PLOS Biology from the Wymann group provides new insights into this process by identifying an alternate route to PI3Kγ activation, and these results enable us to understand how PI3Kγ signaling is regulated in mast cells and is thus important in so many cell, tissue, and disease settings.In the huge collection of molecules that underpin mammalian biology, only a small number stand out as targets for drug development. PI3Kγ is one such molecule that has received substantial investments to assess whether inhibitors can be developed as potential therapeutics. Although many studies have addressed the structure and regulation of PI3Kγ, our understanding of the enzyme is far from complete. A publication in PI3Kγ is expressed strongly in a number of immune cells, including mast cells, neutrophils, and eosinophils. In these cell types, it sits at a key, early, near-receptor stage in pro-inflammatory, intracellular signaling pathways. This is, in part, why genetic loss or selective inhibition of PI3Kγ has little effect on normal mouse development and function but can suppress inflammation in a number of mouse models of disease, including rheumatoid arthritis 2 to yield the signaling lipid PtdInsP3. The class I PI3Ks can all be activated by cell surface receptors to drive accumulation of PtdInsP3 in the inner leaflet of the plasma membrane. This acts as a signal enabling proteins capable of binding PtdInsP3, typically via PH domains, to concentrate at the cytosolic interface of the plasma membrane. The classic example of a PH domain-containing PtdInsP3 effector is PKB (Akt), but there are possibly as many as 60–70 in a mammalian genome and up to at least 20 can be expressed in the same cell. This family of PtdInsP3 effectors transduce the lipid signals into many forms, including changes in protein kinase, Rac-family-GEF and Rho-family-GAP activity, and/or distribution.PI3Kγ (phosphoinositide 3-kinase) belongs to the class I PI3K family of signaling enzymes  that 3-phosphorylate the membrane phospholipid PtdInsPPIKAP) in vivo, although this is only based on the lack of any evidence for the existence of free p110γ. It appears that despite immune cells like neutrophils, expressing p101 and p84, mast cells, the subject of the study reported by the Wymann group, only express p84 PI3Kγ is made up of a p110γ catalytic subunit Our current understanding of PI3Kγ activation centers on how the p101 subunit allows the complex to be substantially activated by G-protein βγ subunits (Gβγs) liberated from G proteins upon activation of GPCRs. This mechanism is thought to explain why PI3Kγ is typically  activated by GPCRs. Frustratingly, we do not know the residues/domains in either p101 or p110γ involved in the interaction with Gβγs. It appears the interaction between p101 and p110γ is very tight and unlikely to have a significant on/off rate under physiological conditions. There is some evidence supporting direct interactions between Gβγs and both p101 and p110γ But the involvement of the p84 in PI3Kγ has been less clear. There is anecdotal evidence in the field that the interaction between p84 and p110γ is less tight than that between p101 and p110γ. Despite this, once bound, p84/p110γ complexes are able to survive gel-filtration chromatography or multiple cycles of washing with traditional detergent-containing lysis buffers through pull-down protocols without significant dissociation, suggesting that their association is not rapidly reversible. It is clear, however, that the p84/p110γ heterodimer is substantially less sensitive to Gβγs (at least to a limited subset of Gβγs that have been tested) than the p101/p110γ complex in vitro or in transfected cells in vivo, as demonstrated by the effect of knocking-in a Ras-insensitive, but Gβγ-sensitive, allele of p110γ in mice (p110γDASAA/DASAA) P110γ contains a classical Ras-binding domain (RBD) that has been demonstrated to bind selectively to Ras-GTP. Ras-GTP can activate p110γ, p101/p110γ, and p84/p110γ 3-Ars) GPCRs, that synergizes with the primary effects of FcεRI activation.Mast cells are tissue-resident immune cells that express high-affinity receptors for IgE (FcεRI). Mast cell FcεRI are effectively permanently bound with IgE. These FcεRI/IgE complexes are activated by ligands that are specific targets for the variable regions of IgEs, including allergens. Once activated, mast cells can release a huge range of inflammatory mediators, including histamine, LTB4, PAF, and PGD2. The intracellular signals generated by activated FcεRIs are primarily based on protein tyrosine kinase–mediated mechanisms that would naturally lead to activation of the class IA PI3Ks such as PI3Kδ PLOS Biology began with several threads, a key one of which was the ability of thapsigargin (a drug that causes a very selective receptor-independent increase in cytosolic Ca2+ by slowly releasing intracellular ER Ca2+ stores into the cytosol and activating Ca2+ influx via the store-operated-Ca2+-entry (SOCE) route) to stimulate phosphorylation of PKB in a PI3Kγ-dependent manner. That result was very unexpected and a variety of controls suggested it was unlikely to be dependent on release of the mast cell paracrine/autocrine factor adenosine acting back on A3 GPCRs.A recent study by Walser et al. in 2+ signal, involving SOCE, that drives activation of PKCβ. Through an interaction between PKCβ and the helical domain of p110γ, S582-p110γ is phosphorylated. This leads to both activation of the kinase activity of PI3Kγ  and a reduction in the affinity of p110γ for p84. The outcome of these events is argued to be a rebalancing in the amount of GPCR-sensitive p84/p110γ and free, GPCR-insensitive p110γ, thus reducing the sensitivity of PI3Kγ to GPCRs. These conclusions represent major conceptual advances for the field. Beyond their major impact on our understanding of mast cell biology, they offer potential molecular explanations for the roles of PI3Kγ in a number of important, but ill-understood, physiological and disease settings. Furthermore, the paper introduces beautiful structural data from deuterium-exchange protection assays that reveal where the presence of p84 masks the surface of p110γ in its helical domain. Further biochemical experiments demonstrate the potential power of interactions with, or post translational modifications of, the helical domain of p110s to regulate class I PI3K function both in terms of changes in catalytic activity and binding of adaptor subunits. This type of mechanism may be a widely important mode of regulation in class I PI3K signaling.Wide-ranging experiments support a model  in whichAs ever with first steps, many questions remain. The role of Ras in the activation of PI3Kγ in mast cells is unclear. Given that RasGrp1 (and RasGrp4) is relatively abundant in mast cells, and activated by a combination of PKC-mediated phoshorylation and diacylaglycerol (DAG) binding, and in lymphocytes is the primary mechanism driving activation of Ras downstream of antibody receptor activation PLOS Biology paper from Walser et al. makes many important contributions. It reveals how intracellular signals from mast cell FcεRIs, classically thought to engage class IA PI3K signaling, can control p110γ directly via the PLC effectors Ca2+ and DAG/PKC. These effects are mediated by an interaction between PKCβ and the helical domain of p110γ that enables S582-p110γ to be phosphorylated and activate the lipid kinase activity of PI3Kγ. The outcome is the emergence of a new mechanism by which class I PI3Ks can be activated. The paper also provides a first insight into the interactions between p110γ and p84, hopefully the first step in the exploitation of deuterium-exchange methodologies to reveal more about the interactions of p101, p84, p110γ, and Gβγs. Finally, the work may offer molecular explanations for some of the many poorly understood roles that PI3Kγ fulfills outside of the immune system.The


Apium graveolens) has the potential to trigger severe allergic reactions in sensitized patients and thus requires labeling on food products. The aim of the study was to determine the sensitization profile to celery allergens and to identify novel IgE binding proteins in this source.Consumption of celery (A. graveolens sensitized patients from Austria (n=23). Purified allergens were obtained from natural source (celeriac) or produced as recombinant proteins in E. coli and IgE reactivity to rApi g 1, rApi g 2, nApi g 5, and nApi g 6 was tested in ELISA. IgE cross-inhibition assays were performed with purified lipid transfer proteins (LTP). Celeriac extract was separated in 2D gel electrophoresis and IgE reactive spots were subjected to mass spectrometry-based analysis.Immunoblots were performed with extracts of celeriac and celery stalks using sera of A. graveolens sensitized patients. Fifty-six percent reacted to Api g 5 while 26-34% of patients were sensitized to Api g 6 (LTP2) and Api g 2 (LTP1) present in celery tuber and stalks, respectively. Interestingly, Api g 6 and Api g 2 sensitization pattern did not correlate and limited IgE cross-reactivity was observed. Eighteen IgE reactive spots from celeriac extract were obtained in 2D gel electrophoresis. Mass analysis identified them as members of the fructose bisphosphate aldolase, malate dehydrogenase, GAPDH, triosephosphate isomerase, chitinase, and manganese superoxide dismutase families.Immunoblots showed distinct IgE reactive bands at 10 and 16 kDa while broad reactivity was observed in the range between 25 and 100 kDa. IgE reactivity to Api g 1, which is solely present in celery tuber and absent in stalks was 60% in In vitro diagnosis using purified A. graveolens allergens is able to substitute extracts that might give rise to unspecific reactivity. In order to further enhance sensitivity and specificity, newly identified allergens should be produced and included in molecule-based diagnosis of celery allergy.None declared.


