The enzyme 5-aminoimidazole ribonucleotide synthetase (AIR synthetase, AIRS, known also as PurM) catalyzes the fifth step in the de novo purine biosynthetic pathway: the ATP-dependent conversion of formylglycinamidine ribonucleotide (FGAM) to aminoimidazole ribonucleotide (AIR). AIRS belongs to the PurM superfamily, sharing ATPase domains. In this study, we employed hyperthermostable AIRS from Pyrococcus abyssi (Pa) and Pyrococcus horikoshii (Ph), members of the thermophilic archaeal genus known for extreme heat tolerance, to resolve the substrate-binding site and structural features. Crystal structures of AIRS were determined in complex with the substrate analog formylglycinamide ribonucleotide (FGAR), the non-hydrolyzable ATP analog adenylyl-imidodiphosphate (AMPPNP), the product ADP, and metal cofactors (Mn2+, Mg2+, and K+), respectively. AMPPNP and FGAR bind to the α1-β1 loop and the disordered N-terminal tail, respectively, stabilizing the active-site conformation. These findings provide a structural framework for understanding substrate positioning and catalysis within AIRS. A unique non-domain-swapping feature observed in the first 54 residues of the N-terminal domain in PaAIRS and PhAIRS, differing from their orthologs. The domain-swappable feature is found to be β1-β2 loop-dependent and can be categorized into two distinct types in the AIRS family: a short-looped, non-swapping archaeal type and a long-looped, swapping bacterial type. Structural, mutational, and biophysical analyses suggest that the thermostability is supported by enhanced hydrophobic core packing, strengthened dimer interfaces, and shortened surface loops. Collectively, these findings not only elucidate the substrate binding, the catalytic mechanism, and domain swapping of AIRS but also contribute to our understanding of the compact and robust architecture characteristics of thermophilic proteins.

Multidrug resistance in Pseudomonas aeruginosa is strongly promoted by the resistance-nodulation-division family tripartite efflux pump MexAB-OprM, whose inner-membrane transporter MexB plays a central role in recognizing and extruding a broad spectrum of antibiotics and detergents. Although crystal structures of MexB have been determined, no structure of MexB bound to an antibiotic has previously been reported. Here, we report crystal structures of drug-free MexB and chloramphenicol-bound MexB crystallized under mildly basic conditions. In the chloramphenicol-bound structure, chloramphenicol binds at the deep end of the distal binding pocket (DBP) groove in the Binding protomer. Based on this structure, we identified DBP residues (Q125, R128, F178, G179, S180 and Q273) that contact chloramphenicol and evaluated their contributions using in vitro chloramphenicol resistance assays of single-substitution MexB variants. Substitutions at these positions reduced cell growth in the presence of chloramphenicol, minocycline, levofloxacin and the detergent CYMAL-7. These findings identify a MexB-specific recognition subsite within the DBP groove and provide a structural basis for understanding how MexB recognizes chloramphenicol and other chemically diverse substrates.

Voltage-sensing phosphatase (VSP) comprises a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), achieving a unique electrochemical signal conversion. Previous studies suggest that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a membrane phospholipid known to be critical for activities of diverse voltage-gated ion channels, associates with a linker connecting the VSD with the CCR of VSP and regulates VSD-CCR coupling. However, the details of PI(4,5)P2 interaction with the linker of VSP remain elusive. Here, we exploit advantage of sensitivity of a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), to changes in local environment to study interaction between PI(4,5)P2 and the linker of Ciona intestinalis VSP (Ci-VSP). We found that a conserved tyrosine residue (Y255) as well as neighboring basic residues interacts with PI(4,5)P2 and this interaction was maintained in G365A Ci-VSP mutant which lacks the substrate PI(4,5)P2 at the active site and Ci-VSP/human phosphatase and tensin homolog (PTEN) chimera which does not dephosphorylate PI(4,5)P2, indicating that the linker interacts with nonsubstrate, regulatory PI(4,5)P2 outside the active site. Molecular dynamics simulations demonstrated that the linker formed stable interaction with PI(4,5)P2 in the activated state. These findings indicate that regulation of coupling to an effector region downstream of the VSD through PI(4,5)P2 binding to the linker is shared among voltage-dependent membrane proteins.

