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Aims: This open-label, phase I study evaluated the pharmacokinetics and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) for the treatment of chemotherapy-induced neutropenia in children with acute leukaemia., Methods: PEG-rhG-CSF was administered as a single 100 mcg/kg (3 mg maximum dose) subcutaneous injection at the end of each chemotherapy period when neutropenia occurred. Blood samples were obtained from patients treated with PEG-rhG-CSF. PEG-rhG-CSF serum concentrations were determined by an enzyme-linked immunosorbent assay. Population pharmacokinetic (PPK) analysis was implemented using the nonlinear mixed-effects model. Short-term safety was evaluated through adverse events collection (registered at clinicaltrials.gov identifier: 03844360)., Results: A total of 16 acute leukaemia patients (1.8-13.6 years) were included, of whom two (12.5%) had grade 3 neutropenia, six (37.5%) had grade 4 neutropenia, and eight (50.0%) had severe neutropenia. For PPK modelling, 64 PEG-rhG-CSF serum concentrations were obtainable. A one-compartment model with first-order elimination was used for pharmacokinetic data modelling. The current weight was a significant covariate. The median (range) of clearance (CL) and area under the serum concentration-time curve (AUC) were 5.65 (1.49-14.45) mL/h/kg and 16514.75 (6632.45-54423.30) ng·h/mL, respectively. Bone pain, pyrexia, anaphylaxis and nephrotoxicity were not observed. One patient died 13 days after administration, and the objective assessment of causality was that an association with PEG-rhG-CSF was "possible"., Conclusions: The AUC of PEG-rhG-CSF (100 mcg/kg, 3 mg maximum dose) in paediatric patients with acute leukaemia were similar to those of PEG-rhG-CSF (100 mcg/kg) in children with sarcoma. PEG-rhG-CSF is safe, representing an important therapeutic option for chemotherapy-induced neutropenia in paediatric patients with acute leukaemia., (© 2021 British Pharmacological Society.)

MicroRNAs and Yes-associated protein (YAP) play an important role in the occurrence and development of hepatic carcinomas. However, the interaction between microRNAs and YAP was seldom elucidated. In this study, we showed that miR-132 could target YAP gene by using dual-luciferase reporter system. Further quantitative polymerase chain reaction analysis and western blotting showed that miR-132 could significantly reduce the expression of YAP at mRNA and protein levels. Results of annexin V-fluorescein isothiocyanate, 5-ethynyl-2'-deoxyuridine staining and transwell assays showed that miR-132 significantly promoted the cell apoptosis and effectively inhibited the proliferation and invasion of hepatoma cells. These results indicated that miR-132 could inhibit the growth of hepatoma cells by targeting YAP gene and reducing its expression level. Taken together, results from this study would help us to understand the mechanisms for occurrence and development of hepatic carcinoma and provide new targets for diagnosis and treatment of liver cancer., (Copyright © 2015 John Wiley & Sons, Ltd.)

LytA is responsible for the autolysis of many Streptococcus species, including pathogens such as S. pneumoniae, S. pseudopneumoniae and S. mitis. However, how this major autolysin achieves full activity remains unknown. Here, the full-length structure of the S. pneumoniae LytA dimer is reported at 2.1 Å resolution. Each subunit has an N-terminal amidase domain and a C-terminal choline-binding domain consisting of six choline-binding repeats, which form five canonical and one single-layered choline-binding sites. Site-directed mutageneses combined with enzymatic activity assays indicate that dimerization and binding to choline are two independent requirements for the autolytic activity of LytA in vivo. Altogether, it is suggested that dimerization and full occupancy of all choline-binding sites through binding to choline-containing TA chains enable LytA to adopt a fully active conformation which allows the amidase domain to cleave two lactyl-amide bonds located about 103 Å apart on the peptidoglycan.

