Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth

During the symbiotic interaction between legumes and rhizobia, the host cell plasma membrane and associated plant cell wall invaginate to form a tunnel-like infection thread, a structure in which bacteria divide to reach the plant root cortex. We isolated four Lotus japonicus mutants that make infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itdl, itd3, and itd4 mutations identified novel loci. Bacterial release into host cells did occur occasionally in the itdl, itd2, and itd3 mutants suggesting that some infections may succeed after a long period and that infection of nodule cells could occur normally if the few abnormal infection threads that were formed reached the appropriate nodule cells.     

New details from the complete life cycle of the red-tide dinoflagellate Noctiluca scintillans (Ehrenberg) McCartney

Noctilucid protozoans are among the dinoflagellates that cause red tides. Sexual reproduction may occur in this group, as they sometimes undergo gametogenesis. However, the life cycle, in particular the developmental process after gamete fusion, has not been fully elucidated. We have been able to maintain clonal cultures of Noctiluca scintillans throughout the whole life cycle and have revealed new details of various stages. In trophont populations, a small fraction of cells spontaneously transform into gametogenic cells, which undergo two successive nuclear divisions, without cellular division, probably corresponding to meiosis. The products of nuclear division migrate to the cell surface with a small amount of cytoplasm, and there further synchronously divide 6-8 times, during which the division products are connected by thin cytoplasmic bridges. Thus, numerous gametes with a semi-spindle body shape are released from the mother cell ghost. They retain two flagella that differ in length and motion, as is typical of dinoflagellates. The presence of longitudinal and transverse grooves indicates that dinoflagellate-like characteristics are conserved in the gametes, although they are not present in the specialized trophonts. Zygotes with four flagella result from the fusion of two isogametes. The zygotes change shape from spindle to spherical, with a reduction in flagellar number. The developing cell acquires a tentacle and crust, similar to large trophonts, and begins to develop a cytoplasmic network, thus completing the transformation into a miniscule trophont. These early trophonts grow to maturity as cell size increases. Our observations of the life cycle of N. scintillans may provide clues for understanding the evolutionary origin of noctilucae.     

4-Aminophenoxyacetic acids as a novel class of reversible cathepsin K inhibitors

We have designed and synthesized a novel series of 3-biphenylamino acid amides as cathepsin K inhibitors based on compound I. In these inhibitors, we have discovered 4-aminophenoxyacetic acids 43 and 47 with good IC(50) values, although lipophilic groups are favorable for the hydrophobic S1' pocket.     

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,ox)) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.     

Mechanism of docosahexaenoic acid-induced inhibition of in vitro Aβ1–42 fibrillation and Aβ1–42-induced toxicity in SH-S5Y5 cells

The mechanism of the effect of docosahexaenoic acid (DHA; C22:6, n -3), one of the essential brain nutrients, on in vitro fibrillation of amyloid β (Aβ_1–42), Aβ_1–42-oligomers and its toxicity imparted to SH-S5Y5 cells was studied with the use of thioflavin T fluorospectroscopy, laser confocal microfluorescence, and transmission electron microscopy. The results clearly indicated that DHA inhibited Aβ_1–42-fibrill formation with a concomitant reduction in the levels of soluble Aβ_1–42 oligomers. The polymerization (into fibrils) of preformed oligomers treated with DHA was inhibited, indicating that DHA not only obstructs their formation but also inhibits their transformation into fibrils. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12.5%), Tris–Tricine gradient(4–20%) gel electrophoresis and western blot analyses revealed that DHA inhibited at least 2 species of Aβ_1–42 oligomers of 15–20 kDa, indicating that it hinders these on-pathway tri/tetrameric intermediates during fibrillation. DHA also reduced the levels of dityrosine and tyrosine intrinsic fluorescence intensity, indicating DHA interrupts the microenvironment of tyrosine in the Aβ_1–42 backbone. Furthermore, DHA protected the tyrosine from acrylamide collisional quenching, as indicated by decreases in Stern–Volmer constants. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction efficiency and immunohistochemical examination suggested that DHA inhibits Aβ_1–42-induced toxicity in SH-S5Y5 cells. Taken together, these data suggest that by restraining Aβ_1–42 toxic tri/tetrameric oligomers, DHA may limit amyloidogenic neurodegenerative diseases, Alzheimer's disease.     

Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism

We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.Hepatitis C virus (HCV) core protein, whose amino acid sequence is highly conserved among different HCV strains, not only is involved in the formation of the HCV virion but also has a number of regulatory functions, including modulation of signaling pathways, cellular and viral gene expression, cell transformation, apoptosis, and lipid metabolism (reviewed in references 9 and 15). We have previously reported that the E6AP E3 ubiquitin (Ub) ligase binds to the core protein and plays an important role in polyubiquitylation and proteasomal degradation of the core protein (22). Another study from our group identified the proteasome activator PA28γ/REG-γ as an HCV core-binding partner, demonstrating degradation of the core protein via a PA28γ-dependent pathway (16, 17). In this work, we further investigated the molecular mechanisms underlying proteasomal degradation of the core protein and found that in addition to regulation by the Ub-mediated pathway, the turnover of the core protein is also regulated by PA28γ in a Ub-independent manner.Although ubiquitylation of substrates generally requires at least one Lys residue to serve as a Ub acceptor site (5), there is no consensus as to the specificity of the Lys targeted by Ub (4, 8). To determine the sites of Ub conjugation in the core protein, we used site-directed mutagenesis to replace individual Lys residues or clusters of Lys residues with Arg residues in the N-terminal 152 amino acids (aa) of the core (C152), within which is contained all seven Lys residues (Fig. ​(Fig.1A).1A). Plasmids expressing a variety of mutated core proteins were generated by PCR and inserted into the pCAGGS (18). Each core-expressing construct was transfected into human embryonic kidney 293T cells along with the pMT107 (25) encoding a Ub moiety tagged with six His residues (His₆). Transfected cells were treated with the proteasome inhibitor MG132 for 14 h to maximize the level of Ub-conjugated core intermediates by blocking the proteasome pathway and were harvested 48 h posttransfection. His₆-tagged proteins were purified from the extracts by Ni²⁺-chelation chromatography. Eluted protein and whole lysates of transfected cells before purification were analyzed by Western blotting using anticore antibodies (Fig. ​(Fig.1B).1B). Mutations replacing one or two Lys residues with Arg in the core protein did not affect the efficiency of ubiquitylation: detection of multiple Ub-conjugated core intermediates was observed in the mutant core proteins comparable to the results seen with the wild-type core protein as previously reported (23). In contrast, a substitution of four N-terminal Lys residues (C152K6-23R) caused a significant reduction in ubiquitylation (Fig. ​(Fig.1B,1B, lane 9). Multiple Ub-conjugated core intermediates were not detected in the Lys-less mutant (C152KR), in which all seven Lys residues were replaced with Arg (Fig. ​(Fig.1B,1B, lane 11). These results suggest that there is not a particular Lys residue in the core protein to act as the Ub acceptor but that more than one Lys located in its N-terminal region can serve as the preferential ubiquitylation site. In rare cases, Ub is known to be conjugated to the N terminus of proteins; however, these results indicate that this does not occur within the core protein.Open in a separate windowFIG. 1.In vivo ubiquitylation of HCV core protein. (A) The HCV core protein (N-terminal 152 aa) is represented on the top. The positions of the amino acid residues of the core protein are indicated above the bold lines. The positions of the seven Lys residues in the core are marked by vertical ticks. Substitution of Lys with Arg (R) is schematically depicted. (B) Detection of ubiquitylated forms of the core proteins. The transfected cells with core expression plasmids and pMT107 were treated with the proteasome inhibitor MG132 and harvested 48 h after transfection. His₆-tagged proteins were purified and subsequently analyzed by Western blot analysis using anticore antibody (upper panel). Core proteins conjugated to a number of His₆-Ub are denoted with asterisks. Whole lysates of transfected cells before purification were also analyzed (lower panel). Lanes 1 to 11, C152 to C152KR, as indicated for panel A. Lane 12; empty vector.To investigate how polyubiquitylation correlates with proteasome degradation of the core protein, we performed kinetic analysis of the wild-type and mutated core proteins by use of the Ub protein reference (UPR) technique, which can compensate for data scatter of sample-to-sample variations such as levels of expression (10, 24). Fusion proteins expressed from UPR-based constructs (Fig. ​(Fig.2A)2A) were cotranslationally cleaved by deubiquitylating enzymes, thereby generating equimolar quantities of the core proteins and the reference protein, dihydrofolate reductase-hemagglutinin (DHFR-HA) tag-modified Ub, in which the Lys at aa 48 was replaced by Arg to prevent its polyubiquitylation (Ub^R48). After 24 h of transfection with UPR constructs, cells were treated with cycloheximide and the