After having identified the acetyltransferase p300 among several acetyltransferases to be associated with Rb2/p130 during S-phase in NIH3T3 cells in vivo, we used this enzyme and the CDK4 protein kinase for in vitro modification of a variety of full length Rb2/p130 and truncated versions with mutations in the acetylatable lysine residues 1079, 128 and 130. Mutation of these residues results in the complete loss of Rb2/p130 acetylation. Replacement of lysines by arginines strongly inhibits phosphorylation of Rb2/p130 by CDK4; the inhibitory effect of replacement by glutamines is less pronounced. Preacetylation of Rb2/p130 strongly enhances CDK4-catalyzed phosphorylation, whereas deacetylation completely abolishes in vitro phosphorylation. In contrast, phosphorylation completely inhibits acetylation of Rb2/p130 by p300. These results suggest a mutual interdependence of modifications in a way that acetylation primes Rb2/p130 for phosphorylation and only dephosphorylated Rb2/p130 can be subject to acetylation. Human papillomavirus 16-E7 protein, which increases acetylation of Rb2/p130 by p300 strongly reduces phosphorylation of this protein by CDK4. This suggests that the balance between phosphorylation and acetylation of Rb2/p130 is essential for its biological function in cell cycle control.The retinoblastoma protein (pRb) and the related proteins Rb2/p130 and 107 represent the “pocket protein” family of cell cycle regulators. A key function of these proteins is the cell cycle dependent modulation of E2F-regulated genes. The biological activity of these proteins is controlled by acetylation and phosphorylation in a cell cycle dependent manner. In this study we attempted to investigate the interdependence of acetylation and phosphorylation of Rb2/p130  Soon after the discovery of the tumor suppressor retinoblastoma protein (pRb), two other proteins sharing the characteristic structural and functional properties of pRb were identified 2-period 0Rb2/p130 is a nuclear phosphoprotein, sharing homology within the pocket domain with both other family members but being more closely related to Rb1/p107 than to pRB. As both other members of the pocket protein family Rb2/p130 is phosphyorylated in a cell cycle dependent manner by cyclin-dependent kinases (CDKs); more than 20 distinct residues have been identified as phosphorylation sites 2-period vice versa.We have recently discovered that hyperphosphorylated Rb2/p130 exists in an acetylated form in NIH3T3 cells which is exclusively located in the nucleus; acetylation is cell cycle dependent, starting in S-phase and persisting until late Gin vivo. We then analyzed acetylation and phosphorylation by p300 and CDK4 of a variety of recombinant Rb2/p130 proteins with mutations in 3 acetylatable lysine residues and in truncated versions.In the present study we addressed the question whether phosphorylation and acetylation of Rb2/p130 are mutually interdependent. By immunoprecipitation we identified the acetyltransferase p300 to be associated with Rb2/p130 in vivo, in order to use this enzyme for our in vitro modification studies.In a first step we attempted to identify an acetyltransferase responsible for acetylation of Rb2/p130 Nuclear extracts of NIH3T3 cells synchronized in S-phase were incubated with antibodies against a variety of histone acetyltransferases or deacetylases. The immuno-precipitates were then analyzed for the presence of Rb2/p130. These experiments only revealed p300 to be associated with Rb2/p130. in vivo complexes contain Rb2/p130, p300 and HDAC II. Although this is not a proof that these three proteins are part of the same protein complexes, the result provides a strong indication.To substantiate this finding we extracted nuclear proteins from S-phase cells and purified high molecular weight protein complexes by sequential separation with anion exchange- and size exclusion chromatography. Size exclusion chromatographic fractions containing protein complexes of ∼750 kDa that contained Rb2/p130 were subjected to immunoprecipitation using antibodies against Rb2/p130, p300 and HDAC II, respectively. in vitroWe could not detect p300 and HDAC II in immunoprecipitates when using nuclear extracts of serum-starved, arrested NIH3T3 cells (result not shown). In line with these results, we had already shown that p300 is able to acetylate Rb2/p130 as well as truncated versions of the protein in vitro modification of Rb2/p130 by the acetyltransferase p300 and the protein kinase CDK4/cyclin D1, we expressed different full length and truncated FLAG-tagged Rb2 proteins in insect cells using the baculovirus expression system. in vitro modification studies.To study 14C-labelled acetylCoA with subsequent autoradiography of SDS-polyacrylamide gels resulted in radioactivity incorporation in Rb2/p130 as already reported Since we had previously demonstrated that K1079 is the main site of Rb2/p130 acetylation 32P-labelled ATP resulted in strong phosphorylation  Rb2/p130 that was phosphorylated in vitro by CDK4. The phosphorylation status was checked by immunoblotting with anti-phospho-serine antibodies. 14C-acetylCoA. in vitro acetylation.To analyze the effect of the phosphorylation status on Rb2/p130 acetylation by p300 we used three Rb2/p130 protein substrates in the acetyltransferase assay. i) Recombinant full length Rb2/p130, ii) Rb2/p130 that was dephosphorylated 2-period, it remained elusive, whether phosphorylation and acetylation occur independent from each other or are mutually affected by each other. The results presented in this paper rather suggest an interdependence of both modifications; the fact that deacetylated Rb2/p130 is not phosphorylated by CDK4, whereas increasing levels of acetylation are paralleled by increasing phosphorylation argues for acetylation to be a prerequisite for phosphorylation. This assumption is corroborated by the finding that only dephosphorylated Rb2/p130 is acetylated by p300, while phosphorylation by CDK4 abolishes the acetylation potential. We therefore speculate that Rb2/p130 acetylation precedes phosphorylation in vivo, although additional, yet unknown mechanisms could be necessary for in vivo phosphorylation.We have recently shown that Rb2/p130 is posttranslationally acetylated in a cell cycle dependent manner e. g. p53 Circumstantial support for acetylation being a prerequisite for phosphorylation also comes from the results presented in Moreover, proline 1077 and lysine 1079 are located in one of two nuclear localization sequences, flanked by phosphorylated serine 1064. Transfection experiments with p130/GFP constructs where lysine 1079 was mutated to arginine or glutamine did not change the nuclear localization of the reporter protein , which is in line with functional deletion studies demonstrating that deletion of one of the two C-terminal nuclear localization sequences was not sufficient to change nuclear localization of Rb2/p130 2 period in the nuclei of NIH3T3 cells in vivo.For Rb2/p130 it has been shown that phosphorylation by CDKs predisposes the protein for ubiquitination and subsequent proteosomal degradation Based on the data presented in this paper and previously published results NIH3T3 cells  were grown in DMEM/HAMs 15-K medium supplemented with 10% (v/v) fetal calf serum and 1 mM L-glutamine. Synchronization was achieved by serum starvation for 72 h; synchronous growth was induced by re-addition of fully supplemented medium.Spodoptera frugiperda) were cultivated in Gracès insect cell medium at 27°C and ambient atmosphere.For expression of recombinant proteins, Sf9 cells  were transfected using Cellfectin Transfection Reagent . Cells were seeded at a density of 1.5×106 per 35 mm dish 1 h before transfection. Cellfectin and the bacmid-minipreparation  were diluted in Grace medium and complex formation was allowed for 45 min before addition of 800 µl of medium and cells; cells were incubated for 5 h at 27°C in the transfection medium, after which cells were shifted to normal growth medium for 72 to 96 h. Several rounds of virus amplification were performed until recombinant protein levels were sufficient.Sf9 cells susceptible to infection with TMNuclear and Cytoplasmic Extraction Reagents, Pierce, No. 78733).For preparation of nuclear extract fractions of NIH3T3 cells a commercially available kit was used  of the affinity matrix  equilibrated 3 times in 1 ml lysis buffer was added to the supernatant. Binding was performed on an overhead shaker at 25 rpm at 4°C for 3 h. Beads were collected by centrifugation and washed 3 times with lysis buffer (800×g for 5 min at 4°C). Purified proteins were either used directly bound to beads or eluted 3 times from the affinity matrix by the addition of FLAG-peptide  at 4°C for 10 min while gently agitating. Eluted proteins were separated from the beads by centrifugation at 1000×g for 5 min.For extraction and purification of recombinant proteins from Sf9 cells, the cell pellets were resuspended in lysis buffer  glycerol, 0.1% (v/v) Nonidet NP-40, 10 mM 2-mercaptoethanol, 2 mM PMSF, 1×protease inhibitor cocktail ; 1 ml lysis buffer per 140 mm round dish). Cells were lysed in a pre-cooled 15 ml douncer. The lysate was centrifuged twice at 1000×Nuclear extracts were loaded onto a 1 ml MonoQ HR 5/5 anion exchange column (Amersham Pharmacia Biotech). After washing the column with chromatography buffer  glycerol, pH 7.8) bound proteins were eluted with a linear gradient from 10 to 700 mM NaCl in chromatography buffer. Fractions were assayed for p130. Fractions around 300 mM NaCl containing high amounts of p130 were pooled and applied to a TosoH TSKG4000PWXL size exclusion chromatography column (TosoH Biosep), equilibrated and eluted with chromatography buffer containing 150 mM NaCl. Fractions corresponding to molecular masses of 700 to 800 kDa containing high amounts of p130 were used for immunoprecipitation experiments.g for 5 min. The supernatant was saved for SDS-PAGE and immunoblotting. Beads were washed 4 times with 0.5 ml IP-buffer and finally resuspended in 50 µl of SDS-sample buffer for further SDS-PAGE and immunoblotting. Control precipitations were performed with nuclear extracts and Protein G Sepharose without antibodies.Nuclear extract or chromatographic fractions (200 µl) were incubated with antibody solution (2.5 µg/ml) for 60 min at 4°C. Antibodies used were: anti-CBP/p300 (Abcam No ab3164), anti-Rb2/p130 , anti-HDAC I , anti-HDAC II , anti-pCAF (Abcam No ab12188) and anti-Gcn5 (Abcam No ab18381). Protein G Sepharose  equilibrated in IP-buffer  Triton-X-100, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonylfluoride, 1 mM NaF) was added and incubated for 2 h at 4°C under gentle shaking, followed by centrifugation at 800×14C-acetylCoA for 30 min at 37°C . For pre-acetylation with subsequent phosphorylation radioactive acetylCoA was replaced by non-radioactive acetylCoA. The acetylation reaction was stopped by chilling on ice and either addition of SDS-sample buffer (for further SDS-PAGE and autoradiography) or by washing the beads 3 times with an excess of buffer K for subsequent phosphorylation reactions.Acetylation of recombinant Rb2 proteins (control or mutated versions) was performed by incubation of recombinant proteins bound to FLAG agarose beads in buffer P  with 20 ng of p300 and 20 µM 2, 30 mM NaCl, 0.2 mM dithiothreitol, 1 mM EGTA), 50 µM non-radiolabelled ATP, 20 ng CDK4/CycD1  and 10 µCi of gamma32P-labelled ATP. For pre-phosphorylation with subsequent acetylation radioactive gamma32P-labelled ATP was omitted. The assay mixture was incubated for 30 min at 37°C. The reaction was terminated by chilling on ice and either addition of SDS-sample buffer (for further SDS PAGE and autoradiography) or by washing the beads 3 times with an excess of buffer P for subsequent acetylation reaction.Phosphorylation of recombinant Rb2 proteins  was performed by mixing 200 ng of recombinant protein (3 µl) with 10 µl of buffer K  at 37°C for 45 min. After incubation 5 mM EDTA was added and the reaction mixture was heated to 70°C for 5 min. Then the mixture was washed 3 times with an excess of ice cold buffer P and used for subsequent acetylation reactions. For deacetylation purified HDAC2 from NIH3T3 cells was used at 30°C for 30 min. After incubation the reaction mixture was chilled on ice and washed 3 times with an excess of ice cold buffer P before used for subsequent phosphorylation reactions.Samples were subjected to SDS-10%-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with Coomassie Blue. For immunoblotting proteins were transfered to nitrocellulose membrane. Visualization of antibody binding was done with alkaline phosphatase conjugated secondary antibodies or electrochemi-luminescence. Autoradiography was performed by exposing gels on phospho-imaging plates that were analysed in a STORM phosphoimager system (Molecular Dynamics). Stained gels and blots were analyzed in a laser densitometer (Molecular Dynamics). Quantitative evaluation was done with Image-Quant software (Molecular Dynamics).


Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear.Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death.These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia. Endoplasmic reticulum (ER) stress, an important pathophysiological component of diseases such as cancer, diabetes mellitus, neurodegeneration and atherosclerosis, triggers complex specific cell signaling known as the Unfolded Protein Response (UPR) The ubiquitin-proteasome system interfaces with and importantly regulates the UPR. This effect, however, is complex and seemingly ambiguous in a number of aspects. Increased proteasome-mediated degradation of un/misfolded proteins merges with the UPR as an adaptive ER homeostatic mechanism ER stress and UPR signaling have been shown to mediate several aspects of the pathogenesis and natural history of atherosclerosis and vascular inflammation In this study, we investigated, in VSMC exposed to the classical ER stressor tunicamycin, the role of proteasome inhibition on cell viability, UPR signaling, oxidative stress and NADPH oxidase expression/activity. Our results indicate that proteasome inhibition, per se at non-lethal levels, suppresses ER stress-induced UPR signaling and Nox4 expression, but intriguingly increases XBP1 mRNA splicing. In parallel, proteasome inhibition sensitizes vascular smooth muscle cells to ER stress-induced death, through mechanisms not clearly dependent on ROS.Initial experiments were directed to establish concentrations of proteasome inhibitor not associated with cell death within the time frame of our experiments, since cell loss might be accompanied by possible secondary redox and other signaling events. VSMC were incubated in the absence of serum with a range of concentrations of the proteasome inhibitor MG132 (0.1 to 10 µM for 24 h). Cell loss detected by MTT assays started to appear at concentrations equal to or above 3 µM, so that results shown in To assess whether increase in proteasome activity is a normal constituent of the UPR, we assessed 20S proteasome activity (chymotrypsin-like) at several times after starting incubation with Tn. As early as 2 (data not shown) and 4 h of incubation , proteasThe pattern of early, but not sustained proteasome activity increase during ER stress shown above prompted us to investigate whether the proteasome might be involved in UPR signaling. Incubation of VSMC with the ER stressor Tn  is known to trigger UPR signaling, including KDEL-containing chaperones such as Grp78 and Grp94, as seen in Expression of Grp- or PDI-like chaperones represents a major branch of proadaptive signaling during the UPR The transcription factor XBP1 has an unusual mode of regulation in which its mRNA is spliced in the cytosolic face of the ER by the endonuclease action of IRE1α, with removal of an intronic sequence, generating the mRNA for the active spliced transcription factor, while the unspliced form acts as a dominant-negative UPR inhibitor To further investigate the consequences of proteasome inhibition in downstream signaling pathways involved with survival and stress, we assessed Akt/protein kinase B and p38 phosphorylation. VSMC incubated with MG132 (1 µM) for time periods varying from 2 - 18 h showed sustained increases in Akt phosphorylation . For p38We next investigated the importance of Akt in cell death induced by Tn, MG132 or their combination. For these studies MTT assay was performed in VSMC incubated with Tn (5 µg/mL) and/or MG132 (1 µM) for 16 h in the presence or absence of an Akt inhibitor (A6730 Sigma-Aldrich). Results showed that Akt inhibition increased cell death vs. control already at baseline conditions, but was particularly lethal after Tn, MG132, or their combination. Also, such increase in cell death was greater with the co-incubation of Tn and MG132 in comparison with Tn alone. This indicates that Akt significantly contributes to cell survival during either ER stress or proteasome inhibition in an additive way.Since ROS production and NADPH oxidase activity are integral components of the UPR, we investigated effects of proteasome inhibition on these variables. Experiments to assess ROS production were performed at 4-h incubation periods, in order to detect early changes that would be likely independent of secondary signaling events, as well as apoptosis itself NADPH oxidase activation in non-phagocytic cells is characterized by increases in mRNA or protein expression of relevant catalytic or regulatory subunits To investigate whether ROS played a direct effect on cell viability during proteasome inhibition in ER-stressed VSMC, we co-incubated VSMC with Peg-CAT plus Peg-SOD in the same conditions and then performed MTT assays . These eOur data showed that proteasome inhibition, per se at non-lethal levels, sensitizes VSMC to cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive and proapoptotic markers of classical UPR signaling, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition induced complete disruption of NADPH oxidase activity and regulation and of Nox4 mRNA triggered by ER stress. Although oxidative stress was increased, ROS scavenging with PEG−SOD + PEG−CAT had no net effect on VSMC death in our model. Despite the increase in ER stress-induced cell death, proteasome inhibition also promoted survival/stress signaling such as phosphorylation of p38MAPK and Akt, the latter shown to partially offset VSMC death. Together, these data indicate mechanisms of the stress response and redox signaling involved in VSMC cell death induced by proteasome inhibitors, which may be of relevance to understand the therapeutic potential of such compounds in vascular diseases.The mechanisms whereby proteasome inhibition sensitizes ER-stressed VSMC to apoptosis may be multiple, but a likely major factor was the suppression of homeostatic UPR signaling such as failure to upregulate classical chaperone markers including Grp78, Grp94, as well as PDI. Disruption of UPR signaling is known to sensitize cells to death in response to ER stressors The UPR-independent pathways that culminate in executioner caspase-3 activation and apoptosis in our VSMC were likely complex, as in most other studies dealing with proteasome inhibitor-mediated apoptosis. Evidence suggests that these triggering pathways are upstream of mitochondrial dysfunction and caspases, and seem to converge to persistent activation of proapoptotic bcl-2 family proteinsA frequent The induction of survival signals such as XBP1 and particularly Akt phosphorylation, concomitant to cell death sensitization by proteasome inhibition is in line with the complex cell stress response triggered by such compounds and significantly limited VSMC loss. The role of Akt in cell survival during ER stress has been described previously IPK, which may precipitate cell death by releasing PERK-dependent inhibition of protein translation The mechanisms whereby proteasome inhibition suppressed the UPR in our VSMC are unknown. In striking contrast with the other UPR markers assessed in our cells, as well as with findings reported for myeloma cells The potential mechanisms of Nox4 repression after proteasome inhibition can be multiple and our data may help understand the intriguing and so far poorly understood regulation of this NADPH oxidase isoform. One possible explanation for this effect is the known transcriptional role of the proteasome, which has been increasingly recognized In summary, our results indicate that proteasome inhibition impairs VSMC viability during ER stress, while largely suppressing proadaptive and proapoptotic UPR signals, although not XBP1 splicing. The mechanism of such death sensitization likely involves a complex cell stress response induced by proteasome inhibitors, as suggested by the concomitant induction of survival signals such as Akt phosphorylation. In addition, Nox4 as well as PDI induction by ER stress were importantly inhibited by proteasome inhibition. Overall, VSMC death sensitization due to proteasome inhibition did not seem directly dependent on ROS, despite increases in ROS production. ER stress plays a significant role in the pathogenesis of atherosclerotic vascular disease, as well as its complications and risk factors such as hyperlipidemia and insulin resistance Antibodies were obtained as follows: PDI and KDEL from Assay Designs/Stressgen ; CHOP/GADD153 from Thermo Scientific ; Akt, phospho-Akt, p38 and phospho-p38 from Cell Signaling Technology ; ubiquitin from Zymed/Invitrogen ; β-actin from Sigma-Aldrich . Secondary antibodies conjugated with horseradish peroxidase, 20S proteasome activity kit and protease inhibitors were from EMB Biosciences . Dihydroethidium (DHE) was from Invitrogen . All other reagents were from Sigma-Aldrich .Rabbit aortic smooth muscle cells (VSMC) were obtained from a previously established selection-immortalized line, as reported previously 4 cells) were seeded and cultured for 24 h in a 96-well plate, followed by further incubation for 24 h in presence or absence of Tn or/and proteasome inhibitor MG132 at the indicated concentrations, while during the last 4 h MTT -2,5-diphenyltetrazolium bromide; 600 µM) was added. VSMC were then washed with PBS (pH 7.4), followed by DMSO, which solubilizes formazan crystals. Absorbance was measured at 570 nm.VSMC (2×102), at 90% confluence, were incubated or not with Tn (5 µg/mL) or MG132 (1 µM) for 16 h. ROS generation was assessed through HPLC analysis of DHE oxidation products, as described in detail previously g for 10 min at 4°C). Supernatants were dried under vacuum  and pellets maintained at −20°C in the dark until analysis. Samples were then resuspended in 120 µl PBS/DTPA and injected (100 µl) into an HPLC system equipped with a C18 column, Photodyode Array Detector  and fluorescence detectors. This allowed simultaneous detection of DHE and its derived oxidation products 2-hydroxyethidium  and ethidium , using DHE as an internal control during organic extraction of each sample Cells grown in 6-well dishes (well area of 9.6 cmMembrane fraction NADPH oxidase assays were performed as described previously 20S proteasome activity (chymotrypsin-like) was measured with a Proteasome Assay Kit (EMD Biosciences) in VSMC homogenates stimulated or not with Angiotensin II , Tn (5 µg/mL), MG132 (1 µM) or Tn+MG132 combinations for 16 h. This assay monitors the release of free AMC from the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC. The rate of AMC release was measured through the increase in fluorescence over time . VSMC homogenates were obtained by cell lysis in RIPA buffer as described above.CATCATGGAAGGAAGGAGA; Nox4 – CCACAGACTTGGCTTTGGAT; PDI – CGGCCCAGGAACTTCTTAAAGCCG; p22phox – GTACTTCATGGCGTAGGTGCCGAAGTAC.Quantitative PCR was performed as described previously 2O2 5.4 mM, Tris 0.1 mM, pH 8.5). Densitometric analysis was performed with ImageQuant™ 2005 software .VSMC homogenates were obtained by cell lysis in RIPA buffer, , with protease inhibitors  and Triton-100 10%. After 20 min on ice, samples were centrifuged  and supernatants analyzed in SDS–PAGE as described 6 cells) grown in 10-mm dishes and exposed to experimental conditions were washed twice with cold PBS, scraped in 1 ml PBS-EDTA and transferred to 1.5 ml tubes. Cells were centrifuged at 2,500 rpm/5 min, pellets resuspended in 200 µl harvest buffer and incubated on ice for 5 min. Samples were centrifuged at 1000 rpm/10 min and nuclei pellet were washed twice with 500 µl buffer A  containing leupeptin (2 µg/ml), pepstatin (2 µg/ml), phenylmethylsulfonyl fluoride (44 µg/ml). Finally, pellets were resuspended in buffer C , pepstatin (2 µg/ml), PMSF (44 µg/ml). Samples were vigorously vortexed for 15 min at 4°C and after final centrifugation at 14000 rpm/10 min, supernatant containing 20 µg nuclear extracts was used for western blotting.Nuclear extracts used for the analysis of CHOP protein levels were obtained as reported 6 VSMC incubated with or without Tn (5 µg/mL) and/or MG132 (1 µM) by using the Illustra mini RNA isolation kit . RT-PCR for samples was performed with Platinum Taq Polymerase from Invitrogen . PCR for XBP1 was performed with 3 µg cDNA and the primer sequences were AGAGAAAACTCATGGCCTTGTCATTG and GAAGAGTCAGCGCCGTCAGAA. PCR products were separated on a 3% agarose gel, which yielded a 238 bp product for unspliced and a 212 bp fragment for spliced XBP1 mRNA. PCR for GAPDH was performed under the same conditions with primer sequences TCACCATCTTCCAGGAGCG and CACAATGCCGAAGTGGTCGT, except that PCR products were separated on conventional agarose gels.XBP1 mRNA splicing was detected according to published protocols Values are expressed as mean ± standard deviation. Statistical comparisons were performed through one-way analysis of variance (ANOVA), followed by Student–Newman Keuls test, at a 0.05 significant level .


