The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept. The phosphorylated derivatives of phosphatidylinositol (PtdIns) are called phosphoinositides and are regarded as important signaling molecules. Their localization pattern at the membrane marks specific subdomains to which they can selectively recruit cytosolic proteins that harbour one of several known phosphoinositide interaction motifs. These attracted proteins function in the first step of downstream signaling cascades, which finally result in a cellular response.This review focuses on phosphatidylinositol 3-phosphate (PtdIns3P), which is mainly generated by the lipid kinase VPS34 (vacuolar sorting protein 34) through the phosphorylation of PtdIns at the 3-hydoxyl group of the inositol ring A. In man3 or PIP3) from phosphatidylinositol 4,5-bisphosphate P2 or PIP2) at the inner leaflet of the plasma membrane . . 106]. NIn a general context, it is important to note that Nedd4 has also been found to be part of the LC3-interaction map of basal autophagy . A systeMoreover, Nedd4 has been described to ubiquitinate the lipid phosphatase PTEN, which is responsible for the conversion of PtdIns3,4,5)P into PtdInsP . PTEN te,4,5P intInterestingly, Nedd4 can function as a negative regulator of p53 . It stabTaken together, these findings indicate that oncogene Nedd4 might function as a negative regulator of the Beclin 1—p53 axis and thereby inhibits the basal protective function of both apoptosis and autophagy.Beclin 1 is a substrate of the RING-type ligase TRAF6 (tumor necrosis factor receptor—associated factor 6) in macrophages . These cThe TRAF6-cataylzed ubiquitination of Beclin 1 is opposed by the deubiquitinating enzyme A20 . A20 hydIt is interesting to note that the described mechanism resembles the principle by which the Beclin 1—Bcl-2 interaction is also disrupted via phosphorylation of the corresponding interaction interfaces . DissociDrosophila melanogaster and mammalian cells has uncovered a link between oxidative stress and autophagy induction . . 153]. TThe WD40-protein Ambra 1 has been identified as a Beclin 1 binding partner, which functions as a crucial activator of the initial steps of phagophore formation in the nervous system . Cells bNutrient deprivation stimulates the induction of autophagy and triggers the association of Ambra 1 with the PI3K-III core complex via its interaction to Beclin 1 . One impRecently, another molecular mechanism underlying WASH-mediated inhibition has been put forward. WASH functions also as a targeting factor for the RING-type E3 ligase RNF2 (ring finger protein 2)/Ring1b) . RNF2 moRNF2 has been known before as a member of the Polycomb group complex (PgC) . The PgCAn interesting indication for the existence of different layers of interdependent ubiquitination and phosphorylation events in the regulation of autophagy at the PI3K-III complex has been reported recently.Ambra 1 has been found to be modified by two different kinases with contrary outcome. While phosphorylation of Ambra 1 by mTOR inhibits autophagy, phosphorylation of Ambra 1 by ULK1 elicits autophagy ,92. InteThe E3 ligase TRAF6 has been identified as a binding partner of dephosphorylated Ambra 1 in HEK293 cells . HoweverHowever, this mechanism is blocked when Ambra 1 is phosphorylated by mTOR, which renders Ambra 1 unable to bind TRAF6 and therefore prevents ubiquitination of ULK1 and thereby inhibits autophagy.Moreover, a recent study uncovered a direct relationship between ULK1 and Beclin 1 . Under aMutations within the E3 ligase Parkin are the most common cause of Parkinson’s disease (PD) . AccumulParkin-mediated mitophagy involves the translocation of cytosolic Parkin to depolarized mitochondria, which is dependent on PINK1. Subsequentially, Parkin ubiquitinates outer mitochondrial membrane proteins, such as Mitofusin 1, Mitofusin 2 or VDAC1 ,167,168.Finally, the clustered mitochondria are engulfed by autophagosomes. This process is negatively regulated by USP15  and USP3A possible additional mechanistic aspect is indicated by the interaction of Parkin with Ambra 1  136]. A. A136]. While translocation of cytosolic Parkin to mitochondria does not depend on Ambra 1, it is blocked by Bcl-2 , the inhIt will be important to unveil the functional interconnection of this interaction network between Parkin, Beclin 1, Bcl-2 and Ambra 1.While Beclin 1 and Ambra 1 have been demonstrated to be involved in several different ubiquitination-dependent regulatory processes see , much leRecently, VPS34 has been shown to be modified with the ubiquitin-like SUMO 1   in MCF7 The SUMO-ligase that targets VPS34 is KAP1 (KRAB-associated protein 1)  Figure . KAP1 biIn general, cancer cells display an imbalance of the SUMOylation signaling patterns . Ubc9 waSeveral studies published in recent years clearly demonstrate the important functional role of ubiquitination and SUMOylation events in the dynamic regulation of the PI3K-III complex. Especially the multivalent adaptor protein Beclin 1 has been shown to be ubiquitinated with several different types of ubiquitin chains by different E3 ligases. Moreover, the multi-functional protein Ambra 1 has not only been shown to be ubiquitinated itself, but also to contribute to several other ubiquitin-dependent processes. Further work will be required to elucidate if the detected ubiquitination reactions and E3 enzymes are potentially active within the same cell type or if they represent cell type specific events. Furthermore, the entire published literature so far concentrates on mammalian cells, while it is currently unclear if ubiquitination plays also a similar important role in yeasts and plants.Based on the already well analyzed phosphorylation reactions that regulate the PI3K-III complex, it is of particular interest to analyze the possible cross-talk between ubiquitination and phosphorylation.Another interesting aspect concerns the possible different layers of complexity in the regulation of PI3K-III complex function and autophagy in general. As pointed out in each chapter, most of the described E3 enzymes contribute to the regulation of autophagy not only by the ubiquitination of PI3K-III complex components but also in additional ways. It is tempting to speculate that the corresponding E3 enzymes are involved in a cascade-like regulation of different steps in the induction and proceeding of autophagy.The elucidation of the molecular as well as systemic mechanism of E3 enzymes involved in the regulation of the PI3K-III complex will contribute to the establishment of a dynamic regulatory network, which will be important to understand the complex interplay of its constituents in the occurrence of neurodegenerative diseases and cancer.


Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation. The process of converting nucleic acid to functional protein, a process involving the precise spatial and temporal arrangement of proteins, protein complexes, and nucleic acids, is known as translation ,2,3. Inii), and GTP (guanosine triphosphate). The PIC can then bind to nascent messenger RNA (mRNA) through the direct interaction of eIF3 and eIF4G, a member of the eIF4F cap-binding complex. The largest of the eIFs, eIF3 is comprised of thirteen distinct subunits with a holoprotein mass of approximately 800 kDa. Numerous studies implicate the holoprotein eIF3 and its many subunits as an essential factor during translation initiation: It is the central assembly on which other factors, proteins, and nucleic acids bind [At the onset of eukaryotic translation initiation, the 40S ribosome binds several factors including eIF2 and eIF3 to form the 43S preinitiation complex (PIC). eIF2  binds as a ternary complex (TC) comprised of eIF2, the initiator methionyl tRNA  for eIF2, exchanges GDP (guanosine diphosphate) for GTP, which then promotes binding of a new Met-tRNAi for further rounds of translation. Phosphorylation of eIF2α at ser-51 reduces the dissociation rate of eIF2 from eIF2B, effectively sequestering eIF2B and preventing eIF2 TC regeneration and thus globally repressing the rate of mRNA translation [in vitro effects of eIF2γ phosphorylation via protein kinase C (PKC) [During initiation, eIF2 TC hydrolyzes GTP then releases Met-tRNAnslation ,16,17,18 C (PKC) .In addition to the identification of novel phosphosites, determining those levels of phosphorylation on specific residues allows researchers to define potentially important phosphosites, thereby distinguishing those sites from potentially less biologically meaningful ones ,24,25,26While identification of novel phosphosites is the essential first step in the eventual evaluation of their biological impact, follow-up studies, even on a few novel sites of phosphorylation, often presents a task too burdensome for subsequent in-depth investigations. Researchers identify phosphosites for further study through evaluation of their structural location. Phosphosites residing in structurally salient locations such as established binding sites, binding pockets, or areas of significant secondary, tertiary, and/or quaternary structure, are considered desirable candidates for further evaluation. However, without such informative data, further investigations into newly discovered phosphosites are often avoided.in vivo effects of eIF3h’s ser-183 phosphorylation during malignant transformation of NIH 3T3 cells [In order to assess the amount of phosphorylation occurring at specific residues, we recently published a mass spectrometry method proficient at measuring phosphosite stoichiometry . Our metT3 cells ,30.This report is the first to highlight the phosphorylation levels of three heavily phosphorylated eukaryotic initiation factors. Additionally, our quantification analysis is specific to the analysis of cells grown at optimal conditions and thus underscores the importance of variability possible with these phosphosites under different environmental circumstances. Knowledge gained from this study provides a platform for future investigations not only for phosphorylated proteins, but also for the inherent variability of specific phosphosites within the protein itself. Hence, the purpose of this study is to add to the growing pool of knowledge of these factors and more importantly, to initiate an investigation primarily aimed at quantified phosphorylated residues and their implication on translation initiation. Future studies into the regulation of these factors may be based on the findings within this study.We isolated eIF2 from HeLa cell lysate in order to quantify its level of phosphorylation. The factor was purified from HeLa cells under optimal growth conditions (log phase growth). As eIF2 is a heterotrimer with a molecular mass of 126 kDa, we analyzed the two factors that have been previously reported as phosphorylated within HeLa cells, eIF2β and eIF2γ. While eIF2α has been heavily studied at its principally phosphorylated residue of ser-51, quantification of that residue does not lead to