Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1.

Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively) compared with 19 nM for the wild type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. SIGNIFICANCE STATEMENT: Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting organic ion transporters (OATs). This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 is found to mediate hydrogen bonding.

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics.

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom-up proteomics using LC-MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC-MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein-coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein-protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.

Fungal effector SIB1 of Colletotrichum orbiculare has unique structural features and can suppress plant immunity in Nicotiana benthamiana.

Fungal plant pathogens secrete virulence-related proteins, called effectors, to establish host infection; however, the details are not fully understood yet. Functional screening of effector candidates using Agrobacterium-mediated transient expression assay in Nicotiana benthamiana identified two virulence-related effectors, named SIB1 and SIB2 (Suppression of Immunity in N. benthamiana), of an anthracnose fungus Colletotrichum orbiculare, which infects both cucurbits and N. benthamiana. The Agrobacterium-mediated transient expression of SIB1 or SIB2 increased the susceptibility of N. benthamiana to C. orbiculare, which suggested these effectors can suppress immune responses in N. benthamiana. The presence of SIB1 and SIB2 homologs was found to be limited to the genus Colletotrichum. SIB1 suppressed both (i) the generation of reactive oxygen species triggered by two different pathogen-associated molecular patterns, chitin and flg22, and (ii) the cell death response triggered by the Phytophthora infestans INF1 elicitin in N. benthamiana. We determined the NMR-based structure of SIB1 to obtain its structural insights. The three-dimensional structure of SIB1 comprises five ß-strands, each containing three disulfide bonds. The overall conformation was found to be a cylindrical shape, such as the well-known antiparallel ß-barrel structure. However, the ß-strands were found to display a unique topology, one pair of these ß-strands formed a parallel ß-sheet. These results suggest that the effector SIB1 present in Colletotrichum fungi has unique structural features and can suppress pathogen-associated molecular pattern-triggered immunity in N. benthamiana.

Evolution-Based Protein Engineering for Antifungal Peptide Improvement.

Antimicrobial peptides (AMPs) have been considered as the alternatives to antibiotics because of their less susceptibility to microbial resistance. However, compared with conventional antibiotics they show relatively low activity and the consequent high cost and nonspecific cytotoxicity, hindering their clinical application. What's more, engineering of AMPs is a great challenge due to the inherent complexity in their sequence, structure, and function relationships. Here, we report an evolution-based strategy for improving the antifungal activity of a nematode-sourced defensin (Cremycin-5). This strategy utilizes a sequence-activity comparison between Cremycin-5 and its functionally diverged paralogs to identify sites associated with antifungal activity for screening of enhanceable activity-modulating sites for subsequent saturation mutagenesis. Using this strategy, we identified a site (Glu-15) whose mutations with nearly all other types of amino acids resulted in a universally enhanced activity against multiple fungal species, which is thereby defined as a Universally Enhanceable Activity-Modulating Site (UEAMS). Especially, Glu15Lys even exhibited >9-fold increased fungicidal potency against several clinical isolates of Candida albicans through inhibiting cytokinesis. This mutant showed high thermal and serum stability and quicker killing kinetics than clotrimazole without detectable hemolysis. Molecular dynamic simulations suggest that the mutations at the UEAMS likely limit the conformational flexibility of a distant functional residue via allostery, enabling a better peptide-fungus interaction. Further sequence, structural, and mutational analyses of the Cremycin-5 ortholog uncover an epistatic interaction between the UEAMS and another site that may constrain its evolution. Our work lights one new road to success of engineering AMP drug leads.

Red-Beet Betalain Pigments Inhibit Amyloid-ß Aggregation and Toxicity in Amyloid-ß Expressing Caenorhabditis elegans.

Betalain pigments are mainly produced by plants belonging to the order of Caryophyllales. Betalains exhibit strong antioxidant activity and responds to environmental stimuli and stress in plants. Recent reports of antioxidant, anti-inflammatory and anti-cancer properties of betalain pigments have piqued interest in understanding their biological functions. We investigated the effects of betalain pigments (betanin and isobetanin) derived from red-beet on amyloid-ß (Aß) aggregation, which causes Alzheimer's disease. Non-specific inhibition of Aß aggregation against Aß40 and Aß42 by red-beet betalain pigments, in vitro was demonstrated using the thioflavin t fluorescence assay, circular dichroism spectroscopy analysis, transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, we examined the ability of red-beet betalain pigments to interfere with Aß toxicity by using the transgenic Caenorhabditis elegans model, which expresses the human Aß42 protein intracellularly within the body wall muscle. It responds to Aß-toxicity with paralysis and treatment with 50 µM red-beet betalain pigments significantly delayed the paralysis of C. elegans. These results suggest that betalain pigments reduce Aß-induced toxicity.