Comparison of NIL (n = 8) and L (n = 8) myometrium revealed a 2-fold increase in h-CaD phosphorylation  during onset of labor in vivo, and was associated with significantly up-regulated ERK2 expression (P = 0.022), however no change in ERK2 phosphorylation was observed (P = 0.475). During in vitro studies (n = 5), transition from non-contracting tissue to tissue at peak contraction was associated with increased phosphorylation of both h-CaD and ERK 1/2. Furthermore, tissue preserved at relaxation phase exhibited diminished levels of h-CaD and ERK 1/2 phosphorylation compared to tissue preserved at peak contraction, thereby producing a phasic phosphorylation profile for h-CaD and ERK 1/2. h-CaD and ERK 1/2 are phosphorylated during myometrial contractions, however their phospho-regulation is dynamic, in that h-CaD and ERK 1/2 are phosphorylated and dephosphorylated in phase with contraction and relaxation respectively. Comparisons of NIL and L tissue are at risk of failing to detect these changes, as L samples are not necessarily preserved in the midst of an active contraction.Human myometrium develops phasic contractions during labor. Phosphorylation of caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) has been implicated in development of these contractions, however the phospho-regulation of these proteins is yet to be examined during periods of both contraction and relaxation. We hypothesized that protein phosphorylation events are implicated in the phasic nature of myometrial contractions, and aimed to examine h-CaD and ERK 1/2 phosphorylation in myometrium snap frozen at specific stages, including; (1) prior to onset of contractions, (2) at peak contraction and (3) during relaxation. We aimed to compare h-CaD and ERK 1/2 phosphorylation  Human myometrial contractions last approximately 60 seconds and are followed by extended periods of relaxation. Whilst protein phosphorylation cascades have been linked to myometrial contractility, little is known about the regulation of these phosphorylation events during the periods of relaxation.2+)-calmodulin 2+ sensitivity, suggesting that h-CaD is a potent inhibitor of Ca2+-induced smooth muscle contraction in vivo, and although the authors reported significantly increased h-CaD phosphorylation in term laboring myometrium, statistical significance was achieved relative to non-pregnant myometrium, with no significant difference evident between term non-laboring and term laboring human myometrium.Within human myometrium, evidence exists linking the phosphorylation of high molecular weight caldesmon (h-CaD) and extracellular signal-regulated kinase 1/2 (ERK 1/2) to the generation of contractions in vitro. Furthermore, we provide the first evidence that h-CaD phosphorylation is increased during the late gestation transition from term, not-in-labor (NIL) myometrium to term, laboring (L) myometrium, and that this transition is associated with increased ERK expression.Given that contraction associated proteins such as h-CaD are regulated by protein phosphorylation, we hypothesised that rapid protein phosphorylation events may be implicated in the phasic nature of spontaneous myometrial contractions. We aimed to test this hypothesis using the novel approach of snap freezing myometrial tissue at specific stages during the development of contractions, including prior to the onset of contractions, at the peak of contraction and during periods of relaxation between contractions. Here we report the first evidence of phasic phospho-regulation of h-CaD and ERK 1/2 in parallel to phasic contractions in human myometrium rd/4th degree tear (2 samples) or placental praevia (1 samples), whilst reasons for emergency cesarean section were non-reassuring cardiotocograph , undiagnosed breach (2 samples), failed forceps delivery (1 sample), poor progress (1 sample) or herpes simplex virus (1 sample). Following delivery of the placenta, all women were administered 5 units of syntocinon directly into an intravenous line as part of standard care for the prevention of post-partum hemorrhage. Myometrial biopsies were then excised 3–5 min after administration of the syntocinon. All samples were therefore exposed to oxytocin however exposure was brief (3–5 min). In addition, two samples from the L cohort also received 10 units of syntocinon during the induction of labor. After biopsy, myometrial samples were dissected from connective tissue and either frozen in liquid nitrogen or placed into ice-cold saline and stored at 4°C (maximum 16 h) for contractility studies.These studies were approved by the Hunter and New England Area Research Committee, adhering to guidelines of the University of Newcastle and John Hunter Hospital, Newcastle, Australia. All participants gave informed written consent. Human myometrial samples (5×5×10 mm) were obtained from the lower uterine segment of term singleton pregnancies undergoing cesarean section. Age of the participants averaged 31.75±2.06 years for NIL studies and 29±1.76 years for L studies . For NIL studies myometrial biopsies were collected from women ranging from 38–39 weeks gestation , whilst for L studies gestation ranged from 36–42 weeks . Subjects underwent elective cesarean section prior to the onset of labor (NIL cohort) or emergency cesarean section after the onset of labor (L cohort). Reasons for elective cesarean section were previous cesarean section (5 samples), 32. Strips were connected to a Grass FT03C force transducer  and 1 g passive tension applied. Buffer was replaced five times during the first hour, with strips re-tensioned to 1 g passive tension following each wash. Thereafter strips were maintained at 37°C (pH 7.4) and continuously bubbled with 95% O2/5% CO2 until spontaneous rhythmic contractions developed. Data were digitized using a Maclab8E data-acquisition system and contraction status visualised in real time using Chart software . Strips were closely monitored until reaching appropriate contraction phases and then snap frozen using liquid nitrogen (i) after 15 mins of washing but prior to the onset of any contractions, (ii) at maximum contraction once rhythmic contractions were established, and (iii) at maximum relaxation between rhythmic contractions. Snap freezing was performed by rapidly raising the tissue strips out of the organ bath and cutting the threads tethering the strips to the transducer. The strips were collected into 5 ml tubes that were immediately capped and plunged into liquid nitrogen. Total time for the snap freezing procedure was 5–7 seconds. The tissue was then stored at −80°C.Myometrial samples from non-laboring women were cut into strips (7×2×2 mm) and suspended in organ baths containing 30 ml Krebs-Henseleit buffer with 1.89 mM CaCl2 and 100 mg tissue homogenized in 1 ml lysis buffer  supplemented with PhosSTOP phosphatase inhibitor  and Complete Mini Protease Inhibitor (Roche). The protein extraction homogenate was incubated at 4°C for 30 mins on a rotary mixer before being centrifuged at 11,000× g, for 15 min at 4°C. Supernatant was collected and protein content determined by 2D Quant Kit . Reagents and equipment were obtained from Invitrogen , unless stated otherwise. Protein extracts (20 µg per lane) were loaded onto 4–12% NuPAGE gels and separated using a Novex Mini-Cell system at constant voltage . For ‘NIL versus L’ studies, myometrial biopsies were pulverized and extracted from eight non-laboring women and eight laboring women. Samples were run across two gels with 4 non-laboring samples, 4 laboring samples and a calibrator sample were included on each gel.Frozen tissue was pulverized under liquid NFollowing SDS-PAGE separation, proteins were labeled with Pro-Q® Diamond Phosphoprotein Gel Stain (Invitrogen) according to the manufacturer's standard instructions for staining minigels. Pro-Q® Diamond Staining was visualised using a FujiFilm LAS-3000 Luminescent Image Analyser. Following visualisation of Pro-Q® Diamond labeling, gels were immediately stained with SYPRO® Ruby Protein Gel Stain (Invitrogen) according to the manufacturers standard procedure for minigel staining. SYPRO® Ruby protein staining was imaged on a FujiFilm LAS-3000 Luminescent Image Analyser. To ensure specific phosphoprotein labeling was achieved, a Peppermint™ Stick molecular weight marker consisting of 2 phosphoproteins and 4 non-phosphorylated proteins was included on all gels.After 1D SDS-PAGE, proteins were transferred to Hybond-C nitrocellulose  using the XCell II Blot Module (Invitrogen). For anti-phosphotyrosine visualization, blots were first stained with SYPRO® Ruby Protein Blot stain (Invitrogen) according to the manufacturers instructions. During immunoblotting all incubations were performed on a rocking platform. Membranes were blocked in 5% skim milk in TBS/T  overnight at 4°C. Blocking solution was decanted and primary antibody applied at previously optimised dilutions in 10 mL 1% skim milk in TBS/T for 2 h at RT. Antibodies against phospho-h-CaD , total-h-CaD , phospho-ERK 1/2 , total-ERK 1/2  and phospho-MLC  were applied at 1∶2000 dilution, whilst antibodies against phospho-tyrosine  and α-smooth muscle actin (α-SMA)  were applied at 1∶2500 and 1∶10,000 respectively. Blots were subjected to 4×5 min washes with 100 mL of TBS/T. Washed blots were then incubated in horseradish peroxidase (HRP)-conjugated anti-rabbit IgG  or anti-mouse IgG  secondary antibody as appropriate. Secondary antibodies were applied at 1∶2500 dilution in 10 mL of 1% skim milk in TBS/T for 1 h at RT. Blots were washed for 3×5 min in 100 mL of TBS/T before immunoreactive products were detected with enhanced chemiluminescence  and visualized using the Fuji Intelligent Dark Box LAS-3000 Image Reader . To ensure internal controls, blots were first probed with the phospho-specific antibodies before being stripped and re-probed with antibodies against the total proteins. Membranes were stripped by 2×4 min incubations in 100 mL of 0.2 M NaOH. Stripped blots were then washed for 3×3 min in TBS/T before being re-probed according to the outlined regime. Phosphorylation of MLC was examined as a proof of concept.Densitometric analysis of immunoreactive protein bands was performed using Multigauge software (Fuji Photo Film) supplied with the LAS-3000. Where necessary, optical density  was normalized between blots using the incorporated calibrator sample on each blot (NIL versus L studies). OD was expressed as a percentage of the phosphorylated target proteins relative to the total protein density. Similarly, OD of total h-CaD and total ERK 1/2 was expressed as a percentage of α-SMA OD, which was used as a loading control given that α-SMA abundance in myometrium remains unchanged throughout pregnancy P≤0.05 was considered significant.The distribution of each outcome variable was assessed using a histogram and the skewness/kurtosis test for normality. The ratio of phospho-h-CaD to total h-CaD was skewed right, hence median and interquartile ranges have been reported. The ratio of total h-CaD/SMA, p-ERK 1/2/total ERK 1/2, and total ERK 1/2/SMA were normally distributed, hence mean and standard deviation are reported. Statistical comparisons were undertaken comparing the NIL (n = 8) and L (n = 8) group. Two-sample t-tests with equal variance were performed for the normally distributed variables and the non-parametric equivalent, Mann-Whitney U test, was performed for the ratio of phospho-h-CaD to total h-CaD. Statistical analyses were carried out using Intercooled Stata 11 . For all tests, P = 0.567; in vivo  prior to the onset of any contractions, (2) at peak contraction and (3) during relaxation between individual contractions  and