Tadashi Matsui

Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site

A mutant α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 μM; Km(2), 135 μM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2′,3′-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below ~300 μM.

THERMOPHILIC F1-ATPASE IS ACTIVATED WITHOUT DISSOCIATION OF AN ENDOGENOUS INHIBITOR, EPSILON SUBUNIT

Subunit complexes (α3β3γ, α3β3γδ, α3β3γε, and α3β3γδε) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of δ and ε subunits in catalysis. The presence of δ subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of ε subunit was more profound. The (−ε) complexes, α3β3γ and α3β3γδ, initiated ATP hydrolysis without a lag. In contrast, the (+ε) complexes, α3β3γε and α3β3γδε, started hydrolysis of ATP (<700 μm) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+ε) complexes became shorter, and it was not observed above 1 mm ATP. Analysis of binding and hydrolysis of the ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+ε) complexes bound substrate only slowly. Differing fromEscherichia coli F1-ATPase, the activation of the (+ε) complexes from the lag phase was not due to dissociation of ε subunit since the re-isolated activated complex retained ε subunit. This indicates that there are two alternative forms of the (+ε) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

Structural Asymmetry of F1-ATPase Caused by the γ Subunit Generates a High Affinity Nucleotide Binding Site

The α3β3γ and α3β3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the α3β3 complex. The results of equilibrium dialysis show that the α3β3γ complex has a high affinity nucleotide binding site and several low affinity sites, whereas the α3β3 complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the β subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the α3β3γ complex is accelerated by the chase addition of excess ATP, whereas that by the α3β3 complex is not. We further examined the complexes containing mutant β subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant β subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K., Yutani, K., and Yoshida, M.(1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the β subunit in the mutant α3β3γ complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant α3β3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the γ subunit is responsible for the generation of a high affinity nucleotide binding site on the β subunit in F1-ATPase where cooperative catalysis can proceed.

The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

The hydrolytic properties of the mutant α3(βT165S)3γ and wild-type α3β3γ subcomplexes of TF1 have been compared. Whereas the wild-type complex hydrolyzes 50 μM ATP in three kinetic phases, the mutant complex hydrolyzes 50 μM ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg2+, the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0.1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnover-dependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg2+ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg2+. A Lineweaver-Burk plot constructed from rates of hydrolysis of 20-2000 μM ATP by the wild-type complex is biphasic, exhibiting apparent Km values of 30 μM and 470 μM with corresponding kcat values of 26 and 77 s−1. In contrast, a Lineweaver-Burk plot for the mutant complex is linear in this range of ATP concentration, displaying a Km of 133 μM and a kcat of 360 s−1.

DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.

RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).

53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis.

Ionizing radiation (IR) induces a variety of DNA lesions. The most significant lesion is a DNA double‐strand break (DSB), which is repaired by homologous recombination or nonhomologous end joining (NHEJ) pathway. Since we previously demonstrated that IR‐responsive protein 53BP1 specifically enhances activity of DNA ligase IV, a DNA ligase required for NHEJ, we investigated responses of 53BP1‐deficient chicken DT40 cells to IR. 53BP1‐deficient cells showed increased sensitivity to X‐rays during G1 phase. Although intra‐S and G2/M checkpoints were intact, the frequency of isochromatid‐type chromosomal aberrations was elevated after irradiation in 53BP1‐deficient cells. Furthermore, the disappearance of X‐ray‐induced γ‐H2AX foci, a marker of DNA DSBs, was prolonged in 53BP1‐deficient cells. Thus, the elevated X‐ray sensitivity in G1 phase cells was attributable to repair defect for IR‐induced DNA‐damage. Epistasis analysis revealed that 53BP1 plays a role in a pathway distinct from the Ku‐dependent and Artemis‐dependent NHEJ pathways, but requires DNA ligase IV. Strikingly, disruption of the 53BP1 gene together with inhibition of phosphatidylinositol 3‐kinase family by wortmannin completely abolished colony formation by cells irradiated during G1 phase. These results demonstrate that the 53BP1‐dependent repair pathway is important for survival of cells irradiated with IR during the G1 phase of the cell cycle.

