PRR14 is a novel activator of the PI3K pathway promoting lung carcinogenesis.

Chromosomal focal amplifications often cause an increase in gene copy number, contributing to the pathogenesis of cancer. PRR14 overexpression is associated with recurrent locus amplification in lung cancer, and it correlates with a poor prognosis. We show that increased PRR14 expression promoted and reduced PRR14 expression impeded lung cancer cell proliferation. Interestingly, PRR14 cells were markedly enlarged in size and exhibited an elevated activity of the PI3-kinase/Akt/mTOR pathway, which was associated with a heightened sensitivity to the inhibitors of PI3K and mammalian target of rapamycin (mTOR). Biochemical analysis revealed that PRR14, as a proline-rich protein, binds to the Src homology 3 (SH3) domains of GRB2 resulting in PI3K activation. Significantly, two cancer patient-derived PRR14 mutants displayed considerably augmented GRB2-binding and an enhanced ability of promoting cell proliferation. Together with the in vivo data demonstrating a strong tumor-promoting activity of PRR14 and the mutants, our work uncovered this proline-rich protein as a novel activator of the PI3K pathway that promoted tumorigenesis in lung cancer.

Interaction of Lysinibacillus sphaericus Cry48Aa/Cry49Aa toxin with midgut brush-border membrane fractions from Culex quinquefasciatus larvae.

The Cry48Aa/Cry49Aa mosquitocidal toxin from Lysinibacillus sphaericus was uniquely composed of a three-domain (Cry) toxin and binary (Bin) toxin-like protein, with high toxicity against Culex spp. However, its mode of action against the target mosquitoes is still unknown. In this study, Cry48Aa, Cry49Aa and its N- and C-terminal truncated proteins were expressed and purified, and the binding affinities of the purified proteins with midgut brush-border membrane fractions (BBMFs) from Culex quin-quefasciatus larvae were performed. The results showed that both Cry48Aa and Cry49Aa have specific and high binding affinity to BBMFs, with dissociation constants of 9.5 ± 1.8 and 25.4 ± 3.8 nM, respectively. Competition assays demonstrated that Cry49Aa C-terminal derivatives were able to bind to the BBMFs, whereas Far-Western dot blot analysis revealed that its N-terminal constructs interacted with Cry48Aa. Nevertheless, larvicidal activity was almost lost when Cry49Aa truncated proteins, either individually or in pairs, combined with Cry48Aa. It is concluded that Cry49Aa is responsible for receptor binding and interaction with Cry48Aa and plays an important role in the mechanism of action of these two-component toxins.

Somatic human ZBTB7A zinc finger mutations promote cancer progression.

We recently reported that ZBTB7A is a bona fide transcription repressor of key glycolytic genes and its downregulation in human cancer contributes to tumor metabolism. As reduced expression of ZBTB7A is found only in a subset of human cancers, we explored alternative mechanisms of its inactivation by mining human cancer genome databases. We discovered recurrent somatic mutations of ZBTB7A in multiple types of human cancers with a marked enrichment of mutations within the zinc finger domain. Functional characterization of the mutants demonstrated that mutations within the zinc finger region of ZBTB7A invariably resulted in loss of function. As a consequence, the glycolytic genes were markedly upregulated in cancer cells harboring ZBTB7A zinc finger mutation, leading to increased glycolysis and proliferation. Our study uncovers the loss-of-function mutation in ZBTB7A as a novel mechanism causing elevated glycolysis in human cancer, which carries important therapeutic implication.

A novel role of PRR14 in the regulation of skeletal myogenesis.