new information or developments, which is the principal aim of this study. Thus, we focused our efforts at quantifying the numerous sites on the remaining two subunits of eIF2 in order to gain further insight into their function and regulation.C-terminal end of the protein, ser-412 and thr-413 exhibit phosphorylation at 7% while ser-418 and thr-435 have phosphorylation at 70% and 60%, respectively  of the protein, ser-1336 and ser-1364. While ser-1336 has a phosphorylation of 18%, ser-1364 has a level of 36% . A level of 31% phosphorylation was observed for eIF3g’s two phosphorylated residues, thr-41 and ser-42, and a high level of phosphorylation comparable to that of eIF3h was observed for thr-109 on eIF3j , the kinase Mnk1, and initiation factors eIF4E, eIF4A, and eIF3. The phosphorylation of eIF4G from HeLa cells has been well documented with evidence of up to 20 sites of phosphorylation corresponding to eIF4G derived from HeLa cells have been identified. However, as with all other eIFs, the quantification of this phosphorylation has yet to be reported.C-terminus of the protein and is ser-1596 that corresponds to the Mnk1 binding region. Quantification of its phosphorylation reveals less than 5% levels of phosphorylation.Currently, considerable investigation has focused on the portion of eIF4G that has been mapped to bind to eIF3 . This poN-terminal portion of the protein. However, high levels of phosphorylation were observed for thr-647 at 65%. Regions near but distinct from the eIF3 binding site had phosphorylation levels at or near 50%. The peptide mapped to ser-1144 and ser-1147 had a combined level of 45% and the peptide mapped to ser-1209 and ser-1211 was at 50%. The peptide mapping to ser-1185 and ser-1187 had a level of 70% and the peptide mapping to ser-1231 had a level of 75%. Peptides mapping in close proximity to the Mnk1 binding region, thr-1425 and ser-1430 were quantified at phosphorylation levels of 62%.As for the remaining known sites of phosphorylation for eIF4G from HeLa cells, insufficiently scored peptides prevented our quantitative analysis of phosphosites residing in the i and several initiation factors, binds to the 5' end of mRNA then scans in the 5' to 3' direction until the initiation codon is recognized. Recruitment of Met-tRNAi requires formation of a ternary complex also containing eIF2 and GTP, while mRNA recruitment to the ribosome complex requires binding of eIF4G and eIF3.The entire process of translation initiation requires a concerted and choreographed effort from numerous proteins to effectively translate the nascent mRNA into functional protein. Although the process of translation may be separated into the four distinct phases of initiation, elongation, termination, and recycling, much of the regulation is centralized to the initiation phase. Translation initiation is the process by which the 40S ribosome, bound to Met-tRNAAs the formation of the ternary complex is critical to translation, a better understanding of the major protein component, eIF2, would help define this process. A closer look into the function of eIF2 reveals that the heterotrimer consists of three subunits, eIF2α and eIF2β bound to eIF2γ but not each other. Classically, eIF2α’s phosphorylation on ser-51 converts the eIF2 heterotrimer into an inhibitor of eIF2B, the heteropentamer responsible for exchanging GDP for GTP on eIF2. Since the phosphorylation of this one residue on eIF2α has a profound impact on the overall process of translation, we hypothesized that the other subunits of eIF2 may also contribute in some part to the overall process of translation.i and GTP, the switch 1 region of the molecule undergoes a conformational change in order to allow for complexation [in vitro. In the current study, we observed that for cells propagated under log-phase growth conditions, thr-66 on eIF2γ is 71%. Given that a majority of thr-66 is phosphorylated for a cancer cell line under optimal conditions, future discernment of the physiological role of this phosphosite should be of paramount importance. Logically, phosphorylation within this region of the molecule may have a significant impact on the protein’s overall structure and consequently its influence on the conformational change experienced upon GTP binding to eIF2γ.Our phosphoanalysis of eIF2 centered on known phosphosites for both eIF2β and eIF2γ derived from HeLa cells. In our previous report for eIF2γ, one of the novel phosphosites identified was thr-66 . Furtherlexation ,35. Our Two other structurally noteworthy phosphorylation sites are ser-55 and thr-56 ,37. BothC-terminal portion of the protein, ser-412 and thr-413 have levels at 7% while ser-418 and thr-435 are at or near 70%. Thus, from the current study, we can see the dynamics of the phosphorylation landscape unfold. Residues 412 and 413 reside on the outer face of the molecule and despite the fact that they may be more readily available to kinases as compared to residues 55, 56, and 66, (which lie on the interior of the molecule), their levels of phosphorylation are significantly smaller. Future investigations will determine the significance of these phosphorylation stoichiometries, but for now, we can speculate that phosphorylation does not play a major role in the binding to other nucleotides and/or proteins. The same appears not to be true for either ser-418 or thr-435. Again, future studies will determine these residues’ phosphorylation significance, but undoubtedly, different physiological circumstances and pressures exist on the phosphorylated residues of the C-terminal of eIF2γ. Such a variation of phosphorylation may imply a structural necessity adopted by the C-terminus during the binding processes. It would be interesting to observe phosphorylation differences not only under different growth conditions, but also when one of the four C-terminal sites is mutated and how this effect translates to the overall phosphorylation landscape across the C-terminal region.In contrast to those phosphosites near the binding pocket and within the switch 1 region, phosphosites associated with the i and is part of the ternary complex that shuttles this polynucleotide to the awaiting mRNA during protein translation initiation, the interplay of the phosphate on thr-109 and the zinc ion may have a meaningful impact on protein translation.Lastly, thr-109’s phosphorylation level at 30% seems to neither weaken nor strengthen the importance of this phosphosite to the overall physiology of eIF2. However, as stated in our previous report, this residue does reside near a zinc binding domain. The electrostatics involved with the zinc cation and the negative charge inherent in a phosphate moiety could be responsible for an interaction that may determine the proper positioning of the zinc ion. Proteins containing zinc binding motifs have been well documented in their binding affinity for nucleotides. As eIF2 binds to met-tRNAConsiderable more research is required to decipher the significance of eIF2β phosphosite structural regions. Previous targeted investigations into eIF2β have established an interaction of the protein with the kinase CK2 and suggest numerous physiological pressures affecting the dynamics of eIF2β phosphorylation warranting further investigation . NonetheAlong with eIF2, eIF3 is a necessary initiation factor as it binds to many of the numerous eIFs including eIF4A, eIF1, eIF1A, eIF2, and eIF4G. A critical function of eIF3 is prevention of premature association to the 60S ribosome by binding to the 40S ribosome along with other initiation factors. Although its function and structure have been previously investigated, to date eIF3 has neither a definitive crystal structure nor have any of its phosphorylation sites been thoroughly investigated. Recent studies have shown the structure of the tridodecamer as a 5 lobed entity comprised of a core with five protrusions . The exaThe final protein whose phosphorylation was quantified in this study was eIF4G. A classic example of a scaffolding protein, much like the core proteins of eIF3, phosphorylation was present at its highest level for residues residing in known areas of protein binding. Three phosphorylated residues lie within the eIF3 binding region of eIF4G and all three exhibited high levels of phosphorylation. Other residues that also showed high levels of phosphorylation are residues between the eIF3 and eIF4A binding regions. Although future investigations will decipher the role of these specific phosphosites, phosphosite ser-1187 is known in HEK293 cells to function as a substrate for the kinase PKCα and modulates Mnk1 binding . Again, g for 20 min at 4 °C. To the resulting supernatant, KCl was added to a final concentration of 450 mM followed by centrifugation in a Beckman Ti-45 rotor for 4 h at 4 °C at 45,000 rpm. The middle two-thirds of the supernatant was carefully removed and stirred at 4 °C while saturated ammonium sulfate was added to a final concentration of 40%. After stirring on ice for 1 h, the suspension was centrifuged at 20,000× g for 10 min at 4 °C and the pellet (referred to as the A cut) was frozen for future use. To the remaining supernatant, ammonium sulfate was added to a final concentration of 70%. After stirring for 1 h, the mixture was centrifuged at 20,000× g for 10 min at 4 °C. The pellet (the B cut) was resuspended in 50 mL of buffer A  containing 50 mM KCl and dialyzed in two liters of the same buffer for 2.5 h at 4 °C. Following dialysis, the lysate was passed through a 0.2 µm syringe filter and loaded onto a MonoQ (10/10) column . The column was eluted with a linear gradient of 100 to 500 mM KCl in buffer A at 2 mL/min with 3 mL fractions collected. The fractions that contained the protein of interest  were then pooled, dialyzed against buffer A containing 100 mM KCl for 2.5 h at 4 °C, and loaded onto a MonoS (10/10) column . The same gradient as that of the MonoQ column was applied and fractions from the MonoS column were analyzed using SDS-PAGE in a similar fashion. Fractions containing the protein of interest were pooled and loaded onto a hydroxyapatite column made in-house using commercial hydroxyapatite . Elution was performed using a linear gradient from 0% to 100% 0.5 M potassium phosphate buffer at pH 7.5. Fractions were again analyzed via SDS-PAGE in a similar manner to those eluting from either the MonoQ or MonoS columns. Fractions containing the now purified protein were pooled and concentrated using Amicon Ultra filtration devices with a MWCO of 10,000 Da to yield a final concentration between 1 and 2 mg/mL.All proteins for this study, eIF2, eIF3, and eIF4G were enriched from HeLa cell lysate prior to nano-LC-MS/MS according to previously published protocols ,29. BrieEach eIF protein was digested with trypsin following the same protocol. Approximately 1 µmol of either eIF2, eIF3, or eIF4G was first reduced at 56 °C for 45 min in 5.5 mM DTT final concentration followed by alkylation for one hour in the