IDH1 gene mutation activates Smad signaling molecules to regulate the expression levels of cell cycle and biological rhythm genes in human glioma U87­MG cells.

Isocitrate dehydrogenase1 (IDH1) mutation is the most important genetic change in glioma. The most common IDH1 mutation results in the amino acid substitution of arginine 132 (Arg/R132), which is located at the active site of the enzyme. IDH1 Arg132His (R132H) mutation can reduce the proliferative rate of glioma cells. Numerous diseases follow circadian rhythms, and there is growing evidence that circadian disruption may be a risk factor for cancer in humans. Dysregulation of the circadian clock serves an important role in the development of malignant tumors, including glioma. Brain­Muscle Arnt­Like protein 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK) are the main biological rhythm genes. The present study aimed to further study whether there is an association between IDH1 R132H mutation and biological rhythm in glioma, and whether this affects the occurrence of glioma. The Cancer Genome Atlas (TCGA) database was used to detect the expression levels of the biological rhythm genes BMAL1 and CLOCK in various types of tumor. Additionally, U87­MG cells were infected with wild­type and mutant IDH1 lentiviruses. Colony formation experiments were used to detect cell proliferation in each group, cell cycle distribution was detected by flow cytometry and western blotting was used to detect the expression levels of wild­type and mutant IDH1, cyclins, biological rhythm genes and Smad signaling pathway­associated genes in U87­MG cells. TCGA database results suggested that BMAL1 and CLOCK were abnormally expressed in glioma. Cells were successfully infected with wild­type and mutant IDH1 lentiviruses. Colony formation assay revealed decreased cell proliferation in the IDH1 R132H mutant group. The cell cycle distribution detected by flow cytometry indicated that IDH1 gene mutation increased the G1 phase ratio and decreased the S phase ratio in U87­MG cells. The western blotting results demonstrated that IDH1 R132H mutation decreased the expression levels of the S phase­associated proteins Cyclin A and CDK2, and increased the expression levels of the G1 phase­associated proteins Cyclin D3 and CDK4, but did not significantly change the expression levels of the G2/M phase­associated protein Cyclin B1. The expression levels of the positive and negative rhythm regulation genes BMAL1, CLOCK, period (PER s (PER1, 2 and 3) and cryptochrom (CRY)s (CRY1 and 2) were significantly decreased, those of the Smad signaling pathway­associated genes Smad2, Smad3 and Smad2­3 were decreased, and those of phosphorylated (p)­Smad2, p­Smad3 and Smad4 were increased. Therefore, the present results suggested that the IDH1 R132H mutation may alter the cell cycle and biological rhythm genes in U87­MG cells through the TGF­ß/Smad signaling pathway.

Structure-Based Design of a Bromodomain and Extraterminal Domain (BET) Inhibitor Selective for the N-Terminal Bromodomains That Retains an Anti-inflammatory and Antiproliferative Phenotype.

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects.

Ensemble docking-based virtual screening toward identifying inhibitors against Wee1 kinase.

Aim:Wee1 kinase plays a key role in the arrest of G2/M checkpoint that prevents mitotic entry in response to DNA damage. This work is to discover potent Wee1 inhibitors which can be considered valuable. Materials & Methods: Herein, Ensemble docking using multiple crystal structures was considered an effective strategy in the virtual screening. The performance of 17 scoring functions obtained from different docking software was evaluated for molecular docking. Results: Two novel compounds B1 and A2 were identified as Wee1 inhibitors with IC50 values of 10.23 ± 0.505 and 8.72 ± 0.323 µM, respectively. Further cell viability assay demonstrated that the two active compounds exhibited good anticancer activities. Conclusion: This provides a meaningful starting point for further structure optimization to discover more potent Wee1 inhibitors.

Optimized Plk1 PBD Inhibitors Based on Poloxin Induce Mitotic Arrest and Apoptosis in Tumor Cells.