Production of ORF8 protein from SARS-CoV-2 using an inducible virus-mediated expression system in suspension-cultured tobacco BY-2 cells.

KEY MESSAGE: Production of ORF8 protein from SARS-CoV-2 in tobacco BY-2 cells.

Met872 is the key residue determining the novel binominal binding of metabotropic glutamate receptor 7 to calmodulin.

Two mGluR7-derived peptides corresponding to residues 856 to 879 and 856 to 875 are known to bind to Ca2+-saturated calmodulin (Ca2+/CaM), and their binding manners are thought to differ. Met872 function is believed as one of the anchor residues for CaM-binding only in the shorter peptide. To uncover the role of Met872 in CaM-binding, we prepared a mutant of the long peptide, mGluR7 (M872A), in which Met872 was replaced with Ala. We used the mutant together with the two peptides to perform NMR-titration experiments to monitor interaction with stable isotope-labeled CaM. Interaction of Ca2+/CaM with mGluR7 (M872A) caused a spectrum that differed from that of Ca2+/CaM with the long peptide, suggesting that Met872 of mGluR7 could be involved in CaM-binding even in the long peptide. Analyses of all NMR data suggested that the binding between Ca2+/CaM and mGluR7 occurs in some conformational equilibrium manner. The unique CaM-binding properties caused by Met872 may be related to mGluR7's function.

Ca2+-dependent interaction between calmodulin and CoDN3, an effector of Colletotrichum orbiculare.

Nuclear magnetic resonance (NMR) data directly indicated a Ca2+-dependent interaction between calmodulin (CaM) and CoDN3, a small effector of the plant pathogenic fungus Colletotrichum orbiculare, which is the causal agent of cucumber anthracnose. The overall conformation of CoDN3 is intrinsically disordered, and the CaM-binding site spans residues 34-53 of its C-terminal region. Experiments employing a chemically synthesized peptide corresponding to the CaM-binding site indicated that the CaM-binding region of CoDN3 in the Ca2+-bound CaM complex takes an α-helical conformation. Cell death suppression assay using a CoDN3 mutant lacking the CaM-binding ability suggested that the wild type CaM-binding site is necessary for full CoDN3 function in vivo.

Isolation of amaranthin synthetase from Chenopodium quinoa and construction of an amaranthin production system using suspension-cultured tobacco BY-2 cells.

Betalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti-inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow-2 (BY-2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 µm (mean ± SEM) amaranthin and 26.60 ± 1.53 µm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV-1 protease activity, whereas betanin did not.

Increased Dynamics of Tricarboxylic Acid Cycle and Glutamate Synthesis in Obese Adipose Tissue: IN VIVO METABOLIC TURNOVER ANALYSIS.

Obesity is closely associated with various metabolic disorders. However, little is known about abnormalities in the metabolic change of obese adipose tissue. Here we use static metabolic analysis and in vivo metabolic turnover analysis to assess metabolic dynamics in obese mice. The static metabolic analyses showed that glutamate and constitutive metabolites of the TCA cycle were increased in the white adipose tissue (WAT) of ob/ob and diet-induced obesity mice but not in the liver or skeletal muscle of these obese mice. Moreover, in vivo metabolic turnover analyses demonstrated that these glucose-derived metabolites were dynamically and specifically produced in obese WAT compared with lean WAT. Glutamate rise in obese WAT was associated with down-regulation of glutamate aspartate transporter (GLAST), a major glutamate transporter for adipocytes, and low uptake of glutamate into adipose tissue. In adipocytes, glutamate treatment reduced adiponectin secretion and insulin-mediated glucose uptake and phosphorylation of Akt. These data suggest that a high intra-adipocyte glutamate level potentially relates to adipocyte dysfunction in obesity. This study provides novel insights into metabolic dysfunction in obesity through comprehensive application of in vivo metabolic turnover analysis in two obese animal models.