Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities . Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity.  Type II L-ASNases are periplasmic, have anaerobiosis-dependent expression, display high affinity vs L-ASN (in the order of μM) and low to negligible activity toward L-GLN [L-asparaginases (L-ASNase) are amidohydrolases EC 3.5.1.1) that primarily catalyze L-asparagine (L-ASN) deamination leading to the formation of L-aspartate (L-ASP) and ammonia. L-ASNases also exhibit the capability of carrying out L-glutamine (L-GLN) hydrolysis, although the efficiency toward this substrate greatly depends on the enzyme source [.1 that prd L-GLN .Echerichia coli and Erwinia chrysantemi type II L-ASNases  have been used in the treatment of acute lymphoblastic leukaemia (ALL), above all in childhood [For over 4 decades hildhood , 4, 5, 6hildhood , 8. Systhildhood .Among the cytotoxic agents employed in regimens for acute lymphoblastic leukemia (ALL), L-ASNase II is one of the most effective and with relatively mild side effects . NeverthHelicobacter pylori CCUG 17874 type II L-asparaginase (HpASNase) displays a strong preference for L-ASN over L-GLN, good heat stability, and, most of all, high cytotoxicity [0.5 toward L-GLN exhibited by HpASNase with respect to the enzymes used as drugs from E. coli and E. chrisantemi could help to reduce the side effect manifestations linked to excessive L-GLN depletion in blood, especially when L-GLNase activity is not essential [E. coli type I and HpASNase, which exhibit cooperativity vs L-ASN [toxicity , 21. Intssential . In genessential , 23, E. Bacterial L-asparaginases are 140–150 kDa homotetramers more accurately described as dimers of intimate dimers, each defined by A/C and B/D monomeric interactions. Each monomer is organized into two well distinct domains, a large N-terminal domain and a smaller C-terminal domain, connected by a linker region . Four inE. coli type II L-ASNase), and the formation of an acylenzyme as intermediate. Moreover, it implies the motion of the so-called flexible loop (EcAII residues 13–40) for correct arrangement of the active site and substrate allocation [The precise reaction mechanism of bacterial L-ASNses is still unknown, although it has been compared to the two-step ping-pong mechanism of classic serine proteases , with thlocation , 25, 26.In order to explore the role of specific residues in the shaping of the catalytic site, site-directed mutants were expressed in recombinant form and characterized from the biochemical point of view. The most relevant ones were also tested for cell cytotoxicity and a serendipitous, double mutant was helpful in defining a critical region able to modulate GLNase activity.H. pylori L-ASNase  and substrates binding and stabilization mutants , were planned  which d planned .H. pylori CCUG 17874 L-ASNase, the pDC1 plasmid, a derivative of pET101 D-TOPO (Invitrogen) containing the ansB wild type gene [In order to obtain enzymatic variants of ype gene  was subjE. coli BL21(DE3) pLysS cells transformed with pDC1 derivatives were grown in a ZYP-5052 medium [The sense and antisense mutagenic oligonucleotides used are reported in 2 medium  containi2 medium .-1 with a spectral band of 2 nm and a sensitivity of 20 millidegrees. Measurements were performed in 10 mM Na-phosphate pH 7.5 at 25°C. The protein concentration was 5 μM. The Dichroweb platform (http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) was used for data analysis obtained from at least 10 different scans.Far-UV circular dichroism (CD) measurements were carried out using a Jasco J-700 spectropolarimeter  with a 1 mm path cell. Scans were performed between 190 and 250 nm at a speed of 20 nm minBoth L-ASNase and L-GLNase activities were determined at 37°C with L-ASN or L-GLN, respectively, as a substrate, by the L-glutamic dehydrogenase-coupled spectrophotometric assay , in the max and Km were determined by Lineweaver-Burk plot, while S0.5 and nH values were determined by Hill plot using, in all cases, the Enzyme Kinetic Module 1.1 for Sigma Plot (SPSS Inc.).For steady-state kinetic studies, enzyme activity was assayed in triplicate under the above conditions using at least 10 different L-ASN or L-GLN concentrations (0–10 mM L-ASN or 0–400 mM L-GLN). The reaction was started adding the substrate to the reaction mixture containing 5–15 μg enzyme. The kinetic parameters Vcat, or turn-over number, is the number of catalytic events per second. Km is the substrate concentration at which the reaction rate is half maximal. S0.5 is the Km equivalent value for a cooperative enzyme. kcat/Km is an index of enzyme catalytic efficiency at sub-saturating substrate concentrations. Hill coefficient (nH) is an empirical parameter related to the degree of cooperativity; values larger than unity indicate positive cooperativity among ligand binding sites.K50 is the temperature at which an enzyme loses 50% of its activity in a given period of time. To evaluate the T50 of HpASNase variants, enzyme solutions (0.5 mg ml-1) were incubated at different temperatures for 10 min in 50 mM Na-phosphate, pH 7.5. After incubation, the enzyme solutions were chilled on ice and assayed for activity. The residual activity was expressed as the percentage of activity of heat-treated enzyme with respect to the untreated one.T-1 penicillin and 100 μg ml-1 streptomycin and added with 10% FBS and 2 mM L-GLN in a 5% CO2 humidified atmosphere. For the viability tests, 50000 cells/well were seeded in a 96-well plate and incubated at 37°C for 24 h in the presence of different concentrations (0.03–3.3- U ml-1) of purified recombinant H. pylori wild type, T16D, Q63E and M121C/T169M L-ASNase variants in phosphate buffered saline (Sigma Aldrich). As for the wild type and Q63E variants, the viability vs L-GLNase activity (0.03–3.8 U ml-1) was also determined. Each treatment was performed at least three times in different days and in triplicate wells for each condition.Human promyeloblast cells (HL-60) were grown at 37°C in RPMI-1640 medium containing 100 IU mlvs non treated sample and the values obtained at different enzyme concentrations were compared by ANOVA and Tukey test. A p-value<0.05 was considered significant. For the dose-response analysis as EC50 value, the data were plotted using the four parameter logistic function. The EC50 value indicates L-ASNase concentration inducing 50% of the maximal cytotoxic effect.Cell survival was determined by 3--2,5-diphenyltetrazolium bromide (MTT) reduction assay. The viability value was expressed as a percentage of living cells ansB gene by site-direct mutagenesis. In the case of the insert planned to encode the M121C mutant, an additional PCR-derived mutation at nucleotide position 506 C>T lead to a T169M substitution in the protein molecule. After a preliminary biochemical characterization of the resulting M121C/T169M enzyme, the T169M and M121C variants were also generated.Four mutants  of HpASNase were initially designed and generated starting from the cloned E. coli BL21(DE3) pLysS cells and purified to homogeneity  were expressed in ogeneity  followinThe mutants were subjected to kinetic analysis using either L-ASN or L-GLN as a