Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn2+, Co2+, and Cu2, but not for Mg2+ or Ca2+. A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca2+ permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe David Clapham and colleagues offer new evidence that blurs the line between ion transporters and channels Based ondosomes . It is i2+, Mn2+, Cu2+, Zn2+, and Co2+ are required cofactors for many essential cellular enzymes. They cannot cross the plasma membrane through simple diffusion, and active uptake requires specific transporters. DMT1 is the only molecule known to mediate cellular iron uptake in higher eukaryotes. It is structurally unrelated to known Zn2+ and Cu2+ transporters, but DMT1 can transport those and other divalent metal ions  induced large inward currents in DMT1-expressing cells (42– to replace most of the Cl– ([Cl–]o = 5 mM) in low-pH bath solutions. With elimination of the background Cl– current, the proton-evoked current was inwardly rectifying (hyperbolic)  E.2+ (data not shown) or Mn2+ induced an additional current with less pronounced slow inactivation , and Fe3+ is not transported by DMT1  was recorded . In the presence of 100 μM Mn2+ (pH 4.2), the additional Mn2+-induced current was typically half the amplitude of the proton-induced current. Addition of Mn2+ alone (100 μM) at pH 7.4 did not induce any additional current. Since H+ or H+/Mn2+ induced two currents with distinct kinetics in DMT1-expressing cells, the underlying charge-carrying ion species and their relative contributions to the macroscopic currents were investigated. We monitored the reversal potential (Erev) and the current amplitude in ion-substitution experiments. Replacement of Na+ with N-methyl-D-glucamine (NMDG+) did not significantly change the Erev of H+ or H+/Mn2+-induced currents, although the net current amplitude was slightly increased  (Mn/PH can be estimated from the slope of Erev versus pH based on an extended Goldman–Hodgkin–Katz equation (+ and Mn2+). At pH 4.2, the slope of Erev versus pH did not differ significantly with or without Mn2+(see + to IDMT1 is much larger than that of Fe2+/Mn2+ (PMn/PH < 0.01), in contrast to the 1:1 stoichiometry proposed previously  Ca2+ solution  . PMn/PH equation  with two Mn2+see F. Therefeviously . Importation see C.2+ and 140 mM Na+) at pH 7.4 (G185R) was stable over minutes with no slow inactivation (see G185R in more than 85% of enhanced green fluorescent protein (EGFP)-positive cells transfected with the pTracer-G185R construct and in stable, doxycycline-induced G185R-HEK-On cells, but never in cells transfected with wild-type DMT1 . The inwardly rectifying current was cationic, since Ca2+ and Na+ substitution by NMDG+ completely abrogated the current  compared to wild-type DMT1 at similar protein expression levels , IG185R was 64 ± 7 pA/pF at −140 mV .Io 105 mM E, strengove 0 mV D, whereaG185R. The amplitude of IG185R was strongly dependent on [Ca2+]o  increased the current, indicating that Ca2+ permeated the plasma membrane of G185R-transfected cells much more readily than Na+. As shown in 2+]o not only augmented the current amplitude but also shifted Erev toward depolarized potentials. The slope of this shift (25 mV per decade) was close to the slope of 29 mV per decade predicted by the Nernst equation for a Ca2+-selective electrode . We determined G185R-specific currents by measuring the reversal potentials of the currents subtracted from two holding potentials  (G185R was less Ca2+-selective (PCa/PNa of approximately 10) than VGCCs . Single iG185R channels were not observed in cell-attached patches. Analysis of membrane current noise at −100 mV predicted a single-channel chord conductance of 0.4 ± 0.1 pS , too small to be observed under most patch–clamp conditions.We next investigated the cation selectivity of I [Ca2+]o F. With 1lectrode C. The reials see A and 4B lculated  and illuand Ba2+ D. While ransient D, the siransient , yieldinK+ > Cs+ E. Under sequence E of IG18 (VGCCs) , but IG12+ indicator dye Fura-2, we demonstrated G185R-mediated Ca2+ influx by monitoring intracellular Ca2+ levels in response to an elevation of [Ca2+]o  were −33 mV and −10 mV for control and isotonic Ca2+ solutions, respectively , nor was it blocked by antagonists of known Ca2+-permeant channels. In particular, the current was not blocked by ruthenium red (RR), Ca2+-release activated Ca2+ channel (CRAC) blockers SKF96365 and 2-APB , inhibited IG185R. La3+  blocked IG185R in a similar voltage-dependent manner , and CRAC currents, based on its current–voltage (I-V) relation, kinetics, permeation properties, and pharmacological sensitivity.Ihibition A and 4B hibition . The halectively B. The vopendence B. IG185Rnd 2-APB   and in mk enterocytes. A putative associated protein, if present in these different cell types, would have to be activated in a G185R-dependent manner. We did not find evidence of an associated protein when we immunoprecipitated wild-type or G185R DMT1 from transfected CHO-K1 cells (unpublished data). Furthermore, a distinct DMT1 mutant, G185K, also displayed Ca2+ permeability, but this mutant was less selective for Ca2+ over Na+ (unpublished data).We conclude that expression of G185R in transfected cells and in vivo in DMT1 mutants with impaired iron transport or more likely to be noticed by those managing the animal colonies. In parallel with these studies, we have generated knockout mice homozygous for a null DMT1 allele . Although detailed phenotypic characterization has not yet been completed, we have noted that –/–Dmt1 mice invariably die by the end of the first week of life, in contrast to mk/mk mice, which are poorly viable but can survive for more than a year . This suggests that the small amount of residual function of G185R DMT1, perhaps in combination with its gain-of-function Ca2+ conductance, contributes to viability.G185R mutations have occurred at least three times in rodents, which suggests that G185R not only inactivates DMT1, but may confer an unknown selective advantage. Because it has arisen in inbred colonies, the postulated selective advantage must either make the animals more viable than other 2+] has been reported to increase nontransferrin-bound iron uptake through an undefined transport system that has characteristics distinct from DMT1  in a 12-TM transporter protein conferred new Calocation . The “ch2+ entry gain of function helps to account for this remarkable pattern of remutation. Further investigation of this hypothesis will require direct and detailed comparison of DMT1-null and mk mice.We initiated these studies to investigate why a unique DMT1 mutation, G185R, has occurred independently at least twice in mice and once in rats ,1998. ThDMT1 cDNA used in this study was derived from one of four alternatively-spliced DMT1 gene transcripts. The G185R mutation was generated by using M13 phage and the oligonucleotide GTCCCCCTGTGGGGCCGAGTCCTCATCACCA. Wild-type DMT1 and the G185R mutant were tagged with a C-terminal FLAG epitope and subcloned into pTracer-CMV2 . CHO-K1 or HEK293T cells transiently transfected with DMT1 and G185R were used for the 55Fe uptake assay and Western blot analysis. To obtain a stable G185R-expressing cell line, the G185R-encoding DMT1 gene was subcloned into pRevTRE , a retroviral vector that drives expression from a Tet-responsive element. All constructs were confirmed by sequencing. DMT1 Western blot analyses were performed with an anti-FLAG M2 monoclonal antibody  and, in some cases, with a goat polyclonal antibody raised against human DMT1 .The TM, or HEK-On) was generated, and expression was induced by adding 1–10 μg/ml doxycycline into the culture medium. Unless otherwise stated, the pipette solution contained 147 mM cesium, 120 mM methane-sulfonate, 8 mM NaCl, 10 mM EGTA, 2 mM Mg-ATP, 20 mM HEPES (pH 7.4). Bath solution contained 140 mM NaCl, 10 mM CaCl2, 10 mM HEPES, 10 mM MES, 10 mM glucose (pH 7.4). Unless otherwise stated, the low pH solutions contained only nominal free Ca2+ (1–10 μM). Data were collected using an Axopatch 2A patch–clamp amplifier, Digidata 1320, and pClamp 8.0 software . Whole-cell currents were digitized at 10 kHz and filtered at 2 kHz.Wild-type and G185R mutant DMT1 were subcloned into an EGFP-containing vector  for transient expression in CHO-K1 and HEK293T cells. Cells were transfected using Lipofectamine 2000 (Invitrogen). Transfected cells, cultured at 37°C, were plated onto glass coverslips and recorded 24 (DMT1) or 30 (G185R) hrs after transfection. A stable cell line (HEK293 Tet-OnNa) was estimated according to rev upon replacing [Na+]o in nominally Ca2+-free bath solution , where X+ was Na+, K+, Cs+, or Li+. For the permeability to divalent cations (relative to PNa), bi-ionic conditions were used; Y2+ was Ca2+, Ba2+, or Sr2+ . The external solution was 140 mM NMDG-Cl, 10 mM Y2+Cl2, 20 mM HEPES (pH 7.4 adjusted with HCl). The permeability ratios of cations were estimated from the following equations  and 50 mM nitrilotriacetic acid. About 30 h after transient transfection, CHO-K1 or HEK293T cells were washed and harvested with PBS . Cells were resuspended in glass test tubes at 0.5–1 million/ml in 490 μl assay buffer at 30°C. The reaction was started by adding 10 μl of 55Fe stock and stopped at 4, 8, and 16 min by quickly filtering the reaction mix on a nitrocellulose filter . Filters were washed twice with 2 ml of assay buffer, dried, and radioactivity counted by liquid scintillation spectrometry.The assay buffer contained 25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl2+] was constructed according to Cells were loaded with 2 μM Fura-2 AM in culture medium at 37°C for 30 min. Low levels of G185R protein were expressed in the absence of doxycycline in G185R HEK-On cells . Therefore, doxycycline-treated HEK-On cells not expressing DMT1 were used as controls in imaging experiments. We recorded Fura-2 ratios (F340/F380) on an UltraVIEW imaging system . A standard curve for Fura-2 ratio versus o = 5 mM) for all low-pH bath solutions. (C) pH-induced outwardly rectifying current in a nontransfected CHO-K1 cell. A similar current was seen also in HEK293T and HEK-On cells, with properties similar to the cloned ClC-7 channel . This cu(718 KB PDF).Click here for additional data file.Figure S2Whole-cell currents were generated by voltage steps from −140 to +80 mV in 20 mV steps, 400 ms. The interval between steps was 1,000 ms. (1 MB PDF).Click here for additional data file.Figure S3+ by NMDG+ slightly increased the proton current (approximately 20%) and this was further augmented by adding 300 μM Mn2+. The concentrations used were Na+ and NMDG+, 140 mM, (pH 4.2); Mn2+, 300 μM. (141 KB PDF).Replacement of extracellular NaClick here for additional data file.www.ncbi.nlm.nih.gov/GenBank/) accession number for DMT1 is AF029758.The GenBank (


Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer.By Surface Enhanced Laser Desorption/Ionisation – Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells.Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells.HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression. The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5–10 % probability of 5 year survival . The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20  (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer  for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer  for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved ) immediately before the screenings.Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer  and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer , followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA.Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current.4HCO3, pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT  and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument.100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column  in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0–100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column . Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution . Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated.For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries  were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes.-16).We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 , were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20  chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) , and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa FIG. . StatistCompared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples . In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum.The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) Table . The mas50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated FIG. . HNP 1-3The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein FIG. . FurtherAbnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection  has been found in plasma, blood and other body fluids , and plaHNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours  and in tPrevious studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils  and neutin vitro and that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement [We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2–4 molecules  and a crmplement ,23 has bmplement  showed tmplement . TogetheCommon to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor . It has in vivo function of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner [-4 M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10-8 to 10-9 M) increases TNF-alpha production by monocytes [in vivo HNP 1-3 purified from colon tumours are capable of lysing MDCK cells. Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis.The exact concentration of HNPs in the tumour microenvironment may decide the t manner . This isonocytes . In a stonocytes  it was sThe high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation  and coulOur results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy.This study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S.Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital.JA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project.The pre-publication history for this paper can be accessed here:


During endochondral bone formation, the hypertrophy of chondrocytes is accompanied by selective expression of several genes including type X collagen and alkaline phosphatase. This expression is stimulated by inducers including BMPs and ascorbate. A 316 base pair region of the type X collagen (Col X) promoter has been previously characterized as the site required for BMP regulation. The intent of this study was to examine the role of Mitogen Activated Protein (MAP) and related kinase pathways in the regulation of Col X transcription and alkaline phosphatase activity in pre-hypertrophic chick chondrocytes.Using a luciferase reporter regulated by the BMP-responsive region of the type X collagen promoter, we show that promoter activity is increased by inhibition of extra-cellular signal regulated kinases 1 or 2 (ERK1/2). In contrast the ability of BMP-2 to induce alkaline phosphatase activity is little affected by ERK1/2 inhibition. The previously demonstrated stimulatory affect of p38 on Col X was shown to act specifically at the BMP responsive region of the promoter. The inhibitory effect of the ERK1/2 pathway and stimulatory effect of the p38 pathway on the Col X promoter were confirmed by the use of mutant kinases. Inhibition of upstream kinases: protein kinase C (PKC) and phosphatidylinositol 3-(PI3) kinase pathways increased basal Col X activity but had no effect on the BMP-2 induced increase. In contrast, ascorbate had no effect on the BMP-2 responsive region of the Col X promoter nor did it alter the increase in promoter activity induced by ERK1/2 inhibition. The previously shown increase in alkaline phosphatase activity induced by ascorbate was not affected by any kinase inhibitors examined. However some reduction in the alkaline phosphatase activity induced by the combination of BMP-2 and ascorbate was observed with ERK1/2 inhibition.Our results demonstrate that ERK1/2 plays a negative role while p38 plays a positive role in the BMP-2 activated transcription of type X collagen. This regulation occurs specifically at the BMP-2 responsive promoter region of Col X. Ascorbate does not modulate Col X at this region indicating that BMP-2 and ascorbate exert their action on chondrocyte hypertrophy via different transcriptional pathways. MAP kinases seem to have only a modest effect on alkaline phosphatase when activity is induced by the combination of both BMP-2 and ascorbate. During skeletal development and growth, bone formation occurs either by intramembraneous or endochondral bone formation. In endochondral bone formation, which occurs at the growth plates of long bones, cartilage is formed first, then the chondrocytes undergo a proliferative phase followed by hypertrophy, changes in gene expression, and matrix calcification, after which the cartilage is replaced by bone. Although generally referred to as chondrocyte hypertrophy, cell enlargement is just one manifestation of the more complex process of chondrocyte maturation, which can be considered an end-stage of chondrocyte differentiation. It is important to define the mechanisms that induce chondrocyte maturation, not only to understand bone development, but also to help prevent hypertrophy and ossification during cartilage tissue engineering.Hypertrophic chondrocytes are characterized by their increased levels of alkaline phosphatase (ALP), reduced levels of type II and IX collagens, and the emergence of type X collagen (Col X), which is a specific marker of hypertrophy ,2. AscorIn osteoblastic cells, BMPs and ascorbate have been shown to operate via mechanisms that at least partly involve mitogen activated protein kinases (MAP kinases). For example, ascorbate promotes extracellular matrix production which, in turn, activates the extracellular-signal regulated kinases, (ERK1/2 or p42/ p44) in an osteoblastic cell line . MAP kinIn general, MAP kinase pathways involving ERK1/2 stimulate proliferation, growth and differentiation, whereas those that stimulate p38 kinase lead to differentiation and apoptosis . In earlChick sternal chondrocytes are a popular model for the study of chondrocyte maturation because under normal development chondrocytes from the cephalic portion of the sternum undergo hypertrophy followed by mineralization and bone formation, whereas the caudal portion remains as cartilage ,8. In thStudies with the ERK1/2 inhibitor U0126 indicated that blocking ERK1/2 signaling increased the activity of the type X collagen promoter but had no effect on alkaline phosphatase (ALP) activity in chick cephalic sternal chondrocytes Fig. . In contALP activity was highly variable between cell isolates and is expressed here normalized to BMP-2 treated controls for the purpose of combining experiments, typical ALP values ranged from approximately 0.5–2 nmol/min/μg DNA in controls to between 4 and 12 nmol/min/μg DNA in BMP-2 treated cultures.The effects of altered ERK1/2 signaling on Col X promoter activity in chick sternal chondrocytes was further studied both by transfection with mutant kinases and by treatment with additional kinase inhibitors. Col X promoter activity was increased, both in the presence and absence of BMP-2, when the mitogen-stimulated ERK pathway was suppressed by transfecting chondrocytes with dominant negative ERK-2 Figs. . ConversAs seen with the ERK1/2 inhibitor U0126, treatment with the more specific ERK1/2 inhibitor PD098059 increased b2/640 Col X promoter activity, in the presence of BMP-2 Fig. . Dose reTransfection with dominant negative p38 caused a decrease in Col X promoter activity in BMP-2 treated cephalic chondrocytes, reducing activity to half of that seen in BMP-2 treated controls (p < 0.005) and eliminating the BMP-2 response Figs.  and 2B. Addition of either PI3 kinase inhibitor or PKC inhibitor resulted in similar stimulation of the collagen type X promoter. Calphostin C, a PKC inhibitor, increased activity in BMP-2 treated cells more than 2-fold, an effect similar to that seen with the ERK1/2 inhibitor PD98059 at 10 μM Fig. . SimilarTo assess the possible effects of protein kinase inhibitors on cell proliferation and survival, we measured relative numbers of live cells using a tetrazolium (MTS) assay. The results indicated that all cultures treated with inhibitors, with and without BMP-2 and/or ascorbate, had cell numbers within 10% of untreated controls (data not shown).We examined the effect of 75 μM ascorbate-2-phosphate on the activity the Col X promoter in cultures treated with kinase inhibitors. Col X promoter activity was unaffected by addition of ascorbate, and 4 μM of the ERK1/2 inhibitor U0126 increased promoter activity to comparable levels both with and without ascorbate Fig. .ALP activity in the absence of exogenous BMP was stimulated at least 2-fold in ascorbate-treated cultures without inhibitors, as previously reported, and this stimulation was not significantly affected by addition of either ERK1/2 or p38 inhibitors Fig. . In cultThe present studies demonstrate that ERK1/2 inhibition increases activity of the BMP responsive region of the type X collagen promoter. This indicates that ERK1/2 signaling interferes with the ability of BMP-induced signals to stimulate type X collagen transcription. Interestingly ERK1/2 has also been shown to inhibit type I collagen expression in an osteoblastic cell line  suggestiIn contrast to the stimulatory effects of inhibiting the ERK pathway, p38 inhibition blocked BMP-stimulated Col X promoter activity. Zhen et al.  and BeieWhile the classical pathway for BMP signaling is via activation of R-Smads, there is also evidence for BMP signaling via a TGF-activated kinase (TAK1) leading to p38 signaling -26. HoweInhibiting either protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3 kinase) increased type X collagen promoter activity both in BMP-2 treated cultures and controls. Both PKC and PI3 kinase have been reported to negatively regulate p38  and posiAlthough alkaline phosphatase expression, like type X collagen expression, increases during chondrocyte hypertrophy and is stimulated by BMPs, its regulation clearly differs from Col X in several respects. ERK1/2 inhibition has little effect on the ALP activity induced by BMP-2 or ascorbate acting alone and reduces the ability of BMP-2 to further stimulate ALP activity in ascorbate treated cultures. Inhibiting p38 does not have a clear effect on BMP-stimulated alkaline phosphatase activity in this model although it has been shown to reduce ALP activity in long-term (12–15 day) micromass cultures .Preliminary studies with cyclohexamide indicate that new protein synthesis is required for the up-regulation of alkaline phosphatase mRNA in response to BMP-2. We propose that these differences reflect a direct Smad-mediated effect of BMPs on type X collagen expression and an indirect effect on ALP expression.A mechanism which could account for the observed effects of ERK and p38 signaling on expression of type X collagen and ALP is presented in Fig. Evidence that BMP stimulation of type X collagen requires both activated R-Smads and Runx2 has been previously reported ,7. LittlAs well as its demonstrated role in Col X expression as found here and by ,31, therRetinoic acid ,8, anothElucidating the signaling pathways by which chondrocytes are driven to hypertrophy is necessary in order to better understand skeletal development, cartilage disease and improve the design of tissue engineered cartilage. We showed here that the ERK1/2 pathway inhibits type X collagen production by either directly or indirectly acting at the BMP responsive region of the promoter. p38 kinase signaling stimulates type X collagen transcription at the same promoter region, probably in conjunction with BMP-2 activated Smads. The factor upstream of p38 in this stimulatory pathway is unknown. Alkaline phosphatase activity is likely to be regulated in a different way from type X collagen since MAP kinases do not contribute in the same way to this pathway. Although ascorbate and BMPs both induce hypertrophy in chondrocytes ascorbate does not act at the same region of the Col X promoter as BMPs.The ERK1/2 inhibitor PD98059, which blocks the upstream kinase (MEK1) of ERK1/2, the p38 inhibitor SB203580, and the PKC inhibitor Calphostin C were obtained from Sigma . UO126, also a MEK1 inhibitor, was obtained from Biomol  and LY294002, a phosphatidylinositol 3- (PI3) kinase inhibitor from Cell Signaling Technology . Plasmids were kindly donated as follows: constitutively active MEK1 from Michael Webber (University of Virginia); dominant negative p38  from RogChondrocytes were cultured as previously described . Cephalirenilla luciferase reporter (0.4 μg/well) (Promega) which served as a transfection control [4 precipitation or Fugene transfection reagent at 6 μl/ml (Promega). Since preliminary experiments using green fluorescent protein showed that Fugene was more effective in terms of numbers of cells transfected, this method was used for the majority of the experiments; however, relative outcomes between controls and treated cells were not affected by the transfection method.On day 1 of secondary culture (24 hrs after plating) the cell layer was washed in HBSS and the media changed to DMEM supplemented with 10% FBS in place of NuSerum. Cells were co-transfected with pGL2 plasmid containing the b2/640 type X collagen promoter region attached to a firefly luciferase reporter (0.2–1 μg/well)  and pRL null plasmid attached to a  control . When apTransfection proceeded for 5 hrs after which the cell layer was rinsed twice in HBSS and cultured with serum free medium . Some wells were supplemented with 75 μM ascorbate-2-phosphate  or 30 ng/ml human recombinant BMP-2 . Where inhibitors were used they were added at this point and cells incubated for 1 hr before the addition of BMP-2. Cells were cultured for a further 48 hours, then lysed and assayed using a dual luciferase assay kit (Promega).2, 0.1 mM MgCl2 and 1% Triton X-100 for 30 mins at 37°C, followed by overnight storage at 4°C. A sample of the cell lysate was reacted with p-nitrophenyl phosphate substrate in 1.5 M Tris buffer pH 9 with 1 mM ZnCl2 and 1 mM MgCl2. Phosphatase activity was measured specrophotometrically at 410 nm with 1 absorbance unit equivalent to 64 nmol of product. For DNA analysis, cells were trypsinized and a subsample of cell suspension centrifuged, the cell pellet lysed with the CyQUANT lysis buffer and the fluorescent DNA dye added. The resulting solution was transferred to a 96 well plate and DNA assayed fluorometrically. The remaining cells were extracted for the alkaline phosphatase assay as above. Alkaline phosphatase enzyme levels were calculated as nmol p-nitrophenol product per minute normalized to MTS units or μg DNA.For alkaline phosphatase assays, cells were switched to serum free medium on day 1 of secondary culture, inhibitors were added and cells incubated for 1 hr before the addition of ascorbate or BMP-2, as described for the luciferase assay. Cells were cultured for a further 72 hrs and then rinsed twice in HBSS. Cells numbers were assayed either by DNA quantification  or by MTS tetrazolium salt assay of mitochondrial activity . When MTS was used, a 1:10 dilution of MTS was applied in phenol red-free media for 30–60 minutes, 200 μl of media plus MTS was transferred to a 96 well plate and assayed in a 'Multiskan ascent' plate reader . The cell layer was then washed twice in HBSS and extracted with 0.15 M Tris, pH 9 with 0.1 mM ZnClStatistics were performed using Minitab™ software. After expressing results as a ratio of experimental/control within each experiment, the data from at least 3 experiments were combined. The combined data was tested for normality using the Anderson-Darling test. Normally distributed or non-parametric data were tested for differences between treatments using two-sample Students t-test or the Mann-Whitney test respectively. Where groups of experiment means were compared a paired t-test was used.ALP – Alkaline PhosphataseBMP – Bone Morphogenetic Proteinbp – base pairsCA – constitutively activecbfa – core binding factor alpha 1Col X – Collagen type XDN – dominant negativeERK – extracellular signal regulated protein kinaseMAP – Mitogen activated proteinMEK – ERK kinaseMTS – 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner saltPI3 – phosphhatidylinositol 3PKC – Protein Kinase CRunx – Runt associated proteinTAK – Transforming growth factor beta activated kinaseThe author(s) declare that they have no competing interests.GCR was involved in experimental design, performed 90% of the experiments, analyzed the data and wrote the first draft of the manuscript. EBG isolated chondrocytes, performed preliminary experiments and contributed to trouble-shooting of the methods. GG-K, performed preliminary experiments and developed methodology. PSL was involved in experimental design and data analysis, wrote portions of and edited the whole manuscript and was the project supervisor.


Escherichia coli are exposed to harsh and non-ideal growth environments—nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper Under conditions of oxidative stress, methionine auxotrophy in  As a consequence of living in an aerobic world, organisms face the challenge of maintaining a favorable cellular redox status. Oxidative stress is caused by imbalances between the production and disposal of reactive oxygen species, which can damage proteins, lipids, and nucleic acids. Bacteria encounter reactive oxygen intermediates that are generated as byproducts of aerobic metabolism or during challenge by the immune systems of hosts. Thus, understanding the effects of oxidative stress on the cell, as well as elucidating cellular defense mechanisms, is of considerable interest.Escherichia coli have been reported that collectively suggest a link between oxidative stress, methionine limitation, and the enzyme that catalyzes the final step in methionine biosynthesis, cobalamin-independent methionine synthase (MetE). In E. coli, the sulfur of methionine comes from cysteine, which in turn obtains the sulfur group from sulfate via the sulfate assimilation pathway  were sensitive to oxidative challenge and had an oxygen-dependent growth requirement for the sulfur-containing amino acids cysteine and methionine (trxA grxA (thioredoxin 1 and glutaredoxin 1) and grxA grxB grxC gor  strains of E. coli, cells required cysteine or methionine for growth, presumably because of oxidative inactivation of 3′-phosphoadenylylsulfate (PAPS) reductase -independent methionine synthase (the metE gene product) or B12-dependent methionine synthase (the metH gene product). Both enzymes employ a catalytic zinc to