this study we demonstrate that the clinically used uricosuric agent benziodarone is highly effective to stabilize the tetrameric structure of transthyretin. An acid-induced aggregation assay showed that benziodarone had strong inhibitory activity similar to that of tafamidis, which is currently used as a therapeutic agent for transthyretin amyloidosis. Moreover, a possible metabolite, 6-hydroxybenziodarone, retained the strong amyloid inhibitory activity of benziodarone. An ex vivo competitive binding assay using a fluorogenic probe showed that benziodarone and 6-hydroxybenziodarone were highly potent for selective binding to transthyretin in human plasma. An X-ray crystal structure analysis revealed that the halogenated hydroxyphenyl ring was located at the entrance of the thyroxine binding channel of transthyretin and that the benzofuran ring was located in the inner channel. These studies suggest that benziodarone and 6-hydroxybenziodarone would potentially be effective against transthyretin amyloidosis.

Design, synthesis, structure-activity relationship studies, and evaluation of novel GLS1 inhibitors.

Glutaminase converts glutamine into glutamic acid and has two isoforms: glutaminase 1 (GLS1) and glutaminase 2 (GLS2). GLS1 is overexpressed in several tumors, and research to develop glutaminase inhibitors as antitumor drugs is currently underway. The present study examined candidate GLS1 inhibitors using in silico screening and attempted to synthesize novel GLS1 inhibitors and assess their GLS1 inhibitory activities in a mouse kidney extract and against recombinant mouse and human GLS1. Novel compounds were synthesized using compound C as the lead compound, and their GLS1 inhibitory activities were evaluated using the mouse kidney extract. Among the derivatives tested, the trans-4-hydroxycyclohexylamide derivative 2j exhibited the strongest inhibitory activity. We also assessed the GLS1 inhibitory activities of the derivatives 2j, 5i, and 8a against recombinant mouse and human GLS1. The derivatives 5i and 8a significantly decreased the production of glutamic acid at 10 mM. In conclusion, we herein identified two compounds that exhibited GLS1 inhibitory activities with equal potencies as known GLS1 inhibitors. These results will contribute to the development of effective novel GLS1 inhibitors with more potent inhibitory activity.

Structural and mutational analyses of decarboxylated osteocalcin provide insight into its adiponectin-inducing activity.

An acidic environment in bone is essential for bone metabolism and the production of decarboxylated osteocalcin, which functions as a regulatory hormone of glucose metabolism. Here, we describe the high-resolution X-ray crystal structure of decarboxylated osteocalcin under acidic conditions. Decarboxylated osteocalcin at pH 2.0 retains the α-helix structure of native osteocalcin with three γ-carboxyglutamic acid residues at neutral pH. This implies that decarboxylated osteocalcin is stable under an acidic environment in bone. In addition, site-directed mutagenesis revealed that Glu17 and Glu21 are important for the adiponectin-inducing activity of decarboxylated osteocalcin. These findings suggest that the receptor of decarboxylated osteocalcin responds to the negative charge in helix 1 of osteocalcin.

Chlorinated Naringenin Analogues as Potential Inhibitors of Transthyretin Amyloidogenesis.

Misfolding and aggregation of transthyretin are implicated in the fatal systemic disease known as transthyretin amyloidosis. Here, we report the development of a naringenin derivative bearing two chlorine atoms that will be efficacious for preventing aggregation of transthyretin in the eye. The amyloid inhibitory activity of the naringenin derivative was as strong as that of tafamidis, which is the first therapeutic agent targeting transthyretin in the plasma. X-ray crystal structures of the compounds in complex with transthyretin demonstrated that the naringenin derivative with one chlorine bound to the thyroxine-binding site of transthyretin in the forward mode and that the derivative with two chlorines bound to it in the reverse mode. An ex vivo competitive binding assay showed that naringenin derivatives exhibited more potent binding than tafamidis in the plasma. Furthermore, an in vivo pharmacokinetic study demonstrated that the dichlorinated derivative was significantly delivered to the eye.

Neutron crystallographic analysis of the nucleotide-binding domain of Hsp72 in complex with ADP.