amounts of core proteins and DHFR-HA-Ub^R48 at the indicated time points were determined by Western blot analysis using anticore and anti-HA antibodies. The mature form of the core protein, aa 1 to 173 (C173) (13, 20), and C152 were degraded with first-order kinetics (Fig. 2B and D). MG132 completely blocked the degradation of C173 and C152 (Fig. ​(Fig.2B),2B), and C152K6-23R and C152KR were markedly stabilized (Fig. ​(Fig.2C).2C). The half-lives of C173 and C152 were calculated to be 5 to 6 h, whereas those of C152K6-23R and C152KR were calculated to be 22 to 24 h (Fig. ​(Fig.2D),2D), confirming that the Ub plays an important role in regulating degradation of the core protein. Nevertheless, these results also suggest possible involvement of the Ub-independent pathway in the turnover of the core protein, as C152KR is more destabilized than the reference protein (Fig. ​(Fig.2C2C and ​and2D2D).Open in a separate windowFIG. 2.Kinetic analysis of degradation of HCV core proteins. (A) The fusion constructs used in the UPR technique. Open boxes indicate the DHFR sequence, which is extended at the C terminus by a sequence containing the HA epitope (hatched boxes). Ub^R48 moieties bearing the Lys-Arg substitution at aa 48 are represented by open ellipses. Bold lines indicate the regions of the core protein. The amino acid positions of the core protein are indicated above the bold lines. The arrows indicate the sites of in vivo cleavage by deubiquitylating enzymes. (B and C) Turnover of the core proteins. After a 24-h transfection with each UPR construct, cells were treated with 50 μg of cycloheximide/ml in the presence or absence of 10 μM MG132 for the different time periods indicated. Cells were lysed at the different time points indicated, followed by evaluation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using antibodies against the core protein and HA. (D) Quantitation of the data shown in panels B and C. At each time point, the ratio of band intensity of the core protein relative to the reference DHFR-HA-Ub^R48 was determined by densitometry and is plotted as a percentage of the ratio at time zero.We have shown that PA28γ specifically binds to the core protein and is involved in its degradation (16, 17). Recent studies demonstrated that PA28γ is responsible for Ub-independent degradation of the steroid receptor coactivator SRC-3 and cell cycle inhibitors such as p21 (3, 11, 12). Thus, we next investigated the possibility of PA28γ involvement in the degradation of either C152KR or C152. Since C152KR carries two amino acid substitutions in the PA28γ-binding region (aa 44 to 71) (17), we tested the influence of the mutations of C152KR on the interaction with PA28γ by use of a coimmunoprecipitation assay. When Flag-tagged PA28γ (F-PA28γ) was expressed in cells along with C152 or C152KR, F-PA28γ precipitated along with both C152 and C152KR, indicating that PA28γ interacts with both core proteins (Fig. ​(Fig.3A).3A). Figure ​Figure3B3B reveals the effect of exogenous expression of F-PA28γ on the steady-state levels of C152 and C152KR. Consistent with previous data (17), the expression level of C152 was decreased to a nearly undetectable level in the presence of PA28γ (Fig. ​(Fig.3B,3B, lanes 1 and 3). Interestingly, exogenous expression of PA28γ led to a marked reduction in the amount of C152KR expressed (Fig. ​(Fig.3B,3B, lanes 5 and 7). Treatment with MG132 increased the steady-state level of the C152KR in the presence of F-PA28γ as well as the level of C152 (Fig. ​(Fig.3B,3B, lanes 4 and 8).Open in a separate windowFIG. 3.PA28γ-dependent degradation of the core protein. (A) Interaction of the core protein with PA28γ. Cells were cotransfected with the wild-type (C152) or Lys-less (C152KR) core expression plasmid in the presence of a Flag-PA28γ (F-PA28γ) expression plasmid or an empty vector. The transfected cells were treated with MG132. After 48 h, the cell lysates were immunoprecipitated with anti-Flag antibody and visualized by Western blotting with anticore antibodies. Western blot analysis of whole cell lysates was also performed. (B) Degradation of the wild-type and Lys-less core proteins via the PA28γ-dependent pathway. Cells were transfected with the UPR construct with or without F-PA28γ. In some cases, cells were treated with 10 μM MG132 for 14 h before harvesting. Western blot analysis was performed using anticore, anti-HA, and anti-Flag antibodies. (C) After 24 h of transfection with UPR-C152KR and UPR-C191KR with or without F-PA28γ (an empty vector), cells were treated with 50 μg of cycloheximide/ml for different time periods as indicated (chase time). Western blot analysis was performed using anticore and anti-HA antibodies. The precursor core protein and the core that was processed, presumably by signal peptide peptidase, are denoted by open and closed triangles, respectively.We further investigated whether PA28γ affects the turnover of Lys-less core protein through time course experiments. C152KR was rapidly destabilized and almost completely degraded in a 3-h chase experiment using cells overexpressing F-PA28γ (Fig. ​(Fig.3C,3C, left panels). A similar result was obtained