In the present study, the gene expression profiles of G. A hypothetical E3 ubiquitin ligase gene, GpDSR7 , was found to be involved in the response to drought stress, and an in vitro ubiquitination assay showed that it functions as an E3 ubiquitin ligase and is localised to the plasma membrane. GpSDR7 production was induced by various abiotic stresses, and its overexpression conferred drought tolerance to Arabidopsis transgenic plants.
These results suggest that GpDSR7 encodes an E3 ubiquitin ligase that plays essential roles in regulating the response to drought tolerance through protein modification. And we want to mention that no specific permissions were required for collecting Grimmia species in Laoshan Mountain in Shandong Province, China because the field where we collected the moss is not a national park or other protected area and and open to scientific research in China. Each G. Beauv clump was carefully separated into single shoots and then washed thoroughly in running water to remove debris.
Hydrated moss was obtained after a h rehydration period [ 21 ]. Arabidopsis thaliana ecotype Columbia was used as the WT in this study. Surface-sterilisation of seeds and conventional culture were performed as described previously [ 24 ].
Recombinant clones were used to establish the subtracted cDNA library. Randomly chosen clones were single-pass sequenced Shanghai Sangong, China. All overlapping sequences were clustered into contigs using the Aligner software CodonCode. Sequences of fewer than nucleotides were excluded from clustering. The pairs of mutagenic oligonucleotide primers for site-directed mutagenesis are shown in S1 Table. For evaluation of drought resistance, 1-week-old seedlings were transplanted to soil for 2 weeks under standard growth conditions.
The plants were then subjected to sustained drought by ceasing watering for the indicated times, followed by re-watering for 7 days. Survival rates were calculated 7 days later. The water loss assay was performed as described by Lee [ 14 ]. Leaf water loss was monitored as a percentage of that at the initial time.
For gene expression analysis, 2-week-old seedlings grown in agar plates were transferred onto Whatman 3-mm filter paper and subjected to drought treatment. Total RNA was extracted from G. Actin was used as the internal control. To determine the expression levels of stress-responsive genes in Arabidopsis under drought stress, quantitative real-time PCR analysis was performed.
Primers are listed in S3 Table. To determine the water status of samples and select appropriate stress conditions for constructing the subtracted cDNA library, the relative water content RWC was monitored during dehydration treatment.
Moreover, the morphology of G. Early and late drought responses may occur at 0. A Relative water contents were measured to determine the water status of dehydrated gametophytes. Arrows indicate 0. B Morphologies of G. C Percentage distribution of unigenes categorised into functional classes. D Expression patterns of several drought-responsive genes identified by SSH.
Only the forward SSH cDNA library was used in further experiments because it represented genes either upregulated or specifically expressed in response to drought.
After cloning and sequencing, readable ESTs were obtained, which represented unigenes containing singletons and contigs S4 Table.
The corresponding proteins were sorted into various groups, including 15 functional categories A—O, Fig 1C , unclassified unknown and unnamed proteins P , and proteins with no matches in the database Q. As shown in Fig 1C , the genes encoding proteins responsible for metabolism A, 5. Most of the genes were induced under drought stress, although their levels of induction differed. In addition, the expression levels of unigenes matched their EST abundance or redundancy because those genes with high EST redundancy showed greater intensities than those with low EST redundancy Fig 1D.
We identified a broad spectrum of partial cDNA clones in G. GpDSR7 is predicted to be of Fig 2 shows the nucleotide and protein sequences of GpDSR7. It possesses three putative transmembrane TM domains near the C-terminal, suggesting that it to be membrane-associated Fig 2A. Solid line denotes the RING motif, and conserved metal ligand positions are indicated by numbered cysteine C and histidine H residues.
Interestingly, the expression pattern of GpDSR7 following application of exogenous ABA is similar to that with drought treatment, however the drought is more effective to trigger the transcription of GpDSR7. Likewise, salt stress enhanced GpDSR7 expression, albeit to a lesser degree than drought stress. GpDSR7 accumulation was not obviously affected by low temperature.
