Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.     

Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.Bisphosphonates (BPs), synthetic analogues of pyrophosphates, are clinically in use for diseases with excessive bone absorption such as osteoporosis and malignancy-associated hypercalcemia. BPs administered in vivo are accumulated in the bone matrix and inhibit activities of osteoclasts.¹ The first generation of BPs, without nitrogen in the structure, is converted into cytotoxic non-hydrolyzable ATP analogues and achieves cytotoxic effects thorough decreased mitochondrial membrane potentials.^2,3 The second and the third generations, containing nitrogen, inhibit farnesyl pyrophosphate synthetase, a key enzyme in the mevalonate pathways, and deplete isoprenoid pools, which subsequently results in decreased prenylation of small guanine-nucleotide-binding regulatory proteins (small G proteins) (Supplementary Figure S1).⁴Isoprenoid lipids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are substrates for prenylation processes that mediate farnesylation and geranylgeranylation of small G proteins, respectively.^5,6 Ras family proteins are either farnesylated by farnsyl transferase or geranylgeranylated by geranylgeranyl transferase I. In contrast, the majority of Rho family proteins and Rab family proteins are geranylgeranylated by geranylgeranyl transferase I and II, respectively. These lipid modifications are essential for most of small G proteins to bind to cytoplasmic and organelle membranes where prenylated small G proteins become functional, whereas unprenylated small G proteins remain in the cytoplasm and non-functional.⁵The nitrogen-containing BPs (N-BPs) also induce cytotoxicity to osteoclasts, which is favorable for enhanced bone mineralization, and recent studies also showed that N-BPs had cytotoxic activities on tumors such as breast and prostate cancer.^7,8 These cytotoxic actions are attributable to a number of mechanisms including apoptosis induction and anti-angiogenesis,^9,10 but it is not well investigated as to which small G proteins produce the cytotoxic effects.We recently showed that zoledronic acid (ZOL), which is one of the N-BPs to inhibit farnesyl pyrophosphate synthetase, produced cytotoxic activities to human mesothelioma.¹¹ ZOL treatments induced apoptotic cell death or S-phase arrest in cell cycle, and moreover caused morphological changes from fibroblast-like to spherical shapes. In the present study, we examined what kinds of small G proteins are responsible to these ZOL-mediated effects using inhibitors or small interfering RNA (siRNA) for the respective small G proteins and for prenylating enzymes.     

Hydrogen Cyanide Production due to Mid-Size Impacts in a Redox-Neutral N2-Rich Atmosphere

Cyanide compounds are amongst the most important molecules of the origin of life. Here, we demonstrate the importance of mid-size (0.1–1 km in diameter) hence frequent meteoritic impacts to the cyanide inventory on the early Earth. Subsequent aerodynamic ablation and chemical reactions with the ambient atmosphere after oblique impacts were investigated by both impact and laser experiments. A polycarbonate projectile and graphite were used as laboratory analogs of meteoritic organic matter. Spectroscopic observations of impact-generated ablation vapors show that laser irradiation to graphite within an N₂-rich gas can produce a thermodynamic environment similar to that produced by oblique impacts. Thus, laser ablation was used to investigate the final chemical products after this aerodynamic process. We found that a significant fraction (>0.1 mol%) of the vaporized carbon is converted to HCN and cyanide condensates, even when the ambient gas contains as much as a few hundred mbar of CO₂. As such, the column density of cyanides after carbon-rich meteoritic impacts with diameters of 600 m would reach ~10 mol/m² over ~10² km² under early Earth conditions. Such a temporally and spatially concentrated supply of cyanides may have played an important role in the origin of life.     

Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

Structure and composition of riparian forests with special reference to geomorphic site conditions along the Tokachi River,northern Japan

The structure and composition of riparian forests were examined along the Tokachi River, northern Japan. Both the hydrogeomorphic gradient and the temporal gradient were analyzed in attempt to explain the present pattern of riparian forests. The stability of floodplain surfaces was estimated on the basis of the elevation above the riverbed and the distance from the river channel. The characteristics of the substratum on which trees were established were also examined by excavation of buried sediment. The results indicated that soil moisture and organic content increased while the size of particles in the substratum decreased with increased elevation and distance from the river channel. Gradient analysis was employed to examine the distribution of dominant species, such as Alnus hirsuta, Toisusu urbaniana, Populus maximowiczii, Picea jezoensis and Abies sachalinensis. The relative dominance of each could be arrayed across the elevation gradient. Although the three broad-leaved pioneers dominated bars and floodplains near the river channel, their modes shifted from lower to higher elevation and amplitudes of distribution curves decreased in the following order: A. hirsuta, T. urbaniana, P. maximowiczii and conifers, which were located on the highest floodplains. Sites could be divided into three classes in terms of stability. There were fewer species at active sites, which favored the three pioneer species, but species richness and diversity increased with stand age. Semi-active and stable sites were more diverse with the establishment of conifers and other broad-leaved trees, which included upland species. However, species richness peaked and then decreased after trees reached 50 to 60 years of age. The growth of dwarf bamboo and the development of conifer-dominant stands impeded the establishment of other species, thereby reducing species richness and diversity in mature stands. Chronologically, floodplains could be differentiated into high- and low-frequency zones of flood disturbance, with pioneer species occupying the former, and late successional species found largely in the latter.     

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.