Polo-like kinase 1 (Plk1) is a central regulator of mitosis and has been validated as a target for antitumor therapy. The polo-box domain (PBD) of Plk1 regulates its kinase activity and mediates the subcellular localization of Plk1 and its interactions with a subset of its substrates. Functional inhibition of the Plk1 PBD by low-molecular weight inhibitors has been shown to represent a viable strategy by which to inhibit the enzyme, while avoiding selectivity issues caused by the conserved nature of the ATP binding site. Here, we report structure-activity relationships and mechanistic analysis for the first reported Plk1 PBD inhibitor, Poloxin. We present the identification of the optimized analog Poloxin-2, displaying significantly improved potency and selectivity over Poloxin. Poloxin-2 induces mitotic arrest and apoptosis in cultured human tumor cells at low micromolar concentrations, highlighting it as a valuable tool compound for exploring the function of the Plk1 PBD in living cells.

Cancer markers: integratively annotated classification.

Translational cancer genomics research aims to ensure that experimental knowledge is subject to computational analysis, and integrated with a variety of records from omics and clinical sources. The data retrieval from such sources is not trivial, due to their redundancy and heterogeneity, and the presence of false evidence. In silico marker identification, therefore, remains a complex task that is mainly motivated by the impact that target identification from the elucidation of gene co-expression dynamics and regulation mechanisms, combined with the discovery of genotype-phenotype associations, may have for clinical validation. Based on the reuse of publicly available gene expression data, our aim is to propose cancer marker classification by integrating the prediction power of multiple annotation sources. In particular, with reference to the functional annotation for colorectal markers, we indicate a classification of markers into diagnostic and prognostic classes combined with susceptibility and risk factors.

Target deletion of the AAA ATPase PpCDC48II in Physcomitrella patens results in freezing sensitivity after cold acclimation.

CDC48 is a highly conserved protein in eukaryotes and belongs to the AAA (ATPase associated with a variety of cellular activities) superfamily. It can interact with many different co-factors and form protein complexes that play important roles in various cellular processes. According to the Physcomitrella patens database, one member of the ATPases, the cell cycle gene PpCDC48II, was cloned. PpCDC48II contains two typical ATPase modules and is highly homologous to AtCDC48A. PpCDC48II was up-regulated in mRNA levels after incubation at 0°C for 36 and 72 h. To further elucidate protein function, we disrupted the PpCDC48II gene by transforming P. patens with the corresponding linear genomic sequences. When treated to the same freezing stress, it was found that PpCDC48II knockout plants were less resistant to freezing treatment than wild type after acclimation. This suggested that PpCDC48II was an essential gene for low-temperature-induced freezing tolerance in P. patens cells.

ATM activation in the presence of oxidative stress.

The Ataxia-Telangiectasia mutated (ATM) kinase is regarded as the major regulator of the cellular response to DNA double strand breaks (DSBs). In response to DSBs, ATM dimers dissociate into active monomers in a process promoted by the Mre11-Rad50-Nbs1 (MRN) complex. ATM can also be activated by oxidative stress directly in the form of exposure to H2O2. The active ATM in this case is a disulfide-crosslinked dimer containing 2 or more disulfide bonds. Mutation of a critical cysteine residue in the FATC domain involved in disulfide bond formation specifically blocks ATM activation by oxidative stress. Here we show that ATM activation by DSBs is inhibited in the presence of H2O2 because oxidation blocks the ability of MRN to bind to DNA. However, ATM activation via direct oxidation by H2O2 complements the loss of MRN/DSB-dependent activation and contributes significantly to the overall level of ATM activity in the presence of both DSBs and oxidative stress.

Characterization of cell cycle specific protein interaction networks of the yeast 26S proteasome complex by the QTAX strategy.

