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Sucrose solutions were prepared in 50 mM Tris–HCl (pH 8.0), 20 mM NaCl. Sucrose gradients from 50–5% were prepared on top of a 300 µl CsCl (1.4 g/ml) cushion. A total of 1 × 10 7 cells, without pre-treatment with cyclohexemide, were lysed in 1 ml lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, 0.1% NP-40, protease inhibitors] at 4°C for 5 min. A total of 20 µg of glass beads were added each and total cell lysates were sonificated 10 times for 1 s. A total of 100 µl of total cell lysate were loaded on top of the sucrose gradients. Subsequently, sucrose gradients were centrifuged at 30 000 r.p.m. for 2.5 h using a Ti60W rotor. Fractions of 200 µl each were collected. A total of 20 µl of each fraction were used for SDS–PAGE electrophoresis.
|
16738141_p14
|
16738141
|
Sucrose gradients
| 4.161634 |
biomedical
|
Study
|
[
0.9994658827781677,
0.00029833015287294984,
0.00023574144870508462
] |
[
0.9974019527435303,
0.0020740500185638666,
0.0004114661132916808,
0.00011246145004406571
] |
en
| 0.999996 |
The day before transfection, 5 × 10 4 to 10 5 U2OS cells were seeded in 6-well plates. A total of 5 µl of 20 µM control or Pes1-specific siRNA were diluted in 150 µl Optimem (Invitrogen). A total of 150 µl Optimem containing 5 µl Oligofectamine (Invitrogen) was added and incubated for 15 min. Finally, 600 µl Optimem was added and applied to cells after aspiration of the culture medium. Cells were incubated for 6 h. The following sequences (sense) were used: Pes1, CCAGAGGACCUAAGUGUGAdTdT; Control (nonspecific siRNA), UUCUCCGAACGUGUCACGUdTdT.
|
16738141_p15
|
16738141
|
siRNA transfection
| 4.115244 |
biomedical
|
Study
|
[
0.9988849759101868,
0.0007956072222441435,
0.0003194962628185749
] |
[
0.9583994746208191,
0.04014439508318901,
0.0009387125610373914,
0.0005174056859686971
] |
en
| 0.999996 |
Cells were directly lysed with 2× SDS-loading buffer (100 mM Tris–HCl, 200 mM DTE, 4% SDS, 10 mM EDTA, 0.2% bromphenol blue, 20% glycerol). Cell lysates were separated by SDS–PAGE and blotted on nitrocellulose membranes (GE Healthcare). Equal loading of samples was controlled by Ponceau S staining. Immunodetection was performed with anti-HA (3F10; Roche), anti-WDR12 (1B8), anti-Bop1 (6H11), anti-Pes1 (8E9) [Holzel et al . ( 14 )], and anti-p53 (Pab-240; Dianova) antibodies.
|
16738141_p16
|
16738141
|
Immunoblotting and immunofluorescence
| 4.11488 |
biomedical
|
Study
|
[
0.9995835423469543,
0.00019934713782276958,
0.00021709433349315077
] |
[
0.9961362481117249,
0.003082440234720707,
0.0006635444588027894,
0.00011774573067668825
] |
en
| 0.999997 |
For immunofluorescence, cells were grown on cover slides, fixed with ice cold methanol or methanol–aceton 1:1 and air dried. Unspecific binding was blocked with PBS/10% FBS. p53 and HA-tagged Pes1 were detected with a 1:100 dilution of anti-p53 antibody (Pab122; Dianova) and a 1:1000 dilution of 3F10 anti-HA antibody, respectively. Primary antibodies were incubated overnight at 4°C in a humidified chamber. Cy3 labelled secondary antibodies (Dianova) were incubated for 1 h at RT. Nuclei were counterstained with DAPI (Sigma–Aldrich). Digital images were acquired using the Openlab acquisition software (Improvision) and a microscope (model Axiovert 200 M; Carl Zeiss MicroImaging, Inc.) with 63 (1.15) and 100 (1.30) plan oil objectives connected to a five charge-coupled device camera (model ORCA- 479; Hamamatsu).
|
16738141_p17
|
16738141
|
Immunoblotting and immunofluorescence
| 4.135786 |
biomedical
|
Study
|
[
0.9993348717689514,
0.0003998902393504977,
0.00026517859078012407
] |
[
0.9849094748497009,
0.014051171019673347,
0.0007627694867551327,
0.0002765868848655373
] |
en
| 0.999997 |
For immunoprecipitations, U2OS cells were seeded at subconfluent density and treated with 1 µg/ml doxycycline for 20 h. Cells were harvested by trypsination and washing three times with PBS. For lysis, cells were resuspended in lysis buffer [50 mM Tris–HCl (pH 8.0), 1% NP-40, 150 mM NaCl, protease inhibitors, phosphatase inhibitors] and incubated on ice for 20 min. After centrifugation, protein G beads (incubated with antibodies for at least 1 h, washed twice after incubation) were incubated with the lysate over night at 4°C. The beads were washed three times with lysis buffer (lacking phosphatase- and protease inhibitor) and resuspended in SDS-loading buffer/lysis buffer 1:1 with the volume equal to the volume of cell lysate used for the IP.
|
16738141_p18
|
16738141
|
Immunoprecipitation
| 4.134936 |
biomedical
|
Study
|
[
0.9993391633033752,
0.00040085529326461256,
0.0002599505241960287
] |
[
0.9749042987823486,
0.023808717727661133,
0.0009463236201554537,
0.00034060305915772915
] |
en
| 0.999997 |
The Pes1 protein is highly conserved in eukaryotes and several domains and motifs have been predicted based on sequence analysis . Pes1 contains an evolutionary highly conserved N-terminal pescadillo-like protein domain at the N-terminus, a BRCT domain located in the middle of the protein, three classical nuclear localization sequences (NLS) distributed over the protein, six bipartite NLS at the C-terminus and two stretches of acidic amino acids near the C-terminus ( 24 ). Pes1 is modified by SUMOylation, but SUMOylation at the consensus site, ψKXE, located at the C-terminus has yet not been experimentally confirmed ( 25 ). Human and mouse Pes1 proteins are 89% identical .
|
16738141_p19
|
16738141
|
Cloning and conditional expression of human Pes1 mutants
| 4.359449 |
biomedical
|
Study
|
[
0.9994620680809021,
0.0001755747216520831,
0.00036234845174476504
] |
[
0.9984405636787415,
0.0007708143093623221,
0.0007040241034701467,
0.00008460540266241878
] |
en
| 0.999998 |
Systematic deletions are an appropriate tool for the investigation of domains involved in subcelluar localization and interactions as well as the generation of dominant-negative mutants. A panel of pes1 deletion mutants covering the entire open reading frame and a point mutation of the lysine in the consensus SUMOylation site were constructed and tagged with the hemagglutinin (HA) epitope at the C-terminus . For the conditional expression in mammalian cells, wild-type (wt) and mutant pes1 alleles were cloned into the pRTS-1 vector ( 14 , 22 ).
|
16738141_p20
|
16738141
|
Cloning and conditional expression of human Pes1 mutants
| 4.13886 |
biomedical
|
Study
|
[
0.9995566010475159,
0.0001967047282960266,
0.0002466524310875684
] |
[
0.9992940425872803,
0.0003517591976560652,
0.0003006360784638673,
0.000053551535529550165
] |
en
| 0.999997 |
The expression of the gene of interest is tightly controlled by a doxycycline-regulable trans-activator and trans-repressor. Thus, this tight control prevents any selection against vectors carrying dominant-negative pes1 alleles during normal tissue culture due to leakiness of the promoter. Moreover, the pRTS-1 vector allows for high expression levels of the gene of interest. In stably transfected polyclonal cell cultures, conditional gene activation is achieved in >95% of cells . As the bidirectional promoter of the pRTS-1 vector accomplishes simultaneous expression of EGFP in addition to the gene of interest, conditional gene activation can be easily monitored by FACS analysis or fluorescence microscopy . Pes wt and mutants M1–M8 were conditionally expressed in the rat fibroblast cell line TGR-1. Expression levels were determined 30 h after addition of doxycycline by western blot analysis. Most of the mutant forms were detected at the expected size, however deletion of the acidic regions in M5 and M6 significantly enhanced migration .
|
16738141_p21
|
16738141
|
Cloning and conditional expression of human Pes1 mutants
| 4.256857 |
biomedical
|
Study
|
[
0.9995137453079224,
0.00028662022668868303,
0.00019954092567786574
] |
[
0.9992832541465759,
0.00029367062961682677,
0.00034610231523402035,
0.00007704420568188652
] |
en
| 0.999997 |
Pes1 protein has been reported to localize predominantly to the nucleolus. To determine the subcellular localization of the Pes1 mutants, we performed indirect immunofluorescence using HA-tag specific antibodies. As expected, the recombinant Pes1 wt protein showed predominantly nucleolar localization and also some nucleoplasmic staining in cells with high expression of Pes1 . The deletion of 54 N-terminal amino acids in mutant M1 did not affect the nucleolar localization whereas the extension of this deletion to amino acid 154 (M2) or 249 (M3) results in a diffuse nuclear staining. Deletion of the central part of Pes1 containing the BRCT domain and adjacent uncharacterized regions in mutant M4 leads to a diffuse nucleoplasmic staining but with nucleolar staining in very few cells . The C-terminal deletion mutants M5 to M7, together with mutant M8 harbouring a mutated consensus SUMOylation site, located to the nucleolus as Pes1. Therefore, our mutational analysis suggests that the region between 55 and 154 amino acid plays an important role for the nucleolar localization of the Pes1 protein. A contribution of the region extending from amino acid 155 to 249 remains elusive for nucleolar localization in this study. Noteworthy, none of the mutants showed cytoplasmic staining suggesting that neither of the deletions affected the nuclear transport of Pes1. Since all mutants retained at least two putative NLS, the functionality of a single NLS could not be addressed by this set of mutants.
|
16738141_p22
|
16738141
|
Subcellular localization of Pes1 mutants
| 4.301403 |
biomedical
|
Study
|
[
0.9993053674697876,
0.00039924378506839275,
0.0002954356314148754
] |
[
0.9993556141853333,
0.00020074265194125473,
0.0003494101401884109,
0.00009423740993952379
] |
en
| 0.999997 |
Next, we tested the effect of Pes1 deletion mutants on cell proliferation. Equal numbers of cells were seeded and expression of wt Pes1, mutants M1 to M8 and luciferase (mock) was induced by addition of doxycycline. Cell numbers were determined after 6d . Overexpression of Pes1 wt reduced the cell count to 62% compared to mock cells. A likewise decrease was observed for the mutants M6, M7 and M8, and to a higher extent for the mutants M2, M3 and M4. Expression of the mutants M1 and M5 resulted in the strongest reduction, 10 and 22% of mock cell count, respectively. FACS analysis revealed a significant increase of cells in the G 0 /G 1 phase of the cell cycle 48 h after expression of mutants M1 and M5 . Hence, the mutants M1 and M5 suppressed cell proliferation in rat fibroblasts most efficiently.
|
16738141_p23
|
16738141
|
N-terminal (M1) and C-terminal (M5) truncation mutants potently inhibit proliferation and trigger a reversible cell cycle arrest
| 4.137518 |
biomedical
|
Study
|
[
0.9994679093360901,
0.0002644090272951871,
0.0002677044249139726
] |
[
0.9994887113571167,
0.0001573620829731226,
0.00030303135281428695,
0.00005091255661682226
] |
en
| 0.999998 |
Further, we studied the ability of mutants M1 and M5 to mediate a reversible cell cycle arrest in a BrdU/light assay. In brief, dividing cells that incorporate BrdU into their DNA become highly photosensitive if they are additionally labelled with the Hoechst dye 33 258 and undergo apoptosis upon irradiation with visible light. Cell cycle arrested cells are protected from increased photosensitivity and survive BrdU/light treatment. Thus, cells that were arrested by conditional expression of an anti-proliferative Pes1 mutant, would subsequently give rise to colonies after withdrawal of doxycycline. Mock cells expressing luciferase showed only little colony outgrowth and thus demonstrating the low background of this assay. Expression of Pes1 wt did not increase the number of colonies in comparison to the mock situation. The Pes1 mutant M1 provoked a pronounced rescue effect and subsequent colony outgrowth, consistent with a strong reversible cell cycle inhibition. The effect of mutant M5 was less intense but still significant. Mutant M3 showed a colony number slightly over the background level, while all other Pes1 mutants remained at the background level. The reduced proliferation rates of Pes1 wt (62% in the proliferation assay) and mutants M2–M4 and M6–M8 is not accompanied by an increased apoptosis rate (data not shown) or altered cell cycle distribution ( Table 1 ), thus the reason for the reduced cell count remains currently unclear. Taken together, the mutants M1 and M5 mediated a potent inhibitory effect on cell proliferation and triggered a reversible G 0 /G 1 -specific cell cycle arrest ( Table 1 ).
|
16738141_p24
|
16738141
|
N-terminal (M1) and C-terminal (M5) truncation mutants potently inhibit proliferation and trigger a reversible cell cycle arrest
| 4.258542 |
biomedical
|
Study
|
[
0.9994354844093323,
0.0003549170505721122,
0.0002095586823998019
] |
[
0.9992609620094299,
0.00019329102360643446,
0.0004520235816016793,
0.00009371685882797465
] |
en
| 0.999998 |
Pes1 is involved in ribosome biogenesis and therefore we tested the ability of mutants M1 and M5 to inhibit maturation of ribosomal RNA. A short overview of the major mammalian rRNA processing pathway is depicted in Figure 4A . Pes1 wt and mutant forms were expressed in TGR-1 cells for 30 h. Total RNA was isolated, and analysed by northern analysis with hybridization probes specific for the internal transcribed spacer 1 and 2 (ITS-1 and ITS-2) of the ribosomal pre-RNA . Expression of M1 and M5-induced a significant accumulation of the 36S precursor as visualized by hybridization with the ITS-1 specific probes . Inhibition of pre-rRNA processing becomes even more pronounced, if rRNAs were hybridized with the ITS-2 specific probe. Mutants M1 and M5 lead to a strong increase of the amount of 32S rRNA precursor . While both mutants, M1 and M5, induced accumulation of the 36S/32S rRNA, their effect on processing of the 12S rRNA intermediate differed. The level of 12S pre-rRNA was reduced for the mutant M1, but appeared unaffected for the mutant M5 . This may indicate separable and common functions of the mutants M1 and M5 in rRNA processing.
|
16738141_p25
|
16738141
|
The Pes1 mutants M1 and M5 inhibit pre-rRNA processing
| 4.32584 |
biomedical
|
Study
|
[
0.9994134902954102,
0.0003543538914527744,
0.0002322332002222538
] |
[
0.9992315769195557,
0.00025731680216267705,
0.000421428179834038,
0.00008967857138486579
] |
en
| 0.999999 |
In addition we studied the impact of mutants M1 and M5 on rRNA processing in TGR-1 cells by metabolic 32 P-labelling . The production of mature 28S rRNA was severely reduced resulting from an inefficient processing of the 32S rRNA precursor, as concluded from the relative high abundance of metabolically labelled 32S rRNA . In contrast, synthesis of mature 18S rRNA was almost unaffected. These results are in line with our northern blot analysis. In conclusion, the deletion of the N-terminus or the C-terminus of Pes1 either compromises ribosome biogenesis by blocking rRNA processing at the level of the 32S rRNA precursor.