Knockdown of stromal interaction molecule 1 (STIM1) suppresses store-operated calcium entry, cell proliferation and tumorigenicity in human epidermoid carcinoma A431 cells

Store-operated calcium (Ca(2+)) entry (SOCE) is important for cellular activities such as gene transcription, cell cycle progression and proliferation in most non-excitable cells. Stromal interaction molecule 1 (STIM1), a newly identified Ca(2+)-sensing protein, monitors the depletion of endoplasmic reticulum (ER) Ca(2+) stores and activates store-operated Ca(2+) channels at the plasma membrane to induce SOCE. To investigate the possible roles of STIM1 in tumor growth in relation to SOCE, we established STIM1 knockdown (KD) clones of human epidermoid carcinoma A431 cells by RNA interference. Thapsigargin, an inhibitor of ER Ca(2+)-ATPase, -induced and phospholipase C-coupled receptor agonist-induced SOCEs were reduced in two STIM1 KD clones compared to a negative control clone. Re-expression of a KD-resistant full-length STIM1, but not a Ca(2+) release-activated Ca(2+) channel activation domain (CAD)-deleted STIM1 mutant, in the KD clone restored the amplitude of SOCE, suggesting the specificity of the STIM1 knockdown. The cell growth of the STIM1 KD clones was slower than that of the negative control clone. DNA synthesis assessed by BrdU incorporation, as well as EGF-stimulated EGF receptor activation, decreased in the STIM1 KD clones. Xenograft growth of the STIM1 KD clones was significantly retarded compared with that of the negative control. Cell migration was attenuated in the STIM1 KD clone and the STIM1 silencing effect was reversed by transient re-expression of the full-length STIM1 but not CAD-deletion mutant. These results indicate that STIM1 plays an important role in SOCE, cell-growth and tumorigenicity in human epidermoid carcinoma A431cells, suggesting the potential use of STIM1-targeting agents for treating epidermoid carcinoma.

PKU-β/TLK1 regulates myosin II activities, and is required for accurate equaled chromosome segregation

Tousled-like kinase 1 (or protein kinase ubiquitous, PKU-beta/TLK1) is a serine/threonine protein kinase that is implicated in chromatin remodeling, DNA replication and mitosis. RNAi-mediated PKU-beta/TLK1-depleted human cells showed aneuploidy, and immunofluorescence analysis of these cells revealed the unequal segregation of daughter chromosomes. Immunoblots indicated a substantial reduction in the phosphorylation level of Ser19/Thr18 on the myosin II regulatory light chain (MRLC) in PKU-beta/TLK1-depleted cells, with no change in total MRLC protein. To confirm the relationship between mitotic aberration and MRLC dysfunction, we expressed wild type MRLC or DD-MRLC (mimics diphosphorylation; substitution of both Thr18 and Ser19 with aspartate) in PKU-beta/TLK1-depleted cells. DD-MRLC expression dramatically reduced the unequal segregation of chromosomes. Our data suggest that human PKU-beta/TLK1 plays an important role in chromosome integrity via the regulation of myosin II dynamics by phosphorylating MRLC during mitosis.

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase β subunit determined by 13C-NMR

Glutamic acid‐190 in the β subunit of F1‐ATPase from thermophilic Bacillus PS‐3 (TF1) was reporte to be essential for the ATPase activity. The mutant TF1β subunit in which Glu‐190 had been substituted by cysteine was carboxymethylated with 13C‐labeled monoiodoacetic acid. The pK a value of the carboxymethylene group at the 190 position was determined as 5.6 ± 0.4 by 13C‐NMR. On the basis of this value, the pK a of the carboxylate of Glu‐190 of the TF1β subunit was estimated to be 6.8 ± 0.5. The unusually high pK a could play a role in the catalytic mechanism of F1‐ATPase.

Characterization of a cancer cell line that expresses a splicing variant form of 53BP1: Separation of checkpoint and repair functions in 53BP1

53BP1 plays important roles in checkpoint signaling and repair for DNA double-strand breaks. We found that a colon cancer cell line, SW48, expressed a splicing variant form of 53BP1, which lacks the residues corresponding to exons 10 and 11. Activation of ATM and phosphorylation of ATM and ATR targets occurred in SW48 cells in response to X-irradiation, and these X-ray-induced responses were not enhanced by expression of full-length 53BP1 in SW48 cells, indicating that this splicing variant fully activates the major checkpoint signaling in SW48 cells. In contrast, the expression of full-length 53BP1 in SW48 cells promoted the repair of X-ray-induced DNA damage, evidenced by faster disappearance of X-ray-induced gamma-H2AX foci, a marker for DNA damage, and less residual chromosomal aberrations after X-irradiation. We conclude that the two major roles of 53BP1, the checkpoint signaling and repair for DNA damage, can be functionally separated.