Ubiquitin-proteasome dependent protein degradation plays a fundamental role in the regulation of the eukaryotic cell cycle. Cell cycle transitions between different phases are tightly regulated to prevent uncontrolled cell proliferation, which is characteristic of cancer cells. To understand cell cycle phase specific regulation of the 26S proteasome and reveal the molecular mechanisms underlying the ubiquitin-proteasome degradation pathway during cell cycle progression, we have carried out comprehensive characterization of cell cycle phase specific proteasome interacting proteins (PIPs) by QTAX analysis of synchronized yeast cells. Our efforts have generated specific proteasome interaction networks for the G1, S, and M phases of the cell cycle and identified a total of 677 PIPs, 266 of which were not previously identified from unsynchronized cells. On the basis of the dynamic changes of their SILAC ratios across the three cell cycle phases, we have employed a profile vector-based clustering approach and identified 20 functionally significant groups of PIPs, 3 of which are enriched with cell cycle related functions. This work presents the first step toward understanding how dynamic proteasome interactions are involved in various cellular pathways during the cell cycle.

The origin of peroxisomes: The possibility of an actinobacterial symbiosis.

The peroxisome is an organelle found in most eukaryotes that is crucial for lipid metabolism. The ability of peroxisomes to divide themselves and transport post-translational proteins suggests that the peroxisome may have had an endosymbiotic origin. However, the localization of peroxisomal proteins to the endoplasmic reticulum (ER) and the similarity of some peroxisomal proteins to those localized in the ER suggest an alternative hypothesis: that the peroxisome was developed from the ER. To study the evolutionary distance between the peroxisome, the ER and prokaryotes, we conducted a phylogenetic analysis of cell division control 48 (CDC48) and its homologs, including the ER-localized CDC48, the CDC48 homologs in prokaryotes and the peroxisome-localized PEX1 and PEX6. We also conducted a similarity search of peroxisomal protein sequences against prokaryotic protein sequences using BLAST at several E-value thresholds. We provide several lines of evidence supporting an actinobacteria symbiotic origin for the peroxisome: (1) PEX1 and PEX6 are more closely related to the CDC48 homologs in actinobacteria than to the ER-localized CDC48; (2) actinobacterial proteins show higher levels of similarity to those of the peroxisome than to those of other prokaryotes.

Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins.

Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1(Ap) results in loss of apicoplast protein import and subsequent death of the parasite.

Suppression of the ER-localized AAA ATPase NgCDC48 inhibits tobacco growth and development.

CDC48 is a member of the AAA ATPase superfamily. Yeast CDC48 and its mammalian homolog p97 are implicated in diverse cellular processes, including mitosis, membrane fusion, and ubiquitin-dependent protein degradation. However, the cellular functions of plant CDC48 proteins are largely unknown. In the present study, we performed virus-induced gene silencing (VIGS) screening and found that silencing of a gene encoding a tobacco CDC48 homolog, NgCDC48, resulted in severe abnormalities in leaf and shoot development in tobacco. Furthermore, transgenic tobacco plants (35S:anti-NgCDC48), in which the NgCDC48 gene was suppressed using the antisense RNA method, exhibited severely aberrant development of both vegetative and reproductive organs, resulting in arrested shoot and leaf growth and sterile flowers. Approximately 57-83% of 35S:anti-NgCDC48 plants failed to develop mature organs and died at early stage of development. Scanning electron microscopy showed that both adaxial and abaxial epidermal pavement cells in antisense transgenic leaves were significantly smaller and more numerous than those in wild type leaves. These results indicate that NgCDC48 is critically involved in cell growth and development of tobacco plants. An in vivo targeting experiment revealed that NgCDC48 resides in the endoplasmic reticulum (ER) in tobacco protoplasts. We consider the tantalizing possibility that CDC48-mediated degradation of an as-yet unidentified protein(s) in the ER might be a critical step for cell growth and expansion in tobacco leaves.

Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins.

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. Although the exchange potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases. Phosphorylation, substitution, or deletion of the blocking helices relieves this autoinhibition. Here we show that two other Dbl-family proteins, Ngef and Wgef, which like Tim contain a C-terminal SH3 domain, are also activated by tyrosine phosphorylation of a blocking helix. Consequently, basal autoinhibition of DH domains by direct steric exclusion using short N-terminal helices likely represents a conserved mechanism of regulation for the large family of Dbl-related proteins. N-Terminal truncation or phosphorylation of many other Dbl-family GEFs leads to their activation; similar autoinhibition mechanisms could explain some of these events. In addition, we show that the C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of the Tim subgroup of Dbl-family proteins provides a unique mechanism of regulated autoinhibition of exchange activity that is functionally linked to the interactions between the autoinhibitory helix and the DH domain.