Dysregulation of genes involved in organizing and maintaining nuclear structures, such as SYNE1, SYNE2, TREM43, EMD and LMNA is frequently associated with diverse diseases termed laminopathies, which often affect the muscle tissue. The PRR14 protein was recently reported to tether heterochromatin to nuclear lamina but its function remains largely unknown. Here, we present several lines of evidence demonstrating a critical role of PRR14 in regulation of myoblast differentiation. We found that Prr14 expression was upregulated during skeletal myogenesis. Knockdown of Prr14 impeded, whereas overexpression of PRR14 enhanced C2C12 differentiation. The pro-myogenesis activity of PRR14 seemed to correlate with its ability to support cell survival and to maintain the stability and structure of lamin A/C. In addition, PRR14 stimulated the activity of MyoD via binding to heterochromatin protein 1 alpha (HP1α). The results altogether support a model in which PRR14 promotes skeletal myogenesis via supporting nuclear lamina structure and enhancing the activity of MyoD.

Low-dose radiation exposure induces a HIF-1-mediated adaptive and protective metabolic response.

Because of insufficient understanding of the molecular effects of low levels of radiation exposure, there is a great uncertainty regarding its health risks. We report here that treatment of normal human cells with low-dose radiation induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis resulting in increased radiation resistance. This metabolic change is highlighted by upregulation of genes encoding glucose transporters and enzymes of glycolysis and the oxidative pentose phosphate pathway, concomitant with downregulation of mitochondrial genes, with corresponding changes in metabolic flux through these pathways. Mechanistically, the metabolic reprogramming depends on HIF1α, which is induced specifically by low-dose irradiation linking the metabolic pathway with cellular radiation dose response. Increased glucose flux and radiation resistance from low-dose irradiation are also observed systemically in mice. This highly sensitive metabolic response to low-dose radiation has important implications in understanding and assessing the health risks of radiation exposure.

Low-dose arsenic induces chemotherapy protection via p53/NF-κB-mediated metabolic regulation.

Most chemotherapeutical drugs kill cancer cells chiefly by inducing DNA damage, which unfortunately also causes undesirable injuries to normal tissues, mainly due to p53 activation. We report a novel strategy of normal tissue protection that involves p53/NF-κB coordinated metabolic regulation. Pretreatment of untransformed cells with low doses of arsenic induced concerted p53 suppression and NF-κB activation, which elicited a marked induction of glycolysis. Significantly, this metabolic shift provided cells effective protection against cytotoxic chemotherapy, coupling the metabolic pathway to cellular resistance. Using both in vitro and in vivo models, we demonstrated an absolute requirement of functional p53 in arsenic-mediated protection. Consistently, a brief arsenic pretreatment selectively protected only normal tissues, but not tumors, from toxicity of chemotherapy. An indispensable role of glycolysis in protecting normal tissues was demonstrated by using an inhibitor of glycolysis, 2-deoxyglucose, which almost totally abolished low-dose arsenic-mediated protection. Together, our work demonstrates that low-dose arsenic renders normal cells and tissues resistant to chemotherapy-induced toxicity by inducting glycolysis.

Elevated snoRNA biogenesis is essential in breast cancer.

Hyperactive ribosomal biogenesis is widely observed in cancer, which has been partly attributed to the increased rDNA transcription by Pol I in cancer. However, whether small nucleolar RNAs (snoRNAs), a class of non-coding RNAs crucial in ribosomal RNA (rRNA) maturation and functionality, are involved in cancer remains elusive. We report that snoRNAs and fibrillarin (FBL, an enzymatic small nucleolar ribonucleoprotein, snoRNP) are frequently overexpressed in both murine and human breast cancer as well as in prostate cancers, and significantly, that this overexpression is essential for tumorigenicity in vitro and in vivo. We demonstrate that when the elevated snoRNA pathway is suppressed, the tumor suppressor p53 can act as a sentinel of snoRNP perturbation, the activation of which mediates the growth inhibitory effect. On the other hand, high level of FBL interferes with the activation of p53 by stress. We further show that p53 activation by FBL knockdown is not only regulated by the ribosomal protein-MDM2-mediated protein stabilization pathway, but also by enhanced PTB-dependent, cap-independent translation. Together, our data uncover an essential role of deregulated snoRNA biogenesis in tumors and a new mechanism of nucleolar modulation of p53.