The 70 kDa heat-shock proteins (Hsp70s) are ATP-dependent molecular chaperones that contain an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain. Hsp70s bind to misfolded/unfolded proteins and thereby prevent their aggregation. The ATP hydrolysis reaction in the NBD plays a key role in allosteric control of the binding of substrate proteins. In the present study, the neutron crystal structure of the NBD of Hsp72, a heat-inducible Hsp70 family member, was solved in complex with ADP in order to study the structure-function relationship with a focus on hydrogens. ADP bound to Hsp72 was fully deprotonated, and the catalytically important residues, including Asp10, Asp199 and Asp206, are also deprotonated. Neutron analysis also enabled the characterization of the water clusters in the NBD. Enzymatic assays and X-ray crystallographic analysis revealed that the Y149A mutation exhibited a higher ATPase activity and caused disruption of the water cluster and incorporation of an additional magnesium ion. Tyr149 was suggested to contribute to the low intrinsic ATPase activity and to stabilize the water cluster. Collectively, these structural studies will help to elucidate the molecular basis of the function of Hsp72.

Usefulness of the whole-body counter for infants and small children (BABYSCAN) as a risk communication tool after the Fukushima Daiichi nuclear power plant incident.

Responding to the radiation-related concerns of parents/guardians with infants/small children is an important public health issue for regional recovery after radioactive contamination. This study summarizes the results of a systematic internal contamination screening of infants/small children, aged 0-6 years, using BABYSCAN and individual counselling sessions with physicians about radiation concerns from 2014 to 2018 in Minamisoma City. Of 3,114 participants, no one was found to have internal contamination with radioactive caesium with a detection limit of 50 Bq/body. The questionnaire survey showed a decreasing trend of concerns about food contamination and playing outside as possible causes of internal contamination over time. Because people's concerns were diverse in counselling sessions, individual responses are required. This study showed that examinations using BABYSCAN provide an opportunity for direct dialogue between the parents/guardians of infants/small children and experts. This can be considered a model case for risk communication conducted by the local government after a radioactive contamination incident.

Structural and thermodynamic characterization of the binding of isoliquiritigenin to the first bromodomain of BRD4.

Bromodomain-containing protein 4 (BRD4) recognizes the acetylated lysine of histone H4 via its bromodomains, leading to the recruitment of positive transcription elongation factor b. Small molecules that inhibit BRD4 have potential as anticancer agents by leading to the downregulation of specific oncogenes. Using X-ray crystallographic screening, we identified the BRD4 inhibitory activity of isoliquiritigenin (ISL), a natural chalcone found in licorice. Structural analysis revealed that ISL bound to BRD4 with a novel binding mode and squeezed out one of the six conserved water molecules that form a strong hydrogen bond network. The thermodynamic analysis revealed that the binding of ISL is enthalpy driven, suggesting that strong hydrogen bonds would compensate for the desolvation penalty. Neutron protein crystallography further suggested that the favorable binding enthalpy originates from the stabilization and optimization of the hydrogen bond network of the conserved water molecules. Here, we describe the novelty and potential of ISL as a template for new BRD4 inhibitors.

Allosteric modulation of the binding affinity between PQBP1 and the spliceosomal protein U5-15kD.

Polyglutamine tract-binding protein 1 (PQBP1) is an intrinsically disordered protein composed of a small folded WW domain and a long disordered region. PQBP1 binds to spliceosomal proteins WBP11 and U5-15kD through its N-terminal WW domain and C-terminal region, respectively. Here, we reveal that the binding between PQBP1 and WBP11 reduces the binding affinity between PQBP1 and U5-15kD. Our results suggest that the interaction between PQBP1 and WBP11 negatively modulates the U5-15kD binding of PQBP1 by an allosteric mechanism.

Segmental isotope-labeling of the intrinsically disordered protein PQBP1.

Polyglutamine tract-binding protein 1 (PQBP1) is an intrinsically disordered protein abundantly expressed in the brain. Mutations in the PQBP1 gene are causative for X-linked mental retardation disorders. Here, we investigated the structure of the C-terminal segment within the context of full-length PQBP1. We produced a segmentally isotope-labeled PQBP1 composed of a non-labeled segment (residues 1-219; N-segment) and a (13)C/(15)N-labeled segment (residues 220-265; C-segment). Our results demonstrate that the segmental isotope-labeling combined with NMR spectroscopy is useful for detecting a very weak intra-molecular interaction in an intrinsically disordered protein.

Mutations in the PQBP1 gene prevent its interaction with the spliceosomal protein U5-15 kD.

A loss-of-function of polyglutamine tract-binding protein 1 (PQBP1) induced by frameshift mutations is believed to cause X-linked mental retardation. However, the mechanism by which structural changes in PQBP1 lead to mental retardation is unknown. Here we present the crystal structure of a C-terminal fragment of PQBP1 in complex with the spliceosomal protein U5-15 kD. The U5-15 kD hydrophobic groove recognizes a YxxPxxVL motif in PQBP1, and mutations within this motif cause a loss-of-function phenotype of PQBP1 in vitro. The YxxPxxVL motif is absent in all PQBP1 frameshift mutants seen in cases of mental retardation. These results suggest a mechanism by which the loss of the YxxPxxVL motif could lead to the functional defects seen in this type of mental retardation.

