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The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1–α2–α3–α4–α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54. [ABSTRACT FROM AUTHOR]

Purpose: The present study evaluated the sex-specific susceptibility to the development of emphysema in patients with smoking histories who underwent lung cancer surgeries. Methods: Lung cancer patients with smoking histories who underwent lung resection at the University of Tsukuba Hospital, Japan, were enrolled. Radiologic emphysematous changes were analyzed using three-dimensional computed tomography (3D-CT). The volume proportion of emphysematous lung per unit of smoking and the relationship between emphysematous change and clinicopathologic factors were evaluated. Results: Radiologic emphysematous changes analyzed using 3D-CT per pack-year smoked, defined as the Smoking-Emphysema Index (SEI), were greater in females than males. The difference was more profound in adenocarcinoma patients than in non-adenocarcinoma patients (0.70 ± 2.30 vs. 0.21 ± 0.28, P = 0.037). Conclusion: Female lung cancer patients are more susceptible to smoking-induced emphysema than males. The SEI may be an effective indicator for evaluating smoking-induced emphysema. [ABSTRACT FROM AUTHOR]

Gaussia princeps luciferase (GLuc 18.2 kDa; 168 residues) is a marine copepod luciferase that emits a bright blue light when oxidizing coelenterazine (CTZ). GLuc is a small luciferase, attracting much attention as a potential reporter protein. However, compared to firefly and Renilla luciferases, which have been thoroughly characterized and are used in a wide range of applications, structural and biophysical studies of GLuc have been slow to appear. Here, we review the biophysical and mutational studies of GLuc's bioluminescence from a structural viewpoint, particularly in view of its recent NMR solution structure, where two homologous sequential repeats form two anti-parallel bundles, each made of four helices, grabbing a short N-terminal helix. Additionally, a long loop classified as an intrinsically disordered region separates the two bundles forming one side of a hydrophobic pocket that is most likely the binding/catalytic site. We compare the NMR-determined structure with a recent AlphaFold2 prediction. Overall, the AlphaFold2 structure was in line with the solution structure; however, it surprisingly revealed a possible, alternative conformation, where the N-terminal helix is replaced by a newly formed α helix in the C-terminal tail that is unfolded in the NMR structure. In addition, we discuss the results of previous mutational analysis focusing on a putative catalytic core identified by chemical shift perturbation analysis and molecular dynamics simulations performed using both the NMR and the AlphaFold2 structures. In particular, we discuss the role of the possible conformational change and the hydrophobic pocket in GLuc's activity. Overall, the discussion points toward GLuc's unexpected and unusual characteristics that appear to be much more flexible than traditional enzymes, resulting in a unique mode of catalysis to achieve CTZ oxidative decarboxylation. [ABSTRACT FROM AUTHOR]

Open in new tab Download slide OBJECTIVES Postoperative prolonged air leakage is a frequent complication following lung resection. We have shown the high adhesive quality of a newly developed sealant based on a hydrophobically modified Alaska pollock-derived gelatin (ApGltn) sealant in acute in vivo settings. The purpose of this study was to investigate the long-term efficacy and safety of ApGltn sealant using rats as a preclinical model. METHODS An air leakage rat model with a 5-mm pleural defect was created, to which ApGltn sealant or fibrin sealant was applied. In both groups, the rats were evaluated on days 1, 7, 14 and 28. In the ApGltn sealant group, days 56 and 84 were added to evaluate absorption as sealant was still present on day 28. The number of rats in each subgroup was 4 (for a total of 40). Lung specimens and blood samples were obtained for histological and haematological assessment. RESULTS No findings suggesting infection or air leakage were observed. ApGltn sealant was absorbed from day 56 to day 84. Histologically, although neutrophil and lymphocyte infiltrations on the lung side did not differ between groups, those on the sealant side were significantly less in the ApGltn sealant group. Blood sample tests revealed no significant findings suggesting inflammation or organ damage in either group. CONCLUSIONS ApGltn sealant showed long-term sealing efficacy and safety with mild inflammation in a pulmonary air leakage rat model. ApGltn sealant is expected to be a safe and effective sealant for clinical applications. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of liver diseases including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV has the multifunctional protein, HBV X protein (HBx, 154 residues), which plays key roles in HBV replication and liver disease development. Interaction of HBx through its BH3-like motif with the anti-apoptotic protein Bcl-xL leads to HBV replication and induction of apoptosis, resulting in HCC development. Our previous nuclear magnetic resonance (NMR) study revealed that the HBx BH3-like motif peptide (residues 101–136) binds to the common BH3-binding groove of Bcl-xL. Importantly, a C-terminal-truncated HBx, e.g., residues 1–120 of HBx, is strongly associated with the increased risk of HBV-related HCC development. However, the interaction mode between the C-terminal-truncated HBx and Bcl-xL remains unclear. To elucidate this interaction mode, the C-terminal-deleted HBx BH3-like motif peptide (residues 101–120) was used as a model peptide in this study. To facilitate the NMR analysis, we prepared a fusion protein of HBx (101–120) and Bcl-xL connected with five repeats of the glycine-serine dipeptide as a linker. Here, we report the 1H, 13C, and 15N resonance assignments of the fusion protein. This is the first step for the elucidation of the pathogenesis of liver diseases caused by the interaction between the C-terminal-truncated HBx and Bcl-xL. [ABSTRACT FROM AUTHOR]