Microcystins, which are the most common cause of hepatotoxicity associated with cyanobacterial water blooms, are assembled in vivo on a large multienzyme complex via a mixed nonribosomal peptide synthetase/polyketide synthetase (NRPS/PKS). The biosynthesis of microcystin in Microcystis aeruginosa PCC 7806 starts with the enzyme McyG, which contains an adenylation-peptidyl carrier protein (A-PCP) didomain for loading the starter unit to assemble the side chain of an Adda residue. However, the catalytic mechanism remains unclear. Here, the 2.45 Å resolution crystal structure of the McyG A-PCP didomain complexed with the catalytic intermediate L-phenylalanyl-adenylate (L-Phe-AMP) is reported. Each asymmetric unit contains two protein molecules, one of which consists of the A-PCP didomain and the other of which comprises only the A domain. Structural analyses suggest that Val227 is likely to be critical for the selection of hydrophobic substrates. Moreover, two distinct interfaces demonstrating variable crosstalk between the PCP domain and the A domain were observed. A catalytic cycle for the adenylation and peptide transfer of the A-PCP didomain is proposed.

The L,D-carboxypeptidase DacB plays a key role in the remodelling of Streptococcus pneumoniae peptidoglycan during cell division. In order to decipher its substrate-binding properties and catalytic mechanism, the 1.71 Å resolution crystal structure of DacB from S. pneumoniae TIGR4 is reported. Structural analyses in combination with comparisons with the recently reported structures of DacB from S. pneumoniae D39 and R6 clearly demonstrate that DacB adopts a zinc-dependent carboxypeptidase fold and belongs to the metallopeptidase M15B subfamily. In addition, enzymatic activity assays further confirm that DacB indeed acts as an L,D-carboxypeptidase towards the tetrapeptide L-Ala-D-iGln-L-Lys-D-Ala of the peptidoglycan stem, with Km and kcat values of 2.84 ± 0.37 mM and 91.49 ± 0.05 s(-1), respectively. Subsequent molecular docking and site-directed mutagenesis enable the assignment of the key residues that bind to the tetrapeptide. Altogether, these findings provide structural insights into substrate recognition in the metallopeptidase M15B subfamily.

Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8-14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF from Microcystis aeruginosa PCC 7806 is reported at 2.7 Å resolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an α+β class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each other via interactions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.

Saccharomyces cerevisiae Coq5 is an S-adenosyl methionine (SAM)-dependent methyltransferase (SAM-MTase) that catalyzes the only C-methylation step in the coenzyme Q (CoQ) biosynthesis pathway, in which 2-methoxy-6-polyprenyl-1,4-benzoquinone (DDMQH2) is converted to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (DMQH2). Crystal structures of Coq5 were determined in the apo form (Coq5-apo) at 2.2 Å resolution and in the SAM-bound form (Coq5-SAM) at 2.4 Å resolution, representing the first pair of structures for the yeast CoQ biosynthetic enzymes. Coq5 displays a typical class I SAM-MTase structure with two minor variations beyond the core domain, both of which are considered to participate in dimerization and/or substrate recognition. Slight conformational changes at the active-site pocket were observed upon binding of SAM. Structure-based computational simulation using an analogue of DDMQH2 enabled us to identify the binding pocket and entrance tunnel of the substrate. Multiple-sequence alignment showed that the residues contributing to the dimeric interface and the SAM- and DDMQH2-binding sites are highly conserved in Coq5 and homologues from diverse species. A putative catalytic mechanism of Coq5 was proposed in which Arg201 acts as a general base to initiate catalysis with the help of a water molecule.

Grx5 from the yeast Saccharomyces cerevisiae is a monothiol glutaredoxin that is involved in iron-sulfur cluster biogenesis. Here, yeast Grx5 was cloned and overproduced in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion method. Diffraction data for Grx5 were collected to 1.67 A resolution. The crystal of Grx5 belonged to space group R3, with unit-cell parameters a = b = 85.12, c = 48.95 A, alpha = beta = 90.00, gamma = 120.00 degrees .

Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 A resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence.

Thioredoxins (Trxs) are a family of small redox-active proteins that are found in all living organisms. In Saccharomyces cerevisiae, two cytosolic Trxs (Trx1 and Trx2) and one mitochondrial Trx (Trx3) have previously been identified. In this work, cytosolic Trx1 containing a C33S mutant was overexpressed, purified, glutathionylated and crystallized using the hanging-drop vapour-diffusion method. A set of X-ray diffraction data was collected to 1.80 A resolution. The crystal belonged to space group P1, with unit-cell parameters a = 38.53, b = 38.81, c = 41.70 A, alpha = 72.91, beta = 87.51, gamma = 60.58 degrees.

A non-Pfam hypothetical protein SCO4226 of molecular weight 9 kDa from Streptomyces coelicolor A3(2) was overexpressed in Escherichia coli and the purified recombinant protein was crystallized using the sitting-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.0 A resolution. The crystal belonged to space group P2(1), with unit-cell parameters a = 29.67, b = 67.00, c = 34.43 A, alpha = gamma = 90.00, beta = 94.26 degrees .

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of the gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates. In addition to biochemical studies, a number of crystal structures of NDPK from various organisms, including both native proteins and complexes with nucleotides or nucleotide analogues, have been determined. Here, the crystal structure of Ynk1, an NDPK from the yeast Saccharomyces cerevisiae, has been solved at 3.1 A resolution. Structural analysis strongly supports the oligomerization state of this protein being hexameric rather than tetrameric.

Thioredoxins play key roles in the cellular response to oxidative stress. Three isoforms of thioredoxin have been identified in Saccharomyces cerevisiae: two that are cytosolic (Trx1 and Trx2) and one that is mitochondrial (Trx3). In the present work, the cytosolic form Trx1 was cloned, expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapour-diffusion method. A data set was collected from a single crystal to 1.7 A resolution. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.29, b = 46.59, c = 64.20 A, alpha = beta = gamma = 90 degrees .

The heat-shock protein Hsp33 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. A crystal was obtained using the hanging-drop vapour-diffusion method and a data set was collected to 2.7 A resolution. The crystal belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 96.43, c = 132.22 A, alpha = beta = gamma = 90 degrees . The asymmetric unit is assumed to contain two subunits of Hsp33, with a V(M) value of 2.96 A(3) Da(-1) and a solvent content of 58.41%.

There are three thioredoxin isoforms in the yeast Saccharomyces cerevisiae: two cytosolic/nuclear thioredoxins, Trx1 and Trx2, and one mitochondrial thioredoxin, Trx3. In the present work, S. cerevisiae Trx3 overexpressed in Escherichia coli was purified and crystallized. The Trx3 crystals were obtained by the hanging-drop vapour-diffusion method. A data set diffracting to 2.0 A resolution was collected from a single crystal. The crystal belongs to space group P3(1), with unit-cell parameters a = b = 49.57, c = 94.55 A, alpha = beta = 90, gamma = 120 degrees. The asymmetric unit is assumed to contain two subunits of Trx3, with a V(M) value of 2.62 A(3) Da(-1) and a solvent content of 53%.

The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml(-1), whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 A resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 A, alpha = 71.405, beta = 73.376, gamma = 89.633 degrees. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.

Tumor necrosis factor receptors (TNFR) signal events in immune responses, Ig class switching, activation of NF-kappaB or regulation of apoptosis. TNFR-associated factors (TRAFs) are adaptor proteins that connect TNFRs to downstream signaling pathways, including the NF-kappaB and c-JUN N-terminal kinase (JNK) pathways. Members of the TRAF family exist as trimers and share a conserved TRAF domain that mediates binding to the cytoplasmic domains of TNFRs. The TRAF domain from TRAF3 has been crystallized. In addition, an N-terminally truncated form of the domain has been crystallized in space group P321 with a shortened c axis and markedly improved diffraction (2.5 A resolution).