Rehydrated G. GpDSR7 expression without treatment was set as 1. In contrast to the wild type WT , all of the mutants lost their Ub ligase activity almost completely Fig 4B. Ubiquitinated proteins were detected by western blot analysis using an anti-GST antibody left panel and anti-Myc antibody right panel. Lines indicate deleted regions.
All images were obtained from one optic section. Because GpDSR7 is a drought-inducible gene, it may function in drought response. Three-week-old Arabidopsis plants were grown under normal conditions before drought treatment. All plants without watered for 20 days exhibited wilt, although the 35S : GpDSR7 plants displayed less severe wilting. After re-watering for 7 days, most WT and plants transformed with empty vector were unable to recover and had a relatively low survival rate of To further evaluate the anti-drought abilities, rosette leaves were excised from plants and the water loss rates were determined.
Decreases in fresh weights were measured over time 0—3 h. As shown in Fig 6C , the greatest rate of water loss occurred during the first 30 min after detachment. Thus, we concluded that overexpression of GpDSR7 conferred drought tolerance on transgenic Arabidopsis. A Seven-day-old seedlings were transferred to soil for a further 2 weeks of normal growth left panel , subjected to progressive drought by withholding water for 20 days middle panel , and then re-watered for 7 days right panel.
C Leaves of day-old plants were excised and weighed at various time points after detachment. We have used the wildtype WT and the plants tranformed with empty vector EV as the control. Proline accumulation has been reported to function as a molecular chaperone that stabilises protein structure [ 28 ]. Taken together, these results suggest that GpDSR7 is involved in positive regulation of drought stress. The mean value of three technical replicates was normalised to the expression level of glyceraldehydephosphate dehydrogenase GAPDH.
Blank columns: control group; slash columns: 1 transgenic plants; black columns: 2 transgenic plants. Using SSH technology, drought-stress-related unigenes were identified in G.
The products of these GpDSRs can be divided into two categories: functional proteins that directly protect plants and regulatory proteins [ 5 ].
In the first group, GpDSR1 encoded the putative chlorophyll a-b binding protein CP26 and was rapidly upregulated under drought stress. LHCB5 also called CP26 has been proposed to regulate the transduction of light energy through the xanthophyll cycle to disperse high irradiance [ 30 ].
In addition, we detected several genes related to osmotic homeostasis. A centriole mask was created using centrin as a marker of centrioles. To obtain a mask for the pericentriolar region, the centriole mask was expanded using two-pixel dilation.
Fluorescence intensity values were measured and corrected for background intensity. Pericentrin-positive structures were segmented based on an adaptive intensity-based threshold. The border perimeter and bounding circle diameter of each detected object was then analyzed supplemental Fig. Segmentation was performed using Volocity software version 6. The cells were cultured for a further 36 h and lysed in 0.
For experiments investigating protein ubiquitylation, 5 m m N -ethylmaleimide Sigma, E was added to lysis buffer. The immunoprecipitates were analyzed by immunoblotting using standard procedures. The baits and associated proteins were eluted with ammonium hydroxide, lyophilized in a SpeedVac, resuspended in 50 m m ammonium bicarbonate pH 8—8.
Each species was sequenced twice within 30 s before being placed on a dynamic exclusion list for up to 90 s exclusion list contains 60 species. Acquired RAW files were converted to mgf format using the recommended parameter from Thermo in the ProteoWizard tool 33 and searched with the Mascot search engine version 2.
Methionine oxidation was allowed as a variable modification, and trypsin specificity with two missed cleavages allowed was selected. Scoring high confidence interaction partners was performed as follows: proteins detected in any of the six negative control runs were first eliminated from any subsequent analysis.