|
16738141_p26
|
16738141
|
The Pes1 mutants M1 and M5 inhibit pre-rRNA processing
| 4.294608 |
biomedical
|
Study
|
[
0.9994456171989441,
0.0003452705277595669,
0.00020915232016704977
] |
[
0.9991970658302307,
0.0003077678557019681,
0.0003954418934881687,
0.00009976315777748823
] |
en
| 0.999998 |
To confirm the role of Pes1 in ribosome biogenesis and cell proliferation, we performed small interfering RNA (siRNA) knockdown experiments of endogenous Pes1. We used human osteosarcoma U2OS cells, because we had generated a monoclonal antibody directed against human Pes1 ( 14 ). Cells were transfected at day 0, 1 and 2 with either control or Pes1-specific siRNA. Expression of endogenous Pes1 was dramatically reduced, as monitored by western blot analysis 2d after the last transfection . Moreover, proliferation of Pes1-depleted cells was severely impaired (data not shown). Further, we investigated the impact of Pes1 knockdown on ribosome biogenesis after in vivo labelling of rRNA. The production of the mature 28S rRNA was strongly compromised . Notably, synthesis of the large 45/47S precursor was not affected, indicating that Pes1 is not involved in rRNA transcription. In conclusion, expression of dominant-negative Pes1 and depletion of endogenous Pes1 block rRNA processing of the 32S rRNA precursor.
|
16738141_p27
|
16738141
|
Endogenous Pes1 is required for rRNA processing and cell proliferation
| 4.307421 |
biomedical
|
Study
|
[
0.9994557499885559,
0.00034823582973331213,
0.0001959918881766498
] |
[
0.9992150068283081,
0.00025421581813134253,
0.00043302905396558344,
0.00009772622433956712
] |
en
| 0.999995 |
As mentioned before, p53 is believed to induce cell cycle arrest following nucleolar stress. This raised the question if the observed impact of mutants M1 and M5 on pre-rRNA processing triggers the accumulation of p53. To address this question, we analysed p53 levels in TGR-1 cells 30 h after expression of wt and mutant Pes1 proteins by western blotting . Pes1 wt and mock cells did not accumulate p53 . Expression of mutant M1 provoked a strong increase of p53 protein (lanes 5 and 6). A less pronounced accumulation was seen for the mutants M3 and M5 (lanes 9, 10 and 13, 14), whereas all other mutants caused no change in p53 levels. If disturbance in ribosome biogenesis is the reason for p53 accumulation, then the accumulation of p53 should be diminished in cells in the absence of active ribosome biogenesis, such as serum-starved cells. To test this assumption, subconfluent TGR-1 cells were cultured with 0.1% FBS for 72 h prior to the addition of doxycycline. The cells were lysed 30 h later. Serum starvation did not affect the induction rate of recombinant proteins , however, neither of the Pes1 mutants was able to trigger the accumulation of p53 .
|
16738141_p28
|
16738141
|
Mutants M1 and M5 induce p53 accumulation in proliferating, but not in starving cells
| 4.19967 |
biomedical
|
Study
|
[
0.9994528889656067,
0.0002907177258748561,
0.0002564217138569802
] |
[
0.9994329810142517,
0.00017935644427780062,
0.0003238001954741776,
0.0000638132041785866
] |
en
| 0.999996 |
To further investigate the p53 accumulation induced by the mutant Pes1 proteins on the single cell level, we analysed p53 accumulation by immunofluorescence . In proliferating cells expressing Pes1 wt, 8.9% of nuclei stained positive for p53. Expression of Pes1 mutants M1 and M5 increased the number of positive cells to 82.0 and 53.4%, respectively . Serum starvation reduced the number of p53-positive cells for Pes1 wt to 0.71%, and for the mutants M1 and M5 to 8.7 and 3.3%, respectively. Thus neither of the mutants were able to induce p53 significantly in serum-starved cells supporting the notion, that ribosome biogenesis is a prerequisite for the dominant-negative action of Pes1 mutants M1 and M5.
|
16738141_p29
|
16738141
|
Mutants M1 and M5 induce p53 accumulation in proliferating, but not in starving cells
| 4.172735 |
biomedical
|
Study
|
[
0.9995019435882568,
0.0002545785391703248,
0.00024338105868082494
] |
[
0.9994105100631714,
0.00021385612490121275,
0.00031512105488218367,
0.00006049717921996489
] |
en
| 0.999995 |
As previously demonstrated, N-terminally deleted WDR12 elicited a p53-dependent cell cycle arrest that was attenuated by the coexpression of the human papillomavirus protein E6 ( 14 ), a ubiquitin ligase targeting p53 for degradation. In line with these studies, mutants M1 and M5-induced cell cycle arrest was alleviated in TGR-1 cells stably transfected with HA-tagged E6 .
|
16738141_p30
|
16738141
|
Mutants M1 and M5 induce p53 accumulation in proliferating, but not in starving cells
| 4.157539 |
biomedical
|
Study
|
[
0.999554455280304,
0.00019890497787855566,
0.0002466050791554153
] |
[
0.9993746876716614,
0.0002903201093431562,
0.0002714396105147898,
0.0000636302211205475
] |
en
| 0.999997 |
The association of Pes1 mutants with pre-ribosomal complexes was studied by sucrose gradient fractionation using TGR-1 total cell lysates . All recombinant proteins accumulated in low molecular fractions, most likely due to an access of overexpressed free protein in total cell lysates. However, Pes1, and mutants M1 and M5, but not the mutant M3, exhibited specific enrichment in high molecular weight fractions that co-fractionated with ribosomal particles. Given the fact that M1 and M5 impair rRNA processing at the level of the 32S pre-rRNA and co-sediment in fractions similar to the 60S peak (see 28S RNA), our data suggests that mutants M1 and M5 accumulate with the 66S pre-ribosomal large subunit.
|
16738141_p31
|
16738141
|
Mutants M1 and M5 associate with the large pre-ribosomal subunit
| 4.240054 |
biomedical
|
Study
|
[
0.9993917942047119,
0.00034468446392565966,
0.000263565918430686
] |
[
0.9993929862976074,
0.00023522754781879485,
0.00029749589157290757,
0.00007434354483848438
] |
en
| 0.999997 |
Previously we showed that Pes1 forms a stable complex with two other nucleolar proteins, Bop1 and WDR12, respectively (PeBoW-complex). Interestingly, several of the deletion mutants generated in this study localized to the nucleolus with or without a dominant-negative phenotype. Thus we asked, if these mutants are nucleolar upon incorporation into the PeBoW-complex or if nucleolar localization is a feature of Pes1 independent of PeBoW-association. Further, it was unclear if the Pes1 mutants interact with Bop1 or WDR12 in conjunction or independently of each other. Moreover, analysing the incorporation of the dominant-negative mutant proteins M1 and M5 into the PeBoW-complex would help to unravel the mechanism of their inhibitory function. To address these issues, we performed a series of vice-versa immunoprecipitation experiments. As our antibodies against the PeBoW components WDR12 and Bop1 are human specific, we performed the experiments in the human osteosacroma cell line U2OS. This cell line also allowed proper conditional expression of Pes1 mutants by the pRTS-1 vector.
|
16738141_p32
|
16738141
|
Incorporation of mutant Pes1 proteins into the PeBoW-complex
| 4.204343 |
biomedical
|
Study
|
[
0.9994041919708252,
0.00026481557870283723,
0.0003310100291855633
] |
[
0.999504566192627,
0.00019389577209949493,
0.0002472127671353519,
0.000054280371841741726
] |
en
| 0.999995 |
U2OS cells were plated at a subconfluent density and the Pes1 constructs were induced for 20 h with doxycycline. The immunoprecipitation experiments were carried out with antibodies against the HA-tag, WDR12 and Bop1, as well as an isotype control. Western blot analysis was then performed with a HA-tag specific monoclonal antibody (3F10) to detect the recombinant Pes1 protein and monoclonal antibodies against WDR12 and Bop1 to detect the association of the recombinant Pes1 protein with endogenous PeBoW members. In the mock cell line, the lack of the HA-tag, WDR12 and Bop1 specific signals in the IP against the HA-tag revealed no unspecific reactivity of the antibodies . The endogenous PeBoW-complex was precipitated in IPs against WDR12 and Bop1, as expected . HA-tagged Pes1 wt protein co-immunoprecipitated high amounts of WDR12 and Bop1 indicating a proper incorporation into the PeBoW-complex . The IPs against WDR12 and Bop1 could co-precipitate only a small fraction of HA-tagged Pes1 protein . This might be due to the fact that 20 h after addition of doxycycline not all PeBoW-complexes have incorporated the HA-tagged Pes1 protein. Hence, the co-immunopreciptation of WDR12 and Bop1 with the anti-HA antibody is the most reliable readout for the incorporation of HA-tagged Pes1 proteins in the BeBoW-complex.
|
16738141_p33
|
16738141
|
Incorporation of mutant Pes1 proteins into the PeBoW-complex
| 4.241365 |
biomedical
|
Study
|
[
0.9994229078292847,
0.0002903803833760321,
0.0002867543662432581
] |
[
0.9993969202041626,
0.000269169919192791,
0.0002717585302889347,
0.00006218333874130622
] |
en
| 0.999997 |
The dominant-negative mutant M1 co-precipitated WDR12 and Bop1 proteins . Mutant M1 was also detectable in IPs against WDR12 and Bop1. This demonstrates that the Pes1 mutant M1 is efficiently incorporated into the PeBoW-complex . The mutant proteins M2 and M3 are not incorporated into PeBoW. Both proteins did not co-precipitate WDR12 and Bop1 and could not be precipitated in IPs against WDR12 and Bop1 . This finding is compatible with a previous report, that the region in Pes1 needed for nucleolar localization is also needed for binding of Bop1 ( 15 ). Pes1 mutant M4 is also not incorporated into the PeBoW-complex according to the failure to co-precipitate significant amounts of WDR12 and Bop1 . The dominant-negative mutant M5,similar to Pes1 wt and mutant M1 protein, co-precipitates WDR12 and Bop1 and is co-precipitated by WDR12 and Bop1 . Mutants M6, M7 and M8 are all incorporated into the PeBoW-complex, as they co-precipitate WDR12 and Bop1 and in reciprocal IPs . Taken together, our immunoprecipitation studies revealed that all mutants are efficiently incorporated in the PeBoW-complex except the mutants M2, M3 and M4. Notably, the mutants M2, M3 and M4 are the only mutants showing no proper nucleolar localization, suggesting that the nucleolar localization of Pes1 is linked to the PeBoW-association. Interestingly, we observed no independent interaction of Pes1 mutants with either Bop1 or WDR12. Further, our results propose that dominant-negative forms of Pes1 require incorporation into the PeBoW-complex for their negative effect on ribosomal rRNA processing and cell cycle progression.
|
16738141_p34
|
16738141
|
Incorporation of mutant Pes1 proteins into the PeBoW-complex
| 4.512647 |
biomedical
|
Study
|
[
0.9993064403533936,
0.0004103955870959908,
0.00028319499688223004
] |
[
0.9987800717353821,
0.0003162706270813942,
0.0007743748719803989,
0.0001292423694394529
] |
en
| 0.999998 |
In mammals, Pes1, Bop1 and WDR12 proteins are components of the evolutionary highly conserved PeBoW-complex, which is required for maturation of the 60S ribosomal subunit. Although PeBoW is highly abundant in the nucleolus, the way and the place of its assembly is unclear. A homologous complex in yeast, composed of Nop7, Erb1 and Ytm1, associates with four consecutive 66S pre-ribosomal subunits containing the 27SA2, 27SA3, 27SB and the 25.5S plus 7S pre-rRNAs ( 18 , 26 ). Similar as in mammals, the yeast complex is required for proper processing of the 27S pre-rRNA to the mature 25S rRNA. Recent evidence suggests that the assembly of the trimeric yeast complex occurs in the nucleolus, with assembly of Nop7 and Erb1 into the pre-ribosomes prior to Ytm1 ( 18 ). Whether Nop7 and Erb1 assemble before binding to 66S pre-ribosomes is not known. A stable complex of Nop7 and Erb1 has not been demonstrated yet, neither in yeast, nor for the homologues Pes1 and Bop1 in mammalian cells. This is intriguing since the interaction of Pes1 and Bop1 can easily be demonstrated in GST-pull-down and yeast two hybrid experiments ( 15 ). This suggests that the interaction of endogenous Pes1 and Bop1 may be inhibited during their passage to the nucleolus and occurs only after association with the pre-ribosome and/or the assembly of WDR12 in the complex.
|
16738141_p35
|
16738141
|
DISCUSSION
| 4.603489 |
biomedical
|
Study
|
[
0.9991940855979919,
0.0004241840506438166,
0.00038175014196895063
] |
[
0.9969937801361084,
0.0006240273942239583,
0.0022143111564219,
0.00016786517517175525
] |
en
| 0.999999 |
In this study we have constructed a set of deletion mutants to characterize the domains of Pes1 required for the nucleolar localization and assembly into the PeBoW-complex. The Pes1 protein has an evolutionary highly conserved N-terminal domain of pescadillo-like proteins of 250 amino acids followed by a distal BRCT domain ( 27 ). While the NPLP-domain is present in all eukaryotes, the BRCT domain is missing in some simple organisms as Plasmodium falciparum . Three of our deletions mutants, M1, M2 and M3, successively lacked parts of the NPLP-domain. The removal of 54 N-terminal amino acids of Pes1 in mutant M1 resulted in a strong dominant-negative phenotype. Sucrose gradient centrifugation, co-immunoprecipitation experiments and native gel-electrophoresis experiments (data not shown) revealed proper incorporation of mutant M1 protein into the PeBoW-complex. Hence, the N-terminal 54 amino acids of the NPLP-domain are neither required for the assembly of the PeBoW-complex nor for the transport of Pes1 to the nucleolus. It is more likely that the N-terminal domain of Pes1 fulfils other essential tasks after the assembly of the complex by recruitment of further factors, or by catalysing specific steps in the maturation of the 32S pre-rRNA precursor. However, an enzymatic activity of the complex has not been demonstrated yet, neither in mammals nor in yeast. The mutants M2 and M3 lacked larger parts of the NPLP-domain. Proteins of both mutants were still transported into the nucleus. A transport into the nucleolus, however, was inhibited. This is in line with the observation that mutant M2 and M3 proteins did not co-immunoprecipitate Bop1 and WDR12, because the assembly of the PeBoW-complex in the nucleolus could not take place. The absence of a dominant-negative phenotype of both mutants also supports the model that incorporation of Pes1 mutants into the PeBoW-complex is a prerequisite for a dominant-negative phenotype. The region deleted in M2 and M3 has been reported to interact directly with Bop1 ( 15 ). The interaction domain with Bop1 has been identified in murine Pes1 after transposon insertion mutagenesis of 19 extra amino acids downstream of amino acid 198 (Pes1-tn2), amino acid 202 (Pes1-tn11) and amino acid 204 (Pes1-tn12) in yeast two hybrid experiments ( 15 ). The failure of mutants M2 and M3 proteins to immunoprecipitate complexes containing Bop1 and WDR12 supports this notion. Anyway, incorporation of both mutant proteins into the PeBoW-complex cannot take place, since they do not localize to the nucleolus. In conclusion, the NPLP-domain is critical for nucleolar localization and assembly of the PeBoW-complex.
|
16738141_p36
|
16738141
|
Nucleolar transport and assembly of Pes1 into the PeBoW-complex
| 4.504173 |
biomedical
|
Study
|
[
0.9989222288131714,
0.0006697513745166361,
0.000408031337428838
] |
[
0.999134361743927,
0.00030135439010336995,
0.0004195452493149787,
0.00014481664402410388
] |
en
| 0.999997 |
The Pes1 mutant M4 harbours a deletion of the BRCT (BRCA1 C-terminal) domain that was first described as an essential domain of the tumour suppressor function of the BRCA1 protein. Similar repeat sequences have been identified in many proteins that function in DNA damage, repair and replication. Many BRCT-containing proteins have phospho-peptide binding activity suggesting that BRCT repeats might mediate phosphorylation-dependent protein–protein interactions in processes that are central to cell cycle checkpoint and DNA repair functions. It is tempting to speculate that the BRCT domain in Pes1 might fulfil similar tasks and links nucleolar processes to cell cycle control and DNA synthesis. Interestingly, a temperature sensitive mutant of yeast Nop7, a homologue of Pes1, can specifically inhibit S phase entry in yeast cells ( 17 ). This points to additional cell cycle control mechanisms by Pes1, which may act independently of ribosome biogenesis. The mutant M4 protein did not co-immunoprecipitate Bop1 and WDR12 from cellular lysates. This lack of interaction suggests that the NPLP-domain in Pes1 is not sufficient for binding of Bop1 in the nucleoplasm, or that the interaction of nucleoplasmic Pes1 and Bop1 is negatively controlled by a yet unknown mechanism. Importantly, in addition to the Bop1 interacting domain, the BRCT domain also plays an important role in nucleolar localization.