substrate. At least ten different concentrations of L-ASN or L-GLN were used for each kinetics .All of them, except T16D and T95E, resulted to be active toward L-ASN, although to a different extent . Circulavs. L-ASN of the wild type enzyme; the M121C, M121C/T169M and T169M mutants showed a slightly cooperative kinetic toward L-ASN .Only the Q63E mutant preserved the hyperbolic kinetic cat, nearly 70% with respect to that of the wild type) and its catalytic efficiency was even comparable to that of the wild type enzyme , owing to a slightly higher affinity toward L-ASN (S0.5 for M121C/T169M: 0.23 vs wt: 0.29 mM). The M121C and T169M mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme), but the Q63E mutant, in spite of its 3-fold lower catalytic rate (kcat for Q63E: 6.53 sec-1vs wild type: 19.26 sec-1), displayed a catalytic efficiency superimposable to that of the wild type enzyme, thanks to its 3-fold higher affinity (S0.5 for Q63E: 0.10 mM vs wild type: 0.29 mM).The double mutant was the less affected in catalytic activity . This high thermostability would be helpful for both industrial and therapeutic purposes.Protein stability was evaluated incubating the variants in a wide range of temperatures (40–70°C) within 10 min and calculating Tmostable  with T50e enzyme . Particu-1) both toward L-ASN and L-GLN. In the case of the inactive T16D mutant, the amount of protein as mg ml-1 was considered. Mutants M121C and T169M were not tested on cells because they showed no GLNase activity and half the ASNase catalytic efficiency of the wild-type and were therefore considered not very informative.The cytotoxic effect of variable concentrations of the wild type, T16D, Q63E and M121C/T169M L-asparaginases on the human promyeloid HL-60 cells is reported in -1 (-1) of ASNase was used  and substrates binding and stabilization mutants  .The T16D and T95E replacements, which have never been experimented before in ASNase, were performed to verify the role of HpASNase threonines in substrate binding and catalysis. L-ASNases carry out a ping-pong like catalysis mechanism that involves transitory and sequential nucleophilic attacks resolved in the release of ammonia and aspartic or glutamic acid. The two threonines of the triads (Thr12 and Thr89 in EcAII and the corresponding Thr16 and Thr95 in HpASNase) and several water molecules are reported to be involved in this mechanism .Crystallographic data show that Thr16 acquires hydrogen bonds through its OG1 atom, exclusively from elements of the side chain of the substrate , while the Thr95 OG1 interacts also with the O of the carboxylic group in α of the main chain . We repl3+ of the substrate (L-ASN or L-GLN). Its replacement with a Glu leads to the introduction of a carboxylic group at position 63 of the polypeptide chain, resulting in a locally increased negative charge, which should allow a better interaction with L-ASN as a substrate. In fact, the characterization of the Q63E mutant confirmed the residue role in both substrates binding and catalysis. The variant had a catalytic efficiency (kcat/S0.5: 65.3 s-1 mM-1) vs L-ASN similar to the wt enzyme , with a reduction of catalytic activity (nearly 3-fold), but increased affinity (nearly 3-fold), and halved glutaminase efficiency (kcat/S0.5: 0.19 vs 0.47 s-1 mM-1) due to the reduction of both catalytic activity and affinity. Several mutants on the corresponding EcAII residue (Q59) have been characterized to have perturbed ASNase and GLNase activities [Regarding the second group of mutants, amino acid replacement concerned the Gln63 residue that, with Asn255 and Glu289, is located around the active site and is involved in substrate stabilization and in modulating the accessibility of the binding pocket [tivities , 19.vs L-ASN unchanged.In order to discuss the M121C mutant, we need to consider that, beside the flexible loop of the active site (HpASNase residues 19–46), at least two other highly conserved loops play a key role in active site stabilization: a left-handed crossover β4-β5 loop (HpASNase residues 119–153) and the active site forming β13–14 loop (HpASNase residues 286–297) . ResidueDuring the mutagenesis experiments to produce the M121C mutant, an unwanted, additional random mutation was introduced, leading to the formation of the M121C/T169M mutant, namely a double mutant. Very little is known about the Thr169 residue, though the corresponding EcAII residue (Thr163) is reported to be involved in intersubunits interactions , 23. To vs L-ASN, underlining alterations within enzyme subunits interactions.Replacements of either Met121, or Thr169, or both (Met121/Thr169) killed the enzyme GLNase activity leaving the affinity toward L-ASN unchanged. Interestingly, in all the three cases mutants displayed positive cooperativity Both mutants carrying each single mutation (M121C or T169M) had a reduced ASNase activity, but the double mutant had an activity quite similar to the wild type enzyme. Its preserved activity toward L-ASN most likely depends on a complementary action of the two mutations affecting residues located in two critical regions of the enzyme structure: residue Met121, near the active site region, and Thr169, in the intersubunit one . The disThe cytotoxic effect of the T16D, Q63E and M121C/T169M mutants was assayed on HL-60 cells. The inactive T16D mutant did not exert any cytotoxic effect, proving that the anti-proliferative efficacy of HpASNase is related only to its enzymatic activity and is independent from unknown features of the protein or from the recombinant nature of the enzyme carrying the N-terminal His-tag. The double mutant, completely devoid of GLNase activity, had a very low cytotoxic effect that was not dependent on the enzyme dose, an effect confirmed by Trypan blue exclusion in slightly different conditions .cat/S0.5vs L-GLN, wt: 0.47 and Q63E, 0.19). In fact, mathematical modelling by COPASI of the relative GLNase activity for the wild type and Q63E mutant, respectively, at similar ASNase units , and in a reduction (Q63E) or complete removal of the glutaminolytic activity, indicating that co-participation of other structural elements besides the triads elements is relevant for the catalytic cycle.The production of ASNase variants with reduced or null glutaminolytic activity has allowed to verify the importance of GLNase activity in the cytotoxicity exterted on HL-60 cells, confirming that modulation of the GLNase kinetic parameters of ASNase is a relevant issue to consider in order to optimize the outcome of its therapeutic applications.S1 Fighttp://espript.ibcp.fr). Robert, X. and Gouet, P. (2014) “Deciphering key features in protein structures with the new ENDscript server”. Nucl. Acids Res. 42(W1), W320-W324. doi: 10.1093/nar/gku316 (freely accessible online).The amino acids sequences derived from each mutant nucleotide sequencing are aligned to the one of the wild type enzyme. The wild type derived secondary structure is as well reported. α-helices, 3 -helices and π-helices are displayed as medium, small and large squiggles respectively. β-strands are rendered as arrows, strict β-turns as TT letters and strict α-turns as TTT. (ESPript -(TIF)Click here for additional data file.


Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen,  Helicobacter pylori (H. pylori) have been associated with several gastric disorders ranging from chronic gastritis and ulceration to lymphoma of the mucosa-associated lymphoid tissue (MALT) system and gastric cancer1H. pylori persistently colonises the epithelial lining of the human stomach, which represents an important barrier against toxins and pathogens. The organisation and maintenance of the gastric epithelium as a protective barrier require functional adherens junctions with the classical cadherin Cdh1 (E-cadherin) as a key molecule. Structurally, E-cadherin consists of five extracellular repeats (EC1-EC5), a single transmembrane domain (TMD) and an intracellular domain (IC). E-cadherin mediates intercellular adhesions between epithelial cells through dynamic, calcium-dependent, homophilic cis- and trans-interactions of its ectodomains46Chronic infections of humans by the pathogen and class-I carcinogen ts EC1-EC, a singlH. pylori-associated metastatic gastric cancer (GC) is the leading cause of cancer-related deaths worldwide because effective treatment strategies are missing. Both the diffuse and intestinal GC types have been correlated with an altered functionality of the E-cadherin complex. The most prominent mechanisms in the gastric system include (i) loss-of-function mutation of the cdh1 gene91116171820H. pylori induces strong E-cadherin shedding, which leads to the disruption of adherens junctionsH. pylori-activated ADAM-10  has been suggested to be responsible for the cleaving of E-cadherin on host cellsH. pylori-mediated E-cadherin shedding, while a major contribution of H. pylori HtrA was detectedCampylobacter jejuni (C. jejuni), enteropathogenic Escherichia coli (EPEC) and Shigella flexneri of the gastrointestinal tract2025Serum levels of soluble E-cadherin are increased in the intestinal GC-type and might serve as a biological markerE. coli expressing the three HtrA homologues DegP, DegQ and DegS. The main function of bacterial HtrA is protein quality control and degradation of misfolded proteins in the periplasm, enhancing bacterial survival under stress conditions. In fact, H. pylori HtrA is highly active under extreme conditionshtrA gene is essential and cannot be deleted or mutagenised in the H. pylori genome2128H. pylori HtrA directly affects the integrity of the gastric epithelium via E-cadherin ectodomain shedding implies that truncated E-cadherin could also interfere with H. pylori-induced signalling in GC.HtrA has already been intensively investigated in 585↓S↓D587  in the EC4 domain as a target site700↓V701 was proposed as an MMP cleavage siteH. pylori HtrA targets the same sites or prefers another amino acid sequence is unknown, but this information is crucially important for understanding the HtrA-E-cadherin interaction and developing HtrA inhibitors.Previous studies investigating MMP-mediated E-cadherin cleavage and Edman sequencing of E-cadherin fragments have suggested the residue pattern LH. pylori transmigration across a polarised monolayer.In the present study, we investigated the molecular mechanism of the HtrA-E-cadherin interaction. We discovered that E-cadherin signature motifs function as target sites for the proteolytic degradation by HtrA, leading to a defined fragmentation pattern of E-cadherin. As HtrA-mediated E-cadherin shedding may possess cancer-promoting properties, we used the identified signature sites to design the first substrate-based peptide inhibitor to impede HtrA-mediated E-cadherin cleavage and 155–D882). Several antibodies are available that selectively recognise individual domains of the E-cadherin protein. SHE78-7 detects the EC1 domain, the HecD1 antibody recognises the EC2 domain and H108 binds to the EC5 domain19H. pylori for the indicated time periods and aliquots of the supernatants were analysed for E-cadherin fragments containing the EC1, EC2 or EC5 domain. After 16 h of infection, the anti-EC1 antibody detected a strong increase in the 90 kDa NTF fragment of E-cadherin (hCdh1NTF). We observed a similar picture for the anti-EC2 antibody. When the supernatant was analysed with the anti-EC5 antibody, the hCdh1NTF fragment enriched during H. pylori exposure was already visible after 4 h of H. pylori exposure and the levels increased during the 16 h of infection  in lysates of H. pylori-infected NCI-N87 cells . rCdh1 contains the five EC domains and the linker region (A698–I707), but lacks the TMD. Instead of the IC domain, rCdh1 is fused to the Fc region and a His6 tag (155–I707). Incubation of rCdh1 with recombinant H. pylori HtrA (rHtrA) for the indicated time periods showed a different cleavage pattern compared to the H. pylori infection experiments. Using the anti-EC1 antibody, we only detected the loss of the full-length E-cadherin protein (rCdh1FL)  . Even afrCdh1FL) . The antrCdh1FL) . A ladderCdh1FL) . SimilarrCdh1FL) , indicatrCdh1FL) . Accordiragments .↓T, A↓K, V↓A, I↓T, A↓G) match the proteomic cleavage site pattern [VITA]↓[VITA]-x-x-D-[DN], which are located within the four calcium-binding sites and which we have named “signature sites” S1, S2, S3 and S4 , V (28%), T (17%), A (9%) and S (9%). Sequence analysis revealed that HtrA cleavage takes place favourably between hydrophobic amino acids . We also3 and S4 . The sightrA gene in the H. pylori genome is not possible, we aimed at the generation of a peptide-based E-cadherin cleavage inhibitor. We synthesised five peptides (P1−P5) around the cleavage signature sequence between E-cadherin domains EC4 and EC5 (positions L583–P593) (in vitro HtrA-mediated E-cadherin cleavage (mut) or N-terminal (P1_NTmut) regions with glycine  to SPR analysis. Although both peptides exhibited concentration-dependent binding  . The 21-83–P593)  effectivcleavage . Shortercleavage  did not cleavage . The dircleavage . N-termicleavage . None ofcleavage . We thencine see . Both P1vage see . Therefoding see , the P1-ding see . This reH. pylori-infected AGS cells that were transfected with hCdh1FL. We observed a significant dose-dependent decrease in the formation of the hCdh1NTF . We then analysed the epithelial transmigration of H. pylori in a transwell filter assay. Partially polarised MKN-28 cell monolayers were infected and the transmigration of E-cadherin-cleaving H. pylori was determined over time. Polarisation of MKN-28 cells