dark with iodoacetamide (IAA) added to a final concentration of 10 mM. Trypsin was added at a final enzyme:substrate mass ratio of 1:50 and digestion carried out overnight at 37 °C. The reaction was quenched by flash freezing in liquid nitrogen and the digest was lyophilized. Prior to TMT labeling, each lyophilized sample was dissolved into 100 µL of 2% acetonitrile supplemented with 0.1% trifluoroacetic acid. All eIF peptides were prepared for TMT labeling following our published protocol . Brieflyv/v) formic acid in acetonitrile) with 98% solvent A at a flow rate of 750 nL/min. Peptides were eluted off the column at 750 nL/min using the following gradient: 2%–10% solvent B for 5 min, 10%–35% solvent B for 65 min, 35%–70% solvent B for 5 min, 35%–70% solvent B for 5 min, 70%–90% solvent B for 5 min, 90% solvent B for 5 min, then reversed to 2% solvent B for 10 min. All ions with TMT labels were analyzed via higher energy collision dissociation (HCD), all others with collision-induced dissociation (CID). Settings for the LTQ-Orbitrap XL were set as follows: data-dependent scan of the top 5 most abundant ions, minimum signal threshold of 55,000, collision energy set to 35%, resolution set to 30,000, dynamic exclusion set to 60 s, repeat count set to 2, and repeat duration set to 30 s. All RAW data were deposited directly into SEQUEST Bioworks 3.3.1  and manually validated. Searches were conducted against an in-house developed database consisting of 584 proteins containing the sequences of all known mammalian eukaryotic initiation factors as well as their reversed sequences. Common contaminants including human keratins, porcine trypsin, bovine serum albumin, bovine beta-casein, as well as their reversed sequences were also included. We performed searches with tryptic specificity and allowed for three missed cleavages at a tolerance of 20 ppm in MS mode and 0.2 Da in MS2 mode. Possible structure modifications included for consideration were N-terminal and lysine TMT labeling, methionine oxidation, carbamidomethylation of cysteine, and serine, threonine, and tyrosine phosphorylation. Peptides with Xcorr scores greater than 2.5 were considered for further manual validation. All TMT ratios were measured individually and experiments were carried out in triplicate.All samples underwent complete labeling and were combined as outlined in our protocol ,27 priorThis study represents the first global attempt to elucidate the levels of phosphorylation for three integral protein complexes in the translation initiation pathway . Prior s


Human DBC1  is a protein implicated in the regulation of apoptosis, transcription and histone modifications. Upon DNA damage, DBC1 is phosphorylated by ATM/ATR on Thr454 and this modification increases its inhibitory interaction with SIRT1, leading to p53 acetylation and p53-dependent apoptosis. Here, we report that the inhibition of SIRT1 by DBC1 in the DNA damage response (DDR) also depends on Chk2, the transducer kinase that is activated by ATM upon DNA lesions and contributes to the spreading of DNA damage signal. Indeed we found that inactivation of Chk2 reduces DBC1-SIRT1 binding, thus preventing p53 acetylation and DBC1-induced apoptosis. These events are mediated by Chk2 phosphorylation of the 11S proteasome activator REGγ on Ser247, which increases REGγ-DBC1 interaction and SIRT1 inhibition. Overall our results clarify the mechanisms underlying the DBC1-dependent SIRT1 inhibition and link, for the first time, Chk2 and REGγ to the ATM-DBC1-SIRT1 axis. In order to preserve genomic stability, eukaryotic cells have evolved a complex network of signaling pathways, collectively known as DNA damage response (DDR), that are activated when cells are exposed to genotoxic lesions .Key components of the DDR cascade in response to double-strand breaks (DSBs) are the apical kinase ATM (Ataxia Telangiectasia Mutated) and its target and effector checkpoint kinase Chk2 , TRF2 33,4 and KThe 11S proteasome activator REGγ binds and activates the catalytic function of the 20S proteasome , promote+-dependent deacetylase able to deacetylate histone and non-histones protein, such as the tumor suppressor p53  was added to cells 20 min before genotoxic treatments to a final 25 μM concentration. The Chk2 inhibitor VRX0466617 , was added 1 h before treatments at 100 μM. Etoposide (TEVA) was used at 20 μM and Neocarzinostatin (Sigma) at 8.8 nM. Cells viability was analyzed by trypan blue (Sigma) staining.Human osteosarcoma U2OS cell line was cultured in DMEM , supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin; cells were maintained at 37°C and 5% COVectors encoding DBC1-WT and DBC1-T454A were reported in , FLAG-SIWestern blot (WB) analyses were performed with the NuPAGE system (Invitrogen) and densitometric evaluations were done with the ImageQuant 5.2 software (Molecular Dynamics). REGγ phosphorylation analyses were performed using Phos-Tag (Wako) at 30 μM in a 10% acrylamide gel . The antEscherichia coli and purified. In vitro kinase reactions with catalytically active GST-Chk2 were performed as previously reported  and phospho-peptides (EKIKRPR(p)SSNAETLY) conjugated to agarose beads were purchased from PrimmBiotech. Beads were incubated with cell lysates prepared in ELB buffer and, after washing, samples were boiled in SDS sample buffer and analyzed by western blot. For phosphopeptides dephosphorylation, FastAP Thermosensitive Alkaline Phosphatase (Fermentas) was used, according to manufacturer's protocol.REGγ-S247 We and others have previously shown that genotoxic stress induces the phosphorylation by ATM/ATR of DBC1-Thr454, promoting DBC1's interaction with and inhibition of SIRT1 deacetylase ,29. As tAs DBC1-induced p53 acetylation promotes p53-dependent transcriptional activity and apoptosis , we examSince also ectopic Chk2 can promote apoptosis ,36, we vTo determine whether Chk2 inhibition has the same effect on DBC1-SIRT1 regulation as Chk2 knockdown, experiments were performed in U2OS cells overexpressing DBC1 that, prior to etoposide exposure, were pre-treated with VRX0466617 (hereafter referred to VRX), a selective inhibitor of Chk2 . The resCollectively, besides ATM/ATR, the activity of Chk2 kinase is also required for DBC1-mediated SIRT1 inhibition, ensuing acetylation/activation of p53, and finally apoptosis.To investigate the mechanism involved in SIRT1 inhibition by DBC1 and Chk2, we initially determined whether Chk2 depletion could affect DBC1 phosphorylation on Thr454, but, as shown in Supplementary Figure S4, no significant differences were found between control and siChk2 transfected cells before and after etoposide exposure. We then analyzed DBC1-SIRT1 association in response to DNA damage in control and Chk2 depleted U2OS cells. Although DBC1 and SIRT1 protein levels were similar in all cell extracts Figure ,29, but We next carried out co-IP experiments to determine whether Chk2 physically interacts with DBC1 and SIRT1 in human cells and to what extent DNA damage regulates this interaction. For this, Chk2 was immunoprecipitated from U2OS cells treated or untreated with neocarzinostatin or etoposide and immunoblotted for DBC1 and SIRT1, and, as shown in Figure We next analyzed whether DBC1 could affect Chk2-SIRT1 association. U2OS cells were therefore transfected with siRNA to knockdown DBC1 and SIRT1 immunocomplexes analyzed for Chk2. As shown in Figure Together, these co-IP studies are consistent with a model where DBC1, SIRT1 and Chk2 form a protein complex in human cells, and loss of any one of these proteins does not impair the binding between the other two.in vitro kinase assays with recombinant GST-Chk2 , we checked in s Figure  evidence of REGγ . To detem Figure , in accom Figure , markedln Figure . TogetheWT or REGγS247A in control or Chk2 silenced U2OS cells, we detected an increase in p53 acetylation, upon etoposide, only with the ectopic wild-type protein and in Chk2 proficient cells (Figure WT, but not of REGγS247A, can promote the accumulation of PUMA and the increase of U2OS cell death (Figure The potential role played by REGγ in DBC1-dependent inhibition of SIRT1 was analyzed in DBC1-overexpressing U2OS cells transfected with siRNA against REGγ or a control sequence, and the results showed that REGγ depletion reduces the DBC1-dependent increase in p53 acetylation Figure . Moreoves Figure , clearlyh Figure . To furth Figure .WT increases in response to DNA damage, the association with REGγS247A is reduced (Figure We then determined the function of REGγ phosphorylation on Ser247 in DBC1-REGγ complex formation and found that, whereas the binding of DBC1 to REGγin vitro, although we cannot definitely exclude the occurrence of these events in vivo, where other factors might favor Chk2 activity toward these substrates. Curiously, when we analyzed the role of DBC1 in Chk2 induced apoptosis, we found that DBC1 depletion does not affect cell death, as previously reported, although it reduces p53 acetylation and PUMA induction, but not PARP1 cleavage. This phenomenon might be ascribed to different experimental conditions, which can result in different effects of DBC1 depletion. Indeed, recently, other papers reported that DBC1 absence, in response to various stimuli, enhances cell death (Here, we report that catalytically active Chk2 is required for DBC1-mediated SIRT1 deacetylase inhibition and p53-dependent apoptosis. This phenomenon is not achieved through Chk2-dependent alterations of DBC1 phosphorylation on Thr454, but through a mechanism in which Chk2 potentiates the DBC1-SIRT1 binding and consequently SIRT1 inhibition. Indeed this event is abolished by depletion or chemical inhibition of Chk2 and, as we demonstrated, it is not influenced by DBC1 expression levels, even if we found that Thr454 phosphorylation is necessary for the Chk2 mediated increase of DBC1-SIRT1 binding. However, although we noticed in Chk2-depleted cells reduced level of both PUMA protein and cleaved caspase-3, as previously reported in ,42, we cll death –45, thusin vitro and in vivo, and that this modification promotes p53 acetylation, PUMA protein induction and apoptosis. Indeed, although REGγ was found not to be implicated in the regulation of SIRT1 activity towards p53 (It was recently found that the activity of the 11S proteasome activator REGγ is regulated by CBP and SIRT1-dependent acetylation/deacetylation  and thatThus, according to our model, following DNA damage ATM phosphorylates p53-S15, DBC1-T454 and Chk2-T68, inducing p53 stabilization, DBC1-SIRT1 binding and Chk2 activation and phosphorylation of REGγ-Ser247. These events enhance DBC1-REGγ complex formation which, in turn, further increases its binding to and inhibition of SIRT1, finally leading to p53-dependent apoptosis (Figure In summary, we demonstrated that DBC1 promotes apoptosis by SIRT1 inhibition and that this phenomenon is regulated by the ATM-Chk2-REGγ axis Figure . With thSupplementary Data are available at NAR Online.