|
16738141_p37
|
16738141
|
Nucleolar transport and assembly of Pes1 into the PeBoW-complex
| 4.649754 |
biomedical
|
Study
|
[
0.9990904331207275,
0.000602482003159821,
0.0003070326638408005
] |
[
0.9982444047927856,
0.00067570258397609,
0.0007986476412042975,
0.0002812892780639231
] |
en
| 0.999995 |
The second mutant with a strong dominant-negative phenotype on cell proliferation and rRNA processing was M5. Similar to M1, mutant M5 protein localized to the nucleolus and co-immunoprecipitated Bop1 and WDR12. However, we noticed a specific difference in the processing of the 5.8S rRNA for mutants M1 and M5. While the amount of 12S pre-rRNA was reduced for mutant M1, the levels of 12S pre-rRNA were unchanged for the mutant M5. This suggests an inhibition of 12S pre-rRNA processing by mutant M5 and an accumulation of this precursor, whereas 12S pre-rRNA processing proceeds in the presence of mutant M1. The data suggests that the PeBoW-complex is required for consecutive rRNA processing steps during maturation of the 60S ribosomal subunit and that mutant M5 can block the processing of the 12S pre-rRNA in addition to the 32S pre-rRNA. Accordingly, the C-terminal domain of Pes1 is required for processing of the 32S and 12S pre-rRNA precursors, whereas the N-terminal domain appears to be dispensable for processing of the latter.
|
16738141_p38
|
16738141
|
Nucleolar transport and assembly of Pes1 into the PeBoW-complex
| 4.450529 |
biomedical
|
Study
|
[
0.9992859959602356,
0.0004669169429689646,
0.0002470050530973822
] |
[
0.9989396929740906,
0.00047264955355785787,
0.0004290781798772514,
0.00015861169958952814
] |
en
| 0.999996 |
The impaired function of the PeBoW-complex evokes a p53 response in proliferating but not in serum-starved cells. All three components of the PeBoW-complex are encoded by Myc target genes and usually are not expressed in quiescent cells ( 14 , 28 , 29 ). This suggests that expression of dominant-negative mutants of Pes1 (this paper), or WDR12 ( 14 ) may be not sufficient for the induction of p53, because they cannot assemble into the PeBoW-complex and block its function. However, a recent study showed that human WI-38 fibroblasts accumulated p53 under growth-restricting conditions, such as serum starvation or confluency ( 30 ).
|
16738141_p39
|
16738141
|
Role of the PeBoW-complex in cell cycle control
| 4.205559 |
biomedical
|
Study
|
[
0.9995484948158264,
0.00019665622676257044,
0.0002548986813053489
] |
[
0.9993576407432556,
0.00031748946639709175,
0.0002660389873199165,
0.00005889791646040976
] |
en
| 0.999996 |
Whether the PeBoW-complex is directly involved in the degradation of p53 remains unclear. Since dominant-negative Pes1 mutants act only in the presence of ongoing ribosome biogenesis it is conceivable that signalling of p53 degradation should be linked to this process. Two non-mutually exclusive mechanisms have been proposed for p53 degradation: nucleolar sequestration of Mdm2, and the blockage of a postulated nucleolar route of p53 export for cytoplasmic degradation ( 13 , 31 ). The Mdm2 sequestration model is supported by the recent finding that several ribosomal proteins, L5, L11 and L23, can bind and inhibit the function of Mdm2. Inhibition of ribosome biogenesis leads to accumulation of free L5, L11 and L23 proteins, which bind to and inhibit Mdm2-mediated p53 ubiquitination and degradation. Inhibiton of Mdm2 restores p53-mediated transactivation, accumulation of p21 protein levels, and induction of cell cycle arrest ( 5 – 9 , 27 ). The second model postulates a nucleolar export route for Mdm2–p53 complexes and subsequent cytoplasmic degradation of p53, termed by Sherr and Weber ( 13 ) as Mdm2–p53 complexes ‘riding the ribosome’. This model is supported by the observation that p53 can be covalently linked to 5.8S rRNA, and associates with a subset of ribosomes ( 32 – 34 ). Further support comes from the finding that inhibition of nuclear export leads to accumulation of p53 in the nucleus. If p53 has to enter the nucleolus for the second model, or is loaded on ribosomal subunits during the passage through the nucleoplasm, is currently an open question. Evidence for the presence of p53 in the nucleolus has recently been shown following cell permeabilization, where most soluble nucleoplasmic p53 was eliminated, but nuclear-bound p53 remained readily detectable in the nucleoli ( 6 ). Accumulation of nucleolar p53 has also been observed after inhibiton of the proteasome ( 35 ). Alternatively, p53 associate with large pre-ribosomal subunits, if they leave the nucleolus. Comparable to processing of the 27S pre-rRNA in yeast ( 25 ), processing of the 32S pre-rRNA in mammalian cells probably occurs at the transition of the large pre-ribosomal subunit from the nucleolus to the nucleoplasm. At this moment, the functionality of the PeBoW-complex is required. Successful processing of the 32S rRNA would cause the release of the PeBoW-complex and loading of Mdm2 and p53. A non-functional PeBoW-complex would inhibit processing of the 32S pre-RNA and thereby nuclear export of p53/Mdm2.
|
16738141_p40
|
16738141
|
Role of the PeBoW-complex in cell cycle control
| 4.894052 |
biomedical
|
Study
|
[
0.998128354549408,
0.0012372221099212766,
0.0006344867288134992
] |
[
0.9888170957565308,
0.0016769689973443747,
0.008771467953920364,
0.0007344644982367754
] |
en
| 0.999996 |
Supplementary Data are available at NAR Online.
|
16738141_p41
|
16738141
|
SUPPLEMENTARY DATA
| 0.985075 |
biomedical
|
Other
|
[
0.7697952389717102,
0.0054101841524243355,
0.22479452192783356
] |
[
0.01419480424374342,
0.982682466506958,
0.0018953380640596151,
0.00122743786778301
] |
en
| 0.571427 |
Prolonged infection with Hepatitis B virus (HBV) has been clearly recognized as a major etiological factor for hepatocellular carcinoma (HCC) ( 1 ). HBx, a virally encoded protein of 154 amino acids, has been shown to have multifunctional activities relevant to HBV-mediated oncogenesis ( 2 ). HBx is involved in neoplastic transformation in cultured cells and can induce liver cancer in transgenic mice. Although HBx does not bind to double-stranded DNA, it regulates transcription of a variety of cellular and viral genes by interacting with cellular proteins and/or components of signal transduction pathways. HBx has been shown to interact with transcriptional factors such as RPB5 of RNA polymerase ( 3 ), TATA-binding protein ( 4 ), basic region/leucine zipper (bZIP) proteins ( 5 ) and the tumor suppressor p53 ( 6 ). Besides, it can also associate with serine protease TL2 ( 7 ) and cellular DNA repair protein ( 8 ). The interaction of HBx with these proteins leads to activation of signal transduction pathways including the Ras/Raf/mitogen-activated protein kinase, protein kinase C, Jak1-STAT and nuclear factor κB pathways ( 9 – 12 ). However, the intracellular signaling pathways in which Hbx is involved are not fully elucidated.
|
16757575_p0
|
16757575
|
INTRODUCTION
| 4.713511 |
biomedical
|
Study
|
[
0.9989790916442871,
0.0005908814491704106,
0.0004300795844756067
] |
[
0.9628477692604065,
0.0009282227256335318,
0.035764843225479126,
0.0004591165925376117
] |
en
| 0.999999 |
Estrogen was shown to suppress HBV replication in male athymic mice transplanted with HBV-transfected HepG2 cells ( 13 ). The fact that HCC is more prevalent in men than in women suggests that estrogen may play an important role in the development of HCC ( 14 – 17 ). Estrogen exerts its function through its two nuclear receptors, estrogen receptor α and β (ERα and ERβ) ( 18 – 21 ). ERα and ERβ share structural similarity characterized by several functional domains. Two distinct activation function (AF) domains, AF-1 and AF-2, located at the N-terminus and the C-terminus, respectively, contribute to the transcriptional activity of the two receptors. The DNA-binding domain (DBD) of the two receptors is well conserved and centrally located. Activation of ERs is responsible for many biological processes, including cell growth, differentiation and apoptosis.
|
16757575_p1
|
16757575
|
INTRODUCTION
| 4.290605 |
biomedical
|
Study
|
[
0.9996495246887207,
0.00018702841771300882,
0.00016355575644411147
] |
[
0.990858256816864,
0.00046104041393846273,
0.00856438372284174,
0.0001163381602964364
] |
en
| 0.999996 |
ERα has been well characterized in human liver ( 22 ). ERα is expressed in the liver of both healthy individuals and patients with HCC, with no differences in the pattern of expression ( 23 , 24 ). In contrast, the mutant form with the entire exon 5 deleted (ERΔ5) is preferentially expressed in patients with HCC compared with patients with normal livers ( 25 ). The presence of the liver ERΔ5 transcript in the tumor was the strongest negative predictor of survival in operable HCC ( 26 – 28 ). Its presence also correlates with a higher clinical aggressiveness of the tumor in comparison with tumors characterized by wild-type ERα (wt ERα) transcript. High rates of ERΔ5 expression have been shown to present in men at high-risk for HCC development. ERΔ5 encodes the hormone-independent AF-1 domain, as well as the DBD. Although ERΔ5 was demonstrated to be coexpressed with wt ERα in HCC, the role of ERΔ5 in ERα signaling remains to be investigated.
|
16757575_p2
|
16757575
|
INTRODUCTION
| 4.329419 |
biomedical
|
Study
|
[
0.9995237588882446,
0.00022359471768140793,
0.000252598081715405
] |
[
0.9956446886062622,
0.00028068438405171037,
0.003982951864600182,
0.00009164927178062499
] |
en
| 0.999996 |
On the basis of in vivo and in vitro functional relevance of the estrogen/ERα axis and HBx in the development of HCC, we hypothesized that HBx may play a role in ERα signaling. Here, we show that ERΔ5 has a dominant negative activity in hepatoma cells when expressed together with wt ERα. HBx decreases ERα transcriptional activity, and HBx and ERΔ5 have additive effect on inhibition of ERα transactivation. We further present in vitro and in vivo evidence that both HBx and ERΔ5 interact with ERα. HBx inhibits ERα signaling possibly through recruitment of histone deacetylase 1 (HDAC1).
|
16757575_p3
|
16757575
|
INTRODUCTION
| 4.229728 |
biomedical
|
Study
|
[
0.9995293617248535,
0.00029258435824885964,
0.00017798053158912808
] |
[
0.9993382096290588,
0.00024246438988484442,
0.00033077236730605364,
0.00008845635602483526
] |
en
| 0.999995 |
The reporter constructs ERE-Luc ( 29 ), C3-LUC ( 30 , 31 ), pS2-LUC ( 32 ) and pS2ΔERE-LUC ( 33 ), and expression vector for ERα have been described previously. For the generation of FLAG-tagged full-length HBx, human HBx DNA was amplified by PCR using pHBV3091 as a template ( 34 ). The amplified HBx DNA was cloned into pcDNA3 vector harboring FLAG epitope sequence (pcDNA3-FLAG). The deletion mutant of HBx (Δ73-120) was constructed by inserting the recombinant PCR-generated fragment from the HBx DNA into the pcDNA3-FLAG vector. The expression vectors for the full-length ERα (1–595), ERα AF1 (1–185), ERα DBD (180–282), ERα AF2 (282-595), ERα AF2 (302–595) and ERΔ5 (370 amino acids with a novel five-amino acid residue COOH terminus) were made by introducing the corresponding cDNAs into pcDNA3 (Invitrogen). Enhanced green fluorescent protein (EGFP)-tagged HBx construct was generated by inserting HBx DNA into pEGFP-C1 (Clontech), and red fluorescent protein (RFP)-tagged ERα construct by inserting ERα cDNA into pDsRed-N1 (Clontech). A cDNA fragment encoding entire coding region of HDAC1 was obtained by RT–PCR using as a template total RNA from the human hepatoma cell line HepG2, and the cDNA fragment was inserted in frame into a pcDNA3 vector linked with HA tag at the amino terminus. Plasmids encoding GST-fusion proteins were prepared by amplification of each sequence by standard PCR methods, and the resulting fragments were cloned in frame into pGEX-KG (Amersham Pharmacia Biotech) using appropriate restriction sites. All of the constructs were confirmed by sequencing. Details of cloning are available upon request.
|
16757575_p4
|
16757575
|
Plasmids
| 4.269895 |
biomedical
|
Study
|
[
0.9995800852775574,
0.0002340106148039922,
0.00018592955893836915
] |
[
0.9983618855476379,
0.0006078699370846152,
0.0009228640701621771,
0.00010740470315795392
] |
en
| 0.999996 |
HepG2 and SMMC-7721 cells were routinely grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS). For transfection, cells were seeded in 12-well plates containing phenol red-free DMEM medium supplemented with 10% charcoal-stripped FBS (Hyclone). The cells were transfected using Lipofectamine 2000 (Invitrogen) with 0.2 µg of ERE-LUC, C3-LUC, pS2-LUC or pS2ΔERE-LUC reporter plasmid, 50 ng of ERα expression plasmid, 250 ng to 2 µg of the expression vector for HBx and 0.1 µg of β-galactosidase reporter as an internal control. The empty vector pcDNA3 was used to adjust the total amount of DNA. After treatment with 10 nM of 17β-estradiol (E 2 ) and 100 nM 4-hydroxytamoxifen (4-OHT) for 24 h, or 100 nM trichostatin A (TSA) for 12 h, the cells were harvested, and luciferase and β-galactosidase activities were determined as described previously ( 35 ). All experiments were repeated at least five times.
|
16757575_p5
|
16757575
|
Transfection and luciferase assay
| 4.127534 |
biomedical
|
Study
|
[
0.9994831085205078,
0.00029333215206861496,
0.00022351613733917475
] |
[
0.9991998076438904,
0.0004039394552819431,
0.00032994180219247937,
0.00006629990821238607
] |
en
| 0.999997 |
GST and GST-fusion proteins were expressed in E.coli DH5α, with the induction of protein expression performed at 20°C overnight ( 36 ). After large-scale preparation, purification of the recombinant proteins were performed according to the manufacturer's instruction (Pharmacia) using glutathione–Sepharose beads. The expression plasmid for the ERα, ERα deletion mutants, HBx or HDAC1 was used for in vitro transcription and translation in the TNT System (Promega). The 35 S-labeled in vitro translated products were incubated with ∼10 µg of GST derivatives bound to glutathione–Sepharose beads in 500 µl binding buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.3 mM DTT, 0.1% NP-40 and protease inhibitor tablets from Roche) at 4°C. The beads were precipitated, washed four times with binding buffer, eluted in SDS–PAGE sample buffer, and analyzed by SDS–PAGE. After electrophoresis, the gel was dried and exposed to X-ray films.