transfer a methyl group from methyltetrahydrofolate to L-homocysteine to form methionine  use only the B12-independent isoform  reversibly oxidizes MetE at a rate that correlates with enzyme inactivation. GSSG was found to specifically glutathionylate MetE and induce a conformational change. In vivo thiol-trapping experiments provide direct evidence that MetE is readily oxidized in cells experiencing a variety of oxidative stress conditions. Moreover, oxidation of MetE is associated with a cellular dependence upon methionine for growth, suggesting that oxidant-mediated inactivation of MetE may have a profound impact on the stressed cell.In this study, we investigated the effects of oxidative stress on MetE and methionine availability within the cell. Transient oxidative stress conditions induced by diamide were found to elicit a methionine auxotrophy in E. coli. We investigated the duration of growth lags induced by oxidative stress in cells growing in glucose-minimal medium containing or lacking methionine. In the experiment shown in E. coli cultures logarithmically growing in glucose-minimal medium with and without methionine experienced a lag in growth upon challenge with diamide  was used to induce oxidative stress. Addition of methionine to the medium significantly decreased the growth lag produced by H2O2 treatment  as loss of activity (0.07 min−1).The methionine auxotrophy observed under oxidative stress conditions could reflect an increased demand for methionine or a decreased rate of de novo synthesis due to inactivation of MetE. To distinguish between these possibilities, we assayed the activity of the purified enzyme upon challenge with GSSG . Nearly While the rate of thiol oxidation could be measured with DTNB, the absolute number of cysteines oxidized was ambiguous. MetE contains seven cysteines, yet DTNB titration of the fully reduced enzyme gave values between 7.2 and 9.0 cysteines for different protein preparations, and oxidation of MetE led to an average decrease of 1.8 cysteines per mol. Thus it was important to use independent means to determine the number of cysteines oxidized by GSSG.Oxidation of MetE by GSSG would be expected to proceed by initial formation of a mixed disulfide between MetE and glutathione .An inter- or intramolecular disulfide bond could then be generated by attack on the mixed disulfide by a second cysteine residue. These two possibilities can be distinguished by using mass spectrometry to determine the molecular mass of the oxidized protein. The reduced protein was found to be 84,530 ± 6 Da , whereas the mass of the protein oxidized by GSSG was 84,835 ± 8 Da. The mass difference of 305 ± 10 Da between oxidized and reduced MetE is consistent with the addition of a single glutathione adduct . Formation of an intramolecular disulfide bond would only decrease the mass by 2 Da, which would not be detected by mass spectrometry in such a large protein.To obtain evidence for stoichiometric glutathionylation of MetE, the oxidized protein was reduced with DTT to release the glutathione adduct. The DTT and GSH were separated from the protein using a Microcon concentrator. The filtrate was assayed for GSH using fluorescamine, which reacts with primary amines to form a fluorescent product. The reduced protein did not release GSH (0.0 mol GSH per mol protein) while oxidized protein released 0.9 mol GSH per mol of protein. Thus both mass spectrometry and the fluorescamine assay were consistent with glutathionylation of MetE by GSSG.In order to determine the site of oxidation and verify that an intramolecular disulfide was not being formed concomitant with oxidation, we performed disulfide mapping experiments based on the method of This approach allows for relatively straightforward mapping of disulfide bonds. The acidic pH employed minimizes the possibility of disulfide bond scrambling. Furthermore, the ability to cleave N-terminally to each cyanylated cysteine allows cleavage between closely spaced, even adjacent, cysteines. These factors provide a significant advantage over traditional methods of peptide mapping, particularly for MetE, which contains cysteines spaced only two residues apart at the active site (cysteines 643 and 645). Moreover, for large proteins, most proteolytic and chemical cleavage methods have the potential to generate numerous peptides, whereas this approach produces a more limited number.I, and β-elimination at cysteine 560 produced a small amount of product H. Identification of these side products provides additional evidence that cysteines 560 and 726 were reduced and available for cyanylation. Disulfide mapping of a mutant MetE, MetEC>A1–4, in which cysteines 323, 353, 516, and 560 were changed to alanines, further confirmed the mass assignments. Only peaks corresponding to cleavage next to the three remaining cysteines  were observed  need to be taken into account. Based on the equilibrium constant for GSSG oxidation, MetE activity at equilibrium was calculated for typical cellular glutathione concentrations  at varioE is –0.252 V at pH 7.2  of 1, 2, or 5 mM, and the calculated [GSH]2/[GSSG], [GSH] and [GSSG] can be determined at each cellular potential. The ratio of [GSH]/[GSSG] may then be used to calculate the relative amount of MetE activity expected at equilibrium bywhere se in pH ), R is 8mix is 1.4.)where KE. coli growing under normal conditions (–250 mV to –280 mV /[GSSG] ratio of less than 1.4 would be required to accumulate that much oxidized MetE . Moreover, in vitro oxidation of MetE by GSSG is rather slow, further suggesting that glutathionylation of MetE via thiol-disulfide exchange is not the primary mechanism functioning in vivo. Rather, the in vitro and in vivo data for MetE are consistent with initial formation of a reactive intermediate followed by glutathionylation of cysteine 645, resulting in enzyme inactivation.The ratios of [GSH] to [GSSG] in wild-type (DHB4) and ectively . Though trxA strain. Since E. coli lacking trxA do not experience a substantial amount of cytosolic disulfide bond formation (2O2) accumulate in the trxA strain, since thioredoxin appears to be required to regenerate the reduced thiol status of these proteins. Our preliminary in vitro experiments indicate that glutathionylated MetE is inefficiently reduced by thioredoxin (data not shown), which is not unexpected since glutaredoxins are generally more specific for glutathione-protein adducts  results in irreversible modification, whereas oxidation in the presence of GSH (as would be expected in vivo) yielded reversible glutathionylation  or sulfonic (–SO3) acid adducts (data not shown). Thus, glutathionylation of MetE following the formation of a reactive intermediate may serve the dual purpose of modulating enzyme activity while protecting the active site from more extensive damage. Reduction by cellular thioreductases could then readily reactivate the enzyme.Protein glutathionylation has been suggested to play a protective role in oxidative stress. Studies with purified eukaryotic proteins have shown that incubation with oxidants  and Grx3 (grxC) are structurally distinct from Grx2 (grxB), yet all three proteins are capable of reducing glutathionylated PAPS reductase . This places the sulfhydryl in direct contact with the solvent, where it is accessible to oxidation and reduction. Glutathionylation of cysteine 645 at the entrance to the active site may sterically prevent substrate binding, leading to the observed inactivation of the enzyme. Addition of the glutathione adduct also appears to correlate with a weakened binding of the catalytic zinc to the enzyme (data not shown). The active site of MetE is located in a cleft formed between two domains . Addition of the glutathione tripeptide to cysteine 645 may provide enough bulk to push the two domains apart, giving rise to the observed conformational change and increased hydrodynamic radius. CD spectroscopy indicates that oxidation does not result in gross unfolding of MetE  as well as wild-type cells challenged with diamide or H2O2. Our results further show that oxidation of MetE is associated with a methionine limitation imposed by oxidative stress. Hence this study provides insight into a previously unknown, but important aspect of the E. coli cellular response to oxidative stress.In conclusion, we have established that MetE, a highly expressed protein in 6S)-5-methyltetrahydropteroyltriglutamate was synthesized from pteroyltriglutamate , as described previously , ammonium persulfate, TEMED, 10 N sodium hydroxide, and 85% phosophoric acid were obtained from Genomic Solutions . Iodoacetate and iodoacetamide were purchased from Fluka . (eviously . L-Homocolactone . PolycloE. coli K-12 strain DHB4 and isogenic E. coli K-12 strains WP843 (trxA gor) and WP778 (trxB gor) (E. coli K-12 strain GW2531 (metE) . Wild-type rxB gor)  were obt1 (metE) . The Met1 (metE)  using Pf1 (metE) , specify600 ∼1). Cultures were incubated at 37 °C with shaking at 250 rpm for approximately 30 h before harvesting. The proteins were purified as described previously  (2O2 concentrations were determined similarly (ɛ240 = 43.6 M−1cm−1)  medium  supplemeM−1cm−1) . H2O2 coM−1cm−1) .