The resulting hits were further filtered by requiring a minimal protein Mascot score of 60 and at least two unique peptides per protein. We required the protein to have been detected in at least two of the three biological replicates with at least one of the baits to be included in the data set. Lastly, to increase the stringency of the data set, we further required that at least a total of 40 total peptides was detected for a given hit across the three biological replicates.
Hits that passed these two last criteria were included in Table I highlighted in bold font and Fig. All of the interactions involving these hits were reported in supplemental Table S1 , whether they passed these last two filters with each bait or not.
A list of unfiltered interactors as well as list of control contaminants can be found in supplemental Tables S2 and S3 , respectively. The numbers listed in the columns for each bait represent the total number of peptides number for each hit. The italic text highlights the total peptide number associated with the bait protein. Protein identifier is the NCBI gi identifier for the protein. The hits that were detected from the control samples were removed from this list. The control samples were as follows: three of the control runs included data from control IPs in HEK cells where rabbit IgG coupled to protein G-Sepharose resin was used.
Identification of novel CP interacting proteins. The control samples were as follows: three of the control runs included data from control IP in HEK cells where rabbit IgG coupled to protein G-Sepharose resin was used. The thicknesses of the connecting lines are proportional to the average number of total peptides associated with the interaction. The gray connecting lines indicate new interactions, whereas the blue connecting lines indicate previously reported interactions.
RCC , regulator of chromosome condensation amino acids —, —, and — ; CYTOCH , cytochrome b 5 heme-binding domain amino acids — ; ZF , zinc finger amino acids — ; DOC , Anaphase-promoting complex, subunit 10 amino acids — ; HECT , homologous to the E6-AP C terminus amino acids — ; NHR , neuralized homology region amino acids 41—, —, —, —, —, and — As a control, IP was performed in parallel with a nonspecific rabbit IgG.
WCE were blotted as a loading control bottom panel. The protein size kDa is indicated for each immunoblot. The nonspecific cross-reactive bands are indicated by an asterisk. C and D , HERC2 is a kDa protein and runs as a very high molecular mass band, and there is no molecular mass standard in that region of the gel.
IB , immunoblot. Antibody production services were purchased from Covance Denver, PA. The reaction was carried out as previously described The ubiquitylation reactions were assessed by subjecting the samples to immunoblot analysis with different ubiquitin antibodies. An interesting feature of CP function appears to be its association in different subcomplexes with distinct functions. RCC1-like domains have been implicated in Ran-mediated nuclear transport and chromatin-based microtubule-nucleation 38 , 39 Fig.
NEURL4 belongs to a family of neuralized-like proteins whose function is not understood in mammalian cells. We note, however, that many of the mammalian proteins bearing NHR domains also contain an E3 ligase domain or suppressor of cytokines signaling domain 43 linking them to putative ubiquitylation-related functions in cells. By immunofluorescence, we observed distinct NEURL4 foci that co-localized in the vicinity of centrin positive structures, a core subdistal component of centrioles 24 Fig.
S3, E and F. S5, A and C. We note that some of these noncentrosomal foci were still present after NEURL4 depletion, indicating that a subset of these structures are due to nonspecific labeling or that they are stable enough to still be present after RNAi. Analysis of NEURL4 distribution at different stages of the cell cycle revealed that it associates with centrosomes during interphase but not during mitosis Fig. We analyzed NEURL4 protein levels at different stages of the cell cycle and found them to be slightly increased during mitosis supplemental Fig.
This suggests that the observed change in localization in mitosis is caused by the dissociation of NEURL4 from the centrosome. Similarly, we observed that HERC2 also associates with centrosomes, as judged by its localization in the vicinity of centrin structures Fig. Further co-localization analyses with additional centrosome markers showed that HERC2 appears to predominantly label the proximal region of centrioles, based on its co-localization with CPAP Fig.
S5, B and D. Taken together, these results suggest that a portion of NEURL4 and HERC2 localize to interphase centrosomes and that they appear to predominantly accumulate at the distal and proximal region, respectively.
