Crystal structural characterization reveals novel oligomeric interactions of human voltage‐dependent anion channel 1

Voltage‐dependent anion channel 1 (VDAC1), which is located in the outer mitochondrial membrane, plays important roles in various cellular processes. For example, oligomerization of VDAC1 is involved in the release of cytochrome c to the cytoplasm, leading to apoptosis. However, it is unknown how VDAC1 oligomerization occurs in the membrane. In the present study, we determined high‐resolution crystal structures of oligomeric human VDAC1 (hVDAC1) prepared by using an Escherichia coli cell‐free protein synthesis system, which avoided the need for denaturation and refolding of the protein. Broad‐range screening using a bicelle crystallization method produced crystals in space groups C222 and P22₁2₁, which diffracted to a resolution of 3.10 and 3.15 Å, respectively. Each crystal contained two hVDAC1 protomers in the asymmetric unit. Dimer within the asymmetrical unit of the crystal in space group C222 were oriented parallel, whereas those of the crystal in space group P22₁2₁ were oriented anti‐parallel. From a model of the crystal in space group C222, which we constructed by using crystal symmetry operators, a heptameric structure with eight patterns of interaction between protomers, including hydrophobic interactions with β‐strands, hydrophilic interactions with loop regions, and protein–lipid interactions, was observed. It is possible that by having multiple patterns of interaction, VDAC1 can form homo‐ or hetero‐oligomers not only with other VDAC1 protomers but also with other proteins such as VDAC2, VDAC3 and apoptosis‐regulating proteins in the Bcl‐2 family.     

Syntheses and Configurational Analysis of Rotenoids,XIX The Total Synthesis of Natural Rotenone

d,l-Derrisic acid (IIa, R′: H) was synthesized from d,l-hydroxy dihydrotubanol (Ve) by Hoesch condensation with a nitrile (VI). The possible optical resolution of d,l-IIa was demonstrated by a conventional “reversed resolution” method.

This communication and previous works constitute the first total synthesis of natural rotenone (Ia).     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Expression and characterization of recombinant human antithrombin III in Pichia pastoris

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin.     

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.     

What are the post-ablation insular residual electrograms in the posterior left pulmonary veins electrically connected to?

A 67-year-old man underwent a third ablation procedure for a recurrent atrial tachycardia (AT) after an extensive pulmonary vein (PV) isolation, linear ablation along the left atrial (LA) roof and posterolateral mitral isthmus (MI), and defragmentation of persistent atrial fibrillation and an induced perimitral AT. High-resolution mapping during the clinical AT using the Rhythmia system (Boston Scientific) suggested that the AT was a ridge-related reentrant AT and exhibited a reconnection of the left PVs (LPVs). The residual electrograms in the posterior LPVs were surrounded by endocardial scar, which was like an island consisting of residual LPV electrograms. Retrograde venography of the vein of Marshall (VOM) demonstrated that the VOM reached the posterior left superior PV through the ridge between the LA appendage and left inferior PV and then the LPV carina. An ethanol infusion into the VOM resulted in a simultaneous AT termination and complete electrical isolation of the LPVs, that is, the disappearance of the residual LPV electrograms. The insular residual LPV electrograms in the present case did not appear to be endocardially connected to the LA, because the LPV electrograms were surrounded by endocardial scar and there was a large time gap between the earliest activation in the posterior LPVs and activation in the surrounding area. The VOM course on the venography and elimination of the residual LPV electrograms with an ethanol infusion into the VOM suggested that the insular residual LPV electrograms were electrically connected to the posterolateral LA via the VOM and its branches.     

Mechanism of How Salt-Gradient-Induced Charges Affect the Translocation of DNA Molecules through a Nanopore

Experiments using nanopores demonstrated that a salt gradient enhances the capture rate of DNA and reduces its translocation speed. These two effects can help to enable electrical DNA sequencing with nanopores. Here, we provide a quantitative theoretical evaluation that shows the positive net charges, which accumulate around the pore entrance due to the salt gradient, are responsible for the two observed effects: they reinforce the electric capture field, resulting in promoted molecule capture rate; and they induce cationic electroosmotic flow through the nanopore, thus significantly retarding the motion of the anionic DNA through the nanopore. Our multiphysical simulation results show that, during the polymer trapping stage, the former effect plays the major role, thus resulting in promoted DNA capture rate, while during the nanopore-penetrating stage the latter effect dominates and consequently reduces the DNA translocation speed significantly. Quantitative agreement with experimental results has been reached by further taking nanopore wall surface charges into account.