a protein with Mr of ∼80×103 were also observed to undergo phospho regulation. A series of other phosphoprotein bands also exhibited increased phosphorylation although to varying lesser extents. The transition from peak contraction at point 2 to relaxation at point 3 was associated with reduced phosphorylation of multiple proteins. This was particularly evident for the two high molecular weight phosphoprotein bands labeled (i) and (ii), and is consistent with the phasic phosphorylation profiles observed for h-CaD and ERK 1/2. In contrast, other proteins were phosphorylated at peak contraction and remained phosphorylated despite the myometrium having transitioned into relaxation, such as phosphoprotein band (iii). Two proteins with Mr of 23×103 and 25×103 (indicated by white arrowheads) exhibited increased phosphorylation during the transition from peak contraction at point 2 to relaxation at point 3. The identities and characterisation of these protein bands is the subject of further investigation.We sought to visualise global changes in protein phosphorylation associated with the development of contractions rowheads . The majr of ∼115×103. As per the Pro-Q Diamond staining, we again observed that the transition from peak contraction at point 2 to relaxation at point 3 was associated with reduced tyrosine phosphorylation of specific proteins, such as band (iv) for example, whilst tyrosine phosphorylation of other proteins, such as band (v), remained high despite the transition into relaxation. These phospho-tyrosine protein bands are now being characterized.Our complementary analyses into the specific detection of tyrosine phosphorylation events yielded similar results . ImmunobAt labor the human uterus develops co-ordinated regular contractions that force the fetus through a softened cervix to achieve delivery. Previous studies of human myometrium have compared tissue obtained from women prior to the onset of labor with samples taken after the onset of labor; yet even during labor the uterus is relaxed for the majority of time. Here we have developed and demonstrated a method for studying the molecular events associated with contraction and relaxation in term myometrial tissue.in vivo . Unlike ordinary tension assays or ‘NIL versus L’ comparisons, we sub-divided our contractile samples into those preserved at peak contraction and those preserved at relaxation. This approach provides additional insight into changes that occur during the transition from peak contraction to intervals of relaxation that exist in between individual contractions. Our data on MLC provides ‘proof of concept’ for this approach, as MLC was phosphorylated in tissue preserved at peak contraction, yet minimally dephosphorylated in non-contractile tissue or tissue preserved during relaxation (Li et al. 2009 have previously reported that h-CaD expression is significantly up-regulated in human myometrium at ≥37 weeks gestation in vivo . Increaslaxation .in vitro, which is consistent with serine-789 being a known ERK phosphorylation site Our studies reveal that phosphorylation of h-CaD parallels the onset and subsidence of contractions. That is, h-CaD is phosphorylated at the peak of myometrial contraction, and de-phosphorylated during periods of relaxation between individual contractions. This pattern of phasic phosphorylation was also observed for ERK 1/2, and provides indirect evidence that ERK 1/2 regulates the phasic phosphorylation of h-CaD on serine-789 during myometrial contractions Results of our global phosphorylation analyses demonstrate that ERK 1/2 and h-CaD are just two members of a cohort of proteins that exhibit phasic phosphorylation during myometrial contractions. We visualised at least 14 phosphorylation events associated with the development of contractions. A sub-set of these proteins underwent dephosphorylation during relaxation, while others remained phosphorylated.in vitro evidence demonstrating that ERK 1/2 is phosphorylated at peak contraction (in vivo, and if so how could it be achieved?Our evidence of phasic phosphorylation in parallel to contractions is of key significance as it demonstrates that phasic phosphorylation events may be missed by traditional experimental approaches. For instance, during our ‘NIL versus L’ analyses we observed increased h-CaD phosphorylation during the onset of labor, as well as significantly up-regulated expression of ERK2 . Examinatraction . The inain vivo. Venturing beyond this, we further demonstrate that although h-CaD is phosphorylated during the development of contractions, the regulation of serine-789 phosphorylation is actually phasic, in that h-CaD is once again de-phosphorylated during periods of relaxation. We have shown that in humans the transition from term, NIL myometrium to term L myometrium is associated with up-regulated ERK2 expression, and that during the development of spontaneous contractions in vitro ERK 1/2 demonstrates the same phasic phosphorylation exhibited by h-CaD. We demonstrate that h-CaD and ERK 1/2 are just two members of a cohort of proteins subjected to this type of phasic regulation, and we reveal that the transient nature of these phosphorylation events impedes our ability to capture evidence of their existence using traditional experimental approaches. Finally, where protein phosphorylation status is of concern, we stress the importance of considering the marked changes that exists between contraction and relaxation phases of human myometrium during experimental design and interpretation of results.This investigation provides a unique insight into phasic phosphorylation events that occur during myometrial contractions in humans. We have demonstrated that h-CaD phosphorylation increases late in gestation during the onset of labor


In the vertebrate body the periodontal ligament is the tissue with the highest turnover of collagen. Three major groups of integrins are of particular interest in the periodontal ligament: (i) I-domain-containing collagen-binding integrins; (ii) non-I-domain integrins linked via ‘bridging’ molecules to collagen  and vascularized soft tissues  . The extThe periodontal ligament is the only ligament in the body that connects two distinct hard tissues. It is a fibrous, complex, soft connective tissue that attaches the tooth root to the inner wall of the alveolar bone. The width of the periodontal ligament in human molars ranges from 0.15–0.38 mm, with the thinnest part around the middle third of the root. The corresponding values for mouse molar periodontal ligament are 0.1–0.13 mm . The perAnother important function of the periodontal ligament is to regulate alveolar bone volume and to serve as a cell reservoir for tissue homeostasis and regeneration . The perin vivo possess alkaline phosphatase activity  synthesis/assembly. However, in the uncharacterized form of Ehlers–Danlos syndrome type VIII, periodontitis is a typical feature . It willIn recent years the role of proteoglycans in tissue-specific collagen reorganization has attracted increasing interest. Data from decorin-deficient animals have convincingly demonstrated an important role for decorin in collagen organization of the periodontal ligament . SeparatFibrillin-1 is part of the oxytalan fibers in the periodontal ligament. Elastic oxytalan fibers identified within the periodontal ligament are described as a three-dimensional meshwork surrounding the root and terminating in the apical complex of arteries, veins and lymphatic vessels. It is believed that oxytalan fibers regulate vascular flow to the tooth. These fibers contain fibrillin-1 and elastin, but in the human periodontal ligament no elastin is present in these fibers , 130. CoThe protein, periostin, is highly expressed in molar and incisor periodontal ligament  and can in vitro  is related to periostin and was, in one study, reported to inhibit the mineralization of periodontal ligament cells in vitro , but latin vitro  and in tin vitro , 167. Rein vitro . Given tTenascin-C has a localized distribution toward the attachment zone to bone in the periodontium and appears not to be expressed throughout periodontal ligament . In a sy−/− mice, which was suggested to reflect a secreted-protein-acidic-and-rich-in-cysteine-dependent regulation of collagen turnover by periodontal ligament fibroblasts Fig. 2).The α-subunit is composed of a seven-bladed β-propeller that forms a head domain which is connected to a thigh domain, a calf-1 domain and a calf-2 domain, together forming the leg structure that supports the integrin , 106. Th2+ ion in the so-called metal-ion-dependent adhesion site , which is composed of approximately 200 amino acids and inserted between blades 2 and 3 in the β-propeller . Integriion site . The αI ion site , which iion site , 80 reguion site .2+ coordinating metal-ion-dependent adhesion site and a site adjacent to the metal-ion-dependent adhesion site that binds an inhibitory Ca2+ ion. It is this site adjacent to the metal-ion-dependent adhesion site that binds an Mn2+ ion, leading to a conformation change resulting in an active form of the integrin  domain, a hybrid domain, a βI domain , four cyintegrin . The β-iintegrin .Some key proteins seem to be essential for inside-out integrin activation. Detailed studies of the interacting regions in the αΙΙb- and β3-subunit transmembrane domains suggest a model for conformation-mediated changes over the membrane . Talins in vivo, either by allosteric changes in the transforming growth factor-beta/latency-associated peptide (αvβ6) complex or by inducing matrix metalloproteinase-14 and causing proteolytic release of transforming growth factor-beta (αvβ8)  , 97, 98.a (αvβ8) . In addia (αvβ8) , αvβ5 (4a (αvβ8)  can all RGD sequences present in triple helical fibrillar collagen sequences are normally not available for fibronectin receptors in the native fibrillar collagen , 46, onlCertain variants of the GFOGER sequence might show specificity for the different collagen-binding integrins, as indicated by a recent finding showing that the bacterial collagen-like peptide, sscl, with the active binding-site GLPGER, is preferred by α11β1-expressing cells over α2β1-expressing cells . More reAlthough the list of integrin ligands is long and we are a decade into the postgenomic era, new integrin ligands are still being characterized. Recently, the widely expressed extracellular matrix protein, polydom, was identified as a high-affinity integrin α9β1 ligand .The first function established for integrins was their function as links between the extracellular matrix and the cytoskeleton. For a majority of integrins, the linkage is to the actin cytoskeleton . Some ofIntegrins are bidirectional signaling receptors involved in outside-in and inside-out signaling. The inside-out signaling mainly acts to bring the integrin into the active conformation. Talin, kindlins, filamins, migfilin, focal adhesion kinase and integrin-linked kinase  can all −/− mice die before reaching adulthood, the role of matrix metalloproteinase-14 in continuous incisor eruption cannot be evaluated in this model. Matrix metalloproteinase-8 and matrix metalloproteinase-13 have also been studied in the context of tooth eruption and tooth movement  western blotting, to confirm the presence of protein; and (iii) immunostaining, to localize specific integrin subunits within the periodontium. For isolated periodontal ligament cells a limited number of studies have performed functional studies using integrin-blocking antibodies. To our knowledge the α11−/− mouse is the only integrin-deficient mouse model described to have a periodontal ligament phenotype  noncollagen binding-integrins