Furthermore, both proteins appear to lose their association with centrosomes during mitosis. Interphase cells are shown in the top panels. The insets are 2-fold magnifications of the outlined areas to better visualize the centrosomal regions. The CEPCP protein complex caps the distal ends of centrioles, physically restricting centriole length 15 , 25 , The depletion of either protein induces the formation of overly long centrioles S3, C and D.
S5, A and B. S4, C and D. To further characterize the pericentrin-containing filaments, we imaged the cells using structured illumination microscopy This allowed us to observe with higher spatial resolution fine details of the modifications in pericentriolar material architecture Fig. These results suggest that different cell types have developed varying sensitivities toward NEURL4 depletion in terms of its impact on centrosome morphology. The cells were imaged using conventional deconvolution microscopy.
NEURL4 depletion caused abnormalities in pericentriolar material, shown in more detail in the insets. The insets are magnifications of the outlined centrosomal regions. The presence of pericentrin filaments was quantified 72 h post-transfection.
At least cells were analyzed under each condition. The increase in the number of pericentrin filaments is statistically significant. C , cells treated as described in A were imaged on a structured-illumination microscope. D and E , wild type and filamentous centrosomes were segmented, based on an adaptive threshold.
The increase in both parameters was highly significant. At least 30 centrosomes were measured in three independent experiments for each condition.
Stable U2OS cell lines were generated that expressed each of these deletion constructs, and protein expression was confirmed by immunoblot analysis with the GFP antibody bottom panel. NS , nonsignificant difference. HERC2 is a kDa protein and runs as a very high molecular mass band, and there is no molecular mass standard in that region of the gel.
Immunostaining analysis revealed that the myc-tagged SENP2 is preferentially located to the nucleus, exhibiting the nuclear plasma Figures 3a and b or dotted staining pattern Figures 3c and d. Occasionally, we found SENP2-S in both the nucleus and cytoplasm data not shown that might contribute to the slight reduction of p53 Figure 1g.
Many sumoylated proteins are targeted to these specific structures, 4 including p Differential localizations of SENP2 isoforms. Immunostained cells were counterstained by DAPI blue.
SUMO conjugation affects the subcellular distribution of Mdm2. Although SENP2 and Mdm2 showed similar patterns of nuclear distribution, their co-localization appeared to be minimal, with only few spots containing both proteins Figures 5a—d.
It seems that SUMO conjugation of Mdm2 facilitates the complex formation between these two molecules. Total cell extracts directly analyzed by immunoblot analysis show the protein expression levels a and b.
Actin level is used as a loading control. First, we investigated if overexpression of SENP2 alters the sumoylation status of endogenous Mdm2 in cells with either transient or stable expression of SENP2 at high levels. The sumoylated levels of endogenous Mdm2 were then analyzed by immunoblot analysis. SENP2-mediated desumoylation of Mdm2 is analyzed by the in vivo a and b and in vitro c and d systems. Total cell extracts directly analyzed by immunoblot analysis show the protein expression levels.
To decipher the regulatory pathway underlying the modulation of p53 by SENP2 and Mdm2, we first examined whether Mdm2 has an essential role in this process. As expected, the knockdown of Mdm2 by RNA interference increased the levels of p53 and its downstream target p21 Figure 8a. However, when Mdm2 was knocked down, high levels of SENP2 were no longer capable of reducing the levels of p53 and p21, suggesting a requirement of Mdm2 in the regulation of p53 mediated by SENP2 Figure 8a.
Nutlin-3 is a potent small-molecule antagonist, which binds to the pbinding pocket of Mdm2 and prevents their interaction, thereby stabilizing p53 and increasing the level of p21 Figure 8b. A reduction of p53 occurred when Mdm2 was expressed Figure 8c. Furthermore, presence of Nutlin-3 not only abolished the reduction of p53 caused by Mdm2, but also significantly elevated the p53 level Figure 8c. To ensure that the observed effects are not due to a disruption of the E3 ligase activity, we examined the ability of the Mdm2 fusion proteins to modify p We first examined whether the subcellular distribution of Mdm2 is altered in DNA damage-induced apoptosis.