|
16757575_p6
|
16757575
|
GST pull-down assay
| 4.140201 |
biomedical
|
Study
|
[
0.9995625615119934,
0.00022695100051350892,
0.00021040241699665785
] |
[
0.9988883137702942,
0.000673564150929451,
0.0003685426199808717,
0.00006960128666833043
] |
en
| 0.999997 |
HepG2 cells were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), washed with phosphate-buffered saline (PBS), lysed in 0.5 ml lysis buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 0.25% NP-40, 1 mM DTT and protease inhibitor tablets from Roche), and immunoprecipitated with anti-FLAG-agarose beads (Sigma) for 3 h at 4°C. The beads were centrifuged, washed four times with the lysis buffer, and eluted in 30 µl of SDS–PAGE sample buffer. The eluted proteins were separated by SDS–PAGE, followed by immunoblotting with anti-ERα (Santa Cruz Biotech), anti-HA (Sigma) or anti-FLAG (Sigma) according to the standard procedures.
|
16757575_p7
|
16757575
|
Coimmunoprecipitation
| 4.104903 |
biomedical
|
Study
|
[
0.999580442905426,
0.0002039680548477918,
0.0002155817928723991
] |
[
0.9977561831474304,
0.0017680184682831168,
0.0003860067226924002,
0.0000898234429769218
] |
en
| 0.999997 |
For reimmunoprecipitation, the immune complexes precipitated with anti-FLAG were eluted under native condition by a competition with 3× FLAG peptide according to the manufacturer's instructions (Sigma). The eluate was precleared with 20 µl of 50% protein A agarose beads (Santa Cruz Biotech) for 30 min. Proteins were reprecipitated with anti-ERα or control serum (Santa Cruz Biotech) plus 20 µl of protein A agarose beads. Reprecipitates were washed four times with lysis buffer, eluted by boiling in SDS–PAGE sample buffer, and resolved by SDS–PAGE, followed by immunoblotting.
|
16757575_p8
|
16757575
|
Coimmunoprecipitation
| 4.117538 |
biomedical
|
Study
|
[
0.9994949102401733,
0.00026223663007840514,
0.00024279051285702735
] |
[
0.9919501543045044,
0.007333437912166119,
0.0005513906362466514,
0.00016501791833434254
] |
en
| 0.999995 |
For detecting interaction of endogenous HBx with ERα, liver tissue from an HBV positive patient (General Hospital of PLA, Beijing) was lysed in 1.0 ml RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor tablets from Roche), and immunoprecipitated with anti-ERα or control serum (Santa Cruz). After extensive washing with RIPA buffer, the immunoprecipitates were resolved by SDS–PAGE, followed by western blot analysis using anti-HBx (Chemicon).
|
16757575_p9
|
16757575
|
Coimmunoprecipitation
| 4.106415 |
biomedical
|
Study
|
[
0.9995909333229065,
0.00020132258941885084,
0.00020775444863829762
] |
[
0.9990033507347107,
0.0006736067589372396,
0.00024899086565710604,
0.00007400641334243119
] |
en
| 0.999996 |
HepG2 cells were seeded in 6-well dishes with glass coverslips containing phenol red-free DMEM medium (Invitrogen) supplemented with 10% charcoal-stripped FBS (Hyclone). Cells were transiently transfected with the indicated plasmids using lipofectamine 2000. Six hours after transfection, cells were treated with 10 nM of 17β-estradiol (E 2 ) for various times. Nuclear DNA was visualized with 4′,6′-diamidino-2-phenylindole (DAPI). The subcellular localization of EGFP-HBx and RFP-ERα was analyzed with a Radiance 2100 confocal microscope (Bio-Rad). Fluorescence was detected with appropriate filter sets (the green signal, excitation 488 nm, dichroic mirror 560 DCLPXR, emission HQ 515/30; the red signal, excitation 543 nm, dichroic mirror 650 DCLPXR, emission HQ590/70).
|
16757575_p10
|
16757575
|
Confocal microscopy
| 4.115505 |
biomedical
|
Study
|
[
0.9995504021644592,
0.0002731947461143136,
0.00017639115685597062
] |
[
0.9986889958381653,
0.0009043305763043463,
0.0003264441038481891,
0.0000802043650764972
] |
en
| 0.999999 |
To examine the effects of wt ER and the ER variant ERΔ5 on E 2 -responsive gene transcription, ERα−negative human liver carcinoma HepG2 cells ( 37 ) were transiently transfected with wt ERα and/or ERΔ5, along with the synthetic estrogen-responsive reporter plasmid ERE-LUC, or the natural estrogen-responsive reporters pS2-Luc and Complement 3-Luc (C3-Luc). As shown in Figure 1 , in the presence of E 2 , ERα stimulated the transcription of these reporter genes, whereas ERΔ5 had little effect. Importantly, when wt ER and ERΔ5 were co-transfected into the HepG2 cells in equal amounts, ERΔ5 was able to reduce the transcriptional activity of wt ER. These data suggest that ERΔ5 is able to interfere with the transcriptional activity of wt ER and to act as a dominant negative receptor.
|
16757575_p11
|
16757575
|
Repression of ERα transcriptional activity by ERΔ5
| 4.179768 |
biomedical
|
Study
|
[
0.9995489716529846,
0.00023927926667965949,
0.00021176175505388528
] |
[
0.9993152618408203,
0.00025050167459994555,
0.00037219288060441613,
0.00006214423046912998
] |
en
| 0.999996 |
The dominant negative property of the ER variant ERΔ5 could involve the formation of a heterodimer between ERΔ5 and wt ERα through protein–protein interactions. To test this possibility, GST pull-down experiments were performed in which in vitro translated 35 S-methionine-labeled ERΔ5 was incubated with full-length GST-ERα or GST. As shown in Figure 1B , in both the absence and presence of E 2 , ERΔ5 bound to GST-ERα, but not to GST, suggesting that ERα physically interacts with ERΔ5 in vitro .
|
16757575_p12
|
16757575
|
Interaction of ERα with ERΔ5 in vitro and in vivo
| 4.143654 |
biomedical
|
Study
|
[
0.9995449185371399,
0.00021765666315332055,
0.0002373057504883036
] |
[
0.9994033575057983,
0.0003120864857919514,
0.00022447598166763783,
0.000060078185924794525
] |
en
| 0.999995 |
To determine whether ERΔ5 interacted with ERα in vivo , HepG2 cells were transfected with ERα and FLAG-tagged ERΔ5, and grown both in the absence and presence of 10 nM E 2 . The cells were then subjected to immunoprecipitation (IP) with FLAG antibody-conjugated agarose beads, followed by immunoblot (IB) with ERα antibody, which recognizes both ERα and ERΔ5 proteins. As shown in Figure 1C , ERα could be co-immunoprecipitated in a ligand-independent manner in the presence, but not in the absence, of FLAG- ERΔ5. These results suggest that ERΔ5 interacts with ERα in hepatoma cells.
|
16757575_p13
|
16757575
|
Interaction of ERα with ERΔ5 in vitro and in vivo
| 4.105988 |
biomedical
|
Study
|
[
0.9995086193084717,
0.00022433312551584095,
0.00026703832554630935
] |
[
0.9994669556617737,
0.0002626006898935884,
0.00021911330986768007,
0.000051313636504346505
] |
en
| 0.999995 |
To gain insight into the functional role of HBx in HCC, the effect of HBx protein on ERα transactivation function was investigated. HepG2 cells were co-transfected with the synthetic estrogen response element (ERE)-containing reporter ERE-LUC, ERα, and increasing amounts of HBx. As shown in Figure 2A , as little as 250 ng of HBx was sufficient to exert a potent repression of ERα transactivation function and the extent of repression increased with increasing amount of HBx expression, suggesting that HBx decreased ERα transcriptional activity in a dose-dependent manner. Similar repression was observed in other liver cancer cells such as SMMC-7721 (data not shown). It should be noted that the decreased transcriptional activity was not a result of reduced ERα protein production .
|
16757575_p14
|
16757575
|
Repression of ERα transcriptional activity by HBx
| 4.125448 |
biomedical
|
Study
|
[
0.9995552897453308,
0.00024918190320022404,
0.00019548888667486608
] |
[
0.9994633793830872,
0.00017139999545179307,
0.0003028211649507284,
0.00006232111627468839
] |
en
| 0.999995 |
To test the effect of HBx on natural estrogen-responsive promoter activity, HepG2 cells were co-transfected with the natural ERE-containing reporter C3-Luc or pS2-Luc, together with expression vectors for ERα and HBx. As shown in Figure 2C and D , activation of the C3 promoter by ERα was not affected by HBx, whereas activation of the pS2 promoter by ERα was significantly repressed by HBx, indicating that the effect of HBx is promoter specific. Interestingly, mutations of the EREs at the pS2 promoter abolished both the E 2 -dependent gene activation and HBx-mediated repression . A similar repressive effect of HBx on ERα-mediated transcription was also observed in SMMC-7721 cells (data not shown). As another control of the effects of HBx on transcription, HBx stimulated Smad-mediated gene transcription as reported previously ( 38 ), when HepG2 cells were co-transfected with the synthetic TGFβ-responsive transcriptional reporter p3TP-Lux (data not shown). Taken together, our data suggest that specific cis - and trans -acting elements are required for the HBx-mediated repression.
|
16757575_p15
|
16757575
|
Repression of ERα transcriptional activity by HBx
| 4.327793 |
biomedical
|
Study
|
[
0.9994992017745972,
0.0003081017639487982,
0.00019262064597569406
] |
[
0.999165415763855,
0.0002563457819633186,
0.0004803470801562071,
0.00009793799836188555
] |
en
| 0.999997 |
Since both HBx and ERΔ5 repressed ERα transcriptional activity, we determined if HBx and ERΔ5 had synergistic or additive effect on ERα transactivation. We co-transfected HepG2 cells with the ERE-Luc, C3-Luc, pS2-Luc or pS2ΔERE-Luc reporter construct, together with HBx or ERΔ5 or in combination. As expected, HBx alone inhibited the transcription of the ERE-Luc and pS2-LUC reporter genes but not the C3-Luc and pS2ΔERE-Luc reporter genes. ERΔ5 alone repressed the transcription of all of reporter genes except pS2ΔERE-Luc . Cotransfection with HBx plus ERΔ5 expression vectors gave an additive effect in repressing the transcription of the pS2-LUC reporter gene but not the other reporter genes. These results indicate that the additive effect of HBx and ERΔ5 on ERα-responsive gene transcription is promoter specific.
|
16757575_p16
|
16757575
|
Additive repression of specific ERα responsive gene transcription by HBx and ERΔ5
| 4.143712 |
biomedical
|
Study
|
[
0.9995500445365906,
0.00022712742793373764,
0.0002227883378509432
] |
[
0.9994545578956604,
0.0002091918868245557,
0.00028192269382998347,
0.00005435090133687481
] |
en
| 0.999996 |
To examine the effect of antiestrogen on suppression of ERα transactivation by HBx and ERΔ5, HepG2 cells were co-transfected with the pS2-Luc reporter, ERα, and HBx or ERΔ5 or in combination, and subsequently treated with the antiestrogen 4-OHT . 4-OHT alone did not have significant effect on HBx- or ERΔ5-mediated repression, whereas combination of 17β-estradiol (E 2 ) and 4-OHT inhibited E 2 -induced ERα transactivation regardless of HBx and ERΔ5.
|
16757575_p17
|
16757575
|
Additive repression of specific ERα responsive gene transcription by HBx and ERΔ5
| 4.117494 |
biomedical
|
Study
|
[
0.9995531439781189,
0.00021831058256793767,
0.0002285849186591804
] |
[
0.999398946762085,
0.0002701000776141882,
0.00027716084150597453,
0.00005380191214499064
] |
en
| 0.999996 |
HBx has been shown to regulate viral and cellular gene transcription by interacting with transcription factors ( 3 – 6 ). Our observation that HBx could function as a co-repressor to repress ERα transactivation raised the possibility that HBx might physically interact with ERα. To test this possibility, GST pull-down experiments were performed using 35 S-labeled full-length ERα and GST-tagged full-length HBx. As shown in Figure 4A , GST-HBx, but not GST, was able to pull down the 35 S-labeled ERα, thus demonstrating an in vitro interaction between HBx and ERα.
|
16757575_p18
|
16757575
|
Interaction of HBx with ERα in vitro and in vivo
| 4.113073 |
biomedical
|
Study
|
[
0.9995692372322083,
0.00021427309548016638,
0.00021648567053489387
] |
[
0.9994845390319824,
0.00022255121439229697,
0.00023686952772550285,
0.00005598462303169072
] |
en
| 0.999999 |
To test if HBx binds to ERα in mammalian cells, HepG2 cells were transfected with ERα and FLAG-tagged HBx, and harvested for coimmunoprecipitation experiments. Figure 4B demonstrates that ERα could be co-immunoprecipitated in a ligand-independent manner in the presence of FLAG-HBx but not FLAG-tagged empty vector. To ascertain the HBx–ERα interaction in a more physiological context, the endogenous ERα protein from liver tissue of an HBV positive patient was immunoprecipitated with an anti-ERα antibody. Subsequent immunoblotting with anti-HBx antibody indicated that the endogenous HBx was coprecipitated with ERα . In the negative control experiment, normal rabbit serum or an irrelevant antibody, anti-FLAG antibody, did not immunoprecipitate HBx . Taken together, these data strongly suggest that HBx interacts with ERα in vivo .
|
16757575_p19
|
16757575
|
Interaction of HBx with ERα in vitro and in vivo
| 4.148002 |
biomedical
|
Study
|
[
0.9995706677436829,
0.0002451171458233148,
0.00018420086416881531
] |
[
0.999381422996521,
0.00022582335805054754,
0.0003245473781134933,
0.00006815564847784117
] |
en
| 0.999995 |
Since HBx and ERΔ5 have additive effect on the ERα transactivation , the effect of ERΔ5 on the HBx–ERα interaction was investigated. HepG2 cells were transfected with FLAG-tagged HBx, ERα, and increasing amounts of ERΔ5, and collected for coimmunoprecipitation assays. As shown in Figure 4D , Both ERα and ERΔ5 were coprecipitated with FLAG-tagged HBx, but not FLAG control vector. Consistent with the functional results , ERΔ5 had little effect on the interaction of ERα with HBx .
|
16757575_p20
|
16757575
|
Interaction of HBx with ERα in vitro and in vivo
| 4.097438 |
biomedical
|
Study
|
[
0.9994344115257263,
0.00021295131591614336,
0.0003526873770169914
] |
[
0.9996108412742615,
0.0001782733015716076,
0.00016610402963124216,
0.00004485594035941176
] |
en
| 0.999996 |
To confirm the protein–protein interaction between HBx and ERα in situ , constructs were made for EGFP-tagged HBx (EGFP-HBx) and RFP-tagged ERα. Based on their ability to regulate the ERE-Luc reporter activity, these fluorescent protein-tagged constructs were similar to those with or without the above mentioned FLAG tag (data not shown). HepG2 cells were then co-transfected with EGFP-HBx and RFP-ERα, and analyzed for co-localization of ERα with HBx. As expected, EGFP-HBx localizes in both the cytoplasm and the nucleus of HepG2 cells ( 39 ) . RFP-ERα localizes essentially in the nucleus of HepG2 cells in both the presence and absence of estrogen . Co-localization studies indicated that EGFP-HBx colocalized with RFP-ERα, but not with the empty vector RFP, predominantly in the cell nucleus in a ligand-independent manner, suggesting that ERα may facilitate the nuclear localization of HBx .
