NMR mapping of the highly flexible regions of 13C/15N-labeled antibody TTAC-0001-Fab.

Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the 13C/15N-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli (E. coli). Here, we notably increased the periplasmic expression of the 13C/15N-labeled TTAC-0001-Fab (13C/15N-TTAC-Fab) through simple isopropyl ß-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the VH and CH1; LI-2 between the VL and CL) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.

Multi-Species Probiotic Strain Mixture Enhances Intestinal Barrier Function by Regulating Inflammation and Tight Junctions in Lipopolysaccharides Stimulated Caco-2 Cells.

Although leaky gut syndrome is not recognized as an official diagnosis for human diseases, it is now believed that dysfunction of the cell barrier causes increased permeability of intestinal epithelial cells leading to this condition. Probiotics have been widely used to improve gut health, and studies have investigated the relevance of protecting the intestinal barrier by taking probiotic strains in vitro and in vivo. However, most studies have restricted the use of single or several probiotic strains and do not consider commercially available probiotic products composed of multi-species. In this study, we provide experimental evidence that a multi-species probiotic mixture composed of eight different strains and a heat-treated probiotic strain is effective in preventing leaky gut conditions. We employed an in vitro co-culture model system utilizing two different differentiated cell lines to mimic human intestinal tissue. The integrity of epithelial barrier function was protected by the preserving the occludin protein level and activating the AMPK signaling pathway, associated with tight junctions (TJs), through treatment with the probiotic strain mixture in Caco-2 cells. Moreover, we confirmed that application of the multi-species probiotic mixture reduced the expression of proinflammatory cytokine genes by inhibiting NFκB signaling pathway when artificial inflammation was induced in an in vitro co-culture model system. Finally, we proved that the epithelial permeability measured by trans-epithelial electrical resistance (TEER) was significantly decreased in the probiotic mixture treated cells, indicating that the integrity of the epithelial barrier function was not compromised. The multi-species probiotic strain mixture exhibited the protective effect on the integrity of intestinal barrier function via enhancing TJ complexes and reducing inflammatory responses in the human intestinal cells.

Kinetic evidence in favor of glyoxalase III and against deglycase activity of DJ-1.

DJ-1, a protein encoded by PARK7 plays a protective role against neurodegeneration. Since its glyoxalase III activity catalyzing methylglyoxal (MG) to lactate was discovered, DJ-1 has been re-established as a deglycase decomposing the MG-intermediates with amino acids and nucleotides (hemithioacetal and hemiaminal) rather than MG itself, but it is still debatable. Here, we have clarified that human DJ-1 directly recognizes MG, and not MG-intermediates, by monitoring the detailed catalytic processes and enantiomeric lactate products. The hemithioacetal intermediate between C106 of 15 N-labeled DJ-1 (15N DJ-1) and MG was also monitored by NMR. TRIS molecule formed stable diastereotopic complexes with MG (Kd , 1.57 ± 0.27 mM) by utilizing its three OH groups, which likely disturbed the assay of deglycase activity. The low kcat of DJ-1 for MG and its MG-induced structural perturbation may suggest that DJ-1 has a regulatory function as an in vivo sensor of reactive carbonyl stress.

Bean Extract-Based Gargle for Efficient Diagnosis of Active COVID-19 Infection Using Rapid Antigen Tests.

The antigen-based rapid diagnostic test (Ag-RDT) using saliva specimens is fast, noninvasive, and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. We evaluated a novel Beanguard gargle (BG)-based virus collection method that can be applied to Ag-RDT as an alternative to the current RT-PCR with an NPS for early diagnosis of COVID-19. This clinical trial comprised 102 COVID-19-positive patients hospitalized after a governmental screening process and 100 healthy individuals. Paired NPS and BG-based saliva specimens from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs, whose diagnostic performance for detecting SARS-CoV-2 was compared. BG-Ag-RDTs showed high sensitivity (97.8%) and specificity (100%) in 45 patients within 6 days of illness and detected all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic active COVID-19 cases, both BG-Ag-RDTs and BG-RT-PCR showed sensitivities and specificities of 100%. Sensitivities of BG-Ag-RDT and BG-RT-PCR toward salivary viral detection were highly concordant, with no discrimination between symptomatic (97.0%), asymptomatic (100%), or SARS-CoV-2 variant (100%) cases. The intermolecular interactions between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from the bean extract (BE), were observed in terms of physicochemical properties. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Using BG-based saliva specimens remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables noninvasive, rapid, and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management. IMPORTANCE An Ag-RDT is less likely to be accepted as an initial test method for early diagnosis owing to its low sensitivity. However, our self-collection method, Ag-RDT using BG-based saliva specimens, showed significantly enhanced detection sensitivity and specificity toward SARS-CoV-2 including the Alpha and Delta variants in all patients tested within 6 days of illness. The method represents an attractive alternative to nasopharyngeal swabs for the early diagnosis of symptomatic and asymptomatic COVID-19 cases. The evidence suggests that the method could have a potential for mass screening and monitoring of COVID-19 cases.

Determinants of PB1 Domain Interactions in Auxin Response Factor ARF5 and Repressor IAA17.