linked to collagen via ‘bridging’ molecules, (ii) transforming growth factor-beta activating integrins and (iii) collagen-binding integrins; we will also discuss, in more detail, the α11β1 integrin.It is important to remember that for periodontal ligament fibroblasts, as with all fibroblasts, the integrin repertoire changes upon cell culture. Furthermore, because periodontal ligament fibroblasts have the capacity to differentiate into other cell types, they display more plasticity compared with normal adult tissue fibroblasts. Primary periodontal ligament fibroblasts, with this plasticity feature, might be more prone to undergo changes in gene expression with time in culture, including integrin expression. Factors to consider as modulators of integrin expression include: stiffness of the culture surface; two-dimensional versus three-dimensional culture; serum content of media; and epigenetic changes. Function-blocking antibodies, as well as antibodies suitable for immunostaining, are available, and it should be relatively easy to sort out more systematically the relevant integrins on periodontal ligament cells in vivo. With accumulating data on the complexity of the interactions in the extracellular matrix demonstrating an increasing diversity of extracellular matrix interactions, not only between extracellular matrix components, but also with growth factors/cytokines and matrix metalloproteinases, it is time to revisit the ‘nectin’ concept. COLINBRI molecules comprise a class of molecules with the potential to bridge the binding of non-I domain integrins, such as fibronectin receptors and vitronectin receptors, to the collagen matrix.Before the isolation of collagen receptors, a widely held belief in the cell-adhesion field claimed that cells did not interact directly with collagen, but via ‘nectins’ , 144. WeFibronectin is a classical COLINBRI molecule, and fibronectin binds both native and denatured collagens, although the binding to denatured collagen is of higher affinity , 123.α5β1 integrin is the classical fibronectin-binding integrin, is present on cultured periodontal ligament fibroblasts , 154 andin vivo  is(are) induced and how these integrins compare with regard to specialized functions normally carried out by αvβ3.The Transforming growth factor-beta1 is present in the murine periodontal ligament , but theIn the tooth context it has been reported that αvβ6 integrin-mediated transforming growth factor-beta1 activation in the junctional epithelium plays a protective role in inflammatory periodontal disease . β6-defiHowever, little is known about transforming growth factor-beta-activating integrins in the periodontal ligament tissue itself. As mentioned above, the current paradigm is that only RGD-binding integrins are transforming growth factor-beta activating, either by binding latency-associated peptide and unfolding the transforming growth factor-beta/latency-associated peptide complex or by inducing matrix metalloproteinase-14 synthesis, which cleaves and activates the complex . RGD-binFibrillin-1 exists in fibrils independent from the collagen network. Data suggest that assembly of fibrillin-1-containing oxytalan fibers may be controlled by αvβ3 on periodontal ligament fibroblasts . αvβ3, bin vivo. Neither α1β1 nor α2β1 are restricted to fibroblasts but are, in other tissues, expressed in epithelial cells, capillaries and osteoblasts  periodontal ligament tissue. From a therapeutic point of view, it would also be important to characterize the dynamic nature of the changing integrin repertoire of these cells during the clinical procedure. One can envision that cell culture of stem cells changes the integrin repertoire. This could be taken advantage of so that the scaffold used has a ligand composition matching the integrin repertoire of the cells to be grafted. It is also possible that culture of cells in a three-dimensional matrix might ‘normalize’ the integrin repertoire to a more in vitro, but in the periodontal ligament a highly organized pattern of collagen organization exists and we predict that periodontal ligament fibroblast integrins assist in this assembly . The organizing role is important in order to maintain the homeostasis of the periodontal ligament tissue with a high turnover rate of collagen. To maintain/restore this organized structure during injury and in pathological situations we believe that it is essential to reorganize the newly synthesized periodontal ligament tissue and, in this respect, facilitate the return to normal tissue architecture. Also, integrins might play important roles in these events by activating transforming growth factor-beta, regulating matrix metalloproteinases and orchestrating the physical reorganization of the matrix.Collagen I can self-assemble


Bisphosphonates interfere with the mevalonate pathway and inhibit the prenylation of small GTP-binding proteins such as ras and rap. We hypothesized that zoledronic acid would synergistically inhibit T24 bladder cancer cell growth in combination with fluvastatin and paclitaxel.Increasing doses of fluvastatin, zoledronic acid, and paclitaxel were investigated as single agents and in combination, and synergistic interactions were evaluated by the Chou-Talalay method. Western blots were used to assess effects on signal transduction pathways.50 values of 2.67 ± 0.61 μM for fluvastatin and 5.35 ± 1.35 μM for zoledronic acid after 72 hours treatment. Geranylgeranyl pyrophosphate and farnesyl pyrophosphate was able to block, in part, this inhibitory activity. The combinations of zoledronic acid and paclitaxel, zoledronic acid and fluvastatin, and fluvastatin and paclitaxel were all synergistic. Both fluvastatin and zoledronic acid inhibited Ras and Rap prenylation, and the phosphorylation of ERK1/2 and AKT. The degree of inhibition of phosphorylation of these key signaling transduction pathways appears to closely correlate with their synergistic interactions.Growth of T24 was significantly inhibited with ICZoledronic acid enhances fluvastatin and paclitaxel activity against T24 in a synergistic manner and this is mediated largely by inhibition of both the Ras/Raf/MEK/ERK and PI3K/AKT signaling pathways via isoprenylation inhibition. Recently, it has been shown that nitrogen-containing bisphosphonates such as zoledronic acid inhibit farnesyldiphosphate synthase . We hypo2.2.1.50 2.67 ± 0.61 μM at 72 h) than to zoledronic acid (IC50 5.35 ± 1.35 μM at 72 h) (Treatment of T24 cells with increasing concentrations of fluvastatin (0.1 to 100 μM) or zoledronic acid (0.1 to 100 μM) for 24–72 hours caused dose and time-dependent cell growth inhibition. T24 cells were slightly more sensitive to fluvastatin (ICat 72 h) .2.2.In these experiments, fluvastatin (3μM) and zoledronic acid (10μM) inhibited cell proliferation by 44 ± 6.2%, and 71 ± 2%, respectively . Geranyl2.3.At lower concentrations of fluvastatin (0.5–1.5 μM), zoledronic acid (0.5–3 μM), and paclitaxel (below 2 nM) additive growth inhibition was achieved (CI = 0.9–1.1), whereas 5–10 μM of zoledronic acid and 1.5–3 μM fluvastatin combined with 4–8 nM of paclitaxel produced greater synergistic effects (combination indexes ranging from 0.3 to 0.6)  was dissolved in ddH2O and fluvastatin was dissolved in DMSO; 100 mM of stock solutions were stored in −20 °C, and were diluted in growth medium before use. Both zoledronic acid and fluvastatin were kindly provided by Novartis Pharmaceuticals Australia. Paclitaxel (Sigma) was dissolved in DMSO and stored in 4 °C as a stock solution. Geranylgeranyl pyrophosphate and Farnesyl pyrophosphate were both purchased from Sigma and stored in −20 °C; Anti-RAP1A antibody, anti-® actin and horseradish peroxidase (HRP) -linked anti-goat antibody were purchased from Santa Cruz Biotechnology (U.S.). Anti-RAP1A antibody can bind to the nongeranylgeranylated form RAP1 [Human bladder cancer cell line T24 cells were grown in RPMI medium (Sigma), supplemented with 10% of fetal calf serum (GIBCOBRL), 100 U/mL of penicillin and 100 μg/mL of streptomycin (GIBCOBRL), at a constant temperature of 37 °C in a humidified atmosphere with 5% COorm RAP1 . Anti-ph3.2.Cell growth inhibition was determined by the sulforhodamine B colorimetric assay (SRB) . Briefly3.3.T24 cells were seeded in 96-well plates at 1000/well for 24 hours, then treated with the drugs (3 μM fluvastatin or 10 μM zoledronic acid) alone. Farneysl pyrophosphate and geranylgeranyl pyrophosphate (10 μM each) were then added within 60 minutes, and growth inhibition was assessed by SRB assay as above.3.4.Drug combination effects were analyzed using “CalcuSyn Windows Software for Dose Effect Analysis” developed by Chou and Talalay . BrieflyX)1 is the dose of drug-1 required to produce x percent effect alone and (D)1 is the dose of drug-1 required to produce the same x percent effect in combination with drug-2; similarly, (DX)2 is the dose of drug-2 required to produce x percent effect alone and (D)2 is the dose of drug-2 required to produce the same x percent effect in combination with drug-1. CI < 1, =1, and >1 indicates synergism, additive effect, and antagonism, respectively.Where (D3.5.3 (2 mM) and PMSF (1 mM). Lysates were sonicated in an ice water bath for 20 seconds; the supernatant containing protein was separated from the insoluble material by centrifugation at 10,000rpm for 10 minutes at 4 °C. The total protein in each sample was then quantified using Bicinchoninic Acid Kit (Sigma) by following the protocol described. Samples containing equal amounts of protein were mixed with loading buffer and boiled for 5 minutes. Proteins were then separated by SDS-PAGE, and the proteins on the gels were transferred to PVDF-Membrane (Amersham Biosciences) and incubated with different antibodies. After incubation with HRP-conjugated secondary antibodies, the primary antibody binding was visualized with Western Lightning™ Chemiluminescence . Density of the bands were quantified by scanning with Gel Doc 200 (Bio-Rad) and normalized by β-actin as loading control (density of the sample /density of β-actin); P-AKT and P-ERK expression levels were further normalized by density of the band of the non-drug treated control. All Western blots were repeated at least once, and similar results were confirmed.Cells were allowed to reach 70% confluence in RPMI containing 10% of FCS for 24 hours, before drugs were added in serum-free medium for another 24 hours. Cells were then washed with cold PBS buffer and lysed in buffer containing 50 mM Tris-HCI (pH7.5), 150 mM NaCI (150 mM), EGTA (1 mM), EDTA (2 mM), 2.5%(V/V) glycerol, NaF (10 mM), 0.2%(V/V) Triton X-100, 0.3% (V/V) NP40, aprotinin (5 μg/mL), Leupeptin (10 μg/mL), pepstatin (10 μg/mL), activated NaVO3.6.Each experiment was performed at least three times, and all values are represented as the means ± SD of triplicates. Student's t-test was used to determine the statistical significance of the results. Values of p < 0.05 were considered statistically significant.4.In conclusion, our paper demonstrates a number of novel observations of the mechanism of the antitumor activity of zoledronic acid on T24 cells. We show that zoledronic acid inhibits isoprenylation, and that this can be rescued by GGPP, but not by FPP. Further, isoprenylation inhibition inhibits the function of Ras, and this results in reduced phosphorylation of downstream targets such as ERK and AKT. The antiproliferative and anti-Ras function of zoledronic acid can be improved by the addition of fluvastatin and paclitaxel in a synergistic manner. The mechanism of this synergy appears to be largely due to drug effects on two major signaling transduction pathways, Ras, and AKT.