We found that the out-of-plane model only holds when buckling is the dominant failure mode, as should be expected. Furthermore, we controlled total pore volume by adjusting solids loading and sintering temperature. This strategy allows us to independently control macroporosity and densification of walls, and the compressive strength of ice-templated materials is exclusively dependent on total pore volume.We show that the honeycomb out-of-plane model derived by Gibson and Ashby can be applied to describe the compressive behavior of unidirectional porous materials. Ice-templating allowed us to process samples with accurate control over pore volume, size, and morphology. These samples allowed us to evaluate the effect of this microstructural variations on the compressive strength in a porosity range of 45–80%. The maximum strength of 286 MPa was achieved in the least porous ice-templated sample (P(%) = 49.9), with the smallest pore size (3  Macroporous ceramics are widely used in applications such as filtration, thermal insulation, scaffolds for tissue engineering, oxygen transport membranes, and solid oxide fuel cells1Strength is also an important parameter. It is usually increased by decreasing the total pore volume, even though it may degrade other functional properties. Morphological parameters such as pore size, morphology, or tortuosity can become crucial to maximize the performance while maintaining high strength. For example, a significant improvement in strength can be achieved by engineering anisotropic structures to mechanically reinforce the direction of the principle stress. However, most of the techniques conventionally used to produce macroporous ceramics with aligned porosity do not offer this level of flexibility.Ice-templating is a processing technique suitable for producing anisotropic macroporous materials. It is based on freezing of colloidal suspensions and segregation of particles by the solidification front. After solidification, the frozen solvent is removed, leaving pores whose morphologies are a replica of the sublimated crystalset al.et al.TiO2 by controlling the pore size through the polyethylene glycol (PEG) viscosity, pH, and isopropanol alcohol (IPA) concentration. Hunger et al.As predicted by Gibson and Ashby4545Our objective is to characterize the compressive strength of ice-templated ceramics in a broad porosity range and link their mechanical behavior to the pore architecture. We will discuss the effects of pore volume, size, and morphology on the failure mechanism. This discussion will allow us to assess the validity of the Gibson and Ashby models for the prediction of the compressive strength of unidirectional porous materials.Ice-templated specimens were produced following the same procedure reported elsewhereThe ice templating process consisted of pouring 10 ml of slurry into a PTFE mould (20 mm diameter and 25 mm height) placed on a copper plate and freezing from the bottom to the top. The top of the samples was exposed to air and kept at room temperature. The freezing temperature was controlled by circulating silicone oil regulated by a cryothermostat . The cooling rate was set at 2 °C/min. A faster cooling rate was achieved dipping a copper rod with the mould on top in liquid nitrogen. The cooling rate was monitored with a thermocouple and it was determined to be 25 °C/min on average. After solidification, samples were demoulded and sublimated for at least 48 h in a commercial freeze-dryer .Binder was removed from the green bodies by heating to 500 °C at 3 °C/min with a 5 h hold. Then, samples were sintered between 1300 °C and 1400 °C at 5 °C/min and held 3 h. The cooling rate was kept constant at 5 °C/min until room temperature.Samples with a non-oriented porosity were also prepared. The same zirconia powder was mixed with a commercially available pore former polypropylene  at different ratios  and mixed with distilled water. The slurry was magnetically stirred and ball milled for a minimum of 24 h to break up the agglomerates. Afterwards, the slurry was frozen by dipping the container in liquid nitrogen and freeze-dried to obtain an homogeneous mix. Then, 8 g of the powder was pressed at 0.8 MPa in a mould of 20 mm diameter. The sintering temperature and dwell time were the same as those used for the ice-templated samples. To ensure a proper burn-out and avoid the formation of cracks an extra hold was added at 900 °C.m) and volume (V) of the samples with respect to that of fully dense TZ-3YS (ρysz = 5.8 g/cm3), as:The overall porosity, P(%), was calculated based on the mass (The results were confirmed in at least two samples per condition by the Archimedes method (ASTM B962-13). The deviation between the overall porosity obtained by Archimedes and by The mechanical properties of specimens with isotropic (pore formers) and anisotropic (ice-templated) porosity were measured by a compression test  at a crosshead speed of 0.5 mm/min. The bottom and the top of the samples were removed with a slow speed saw leaving the final dimensions around 15 mm diameter and 17 mm height. Samples were tested with a cardboard pad on both sides to minimize the effect of superficial defects and misalignment. In all the tests, the maximum load at the end of the elastic stage was used to calculate the compressive strength.We investigated the effects of pore volume, size, morphology, and directionality on the compressive behavior of porous materials. The typical lamellar microstructure obtained in ice-templated samples when water is used as a solvent it is shown in 12131415μm) and a narrower distribution compared with the sample frozen at 2 °C/min (13.7 ± 4.8 μm), For ice-templated materials, the freezing rate is the most important feature controlling pore size. dpIA) and mercury porosimetry (dpHg), We measured pore size distribution using two different techniques: image analysis  into the initial slurry turns the pore morphology into a honeycomb-like structure with smooth surfacesμm, similar to the samples obtained by ice-templating. However, A set of samples with isotropic porosity  was alsoThe most widely used models to predict the mechanical response of cellular materials were developed by Gibson and Ashby4• Closed-cell (brittle crushing)C6 = 0.65 and ϕ represents the solid fraction in the edges, ρs and ρ* are the apparent density of the dense and cellular material respectively, and σp the modulus of rupture of the solid material within the walls.where • Open-cell (brittle crushing)C4 = 0.2, and where σp, ρs, and ρ* have the same meaning as in the closed-cell model.with • Honeycomb (out-of-plane)Es is the Young’s modulus of the corresponding dense material, and ρs and ρ* are again the apparent density of the dense and cellular material respectively.where 313233On the other hand, 4327μm). This effect is particularly remarkable at high relative density (i.e. high strength) where the amount of energy stored during compression is higher and materials tend to be more brittle. In contrast, at low values of relative density, the total pore volume and the presence of microstructural defects increase and favor the progressive damage by buckling of the struts and the fit by Compressive strength is the most reported mechanical property for ice-templated materials due to its simplicity in testing and sample preparation. Nonetheless, it is difficult to compare with the results in the literature because structures are notably different in terms of size, morphology, directionality, and even microstructure of the walls. However, it is possible to point out several trends common in the majority of samples.μm, and lamellar morphology. All specimens were tested with the pores aligned in the direction of the applied load. The P(%) = 45% and 62% samples exhibited an initial linear stage up to a sudden rupture, typical of brittle behavior. In contrast, samples with higher porosity (P(%) = 71%) presented a cellular-like rupture commonly observed in ceramic foams: after an initial elastic phase, the stress reaches a steady value caused by the progressive collapse of the strutsRepresentative stress-strain curves recorded during the compression test are shown in Although samples with isotropic porosity (pore formers) processed in this work have lower strength than the ice-templated ones , we mustHowever, there are other microstructural factors that also affect the mechanical performance of the isotropic samples such as the presence of large agglomerates  or the bSamples with lamellar and honeycomb structure  followed815414210Interestingly, when the pore volume increases the experimental strength values tend to those predicted by the honeycomb out-of-plane model, regardless of the pore structure. This behavior might be related with the transition between failure mechanisms (brittle and cellular-like fracture)Total pore volume can also be tailored by the sintering temperature. The higher densification of the walls at 1400 °C increases the observed shrinkage, Interestingly, the relative reduction of pore volume caused by the sintering temperature is remarkably different depending on the initial wall thickness, and hence the particle concentration used. For example, samples in et al.However, no additional increment in the strength has been observed caused by the reduction on the residual porosity in the walls at a given total pore volume, . All theThis work reveals that the compressive behavior of unidirectional porous ceramics can be predicted by the honeycomb out-of-plane model in a wide porosity range, from 45% to 80%. However, the use of the model is limited to structures with a continuous array of unidirectional walls and where buckling is the main failure mode. Further improvements of the model considering microstructural effects on the strength like the pore size reduction could lead to more accurate results.In addition, ice-templating exhibits a high versatility to control almost independently the main pore descriptors . The possibility to aim for a specific porosity, and hence strength, (through the solids loading) and tailor the densification of the walls (through the sintering temperature) is a powerful tool to adjust the specific surface area of the walls while keeping a constant pore volume. This flexibility can be useful in applications like SOFC cathodes or tissue engineering scaffolds, where a sufficient amount of macroporosity (interlamellar porosity) is required to guide the flow of the fluid, coupled with intralamellar porosity to improve conversion efficiencyHow to cite this article: Seuba, J. et al. Mechanical properties and failure behavior of unidirectional porous ceramics. Sci. Rep. 6, 24326; doi: 10.1038/srep24326 (2016).


Obesity is a known risk factor for breast cancer. Sphingosine kinase 1 (SK1) is an oncogenic lipid kinase that is overexpressed in breast tumours and linked with poor prognosis, however, its role in obesity-driven breast cancer was never elucidated.Human primary and secondary breast cancer tissues were analysed for SK1 and leptin receptor expression using quantitative real-time polymerase chain reaction (qRT-PCR) assay. Leptin-induced signalling was analysed in human oestrogen receptor (ER)-positive and negative breast cancer cells using Western blotting, qRT-PCR and radiolabelling assays.R = 0.78 and R = 0.77, respectively, P <0.001). Both these genes are elevated in metastases of ER-negative patients and show a significant increase in patients with higher body mass index (BMI). Leptin induces SK1 expression and activation in ER-negative breast cancer cell lines MDAMB-231 and BT-549, but not in ER-positive cell lines. Pharmacological inhibition and gene knockdown showed that leptin-induced SK1 activity and expression are mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and Src family kinase (SFK) pathways, but not by the major pathways downstream of leptin receptor (LEPR) - janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Src-homology 2 domain-containing phosphatase 2 (SHP2) appeared to be key to SK1 activation, and may function as an adaptor protein between SFKs and LEPR. Importantly, leptin-induced breast cancer cell proliferation was abrogated by SK1-specific small interfering RNA (siRNA).Our findings show for the first time that human primary breast tumours and associated lymph node metastases exhibit a strong correlation between SK1 and leptin receptor expression  contains supplementary material, which is available to authorized users. Wortmannin, LY294002, and SU6656 were from Calbiochem , UO126 was from New England Biolabs . Antibodies for p-SFK (Tyr416), total Src (cross-reacts with other SFK family members), ERK 1/2, phospho (p)-STAT 3, p-Akt (Ser473), Akt, JAK2 were obtained from New England Biolabs . p-ERK 1/2 from Sigma-Aldrich , STAT-3 from Santa Cruz Biotechnology . Other reagents and chemicals used were purchased from Sigma-Aldrich  unless otherwise specified.Recombinant human leptin was obtained from Sigma-Aldrich . . Ce. Ce20]. 2, 20 mM ATP and 10 μCi [γ-32P]-ATP (6000 Ci/mmol) were added and incubated for 1 h at 37°C. The reaction was stopped by addition of 50 μl 1 M HCl, 800 μl chloroform/methanol/HCl, 240 μl chloroform and 240 μl 2 M KCL. After centrifugation, the lower organic phase was collected and vaporised. Dried lipids were solubilised with 40 μl chloroform/methanol  and separated by thin layer chromatography on silica gel G60 plates using 1-butanol/ethanol/acetic acid/water  as migration solution. Plates were air-dried, exposed to X-ray film and quantified using Image J software.SK1 assay was performed using radiolabelling as previously described . Cell lyCells were seeded at a density to reach 30 to 50% confluence by the day of transfection. Forty nm siRNA oligonucleotides combined with oligofectamine in Opti-MEM™  were used. siRNA directed against SK1 was obtained from Applied Biosystems. All other siRNAs were purchased from Thermo Fisher Scientific  as pooled four independent sequences. Non-targeting siRNA, were used as a negative control . The Pearson’s correlation coefficient was calculated between the expression levels of two target genes. Multivariate analysis was performed to find the parameters fitting the best linear regression model based on the minimization of Akaike information criterion. The odds ratio (OR) for all pairs of parameters has been calculated via logistic regression.Data are presented as the mean values normalised to control ± standard error of the mean (SEM) calculated using GraphPad Prism . Statistical significance between two groups was conducted by unpaired Student’s P <0.001, R = 0.78) and LNs   and LNs (not significant)  Figure . MultivaAssessment of cellular proliferation by MTT assay showed that MDAMB-231 cells treated with 10, 100 and 1,000 ng/ml leptin exhibited a significant increase in proliferation from 72 to 120 h, with the maximum effect obtained at 1,000 ng/ml   followed by a second peak at 6 h   in MDAMB-231 . In contrast, SFKs inhibition significantly decreased STAT3 phosphorylation, which is also noticed to a lesser extent in combination with UO126 (P <0.001) Additional file 1: Table S1.: Patient characteristics with clinicopathological parameters of breast cancer patients. Figure S2. Leptin does not increase the proliferation of BT-549 cells in the absence of SK1 signalling. Figure S3. Leptin activates p-STAT3 and P-SFK in MDAMB-231 cells. Figure S4. Leptin activates p-STAT3 and P-SFK in BT-549 cells. Figure S5. Leptin increases SK1 expression and enzymatic activity and VEGF expression in BT-549 cells. Figure S6. Leptin activates p-STAT3 in a dose-dependent manner In MCF-7. Figure S7. Leptin does not increase SK1 expression and enzymatic activity and VEGF expression in MCF-7 cells. Figure S8. Leptin activates p-STAT3 in BT-474 cells. Figure S9. JAK2 silencing or PI3K/Akt inhibition does not abrogate STAT3 phosphorylation. Figure S10. STAT3 silencing potentiates SK1 expression. Figure S11. STAT3 siRNA silenced STAT3 and induced SK1 expression. Figure S12. Inhibition of ERK1/2 and SFK modulates STAT3 phosphorylation. Figure S13. ERK silencing increases STAT3 phosphorylation. Figure S14. ERK silencing attenuates SK1 expression and enzymatic activity and VEGF expression. Figure S15. SFK phosphorylation at site 416 is important for leptin signalling. Figure S16. Inhibition of ERK1/2 and SFK modulates STAT3 phosphorylation and attenuates SK1 and VEGF expression. Figure S17. Knockdown of SHP2 decreases SFK phosphorylation. Figure S18. Knockdown of SHP2 decreases SFK phosphorylation and SPHK1 expression. (PDF 2 MB)Additional file 2: Figure S1.: LEPR-Long and SK1 expression is slightly elevated in triple-negative breast cancer patients. Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Authors’ original file for figure 7Below are the links to the authors’ original submitted files for images.


Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr2+-binding sites and one Cs+-binding site were discovered in the HaBLA molecule.The tertiary structure of a β-lactamase derived from the halobacterium  2+ and Cs+, as the removal of the radioactive Sr2+ and Cs+ that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs+ or Sr2+. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr2+ and Cs+ ions were identified by anomalous X-ray diffraction. The location of one Cs+-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na+ (90 mM Na+/10 mM Cs+). From an activity assay using isothermal titration calorimetry, the bound Sr2+ and Cs+ ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs+-binding site provides important information that is useful for the design of artificial Cs+-binding sites that may be useful in the bioremediation of radioactive isotopes.Environmentally friendly absorbents are needed for Sr For example, alkaline phosphatases from Halobacterium salinarum  and from Halomonas sp. 593  require Na+ for quaternary-structure formation and Mg2+ and Zn2+ for enzyme activity  requires Mg2+ or Ca2+ for the recognition of a nucleoside as a substrate  requires Na+, Mg2+, K+, Sr2+ and Cd2+ for stabilization of the subunit conformation  is fixed in soil particles, mainly weathered biotite  derived from the moderate halophile Chromohalobacter sp. 560  was chosen as a model system. This selection was based on the following reasons.Burkholderia sp. Ch1-1  and Serratia marcescens . With this abundant content of acidic amino acids, the HaBLA molecule may provide scaffolds for metal ion-binding sites.(i) Abundant content of acidic amino acids. HaBLA, although a moderately halophilic periplasmic protein, has a particularly abundant content of acidic amino acids similar to those in extremely halophilic proteins. The ratio of acidic to basic amino acids in HaBLA [(Glu + Asp)/(Lys + Arg + His) = 57/32] is significantly higher than those in homologous nonhalophilic class C BLAs derived from et al., 2004et al., 2010et al., 2010Escherichia coli cytoplasm in a fully active form  High solubility and mesophilic heterologous expression. HaBLA shows high solubility, which results from the high content of acidic amino acids, which prevents the aggregation of the HaBLA molecules  has Asn-Gly dipeptide sequences at residue positions 288–289 and 321–322 which can promote deamidation. Because deamidation often causes heterogeneity of protein molecules in crystals and deteriorates the diffraction resolution, the N288Q/N321Q mutant (NQ-HaBLA) was prepared and used as the standard HaBLA in this study.et al., 2004E. coli BL21 Star (DE3) cells. The HaBLA gene was inserted into the pET-15b His-tag fusion vector  to construct pET-15b-bla. The expressed HaBLA proteins were purified to homogeneity using an Ni–IMAC column ; subsequently, the His tag of HaBLA was removed by thrombin digestion. The resulting non-His-tagged HaBLA protein was purified using a Resource Q anion-exchange column .Recombinant expression of the HaBLA proteins was performed essentially as described previously  and Wizard Screen I and II . After initial screening identified useful crystallization conditions, the protein concentration and precipitant conditions were optimized to obtain diffraction-quality crystals.Initial screening for crystallization conditions for WT-HaBLA and NQ-HaBLA was performed by the sitting-drop vapour-diffusion method using a 96-well Intelli-Plate  and a Hydra II Plus One crystallization workstation  at 293 K. Before crystallization, HaBLA was dialyzed against 50 mM HEPES buffer pH 7.0 containing 200 mM NaCl, 200 mM magnesium formate, 30%(w/v) polyethylene glycol (PEG) 3350 (which originated from PEG/Ion solution No. 20) with 47 mg ml−1 protein at 293 K. The NQ-HaBLA crystals were grown in 100 mM MES buffer pH 6.5 containing 200 mM calcium acetate, 18%(w/v) PEG 8000  with 30 mg ml−1 protein at 293 K.The WT-HaBLA crystal was grown in 50 m+ or Sr2+ in order to test their affinities for these ions: solutions 1A, 1B and 1C contained NaCl/CsCl at 0/100, 75/25 and 90/10 mM, respectively (in order to compare the affinity between Na+ and Cs+), while solutions 2A and 2B contained calcium acetate/strontium acetate at 0/200 and 100/100 mM, respectively, in order to compare the affinity between Ca2+ and Sr2+.The obtained NQ-HaBLA crystals were soaked in five different solutions containing Cs2.3.M Na+/100 mM Cs+), 1B (75 mM Na+/25 mM Cs+), 1C (90 mM Na+/10 mM Cs+), 2A (0 mM Ca2+/200 mM Sr2+) and 2B (100 mM Ca2+/100 mM Sr2+)] were measured using a wavelength of 1.000 Å. To identify the presence of Cs+ by anomalous diffraction, wavelengths of 2.066 and 2.166 Å were used for crystals from conditions 1A, 1B and 1C , Tsukuba, Japan, beamlines BL38B1 and BL41XU at SPring-8, Hyogo, Japan and beamline BL7 at the SAGA Light Source (SAGA-LS). All crystals tot is the molar concentration of the substrate being hydrolysed. The substrate concentration at any time point ([S]t) during the enzymatic reaction is calculated fromhydr is the substrate concentration that has been hydrolysed at any time point during the enzymatic reaction. From the measured dQ/dt and (2)t, Rt) data points is obtained. A continuous set of  is fitted by the Michaelis–Menten equation  in order to obtain Kcat and Km values, A brief description of the equations utilized for ITC analyses . Both WT-HaBLA and NQ-HaBLA crystallized isomorphously, and both contained three HaBLA molecules in the asymmetric unit. The overall structure of Ca2+-bound and Cs+-bound NQ-HaBLA from condition 1B (75 mM Na+/25 mM Cs+) determined at 1.8 Å resolution and Sr2+-bound NQ-HaBLA from condition 2A (0 mM Ca2+/200 mM Sr2+) determined at 1.9 Å resolution are shown in Fig. 1The crystallization of WT-HaBLA and NQ-HaBLA required divalent metal ions  chosen as the search model for molecular replacement are less than 1.2 Å. The surface charge distribution of the WT-HaBLA molecule is shown in Fig. 2a) and is compared with that of the EaBLA. The molecular surface of WT-HaBLA is almost fully occupied by negative charges, whereas equivalent positive and negative charges appear on the molecular surface of EaBLA, as shown in Fig. 2b).WT-HaBLA crystallized in the presence of Nan Table 1. The sol+ and Ca2+ and the structure was determined to 1.8 Å resolution. The solvent content in the NQ-HaBLA crystal is 43.0%. The r.m.s.d. on Cα atoms between the monomers of WT-HaBLA and NQ-HaBLA are less than 0.5 Å; therefore, the N288Q/N321Q mutation did not significantly alter the overall structure of HaBLA.NQ-HaBLA was initially crystallized with Na3.2.et al., 2005et al., 2010Ser64, Lys67, Tyr150, Glu272 and Lys315 are involved in the catalytic action of nonhalophilic class C BLAs  in each asymmetric unit (Supplementary Table S1). The site for Ca1 consists of Asp56 and Glu170, the site for Ca2 consists of Asp58 and Asp128, the site for Ca3 consists of Asp85, Asp87 and Asp187, the site for Ca4 consists of Asp124, the site for Ca5 consists of Asp199, the sites for Ca6 (in chain A), Ca7 (in chain B) and Ca8 (in chain C) consist of Asp219 and Asp220 and the sites for Ca9 (in chain A), Ca10 (in chain B) and Ca11 (in chain C) consist of Asp291, Glu295 and Glu352. These 11 binding sites for Ca2+ were integrated into seven sites per molecule for NQ-HaBLA .The binding sites for Na3.4.+, Cs+ and Ca2+  diffracted to resolutions of 2.0, 1.8 and 2.1 Å, respectively. All measured HaBLA crystals retained the same space group (P31) and similar unit-cell parameters  of other atoms. The structure of the Cs+-binding site in NQ-HaBLA is shown in Fig. 4The NQ-HaBLA crystals soaked into solutions containing Nas Table 1. The peaM Na+/100 mM Cs+) showed ten locations for bound Ca2+ (Ca1–Ca10) and two locations for Cs+ (Cs1 and Cs2) in an asymmetric unit (Supplementary Table S1). The peak heights of the anomalous difference Fourier maps were 17.5σ for Cs1 and 8.0σ for Cs2. The binding sites of Ca2+ were integrated into five sites in one molecule of NQ-HaBLA, which were the same locations as the site 2, site 3, site 5, site 6 and site 7 Ca2+-binding sites in NQ-HaBLA without soaking (Table 2+ ions were integrated into one site (site 8) per molecule of NQ-HaBLA.Structural analysis of NQ-HaBLA in condition 1A (0 mg Table 2. MoreoveM Na+/25 mM Cs+) showed ten locations for Ca2+ (Ca1–Ca10) and two locations for Cs+ (Cs1 and Cs2) in an asymmetric unit (Supplementary Table S1). The peak heights of the anomalous difference Fourier maps were 17.0σ for Cs1 and 7.5σ for Cs2. The binding sites of Ca2+ ions were integrated into six sites in one molecule of NQ-HaBLA, which were the same locations as the site 2, site 3, site 4, site 5, site 6 and site 7 Ca2+-binding sites in NQ-HaBLA without soaking (Table 2+ ions were integrated into one site (site 8) per molecule of NQ-HaBLA. This Cs+-binding site was in the same location as the site 8 Cs+-binding site in condition 1A  showed ten locations for Ca2+ (Ca1–Ca10) and one location for Cs+ (Cs1) in an asymmetric unit (Supplementary Table S1). The peak height of the anomalous difference Fourier map was 9.0σ for Cs1. The binding sites of the Ca2+ ions were integrated into six sites in one molecule of NQ-HaBLA, which were the same locations as the site 2, site 3, site 4, site 5, site 6 and site 7 Ca2+-binding sites in NQ-HaBLA without soaking  was discovered for conditions 1A (0 mM Na+/100 mM Cs+), 1B (75 mM Na+/20 mM Cs+) and 1C (90 mM Na+/10 mM Cs+)  Table 2.d, 4e and 4f). The aromatic ring of Trp189 interacts with Cs+ by a cation–π interaction. The distances between the identified Cs+ ion and the chelating main-chain O atoms in site 8 are slightly different in each molecule in the asymmetric unit and fall between 3.1 and 3.7 Å (Supplementary Table S1). In addition, the distance between the identified Cs+ ion and C atoms  of Trp189 are also slightly different in each molecule of the asymmetric unit and fall between 3.2 and 4.0 Å (Supplementary Table S1).Site 8 is located at the surface of a single HaBLA molecule and is comprised of two main-chain O atoms of Gln186 and Thr188 and the aromatic ring of Trp189  and 2B (100 mM Ca2+/100 mM Sr2+)] diffracted to resolutions of 1.9 and 2.8 Å, respectively. All measured HaBLA crystals retained the