|
16757575_p21
|
16757575
|
Co-localization of ERα with HBx
| 4.160051 |
biomedical
|
Study
|
[
0.9995357990264893,
0.00025187633582390845,
0.00021227820252534002
] |
[
0.9994291663169861,
0.00022720699780620635,
0.0002844345581252128,
0.00005922358832322061
] |
en
| 0.999997 |
To define the interacting region(s) of HBx on ERα, GST-fusion proteins containing various regions of HBx were prepared and the ability of each of these to interact with 35 S-methionine-labeled in vitro translated full-length ERα were determined by GST pull-down assay . Deletion of only the first 51 or last 11 amino acids of HBx did not affect the ability to interact with ERα. The GST-HBx(73–120) containing part of the transactivation domain bound specifically to ERα, but the GST-HBx(1–72) and the GST-HBx(121–154) did not.
|
16757575_p22
|
16757575
|
Mapping of the ERα and HBx interaction regions
| 4.138111 |
biomedical
|
Study
|
[
0.9995800852775574,
0.00020668251090683043,
0.0002131534565705806
] |
[
0.9993744492530823,
0.0002809344441629946,
0.0002894621866289526,
0.00005513397991308011
] |
en
| 0.999997 |
To map the domain of ERα responsible for interaction with HBx, a series of 35 S-methionine-labeled in vitro translated ERα mutants were used in GST pull-down experiments . The ERα(282–595) and the ERα(302–595) containing the AF2 domain were found to associate with HBx, whereas the ERα(1–185) containing the AF1 and the ERα(180–282) containing the DBD did not. ERΔ5, which has amino acid residues 1–365 of ERα also interacted with HBx, although with weak binding affinity.
|
16757575_p23
|
16757575
|
Mapping of the ERα and HBx interaction regions
| 4.173536 |
biomedical
|
Study
|
[
0.9994788765907288,
0.0002500485861673951,
0.0002710759290494025
] |
[
0.9994205236434937,
0.0002781279617920518,
0.00024476126418448985,
0.00005670707469107583
] |
en
| 0.999997 |
To test the possibility that the interaction of HBx with ERα is required for the repression of ERα transactivation function, the HBx mutant [HBx(Δ73–120)] in which the interaction region from amino acids 73 to 120 of HBx was deleted was constructed. HepG2 cells were co-transfected with the ERE-LUC reporter, ERα, and FLAG-tagged full-length HBx or HBx(Δ73–120). As shown in Figure 5A , the mutation lacking the ERα-binding site abrogated the repression of ERα transactivation function by HBx. Notably, FLAG-tagged HBx and HBx(Δ73–120) were expressed at comparable levels . To determine if HBx(Δ73–120) did lose the ability to interact with ERα in HepG2 cells, coimmunoprecipitation experiments were performed. As expected, HBx(Δ73–120) did not interact with ERα . Taken together, these findings suggest that interaction of HBx with ERα is required for repression of ERα transactivation function.
|
16757575_p24
|
16757575
|
Interaction of HBx with ERα is required for inhibition of ERα transactivation function
| 4.193316 |
biomedical
|
Study
|
[
0.9995243549346924,
0.00026895300834439695,
0.00020674530242104083
] |
[
0.9994383454322815,
0.00022101198555901647,
0.00026752144913189113,
0.00007317334529943764
] |
en
| 0.999997 |
To investigate whether the observed repression of ERα-responsive gene transcription by HBx was associated with recruitment of HDAC complexes, we examined the interaction between HBx and HDAC1 by GST pull-down assay. As shown in Figure 6A , in vitro translated HDAC1 interacted with GST-HBx, but not with GST alone, indicating that HBx associated with HDAC1 in vitro .
|
16757575_p25
|
16757575
|
HBx, ERα and HDAC1 form a complex
| 4.093981 |
biomedical
|
Study
|
[
0.9995630383491516,
0.00020994371152482927,
0.000226935968385078
] |
[
0.9995017051696777,
0.0002526317839510739,
0.0001915215398184955,
0.0000541471345059108
] |
en
| 0.999999 |
To determine whether the interaction of HBx with HDAC1 occurred in vivo , we performed coimmunoprecipitation and immunoblotting . Transient expression of FLAG-tagged HBx, but not control FLAG vector, in HepG2 cells was accompanied by interaction with HA-tagged HDAC1. These results suggest that HBx interacts with HDAC1 in vivo .
|
16757575_p26
|
16757575
|
HBx, ERα and HDAC1 form a complex
| 4.071887 |
biomedical
|
Study
|
[
0.9995470643043518,
0.0001953070895979181,
0.00025758714764378965
] |
[
0.9993507266044617,
0.00036863746936433017,
0.00022153384634293616,
0.00005910192339797504
] |
en
| 0.999997 |
To examine whether HBx, ERα and HDAC1 formed a complex, HepG2 cells were transfected with ERα and HA-tagged HDAC1 with or without FLAG-tagged HBx. The cells were then subjected to immunoprecipitation with FLAG antibody-conjugated agarose beads, followed by immunoblot with ERα and HA antibodies . Both ERα and HDAC1 were coprecipitated with FLAG-tagged HBx, but not FLAG control vector. To further confirm the possibility of a ternary complex among HBx, ERα, and HDAC1, the immune complexes precipitated with FLAG antibody were eluted with a FLAG peptide, and subjected to a second immunoprecipitation with an anti-ERα antibody. The anti-ERα immunoprecipitates were then subjected to immunoblotting with anti-HA to detect HA-tagged HDAC1. HA-tagged HDAC1 was present after sequential immunoprecipitation . In contrast, HA-tagged HDAC1 was absent after a second immunoprecipitation with control antibody. These data provide strong evidence that HBx, ERα and HDAC1 together can form a ternary complex in vivo .
|
16757575_p27
|
16757575
|
HBx, ERα and HDAC1 form a complex
| 4.241615 |
biomedical
|
Study
|
[
0.9995606541633606,
0.0002506886958144605,
0.0001886524260044098
] |
[
0.9992061257362366,
0.00033916073152795434,
0.00037688500015065074,
0.00007781727617839351
] |
en
| 0.999998 |
Our observation that HBx interacts with HDAC1 raises the possibility that the repression of ERα functions by HBx could be HDAC dependent. To this end, we examined the effect of TSA, a specific inhibitor of HDAC enzyme, on HBx-induced repression of ERE transcription in HepG2 cells . We found that HBx-mediated repression of ERα transcriptional activity could be effectively relieved by inhibiting HDAC activity. Interestingly, E 2 and TSA have additive or synergistic effect in stimulating ERα transcriptional activity. These data suggest that HBx may recruit HDAC enzyme to repress ERE-mediated transcription.
|
16757575_p28
|
16757575
|
HDAC inhibitor relieves repression of ERα transactivation function by HBx
| 4.177924 |
biomedical
|
Study
|
[
0.9995591044425964,
0.0002460891264490783,
0.00019480734772514552
] |
[
0.9993982315063477,
0.00024610679247416556,
0.00028582694358192384,
0.00006984719220781699
] |
en
| 0.999997 |
In the present study, we demonstrated for the first time that both ERΔ5 and HBx can inhibit ERα transcriptional activity in human liver cancer cells. We found that the repression of ERα transactivation function by ERΔ5 and HBx is mediated by their physical interaction with ERα. The binding of HBx with ERα is important for HBx-induced repression of ERα transactivation function because the HBx deletion mutant that lacks ERα-binding site completely abolished the repression of ERα transcriptional activity by HBx. Furthermore, we have shown that ERΔ5 and HBx have additive but not synergistic effect on repression of ERα-responsive gene transcription, suggesting that ERΔ5, ERα and HBx may not form a complex.
|
16757575_p29
|
16757575
|
DISCUSSION
| 4.194822 |
biomedical
|
Study
|
[
0.9995321035385132,
0.00027648304239846766,
0.0001914446329465136
] |
[
0.9993933439254761,
0.00022682322014588863,
0.0003063374024350196,
0.00007353857654379681
] |
en
| 0.999998 |
Estrogen has been reported to promote the growth of certain human neoplasms, notably tumors of the breast, endometrium and pituitary ( 40 , 41 ). In sharp contrast, Estrogen was shown to suppress the replication of HBV that has been clearly recognized as a major etiological factor for HCC ( 13 ). Studies of chemical carcinogenesis also suggested that ERα may modulate HCC risk by inhibiting the malignant transformation of pre-neoplastic liver cells. The ERα variant ERΔ5 was shown to be preferentially expressed in patients with HCC compared with parents with normal livers and to be associated with poor clinical outcome ( 25 – 28 ). Therefore, it is important to determine whether the ERΔ5 is able to interfere with the transcriptional activity of wt ERα. When ERΔ5 was expressed alone in human ERα-negative hepatoma HepG2 cells, it had little effect on either basal or E 2 -mediated ERE-containing reporter activity. However, when ERΔ5 was coexpressed with wt ERα, the reporter activity was significantly decreased, similar to that in human breast cancer cells ( 42 ). Our data suggest that ERΔ5 functions as a dominant negative receptor in human liver cancer cells. Although both in breast and liver cancer cells, ERΔ5 displays dominant negative activity, ERΔ5 was found to act as a dominant positive receptor isoform and facilitate both basal and E 2 -stimulated ERE-mediated transcription of wt ERα when coexpressed in ERα-negative human osteosarcoma U2-OS and human endometrial cancer Ishikawa cells ( 43 , 44 ). These discordant results could be due to the different cell types used, suggesting that some factors required for ERα transcriptional activity may be tissue specific.
|
16757575_p30
|
16757575
|
DISCUSSION
| 4.380504 |
biomedical
|
Study
|
[
0.9995137453079224,
0.00028393309912644327,
0.0002022809931077063
] |
[
0.9985747337341309,
0.0002787135017570108,
0.001033505075611174,
0.00011306509259156883
] |
en
| 0.999996 |
A number of studies have shown that HBx interacts with proteins involved in transcriptional regulation ( 3 – 5 , 45 ). Most of the studies identify HBx as a co-activator of transcription. For example, HBx enhances the transcription efficacy of the CREB transcription factor through interaction with CREB ( 46 ). HBx associates with hypoxia inducible factor-1α (HIF-1α), a major transcriptional factor that regulates expression of angiogenic factors such as vascular endothelial growth factor (VEGF), and enhances the transactivation function of HIF-1α ( 47 , 48 ). HBx was also shown to stimulate transcription by activating cellular signal transduction pathways. For instance, HBx is involved in activating Wnt/β-catenin signaling by stabilizing cytoplasmic β-catenin. HBx can stimulate activator protein 1 (AP-1) via two distinct pathways, the Ras-Raf-mitogen-activated protein kinase (MAPK) and the c-Jun amino-terminal kinase (JNK) cascades ( 49 ). On the other hand, HBx was found to act as a co-repressor of transcription. HBx interferes with p53 by direct binding and by sequestering p53 in the cytoplasm, resulting in the abrogation of p53-mediated transcriptional activity ( 6 ). HBx binds to DBD of peroxisome proliferator-activated receptor γ (PPARγ), a member of the steroid hormone receptor superfamily, and suppresses PPARγ-mediated transactivation ( 50 ). Our results showed that HBx represses ERα transcriptional activity through interaction with ERα and recruitment of HDAC1, which belongs to a class of enzymes involved in deacetylation of hyperacetylated histone tails, leading to compaction of chromatin and transcriptional repression ( 51 ). Importantly, the inhibitory effect of HBx on ERα transcriptional activity was antagonized by the HDAC inhibitor TSA. Interestingly, E 2 and TSA are additive or synergistic in inducing ERα transcriptional activity. The fact that HBx represses ERα transcriptional activity in the presence or absence of E 2 suggests that HBx regulates ERα transcriptional activity in a ligand-independent manner. Recently, the tumor suppressor BRCA1 has been shown to mediate ligand-independent transcriptional repression of ERα in a manner dependent on HDAC activity ( 52 ). Our observation that HBx, ERα and HDAC1 can form a complex and TSA can effectively reverse ligand-independent repression mediated by HBx suggests that one of the underlying mechanisms by which HBx mediates ligand-independent repression of ERα transcriptional activity may involve targeted recruitment by unliganded, promoter-bound ERα of a HBx-associated HDAC activity.
|
16757575_p31
|
16757575
|
DISCUSSION
| 4.779448 |
biomedical
|
Study
|
[
0.9986054301261902,
0.0009607922402210534,
0.0004338138969615102
] |
[
0.9964160919189453,
0.0008004767587408423,
0.002344020875170827,
0.0004393199924379587
] |
en
| 0.999998 |
Tamoxifen is considered to be relatively more estrogenic than antiestrogenic in the urine, bone and liver tissues ( 37 ). Thus, tamoxifen has been used for the treatment of liver cancer. Indeed, initial studies with a relatively small population of patients with HCC show regression of liver tumor mass and improved survival in some of the tamoxifen-treated patients ( 53 ). However, more and more recent controlled trials with this drug were disappointing ( 54 – 56 ). Tamoxifen does not prolong survival in patients with HCC and has an increasingly negative impact with increasing dose. There is also no appreciable advantage to quality of life with tamoxifen. These studies showed conclusively that, although the mechanisms by which tamoxifen negatively impacts HCC are not known, tamoxifen does not benefit patients with HCC and is likely to be detrimental. Thus, the use of tamoxifen in patients with HCC is not recommended. Our study indicated that 4-OHT, a metabolite of the tamoxifen with a more potent estrogen agonist/antagonist activity than tamoxifen, acts as a pure estrogen antagonist in HepG2 cells, which is in agreement with the previous study showing that tamoxifen functions as a pure estrogen antagonist in HepG2 cells ( 37 ). This may at least in part explain why tamoxifen is ineffective in the treatment of HCC.
|
16757575_p32
|
16757575
|
DISCUSSION
| 4.237666 |
biomedical
|
Study
|
[
0.9995809197425842,
0.0002562809968367219,
0.00016277212125714868
] |
[
0.9957160353660583,
0.00029837441979907453,
0.003862872486934066,
0.00012263785174582154
] |
en
| 0.999996 |
Recently, HDAC inhibitors have been used successfully to inhibit cancer cell growth in vitro and in vivo ( 57 – 59 ). TSA specifically inhibits classes I and II HDACs by binding directly to their catalytic site ( 3 ). Class I HDACs include HDAC1, HDAC2, HDAC3, HDAC8 and HDAC11, and Class II HDACs include HDACs 4–7 and HDACs 9–10. TSA regulates the expression of small subsets of growth-related genes and has potent antitumor activity in vitro and in vivo . In hepatoma cells, TSA induces a G 2 /M cell cycle arrest followed by apoptosis ( 60 , 61 ). Since E 2 and TSA are additive or synergistic in inducing ERα transcriptional activity in hepatoma cells, it is important to develop more effective therapeutic agents for HCC that increase ERα transactivation function, with no obvious side effects.
|
16757575_p33
|
16757575
|
DISCUSSION
| 4.172459 |
biomedical
|
Study
|
[
0.9996066689491272,
0.00020647718338295817,
0.00018690075376071036
] |
[
0.9802071452140808,
0.0005580640281550586,
0.01909315399825573,
0.00014159391866996884
] |
en
| 0.999996 |
Ribozymes isolated through in vitro selection catalyze reactions ranging from ligation and peptide bond formation to oxidation/reduction and Michael addition ( 1 – 5 ). Efforts are under way in several laboratories to use in vitro selected ribozymes to understand intrinsic constraints for RNA World evolution and to create artificial metabolisms either in extant cells or in artificial cells. Fully artificial ribozymes and aptamers can also be harnessed for the fabrication of molecular scale mechanical devices and to improve chemical process engineering. For example, the Lu group has described lead-sensing DNAzymes ( 6 ) and aptamer-conjugated gold nanoparticles that act as biosensors for free ATP ( 7 ), and the Jaeschke group has described enantioselective synthesis of a Diels–Alder cycloaddition product by passing substrates over a catalytic column of immobilized Diels-Alderase ribozyme ( 8 – 11 ). Ribozymes that transduce stored chemical energy into multiple cycles of mechanical work would represent a significant advance in nucleic acid nanotechnology.