Localization of serine racemase and its role in the skin.

D-serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier.

Hydrogen-bond network and pH sensitivity in human transthyretin.

Transthyretin (TTR) is a tetrameric protein. TTR misfolding and aggregation are associated with human amyloid diseases. Dissociation of the TTR tetramer is believed to be the rate-limiting step in the amyloid fibril formation cascade. Low pH is known to promote dissociation into monomer and the formation of amyloid fibrils. In order to reveal the molecular mechanisms underlying pH sensitivity and structural stabilities of TTR, neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. Crystals for the neutron diffraction experiments were grown up to 2.5 mm(3) for four months. The neutron crystal structure solved at 2.0 Å revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is involved in monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. Structural comparison with the X-ray crystal structure at acidic pH identified the three amino acid residues responsible for the pH sensitivity of TTR. Our neutron model provides insights into the molecular stability related to amyloidosis.

Structural characterization of a trapped folding intermediate of pyrrolidone carboxyl peptidase from a hyperthermophile.

The refolding of cysteine-free pyrrolidone carboxyl peptidase (PCP-0SH) from a hyperthermophile is unusually slow. PCP-0SH is trapped in the denatured (D1) state at 4 °C and pH 2.3, which is different from the highly denatured state in the presence of concentrated denaturant. In order to elucidate the mechanism of the unusually slow folding, we investigated the structure of the D1 state using NMR techniques with amino acid selectively labeled PCP-0SH. The HSQC spectrum of the D1 state showed that most of the resonances arising from the 114-208 residues are broadened, indicating that conformations of the 114-208 residues are in intermediate exchange on the microsecond to millisecond time scale. Paramagnetic relaxation enhancement data indicated the lack of long-range interactions between the 1-113 and the 114-208 segments in the D1 state. Furthermore, proline scanning mutagenesis showed that the 114-208 segment in the D1 state forms a loosely packed hydrophobic core composed of α4- and α6-helices. From these findings, we conclude that the 114-208 segment of PCP-0SH folds into a stable compact structure with non-native helix-helix association in the D1 state. Therefore, in the folding process from the D1 state to the native state, the α4- and α6-helices become separated and the central ß-sheet is folded between these helices. That is, the non-native interaction between the α4- and α6-helices may be responsible for the unusually slow folding of PCP-0SH.

Quaternary structure, aggregation and cytotoxicity of transthyretin.

Transthyretin (TTR) with a Ser112-to-Ile mutation is known to cause amyloidosis with severe cardiomyopathy. We investigated the quaternary structure, aggregation and cytotoxicity of the S112I variant. This variant exists as a dimer at physiological pH, self-assembles into spherical aggregates and induces cell death in human neuroblastoma IMR-32 cells. In addition, we determined the neutron crystal structure of TTR at 2.0 Å resolution. The neutron structure revealed that the hydrogen-bond network involving His88 is important for the stabilization of the dimer-dimer and monomer-monomer interfaces.

Hydrogen-bond network and pH sensitivity in transthyretin: Neutron crystal structure of human transthyretin.

Transthyretin (TTR) is a tetrameric protein associated with human amyloidosis. In vitro, the formation of amyloid fibrils by TTR is known to be promoted by low pH. Here we show the neutron structure of TTR, focusing on the hydrogen bonds, protonation states and pH sensitivities. A large crystal was prepared at pD 7.4 for neutron protein crystallography. Neutron diffraction studies were conducted using the IBARAKI Biological Crystal Diffractometer with the time-of-flight method. The neutron structure solved at 2.0Å resolution revealed the protonation states of His88 and the detailed hydrogen-bond network depending on the protonation states of His88. This hydrogen-bond network is composed of Thr75, Trp79, His88, Ser112, Pro113, Thr118-B and four water molecules, and is involved in both monomer-monomer and dimer-dimer interactions, suggesting that the double protonation of His88 by acidification breaks the hydrogen-bond network and causes the destabilization of the TTR tetramer. In addition, the comparison with X-ray structure at pH 4.0 indicated that the protonation occurred to Asp74, His88 and Glu89 at pH 4.0. Our neutron model provides insights into the molecular stability of TTR related to the hydrogen-bond network, the pH sensitivity and the CH···O weak hydrogen bond.