same space group (P31) and similar unit-cell parameters . The structure of the Sr2+-binding site in NQ-HaBLA is shown in Fig. 4NQ-HaBLA crystals soaked in solutions containing Nas Table 1. The peaM Ca2+/200 mM Sr2+) showed eight locations for Sr2+ (Sr1–Sr8) in an asymmetric unit (Supplementary Table S1). The peak heights of the anomalous difference Fourier maps were 7.5σ for Sr1 and Sr2, 9σ for Sr5, 12σ for Sr4, 13σ for Sr3, 14σ for Sr7, 15σ for Sr8 and 16σ for Sr6. The binding sites of Sr2+ ions were integrated into three sites per molecule of NQ-HaBLA, which were the same locations as the site 2, site 6 and site 7 Ca2+-binding sites in NQ-HaBLA without soaking  showed one location for Ca2+ (Ca1) and one location for Sr2+ (Sr1) in the asymmetric unit (Supplementary Table S1). The peak height of the anomalous difference Fourier map was 7.0σ for Sr1. The binding site for the Ca2+ ion was in the same location as the site 7 Ca2+-binding site in NQ-HaBLA without soaking  were discovered in conditions 2A (0 mM Ca2+/200 mM Sr2+) and 2B (100 mM Ca2+/100 mM Sr2+) . The coordination number of the identified Sr2+ at site 2 is five. The distance between the identified Sr2+ and chelating O atoms in site 2 lies between 2.4 and 2.7 Å (Supplementary Table S1). Site 6 is located on the surface of a single HaBLA molecule and is constructed of the Oδ1 atoms of Asp219 and Asp220 . The observed coordination number of the identified Sr2+ at site 6 is distributed between four and six. The distance between the identified Sr2+ and chelating O atoms in site 6 lies between 2.3 and 2.9 Å (Supplementary Table S1). Site 7 is located at the interface with a neighbouring chain in the asymmetric unit and is constructed of the Oδ1 atom of Asp291 and the O∊1 atoms of Glu295 and Glu352 . The observed coordination number for the identified Sr2+ at site 7 is distributed between three and seven. The distance between the identified Sr2+ and chelating O atoms in site 7 is different in each molecule in the asymmetric unit and lies between 2.3 and 2.8 Å (Supplementary Table S1).As mentioned above, three Sr) Table 2. Site 2 28 Fig. 4a. The c20 Fig. 4b. The o52 Fig. 4c. The o2+ and chelating O atoms in site 2, site 6 and site 7 of NQ-HaBLA are each 2.6 Å, which is very similar to those of known Sr2+-binding sites, including 2.6 Å in ribonuclease T1 , 2.6 Å in P-selectin , 2.5 Å in PLAP  and 2.6 Å in the oligomerization domain of rotavirus NSP4 .The average distances between Sr3.8.kcat, Km and kcat/Km) estimated from Michaelis–Menten plots are summarized in Fig. 6M. Interestingly, HaBLA maintained enzymatic activity without metal ions (0 M NaCl), where the kcat, Km and kcat/Km values were 4.25 ± 0.04 s−1, 0.133 ± 0.003 µM and 32.1 ± 0.9 s−1 µM−1, respectively. The value of kcat decreased to 2.72 ± 0.05 s−1 on an increase in NaCl concentration to 4.0 M. The Km value decreased to 0.023 ± 0.007 µM on an increase in NaCl concentration to 2.0 M and increased again to 0.043 ± 0.014 µM at 4.0 M. The maximum kcat/Km value (142.8 ± 20.4 s−1 µM−1) was estimated to be at 2.0 M NaCl  and 6(d)kcat, Km and kcat/Km were 3.18 ± 0.05 s−1, 0.031 ± 0.005 µM and 105.2 ± 11.0 s−1 µM−1, respectively).The salt dependency of penicillin G hydrolysis by HaBLA was assessed by ITC. Exothermic calorimetric thermograms of the enzymatic reaction and Michaelis–Menten plots are shown in Fig. 5M NaCl/40 mM MgCl2, 1.0 M NaCl/40 mM CaCl2 and 1.0 M NaCl/40 mM SrCl2 were also compared in order to assess the divalent metal-ion dependency of the enzymatic activity. The kcat, Km and kcat/Km values in the presence of 1.0 M NaCl/40 mM SrCl2 were similar to the values obtained in 1.0 M NaCl/40 mM MgCl2 and 1.0 M NaCl/40 mM CaCl2, as shown in Figs. 6(c) and 6(d)kcat, Km and kcat/Km are 3.17 ± 0.03 s−1, 0.040 ± 0.005 µM and 79.6 ± 3.7 s−1 µM−1, respectively).The enzymatic activities of HaBLA in 1.0 + is present instead of Na+ and even if Sr2+ is present instead of Mg2+ and Ca2+.From these results, it is demonstrated that HaBLA maintains enzymatic activity even if Cs4.M) even in the negatively charged circumstances of the overall structure. Seven binding sites (site 1 to site 7) for divalent metal ions (Ca2+ and/or Sr2+) and one binding site (site 8) for Cs+ per HaBLA molecule were identified by X-ray diffraction studies including anomalous dispersion. Although metal ions belonging to the same group in the periodic table have similar chemical characteristics, at least two metal ion-binding sites exhibited specificity for different metal ions (i.e. site 6 selects Sr2+ rather than Ca2+ and site 8 selects Cs+ rather than Na+). To understand the mechanism of the metal-ion selectivity, the structural characteristics of the metal ion-binding sites are discussed.HaBLA has an abundant content of surface acidic amino acids that have enabled adaptation to high salt concentrations. The crystal structure of HaBLA shows a unique active-site structure comprising the positively charged residues Lys68, His113, Arg149 and Lys316 and a hydrophobic cluster , which helps to maintain the enzymatic function over a wide range of salt concentration  (Table 32) is similar to those of nonhalophilic homologous BLAs (13 928–14 797 Å2), the acidic residue content [(Asp + Glu)/(Arg + Lys + His)] at the solvent-accessible surface of HaBLA (1.68) is about twice as large as those of the nonhalophilic BLAs (0.70–0.88). The number of negative charges [(Asp + Glu) − (Arg + Lys + His)] at the surface of HaBLA is calculated to be 21, which is also larger than those of nonhalophilic homologous BLAs (−5 to −12). From the ASA and the number of negative charges, the negative charge density at the surface of HaBLA is calculated to be 1.49 × 10−3 e Å−2, which is larger than those of nonhalophilic homologous BLAs (−0.34 × 10−3 to  −0.86 × 10 3 e Å−2)  Table 3. While t) Table 3. In addiM salt). However, the results of the enzymatic assay indicated that HaBLA can hydrolyse penicillin G over a wide salt concentration range (e.g. 0–4.0 M NaCl). In order to understand the relationship between the structure and function of HaBLA, we compared the crystal structure of HaBLA with those of other halophilic proteins.On the other hand, a high content of acidic residues in halophilic proteins may destabilize their structure and remove their enzymatic function under low salt concentrations owing to charge repulsion  from the extreme halophile Haloferax volcanii  to 4.0 M NaCl, the tertiary structure of HaBLA including the positive charges and the hydrophobic cluster is maintained under these solution conditions.Since the enzymatic activity of HaBLA was retained in solutions containing 1.0 4.2.2+ or Sr2+ were successfully identified in NQ-HaBLA , <1.63 Å for site 6 (Asp219 and Asp220) and <2.65 Å for site 7 . Therefore, it can be concluded that side-chain structural adaptations to the ionic radius of the specific metal ions at sites 2, 6 and 7 appear to be responsible for enabling suitable coordination of the ligand O atoms.Three binding sites  for the recognition of CaA Table 2. There aM.Divalent metal-ion binding to HaBLA may also contribute to the association of HaBLA molecules. Several binding sites for divalent metal ions  are located at the interface of HaBLA molecules; furthermore, HaBLA precipitated in crystallization mother liquor containing divalent metal ions at concentrations above 200 m2+ in solution, since crystal packing is necessary to construct these sites. On the other hand, site 6 in monodisperse HaBLA may bind Sr2+ in solution, since the crystal-packing arrangement is not needed to construct this site. Therefore, structural information on site 6 might be useful in creating an artificial Sr2+-binding site.From these results, it is suggested that HaBLA has a broad spectrum of specificity for alkaline-earth metal ions and that the association of HaBLA molecules may be promoted by alkaline-earth metal ions. Sites 2 and 7 in monodisperse HaBLA may not bind Sr4.3.+-binding site is not located at the interface of HaBLA molecules but is constructed on the surface of a single HaBLA molecule , the guanine nucleotide-binding protein G(I) , subtilisin  and the potassium inwardly rectifying channel . The Cs+ selectivity of these proteins was not determined, since the location of Cs+ in these crystal structures was identified only in the presence of a high concentration of Cs+ and low (or zero) concentrations of other metal ions .As mentioned previously, the site 8 Csle Fig. 4, indicat89 Fig. 4. Similar+ selectively from the mother liquor even when it contained a ninefold greater concentration of Na+ over Cs+ , indicating that site 8 shows a greater affinity for Cs+ than Na+. Because the r.m.s.d. for the main-chain O atoms and the side chain of Trp189 at site 8 among all observed HaBLA structures is small (<0.18 Å), it can be concluded that the rigid conformation of the site 8 Cs+-binding site must be conserved in the presence of Na+, Mg2+, Ca2+, Sr2+ and/or Cs+, and this retention of the conformation is postulated to be responsible for the specificity for Cs+.On the other hand, we found that site 8 can recognize Cs+-selective binding site in the halophilic protein HaBLA. Future studies include the following. (i) Transplantation of the structure of the Cs+-selective binding site from HaBLA to other proteins: since it may be possible to create an artificial metal ion-binding site using this structural information  Improvement of the Cs+ affinity and/or selectivity of HaBLA: if the number of chelating O atoms with suitable ionic bond distances to Cs+  A search for proteins having a Cs+-selective binding site: if proteins having a structure similar to the Cs+-selective binding site of HaBLA are identified in the PDB, these proteins might be useful as a scaffold to create a protein absorbent for Cs+.In conclusion, we succeeded in discovering a Csβ-lactamase, wild type, 3wrtPDB reference: N288Q/N321Q mutant, 3wrzPDB reference: 3ws0PDB reference: 3ws1PDB reference: 3ws2PDB reference: 3ws4PDB reference: 3ws5PDB reference: 10.1107/S1399004714027734/mn5080sup1.pdfSupporting Information.. DOI:


Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc.The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27 Myc family, c-Myc, N-Myc and L-Myc, are master regulators of cell proliferation [Oncogenes of the feration –5. The Mferation –9. In adferation –5, 10. Iferation .Fbxw7 [Skp2, Huwe1/HectH9, SCFβ-TrCP and SCFFbxo28 [The Myc protein is short-lived, having a half-life of around 30 min and is degraded through the ubiquitin-proteasome pathway . PhosphoFbxw7 , 20. TheFbxw7 . In addiCFFbxo28 , 21–24.Kip1, from now on referred to as p27. Here we show that p27 in addition to its inhibition of Cdk2 also overrides Myc's suppression of senescence independent of Ser-62 and induces Myc degradation independent of Cdk2 by binding Myc via its C-terminus. Further, high expression of active p27 protein significantly correlated with low Myc protein level in human breast cancer.We recently demonstrated that Cdk2-mediated phosphorylation of Myc at Ser-62 plays an important role in regulating a subset of Myc target genes involved in suppression of cellular senescence and differentiation, thereby contributing to malignant transformation , 25. SenWe have previously reported that Myc cooperates with Ras in transformation of primary rat embryo fibroblasts (REFs) by suppressing Ras-induced senescence, and that this function of Myc depends on Cdk2-mediated phosphorylation of Ser-62 . FurtherTo address this issue, primary REFs were cotransfected with H-Ras together with wt Myc or a phospho-mimicking Myc S62D mutant and exposed to the Cdk2 inhibitor CVT-313 or enforced expression of p27, after which senescence was measured. Ras alone induced senescence-associated β-Gal  activity and coexpression of c-Myc suppressed senescence induction, while treatment with CVT-313 or p27 cotransfection restored senescence as expected Figure , 1B. HowFbxw7−/− cells, in which c-Myc was stabilized  observed in response to TPA in parental U-937-GTB cells was strongly inhibited in U-937-myc-6 cells. However, co-stimulation with IFN-γ resulted in increased CD11c and reduced proliferation in U-937-myc-6 cells to a similar level as in TPA-treated parental cells, in accordance with previous results [p27 expression is induced by growth inhibitory cytokines like IFN-γ and TGF-β , 26–28.  results . Treatme results .p27 mRNA level was essentially unaffected (data not shown). We therefore addressed whether IFN-γ + TPA or IFN-γ alone might regulate v-Myc and/or endogenous c-Myc protein turnover by induction of p27. v-Myc runs slightly faster than endogenous human c-Myc in SDS-page gels [Both IFN-γ + TPA and IFN-γ treatments induced p27 expression in U-937-myc-6 cells Figure  in agreeage gels   followed by chase. In response to IFN-γ + TPA, the turnover rate of v-Myc and endogenous c-Myc began to increase already within 4 hrs and was further accelerated after 8 hrs of IFN-γ + TPA treatment as demonstrated by western blot analysis . The incAs expected, the increased turnover rate of Myc in response to IFN-γ and IFN-γ + TPA was accompanied by increased Myc ubiquitylation Figure . Further−/− MEF cells, verifying that IFN-γ mediated turnover of c-Myc is indeed p27-dependent  were treated with IFN-γ after which Myc levels were determined by western blot analysis. While IFN-γ treatment strongly reduced Myc protein levels in wt MEFs, no effect on the steady state levels of c-Myc was seen in p27t Figure . IFN-γ t wt MEFs .Taken together, these results suggest that IFN-γ induces Myc ubiquitin/proteasome-mediated degradation through induction of p27.To study Myc turnover and intracellular localization after IFN-γ in living cells, we expressed a c-Myc-eGFP fusion protein together with an eCFP reporter protein in 2fTGH cells. IFN-γ treatment decreased the c-Myc-eGFP fluorescence signal to approximately 40% of control within 4 hrs relative to the eCFP fluorescence signal Figure , 3C. No To study where ubiquitylation of Myc occurs in response to IFN-γ, a BiFC experiment was performed in living 2fTGH cells using the c-Myc-YC and YN-ubiquitin (Ub) fusion proteins to visualize conjugation of ubiquitin to c-Myc. Increased c-Myc/Ub BiFC fluorescence intensity, indicative of increased c-Myc ubiquitylation, was observed in IFN-γ stimulated compared to non-stimulated cells after proteasome inhibition Figure , 3G. TheMany of the biological effects of signaling via the IFN-γ receptor are mediated through activation of the Jak/Stat1 pathway . Indeed As mentioned in the Introduction, the Thr-58 phosphorylation site in MB1 plays a pivotal role in the regulation of c-Myc degradation , 17, 18.Taken together these results suggest that IFN-γ-induced Myc degradation is Jak/Stat1- dependent but Myc Thr-58-independent.Skp2, which promotes c-Myc degradation independent of MB1 [As shown in Figure t of MB1 , 24 and t of MB1 –28. To scyclin D2 promoter  and the C-terminal part of p27 covering amino acids 82–198 (N101)  constructs. N101, but not N100 or the control vector, increased c-Myc turnover Figure , indicatA levels .p = 2.44e−13), and a strong correlation (r = 0.5) between Myc protein level and p27 T157 phosphorylation was observed  data portal. The TCGA breast invasive carcinoma (BRCA) data set  containsobserved .p = 4.74e–02), while we observed the same trend for overall patient survival . survival . The grosurvival . UnfortuFbxw7 and of SCFSkp2, which have been connected to Myc turnover previously, the former through MB1 and the latter connected to p27 [The activity and stability of the oncoprotein Myc is controlled by posttranslational modifications and protein-protein interactions –5, 37. Wd to p27 , 23, 24.Cdk2 is the best-known target for the anti-proliferative activity of p27. An important point of this work is that p27 not only affects Myc by binding to and inhibiting Cdk2, but also by interacting directly or indirectly with Myc Figures  and 6, tBinding of Myc to p27 was shown to be dependent on the presence of amino acids 294–366 of Myc, which includes MB4, the NLS and the basic DNA-binding region. This region contains several ubiquitylation/acetylation sites implicated both in positive and negative regulation of Myc , 40, 41.Skp2 [The binding of p27 to Myc required the C-terminal half of p27, but not the N-terminus that binds to and inhibits Cdk2. The C-terminal region contains a number of regulatory phosphorylation sites and is involved in p27 protein stability, nuclear localization, protein interactions as well as CDK-independent functions of p27 in the cytoplasm , 44, 45.Skp2 –28. The Skp2 –28. CytoInterestingly, C-terminal part of p27 was sufficient to induce Myc degradation. It is at present unclear by what mechanism the C-terminus regulates Myc turnover. It is conceivable that the binding of p27 to Myc either involves recruitment of an E3 ligase that degrades Myc or alternatively excludes binding of a protein that stabilizes Myc, but at present we do not have any evidence that the binding of the C-terminus p27 to Myc is required for Myc degradation or whether this fragment acts through some other mechanism.Myc and p27 is thus involved in a mutually antagonistic relation to each other. It has been shown previously that Myc antagonizes p27. Myc directly represses transcription from the p27 promoter  and inhiin situ and invasive breast cancer [High Myc copy number/expression in tumors is known to correlate with high tumor grade and poor prognosis in a number of different tumors –5, 26–28t cancer . High p2t cancer . HoweverH. pylori eradication in chronic gastritis [Although we cannot say whether there is a causal relationship between high p27 and low Myc protein in these tumors, this would be compatible with a scenario where high p27 expression causes downregulation of Myc protein levels. Inverse relation between p27 and Myc protein levels has previously been observed in tumor biopsies from chronic lymphocytic leukemia (CLL)  as well astritis . It was astritis , which iin vivo by inducing cellular senescence [In summary, we show here that p27 targets Myc both indirectly through Cdk2 and directly by binding Myc  or Dulbecco's modified essential medium (DMEM)  supplemented with 10% fetal calf serum and antibiotics. The U937 clone myc-6 expresses the OK10 v-myc gene [5/mL) and Colo-320 cells were treated with 1.6 × 108 mol/L TPA  and/or 100 U/mL IFN-γ (1000 U/ml for 2fTGH cells) . 3H-thymidine incorporation assays and FACS analysis of CD11c expression were performed as described earlier [Cells were cultured in RPMI-1640 medium , Lipofectamine (BD Biosciences) or Superfect (Qiagen) according to manufacturer's instructions, or were electroporated as described . The folPulse chase, immunoprecipitation and immunoblot analyses were performed as described previously , 67. TheThe proteasome inhibitor Z-leu-leu-leu-H aldehyde (MG115)  and N-acetyl-leucinyl-leucinyl-norleucinal-H (LLnL) (Sigma) or vehicle were added to the cells 2 hrs before harvest at a concentration of 50 μM. To block protein synthesis, cycloheximide (CHX) was applied at a concentration of 100 μg/ml.7 cells was immunoprecipitated with 2 μg antibody. The following antibodies were used in CHIP: α-pan-Myc (IG-C), and pre-immune serum (IG-0). Primer sequences for the cyclin D2 promoter (E-box binding site region) used in Quantitative real-time PCR: forward primer 5′ CCCCTTCCTCCTGGAGTGAAATAC, reverse primer 5′ CGTGCTCTAACGCATCCTTGA-GTC. RT-qPCR analysis of mRNA expression of CCND2 and MYC was performed as described [Chromatin immunoprecipitations were performed as detailed . Brieflyescribed . Primer 2fTGH cells were cotransfected with constructs containing Myc-GFP together with CFP. BiFC was performed essentially as described  using coProtein expression data for breast invasive carcinoma (BRCA) from The Cancer Genome Atlas (TCGA)  were accAll analyses were performed in R (version 3.1.1). Associations between the expression levels of the target proteins were evaluated using Spearman's rank correlation coefficient. The correlations were derived using the rcorr function from the Hmisc package. Differences in Myc protein levels between patients with high p27 expression but low expression levels of p27PT157 and the remaining patient samples were analyzed using the Wilcoxon rank sum test from the R stats package. Differences in survival time between the indicated patient groups were assessed using the R survival package. A significance level of 5% was considered as statistically significant.