−1cm−1 using the Endelhoch method -5-methyltetrahydropteroyltriglutamate, and enzyme in a total volume of 400 μl. After a 30-s incubation at 37 °C, the reaction was quenched by addition of 100 μl of 5 N hydrochloric acid/60% formic acid, followed by heating at 88 °C for 10 min. After cooling on ice, the reaction was centrifuged at 14,000g for 5 min at 4 °C to remove precipitated protein. The amount of tetrahydrofolate product (converted to methenyltetrahydrofolate by formic acid) was quantitated by its absorbance at 350 nm. Excess GSSG used to oxidize the enzyme did not significantly alter activity during the 30-s assay.The molar absorption coefficient for MetE at 280 nm was determined to be 157,000 ± 1000 Mh method . The speh method , but omig for 30 min at 4 °C. The supernatant was removed, and the pellet was dissolved in 100 mM potassium phosphate buffer (pH 7.3) containing 0.25 mM DTNB, 6 M guanidinium chloride, and 1 mM EDTA. Complete dissolution of the pellet required vortexing for 15–20 min. Reduced cysteines were quantitated by measuring the absorbance at 412 nm after subtraction of a blank containing DTNB and buffer but no protein. The extent of oxidation was determined using the fraction of reduced cysteines at the indicated time compared to the value at time zero (taken as 100%).MetE (50 μM) in 100 mM Tris chloride (pH 7.2) was equilibrated to 37 °C, and GSSG was added  to initiate the oxidation reaction. The GSSG concentration was established by assay with glutathione reductase . At timePurified MetE was desalted into 100 mM Tris chloride (pH 7.2) to remove DTT from the storage buffer. The reduced protein (50 μM) was then incubated with 10 mM GSSG for 90 min at 37 °C. Samples were concentrated using Microcon concentrators , and GSSG was removed using Bio-Gel P-6 columns  equilibrated with 100 mM Tris chloride (pH 7.2). Reduction and reactivation of GSSG-oxidized MetE was accomplished by incubation of the oxidized enzyme with 2 mM reductant at 37 °C. For preparation of samples of diamide-oxidized MetE to be used as standards for in vivo thiol-trapping assays, diamide (1 mM) was incubated with MetE (10 μM) in 100 mM Tris chloride (pH 7.2) at 37 °C. After 45 min, diamide was removed by gel filtration using Bio-Gel P-6 columns.g for 5 min at room temperature. Aliquots (100 μl) of the filtrate were added to 1.7 ml of 200 mM borate buffer (pH 9.0) containing 1 mM p-(hydroxymercuri)benzoic acid. While vortexing, 0.6 ml of 1 mg/ml fluorescamine (in acetone) was added. The fluorescence emission of the samples was measured at 485 nm with excitation at 390 nm. A range of GSH concentrations (0–125 mM) was assayed to generate a standard curve. GSH concentrations were determined by DTNB titration (Reduced and GSSG-oxidized MetE preparations were exchanged into 50 mM potassium phosphate (pH 7.2) using Bio-Gel P-6 columns. Proteins were then washed three times with the same buffer using Centricon concentrators. MetE (50 μM) was incubated with 2 mM DTT in 50 mM potassium phosphate (pH 7.2) in a volume of 200 μl for 1 h at 37 °C. Each solution was then loaded into a Microcon concentrator and centrifuged at 12,000itration . The stoCDAP dissolved in 200 μl of 100 mM citrate buffer (pH 3) containing 6 M guanidine hydrochloride was added to 100 μl of MetE in 100 mM Tris chloride (pH 7.2) and incubated for 15–20 min at room temperature. The stock concentrations were adjusted so that CDAP was approximately 25-fold in excess over the total thiol concentration. CDAP was removed from the solution by gel filtration, using Bio-Gel P-6 columns equilibrated with 6 M guanidine hydrochloride solution (brought to pH ∼7–8 with potassium hydroxide). Concentrated ammonium hydroxide was added , and the reaction was incubated at room temperature for approximately 75 min. Samples were then lyophilized to remove the ammonia and stored at –80 °C. Prior to LC-MS analysis, DTT was added  and allowed to incubate at room temperature for 30 min. Solutions were then diluted into 6 M guanidine hydrochloride solution (adjusted to pH 3 with acetic acid) and characterized by LC-MS.Samples (250–500 pmol) were injected onto a Vydac  250 mm × 2.1 mm C4 column (214MS52) using an analytical HPLC Surveyor from ThermoFinnigan . The column was equilibrated with 97% solvent A (water with 0.1% v/v acetic acid and 0.02% v/v TFA) and 3% solvent B (acetonitrile with 0.1% v/v acetic acid and 0.02% v/v TFA), and samples were eluted at a flow rate of 0.25 ml/min with a three-step linear gradient: 3% B for 15 min, 3% to 30% B in 9 min, and 30% to 65% B in 35 min. For LC-MS of the holo-protein, samples were desalted using a two-step linear gradient: 20% B for 10 min, and 20% to 70% B in 25 min. Fractionation was monitored by the UV chromatogram recorded at 280 nm. The eluent was diverted to waste for the first 15 min, and then directly infused into the electrospray ionization source of a ThermoFinnigan LCQ mass spectrometer. Mass spectral data were obtained in the positive mode, and ESI spectra were deconvoluted using the BioMass software provided by ThermoFinnigan.Oxidation of MetE by diamide was monitored at 37 °C using a Hi-Tech Scientific  SF-61DX2 stopped-flow spectrophotometer equipped with a xenon light source for single wavelength detection. Diamide (1 mM) and MetE (100 μM) in 100 mM Tris chloride (pH 7.2) were mixed, and reduction of diamide was monitored at 325 nm.MetE (20 pmol) was injected onto an Alltech  macrosphere size exclusion HPLC column (250 mm × 4.6 mm) with a 300-Å pore size (7-μm particle size) and run at 0.5 ml/min with 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride. For GroEL interaction experiments, GroEL (2 μM) was incubated with MetE (1 μM) in 20 mM MOPS (pH 7.5) containing 100 mM potassium chloride and 5 mM magnesium chloride for 30 min at 37 °C before analysis by size exclusion chromatography.g at 4 °C and then diluted to 0.4 mg/ml. Far UV CD spectra were recorded using a JASCO  J-810 spectropolarimeter and 0.1 cm cuvettes. Absorbance spectra of the same sample were recorded on a JASCO V-550 UV/Vis spectrophotometer to determine the precise protein concentration. The data were then normalized to a protein concentration of 4.5 μM MetE.Reduced and oxidized MetE were buffer exchanged into 10 mM potassium phosphate buffer (plus 500 μM DTT for the reduced protein) (pH 7.2). The final filtrates were collected and used as a blank. Samples were centrifuged for 1 h at 14,000Aliquots containing 2.5 mg/ml reduced or oxidized MetE in 50 mM potassium phosphate (pH 7.2) were digested with 0.02% (w/v) trypsin at room temperature. At indicated times, 5-μl samples were quenched with 10 μg of 1-tosylamido-1-lysyl chloromethylketone, and then analyzed by SDS-PAGE (10% gel).350 directly correlates to the amount of product in the activity assay, relative MetE activity was calculated by:MetE (10 μM) was incubated with varying concentrations of GSH and GSSG in 100 mM Tris chloride (pH 7.2) at 37 °C. GSH  and GSSG350(x) is the absorbance at 350 nm for sample x, A350(oxid) is for completely oxidized MetE , and A350(red) is for completely reduced MetE . The equilibrium constant, Kmix, was then determined by plotting relative MetE activity versus the [GSH]/[GSSG] ratio.where AE. coli cultures used for in vivo thiol-trapping experiments were grown aerobically at 37 °C. E. coli wild-type (DHB4) and trxA gor (WP843) strains were grown in glucose-minimal MOPS medium supplemented with 10 μM thiamine and all amino acids except methionine  was transformed into electrocompetent trxB gor (strain WP778) cells by electroporation. An overnight culture of WP778/pJG816 in LB medium containing 20 μg/ml kanamycin, 50 μg/ml ampicillin, and 4 mM DTT was diluted to an OD600 of approximately 0.02 into the same medium (without kanamycin). Care was taken to ensure that an overnight culture from the same inoculum failed to grow when DTT was not included in the medium. The 50-ml culture was grown to an OD600 of approximately 0.4, and then filtered into a prewarmed sterile filter flask. The cells were washed twice with prewarmed sterile LB medium to remove residual DTT, and resuspended in 50 ml of prewarmed LB medium. At the indicated time points following DTT removal, samples were taken for alkylation as described.For in vivo thiol-trapping experiments performed on the E. coli were grown in glucose-minimal MOPS medium without methionine as in the growth experiments described above, except that diamide  was added at an OD600 of approximately 0.4. Aliquots were removed at intervals following diamide addition for treatment with alkylating agents.To assay the oxidation status of MetE in diamide-treated cells, wild-type g) for 30 min at 4 °C, washed with cold 10% TCA (once) followed by ethanol (twice), and then dried by incubating approximately 20 min in a 37-°C incubator. The pellet could then be stored at –80 °C prior to secondary alkylation with iodoacetic acid. Pellets were dissolved in 50 μl of alkylation buffer containing DTT , and incubated 15 min at room temperature. Newly exposed cysteines were trapped by adding 12.5 μl of 0.5 M iodoacetate  and incubating 2 min at room temperature. For some samples, excess DTT was added to quench the reaction; however, this was found to not be necessary. Alkylated proteins were then frozen in liquid nitrogen and stored at –80 °C prior to analysis by isoelectric focusing.In order to trap oxidized thiols in vivo, 1 ml of culture was added to 250 μl of 0.5 M iodoacetamide in 100 mM Tris chloride (pH 7.2) that had been pre-equilibrated at 37 °C. Samples were incubated 2 min at 37 °C, quenched by the addition of 140 μl of 100% (w/v) ice-cold TCA, and incubated on ice for at least 30 min. Precipitated proteins were pelleted by centrifugation  at 4 °C for 30 min and then washed with cold 10% TCA (once) and either ether (3 times) or 1 M Tris chloride (pH 7.2) followed by water. Pellets were then alkylated with iodoacetate as described for the in vivo culture samples.For the reduced and oxidized MetE standards, 25 μl of 0.5 M iodoacetamide was added to 100 μl of 10 μM MetE in 100 mM Tris chloride (pH 7.2) and incubated for 1 min at 37 °C. The reaction was quenched by addition of 125 μl of 20% (w/v) ice-cold TCA and incubation on ice for at least 30 min. The proteins were pelleted by centrifugation , 50 mM DTT, and 0.15% SDS. Care was taken not to increase the solution temperature above 30 °C to avoid breaking down urea, which could then carbamylate proteins. Aliquots of the sample buffer were snap-frozen in liquid nitrogen and stored at –20 °C.An Xcell Surelock Mini-cell gel apparatus from Invitrogen was assembled with the freshly prepared gel, and degassed cathode  and anode  solutions were added. Samples were diluted at least 10-fold into sample buffer and loaded. Power was applied for 1 h at 100 V followed by 1 h at 200 V and finally 30 min at 500 V. Gels were then stained as described previously  or blottGels were electroblotted onto PVDF membrane  according to the manufacturer's (Invitrogen) protocol using 1/2X Towbin buffer. Proteins were immunoblotted with a polyclonal rabbit antibody to MetE  and immune complexes were revealed using a fluorescein-linked anti-rabbit antibody  followed by direct detection using a Molecular Dynamics  Storm phosphorimager.http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are AhpC (P26427), glutaredoxin 1 (P00277), glutaredoxin 2 (P39811), glutaredoxin 3 (P37687), glutathione reductase (P06715), GroEL (P06139), iron superoxide dismutases (P09157), manganese superoxide dismutase (P00448), MetA (P07623), MetE  (P82610), MetE (E. coli) (P25665), MetE (T. maritima) (Q9X112), MetH (P13009), OxyR (P11721), PAPS reductase (P17854), thioredoxin 1 (P00274), thioredoxin reductase (P09625), and Tpx (P37901).The Swiss-Prot (