Aberrant immune responses to viral pathogens contribute to pathogenesis, but our understanding of pathological immune responses caused by viruses within the human virome, especially at a population scale, remains limited. We analyzed whole-genome sequencing datasets of 6,321 Japanese individuals, including patients with autoimmune diseases (psoriasis vulgaris, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), pulmonary alveolar proteinosis (PAP) or multiple sclerosis) and coronavirus disease 2019 (COVID-19), or healthy controls. We systematically quantified two constituents of the blood DNA virome, endogenous HHV-6 (eHHV-6) and anellovirus. Participants with eHHV-6B had higher risks of SLE and PAP; the former was validated in All of Us. eHHV-6B-positivity and high SLE disease activity index scores had strong correlations. Genome-wide association study and long-read sequencing mapped the integration of the HHV-6B genome to a locus on chromosome 22q. Epitope mapping and single-cell RNA sequencing revealed distinctive immune induction by eHHV-6B in patients with SLE. In addition, high anellovirus load correlated strongly with SLE, RA and COVID-19 status. Our analyses unveil relationships between the human virome and autoimmune and infectious diseases. Analysis of the blood DNA virome in patients with COVID-19 and autoimmune disease associates endogenous HHV-6 (eHHV-6) and high anellovirus load with increased disease risk, most notably for systemic lupus erythematosus. eHHV-6 carriers show a distinct immune response.

Bacteriophage Mu is a temperate phage known to infect various species of Enterobacteria, playing a role in bacterial mutation induction and horizontal gene transfer. The phage possesses two types of tail fibers important for host recognition, which enable it to expand its range of hosts. The alternate tail fibers are formed through the action of genes 49-50 or 52-51, allowing the Mu phage to recognize different surfaces of host cells. In a previous study, we presented the X-ray crystal structure of the C-terminal lipopolysaccharide (LPS)-binding domain of gene product (gp) 49, one of the subunits comprising the Mu tail fiber. In this study, we have determined the structure of the alternative tail fiber subunit, gp52, and compared it with other tail fibers. The results revealed that Mu phage employs different structural motifs for two individual tail fibers for recognizing different hosts.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

AbstractIntroductionParaneoplastic neurological syndrome (PNS) is associated with small‐cell lung cancer (SCLC). However, the frequency and characteristics of PNS and the efficacy of anticancer treatment for these patients have not been investigated in the Japanese/Asian population previously. Therefore, we aimed to better understand PNS by evaluating real‐world data from patients with PNS complicated by SCLC.MethodsPatients diagnosed with Stage II–IV SCLC at a single center between August 2007 and April 2021 were retrospectively analyzed. The primary outcome was the incidence of PNS. The secondary outcomes were the change in performance status (PS) after treatment commencement and outcomes following anticancer treatment, including objective response rate (ORR), progression‐free survival (PFS), and overall survival (OS).ResultsA total of 318 patients were evaluated; PNS was present in 2.8% (n = 9) of the overall population. All patients with PNS exhibited poor Eastern Cooperative Oncology Group PS (≥2); moreover, 78% of patients had a PS score of 3–4. An improvement in PS was observed in 56% (n = 5) of patients. Patients with PNS exhibited treatment efficacies similar to patients without PNS (ORR: 89% vs. 83%, p = 1.0; PFS: 7.6 vs. 5.7 months, p = 0.69; OS: not reached vs. 15.6 months, p = 0.23).ConclusionsA total of 2.8% of patients had SCLC complicated by PNS, with poor PS observed. However, anticancer therapy led to an improvement in PS and comparable ORR, as well as PFS and OS similar to those observed in patients without PNS. Thus, anticancer therapy should be considered in patients with PNS.

Key Clinical MessageRecently, hypoxia‐inducible factor prolyl hydroxylase (HIF‐PH) inhibitors have been used for renal anemia, but side effects have also been reported. We report on the association of central hypothyroidism and cholesterol with roxadustat. Based on this case and previous reports, we believe that patients receiving roxadustat should have their thyroid function and cholesterol levels checked regularly.