High-accuracy splice site prediction based on sequence component and position features.

Identification of splice sites plays a key role in the annotation of genes. Consequently, improvement of computational prediction of splice sites would be very useful. We examined the effect of the window size and the number and position of the consensus bases with a chi-square test, and then extracted the sequence multi-scale component features and the position and adjacent position relationship features of consensus sites. Then, we constructed a novel classification model using a support vector machine with the previously selected features and applied it to the Homo sapiens splice site dataset. This method greatly improved cross-validation accuracies for training sets with true and spurious splice sites of both equal and different proportions. This method was also applied to the NN269 dataset for further evaluation and independent testing. The results were superior to those obtained with previous methods, and demonstrate the stability and superiority of this method for prediction of splice sites.

Expression of mel gene improves the UV resistance of Bacillus thuringiensis.

AIMS: To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory. METHODS AND RESULTS: Melanin quantitative analysis revealed that the recombinant strain Bt TD841 could synthesize 0.15 mg melanin ml(-1) sporulated culture. Atomic force microscopy confirmed the production of diamond crystal and SDS-PAGE results showed the expression of the 130 kDa Cry1A protein. Bioassay results demonstrated that the LC(50) value of Bt TD841 was 3.69 microl ml(-1) against Helicoverpa armigera and the UV resistance of this recombinant was enhanced 9.7-fold compared to its parental strain Bt HC42 after 4-h UV irradiation. CONCLUSION: Expression of the mel gene can significantly increase UV resistance of B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on genetically engineered Bt strain with co-expression of melanin and the insecticidal crystal proteins gene, and the results may offer a practical solution for improving the photoprotection of Bt products in field application.

Modulation of p53 and MDM2 activity by novel interaction with Ras-GAP binding proteins (G3BP).

Inactivation of the p53 tumor suppressor pathway is a critical step in human tumorigenesis. In addition to mutations, p53 can be functionally silenced through its increased degradation, inhibition of its transcriptional activity and/or its inappropriate subcellular localization. Using a proteomic approach, we have found that members of the Ras network of proteins, Ras-GTPase activating protein-SH3-domain-binding proteins 1 and 2 (G3BP1 and 2), bind to p53 in vitro and in vivo. Our data show that expression of G3BPs leads to the redistribution of p53 from the nucleus to the cytoplasm. The G3BP2 isoform additionally associated with murine double minute 2 (MDM2), a negative regulator of p53. G3BP2 expression resulted in significant reduction in MDM2-mediated p53 ubiquitylation and degradation. Interestingly, MDM2 was also stabilized in G3BP2-expressing cells and its ability to ubiquitylate itself was compromised. Accordingly, short hairpin RNA (shRNA)-mediated knockdown of G3BP2 caused a reduction in MDM2 protein levels. Furthermore, expression of shRNA targeting either G3BP1 or G3BP2 in human cancer cell lines resulted in marked upregulation of p53 levels and activity. Our results suggest that both G3BP isoforms may act as negative regulators of p53.

Kinetics of plasmid transfer among Bacillus cereus group strains within lepidopteran larvae.

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44-56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 x 10(-6) CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.

The synergistic activity between Cry1Aa and Cry1c from Bacillus thuringiensis against Spodoptera exigua and Helicoverpa armigera.

AIMS: To investigate the interaction between two crystal proteins, Cry1Aa and Cry1C, for future development of biopesticides based on Bacillus thuringiensis, toxicities of the two individual proteins and in combinations have been determined against Spodoptera exigua and Helicoverpa armigera larvae, and synergism between the proteins has been evaluated using synergistic factor. METHODS AND RESULTS: SDS-PAGE showed that Cry1Aa and Cry1C proteins could be expressed in acrystalliferous B. thuringiensis 4Q7 strain, with molecular weights of 135 and 130 kDa respectively. The bioassay results indicated a synergistic activity between Cry1Aa and Cry1C against S. exigua and H. armigera, and the highest toxicities could be observed in the combination of Cry1Aa and Cry1C at a ratio of 1 : 1. CONCLUSION: The two toxins, Cry1Aa and Cry1C, interact synergistically to exhibit higher toxicity against S. exigua and H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the investigation on the synergistic activity between two B. thuringiensis Cry1 toxins. It can be applied to the rational design of new generations of B. thuringiensis biopesticides and to strategies for management of resistant insects.