Potential signal components are MAP kinases, phosphatidylinositol 3-kinase and phospholipase Cγ2. The latter can undergo tyrosine phosphorylation, and Src family kinases might carry out this phosphorylation. Btk, a Tec family kinase, could also be important. Our aim was to further elucidate the role of Src family kinases and Btk.Yeast and bacteria elicit arachidonate release in macrophages, leading to the formation of leukotrienes and prostaglandins, important mediators of inflammation. Receptors recognising various microbes have been identified, but the signalling pathways are not entirely understood. Cytosolic phospholipase AArachidonate release from murine peritoneal macrophages was measured by prior radiolabeling. Furthermore, immunoprecipitation and Western blotting were used to monitor changes in activity/phosphorylation of intermediate signal components. To determine the role of Src family kinases two different inhibitors with broad specificity  were used as well as the Btk inhibitor LFM-A13.Staphylococcus aureus or yeast-derived zymosan beads was shown to depend on members of the Src kinase family as well as Btk. Src kinases were found to act upstream of Btk, phosphatidylinositol 3-kinase, phospholipase Cγ2 and the MAP kinases ERK and p38, thereby affecting all branches of the signalling investigated. In contrast, Btk was not involved in the activation of the MAP-kinases. Since the cytosolic phospholipase A2 in macrophages is regulated by both phosphorylation (via ERK and p38) and an increase in intracellular Ca2+, we propose that members of the Src kinase family are involved in both types of regulation, while the role of Btk may be restricted to the latter type.Arachidonate release initiated by either Staphylococcus aureus or zymosan was found to depend on Src family kinases as well as Btk. While members of the Src kinase family were shown to act upstream of Btk and the MAP kinases, Btk plays another role independent of MAP kinases, but down-stream of the Src family kinases.Arachidonate release induced by either  Antibodies against p-JNK, PLC-γ2, ERK-2 and phosphotyrosine as well as HRP conjugated secondary antibodies, were from Santa Cruz Biotechnology . Antibodies against p-ERK, p-p38 and pAkt (Thr308) were from New England Biolabs . Antibodies against pAkt (Ser473) and pBtk (Tyr223) were from Cell Signaling Technology . The Mnk-1 inhibitor CGP057380 was a kind gift from Dr Hermann Gram, Novartis Pharma AG, Basel, Switzerland. Zymosan and bacteria were dispersed in PBS. Zymosan was used at 0,2 mg/ml and bacteria at a concentration of 2,5*10^7/ml. The inhibitors LFM-A13, PP2, CGP057380, SU6656, SKI-1 and wortmannin were dissolved in dimethyl sulphoxide (DMSO) and added to cells resulting in a final concentration of DMSO of less than 0.3%.Zymosan, SU6656 and wortmannin, were from Sigma Chemical Co. . Heat-killed 2 in air at 37°C. Non-adherent cells were removed after 2 h and the rest of the cells were incubated in medium 199 supplemented with 1% fetal bovine serum for 18–20 h. During this incubation cells were labelled with [3H]arachidonic acid in some experiments.Peritoneal macrophages were isolated from female outbred NMRI mice by adherence in a humidified atmosphere of 5% CO3H]arachidonic acid/well for 18–20 h, were washed three times with PBS, and stimulated in serum-free medium. At the end of the experiment the medium was collected and the cells scraped off the dishes in 0,1% Triton X-100 in water. The collected medium was centrifuged and the arachidonate released from cellular phospholipids was determined by liquid scintillation counting. The release into the collected medium is expressed as a percentage of total recovered radioactivity in cells and medium.Cells labelled with 0,5 μCi [2 dishes and stimulated in a serum-free medium. When inhibitors were used, they were added 15 min. before stimulus. After stimulation the cells were scraped off the dishes in 110 μl sample buffer and equal amounts of cell lysate were subjected to SDS-PAGE. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes which were blocked in 3% or 5% non-fat milk/gelatin for 1 h followed by incubation with different antibodies. Bound antibodies were detected with secondary horseradish peroxidase-labeled antibodies and enhanced chemiluminescence using LAS 1000 Plus . To assure equal loading of the gels the membranes were stripped and reprobed with ERK-2 antibody, or as indicated.Cells were cultured in 10 cm6 cells) were stimulated in serum-free medium. When inhibitors were used, they were added 15 min. before the stimulus. At the end of the experiment cells were washed with ice-cold PBS and scraped off the dish in lysis buffer , 20 mM NaF, 1 mM Na3Vo4, 1 mM PMSF and 1 μg/ml each of pepstatin and leupeptin). After centrifugation (104 × g) for 10 min at 4°C the supernatant was incubated with an antibody against PLC-γ2. The immune complexes were captured using protein A-Sepharose (50% w/v). The samples were centrifuged and the immune complexes were washed three times in lysis buffer. The immunoprecipitated proteins were dissolved in 2X sample buffer and subjected to SDS-PAGE (7% acrylamide) and analyzed by Western blotting.Macrophages  inhibited the zymosan-induced release by approximately 82% and the S. aureus-induced release by 83%.Both zymosan and Gram-negative bacteria induce arachidonate release in mouse macrophages , zymosanner Fig. . The dec2 in response to zymosan or bacteria [The MAP kinases ERK and p38 are both known to contribute to the activation of cPLAbacteria . Both zy2 SP600125, a JNK inhibitor [To establish whether also JNK might play a role in the activation of cPLAnhibitor  was trie2 [It has been suggested that the kinase Mnk-1, downstream of both ERK and p38, might transmit the signal to some of the phosphorylations necessary for the activation of cPLA2 . Treatme2 activation and arachidonate release [S. aureus, induced Akt phosphorylation at both Ser 473 and Thr 308 in macrophages, as detected by phosphospecific Akt antibodies  causing gel-shift . Tyr 551 is situated in the activation loop and its phosphorylation may be initiated by SFK, leading to autophosphorylation of Tyr 223, which appears necessary for full activation ,21. Deteift Fig. . Pretreaift Fig. . A Src kift Fig. . A diffelus Fig. . Btk pholus Fig.  and PLCγlus Fig.  argue foS. aureus . However, in contrast to the effect of SFK inhibitors described above, the inhibition of Btk did not affect the zymosan- and bacteria-induced phosphorylation of ERK and p38  caused a decrease in the release of arachidonate induced by both zymosan and eus Fig. . The decp38 Fig. , neitherp38 Fig. . A partip38 Fig. , but onlp38 Fig. .These data indicate that Btk has a regulatory role for arachidonate release, is acting downstream of inhibitor-sensitive SFK, but not involved in signalling to MAP kinase activation.S. aureus, is differentially dependent on SFK and the Tec kinase Btk. Src kinases act upstream of both Btk and the MAP kinases ERK and p38, thereby also of activating phosphorylation(s) of cPLA2. They are also most likely responsible for the tyrosine phosphorylation of PLCγ2 that occurs in response to zymosan, as shown here and previously [In this report we provide evidence that signalling to release of arachidonate induced in resident mouse peritoneal macrophages by non-opsonized zymosan  and the Gram-positive bacterium 2, which is regulated by both phosphorylation(s) and an increase in intracellular Ca2+ [2, especially in the C-terminal cluster of serine residues [The major enzyme responsible for release of arachidonate in the cells used in the present study is cPLAlar Ca2+ ,2. Severresidues . Our finresidues . Howeverresidues . The SFKresidues  which coresidues  for reviPI3K has an important role in zymosan- and bacteria-induced signalling in macrophages . SFK appXenopus egg extracts [S.aureus did not induce detectable tyrosine phosphorylation of PLCγ2. Nevertheless both phosphorylation of ERK and arachidonate release induced by this bacterium were sensitive to wortmannin (data not shown) and therefore most likely mediated via PI3K and accompanied by activation of PLCγ2.We now demonstrate that the tyrosine phosphorylation of PLCγ2 induced by zymosan is dependent on SFK, as shown by its sensitivity to the inhibitors PP2 and SKI-1. PLCγ is a possible substrate for Src  and the extracts . These rextracts , the effextracts  for revi2+ levels and Btk activation results in a rise in the level of IP3 and depletion of intracellular calcium stores [2 synthesis which may affect both Ca2+-signaling and PI3K activity [2, and upon activation generate local PtdIns4,5P2 synthesis [2 is a substrate not only for PI3K but also for PLCγ2 and increased synthesis may well be necessary to provide substrate for PLCγ2 and also be of importance for optimal generation of PtdIns3,4,5P3 [via the generation or further metabolism of PtdIns4,5P2 and the cellular Ca2+-homeostasis.We also found that inhibition of Btk did not affect the zymosan-induced tyrosine phosphorylation of PLCγ2 in macrophages. Most studies on Btk have been carried out in B cells, while information about the role of Btk in macrophage signaling is scarce. Btk activation in B-cells is known to affect both PI3K and Cam stores . Furtheractivity . Btk canynthesis . PtdIns4s3,4,5P3 . A diffes3,4,5P3 ,28. In vS. aureus or yeast-derived zymosan beads was found to depend on SFK members, in part with agonist-specific differences, as well as the Tec kinase Btk. While Src kinases were shown to act upstream of Btk, PI3K, PLCγ2 and the MAP kinases ERK and p38, Btk was not involved in the activation of ERK and p38. An attempt to summarise the findings is provided  declare that they have no competing interests.SO participated in the design and execution of all experiments and helped to draft the manuscript. RS participated in the design of the study and in the preparation of the manuscript. Both authors read and approved the final manuscript.