Plumbago zeylanica L., has drawn a lot of attention for its plenty of pharmacological properties including antidiabetes and anti-cancer. The aim of this study was to investigate the effects of plumbagin on CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver and evaluate the potential herb-drug interactions using the cocktail approach. All CYP substrates and their metabolites were analyzed using high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). Plumbagin presented non-time-dependent inhibition of CYP activities in both human and rat liver. In humans, plumbagin was not only a mixed inhibitor of CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, but also a non-competitive inhibitor of CYP1A2, with Ki values no more than 2.16 μM. In rats, the mixed inhibition of CYP1A2 and CYP2D1, and competitive inhibition for CYP2B1, CYP2C11 and CYP2E1 with Ki values less than 9.93 μM were observed. In general, the relatively low Ki values of plumbagin in humans would have a high potential to cause the toxicity and drug interactions involving CYP enzymes.Plumbagin , a natural naphthoquinone compound isolated from roots of  Plumbago zeylanica L. (family: Plumbaginaceae), also known as Chitrak, is a medicinal plant and mainly distributed around Africa and Asia including India and China1Plumbago zeylanica L. is an active naphthoquinone and responsible for its therapeutic effects1in vitro to predict the potential HDIs, which is the major attrition in drug developmentThe cytochrome P450 (CYP) enzymes play an important role in metabolism of xenobiotics and endogenous substances. Almost 90% drugs are metabolized by CYP superfamily9111214The aim of this study was to investigate the effects of plumbagin on several major CYP activities both in human and rat liver, including CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4, and further explore the mechanisms of plumbagin on CYP inhibitory properties. In this report, the cocktail approach using classical substrates including phenacetin (CYP1A2), bupropion (CYP2B1/6), tolbutamide (CYP2C9/11), dextromethorphan (CYP2D1/6), chlorzoxazone (CYP2E1) and midazolam (CYP3A2/4)1050  .Ki values were determined using the secondary Lineweaver–Burk plot, and then calculated at 0.15 μM, 1.62 μM, 2.16 μM, 1.46 μM, 0.65 μM and 0.88 μM for CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, respectively. From the secondary plot of Lineweaver–Burk plot for αKi, αKi values were 0.16 μM, 2.92 μM, 1.46 μM, 2.22 μM, 2.64 μM and 0.57 μM for CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, respectively. Since α values of CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 were not equal to 1, the types of inhibition by plumbagin for these five CYPs were mixed inhibition. However, the type of inhibition by plumbagin for CYP1A2 was non-competitive inhibition due to α value of CYP1A2 approximately equal to 1  which is used to treat leucoderma in Uighur medicine. Nowadays, herbal medicines, especially being consumed along with modern drugs, are increasingly popular when patients are in several diseases or conditions1120142223222425232731Plumbago zeylanica L.Km values of each CYP-catalyzed reaction according to published papers16To evaluate the potential HDIs, the cocktail approach, frequently used for CYP inhibition studies333425, which was the most sensitive point for detecting a time-dependent inhibitor3636To investigate whether inhibition of CYPs by plumbagin was time-dependent, time-dependent inhibition assays were performed with various plumbagin concentrations close to ICKi values were 1.62 μM, 2.16 μM, 1.46 μM, 0.65 μM, 0.88 μM and 0.15 μM, respectively. Correspondingly, mixed inhibition on CYP1A2 and CYP2D1, but competitive for CYP2B1, CYP2C11 and CYP2E1 were observed in RLMs and the Ki values were 2.64 μM, 9.49 μM, 9.93 μM, 7.85 μM, and 6.28 μM, respectively. Thus, plumbagin was a potent inhibitor on the above CYPs in both humans and rats.The present study showed that the types of inhibition by plumbagin were mixed inhibition on CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, but non-competitive for CYP1A2 in HLMs and the Ki values of inhibition on CYPs by plumbagin in HLMs are generally lower than those in RLMs. It may be attributed to variations in CYP levels, inducibilities and the existence of different CYP isoforms of the same protein family or subfamily in different speciesBased on the inhibition results of HLMs and RLMs, some differences should be noticed such as the types of CYP inhibition and inhibitory intensity. The As mentioned above, when investigating the enzyme inhibition kinetic of CYP3A2, we found it did not conform to the classical Michaelis–Menten kinetics in RLMs. In fact, metabolic activation was achieved by incubation of plumbagin in the presence of CYP3A2 when the concentrations of midazolam are less than 5 μΜ in RLMs42max), half-life (t1/2) and area under the curve (AUC) of plumbagin in the plasma was 0.35 μg/ml (1.86 μM), 1028 min and 271.9 min*μg/ml, respectively50 and Ki values determined in this study, thus suggesting plumbagin could quite probably decrease the activities of CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1 and CYP3A2 in vivo. In fact, these CYP homologues in humans are responsible for 80% of the CYP-mediated metabolism including a number of drugs with narrow therapeutic ranges. Therefore the potential HDIs should be brought to attention and further studies in vivo are needed to confirm the interactions of plumbagin with CYP isoforms in humans.Previous pharmacokinetic study of plumbagin by oral administration (100 mg/kg) in rats showed that the peak concentration  were obtained from Corning Gentest Corporation  and stored at −150 °C until use. All the experimental procedures involving humans have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) guidelines. Phenacetin, bupropion, tolbutamide, dextromethorphan, midazolam, 3-acetamidophenol , chlorpropamide , glucose 6-phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH), β-nicotinamide adenine dinucleotide phosphate (NADP) and tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) were purchased from Sigma Chemical Co. . Chlorzoxazone was purchased from Alfa Aesar . 4-acetamidophenol, hydroxybupropion, 4-hydroxytolbutamide, dextrorphan, 6-hydroxychlorzoxazone and 1-hydroxymidazolam were obtained from Toronto Research Chemical . Mebendazole  was obtained from Aladdin Industrial Co. . Plumbagin (purity > 98%) was purchased from Dalian Meilun Biotech Co., Ltd . Acetonitrile and methanol  were purchased from Fisher Chemicals . Formic acid (HPLC grade) was purchased from TEDIA . Distilled water was purified in a Millipore system Milli Q .Male Sprague-Dawley rats (200–250 g) were supplied by National Rodent Laboratory Animal Resources, Shanghai Branch of China. The animals were kept in a specific pathogen-free facility and with access to rodent cubes and sterile water, with 12 h light-dark cycles. All the methods in animals were carried out in accordance with the National Institutes of Health standards established in the ‘Guidelines for the Care and Use of Experimental Animals’. All experimental protocols in animals were approved by the Ethics Committee on Animal Experimentation of East China Normal University .g at 4 °C for 30 min. The supernatant was then centrifuged at 105,000 × g at 4 °C for 60 min. The pellet was reconstituted with 0.05 mM Tris/KCl buffer (pH 7.4) and stored at −150 °C until use. The protein concentration of the liver microsomes was determined by a protein quantitative assay using bicinchoninic acidIn this study, the rats were fasted overnight and killed by cervical dislocation before removal of the liver. The liver was excised, rinsed with ice-cold saline (0.9% NaCl w/v), weighed and homogenized in a 0.05 mM Tris/KCl buffer (pH 7.4). Subsequently, the homogenate was centrifuged at 10,000 × 2 (5 mM), G6P (10 mM), G6PDH (0.4 U/ml) and NADP (1 mM). Substrates were added into the incubation mixture and obtained the final concentrations , which were close to Km respectively. The proportion of organic solvent was not higher than 1% (v/v) in the incubation mixture18g at 4 °C. The supernatant (60 μl) was transferred to autosampler vials, and 2 μl was injected into LC-MS/MS system.Microsomal incubation was carried out at 37 °C in a mixture of 200 μl in 0.05 M Tris/HCl buffer (pH 7.4) consisting of pooled HLMs (0.5 mg/ml) or RLMs (1 mg/ml) and an NADPH-regenerating system including MgCl50  values were determined with the same incubation condition above. The specific concentrations of plumbagin were ranged from 0.1 μM to 50 μM. The IC50 values were calculated by plotting relative activities over the logarithm of plumbagin concentrations using GraphPad Prism 5.0 .The IC25 2 (5 mM), G6P (10 mM), G6PDH (0.4 U/ml), NADP (1 mM) and 0.05 M Tris/HCl buffer (pH 7.4) was performed for 0, 5, 10 and 20 min at 37 °C. Cocktail probe substrates were then added and incubated for 20 min. Reactions were stopped with 200 μl ice-cold acetonitrile. The samples were treated according to the microsomal incubation method and then injected into LC-MS/MS for analysis.To explore the time-dependent inhibition of plumbagin on CYPs, inactivation incubation containing plumbagin whose final concentration close to ICKinetic studies were performed for determining the types of inhibition on CYP by plumbagin. Pooled HLMs (0.5 mg/ml) or RLMs (1 mg/ml) with plumbagin  were incubated in 0.05 M Tris/HCl buffer (pH 7.4) for 20 min at 37 °C. A series of concentrations for cocktail probe substrates were used: 2.5–20 μM phenacetin for CYP1A2; 5–40 μM bupropion for CYP2B1/6; 5–40 μM tolbutamide for CYP2C9/11; 1.25–10 μM dextromethorphan for CYP2D1/6; 5–40 μM chlorzoxazone for CYP2E1; 2.5–80 μM midazolam for CYP3A2; and 0.625–5 μM midazolam for CYP3A4. Enzyme kinetics as well as statistical analysis were performed using GraphPad Prism 5.0.2), 1.6 MPa. A divert valve was used to divert the eluent to waste from 0 to 2 min, 10 to 11.5 min, and to MS from 2 to 10 min. Representative multiple reaction monitoring chromatograms of analytes were shown in LC-MS/MS is a powerful tool to detect the concentrations of several CYP substrates and metabolites in a short time, and to calculate the enzymatic reaction rate, which is used to evaluate the corresponding CYP enzyme activities. In this study, an Agilent 1290 LC system consisting of a degasser, a binary pump, an autosampler and a thermostatic column compartment was coupled with a 6460 triple-quadrupole mass spectrometer , which was equipped with an Agilent Jet Stream electrospray ionization (ESI) source and operated with Agilent MassHunter version 5.0.280.1 software . Chromatography separation was performed on a Phenomenex Kinetex XB-C18 column  protected by a Phenomenex C18 guard column . The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) using gradient elution at a flow rate of 0.3 mL/min. The optimum condition for elution was as follows: 0–2.2 min, 10–11% B; 2.2–8.5 min, 11–90% B; 8.5–9 min, 90–92% B; 9–9.3 min, 92% B; 9.3–9.6 min, 92–10% B; 9.6–11.5 min, 10% B. The temperature of column oven was maintained at 30 °C, and the injection volume was 2 μL. The mass spectrometer was operated in the positive/negative ion-switching ESI mode, and the detection of the ions was performed in the multiple reaction monitoring (MRM) mode. Each MRM transition and the compound dependent parameters are presented in 50 values were fitted by non-linear regression analysis using GraphPad Prism 5.0. The kinetic parameters were obtained by fitting velocity data to kinetic models (Michaelis-Menten) using GraphPad Prism 5.0. The Lineweaver-Burk Plot  was used to determine the quality of fit to a specific inhibition model. The inhibition constant (Ki) was obtained by a secondary plot using the slopes of the primary Lineweaver-Burk Plot (Km/Vmax versus inhibitor concentrations). αKi was obtained from a secondary plot using the y-intercepts of the Lineweaver-Burk plot (1/Vmax versus inhibitor concentrations). All results were analyzed in quadruplicate. One-way analysis of variance was used to estimate the significance of differences. There was statistical significance between control and test groups if p < 0.05.All data were presented as mean ± SD. The ICHow to cite this article: Chen, A. et al. Evaluation of the inhibition potential of plumbagin against cytochrome P450 using LC-MS/MS and cocktail approach. Sci. Rep.6, 28482; doi: 10.1038/srep28482 (2016).