|
16790565_p0
|
16790565
|
INTRODUCTION
| 4.339945 |
biomedical
|
Review
|
[
0.9975005984306335,
0.0009909651707857847,
0.0015083930920809507
] |
[
0.13649871945381165,
0.0029048637952655554,
0.8600403070449829,
0.0005560165736824274
] |
en
| 0.999995 |
An important function of enzymes is to capture stored chemical energy to drive thermodynamically unfavorable processes, such as a disfavored chemical transformation or the production of mechanical work. A common energy source for cellular enzymes is the hydrolysis of the β–γ phosphoanhydride bond of nucleotide triphosphates to yield the nucleotide diphosphate and inorganic phosphate. Nitrogenase reductase and receptor-coupled GTPases and other enzymes exploit differential binding energy between the NTP and NDP to drive conformational changes that either activate or inactivate the enzyme. Similarly, covalent phosphorylation regulates many enzymes, or drives conformational changes that yield mechanical work. The reversibility of such phosphorylation is essential for biologically responsive enzyme regulation and for completing the work cycle for energy-transducing molecular motors. The P-type ATP hydrolase enzymes comprise a large protein family of ATPases that generate essential ion gradients, which are the basis for such diverse functions as signaling, energy storage and secondary transport. They form a phosphorylated enzyme intermediate during ion transport (hence the name ‘P-type’), and then hydrolyze the phosphate to return to their resting conformation.
|
16790565_p1
|
16790565
|
INTRODUCTION
| 4.679944 |
biomedical
|
Study
|
[
0.9986213445663452,
0.0006766233709640801,
0.0007020421326160431
] |
[
0.8507446050643921,
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0.0010131588205695152
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en
| 0.999998 |
We previously described a set of ribozymes that were selected for autothiophosphorylation activity ( 12 ). The RNAs in the selected population use either ATP or ATPγS to (thio)phosphorylate internal 2′ hydroxyls, leading to the accumulation of (thio)phospho-ribozyme products. A truncated form of one of these ribozymes was separated into a 27 nt catalytic strand (2PTmin3.2) and a 26 nt substrate strand. Replacing most of the ribose with deoxyribose yielded a chimeric DNA–RNA–DNA species (DRD26) wherein eight ribonucleotides are flanked by deoxynucleotides on both sides. Detailed analysis of this complex mapped the site of phosphorylation to the second (G) within the unpaired sequence 5′-G G AAAA-3′ of DRD26 . Most of the deoxynucleotides of the substrate strand can be replaced by other nucleotides as long as Watson–Crick base pairing is maintained ( 12 ). In the present work we demonstrate that the 2PTmin3.2/DRD26 complex can release its covalent modification through hydrolysis. We first delineate the parameters that control this hydrolysis, then establish that the complex can participate in multiple cycles of thiophosphorylation and de-thiophosphorylation, thereby acting as a thio-ATPase (thio-ATP hydrolase). Because the catalytic cycle includes a thiophospho-ribozyme intermediate, this complex can loosely be thought of as a P-type riboATPase.
|
16790565_p2
|
16790565
|
INTRODUCTION
| 4.451188 |
biomedical
|
Study
|
[
0.9992870688438416,
0.0004679195408243686,
0.000245009723585099
] |
[
0.9990038275718689,
0.00042975731776095927,
0.0004058083286508918,
0.00016061231144703925
] |
en
| 0.999996 |
Oligonucleotides were purchased from Integrated DNA Technologies or were transcribed in vitro from synthetic templates. Ribozyme strand 2PTmin3.2 gave equivalent results when used as synthetic strand or when purified from in vitro transcriptions. ATPγS was purchased from Sigma, [γ- 32 P]ATP was purchased from MP Biomedicals.
|
16790565_p3
|
16790565
|
Materials
| 3.036651 |
biomedical
|
Study
|
[
0.9986515641212463,
0.0002776260080281645,
0.0010708863846957684
] |
[
0.8912387490272522,
0.10639629513025284,
0.001562311197631061,
0.000802616763394326
] |
en
| 0.999997 |
Substrate strands were 5′ radiolabeled with 32 P by incubating the initial strand with [γ- 32 P]ATP and T4 polynucleotide kinase. Ribozyme-catalyzed auto-(thio)phosphorylation reactions were performed under conditions described previously ( 12 ). In brief, the two strands of the complex were denatured at 82°C in a solution containing monovalent ions and pH buffer, allowed to cool, and then divalent metal ions were added. Final composition of the ‘selection buffer’ is 20 mM Mg 2+ , 5 mM Ca 2+ , 2.5 mM Mn 2+ , 10 μM each for Co 2+ , Cu 2+ , Zn 2+ and Ni 2+ , 50 mM K + , 150 mM Na + in 50 mM PIPES buffer at pH 6.4. Reactions were initiated by addition of ATPγS to a final concentration of 5 mM, stopped by addition of denaturing gel-loading buffer and quickly frozen at −80°C. De-thiophosphorylation reactions were performed by first purifying the thiophosphorylated substrate strand from the APM layer of a trilayer organomercurial gel ( 12 , 13 ) and then incubating the thiophosphorylated substrate strand DRD26 with or without the ribozyme strand 2PTmin3.2 under similar buffer conditions to those in which it had been thiophosphorylated except as noted in the text. All reactions were performed at 37°C unless otherwise noted. The fraction of the input material that had become de-thiophosphorylated at each time point was determined by separating the reaction products on a trilayer APM gel, exposing the dried gel to a phosphorimager screen (Typhoon, Molecular Dynamics), and quantifying the fraction in the APM layer using ImageQuant software.
|
16790565_p4
|
16790565
|
(thio)Phosphorylation and de-thiophosphorylation
| 4.207582 |
biomedical
|
Study
|
[
0.999395489692688,
0.0003718977386597544,
0.00023257656721398234
] |
[
0.9991152882575989,
0.0003773409116547555,
0.0004253859515301883,
0.00008186659397324547
] |
en
| 0.999997 |
To monitor ribozyme-catalyzed thiophosphate release, active complex was assembled by annealing 25 μM DRD26 and 75 μM 2PTmin3.2 in selection buffer with 5 mM ATPγS and incubated overnight. Control reactions included all-DNA versions of both strands. Samples were removed at 0, 3, 7, 11, 14 and 24 h, and quenched by rapid freezing on dry ice. Assays for free thiophosphate used malachite green, which is a dye that has been shown previously to exhibit greatly increased absorbance at 650 nm upon binding to inorganic phosphate. Calibration with Na 3 PO 3 S showed a similar linearity for thiophosphate . For each time point from the RNA-catalyzed reactions, 2 μl of the reaction mixture was diluted to 800 μl, followed by addition of 200 μl of malachite green solution supplied in the kit (Bioassay Systems). This dilution brought the free thiophosphate concentration into the linear range of the assay (0.1–5 μM) and prevented metal ion-induced precipitation of Malachite Green at concentrations of free phosphate above 10 μM. Dilution mixtures were incubated at room temperature for 11 min before measuring absorbance at 650 nm. Absorbance was converted to the concentration of the thiophosphate in the experimental sample using the linear relation between absorbance and concentration. To keep the final thiophosphate concentrations within the linear range of the assay, the 14 and 24 h time points were diluted 500 and 600 times and therefore a correction factor of 1.25 and 1.5 was applied to these time points in Figure 6. The commercial ATPγS (90% purity) contained a significant amount of ADP (by high-performance liquid chromatography, data not shown) and free thiophosphate, presumably from hydrolysis.
|
16790565_p5
|
16790565
|
Multiple-turnover ATPase activity analysis
| 4.248353 |
biomedical
|
Study
|
[
0.9994028806686401,
0.0003803246363531798,
0.00021683517843484879
] |
[
0.9992152452468872,
0.00035876536276191473,
0.0003283926926087588,
0.00009752225741976872
] |
en
| 0.999997 |
When ribozyme 2PTmin3.2 is annealed with 5′ radiolabeled substrate strand DRD26 and incubated in 5 mM ATPγS, thiophosphorylation of the substrate proceeds with a first-order rate constant, k obs ≈ 0.21 h −1 , similar to values obtained previously ( 12 ). Thiophosphorylated DRD26 was purified from the organomercurial layer of a trilayer gel containing acryloylamino-phenylmercuric chloride (APM) ( 13 , 14 ), annealed to ribozyme strand, and incubated in selection buffer in the presence of 5 mM ADP. When the products of this reaction were separated on a second trilayer organomercurial gel, the fraction of the radioactivity that shifted into the APM layer decreased with time, indicating loss of thiophosphate from the radiolabeled strand . Interestingly, there is a highly reproducible lag in the reaction, with little de-thiophosphorylation occurring within the first hour followed by an increased rate of loss of thiophosphate. Similar biphasic kinetics were observed throughout this study.
|
16790565_p6
|
16790565
|
Phosphorylation and de-thiophosphorylation of substrate strand
| 4.243099 |
biomedical
|
Study
|
[
0.9994156360626221,
0.0003393899241928011,
0.00024490998475812376
] |
[
0.9993565678596497,
0.00023901952954474837,
0.0003218857164029032,
0.00008252781844930723
] |
en
| 0.999996 |
To discern whether the loss of thiophosphate from the DRD26 strand indicates thiophosphate hydrolysis or ATPγS synthesis, thiophosphorylated RNA was incubated in selection buffer either with or without ribozyme (no ADP in either reaction). De-thiophosphorylation kinetics under these conditions were essentially identical to those observed for the complete reaction . The substrate strand is predicted to be able to form alternative dimerized structures with as many as 10 Watson–Crick base pairs. Several of these structures place the thiophosphorylated nucleotide in a structural context that is similar to that of the ribozyme–substrate complex . To rule out the possibility that alternative structures were responsible for the ribozyme-free reaction observed in Figure 2A , de-thiophosphorylation of 0.5 μM radiolabeled DRD26 strand was monitored in the presence of increasing concentrations of unlabeled DRD26. Rather than promoting the reaction, the unlabeled strand was inhibitory, with little de-thiophosphorylation observed when the total DRD26 concentration reached 2.5 μM . In contrast, de-thiophosphorylation proceeded normally when dimerization was prevented by including 10 μM ribozyme strand along with 2.5 μM DRD26 , or when DRD26 was annealed to a fully complementary DNA oligonucleotide (data not shown). Normal de-thiophosphorylation was also observed when the substrate strand was incubated alone in the presence of 8 M urea (data not shown). Taken together with the results of Figure 2A , these results indicate that de-thiophosphorylation is most efficient in unstructured RNA, that substrate self-dimerization is inhibitory, and that the reaction does not require either ADP or the folded secondary structure of the assembled complex, although it can be inhibited by some specific secondary structural contexts (potential self-dimers).
|
16790565_p7
|
16790565
|
Phosphorylation and de-thiophosphorylation of substrate strand
| 4.396469 |
biomedical
|
Study
|
[
0.9992710947990417,
0.0005033847410231829,
0.00022546832042280585
] |
[
0.9989405274391174,
0.0004034142766613513,
0.0005045087891630828,
0.00015151397383306175
] |
en
| 0.999997 |
When DRD26 de-thiophosphorylation kinetics were monitored over the temperature range of 20–55°C, the amount of de-thiophosphorylation after 4 h increased with increasing temperature . Some of this effect may be due to enhanced intrinsic chemical reactivity with increasing temperature. However, the magnitude of the de-thiophosphorylation increased markedly between 30 and 40°C . This apparently cooperative transition supports a model in which inhibitory secondary structures melt over this range to expose the 2′-thiophosphate to hydrolysis. The shortening of the lag phase of the reaction at elevated temperatures is especially evident when yield after 2 h is compared across the temperature range .
|
16790565_p8
|
16790565
|
Temperature and pH dependence of the de-thiophosphorylation reaction
| 4.195838 |
biomedical
|
Study
|
[
0.9993921518325806,
0.00030643842183053493,
0.00030144021729938686
] |
[
0.9994788765907288,
0.00022288448235485703,
0.00023719888122286648,
0.00006103966006776318
] |
en
| 0.999995 |
When the pH of de-thiophosphorylation reactions was increased from 6.3 to 8.5, there was only a slight diminution (∼20%) in the extent of the reaction after 4 h at the highest pH values, in spite of the >150-fold increase in hydroxide ion concentration over this pH range . Thus, proton transfer does not control the rate of de-thiophosphorylation. If the chemical step of the reaction determines the rate, these observations are consistent with an S N1 -like, dissociative reaction mechanism in which the limiting step is the formation of a planar, metaphosphate transition state, rather than the deprotonation of an attacking nucleophile such as water. Consistent with this model, hydrolysis of organic phosphate monoesters—such as sugar phosphates, serine or tyrosine phosphates and the γ-phosphoryl group of ATP in aqueous solution—also progresses through a predominately dissociative reaction pathway that is largely independent of pH ( 15 – 18 ). Alternatively, the rate limiting step for de-thiophosphorylation of DRD26 may be a slow, pH-independent conformational change that occurs prior to the chemical step.
|
16790565_p9
|
16790565
|
Temperature and pH dependence of the de-thiophosphorylation reaction
| 4.414992 |
biomedical
|
Study
|
[
0.9991644620895386,
0.0004999849479645491,
0.0003355430962983519
] |
[
0.9990134239196777,
0.0003673593164421618,
0.000494228966999799,
0.00012492014502640814
] |
en
| 0.999996 |
De-thiophosphorylation of the DRD26 strand was carried out in the presence of several combinations of metal ions. In the first series of reactions, the concentrations of all the divalent metal ions were gradually increased to triple that of the original selection buffer. Over this concentration range there is little change in de-thiophosphorylation, although the overall trend is toward slightly reduced rates and reduced final extent of the reaction at the highest divalent ion concentration . The second series of reactions included various subsets of the ions from the selection buffer, each at their original concentrations. When all components were present, de-thiophosphorylation was essentially complete by 5 h. Both the rate and the final extent of de-thiophosphorylation was reduced when all of the trace transition metals (Zn 2+ , Co 2+ , Ni 2+ , Cu 2+ ) were omitted. Further reductions were observed when Mn 2+ or all added divalent ions were omitted . Interestingly, Mg 2+ by itself appeared to protect against de-thiophosphorylation . For the third set of reactions, the trace transition metal ions were omitted individually while the others were maintained at a combined total of 40 μM. All the other components of the reaction mixture were unaltered. Reactions that included combinations of two or three of the trace transition metal ions yielded complete de-thiophosphorylation of the substrate strand within 4 h. Interestingly, the de-thiophosphorylation yield was reduced by nearly half when only one trace metal was included at a time . Finally, in the fourth set of reactions, the total transition metal ion concentration was increased from 40 to 100 μM, again with the other reaction components unaltered . For each reaction in this set, a similar sigmoidal pattern is observed, with slightly lesser extent of de-thiophosphorylation with increasing concentrations of transition metal ions, possibly due to non-specific binding of these soft metal ions to soft ligands in the RNA. Thus, the de-thiophosphorylation reaction depends heavily on the combination of metal ions present, with significant stimulation by low concentrations of the trace transition metals, slight reduction of yield at higher concentrations, some dependence on the identity of the transition metal ion utilized, and apparent protection by Mg 2+ .
|
16790565_p10
|
16790565
|
Metal ion dependence of the de-thiophosphorylation reaction
| 4.407079 |
biomedical
|
Study
|
[
0.9992309808731079,
0.000472041720058769,
0.00029690295923501253
] |
[
0.9984702467918396,
0.000288000563159585,
0.0011284230276942253,
0.00011338906188029796
] |
en
| 0.999996 |
If the de-thiophosphorylation reaction is a simple hydrolysis, it should regenerate the original DRD26 strand, which should then be capable of re-thiophosphorylation with ATPγS. To test this hypothesis, ribozyme/substrate complex was allowed to de-thiophosphorylate for 5 h, after which ATPγS was added to a final concentration of 5 mM . Re-thiophosphorylation kinetics during the second phase of the reaction were nearly identical ( k = 0.24 h −1 ; plateau value 55%) to those observed when naïve complex was incubated with ATPγS . Thus, the de-thiophosphorylation reaction does not damage or modify the DRD26 strand in any way that impedes its reactivity with ATPγS. Surprisingly, a parallel reaction that included ATPγS from the beginning showed no change in DRD26 thiophosphorylation over the course of the reaction . The fact that this mixture does not de-thiophosphorylate to the same 60% value obtained upon thiophosphorylation of naïve or fully de-thiophosphorylated species may suggest conformational dynamics of the annealed complex (see discussion).