S. pneumoniae through signal transduction. This study was undertaken to evaluate the relation between the structure and cytotoxic activity of Pep27 and its analogues on cancer cells.A secreted peptide Pep27 initiates the cell death program in 2R→W), (4E→W), (11S→W) and (13Q→W) in native Pep27, exhibited greater hydrophobicity and anticancer activity than Pep27 and other analogues. The IC50 values of Pep27anal2 were approximately 10 – 30 μM in a number of cell lines . Confocal microscopy showed that Pep27anal2-FITC was localized in the plasma membrane, and then moving from the membrane to subcellular compartments with the initiation of membrane blebbing. Flow cytometric analysis using propidium iodide and Annexin V also revealed that Pep27anal2 induced apoptosis with minor membrane damage. Electron microscopy revealed that Pep27 induced apoptosis in Jurkat cells. The anticancer activity of Pep27anal2 was neither abrogated by pan-caspase inhibitor (Z-VAD-fmk) nor related to cytochrome c release from mitochondria. The 3D solution structures of these two Pep27 peptides revealed that both form a random coil conformation in water; however, they adopted stable α-helical conformations in solutions.Pep27anal2 characterized substituting (The results indicate that Pep27anal2 can penetrate the plasma membrane, and then induce apoptosis in both caspase-and cytochrome c-independent manner. The hydrophobicity of Pep27anal2 appears to play an important role in membrane permeabilization and/or anticancer properties. The structure-functional relationships of these peptides are also discussed. It is proposed that Pep27anal2 is a potential candidate for anticancer therapeutic agents. Newly discovered peptides are becoming increasingly important, not only as molecular tools for the understanding of protein-protein interactions, but also as lead compounds. Cationic amphipathic peptides, such as cecropins, defensins, and mellitin, induce cell death in prokaryotic and eukaryotic cells by increasing membrane permeability . This inS. pneumoniae through signal transduction triggered via the two-component system (VncR/S) consisting of a membrane-bound histidine protein kinase  and a cytoplasmic effector termed a response regulator   assay, as described by [50 values were determined directly from semilogarithmic dose-response curves. Experiments were carried out at least in duplicate.The ribed by . IC50 vaPeptide hemolytic activities were measured as described by , by deteFITC was freshly dissolved in DMSO to 10 mg/ml, and added to 1 mg/ml of peptide in 100 mM sodium bicarbonate, pH 9.3 to a final concentration of 1 mg/ml FITC. After incubation for 4 hr in the dark at room temperature, 1 M ethanolamine was added to inactivate the residual FITC. The solution was then left in the dark for an additional 2 hr, and chromatographed sequentially on a gel-filtration column followed by a 120 cm Sephadex G-50 column to remove unconjugated dye. The FITC-labeled peptide  was obtained by reverse phase HPLC using a C18 column. Conjugation between FITC and the peptide of interest was confirmed by SDS-PAGE gel as intense peptide fluorescence under UV light.Jurkat cells grown on glass coverslips overnight were treated with FITC-labeled Pep27anal2 at a concentration of 5 μg/ml. The cells were washed with PBS at 30, 60 and 120 min being treated of Pep27anal2, and then fixed using 3.7% formaldehyde for 15 min. Coverslips were mounted on glass slides, FITC was excited using a 488-nm laser, and images were manipulated using the manufacture's software.5/ml) were exposed to etoposide (100 μM), mellitin (12.5 μM) and Pep27anal2 (12.5 μM and 62.5 μM) for 4 hr. Cells were washed with PBS, resuspended in 400 μl of binding buffer , and incubated with 10 μl of annexin V-FITC  for 10 min at room temperature. After incubation, cells were washed with binding buffer and exposed to 1 μg of PI in a final volume of 400 μl. FITC and PI fluorescences were determined using a flow cytometer Annexin V binding was assessed by flow cytometry, and cell staining was evaluated using FITC-labeled annexin V (green fluorescence), and propidium iodide (PI) (negative for red fluorescence) simultaneously. This test was able to discriminate between intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+) . Jurkat cells  for 2 hrs and then fixed in 1% buffered osmium tetroxide for 2 hrs at 4°C. After processing through a graded series of ethanol and propylene oxide, the cells were embedded in Epon. A semi-thin (1 μm) section was cut and stained with toluidine blue for the ultrastructural examination. Ultra-thin (60–90 nm) sections were also prepared from the Epon block above using an ultramicrotome , stained with uranyl acetate and lead citrate, and examined under a Hitachi H-7600 electron microscope . Apoptosis detection was done by taking photographs  of the entire field of each ultra-thin sectioned sample. The degree of apoptosis (apoptosis index) was defined as the number of apoptotic cells expressed as a percentage of the total cell count.2, 1 mM dithiothreitol and 0.1 mM phenylmethylsulfonyl fluoride.Cytochrome c immunoblotting was performed using a slight modification of a previously described method . Jurkat After incubation for 30 min on ice, the cells were homogenized (20 strokes), spun at 14,000 g for 15 min, supernatants were separated from the mitochondrial fraction, and both were stored at -70°C, until required for gel electrophoresis. The mitosol fraction was obtained by adding 0.1% SDS. Fifty μg of cytosolic protein extracts and the mitosol extracts were loaded into the lanes of an 15% SDS-polyacrylamide gel, separated and blotted onto a nitrocellulose membrane  at 150 mA overnight in transfer buffer . Non-specific binding was blocked by incubating the membrane in 5% non-fat milk in TBS-T  for 3 h at RT. The membrane was incubated in anti-cytochrome c monoclonal antibody (7H8.2Cl2) solution  containing 0.5% non-fat milk in TBS-T. After overnight incubation with agitation at 4°C, the membrane was washed three times with 0.5% non-fat milk in TBS-T. The secondary antibody, a horseradish peroxidase-coupled anti-mouse antibody , was incubated at a 1:1000 dilution for 2 h at RT. The membrane was then washed four times (5 min) with 5% non-fat milk in TBS-T.d3)-ethanol (TFE) mixture  at pH 7.2, which was prepared after lyophilizing samples in an aqueous solution. NMR spectra were recorded at 15°C on a Bruker DRX-500 spectrometer equipped with a triple-resonance probe and an x, y, z-shielded pulsed-field gradient coil. Two-dimensional (2D) NMR spectra were recorded in the phase-sensitive mode using time-proportional phase incrementation [t1 domain. The 2D experiments such as TOCSY was performed using a MLEV-17 spin-lock pulse sequence with a mixing time of 69.7 ms and 2D-NOESY with mixing time of 300 ms. Solvent suppression during TOCSY and NOESY experiments was achieved using a WATERGATE pulse sequence [t2 data points and 256 increments in the t1 dimension, with 32 scans per increment. Slowly exchanging amide protons were identified by lyophilization of a fully protonated sample in 50% H2O/ 50% TFE, redissolution in 50% D2O/ 50% TFE solution, and the immediate acquisition of a series of one-dimensional NOESY and 2D-NOESY spectra. 3JHNα coupling constants were measured from the 1D spectrum. NMR data were processed on a Silicon Graphics Indigo II workstation using XWINNMR  software. Proton chemical shifts were expressed relative to the methyl resonance of sodium 2, 2-dimethyl-2-silapentane-5-sulfonic acid (DSS), used as an internal standard.The peptide samples used for NMR experiments contained 1.5 mM peptide in water /2, 2, 2-trifluoro- for Windows, version 7.5 . Results were considered significant when p was <0.05.Data are expressed as means ± SEM. One-way ANOVA and the Student's The amphipathic features of α-helical antimicrobial peptides plays an important role against target cells. Furthermore, a number of parameters, including a net positive charge, α-helicity, and overall hydrophobicity have been shown to modulate the antibiotic activity of the α-helical amphipathic antimicrobial peptides. Recent investigations using amphipathic α-helical model peptides have revealed that the modulation of the hydrophilic-hydrophobic balance of α-helical antimicrobial peptides is a crucial factor in the design of peptides with potent antibiotic activity . In a prRP-HPLC has often been used to experimentally determine the quantitative hydrophobic-hydrophilic balance of amphipathic peptides. Peptides are separated by RP-HPLC according to their hydrophobic interactions with C-18 of the stationary phase. Such hydrophobic interactions can be considered comparable to the interaction between amphipathic antimicrobial peptides and the lipid bilayer of plasma membranes. In order to investigate the possibility of a correlation between peptide hydrophobicity and the biological activities on cancer cells, the hydrophobic characteristics of the peptides were investigated by comparing their retention times on RP-HPLC  and (4E→W) to form Pep27anal1 showed less activity than Pep27anal2, the additional substitution of (11S→W) and (13Q→W) in Pep27anal2 was viewed as being important for anticancer activity. On the other hand, other Pep27 analogues substituted with more than two tryptophans at different sites did not show more anticancer activity than Pep27anal2 in 5 cell lines tested. Moreover, of the Pep27 analogues produced, Pep27anal2 had the greatest hydrophobicity, based on its RP-HPLC retention time  in a time-dependent manner. Moreover, 62.5 μM Pep27anal2 induced more apoptosis than 100 μM of etoposide  were treated with Pep27anal2 at a concentration of 12.5 μM for 1 h, 2 h, and 4 h and at 62.5 μM for 4 h. After these incubations, cytochrome c in the cytosol fraction, obtained by 14,000 g centrifugation for 15 min, and in the mitosol, obtained by adding 0.1% SDS to the mitochondrial fraction, was determined by Western blotting. Pep27anal2 at 62.5 μM did not induce cytochrome c release from mitochondria into the cytosol within 4 hrs  NOE connectivities in the 2D-NOESY spectra. Figure 2O (1v/1v). The observations of continuous dNN contacts, together with the characteristic dαN and dαβ  NOEs strongly support the existence of α-helices . The NMR structures of Pep27 and Pep27anal2 were calculated using the experimental restraints derived from the 2D-NOESY spectra. A total of 50 distance geometry geometric structures served as starting structures for the dynamic simulated-annealing calculations for the peptides in TFE/H2O solution. The 20 lowest-energy structures (<SA>k) of 50 simulated