requires functional tight junctions, which can be monitored by the measurement of the transepithelial electrical resistance (TEER). Corresponding to previous studies2125332 after 14 days of cultivation  has been suggested as a protease responsible for the cleavage of E-cadherin on host cellsH. pylori induces the expression of several MMPs, including MMP-3 and MMP-7, which can also contribute to E-cadherin shedding21H. pyloriH. pylori proteases is still rare and no additional H. pylori protease has yet been identified as an E-cadherin-cleaving protease. H. pylori expresses an active collagenase (Hp0169), which is crucially important for in vivo colonisationH. pylori proteases with an extracellular localisation39E-cadherin is an important cell adhesion molecule and tumour suppressor. Ectodomain shedding and an increase in soluble E-cadherin is associated with several cancer types, such as breast cancer, pancreatic cancer and GC585↓S↓D587 in the EC4 domain and P700↓V701 have been suggested as putative cleavage sites17H. pylori HtrA, which is in line with the L585↓S↓D587 cleavage site for MMPsH. pylori HtrA and possibly for H. pylori-regulated MMPs might explain why we observed different fragmentation patterns in H. pylori infections and in vitro  can interact with the IC of E-cadherin, interfering with its tumour-suppressing function47H. pylori-dependent carcinogenesis. In conclusion, our results provide an inspiring novel entry point for innovative anticancer drug discovery.E-cadherin could play a central role in H. pylori wild-type strains P12Hp26695H. pylori at a MOI of 100. Adherence of H. pylori was quantitated in a cfu (colony-forming unit) assay. Briefly, bacteria were suspended in culture medium, added to the cells at a MOI of 100 and co-incubated with host cells for 16 h. The number of cfu was determined by growth on agar plates for 72 h. To investigate the influence of the P1 inhibitor peptide on bacterial transmigration, transwell infections were performed in the presence or absence of 100, 200 or 500 μM peptide. The human gastric adenocarcinoma cell line MKN-28 was grown to confluence on transwell filters and polarised for 14 days. Establishment of functional tight epithelial cell monolayers was further confirmed by measuring the transepithelial electrical resistance (TEER). Infection of these cells was carried out with bacteria at a MOI of 50 for the indicated periods of time per experiment. The number of transmigrated bacteria was determined as cfu by taking aliquots from the basal chambers21The human gastric adenocarcinoma cell line AGS is E-cadherin-negative and was derived from an adenocarcinoma of the stomach of a 54 year-old Caucasian female . AGS cells expressing hCdh1 were generated by transfection using a plasmid containing E-cadherin wild-type cDNA202123246-tagged proteins were analysed using an anti-His5-antibody (Qiagen). GAPDH  and β-actin (Sigma) were detected as loading controls. Where indicated, signals of protein bands were quantified using the ImageLab software (BioRad). Graphical presentation and statistical evaluations (Student’s t-test) were performed with GraphPad Prism 5 and p-values of ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05 were considered statistically significant.Proteins were separated by SDS PAGE and analysed by Western blot using specific antibodies against HtrAH. pylori HtrA and in vitro cleavage assays have been described elsewhere21Purification of recombinant 6  in 50 mM HEPES buffer , 0.5 mM MgCl2 (Sigma-Aldrich Chemie GmbH) and 1 mM CaCl2 . Sample 2 contained HtrA . Sample 3 included HtrA and human recombinant E-cadherin Fc His6 at a ratio of 1:10 . All samples were incubated for 12 h at 37 °C in a horizontal shaker. Samples 1 and 2 were applied in sextuplicate, and sample 3 was done in triplicate. Finally, 2 μl of sequencing-grade modified porcine trypsin was added . Protease digestion was carried out overnight at 37 °C in a horizontal shaker. After tryptic digestion, Samples 2 and 1 were mixed at a ratio of 1:10 to obtain Sample 4. Peptides were desalted, purified and concentrated using C18 microcolumns OMIX tips (Agilent Technologies). After lyophilisation, peptides were stored at –20 °C.Tryptic digestion was performed on three different samples (sample volume: 200 μl). Sample 1 contained human recombinant E-cadherin Fc His9]-bradykinin, neurotensin, angiotensin I and adrenocorticotropic hormone fragment 1–17; all from Sigma), 0.1% TFA and 70% acetonitrile in water and deposed on a blank MALDI target plate (416 spots per sample) using an online SunCollect system (SunChrom). The final concentration of each internal peptide standard was 50 fmol per spot.Peptides were separated by reverse-phase high-performance liquid chromatography using an EASY-nLC system  with mobile phase A: 0.1% trifluoroacetic acid (TFA) in water; mobile phase B: 0.1% TFA in acetonitrile; flow rate of 300 nl/min. Lyophilised peptides were dissolved in 23 μl of buffer A, of which 18 μl were loaded onto the column . The peptides were eluted with a gradient of 5–33% B for 62 min, 33–48% B for 15 min, 48–100% B for 2 min and 100% B for 10 min. The column was then equilibrated with 5% B for 20 min before the next sample was analysed. Eluting fractions were mixed with a solution of 3 mg/ml α-cyano-4-hydroxycinnamic acid, 187.5 pmol/ml of each of the four internal standard peptides ( and 50 mM N-hydroxysuccinimide [NHS]). The SPR sensor surface was loaded with 100 μl protein and subsequently treated by injecting 192 μl of 1 M ethanolamine at pH 8.5 for inactivation of excess coupling groups. HBS-P  served as running and sample buffer for peptide measurements. Peptide samples were directly dissolved in HBS-P buffer. The experiments with the non-peptidic compounds were performed utilising HBS-PD running buffer .155–I707; UniProt ID: P12830) with ClustalW 57www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html). The best model had 434 (90%) residues in the most favoured regions, with only one problematic residue (V310). We did not perform subsequent molecular dynamics relaxation because we only used the model for visual inspection of H. pylori HtrA cleavage sites.We generated a preliminary protein “homology” model using a mouse E-cadherin crystal structure as template . We used the software Modeller 9.9 after aligning the template with the human E-cadherin sequence .


Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease  and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R2N2 stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6–7 nucleotides) downstream of the asymmetric recognition sequence 5′-GCCGC-3′. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.The stress-sensitive restriction-modification (RM) system CglI from  Restriction-modification (RM) systems protect bacterial cells against invasion by foreign DNA (e.g. phage or plasmid DNA). RM systems are composed of two principal enzymatic activities: an endonuclease and a methyltransferase. Restriction endonucleases (REases) cleave DNA that do not contain characteristic modifications (methyl groups) at defined sites within a specific recognition sequence. The host DNA is protected against nucleolytic attack because it is methylated within the recognition sequences by the cognate methyltransferase activity of the RM system. According to the genetic organization, subunit composition, cofactor requirement and mode of action, the RM systems were subdivided into four types: Type I, II, III or IV . Here weCorynebacterium glutamicum (ATCC 13032) is inactivated under various stress conditions , enabling the transfer of plasmid DNA from a donor (e.g. Escherichia coli) by either conjugation or phage infection  (Neisseria gonorrhoeae FA1090 (ATCC 700825) was also assigned as a stress-sensitive restriction system (BLAST), and correspondingly is composed of three genes homologous to the CglI RM system: M.NgoAVII, R.NgoAVII and N.NgoAVII, respectively  . Howeverneb.com) . The Cglneb.com) . The Ngop-nitrophenyl) phosphate, in contrast to BfiI ; a domain similar to the Z1-superfamily; and a distinct C-terminal domain . Similar DEAD domains are found in SF2 helicases involved in ATP-dependent RNA or DNA unwinding/translocation  and are Type II 5-methylcytosine methyltransferases that recognize the 5′-GCSGC-3′ DNA sequence (where S represents either G or C)  ,17. LikeE. coli, we purified individual proteins and their domains to apparent homogeneity. Using co-expression of R.CglI and N.CglI, the RN.CglI complex with an R2N2 stoichiometry was also isolated. We demonstrate that both R.CglI and R.NgoAVII are endonucleases that require ATP hydrolysis by the corresponding N-proteins to produce site-specific DNA cleavage at fixed positions downstream of the asymmetric recognition sequence 5’-GCCGC-3’. On the basis of their activities, we propose that CglI and NgoAVII represent a new sub-class of RM enzyme with characteristics of both Type I and II RM systems.In this paper, we report a biochemical characterization of the putative REases R.CglI and R.NgoAVII  and cognate ATPases N.CglI and N.NgoAVII . Following recombinant gene expression in Corynebacterium glutamicum ATCC 13032 and Neisseria gonorrhoeae FA1090 genomic DNA (both from LGC Standards) were used to produce recombinant genes of R.CglI/N.CglI, R.NgoAVII/N.NgoAVII or DNA fragments corresponding to individual domains that later were ligated into expression and co-expression vectors (Supplementary Tables S1 and S2). Complete gene sequences were confirmed. His6- or StrepII-tagged proteins were expressed and purified using liquid chromatography as described in Supplementary Tables S1 and S2. The homogeneity of the protein preparations (>90%) was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel densitometry. The identity of the purified proteins was confirmed by mass spectrometry (data not shown). Protein concentrations were determined by measuring absorbance at 280 nm using appropriate extinction coefficient (Supplementary Tables S1 and S2).The R.CglI (H105A), N.CglI (D158A+E159A), R.NgoAVII (H104A) and N.NgoAVII (D150A+E151A) mutants were produced by QuikChange Site-Directed Mutagenesis . SequencGel filtration was carried out at room temperature on an AKTA Avant system using a Superdex 200 HR column (GE) pre-equilibrated with 20 mM Tris–HCl (pH 8.0 at 25°C), 300 mM NaCl and 5 mM 2-mercaptoethanol. Protein samples at 20 μM (monomer) loading concentration were prepared in 50 μl of the above buffer. Elution from the column was monitored by measuring absorbance at 280 nm. The apparent molecular weights of proteins were evaluated from the elution volume using a series of standards .g for 15–20 min.The wild-type (wt) RN.CglI complex was dialyzed against 20 mM Tris–HCl (pH 8.0 at 25°C), 400 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT). R.CglI, R.NgoAVII, N.CglI and N.NgoAVII were transferred into the same buffer using desalting NAP columns . Proteins were concentrated using centrifugal concentrators . All samples were processed at 15 000 s (s = 4π sin(θ)/λ, where 2θ is a scattering angle and λ is x-ray wavelength, 0.124 nm) range from 0.07579 to 4.66533 nm−1, except the R.CglI samples, which were measured in s range from 0.02906420 to 4.491070 nm−1. Data were truncated at the first point of the Guinier region, as calculated by AUTORG (Rg and I(0) were calculated using GNOM  with subtracted buffer scattering, Kratky plots and distance distribution functions P(r) of protein samples are presented in Supplementary Figure S4. Structural parameters derived from the SAXS measurements are presented in Supplementary Table S3.Small angle X-ray scattering (SAXS) data were collected at P12 EMBL beam-line at PETRAIII storage ring of the DESY synchrotron in Hamburg (Germany). The sample-to-detector (Pilatus 2M) distance used in the measurements was 3.1 m. Data were collected for the momentum transfer y AUTORG . Real sp DATGNOM . Unprocehttp://www.if.sc.usp.br/∼saxs/saxsmow.html).Molecular weight estimations of individual proteins and the corresponding complexes are presented in Table GCCGCCGTGCGAGCCCATT-3′/3′-ATGTTAGCTATTATCGGCGGCACGCTCGGGTAA-5′, the recognition sequence is underlined) or a 33 bp non-cognate DNA duplex (5′-TACAATCGATAATACCAGGCGTGCGAGCCCATT-3′/3′-ATGTTAGCTATTATGGTCCGCACGCTCGGGTAA-5′). DNA  was incubated with proteins  for 15 min in 20 μl of 40 mM Tris-acetate (pH 8.3 at 25°C), 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin (BSA) and 10% (v/v) glycerol at room temperature. Free DNA and protein–DNA complexes were separated by electrophoresis using 8% (w/v) acrylamide gels . In the case of the NgoAVII proteins, EDTA in the binding buffer and loading gels was replaced by 5 mM Ca-acetate. Samples were separated at room temperature for 3 h at ∼6 V/cm. Radiolabeled DNA was detected and quantified using the Cyclone phosphorimager and the OptiQuant software (Packard