|
16790565_p11
|
16790565
|
De-thiophosphorylation and re-thiophosphorylation
| 4.271299 |
biomedical
|
Study
|
[
0.999407172203064,
0.0003501896280795336,
0.0002426888677291572
] |
[
0.9992538094520569,
0.00034781204885803163,
0.0003121252520941198,
0.00008628271461930126
] |
en
| 0.999996 |
The data above suggest that the complex is capable of undergoing multiple cycles of de-thiophosphorylation and re-thiophosphorylation at the expense of ATPγS, thereby acting as a multiple-turnover thio-ATPase ribozyme (ATPγS hydrolase). To test this possibility directly, inorganic thiophosphate release was monitored using malachite green, a dye that exhibits greatly increased absorbance at 650 nm upon binding to inorganic phosphate ( 19 – 21 ). We find that the response of the malachite green signal is also linear with thiophosphate concentration , making it appropriate for monitoring thiophosphate release from ATPγS.
|
16790565_p12
|
16790565
|
A multiple-turnover thio-ATPase ribozyme (ribo thioATPase)
| 4.243124 |
biomedical
|
Study
|
[
0.9994682669639587,
0.0002625289198476821,
0.00026931188767775893
] |
[
0.999255359172821,
0.00039840282988734543,
0.00026827718829736114,
0.00007796120917191729
] |
en
| 0.999998 |
Unlabeled substrate strand DRD26 and ribozyme strand 2PTmin3.2 were annealed and incubated overnight in selection buffer with 5 mM ATPγS. A parallel reaction in which both strands were present in all-DNA (inactive) form was included as control. Aliquots were taken from both reactions at various times, diluted to the linear concentration range of the malachite green assay and monitored spectrophotometrically for free thiophosphate. The excess of released thiophosphate in the experimental sample versus the control increased with time . After a short lag, product formation was linear during the 24 h of the assay and showed no sign of plateauing. These observations are consistent with a model in which the annealed complex was actively liberating thiophosphate from ATPγS via a thiophospho-ribozyme intermediate. By the end of the assay, free thiophosphate concentrations reached 940 ± 230 μM. Because the ribozyme/substrate complex was only 25 μM, this indicates that the complex had catalyzed 29–46 turnovers in 24 h, corresponding to a net ATPγS hydrolysis rate of 1–2 h −1 . The annealed ribozyme/substrate complex is therefore a multiple-turnover thio-ATP hydrolase.
|
16790565_p13
|
16790565
|
A multiple-turnover thio-ATPase ribozyme (ribo thioATPase)
| 4.442526 |
biomedical
|
Study
|
[
0.999189555644989,
0.0006026469054631889,
0.00020780356135219336
] |
[
0.9985363483428955,
0.0007053800509311259,
0.000548241485375911,
0.00020999064145144075
] |
en
| 0.999997 |
In addition to the originally selected autothiophosphorylation activity of ribozyme 2PTmin3.2 ( 12 ), we show here that this ribozyme undergoes multiple cycles of thiophosphorylation/de-thiophosphorylation to yield a net conversion of ATPγS into ADP and thiophosphate (riboATPγS hydrolase). The reaction generates a thiophospho-ribozyme intermediate, leading us to refer to the annealed complex as a ‘P-type riboATPase (thio-ATP hydrolase) ribozyme.’ Formation of the thio-phosphoribozyme intermediate requires divalent metal ions ( 12 ) and is inhibited by ADP (data not shown), while thiophosphate hydrolysis yields the original complex.
|
16790565_p14
|
16790565
|
Multiple-turnover catalysis
| 4.301225 |
biomedical
|
Study
|
[
0.9994542002677917,
0.0003006028418894857,
0.0002451615291647613
] |
[
0.9991957545280457,
0.0004438894393388182,
0.0002692749258130789,
0.0000910712915356271
] |
en
| 0.999996 |
The multiple turnover nature of this complex is fundamentally different from that of previously described trans -acting ribozymes. For example, the catalytic cycle of ribozyme Kin.46 is defined by five steps: (i) annealing of a 7 nt RNA substrate to an internal guide sequence in the ribozyme, (ii) binding of ATP (or ATPγS), (iii) (thio)phosphoryl transfer to the substrate 5′ hydroxyl group, (iv) ADP release and (v) dissociation of the 7 nt product strand ( 18 ). For the DRD26/2PT3.2 complex, the nucleic acid strands do not dissociate. Instead, the catalytic cycle is defined by four steps: (i) binding of ATPγS, (ii) thiophosphoryl transfer to a specific guanosine 2′ hydroxyl, (iii) ADP release and (iv) hydrolysis of the 2′ thiophosphate.
|
16790565_p15
|
16790565
|
Multiple-turnover catalysis
| 4.448807 |
biomedical
|
Study
|
[
0.9994237422943115,
0.00034346425672993064,
0.0002328068221686408
] |
[
0.9979089498519897,
0.0011020301608368754,
0.0008339700871147215,
0.00015499354049097747
] |
en
| 0.999996 |
Organic thiophosphates are normally much more stable than what we observe here for 2′-thiophosphorylated RNA. For example, 5′-thiophosphoryl groups on RNA and DNA remain attached for many hours at temperatures as high as 70°C in aqueous solutions that contain K + and Mg 2+ ( 13 ). We have observed similar stability for 5′ thiophosphorylated RNAs incubated at 37°C in the selection buffer used to identify 2PT3.2 (data not shown). The 2′ thiophosphorylated RNA therefore behaves differently from RNA modified at the 5′ position. Similarly, substituted O -aryl phosphorothioates, such as 3,4-dinitro substituted or 4-nitro substituted benzyl phosphorothioates, were previously shown to be stable in aqueous solution ( 17 , 22 , 23 ), and Sekine et al . found that 2′ O -thiophosphate RNA derivatives were stable for >24 h in aqueous and non-aqueous solvents ( 24 ). However, none of these last experiments included any divalent metal ions, while the original selection of isolate 2PT3.2 included various alkaline earth and transition metal ions to provide the evolving library the opportunity to exploit the unique chemical reactivity of each ion ( 12 ).
|
16790565_p16
|
16790565
|
De-thiophosphorylation chemistry
| 4.198628 |
biomedical
|
Study
|
[
0.9995180368423462,
0.00021064982865937054,
0.00027125803171657026
] |
[
0.999198853969574,
0.00023672683164477348,
0.0005019753589294851,
0.00006249010766623542
] |
en
| 0.999997 |
The mechanism of 2′ de-thiophosphorylation is suggested by several observations of the parameters that regulate reactivity. First, the reaction does not require a specific secondary structure, proceeds normally in 8 M urea, and is inhibited by duplex formation and by self dimerization of the substrate strand, thus ruling out a ribozyme-catalyzed hydrolysis. Second, the reaction is highly dependent on the metal ions used in the assays, with significant stimulation by Mn 2+ and low concentrations of soft transition metal ions. Third, in contrast to the de-thiophosphorylation reaction, hydrolysis of normal phosphate (from the product of a [ 32 P]ATP reaction) is essentially undetectable under similar reaction conditions . Fourth, release of thiophosphate from 2′ modified RNA is much more rapid than release from 5′-modified RNA or DNA under similar conditions ( 13 , 18 ).
|
16790565_p17
|
16790565
|
De-thiophosphorylation chemistry
| 4.386745 |
biomedical
|
Study
|
[
0.9994739890098572,
0.0003240843943785876,
0.0002019693492911756
] |
[
0.9986557960510254,
0.0004935617907904088,
0.0007371695246547461,
0.00011352571164024994
] |
en
| 0.999997 |
Each of these observations is consistent with a metal ion-catalyzed reaction in which the metal ion binds directly to the sulfur of the thiophosphate through inner sphere coordination. The metal ion may promote the reaction by reducing the negative charge density in the transition state, by stabilizing the leaving group, by increasing the electrophilicity of the phosphorous atom for attack by water, or by a combination of these effects. The metal ion may also interact with an adjacent backbone phosphate oxyanion, either through direct inner sphere coordination or by indirect outer sphere effects (Coulombic or water-mediated), either of which would serve to increase its occupancy at this site. This secondary coordination is not readily available at the 5′ end, which may contribute to the differential stability of 5′ versus 2′-thiophosphates. Coordination between the soft metal ion and soft ligand is eliminated when the transition metals are replaced by hard metals (such as magnesium), and when the thiophosphate is replaced by phosphate.
|
16790565_p18
|
16790565
|
De-thiophosphorylation chemistry
| 4.52629 |
biomedical
|
Study
|
[
0.9992281198501587,
0.0005340984207578003,
0.00023770997358951718
] |
[
0.9961003065109253,
0.001826619147323072,
0.0017726385267451406,
0.0003004948375746608
] |
en
| 0.999998 |
The 2PTmin3.2/DRD26 complex reproducibly attains ∼60% thiophosphorylation upon extended incubations with ATPγS. Plateau values significantly <100% are commonly observed for other small ribozymes, and are usually interpreted to indicate either that the ribozyme is partially misfolded, or that both the forward and reverse reactions (e.g. cleavage and ligation for small nuclease ribozymes) contribute to establishing an internal equilibrium ( 25 – 27 ). A third possibility is that the complex arrives at a steady state between thiophosphorylation at the expense of ATPγS and de-thiophosphorylation to release thiophosphate. These models may contribute to some of the behavior of the DRD26/2PT3.2 complex. Although internal equilibration of the catalysis is ruled out—the reverse reaction (ATPγS synthesis) does not occur to an appreciable extent—the turnover rate of the annealed complex (∼1–2 h −1 ) is approximately an order of magnitude higher than the apparent forward rate of the reaction , suggesting that the latter value probably reflects an approach to a steady state, rather than a true rate of thiophosphorylation.
|
16790565_p19
|
16790565
|
Potential conformational dynamics during the catalytic cycle
| 4.47321 |
biomedical
|
Study
|
[
0.9992387294769287,
0.00047395803267136216,
0.00028726618620567024
] |
[
0.9987718462944031,
0.00046197802294045687,
0.0006236042827367783,
0.00014244485646486282
] |
en
| 0.999995 |
However, these models fail to account for the lack of appreciable net de-thiophosphorylation in the presence of APTγS . An interesting possibility is that the persistence of a fully thiophosphorylated population may result from conformational dynamics of the ribozyme. Specifically, (thio)phosphorylation is proposed to lock the ribozyme into a catalytically active conformation, effectively preventing it from re-equilibrating into alternative, inactive states. A complex that assembles by incorporating an unmodified DRD26 strand is expected to equilibrate between active and inactive states, with ∼60% in the active conformation . In contrast, a complex that assembles by incorporating a pre-thiophosphorylated DRD26 strand is expected to fold essentially completely into the active complex . If conformational re-equilibration is slow upon de-thiophosphorylation, the complex will be left for a short time in the catalytically competent conformation. During this time it can either re-thiophosphorylate rapidly without re-equilibration if ATPγS is present, , or it can slowly re-equilibrate between active and inactive conformations in the same 60:40 ratio as for the naïve complex if ATPγS is absent. Subsequent addition of ATPγS would then yield the same 60% product as that observed for naïve complex .
|
16790565_p20
|
16790565
|
Potential conformational dynamics during the catalytic cycle
| 4.468615 |
biomedical
|
Study
|
[
0.9992586970329285,
0.00045668985694646835,
0.00028452821425162256
] |
[
0.9983083009719849,
0.0008616454433649778,
0.0006726423744112253,
0.00015737078501842916
] |
en
| 0.999996 |
The existence of a thio-ATP-powered catalytic cycle raises interesting possibilities for nucleic acid nanotechnologies. The active complex can clearly exist in at least two states during its catalytic cycle (with and without thiophosphate). If the angle and/or distance between the distal helical elements is altered by the presence of the (thio)phosphate—which seems likely given the introduction of bulk and charge—then the catalytic cycle could be used to transduce chemical energy into mechanical work. Additional kinase ribozymes that form 2′ thiophosphoryl intermediates ( 12 , 28 , 29 ) may be capable of similar cycles. A new challenge will therefore be to exploit this catalytic activity to capture the energy of ATPγS (or ATP) hydrolysis to drive otherwise unfavorable events, such as to power RNA-based molecular motors and nanodevices.
|
16790565_p21
|
16790565
|
Potential conformational dynamics during the catalytic cycle
| 4.352745 |
biomedical
|
Study
|
[
0.9994868040084839,
0.0002443903067614883,
0.00026882291422225535
] |
[
0.9903535842895508,
0.001958617940545082,
0.00752686895430088,
0.0001609229511814192
] |
en
| 0.999996 |
DNA polymerases of the Y-family have in recent years been shown to play a predominant role in synthesis past DNA bulky lesions, such as those derived from polycyclic aromatic chemicals present in tobacco smoke, automobile exhaust, and broiled meat and fish ( 1 – 5 ). High fidelity replicative DNA polymerases are usually impeded by such damage, leading to a switch to one or more low fidelity bypass polymerases for translesion synthesis ( 6 – 11 ).
|
16820532_p0
|
16820532
|
INTRODUCTION
| 4.239744 |
biomedical
|
Study
|
[
0.9995483756065369,
0.00021461959113366902,
0.00023694813717156649
] |
[
0.9656217098236084,
0.001863231766037643,
0.03232920914888382,
0.00018588789680507034
] |
en
| 0.999998 |
The model Y-family polymerase, DNA polymerase IV (Dpo4), is from the archaeon bacterium Sulfolobus solfataricus P2. It is a member of the DinB family ( 12 ), of which human pol κ is also a member ( 13 ). It has been extensively investigated by X-ray crystallography in binary complexes with primer/template DNA and in ternary complexes in the presence of dNTP, both with and without DNA damage ( 14 – 23 ). These structures have revealed a spacious, water accessible active site that is capable of accommodating two templating bases, in contrast to high fidelity replicative polymerases whose ternary complexes show tight fit of the nascent base pair with exclusion of solvent ( 24 ). Dpo4 has an open pocket on the major groove side of the template, as well as a smaller open space on the minor groove side; this is in contrast to replicative polymerases, which only have an open pocket on the major groove side, while the minor groove side is packed with protein–DNA interactions critical for polymerase fidelity ( 25 – 29 ). In addition like other Y-family polymerases, Dpo4 has a unique little finger domain, also called wrist or polymerase associated domain (PAD) at the C-terminus ( 15 , 30 , 31 ). The flexibility of this little finger domain is believed to play an important role in accommodating specific types of DNA lesions ( 14 , 15 , 21 ). Crystal structures of Dpo4 binary and ternary complexes also reveal that the little finger domain plays a key role in translocation ( 23 ).