Auxin is a plant hormone that is central to plant growth and development from embryogenesis to senescence. Auxin signaling is mediated by auxin response transcription factors (ARFs) and Aux/IAA repressors that regulate the expression of a multitude of auxin response genes. ARF and Aux/IAA proteins assemble into homomeric and heteromeric complexes via their conserved PB1 domains. Here we report the first crystal structure of the PB1 complex between ARF5 and IAA17 of Arabidopsis thaliana, which represents the transcriptionally repressed state at low auxin levels. The PB1 domains assemble in a head-to-tail manner with a backbone arrangement similar to that of the ARF5:ARF5 PB1 complex. The ARF5:IAA17 complex, however, reveals distinct points of contact that promote the ARF5:IAA17 interaction over the ARF5:ARF5 interaction. Specifically, surface charges at the interface form salt-bridges that distinguish the homomeric and heteromeric complexes, revealing common and specific interfaces between transcriptionally repressed and derepressed states. Further, the salt-bridges can be reconfigured to switch the affinity between homomeric and heteromeric complexes in an incremental manner. The complex structure combined with quantitative binding analyses would be essential for deciphering the PB1 interaction code underlying the transcriptional regulation of auxin signaling.

Structural Basis for Cell-Wall Recognition by Bacteriophage PBC5 Endolysin.

Phage endolysins are hydrolytic enzymes that cleave the bacterial cell wall during the lytic cycle. We isolated the bacteriophage PBC5 against Bacillus cereus, a major foodborne pathogen, and describe the molecular interaction between endolysin LysPBC5 and the host peptidoglycan structure. LysPBC5 has an N-terminal glycoside hydrolase 25 domain, and a C-terminal cell-wall binding domain (CBD) that is critical for specific cell-wall recognition and lysis. The crystal and solution structures of CBDs reveal tandem SH3b domains that are tightly engaged with each other. The CBD binds to the peptidoglycan in a bidentate manner via distal ß sheet motifs with pseudo 2-fold symmetry, which can explain its high affinity and host specificity. The CBD primarily interacts with the glycan strand of the peptidoglycan layer instead of the peptide crosslink, implicating the tertiary structure of peptidoglycan as the recognition motif of endolysins.

Zinc-mediated Reversible Multimerization of Hsp31 Enhances the Activity of Holding Chaperone.

Hsp31 protein, belonging to the DJ-1/ThiJ/PfpI superfamily, increases the survival of Escherichia coli under various stresses. While it was reported as a holding chaperone, Hsp31 was also shown to exhibit the glyoxalase III activity in subsequent study. Here, we describe our finding that Hsp31 undergoes a Zn+2-mediated multimerization (HMWZinc), resulting in an enhanced chaperone activity. Furthermore, it was shown that the formation of HMWZinc is reversible such that the oligomer dissociates into the native dimer by EDTA incubation. We attempted to determine the structural change involving the transition between the native dimer and HMWZinc by adding Ni+2, which is Zn+2-mimetic, producing a potential intermediate structure. An analysis of this intermediate revealed a structure with hydrophobic interior exposed, due to an unfolding of the N-terminal loop and the C-terminal ß-to-α region. A treatment with hydrogen peroxide accelerated HMWZinc formation, so that the Hsp31C185E mutant rendered the formation of HMWZinc even at 45 °C. However, the presence of Zn+2 in the catalytic site antagonizes the oxidation of C185, implying a negative role. Our results suggest an unprecedented mechanism of the enhancing chaperone activity by Hsp31, in which the reversible formation of HMWZinc occurs in the presence of heat and Zn+2 ion.

Total Synthesis of Xanthoangelol B and Its Various Fragments: Toward Inhibition of Virulence Factor Production of Staphylococcus aureus.

As an alternative strategy to fight antibiotic resistance, two-component systems (TCSs) have emerged as novel targets. Among TCSs, master virulence regulators that control the expression of multiple virulence factors are considered as excellent antivirulence targets. In Staphylococcus aureus, virulence factor expression is tightly regulated by a few master regulators, including the SaeRS TCS. In this study, we used a SaeRS GFP-reporter system to screen natural compound inhibitors of SaeRS, and identified xanthoangelol B 1, a prenylated chalcone from Angelica keiskei as a hit. We have synthesized 1 and its derivative PM-56 and shown that 1 and PM-56 both had excellent inhibitory potency against the SaeRS TCS, as demonstrated by various in vitro and in vivo experiments. As a mode of action, 1 and PM-56 were shown to bind directly to SaeS and inhibit its histidine kinase activity, which suggests a possibility of a broad spectrum inhibitor of histidine kinases.

Evidence of link between quorum sensing and sugar metabolism in Escherichia coli revealed via cocrystal structures of LsrK and HPr.

Quorum sensing (QS), a bacterial process that regulates population-scale behavior, is mediated by small signaling molecules, called autoinducers (AIs), that are secreted and perceived, modulating a "collective" phenotype. Because the autoinducer AI-2 is secreted by a wide variety of bacterial species, its "perception" cues bacterial behavior. This response is mediated by the lsr (LuxS-regulated) operon that includes the AI-2 transporter LsrACDB and the kinase LsrK. We report that HPr, a phosphocarrier protein central to the sugar phosphotransferase system of Escherichia coli, copurifies with LsrK. Cocrystal structures of an LsrK/HPr complex were determined, and the effects of HPr and phosphorylated HPr on LsrK activity were assessed. LsrK activity is inhibited when bound to HPr, revealing new linkages between QS activity and sugar metabolism. These findings help shed new light on the abilities of bacteria to rapidly respond to changing nutrient levels at the population scale. They also suggest new means of manipulating QS activity among bacteria and within various niches.