Instrument). Apparent Kd values for DNA binding were determined as described in  using a 33 bp cognate duplex  and 0.01 μg/μl of various DNAs (as indicated): (i) phage λ DNA (dam+dcm+), (ii) pBR322, (iii) pBR322 methylated at the CglI/NgoAVII recognition sites, (iv) cognate fragment, (v) noncognate fragment, (vi) cognate oligoduplex , (vii) non-cognate oligoduplex (5′-GATCGACCAGGCTACG-3′/3′-CTAGCTGGTCCGATGC-5′) and (viii) single-stranded M13mp18 (Supplementary Data). Reactions were initiated by adding the CglI (40 nM) or NgoAVII (40 or 100 nM) proteins and were stopped by addition of EDTA to 30 mM. An aliquot (1 μl) was spotted onto a polyethyleneimine-cellulose thin-layer plate, the substrate ATP and product ADP separated by chromatography in a 0.325 M KH2PO4 (pH 3.5 at 25°C) and the plates visualized using Fujifilm FLA-5100 fluorescent image analyzer .ATPase reactions were performed at 37°C for 1 h in 33 mM Tris-acetate (pH 7.0 at 25°C), 0.1 mg/ml BSA, 10 mM Mg-acetate and 150 mM K-acetate (only for CglI proteins), supplemented with 50 μM [α2+, Mn2+, Ca2+ or Ni2+) for NgoAVII and CglI, respectively. Reactions were initiated by adding N.CglI or N.NgoAVII (10 nM) plus R.CglI or R.NgoAVII (200 nM). Aliquots were removed at fixed time intervals and the reactions stopped by adding EDTA to 9.5 mM. The relationship between the absorbance and phosphate concentration was established by using KH2PO4 as a standard. ATPase rates are quoted as the mean of three independent experiments.Malachite green assays (BioAssay Systems) were used to measure the ATP hydrolysis rate by detection of liberated free phosphate. Reactions were performed at 37°C in the same reaction buffer as in the radioactivity assay, and containing 0.02 μg/μl phage λ DNA, ATP , 1 mM (d)NTP and 2.5 or 10 mM divalent metal ions ; 1 mM DTT; 2 or 4 mM adenylyl-imidodiphosphate (AMP-PNP) or ADP; 2 or 4 mM (d)NTP; 25 mM phosphocreatine; and 5 U/ml creatine kinase. The reactions were supplemented with 0.01 μg/μl of various dsDNAs (as indicated): (i) phage λ DNA (dam+dcm+), (ii) pBR322, (iii) pBR322 methylated at the CglI/NgoAVII recognition sites, (iv) pUC19 or (v) 0-site plasmid (without CglI/NgoAVII recognition sequences and generated from pMDS36a by the site-directed mutagenesis (DNA cleavage reactions were performed for 1 h at 37°C in reaction buffer: 33 mM Tris-acetate (pH 7.0 at 25°C); 0.1 mg/ml BSA; either 50 mM (for NgoAVII) or 150 mM (for CglI) K-acetate; either 2.5 mM (for NgoAVII) or 10 mM (for CglI) divalent metal ions (Mgagenesis ). ReactiTo analyze the DNA cleavage position of CglI and NgoAVII, the 1-site plasmid , generated from pMDS36a by the site-directed mutagenesis , was inc6- or StrepII-tags . A set of alanine-substitution mutations of the putative active sites within the R- and N-proteins was generated: H105 (R.CglI) and H104 (R.NgoAVII) belonging to the predicted HXK active site motif of the PLD-superfamily nucleolytic domain; and D158+E159 (N.CglI), and D150+E151 (N.NgoAVII) located in the DEAD domain and involved in Mg2+ coordination . Also, both R- and N-proteins from either system were co-expressed using a single recombinant vector . All proteins were expressed in E. coli and were purified to an apparent homogeneity (>90%) by liquid chromatography. The co-expressed R.CglI (containing a StrepII-tag) and N.CglI (containing a His6-tag) formed the RN.CglI complex that was purified using serial affinity chromatographic columns (HisTrap HP and StrepTrap HP) (Supplementary Table S1). In the case of the co-expressed R.NgoAVII and N.NgoAVII proteins, a complex was not obtained (data not shown). The identity of all purified proteins was confirmed by mass spectrometry (data not shown).The R- and N-proteins and their domains were expressed with either HisThe oligomeric assembly of the individual R- and N-proteins and their domains was analyzed in solution by gel filtration Table . N.CglI,2N2 stoichiometry. In addition, the stoichiometry of the RN.CglI complex was examined using densitometric analysis of the purified complex following SDS/PAGE electrophoresis . The calculated molar ratio of the R.CglI and N.CglI proteins within the complex was 1:0.75. Since both proteins are dimers in solution, this ratio supports the R2N2 stoichiometry. The R2N2 CglI complex was also obtained by mixing the individual R.CglI and N.CglI proteins in vitro (data not shown). Higher-order complex formation was also observed upon mixing of the full-length R.CglI protein with either the N.CglI-DEAD domain or the N.CglI-DEAD-Z1 domain . In contrast, a higher-order complex was not obtained if the PLD domain or B3 domain of R.CglI were mixed with the corresponding domains of N.CglI (data not shown). As with the co-expression study, a stable complex between the R.NgoAVII and N.NgoAVII was not observed upon mixing the proteins in vitro .The molecular weight of the RN.CglI complex, obtained by co-expression of both the R.CglI and N.CglI proteins, was determined by gel filtration and SAXS to be close to the sum of molecular weights of the R.CglI and N.CglI dimers . To test this, the ATPase activity of the N-proteins was first analyzed by thin-layer chromatography using radioactive [αe Figure . The act2N2 complex is only supported by double-stranded DNA containing one or more unmodified recognition sites and with a length >16 bp.The ATPase activity of the RN-complexes was further analyzed in the presence of: various circular or linear double-stranded DNA substrates , either lacking or containing different numbers of recognition sites (unmodified or modified by M.NgoAVII where stated); or circular single-stranded M13mp18 Figures . InteresIn order to test the nucleotide triphosphate specificity of the RN-complexes, we compared the catalytic activity using either ribo- and deoxyribonucleotide cofactors  was determined with Mn2+ or Ca2+ . No significant ATPase activity was detected in reactions containing EDTA .We also studied the divalent metal ion dependence on the ATPase activity. The highest ATP hydrolysis rate was observed with Mg2+, Mn2+ and Ca2+) supported DNA cleavage, whereas DNA hydrolysis was not observed in the presence of EDTA which prevents ATP hydrolysis (data not shown). DNA cleavage was also observed in the presence of other (d)NTPs (data not shown). Thus, the catalytically active ATPases (N-proteins) and ATP hydrolysis are required for the DNA cleavage by the R-proteins.DNA cleavage by the R-proteins was investigated using linear phage λ DNA as a substrate (which contains 181 