|
16820532_p1
|
16820532
|
INTRODUCTION
| 4.557771 |
biomedical
|
Study
|
[
0.9992302656173706,
0.00044700931175611913,
0.00032269206712953746
] |
[
0.9981016516685486,
0.00043533294228836894,
0.0013090333668515086,
0.00015396426897495985
] |
en
| 0.999997 |
Recently, it has been suggested that the DinB family polymerases may be specifically suited for bypass of N 2 -dG adducts ( 32 ). The presence of a conserved ‘steric gate’, usually phenylalanine or tyrosine, is shown to be crucial in bypass of N 2 -dG minor groove adducts ( 32 ). However, the DinB polymerases may be less well suited for bypassing C8-dG bulky adducts. Such adducts would normally be expected, from their position of substitution on guanine, to reside in the major groove of double-stranded DNA. However, rotation of the modified guanine from anti to syn would place the adduct in the minor groove area, in a position roughly similar to an N 2 -substituted guanine. Despite this formal possibility, experimental primer extension data for two C8-dG adducts derived from 2-acetylaminofluorene (AAF), namely N -(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) with Dpo4 ( 12 ) and N -(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) with Escherichia coli DinB and human pol κΔc (33), indicates that these adducts cause polymerase stalling or blockage, with only small amounts of primer extension beyond the lesion site. A recent molecular dynamics (MD) study from our group has provided structural rationale for the case of dG-C8-AAF in Dpo4 ( 34 ). However, this adduct contains an acetyl group, which adds to the steric hindrance in C8 adducts.
|
16820532_p2
|
16820532
|
INTRODUCTION
| 4.403841 |
biomedical
|
Study
|
[
0.9993264675140381,
0.0003813571238424629,
0.0002922569983638823
] |
[
0.9990485310554504,
0.0002919765829574317,
0.0005442514666356146,
0.00011517371603986248
] |
en
| 0.999996 |
Here, we investigate a C8-dG adduct derived from the most prevalent heterocyclic aromatic amine formed by cooking proteinaceous food, 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) ( 35 – 43 ). This substance causes predominantly G to T transversions in mammalian cells, with some G to A transitions, and a few deletions ( 44 – 52 ). In a previous molecular modeling and MD study of this adduct in the Pol α family replicative polymerase RB69, we found considerable active site distortion caused by this lesion at the templating base or in the first double-stranded extension position, when partnered by C or A ( 53 ). These results suggested that the replicative polymerase would be stalled by the lesion, affording opportunity for switch to one or more bypass polymerases. We now use this dG-C8-PhIP adduct, N 2 -(2′-deoxyguanosin-8-yl)-PhIP, as a model to study the structural feasibility of accommodating a C8-dG lesion lacking the acetyl group in Dpo4, employing Dpo4 type I ( 16 ) and type II ( 15 ) crystal structures as initial models .
|
16820532_p3
|
16820532
|
INTRODUCTION
| 4.310583 |
biomedical
|
Study
|
[
0.9992838501930237,
0.00041326237260363996,
0.0003029877261724323
] |
[
0.9994288086891174,
0.00020778794714715332,
0.00026995385996997356,
0.00009337126539321616
] |
en
| 0.999998 |
Our results show that this dG-C8 adduct, like dG-C8-AAF, if accommodated on the Dpo4 minor groove side pocket, would cause serious distortions to the active site region. However, the PhIP ring system can be accommodated in the spacious major groove pocket of this polymerase, and Dpo4 appears capable of incorporating dCTP, dTTP or dATP reasonably well. In addition, our results show that the PhIP-modified lesion produces less distorted structures for anti -G* ·anti -dTTP and anti -G* ·syn -dATP than for their respective unmodified controls ( anti -G ·anti -dTTP and anti -G ·syn -dATP). These findings may be relevant to observed mutagenic behavior of PhIP in inducing G to T transversions and G to A transitions in mammalian systems ( 44 – 52 ), if the human DinB polymerase pol κ is involved in the error-prone incorporation opposite the lesion, and its structural properties prove to be similar to those of its prokaryotic homolog Dpo4. Finally, based on Dpo4 binary and ternary complex structures ( 23 ), we suggest that translocation may be seriously inhibited by bulky dG-C8 adducts positioned on the major groove side of the DNA duplex region, while N 2 -dG adducts residing on the minor groove side ( 54 ) would be less hindering.
|
16820532_p4
|
16820532
|
INTRODUCTION
| 4.512414 |
biomedical
|
Study
|
[
0.9990471005439758,
0.0006653072778135538,
0.0002875594364013523
] |
[
0.9987949132919312,
0.00048506882740184665,
0.00048639136366546154,
0.00023356227029580623
] |
en
| 0.999997 |
G·dNTP initial models. The crystal structure of Dpo4 polymerase with 10 R (+)- cis-anti -benzo[ a ]pyrene(BP)- N 6 -dA located on the major groove side of the modified DNA (BP-2 complex) ( 16 ) was used to obtain initial structures for the G·dNTP models (PDB ID: 1S0M) ( 55 ). Of the many type I structures of Dpo4, the BP-2 complex has the most reaction-ready active site, since it contains two metal ions in the active site and the 3′ hydroxyl group at the primer end. This structure was further remodeled, as described in previous work from our group ( 34 ), to achieve ideal Mg 2+ coordination (Supplementary Table S1) and an O3′-Pα distance of 3.1 Å, in the reaction-ready range.
|
16820532_p5
|
16820532
|
Molecular modeling
| 4.280342 |
biomedical
|
Study
|
[
0.999406099319458,
0.0003247520071454346,
0.00026916415663436055
] |
[
0.9993329644203186,
0.00032651101355440915,
0.0002608620561659336,
0.00007955440378282219
] |
en
| 0.999997 |
The DNA sequence was then adjusted to match the sequence in the adenomatous polyposis coli ( Apc ) gene mutational hotspot for PhIP, codon 635 , with the guanine selected for PhIP modification situated at the templating position opposite the incoming dNTP. The initial models for dynamics were then constructed by locating structures with minimal crowding between the PhIP moiety and the polymerase, by rotating α′ and β′ at 10° intervals, in combination . γ′ was initiated at 26° as in the NMR solution structure ( 56 ). Both anti and syn conformations of the glycosidic torsion χ were investigated, with χ adjusted to achieve optimal hydrogen bonding and stacking in the nascent base pair for each χ domain. The torsion angles in the initial models are summarized in Supplementary Table S2.
|
16820532_p6
|
16820532
|
Molecular modeling
| 4.240675 |
biomedical
|
Study
|
[
0.9994487166404724,
0.0003231756272725761,
0.00022814639669377357
] |
[
0.9993987083435059,
0.0002319944615010172,
0.0002906204608734697,
0.00007877084135543555
] |
en
| 0.999996 |
For the G*·dATP mismatch, three starting models were obtained. Two of them featured the PhIP-modified guanine (G*) anti and the PhIP rings on the major groove side of the template, with the incoming nucleotide dATP either anti or syn . A syn -dATP was investigated since it has been observed opposite a lesion in a Dpo4 crystal structure ( 17 ). A high resolution NMR solution structure containing a dG-C8-PhIP in an 11mer duplex shows a base-displaced intercalation model ( 56 ). In this conformation, the modified guanine is syn and displaced into the major groove, while the PhIP rings intercalate within the DNA duplex. However, the PhIP rings in such a base-displaced intercalation conformation would occupy the position of the incoming nucleotide in Dpo4. A PhIP-modified syn -guanine model could be built with the PhIP rings on the minor groove side of the dNTP, which does allow the accommodation of the dNTP at the active site. With a syn -G* as the template, only anti -dATP is favorable, since the bases in the syn -G*· syn -dATP pair are too far apart for hydrogen bonding. Hydrogen bonding in the nascent base pair is an important consideration in building the initial models, since Dpo4 as well as other DinB family polymerases, such as Dbh and pol κ rely more on hydrogen bonding for catalytic efficiency than the high fidelity polymerases ( 57 , 58 ).
|
16820532_p7
|
16820532
|
Molecular modeling
| 4.434576 |
biomedical
|
Study
|
[
0.9992295503616333,
0.0004322325112298131,
0.0003381648857612163
] |
[
0.999056875705719,
0.00034959978074766695,
0.00046952537377364933,
0.00012407165195327252
] |
en
| 0.999998 |
In the G*·dCTP/dTTP/dGTP series, the G*s were all constructed in the same anti conformation as in the G*·dATP mismatch. A syn -G* was eliminated based on results for the syn -G*· anti -dATP simulation; these show that a syn -G* generated much more disturbance to the polymerase active site than an anti one, as described in Results, and these disturbances are essentially independent of the dNTP. In the G*·dCTP model, the incoming dCTP is anti , in order to form Watson–Crick hydrogen bonds with the anti -G*. In the G*·dTTP model, the dTTP is anti to achieve a wobble pair with the anti -G*. A wobble paired T·dGTP has been observed in a Dpo4 ternary complex ( 18 ). A syn -dTTP can form no hydrogen bonds with anti -G* and syn pyrimidines have scarcely been observed ( 59 ). In the G*·dGTP model, a syn -dGTP was employed, since an anti -dGTP could not form hydrogen bonds with anti -G* in the polymerase, and would collide with either the anti -G* or the minor groove side protein residues.
|
16820532_p8
|
16820532
|
Molecular modeling
| 4.363783 |
biomedical
|
Study
|
[
0.9992456436157227,
0.00042494654189795256,
0.00032948621083050966
] |
[
0.9991984963417053,
0.0003046361671295017,
0.0003980414185207337,
0.00009882696758722886
] |
en
| 0.999997 |
As an unmodified control for all the simulations, an unmodified anti -G opposite a Watson–Crick paired anti -dCTP was used. Additional controls containing an unmodified anti -G opposite anti -dATP for the G*·dATP mismatch, anti -dTTP for the G*·dTTP mismatch and syn -dGTP for the G*·dGTP mismatch were also obtained. Stereoviews of the active sites of the initial models are shown in Supplementary Figure S1.
|
16820532_p9
|
16820532
|
Molecular modeling
| 4.089991 |
biomedical
|
Study
|
[
0.9993820190429688,
0.00024073105305433273,
0.0003771725168917328
] |
[
0.999264657497406,
0.00046694951015524566,
0.0002013186749536544,
0.00006709710578434169
] |
en
| 0.999998 |
−1 deletion initial models. The crystal structure of the Dpo4 type II ternary complex (PDB ID: 1JXL) ( 15 ) was used to obtain the starting models for the −1 deletion structures . In this crystal structure, the coordinates of the first base at the 5′ end of the template could not be resolved, probably due to its flexible conformation outside the polymerase. We therefore modeled it into the structure, using the conformation of an analogous terminal base in the Dpo4 type I structure (PDB ID: 1JX4). Hydrogen atoms absent in the crystal structures were added by the AMBER suite. The dideoxy group at the 3′ end of the primer was replaced by a hydroxyl group. The Ca 2+ ion, residing in the position of the nucleotide binding metal ion, was replaced by a Mg 2+ ion, and repositioned for proper octahedral coordination with water molecules and amino acid residues (Supplementary Table S1). The DNA sequence was also remodeled to match the sequence in the Apc gene mutational hotspot codon 635 . The PhIP moiety was linked to the unpartnered guanine in the active site, while the incoming dCTP paired with the guanine on the 5′ side of the adduct. Starting models were obtained using the same approach as for G*·dNTP models, by rotating torsion angles α′ and β′ at 10° intervals, in combination. Structures were selected for anti and syn domains of χ based on minimal steric close contacts and optimal stacking. The torsion angles in these starting models are summarized in Supplementary Table S2 and stereoviews of the active sites of the initial models are shown in Supplementary Figure S1.
|
16820532_p10
|
16820532
|
Molecular modeling
| 4.369655 |
biomedical
|
Study
|
[
0.9992119073867798,
0.0005567636690102518,
0.00023127210442908108
] |
[
0.9990078806877136,
0.00033750516013242304,
0.0004964634426869452,
0.00015819093096069992
] |
en
| 0.999997 |
Parameters for the anti -dG-C8-PhIP adduct and the incoming anti -dCTP, anti -dATP, syn -dATP and anti -dTTP were the same as in earlier work ( 53 , 54 ). The parameters for the syn -dG-C8-PhIP adduct and the syn -dGTP were obtained using the same method described previously ( 53 ). Supplementary Tables S3 and S4 shows the AMBER atom type, connection type and partial charge assignment for these cases.
|
16820532_p11
|
16820532
|
Force field parameterization
| 4.106915 |
biomedical
|
Study
|
[
0.9992840886116028,
0.0001976343774003908,
0.0005183448665775359
] |
[
0.9993115663528442,
0.00042007313459180295,
0.00021691712026949972,
0.0000514572202519048
] |
en
| 0.999998 |
Simulations were carried out using the SANDER module of the AMBER 6.0 MD software package (University of California, San Francisco), the Cornell et al. force field ( 60 ) and the parm99 parameter set ( 61 ). Electrostatic interactions were approximated by the particle mesh Ewald method ( 62 ), and a 10 Å cutoff was applied to Lennard–Jones interactions. All bonds involving hydrogen atoms were constrained by the SHAKE algorithm ( 63 ) with a tolerance of 0.0005 Å, and a 2 fs time-step was used in the dynamics simulation. Periodic boundary conditions were applied, and all MD simulations were carried out under constant temperature, 300 K, with a temperature coupling parameter of 4.0 ps and at constant atmospheric pressure with a 1.0 ps coupling parameter. The translational motion of the center of mass was removed every 1 ps. No obvious overall rotation of the system was observed during the simulation; thus, energy leakage from internal motion to global rotation through the ‘flying ice cube effect’ did not happen here ( 64 ).
|
16820532_p12
|
16820532
|
MD simulation and data analyses
| 4.227146 |
biomedical
|
Study
|
[
0.9992592930793762,
0.0003128114331047982,
0.0004278951673768461
] |
[
0.9991519451141357,
0.000292394426651299,
0.0004822240152861923,
0.00007333453686442226
] |
en
| 0.999997 |
Fifteen Na + ions were added to each system for neutralization using the LEaP module of AMBER, followed by 600 steps of steepest descent (SD) and 400 steps of conjugate gradient (CG) to relax the added Na + ions and crystal waters. Then ∼15 000 TIP3P water molecules were added to solvate each system creating a rectangular periodic box containing a total of ∼50 000 atoms. The systems were further minimized and heated up to 300 K. The equilibration protocol was conducted in the same manner as in earlier work ( 53 ) and the details are given in Supplementary Data. Production MD simulation was carried out for 3 ns.
|
16820532_p13
|
16820532
|
MD simulation and data analyses
| 4.231723 |
biomedical
|
Study
|
[
0.9992759823799133,
0.0003656946064438671,
0.0003583624493330717
] |
[
0.9988541603088379,
0.0007531359442509711,
0.00029928822186775506,
0.00009344706631964073
] |
en
| 0.999999 |
The PTRAJ, ANAL and CARNAL modules of AMBER were employed to analyze the trajectories. The root-mean-square deviations (RMSDs) of each system were calculated relative to the starting structures as shown in Supplementary Figure S2. The overall structure and the active site region of the protein–DNA complexes became reasonably stable after ∼500 ps production MD simulation in each system. Therefore, the last 2.5 ns of MD simulation were used for structural analyses. Stereoviews of unmodified and modified systems after the total 3 ns of production MD simulations are shown in Supplementary Figure S3, and the active sites of these systems are shown in Figures 2 and 3 , and Supplementary Figure S4.
|
16820532_p14
|
16820532
|
MD simulation and data analyses
| 4.11345 |
biomedical
|
Study
|
[
0.9995065927505493,
0.00027918146224692464,
0.00021420675329864025
] |
[
0.9994508624076843,
0.00019221770344302058,
0.0002863382687792182,
0.00007064675446599722
] |
en
| 0.999998 |
Molecular modeling was carried out with Insight II 97.0 (Accelrys, Inc., a subsidiary of Pharmacopeia, Inc.). Figures of structures were prepared with PyMOL (DeLano Scientific, LLC.).
|
16820532_p15
|
16820532
|
MD simulation and data analyses
| 1.949749 |
biomedical
|
Other
|
[
0.9931206107139587,
0.0009222293738275766,
0.005957115441560745
] |
[
0.2895718812942505,
0.7036153674125671,
0.004400459583848715,
0.0024123210459947586
] |
en
| 0.999997 |
Subsets and Splits
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