CUB domain containing protein-1 (CDCP1, also known as Trask or CD318) is a glycosylated, single pass type 1 transmembrane protein.CDCP1 is overexpressed in breast, colorectal and lung cancer [1] and is expected as a biomarker of cancer.Based on amino acid sequence, CDCP1 is predicted to have 3 CUB domains (CUB1, CUB2 and CUB3) in the extracellular region, 2 lipidation sites and 5 tyrosine which are phosphorylated by kinases in the intercellular region.CDCP1 is cleaved by Matriptase/ST-14 at Arg368-Lys369 to generate approximately 65 kDa amino terminal fragment and approximately 70 kDa carboxyl terminal fragment.This cleavage by Matriptase/ST-14 activates CDCP1 to induce CDCP1 homodimerization and CDCP1-SRC kinase-PKCδ tripartite complex which plays important role for downstream signalling [2,3].SRC kinase is recruited to the cytoplasmic region of phosphorylated Tyr734 of CDCP1 and SRC phosphorylates other tyrosine of CDCP1.This cleaved CDCP1 is also enhanced to interact with MET, EGFR or Integrin β1 which do not have common domain structures [4].It is shown that CDCP1 is important for regulating HGF-MET signalling and that the carboxyl terminal fragment of CDCP1 directly interacts with MET and promotes the activation of MET-STAT3 pathway which induces cell invasion [5].Recently, the low resolution structures of CDCP1 extracellular domain (ECD) complexed with Fab by negative stain electron microscopy have been reported [6].However, the detail structural information of CDCP1 has not been reported, yet.To decipher the molecular mechanism why CDCP1 interacts with various receptors induced by Matriptase/ST-14 digestion and makes signal transduction through Src kinase, we determined the crystal structures of extracellular region of CDCP1.It is predicted that CDCP1 has three CUB domain in the extracellular region from its amino acid sequence, however, the crystal structure of CDCP1 ECD showed two domains (N terminal domain (NTD) and C terminal domain (CTD)).The NTD and the CTD have similar structures, each consisting of two CUB domains.Three crystal structures of different constructs of CDCP1 ECD are similar, but domain orientation was different.This suggests that CDCP1 could be fluctuating.CDCP1 cleavage by Matriptase/ST-14 does not separate N-terminal fragment and C-terminal fragment, because cleavage site by Matriptase/ST-14 is located in the loop of CTD and N-terminus region of CTD has many interactions in the CTD.These results provide new insights into activating mechanism and signal transduction of CDCP1.

C–X–C motif chemokine ligand 9 (CXCL9), a candidate biomarker, reflects type 1 (T1) inflammation pathology. Here, we report the analytical performance and clinical characteristics of a new CXCL9 reagent for a fully automated immunoassay device. We evaluated the limits of blank, detection, and quantitation (LoQ) along with other efficacy parameters, and the ability of the assay to report patient health, COVID-19 status, and the presence of asthma and/or interstitial lung diseases (ILDs). The coefficient of variation for 5-day total precision using two instruments was 7% across two controls, serum, and plasma panels. LoQ of 2.2 pg/mL suggested the efficacy of the assay in detecting T1 inflammation in plasma or serum; no cross-reactivity or interference was observed. We identified high serum CXCL9 levels in samples from patients with acute COVID-19 infections ( n = 57), chronic bird-related hypersensitivity pneumonitis ( n = 61), asthma ( n = 194), and ILDs ( n = 84) compared to healthy individuals (< 39.0 pg/mL). Furthermore, CXCL9 levels increased with age in asthma patients, and an opposite trend was observed for T2 inflammatory factors. These results suggest the utility of the automated CXCL9 immunoassay for measuring CXCL9 in clinical samples and reflect its role in T1 inflammation.

We synthesized Co-Pt nanoparticles in a crystal composed of proteins with a cage structure [i.e., Pyrococcus furiosus virus-like particle (PfV)] and investigated their magnetic interactions. When the soaking concentrations of metal, Co, and Pt ions in the PfV crystal were below 5.5 mM, isolated Co-Pt nanoparticles were formed. The size of the primary particles was 2–3 nm, which was smaller than the inner core size of the cage structure. When the soaking concentrations increased above 11 mM, the Co-Pt nanoparticles formed aggregates exceeding 30 nm in size. The synthesized nanoparticles showed superparamagnetic behavior at 300 K, independent of the soaking concentration. The temperature dependence of the ac magnetic susceptibility and remanent magnetization curve verified the occurrence of magnetic interactions between the Co-Pt nanoparticles. These measurements revealed that the dipolar-dipolar interaction was significant in the closely packed isolated nanoparticles, whereas it was weaker in the aggregated Co-Pt nanoparticles, probably due to the increased distance between the particles.