using an analogous Lys-less mutant of the full-length core protein C191KR (Fig. ​(Fig.3C,3C, right panels), thus demonstrating that the Lys-less core protein undergoes proteasomal degradation in a PA28γ-dependent manner. These results suggest that PA28γ may play a role in accelerating the turnover of the HCV core protein that is independent of ubiquitylation.Finally, we examined gain- and loss-of-function of PA28γ with respect to degradation of full-length wild-type (C191) and mutated (C191KR) core proteins in human hepatoma Huh-7 cells. As expected, exogenous expression of PA28γ or E6AP caused a decrease in the C191 steady-state levels (Fig. ​(Fig.4A).4A). In contrast, the C191KR level was decreased with expression of PA28γ but not of E6AP. We further used RNA interference to inhibit expression of PA28γ or E6AP. An increase in the abundance of C191KR was observed with PA28γ small interfering RNA (siRNA) but not with E6AP siRNA (Fig. ​(Fig.4B).4B). An increase in the C191 level caused by the activity of siRNA against PA28γ or E6AP was confirmed as well.Open in a separate windowFIG. 4.Ub-dependent and Ub-independent degradation of the full-length core protein in hepatic cells. (A) Huh-7 cells were cotransfected with plasmids for the full-length core protein (C191) or its Lys-less mutant (C191KR) in the presence of F-PA28γ or HA-tagged-E6AP expression plasmid (HA-E6AP). After 48 h, cells were lysed and Western blot analysis was performed using anticore, anti-HA, anti-Flag, or anti-GAPDH. (B) Huh-7 cells were cotransfected with core expression plasmids along with siRNA against PA28γ or E6AP or with negative control siRNA. Cells were harvested 72 h after transfection and subjected to Western blot analysis.Taking these results together, we conclude that turnover of the core protein is regulated by both Ub-dependent and Ub-independent pathways and that PA28γ is possibly involved in Ub-independent proteasomal degradation of the core protein. PA28 is known to specifically bind and activate the 20S proteasome (19). Thus, PA28γ may function by facilitating the delivery of the core protein to the proteasome in a Ub-independent manner.Accumulating evidence suggests the existence of proteasome-dependent but Ub-independent pathways for protein degradation, and several important molecules, such as p53, p73, Rb, SRC-3, and the hepatitis B virus X protein, have two distinct degradation pathways that function in a Ub-dependent and Ub-independent manner (1, 2, 6, 7, 14, 21, 27). Recently, critical roles for PA28γ in the Ub-independent pathway have been demonstrated; SRC-3 and p21 can be recognized by the 20S proteasome independently of ubiquitylation through their interaction with PA28γ (3, 11, 12). It has also been reported that phosphorylation-dependent ubiquitylation mediated by GSK3 and SCF is important for SRC-3 turnover (26). Nevertheless, the precise mechanisms underlying turnover of most of the proteasome substrates that are regulated in both Ub-dependent and Ub-independent manners are not well understood. To our knowledge, the HCV core protein is the first viral protein studied that has led to identification of key cellular factors responsible for proteasomal degradation via dual distinct mechanisms. Although the question remains whether there is a physiological significance of the Ub-dependent and Ub-independent degradation of the core protein, it is reasonable to consider that tight control over cellular levels of the core protein, which is multifunctional and essential for viral replication, maturation, and pathogenesis, may play an important role in representing the potential for its functional activity.     

Interactive effects of synthetic nitrogen fertilizer and composted manure on ammonia volatilization from soils

The mutualism between legumes and nitrogen-fixing soil bacteria (rhizobia) is a key feature of many ecological and agricultural systems, yet little is known about how this relationship affects aboveground interactions between plants and herbivores. We investigated the effects of the rhizobia mutualism on the abundance of a specialized legume herbivore on soybean plants. In a field experiment, soybean aphid (Aphis glycines) abundances were measured on plants (Glycine max) that were either (1) treated with a commercial rhizobial inoculant, (2) associating solely with naturally occurring rhizobia, or (3) given nitrogen fertilizer. Plants associating with naturally occurring rhizobia strains exhibited lower aphid population densities compared to those inoculated with a commercial rhizobial preparation or given nitrogen fertilizer. Genetic analyses of rhizobia isolates cultured from field plants revealed that the commercial rhizobia strains were phylogenetically distinct from naturally occurring strains. Plant size, leaf nitrogen concentration, and nodulation density were similar among rhizobia-associated treatments and did not explain the observed differences in aphid abundance. Our results demonstrate that plant–rhizobia interactions influence plant resistance to insect herbivores and that some rhizobia strains confer greater resistance to their mutualist partners than do others.