N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and  The biosynthesis and the mode of action of McC have already been reviewed comprehensively elsewhere -sulfamoyl]adenosine  is not a substrate for either MccF or MccE -sulfamoyl]adenosine (2) and sarcosyl-sulfamoyladenosine (3) (i.e. 5’-O-[N-[N-methyl-glycyl]-sulfamoyl]adenosine)   were syn schemes  and S4).4-8 were created to investigate the ability i) of cellular peptidases to process peptide bonds containing (D)-amino acids and ii) of the YejABEF transporter to recognize and facilitate entry of McC-like compounds with (D)-amino acids. In addition, we wanted to investigate whether compounds 8 and 9 ((D)-D-SA) would still inhibit the aminoacylation reaction and would have the potential to escape hydrolysis/acetylation by MccF and MccE respectively. McC analogues comprising (D)-amino acids  were likE. coli in wells of microtiter plates in the presence of various concentrations of the respective inhibitors. As shown earlier, the intracellular target of McC analogs is determined by the C-terminal amino acid, which remains attached onto the sulfamoyladenosine following intracellular metabolism -sulfamoyl]adenosines: Pro-SA, Gly-SA, Ala-SA and Leu-SA [The growth inhibitory properties for all new compounds were determined by measuring the optical density reached by cultures of McC-sensitive tabolism . To faciompounds . In addied aaSAs  and 3 prd Leu-SA . These c8 showed significant activity in whole-cell activity screenings against Ara-Yej strain .Of the newly synthesized McC analogues (4-8), only compound j strain . The effin vitro studies were performed to test the ability of all compounds to inhibit tRNA aminoacylation in cell extracts prepared either from McC hypersensitive E. coli ΔrimL cells or from McC-resistant ΔpepABN cells. From 8 behaved as tRNA aminoacylation inhibitor in extracts lacking Rim proteins, but had no effect in extracts lacking N-peptidases, confirming the requirement for at least one of these peptidases to release the active moiety. Since none of the McC analogues containing (D)-amino acids at positions two to six showed clear inhibition of AspRS or IleRS in extracts lacking RimL, relative to compounds 9 and 10 which do not require metabolism, we conclude that the presence of a (D)-amino acid within the McC peptide interferes with processing.To further corroborate these results and to explain the absence of biological activity, 8 would be more resistant than compound 1f to MccF-catalyzed hydrolysis and/or MccE/RimL-catalyzed acetylation, and to investigate a possible role of DTD, a time-course of tRNAAsp aminoacylation inhibition was followed in two different cell extracts, one prepared from cells lacking Rim proteins, and another from cells lacking DTD. From 8 this effect was more pronounced than for compound 1f, especially in cell extracts prepared from cells lacking DTD proteins. Using the latter cell extracts, after 60 min, esterification of Asp-C14 to tRNAAsp was reduced only 44% (compared to 89% inhibition or only 11% incorporation at 15 min). In case RimL was absent, both compounds largely remained inhibitory.To investigate whether compound 8 could still reach full activity, comparable to that of the (L)-D-SA analogue, in extracts prepared from cells lacking DTD, suggesting that both (D)-D-SA and (L)-D-SA are equally inhibitory to AspRS and therefore (D)-D-SA does not need to be isomerised by DTD to inhibit AspRS. This is in agreement with the observation of Soutourina et al. [8 suffers more from inactivation than compound 1f in RimL deficient extracts. Compound a et al. , that de8 also showed whole cell activity against (L)-arabinose induced Ara-Yej cells, albeit to a lower extent than its (L)-analogue (8 or 1f showed activity against Δyej or ΔpepABN cells (data not shown), confirming their Trojan horse mode of action.Compound analogue . None ofin vitro aminoacylation experiments with compounds 2 and 3 it can be concluded that these compounds lack inhibitory activity against their hypothesized targets  was found to be inactive against LeuRS. Again, it must be concluded that the Nα-methyl group in this compound also prevents inhibitory activity. This then suggests that the Nα-amine is an important recognition point inside the active site of the enzyme which cannot be modified. The recent results of Cusack et al. [E. coli LeuRS in complex with the leucyl-adenylate analogue Leu-SA, the α-amine of the latter is hydrogen bonded to both the CO main chain of Leu41 and provides an ionic bond to the Asp80 side chain. N-methylation therefore not necessarily will prevent one of these H-bonds as the amine still can be protonated, but methylation is prohibited as it most probably would introduce unavoidable clashes -methionine, whereas amino acids at positions two to six are (D)-amino acids, it must be concluded that the peptidases are not capable of hydrolyzing (D)-amino acids and can therefore not liberate the active moiety. This is a somewhat expected result as it has frequently been shown that the introduction of (D)-amino acids can prevent the hydrolysis by peptidases [Compound ptidases .5-7 were especially designed to circumvent this problem; all three compounds contain L-amino acids at the sixth and seventh position, allowing endopeptidases to cleave between these two L-amino acids, resulting in release of the active moiety. Despite this modification, no activity could be observed. Also the modification in compound 6, inspired by albomycin whereby pepN hydrolyzes the peptide bond between a serine and a modified amino acid carrying an acidic side chain [in vitro aminoacylation experiments it can also be concluded that lack of whole cell activity is due to inability of the peptidases to metabolize peptides containing (D)-amino acids. Hence, this shows that the peptidases PepA, PepB and PepN, commonly known to be responsible for processing of McC and its analogues [via endopeptidic cleavage (i.e. convergently). Whether or not incorporation of (D)-amino acids in the transport peptide part interferes with uptake of the McC derivatives by the YejABEF transporter is less relevant here, as in vitro tests already show lack of activity. Additional reduced uptake of the analogues 4-7 however cannot be excluded.Compounds de chain , could nnalogues , only ca8 can inhibit AspRS, shows that the peptidases can metabolize peptide bonds between two amino acids, whereby the C-terminal amino acid has the (D)-configuration (as in this case a (D)-Asp). This suggests that the peptidases involved in this reaction are only stereoselective for the N-terminal amino acid. The observation that (D)-D-SA (9) and its McC derivative Asp with a rate of 1:4000 for (D)-Asp vs (L)-Asp.Since it was already frequently observed that (D)-Asp can be esterified to tRNA, the finding that (D)-D-SA (9) can also inhibit AspRS can be considered being an expected result. This also shows that the absolute configuration of the amino acid is not required for recognition inside the active site of AspRS, and most probably the same holds for other aaRSs. This finding matches with the results of Thompson et al.  who concvia semi-empirical calculation methods. From their [in vitro inhibitory effects and hence recognition of our (D)-amino acid containing aminoacyl adenylate analogues.These results are conflicting however with the views of Banik and Nandi -27 who som their theoretic1f, compound 8, was more sensitive to inactivation by a mechanism that was not solely related to RimL. This can probably be ascribed to the intrinsic capacity of AspRS, favouring (L)-Asp over (D)-Asp. It is however unlikely that the editing site of AspRS is involved in this mechanism as it has often been shown to hydrolyze only non-cognate amino acids from tRNA. The inactivation of (D)-D-SA is also reflected in a lower whole cell activity for this compound, compared to its (L)-analogue (1f) (Compared to compound gue (1f) . Nα-methylation of Gly-SA and Leu-SA does not result in inhibition of the respective aaRSs and is therefore most probably not well tolerated in other aaRSs as well. N-methylation can therefore also not be considered as a preventive step against acetylases inactivating most aaSAs. Secondly, we showed that when one or more of the amino acids at positions two to six have a (D)-configuration, metabolism by the peptidases is abolished, thus preventing release of the active moiety and inhibition of the respective aaRS. In addition, it was shown that the peptidases primarily function as exopeptidases, as we did not observe significant inhibition of AspRS or IleRS with McC analogues containing an (L)-amino acid at the pre-C-terminal (or sixth) position. Third, if the C-terminal aspartic acid has a (D)-configuration and amino acids at positions one to six are in (L)-configuration, the active moiety was released, resulting in inhibition of AspRS. This shows that metabolism is independent of the C-terminal amino acid and only depends on the configuration of the N-terminal amino acid. Fourth, we have shown that (D)-D-SA is perfectly capable of inhibiting AspRS. However, this proved a relatively short lasting inhibition as over time the compound is inactivated by (predominantly) RimL. A less surprising finding, and therefore of lesser importance, is that DTD is not involved in the inhibitory activity of (D)-D-SA. Four important observations were made in this study. First, we showed that All synthetic procedures, materials used and analysis of the synthesized compounds can be found in the supplementary pages.The respective bacteria were grown overnight in Luria Broth (LB) medium and cultured again the following day in fresh LB medium or LB-medium containing 5 mM (L)-arabinose. Compounds were titrated in a 96-well plate using either LB-medium +/- 5 mM (L)-arabinose to dilute the compounds. To each well, 85 µL LB-medium +/- 5 mM (L)-arabinose was added to a total volume of 90 µL. Next, 10 µL of bacterial cell culture grown to an OD600 of 0.1 was added. The cultures were next placed into a Tecan Infinite M200® incubator and shaken at 37 °C, subsequently the OD600 was determined after 8 h. All experiments have been performed in triplicate.E. coli Ara-Yej (BW39758), expressing the yejABEF transporter upon (L)-arabinose induction; E. coli K-12 (BW28357), used as the wild type control; E. coli ΔyejA, lacking subunit A of the YejABEF transporter; and E.coli ΔABN, lacking all three peptidases pepA, pepB and pepN. ; E. coli Δdtd, lacking (D)-aminoacyl-tRNA deacylaseBacterial strains used for the evaluations: in vitro tests were performed using the relevant S30 cell extracts.To assess the degree of inhibition of the aminoacylation reaction, 2 (10 mM), KCl (100 mM). The cell-suspension was centrifuged again at 4800 rpm. This procedure was repeated 2 times. The pellet was resuspended in 1 mL of the following buffer Tris.HCl or Hepes.KOH (pH = 8.0) (20 mM), MgCl2 (10 mM), KCl (100 mM), DTT (1 mM) and kept at 0 °C. Subsequently, the cells were sonicated for 10 sec. and left at 0 °C for 10 min. This procedure was repeated for 5-8 times. The lysate was centrifuged at 15000 g for 30 min at +4 °C. Cells were grown in 50 mL LB-medium. After centrifuging at 3000 × g for 10 min. the supernatant was discarded and the pellet was resuspended in 40 mL buffer containing: Tris.HCl or Hepes.KOH (pH = 8.0) (20 mM), MgClE. coli S30 extracts was added. Next, 16 μL of the following aminoacylation mixture was added: Tris.HCl , DTT (1 mM), bulk of E. coli tRNA (5 g/l), ATP (3 mM), KCl (30 mM), MgCl2 (8 mM), and the specified, radio labeled amino acid (40 μM). The reaction products were precipitated in cold 10% TCA on Whatman 3MM papers, 5 min. after the aminoacylation mixture was added. The aminoacylation reaction was carried out at room temperature. Depending on whether or not processing was needed, variable time intervals were included between the addition of the cell-extract and the addition of the aminoacylation mixture. After thorough washing with cold 10% TCA, the papers were washed twice with acetone and dried on a heating plate. Following the addition of scintillation liquid, the amount of radioactivity was determined in a scintillation counter.1 μL of solution containing inhibitor, 3 μL of Figure S1AlaRS starting structure from 3hxu.pdb with sarcosine substituting for alanine in the acive site.(TIFF)Click here for additional data file.Figure S2N-methylleucine derivative in the acive site.LeuRS starting structure from 4aq71.pdb with a modelled  Top: introducing the additional methyl moiety on the inhibitor (magenta) gives clashes with the nucleotide 76b (shown in green). ionic/H-bond distances are indicated to asp80, to leu41 and to nucleotide 76b.O2'. Bottom: the tRNA is removed from the model and CA-CB torsion angle of asp80 is adjusted slightly to accommodate the methyl moiety without clashes. (TIFF)Click here for additional data file.Figure S3N-methylated buiding blocks.Synthetic scheme for synthesis of the protected  Upper part: Synthesis of N-methylated and succinimide activated Boc-Leu-OH. i) NaH, MeI in THF, 0 °C, 30 min. ii) EDCI.HCl, HOSu, DIPEA in DMF, rt, 16 h. Lower part: Synthesis of Cbz-protected and succinimide activated sarcosine. i) benzyloxycarbonyloxyl succinimide, NaHCO3 in H2O/dioxane, 0°C to rt, 7 h. ii) HOSu and EDCI.HCl in DMF, rt, 16 h. (TIFF)Click here for additional data file.Figure S4General scheme affording the various McC analogues.i) N-α-CBZ-(L)-aminoacyl-(tBu or Boc)-succinimide, DBU in DMF, 6h, rt. ii) for R2 = Z-group, H2, Pd/C in MeOH, 3h, rt. iii) for R2 = Boc-group, TFA/H2O (5:2), 4h, 0 °C to rt. iv) Et3N.3HF in THF, 16h, rt. v) the respective protected peptide (1eq.), HOBt (4 eq.), DIC (4eq.) and DIEA (2 eq.) in DMF, 16h, rt. vi) TFA/thioanisole/H2O (90/2.5/7.5), 2h, rt. (TIFF)Click here for additional data file.