Voltage-sensing phosphatase (VSP) consists of the voltage sensor domain (VSD) similar to that of voltage-gated ion channels and the cytoplasmic phosphatase region with remarkable similarity to the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Membrane depolarization activates VSD, leading to dephosphorylation of three species of phosphoinositides (phosphatidylinositol phosphates (PIPs)), PI(3,4,5)P3, PI(4,5)P2, and PI(3,4)P2. VSP dephosphorylates 3- and 5-phosphate of PIPs, unlike PTEN, which shows rigid 3-phosphate specificity. In this study, a bioinformatics search showed that some mammals have VSP orthologs with amino acid diversity in the active center motif, Cx5R, which is highly conserved among protein tyrosine phosphatases and PTEN-related phosphatases; lysine next to the active site cysteine in the Cx5R motif was substituted for methionine in VSP orthologs of Tasmanian devil, koala, and prairie deer mouse, and leucine in opossum. Since lysine at the corresponding site in PTEN is known to be critical for enzyme activities, we attempted to address the significance of amino acid diversity among VSP orthologs at this site. K364 was changed to different amino acids in sea squirt VSP (Ci-VSP), and voltage-dependent phosphatase activity in Xenopus oocyte was studied using fluorescent probes for PI(4,5)P2 and PI(3,4)P2. All mutants retained both 5-phosphatase and 3-phosphatase activity, indicating that lysine at this site is dispensable for 3-phosphatase activity, unlike PTEN. Notably, K364M mutant showed increased activity both of 5-phosphatase and 3-phosphatase compared with the wild type (WT). It also showed slower kinetics of voltage sensor motion. Malachite green assay of K364M mutant did not show significant difference of phosphatase activity from WT, suggesting tighter interaction between substrate binding and voltage sensing. Mutation corresponding to K364M in the zebrafish VSP led to enhanced voltage-dependent dephosphorylation of PI(4,5)P2. Further studies will provide clues to understanding of substrate preference in PIPs phosphatases as well as to customization of a molecular tool.

AbstractAlthough remdesivir, a prodrug of nucleoside analog (GS‐441524), has demonstrated clinical benefits in coronavirus disease 2019 (COVID‐19) treatment, its pharmacokinetics (PKs) in patients with COVID‐19 remain poorly understood. Therefore, in this study, the PKs of remdesivir and its major metabolite, GS‐441524, were evaluated using a population PK (PopPK) approach to understand the PK aspect and exposure–clinical outcome relationship. The serum concentrations of remdesivir and GS‐441524 (102 points in 39 patients) were measured using liquid chromatography–tandem mass spectrometry. All patients received 200 mg remdesivir on the first day, followed by 100 mg on 2–5 days, except for one patient who discontinued remdesivir on day 4. The median (range) age, body surface area, and estimated glomerular filtration rate (eGFR) were 70 (42–85), 1.74 m2 (1.36–2.03), and 68 mL/min/1.73 m2 (33–113), respectively. A compartment model with first‐order elimination combined with remdesivir and GS‐441524 was used for nonlinear mixed‐effects model analysis. Remdesivir was rapidly eliminated after infusion, whereas GS‐441524 was eliminated relatively slowly (half‐time = 17.1 h). The estimated apparent clearance (CL) and distribution volume of GS‐441524 were 11.0 L/h (intersubject variability [ISV]% = 43.0%) and 271 L (ISV% = 58.1%), respectively. The CL of GS‐441524 was significantly related to the eGFR (CL × [eGFR/68]0.745). The post hoc area under the curve of GS‐441524 was unrelated to the recovery rate or aspartate aminotransferase/alanine aminotransferase elevation. Overall, PopPK analysis showed the rapid elimination of remdesivir in the blood, and GS‐441524 accumulation depended on eGFR in patients with COVID‐19. However, no relevance of exposure–clinical outcome was not suggestive of the dose adjustment of remdesivir.

When pleural empyema is treated with open-window thoracostomy, only 35% of cases are successfully closed, requiring an average of 4.5 months of treatment until wound closure. Conventional treatments such as daily gauze replacement often result in insufficient re-expansion of the collapsed lung, and surgical procedures such as thoracoplasty, muscle flap transposition, and omentoplasty are often necessary. We report a case of acute pleural empyema secondary to coronavirus disease 2019 treated with negative-pressure wound therapy (NPWT). After a month of conservative treatment following open-window thoracostomy, NPWT was performed for three weeks, and the thoracostoma was completely closed with the expanded lung. Approximately one week later, epithelialization of the exposed pleura was completed, and the patient was discharged. Six months after healing, there was no recurrence of pleural empyema. In this case, NPWT resulted in good pulmonary expansion and likely shortened the healing time. Although NPWT is contraindicated in wounds with exposed organs, the use of a contact layer and appropriate negative-pressure settings ensured safe treatment in our patient. We report the results of this study, along with a review of the available literature.