annealing structures were selected for detailed structural analysis. A best-fit superposition of 20 <SA>k structures and an energy-minimized average structure  are shown in Fig. 2O (1v/1v), whereas Pep27anal2 has a more stable α-helix spanning Trp 4-Leu15 , (4E→W), (11S→W), and (13Q→W) in Pep27 anal2 plays an important role in its hydrophobicity and/or anticancer activity. In particular, the latter two substitutions contribute hydrophobicity or anticancer activity to Pep27anal2, by virtue of the lower anticancer activity of Pep27anal1, containing (2R→W) and (4E→W) substitutions. In addition, substitutions with the more hydrophobic amino acid tryptophan were not always reflected by a higher hydrophobicity or a greater anticancer activity. Pep27anal2 had greater hydrophobicity than other analogues as assessed by RP-HPLC. A positive linear correlation was observed between apoprotein hydrophobicity and column retention time obtained by RP-HPLC [Drosophila antennapedia homeobox gene [In this study, we investigated the anticancer activities of Pep27 analogues. Although Pep27 did not show anticancer activity at concentrations greater than 70 μM, its analogues were found to have various cytotoxicities in a number of cancer cell lines. Of the five Pep27 analogue peptides synthesized, Pep27anal2 showed both greatest hydrophobicity and anticancer activity. Our results suggest that substitutions of -ethanol; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; SDS, sodium dodesyl sulfateRP, reverse phase ; MALDI, matrix-assisted laser desorption ionization; PI, propidium iodide; DSS, sodium 2, 2-dimethyl-2-silapentane-5-sulfonic acid; MTT, 3--2,5-diphenyl tetrazolium bromide; TFE, 2, 2, 2-trifluoro-(d


Mammalian reoviruses naturally infect their hosts through the enteric and respiratory tracts. During enteric infections, proteolysis of the reovirus outer capsid protein σ3 is mediated by pancreatic serine proteases. In contrast, the proteases critical for reovirus replication in the lung are unknown. Neutrophil elastase (NE) is an acid-independent, inflammatory serine protease predominantly expressed by neutrophils. In addition to its normal role in microbial defense, aberrant expression of NE has been implicated in the pathology of acute respiratory distress syndrome (ARDS). Because reovirus replication in rodent lungs causes ARDS-like symptoms and induces an infiltration of neutrophils, we investigated the capacity of NE to promote reovirus virion uncoating.trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane] and with agents that increase vesicular pH did not inhibit reovirus replication. Even when these inhibitors were used in combination, reovirus replicated to significant yields, indicating that an acid-independent non-cysteine protease was capable of mediating reovirus uncoating in U937 cell cultures. To identify the protease(s) responsible, U937 cells were treated with phorbol 12-myristate 13-acetate (PMA), an agent that induces cellular differentiation and results in decreased expression of acid-independent serine proteases, including NE and cathepsin (Cat) G. In the presence of E64, reovirus did not replicate efficiently in PMA-treated cells. To directly assess the role of NE in reovirus infection of U937 cells, we examined viral growth in the presence of N-Ala-Ala-Pro-Val chloromethylketone, a NE-specific inhibitor. Reovirus replication in the presence of E64 was significantly reduced by treatment of cells with the NE inhibitor. Incubation of virions with purified NE resulted in the generation of infectious subviron particles that did not require additional intracellular proteolysis.The human promonocyte cell line U937 expresses NE. Treatment of U937 cells with the broad-spectrum cysteine-protease inhibitor E64 [Our findings reveal that NE can facilitate reovirus infection. The fact that it does so in the presence of agents that raise vesicular pH supports a model in which the requirement for acidic pH during infection reflects the conditions required for optimal protease activity. The capacity of reovirus to exploit NE may impact viral replication in the lung and other tissues during natural infections. Reoviridae family, which also includes the pathogenic rotaviruses, coltiviruses, seadornaviruses and orbiviruses. These viruses share elements of their replication cycle as well as structural features, including a non-enveloped multi-layered capsid that surrounds a segmented dsRNA genome. In humans, mammalian reoviruses are typically associated with mild and self-limiting enteric and respiratory infections. However, studies in neonatal mice reveal that reoviruses can spread to distant tissue sites in immunocompromised hosts . The faviewed in,3.in vitro-generated intermediate subvirion particles (ISVPs) . ISVPs were prepared by treating purified virions with chymotrypsin as described elsewhere .6 each) were incubated in the presence or absence of 300 μM E-64, 5 nM LHVS, or 5 μM CA074 for the times indicated. After incubation, cells were trypsinized, collected by centrifugation at 179 × g for 10 min at 4°C and washed once in PBS. Cell pellets were resuspended in 100 μl of lysis buffer , incubated on ice for 30 min and cell debris was pelleted by centrifugation at 89 × g for 10 min at 4°C. For each sample, 20 μl of clarified cell lysate was added to 80 μl of reaction buffer  in a well of a black 96-well plate (Corning). To measure Cat B activity, 100 μM Z-Arg-Arg-7-amido-4-methylcoumarin (Z-Arg-Arg MCA)  was included in the reaction buffer. To measure Cat L and Cat B activity, 100 μM Z-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-MCA)  was added to the reaction buffer. Reactions were incubated for 30 min at room temperature with gentle tapping every 10 min. Fluorescence was measured using an FL600 microplate reader  with an excitation of 390 nm and emission at 460 nm.Cysteine protease activity was measured as described previously  with som6 U937 cells in the presence or absence of 200 μM NE inhibitor (N-(methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone) (Sigma) for the indicated times. After treatment, cells were collected by centrifugation at 179 × g for 10 min at 4°C, washed twice in PBS and lysed in TLB  for 30 m on ice. The lysate was clarified by centrifugation at 89 × g for 10 min at 4°C. For each sample, 20 μl of cell lysate was added to 80 μl of virion dialysis buffer (VDB)  containing 500 μM of NE substrate   and incubated for 30 min at room temperature with gentle tapping every 10 min. Absorbance was measured at 405 nm using an EL340 BioTek microplate reader (Bio-Tek Instruments).NE activity was determined by incubating 3 × 105 cells/vial) containing 1 ml of chilled medium. Prior to infection, some cells were pre-treated for 3 h with 300 μM E64 and/or 25 nM Baf (Sigma), 20 mM NH4Cl (Sigma) and 200 μM NE inhibitor. Inhibitors were included in the medium throughout the time course for treated samples. Time zero samples were immediately frozen at -20°C and remaining samples were incubated at 37°C until the desired time point was reached. Samples were frozen and thawed three times and titrated by plaque assay on L929 cells as described elsewhere [10(PFU/ml)t = x hr - log10 (PFU/ml)t = 0 +/- standard deviation (SD).Cells were infected (in triplicate) at the indicated MOI and adsorption was allowed to proceed for 1 h on ice at 4°C. After adsorption, cells were pelleted by low speed centrifugation and resuspended in fresh media. Virus and cells were then added to dram vials  were incubated with 25 μg/ml of purified NE  in 20 μL VDB at 37°C for the times indicated. Mock-treated samples were incubated in VDB for the longest time point. 1 mM PMSF and 200 μM of NE inhibitor were added to the samples to terminate the reactions. Protein sample buffer  was added to each reaction mixture and samples were resolved on SDS-12% polyacrylamide gels. The protein gels were stained with Coomassie Brilliant Blue.NE digestions were performed as follows. Purified virions  . Membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated anti-goat IgG (1:5000 in TBST). Bound antibody was detected by treating the nitrocellulose filters with enhanced chemiluminescence (ECL) detection reagents (Amersham) and exposing them to Full Speed Blue X-ray film .To analyze NE expression, cell lysates were generated from U937 cells, either treated or untreated for 48 h with 150 nM PMA as described for the analysis of viral protein expression. Lysate from the equivalent of 1 × 106/well in a 6-well plate 18–24 h prior to infection. Virus was allowed to adsorb to cells for 1.5 h at 4°C. At this temperature, virus binds to cells but is not internalized [4Cl. In those instances inhibitors were also included in the post-adsorption culture medium. At the indicated times p.i., cells were collected by centrifugation at 179 × g, washed twice in chilled PBS and lysed in TLB. After centrifugation at 179 × g to remove cellular debris, samples were resuspended in sample buffer. Protein samples (representing 1 × 105 cells) were analyzed by electrophoresis on SDS-12% polyacrylamide gels and transferred to nitrocellulose membranes for 2 h at 100 V in 25 mM Tris-192 mM glycine-20% methanol. Nitrocellulose membranes  were blocked overnight at 4°C in TBST  containing 5% nonfat dry milk, rinsed with TBST, and incubated with a rabbit anti-μNS polyclonal antiserum [Cells were plated at 10rnalized . After antiserum  (1:1250011) were incubated with 25 μg/ml of purified neutrophil elastase  in 40 μL of VDB at 37°C for 3 h. Reactions were terminated by adding 1 mM PMSF and 200 μM NE inhibitor to the reaction mixture. 5.0 × 1010 particles were run on SDS-12% polyacrylamide gels stained with Coomassie Brilliant Blue to confirm the removal of σ3. Viral infectivity was determined by plaque assay on L929 cell monlayers.Purified virions (1.4 × 1011) were treated with 25 μg/ml of NE in 40 μL of VDB at 37°C for the times indicated. Reactions were terminated as described above. To verify σ3 removal, the proteins from 5.0 × 1010 particles were separated on SDS-12% polyacrylamide gels and visualized with Coomassie Brilliant Blue staining. Viral infectivity for each time point was determined by plaque assay on L929 cell monolayers.Purified Lang virions (1.4 × 10The author(s) declare that they have no competing interests.J.W.G. performed all experiments and was responsible for the experimental design and data analysis. J.W.G. also wrote the initial draft of the manuscript. L.A.S. is corresponding author, participated in experimental design, data analysis and critically edited the manuscript. Both authors read and approved the final manuscript.