Cross-resistance between strains of Bacillus sphaericus but not B. thuringiensis israelensis in colonies of the mosquito Culex quinquefasciatus.

Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145,000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145,000-fold) to B. sphaericus strains C3-41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59. B. sphaericus-resistant mosquito colonies remained highly susceptible to B. thuringiensis israelensis, suggesting that B.t.i. would be of value in the management of B. sphaericus-resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross-resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.

Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity.

AIMS: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. METHODS AND RESULTS: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. CONCLUSION: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.

Identification of p53 sequence elements that are required for MDM2-mediated nuclear export.

It has been demonstrated that MDM2 can differentially regulate subcellular distribution of p53 and its close structural homologue p73. In contrast to MDM2-mediated p53 nuclear export, p73 accumulates in the nucleus as aggregates that colocalize with MDM2. Distinct distribution patterns of p53 and p73 suggest the existence of unique structural elements in the two homologues that determine their MDM2-mediated relocalization in the cell. Using a series of p53/p73 chimeric proteins, we demonstrate that three regions of p53 are involved in the regulation of MDM2-mediated nuclear export. The DNA binding domain (DBD) is involved in the maintenance of a proper conformation that is required for functional activity of the nuclear export sequence (NES) of p53. The extreme C terminus of p53 harbors several lysine residues whose ubiquitination by MDM2 appears to be the initial event in p53 nuclear export, as evidenced by the impaired nucleocytoplasmic shuttling of p53 mutants bearing simultaneous substitutions of lysines 370, 372, 373, 381, 382, and 386 to arginines (6KR) or alanines (6KA). Finally, the region between the DBD and the oligomerization domain of p53, specifically lysine 305, also plays a critical role in fully revealing p53NES. We conclude that MDM2-mediated nuclear export of p53 depends on a series of ubiquitination-induced conformational changes in the p53 molecule that lead to the activation of p53NES. In addition, we demonstrate that the p53NES may be activated without necessarily disrupting the p53 tetramer.

Dual role of p300 in the regulation of p53 stability.

While the function of p300 as a transcriptional co-activator of p53 is well documented, its role in the regulation of p53 stability remains ill-defined since opposite effects of p300 on p53 levels have been reported. We show here that p300 stabilizes both p53 and its negative regulator MDM2, thereby enhancing the p53/MDM2 negative regulatory loop. Binding of p300 is associated with the retention of p53 in the nucleus, which results in the accumulation of p53 in an acetylase-independent manner. Stabilization of MDM2, on the other hand, requires the acetylase activity of p300. Importantly, MDM2, once expressed, is able to reverse the stabilizing effect of p300 on p53. A temperature-permissive p53-expressing cell line enabled us to demonstrate the completely opposite roles of p300 in the regulation of p53 stability, depending on the expression of MDM2. Prior to p53 activation, when MDM2 levels are low, p300 acts as a positive regulator to increase p53 levels. Upon shifting to permissive temperature, however, when MDM2 expression is induced, p300 becomes a negative regulator of p53 by stabilizing MDM2 and thereby augmenting MDM2's ability to target p53 for degradation.

Subcellular distribution of p53 and p73 are differentially regulated by MDM2.

The binding of MDM2 targets p53, but not p73, for degradation, whereas it suppresses the transactivation function of both proteins. MDM2 also mediates p53 nuclear export, but its role in the regulation of p73 distribution is unknown at the present time. We show here that, in sharp contrast to p53, MDM2 induces p73 to form nuclear aggregates that colocalize with MDM2 but are distinct from the promyelocytic leukemia dots. The MDM2 ring-domain that is necessary for mediating p53 nuclear export is not required for the induction of the p73 nuclear aggregates. Using a domain-swapping approach, we demonstrate that the inability of p73 to nuclear-export is attributable to its nonfunctional nuclear-export sequence.