CglI/NgoAVII recognition sites) Figure  and pBR3In order to determine the cleavage position of the CglI and NgoAVII endonucleases, the DNA fragments obtained after ATP-dependent cleavage were analyzed using run-off sequencing  and NgoAVII (from Neisseria gonorrhoeae FA1090) were originally assigned as Type II RM systems. Here, we overproduced, purified and characterized in vitro the wt and mutant R- and N-proteins and their isolated domains from both systems. Our results show that, in contrast to other Type II enzymes, CglI and NgoAVII possess three enzyme activities: namely, a methyltransferase, an endonuclease and an ATPase, the last two of which are coupled  for ATPase activity and were inactive in the presence of EDTA  (2+ coordination (mutations D158A+E159A in N.CglI and D150A+E151A in N.NgoAVII) abolished ATP hydrolysis and cleavage  . Helicasure S14) . Mutatio2N2 stoichiometry could be expressed and isolated  that use ATP hydrolysis (1–1.5 ATP per bp step) for translocation on intact DNA  phosphate, as demonstrated for BfiI. However, in contrast to BfiI, the R-proteins dimers in isolation were unable to cleave cognate DNA   ,34. As oresults) . We showresults) . MutatioBoth R-proteins cleaved DNA at fixed positions downstream of the asymmetric recognition sequence 5′-GCCGC-3′: R.CglI cuts the top and bottom DNA strands 7 and 6/7, R.NgoAVII - both strands 7 nucleotides downstream of the target, respectively Figure . Thus th2N2 complex was not obtained in vitro, we suggest a similar complex is likely exist in vivo. R.CglI dimerizes through its PLD domains, to form a single nuclease active site. The B3 domains within this dimer are situated on the opposite sides of the PLD domain core and make no contacts to each other. N.CglI uses its Z1 domains for dimerization, with the DEAD domains responsible for interaction with R.CglI. On cognate DNA, an R-protein within the complex will first should bind the recognition sequence. This triggers the N-protein to hydrolyze ATP; we suggest that the ATP hydrolysis levels are consistent with translocation of the complex along DNA. DNA cleavage should proceed after translocation because the catalytically inactive R-protein stimulates the ATPase activity of the N-protein and not vice versa.According to the experimental data presented here, the R- and N-proteins act together as a heterotetrameric complex to hydrolyze ATP and thus cleave DNA. Based on the crystal structure of R.NgoAVII (see the accompanying paper) , the gel2N2 complex should contain a single nuclease active site that could produce a double-stranded DNA break through sequential strand cleavage. Instead one could speculate that two RN-complexes are required to interact and form a catalytically competent supercomplex . In this model the two interacting R-protein dimers would each cut one strand to introduce a double-stranded DNA break near to the occupied target site. Such a mechanism is reminiscent of that suggested for the Type III restriction enzymes, although communication between the sites in the Type III case is likely by diffusion and not translocation , have been biochemically characterized   . From thneb.com) . The mete MTases ,17. Alsoe MTases  .,39.http:e MTases . Taken tSupplementary Data are available at NAR Online.


IL-6 (promoter) rs1800795 and the IL-6R (receptor) rs8192284 single nucleotide polymorphisms (SNP) was studied in patients with advanced gastric cancer treated with palliative chemotherapy.IL-6 triggers oncogenic/angiogenic signals and the cytokine-dependent pro-cachexia cascade. The prognostic role of the functional One-hundred-sixty-one patients were genotyped for rs1800795 and rs8192284 SNPs using polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) analysis assay. These results were studied for association with overall survival (OS).In 161 assessable patients, frequencies of rs1800795 G/G, G/C and C/C genotypes were 46%, 42% and 12%, respectively. Frequencies of rs8192284 A/A, A/C and C/C genotypes were 36%, 45% and 19%, respectively. Carriers of the rs1800795 G/G and rs8192284 C/C genotypes showed the worst OS. In the multivariate model, rs1800795 G/G , and rs8192284 C/C  confirmed an adverse prognostic impact.In this population, genetic variants that up-regulate the IL-6 system showed impact on OS. This findings sustain the hypothesis that anti-IL-6 compounds deserve clinical studies as novel therapeutics in the palliative treatment of cancer patients. Interleukin-6 (IL-6) is a four-helical protein of 184 amino acids that belongs to a large family of pleiotropic cytokine involved in numerous functions . There iin vivo[Dysregulation of the IL-6/IL-6R system has been associated with the pathogenesis of several autoimmune and inflammatory diseases in humans, and anti-IL-6 monoclonal antibodies (moAbs) have been successfully developed for the medical treatment of chronic inflammatory diseases, like rheumatoid arthritis . Recentlin vivo-16.IL-6 gene promoter showed higher transcriptional activity in gene reporter assays [In vivo, higher IL-6 levels were determined in carriers of the common allele in studies including both healthy subjects and patients with inflammatory diseases [IL-6R  causes an Asp358Ala amino acid substitution within the extracellular cleavage domain of the IL-6R causing proteolytic cleavage of the membrane-bound IL-6R [in vivo studies, 358Ala carriers showed higher concentrations of the so-called soluble IL-6 receptor (sIL-6R), which is responsible of trans-signaling [There is compelling evidence that circulating IL-6 levels and the levels of its trans-signaling promoting receptor (sIL-6R) are genetically-driven . The sinr assays . In vivodiseases . A commoIn several reports, an up-regulated IL-6/IL6R system has shown a prognostic impact in patients with hematologic malignancies and with solid tumors . This baThe study population consisted of consecutive patients with locally advanced, relapsed of metastatic gastric cancer who were treated with palliative chemotherapy at three participating Institution in Central Italy. One-hundred-seventy-five patients were homogeneously treated with both first-line and second-line palliative chemotherapy between 1998 and 2006 . In 161 IL-6R rs8192284 A/C and the IL-6 rs1800795 G/C genotypes. Genome DNA (10 ng) was used as a template and PCR was carried out using the Diatheva 2×PCR Master Mix  with the following conditions: 95°C 10 min ; 95°C 15 sec, 60°C 30 sec, 72°C 30 sec (35 cycles). The two PCR were performed using the following primer