Genome-wide transcriptional analysis of the human cell cycle identifies genes differentially regulated in normal and cancer cells.

Characterization of the transcriptional regulatory network of the normal cell cycle is essential for understanding the perturbations that lead to cancer. However, the complete set of cycling genes in primary cells has not yet been identified. Here, we report the results of genome-wide expression profiling experiments on synchronized primary human foreskin fibroblasts across the cell cycle. Using a combined experimental and computational approach to deconvolve measured expression values into "single-cell" expression profiles, we were able to overcome the limitations inherent in synchronizing nontransformed mammalian cells. This allowed us to identify 480 periodically expressed genes in primary human foreskin fibroblasts. Analysis of the reconstructed primary cell profiles and comparison with published expression datasets from synchronized transformed cells reveals a large number of genes that cycle exclusively in primary cells. This conclusion was supported by both bioinformatic analysis and experiments performed on other cell types. We suggest that this approach will help pinpoint genetic elements contributing to normal cell growth and cellular transformation.

ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation.

Members of the ING family of tumor suppressors regulate cell cycle progression, apoptosis, and DNA repair as important cofactors of p53. ING1 and ING3 are stable components of the mSin3A HDAC and Tip60/NuA4 HAT complexes, respectively. We now report the purification of the three remaining human ING proteins. While ING2 is in an HDAC complex similar to ING1, ING4 associates with the HBO1 HAT required for normal progression through S phase and the majority of histone H4 acetylation in vivo. ING5 fractionates with two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs. These ING5 HAT complexes interact with the MCM helicase and are essential for DNA replication to occur during S phase. Our data also indicate that ING subunits are crucial for acetylation of chromatin substrates. Since INGs, HBO1, and MOZ/MORF contribute to oncogenic transformation, the multisubunit assemblies characterized here underscore the critical role of epigenetic regulation in cancer development.

Shugoshin, a guardian for sister chromatid segregation.

To ensure sister chromatids to be equally transmitted to daughter cells, it is imperative that physical association of sister chromatids is maintained during S, G2, and early mitosis until the onset of anaphase. Cohesion of sister chromatids in eukaryotes is largely achieved by the cohesin complex. In vertebrates, cohesin molecules are dissociated from chromosome arms but not from centromeres during prophase by the so-called prophase pathway. Although it remains unclear what is the molecular basis by which centromeric cohesin is retained, a flurry of recent studies have shed light on a family of proteins named Shugoshin (Sgo) that are evolutionarily conserved across eukaryotes. Sgo1 functions as a protector of centromeric cohesin during meiosis in yeast and during mitosis in high eukaryotes. Suppression of Sgo1 function results in premature separation of sister chromatids in both meiosis and mitosis. The discovery of members of the Sgo family may help to explain how centromeric cohesin is protected from dissociation from DNA until the onset of anaphase. Given the importance of chromosome cohesion in the maintenance of genomic stability, further characterization of Sgo1 and related molecules may also open up new avenues of research for developing new strategies for cancer intervention.

Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3.

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid-induced kinase 1), but not by protein kinase Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro. All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332 and Thr348 of NDRG2), as well as Thr356 and Thr366. Residues Thr346, Thr356 and Thr366 are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1-/- mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also suppressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser342, Ser352 and Ser362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.

Molecular mechanisms of E2F-dependent activation and pRB-mediated repression.

Alterations in transcription of genes regulated by members of the E2F family of transcription factors can be viewed as a measure of the ebb and flow in a constantly evolving battle between repressor and activator complexes. Various chromatin regulatory complexes have been linked to Rb/E2F proteins, and changes in histone modifications correlate with states of E2F-dependent transcription. E2F has traditionally been viewed in the context of cell-cycle control. However, several recent studies have revealed a new aspect of E2F function in which pRB/E2F-family proteins confer stable repression of transcription. Such repression is evident in both actively proliferating cells and in cells that have withdrawn from the cell cycle.