A sequence element of p53 that determines its susceptibility to viral oncoprotein-targeted degradation.

The molecular basis that the viral oncoproteins, including HPV16 E6 and E1B55k/E4 34k complex, differentially target p53 but not its homolog p73 for degradation remains elusive. Using a series of p53/p73 chimeras, we demonstrated that despite binding to the different regions of p53, both HPV16 E6 and E1B55k/E4 34k required a very same p53 sequence, amino acid residues 92 to 112 [p53(aa.92-112)], previously identified as a necessity for Mdm2-mediated degradation, to target p53 for degradation. Removal of the p53(aa.92-112) by either substitution or deletion resulted in a p53 protein that was no longer degradable by the viral proteins. More significantly, swapping the oncoprotein-binding motif and the p53(aa.92-112) rendered p73 susceptible to oncoprotein-mediated degradation. Collectively, our data supports a model in which the p53(aa.92-112) functions as a determinant for p53 stability while the binding of the oncoproteins directs p53 into the specific pathway for proteolysis.

Evidence for a distinct inhibitory factor in the regulation of p53 functional activity.

Under normal conditions, tumor suppressor protein p53 exists in the cell in its latent form and is unable to function as a transcription factor. The allosteric model of p53 regulation postulates that the extreme portion of p53 carboxyl terminus (aa 364-393) binds to the core domain of the protein, thereby abrogating specific DNA binding in that region. In this study we propose an alternative mechanism of p53 functional regulation, which involves a separate molecule acting in trans to inhibit p53 transcriptional activity. Through the use of chimeric proteins of p53, p63gamma and p73beta, we show that the extreme COOH-terminal domain of p53 exerts a powerful and specific inhibitory effect on the p73- and p63-driven expression of a reporter gene. Moreover, fusion of p53 extreme COOH terminus to a completely unrelated transcriptional activator Gal4-VP16 also results in significant inhibition of transactivation activity. Since p73, p63, or Gal4-VP16 cannot associate with any part of the p53 molecule, we conclude that p53(aa 364-393) represses transcriptional activity of chimeric proteins and p53 itself through the binding of external negative modulator(s) in that region and not by the allosteric mechanism of regulation. In accordance with the "distinct inhibitor" hypothesis, the activity of wild type p53 is substantially increased by overexpression of chimeric proteins bearing p53(aa 364-393), which might be due to the competitive removal of transcriptional inhibitor(s). Our findings provide the basis for the identification of such negative modulators of p53 transcriptional activity.

Mechanism of functional inactivation of a Li-Fraumeni syndrome p53 that has a mutation outside of the DNA-binding domain.

The majority of p53 mutations are located in the DNA-binding domain of the protein. However, recently a family suffering from Li-Fraumeni syndrome (LFS) has been discovered, some of whom harbor a p53 mutation in exon 4, outside of the core domain. How this mutation affects p53 function and subsequently leads to malignant transformation is not yet clear. Interestingly, the p53 mutation found in this LFS family is localized to the p53 region that we have recently identified as necessary for Mdm2-mediated p53 degradation. We therefore endeavored to study further the LFS-associated p53 mutation at the molecular level by creating an equivalent lesion in a p53 expression construct and functionally characterizing it. Here we demonstrate that a mutation in this region is associated not only with resistance of the mutant p53 to Mdm2-mediated degradation, but also with an impaired response of mutant protein to DNA damage. In addition, the p53(LFS) mutant was found to be defective in its transactivation function, which correlated with its inability to suppress cell growth and to induce apoptosis. The molecular basis for p53(LFS) functional impairment appears to be its predominantly cytoplasmic localization caused by faulty nuclear import mechanism, which, at least in part, resulted from the mutant's decreased affinity to importin.