Ribosome biogenesis is a complicated, multistage process coordinated by ribosome assembly factors. Ribosome binding factor A (RbfA) is a bacterial one, which possesses a single structural type-II KH domain. By this domain, RbfA binds to a 16S rRNA precursor in small ribosomal subunits to promote its 5′-end processing. The human RbfA homolog, mtRbfA, binds to 12S rRNAs in the mitoribosomal small subunits and promotes its critical maturation process, the dimethylation of two highly conserved consecutive adenines, which differs from that of RbfA. However, the structural basis of the mtRbfA-mediated maturation process is poorly understood. Herein, we report the 1H, 15N, and 13C resonance assignments of the KH domain of mtRbfA and its solution structure. The mtRbfA domain adopts essentially the same α1–β1–β2–α2(kinked)–β3 topology as the type-II KH domain. Comparison with the RbfA counterpart showed structural differences in specific regions that function as a putative RNA-binding site. Particularly, the α2 helix of mtRbfA forms a single helix with a moderate kink at the Ser-Ala-Ala sequence, whereas the corresponding α2 helix of RbfA is interrupted by a distinct kink at the Ala-x-Gly sequence, characteristic of bacterial RbfA proteins, to adopt an α2-kink-α3 conformation. Additionally, the region linking α1 and β1 differs considerably in the sequence and structure between RbfA and mtRbfA. These findings suggest some variations of the RNA-binding mode between them and provide a structural basis for mtRbfA function in mitoribosome biogenesis. [ABSTRACT FROM AUTHOR]

Glutathione peroxidase 4 (GPx4) behaves as an antioxidant enzyme capable of directly reducing peroxidized phospholipids within cell membranes. Recently, GPx4 has attracted attention as a target molecule for cancer therapy because it induces the immortalization of cancer cells suppressing ferroptosis. In this study, to analyze the function and structure of GPx4 by solution NMR, we performed resonance assignments of GPx4 and assigned almost all backbone 1H, 13C, and 15N resonances and most of the side chain 1H and 13C resonances. Using these assignments, the secondary structure of GPx4 was analyzed by the TALOS + program. GPx4 has six helices and seven strands. Then, the backbone dynamics were examined by the {1H}–15N heteronuclear NOE experiment. GPx4 was found to be rigid except for a short loop region. These results will provide basis for functional analysis and the first solution structure determination of GPx4. [ABSTRACT FROM AUTHOR]

SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–310–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2. PDB Code(s): 7VH9; [ABSTRACT FROM AUTHOR]

MAFB is a basic leucine zipper (bZIP) transcription factor specifically expressed in macrophages. We have previously identified MAFB as a candidate marker for tumor-associated macrophages (TAMs) in human and mouse models. Here, we analyzed single-cell sequencing data of patients with lung adenocarcinoma obtained from the GEO database (GSE131907). Analyzed data showed that general macrophage marker CD68 and macrophage scavenger receptor 1 (CD204) were expressed in TAM and lung tissue macrophage clusters, while transcription factor MAFB was expressed specifically in TAM clusters. Clinical records of 120 patients with lung adenocarcinoma stage I (n = 57), II (n = 21), and III (n = 42) were retrieved from Tsukuba Human Tissue Biobank Center (THB) in the University of Tsukuba Hospital, Japan. Tumor tissues from these patients were extracted and stained with anti-human MAFB antibody, and then MAFB-positive cells relative to the tissue area (MAFB+ cells/tissue area) were morphometrically quantified. Our results indicated that higher numbers of MAFB+ cells significantly correlated to increased local lymph node metastasis (nodal involvement), high recurrence rate, poor pathological stage, increased lymphatic permeation, higher vascular invasion, and pleural infiltration. Moreover, increased amounts of MAFB+ cells were related to poor overall survival and disease-free survival, especially in smokers. These data indicate that MAFB may be a suitable prognostic biomarker for smoker lung cancer patients. [ABSTRACT FROM AUTHOR]