Neither Diprotin A administered in vivo at the time of BMT and/or used for in vitro pretreatment of BM nor sitagliptin administered in vivo had a detectable effect on the level of multilineage chimerism (follow-up >20 weeks). Similarly, sitagliptin did not enhance chimerism after allogeneic BMT, even though DPPIV enzymatic activity measured in serum was profoundly inhibited (>98% inhibition at peak exposure). Our results provide evidence that DPPIV inhibition via Diprotin A or sitagliptin does not improve engraftment of unseparated BM in a nonmyeloablative BMT setting.In order to develop minimally toxic bone marrow transplantation (BMT) protocols suitable for use in a wider range of indications, it is important to identify ways to enhance BM engraftment at a given level of recipient conditioning. CXCL12/stromal cell-derived factor-1α plays a crucial physiological role in homing of hematopoietic stem cells to BM. It is regulated by the ectopeptidase dipeptidyl peptidase IV  known as CD26, which cleaves dipeptides from the N-terminus of polypeptide chains. Blocking DPPIV enzymatic activity had a beneficial effect on hematopoietic stem cell engraftment in various but very specific experimental settings. Here we investigated whether inhibition of DPPIV enzymatic activity through Diprotin A or sitagliptin (Januvia) improves BM engraftment in nonmyeloablative murine models of syngeneic  and allogeneic  BMT (1 Gy total body irradiation, 10–15 × 10 Allogeneic bone marrow transplantation (BMT) has therapeutic potential for a wide range of indications. Its clinical application remains limited mainly to the treatment of life-threatening diseases because of substantial toxicities associated with currently available BMT regimens.Transplantation of donor BM to induce mixed hematopoietic chimerism is an attractive experimental approach to induce robust and lasting donor-specific tolerance in organ transplantation In the experimental setting, less toxic mixed chimerism protocols have been generated gradually during the last several decades. The use of costimulatory blockers—some of which are already under clinical development as immunosuppressive drugs The chemoattractant stromal cell–derived factor-1 (CXCL12) binding to CXCR4 on hematopoietic stem cells (HSCs) plays an important role in regulating trafficking of HSCs to BM An immunosuppressive role of DPPIV inhibition has also been suggested in organ transplantation models (not involving BMT), as an irreversible inhibitor of DPPIV abrogated acute rejection in rat lung and heart transplantation models We report that neither in vitro DPPIV/CD26 enzymatic inhibition of donor BM using Diprotin A nor additional systemic inhibition led to enhanced BM engraftment in a congenic murine model using 1 Gy total body irradiation (TBI) and conventional doses of BM. Moreover, we provide evidence that the clinically approved DPPIV inhibitor sitagliptin b, CD45.2, denoted B6 herein), Balb/c (H-2d), and C3H/N (H-2k) were purchased from Charles River Laboratories , female B6.SJL-Ptprca Pep3b/BoyJ mice  were purchased from the Jackson Laboratory . All mice were housed under specific pathogen-free conditions and were used between 6 and 12 weeks of age. All experiments were approved by the local review board of the Medical University of Vienna, and were performed in accordance with national and international guidelines of laboratory animal care.Female C57BL/6NCrl , as indicated. Allogeneic recipients received costimulation blockade consisting of anti-CD154 monoclonal antibody , and hCTLA4Ig  Treatment protocols per group are listed in Donor BM was harvested, washed, and resuspended in BM medium containing M199 (Sigma M 4530), DNAse (Sigma D-4527), gentamycin, and HEPES (1M) as described ex = 340 nm, λem = 430 nm) from the substrate using an Infinite 200  . Fluorescence intensity was related to a 4-Me-β-NA standard curve. The reversibility of the inhibitors in the serum samples and the dilution of these samples in the assay make it necessary to create a calibration curve with known concentrations of the inhibitors in murine serum to estimate the percentage in vivo inhibition of DPPIV enzymatic activity in the serum. Percentage inhibition was calculated by comparing DPPIV enzymatic activity of treated mice to control mice, which were not enzymatically inhibited (defined as 100% activity). In order to limit the number of blood draws per mouse, groups were split in two and blood was taken only once a day for each mouse (either at 2 hours or 12 hours post–DPPIV inhibitor administration).DPPIV enzymatic activity was assayed by using glycyl-prolyl-4-methoxy-β-naphthylamide (Gly-Pro-4-Me-β-NA) as fluorogenic substrate as described previously d, detected with phycoerythrin-streptavidin) and irrelevant isotype controls + or 34-2-12+ live cells among different cell lineages was calculated. Mice were considered chimeric if they demonstrated at least 2% of donor cells within the myeloid lineage plus at least one lymphoid lineage. Surface staining was performed according to standard procedures and flow cytometric analysis was done on a Coulter Cytomics FC500. CXP software  was used for acquisition and analysis. Antibodies were purchased from Becton Dickinson .Two-color flow cytometric analysis was used to distinguish donor and recipient cells of particular lineages by staining with fluorescein isothiocyanate–conjugated antibodies against CD4, CD8, B220, MAC-1, and biotinylated CD45.2 or 34-2-12  was grafted 2 to 8 weeks after allogeneic BMT and visually inspected thereafter at short intervals. Grafts were considered rejected when <10% remained viable.t test was used to compare chimerism levels between the groups. Skin graft survival was calculated according to the Kaplan–Meier product limit method and compared between groups using the log-rank test.A two-sided Student’s 6 unseparated CD45.2 BMCs that were or were not pretreated in vitro with the DPPIV inhibitor Diprotin A (n = 9/group) p = NS for all lineages). Similarly, there were no differences in chimerism levels between groups in BM and spleen   A. At theCollectively, these experiments demonstrate that DPPIV inhibition with Diprotin A does not lead to a detectable improvement of the engraftment of unseparated congenic BM in nonmyeloablatively conditioned recipients.To assess whether the failure to detect an engraftment effect is due to insufficient DPPIV inhibition achieved with in vivo Diprotin A treatment, serum DPPIV activity was measured.At peak exposure , DPPIV serum activity decreased to 78.1% and 55.1% in two (randomly selected) mice, whereas DPPIV activity remained essentially unchanged in one mouse A. At theSitagliptin (Januvia) is a specific DPPIV inhibitor that has recently been approved for the treatment of type 2 diabetes p = NS at all time points). Twenty-two weeks post-BMT mean chimerism levels were 48.8% (±5.7%) vs 43.7% (±6.4%) among CD4 cells, 34.5% (±3.0%) vs 29.3% (±6.5%) among CD8 cells, 60.1% (±9.8%) vs 54.9% (±9.5%) among B cells, and 48.6% (±11.1%) vs 41.6% (±11.5%) among myeloid cells (treated group E vs control group C) (p = NS). Thus, with the more specific, clinically approved DPPIV inhibitor sitagliptin, no beneficial effect was detectable on the engraftment of congenic BM in nonmyeloablated recipient mice.To assess whether sitagliptin affects engraftment, we followed hematopoietic chimerism in sitagliptin-treated recipients of unseparated congenic BM . Again, no enhanced engraftment was detected on DPPIV inhibition (group C) A. Chimergroup C) B were al6 fully mismatched Balb/c BMCs (day 0), and treated with anti-CD154 monoclonal antibody (day 0) and CTLA4Ig (day +2), as described previously DPPIV inhibition was shown to affect alloreactivity in models of heart and lung transplantation p = NS). Similarly, chimerism levels and rates were also comparable between groups in spleen  and activity remained substantially decreased in 2 of 4 mice  after 12 hours C. As expn spleen B.p = 0.5 sitagliptin-treated vs untreated recipients) Donor and third-party skin was transplanted to assess donor-specific tolerance. Third-party grafts were promptly rejected in both groups. Donor skin graft survival was significantly prolonged in both groups but tolerance was not achieved, consistent with the poor T-cell chimerism  Inhibition or genetic ablation of DPPIV/CD26 showed promising engraftment-promoting effects in several murine models especially when limited numbers of donor cells were used In order to dissect whether a potential effect of DPPIV inhibition is due to modulating alloreactivity or HSC engraftment, we used two established models of nonmyeloablative BMT. The CD45.2 → CD45.1 congenic model is almost free of relevant immunological barriers Several DPPIV inhibitors have recently been approved for treatment of type 2 diabetes. We therefore tested whether sitagliptin enhances the engraftment of congenic or allogeneic BM after nonmyeloablative TBI. As compared to Diprotin A, sitagliptin is both a more potent and specific DPPIV inhibitor Maximum inhibition of DPPIV serum activity (measured 2 hours after in vivo administration) was more profound with sitagliptin—achieving almost 100% inhibition—than with Diprotin A . A rapidWe think that several factors might be responsible for the lack of an effect of DPPIV inhibition in our present study. Models in which an engraftment-enhancing of DPPIV inhibition was found used either myeloablative conditioning or nonmyeloablative conditioning of immunodeficient recipients In summary, although DPPIV enzymatic inhibition had been demonstrated to have engraftment-promoting effects in several specific models of HSC transplantation, our studies provide evidence that DPPIV inhibition with Diprotin A or with sitagliptin does not lead to improved engraftment of unseparated BM after nonmyeloablative recipient conditioning.