Classification of the caspase-hemoglobinase fold: detection of new families and implications for the origin of the eukaryotic separins.

A comprehensive sequence and structural comparative analysis of the caspase-hemoglobinase protein fold resulted in the delineation of the minimal structural core of the protease domain and the identification of numerous, previously undetected members, including a new protease family typified by the HetF protein from the cyanobacterium Nostoc. The first bacterial homologs of legumains and hemoglobinases were also identified. Most proteins containing this fold are known or predicted to be active proteases, but multiple, independent inactivations were noticed in nearly all lineages. Together with the tendency of caspase-related proteases to form intramolecular or intermolecular dimers, this suggests a widespread regulatory role for the inactive forms. A classification of the caspase-hemoglobinase fold was developed to reflect the inferred evolutionary relationships between the constituent protein families. Proteins containing this domain were so far detected almost exclusively in bacteria and eukaryotes. This analysis indicates that caspase-hemoglobinase-fold proteases and their inactivated derivatives are widespread in diverse bacteria, particularly those with a complex development, such as Streptomyces, Anabaena, Mesorhizobium, and Myxococcus. The eukaryotic separin family was shown to be most closely related to the mainly prokaryotic HetF family. The phyletic patterns and evolutionary relationships between these proteins suggest that they probably were acquired by eukaryotes from bacteria during the primary, promitochondrial endosymbiosis. A similar scenario, supported by phylogenetic analysis, seems to apply to metacaspases and paracaspases, with the latter, perhaps, being acquired in an independent horizontal transfer to the eukaryotes. The acquisition of the caspase-hemoglobinase-fold domains by eukaryotes might have been critical in the evolution of important eukaryotic processes, such as mitosis and programmed cell death.

Expression and stability of Arabidopsis CDC6 are associated with endoreplication.

Studies on the CDC6 protein, which is crucial to the control of DNA replication in yeast and animal cells, are lacking in plants. We have isolated an Arabidopsis cDNA encoding the AtCDC6 protein and studied its possible connection to the occurrence of developmentally regulated endoreplication cycles. The AtCDC6 gene is expressed maximally in early S-phase, and its promoter contains an E2F consensus site that mediates the binding of a plant E2F/DP complex. Transgenic plants carrying an AtCDC6 promoter-beta-glucuronidase fusion revealed that it is active in proliferating cells and, interestingly, in endoreplicating cells. In particular, the extra endoreplication cycle that occurs in dark-grown hypocotyl cells is associated with upregulation of the AtCDC6 gene. This was corroborated using ctr1 Arabidopsis mutants altered in their endoreplication pattern. The ectopic expression of AtCDC6 in transgenic plants induced endoreplication and produced a change in the somatic ploidy level. AtCDC6 was degraded in a ubiquitin- and proteosome-dependent manner by extracts from proliferating cells, but it was degraded poorly by extracts from dark-grown hypocotyl endoreplicating cells. Our results indicate that endoreplication is associated with expression of the AtCDC6 gene and, most likely, the stability of its product; it also apparently requires activation of the retinoblastoma/E2F/DP pathway. These conclusions may apply to endoreplicating cells in other tissues of the plant and to endoreplicating cells in other eukaryotes.

Cell cycle regulatory proteins--an overview with relevance to oral cancer.

The cell cycle is controlled by a number of highly conserved proteins, found in species as diverse as yeast and mammals. The study of these proteins is a rapidly advancing field that is increasing our understanding of normal and abnormal cell division. Disruption of the cell cycle has been demonstrated in several different types of neoplasm, and there is increasing evidence that, in head and neck tumours, there is aberrant control of cyclins, cell cycle protein kinases and their inhibitors. Because of the phase specificity of some of the control proteins, antibodies to them are proving to be of value in studying cell kinetics of both normal tissues and malignant tumours.