Upon doxorubicin treatment, sumoylation of Mdm2 is enhanced, which is accompanied by elevated levels of p53 and p21 Figure 9e. The data suggest that the SUMO-dependent regulation of Mdm2 distribution may have a crucial role in genotoxic stresses mediated by p Mdm2 co-localized with PML in cells undergoing apoptosis identified by immunostaining of activated caspase This effect is likely attributed to the differential compartmentalization of SENP2 isoforms within the cells.
The longest form of SENP2 is necessary and sufficient for modulating the stability of p53, and the pdependent transcription and stress responses. Biochemical studies further show that SENP2 promotes the desumoylation of Mdm2, contributing to the pdependent regulations.
Although both SENP2 and Mdm2 preferentially accumulate in the nucleus, their localizations do not seem to overlap significantly. If true, this would add complexities to this multilayered regulatory network. The diagram illustrates the mechanism underlying the regulation of Mdm2 by the SUMO pathway to control the cellular level of p SENP2 regulates p53 through modulation of Mdm2.
Desumoylation of Mdm2 permits its binding and ubiquitination of p53, which is then degraded through the proteolysis system. We have previously reported that Nutlin-3 treatment is still capable of increasing the p53 level in SENP2-null cells. One possibility is that both Mdm2 and sumoylated Mdm2 form complexes with p Desumoylation of Mdm2 then activates its E3 ligase activity.
Alternatively, desumoylation of Mdm2 promotes the cytoplasmic translocation of p53 for degradation. Mdm2-SUMO and p53 could form a sequestration complex accumulating in the nucleus without initiating the degradation and nuclear—cytoplasmic translocation processes. Stabilization of p53 then is capable of promoting the subsequent responses to cellular stresses. The findings support our hypothesis, further implying that Mdm2-SUMO is sequestered at the PML bodies unable to promote p53 degradation under hyper-sumoylated conditions Figure Indeed, we have found that the cellular levels of sumoylated Mdm2 are increased upon doxorubicin treatment.
However, due to the lack of information on identification of the sumoylation site of Mdm2, this hypothesis cannot be definitively assessed. Once the sumoylation site has been identified, it would be important to determine whether mutation of the sumoylation site interferes with the PML body distribution of Mdm2. An important function of p53 as a guardian of the genome ensures that the genetic information is properly propagated during these processes.
Although recent evidence has challenged the classical model of p53 activation, 26 its stabilization remains to be a key regulatory step.
SENP2, involved in the pinduced regulations, may have a role in genotoxic stresses. Further investigation focusing on the action of SENP2 promises new insights into the mechanism underlying cellular stress mediated by the SUMO pathway. As our prior study indicated that the SENP2—Mdm2—p53 pathway is critical for the G—S checkpoint of mitotic divisions and endoreduplication, 7 it is possible that aberrant stimulation of SENP2 causes cell cycle defects, leading to the prevention of cell death and senescence.
Mutations in the p53 gene have been linked to familial and sporadic forms of human cancer. Li—Fraumeni syndrome is a rare familial disorder that greatly increases the risk of developing several types of cancers in children and young adults. Previous reports also suggest that SENP1 might be associated with development of prostate cancers. Therefore, it is conceivable that SENP2 has a crucial role in pinduced tumorigenesis, which remains to be explored.
However, embryonic lethality associated with the SENP2 deletion in mice prevents investigation of its oncogenic role. Colonies, which grow under drug resistance condition, were picked to establish cell clones. For growth factor deprivation, cells were cultured in 0.
The number of cells was counted each day to assess the growth curve. The stained images were taken randomly to determine the percentage of apoptotic cells by counting the activated caspase-3 or TUNEL-positive and -negative cells.
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