sets: rs1800795, forward 5’-TTCCCCCTAGTTGTGTCTTGC-3’ and reverse 5’-TGGGGCTGATTGGAAACCT-3’; rs8192284 forward 5’-CCTCTTCCTCCTCTATCTTCAATTTT-3’ and reverse 5’-AATGTGGGCAGTGGTACTGAA-3’. Primer pairs were designed using the PRIMER3 program (primer3plus.com). The PCR products were run on a 2% agarose gel after digestion with Nla-III  or Hind-III  restriction enzymes. The predicted band sizes for the rs1800795 G/C genotypes after Nla-III digestion were G/G = 75 bp; G/C = 75 bp plus 50/25 bp; C/C = 50/25 bp; the predicted band sizes for the rs8192284 A/C genotypes after Hind-III digestion were A/A = 73; A/C = 73 bp plus 43/30 bp; C/C = 43/30 bp. Samples with ambiguous results were analyzed by direct sequencing using ABI PRISM 310 Genetic Analyzer .Patients’ characteristics and their outcomes were unknown to investigators performing genetic analyses. Genomic DNA extraction using the QiaAmp kit . A polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) technique was used for determining the 2 test. The X2 test and the Fisher's exact test were used to test associations between genotypes and categorical variables describing the clinico-pathologic features of the study population. Survival curves were plotted using the Kaplan-Meier and compared using the log-rank test. The Cox proportional hazards model was used for multivariate analysis to estimate and test demographic characteristics, clinical and genetic features for their associations with OS. In this exploratory study, no formal correction for multiple comparisons was adopted. However, all the following variables were included in multivariate Cox model: age, sex, ECOG performance status, weight loss (>5% in the four weeks before starting chemotherapy), anemia, albumin level, CEA level, tumor grading, histologic subtype according to Lauren’s classification, tumor location, liver involvement, presence of peritoneal carcinomatosis, number of metastatic sites and response to first-line chemotherapy. Assuming a 20% lowest frequency for an unfavorable genotype, 157 events would allow to detect an Hazard Ratio (HR) of 1.75 associated with this group (80% power and 5% type I error for a two-tailed test). All results were considered significant at two-sided p < .05 value. All analyses were performed by using the MedCalc software version 11.1 .The primary endpoint of the study was the association between genotypes and overall survival (OS), as calculated from the start of first-line palliative chemotherapy until death. Genotypes were checked for deviation from the Hardy-Weinberg equilibrium using the Pearson XOne-hundred-sixty-one patients were analyzed. All of them received first and second-line chemotherapy and died after gastric cancer progression. First-line chemotherapy was oxaliplatin or cisplatin plus a fluoropyrimidne in 150 patients, or bolus/infusional 5-Fluorouracil in 11 patients. Second-line chemotherapy was 5-Fluorouracil coupled with cisplatin or oxaliplatin in 48 patients, with CPT-11 in 45 patients, with anthracycline in 33 patients, with paclitaxel or docetaxel in 25 patients, with VP-16 in 10 patients. Median survival time in the whole group was 9.4 months (range 0.4-34 months).Carriers of the rs1800795 G/G, G/C and C/C genotypes were 74 (46%), 68 (42%) and 19 (12%), respectively. Carriers of the rs8192284 A/A, A/C and C/C genotypes were 58 (36%), 73 (45%) and 30 (19%), respectively. These frequencies did not show deviation from Hardy-Weinberg equilibrium and they are comparable with frequencies commonly observed in Caucasian populations.Details of the characteristics of enrolled patients together with their distribution according to rs1800795 and rs8192284 genotypes are shown in Table Survival curves of carriers of the rs1800795 and rs8192284 genotypes are shown in Figure The recessive model was adopted in the multivariate analysis with rs1800795 G/G and rs8192284 C/C defined as the risk genotypes. As shown in Table An additional explorative survival analysis was addressed to the distribution of the rs1800795 G and rs8192284 C risk alleles. There were 16 patients (10%) who were carriers of both unfavorable rs1800795 G/G and rs8192284 C/C genotypes . Eight patients (5%) with rs1800795 C/C and rs8192284 A/A genotypes were classified without risk alleles . Twenty-nine (18%), 68 (42%) and 40 (25%) patients were grouped as carriers of 1 risk allele, 2 risk alleles, or 3 risk alleles. As shown in Figure Clinical studies have demonstrated that increased serum IL-6 concentrations are associated with advanced tumor stages and short survival in patients with solid neoplasms . IL-6 isIL-6 promoter (rs1800795 G > C) enhances serum concentrations of IL-6 [IL6R (rs8192284 A > C) is a strong inducer of the soluble form of the IL-6 receptor (sIL-6R) [IL6R allele also causes an increase in IL-6 circulating levels, but it seems an indirect effect resulting from reduced IL-6 clearance through membrane-bound IL-6R [IL-6 promoter variant (rs1800795) showed association with poor survival of patients with advanced gastric cancer treated with palliative chemotherapy. The minor IL-6R C allele (rs8192284) showed a weaker prognostic role than the IL-6 promoter variant. However, in support of a “dynamic” modulation of the IL-6/sIL-6R system, we observed a possible additive effect with worst survival outcomes in the presence of both IL-6 and IL-6R unfavorable genotypes (Figure There is evidence that the level of activity of IL-6 and its receptor are regulated by functional polymorphisms in the corresponding genes . The comL6R rs819284 A > CsIL-6R genotypes would also support the role of the IL-6/IL-6R system in the acquisition of a specific pattern of metastatic spread [in vivo models, IL-6 increases the metastatic potential of circulating tumor cells and modulates tissue homeostasis in a target organ of metastasis such as the liver [IL-6R minor allele carriers and it is likely to promote hematogenous spread, causing a specific pattern of metastatic disease [The different distribution of patients with and without liver metastasis according to the c spread . In expehe liver . Also, she liver ,2. It is disease .G allele of the rs1800795 IL-6 promoter variant showed association with unfavorable survival outcomes of patients with ovarian cancer [IL-6 polymorphisms in gastric cancer patients [IL-6 variant did not show prognostic role. Notably, they could not investigate the IL-6 rs1800795 because of the rarity of the variant allele in Asiatic populations [IL-6 rs1800796 are less extensively studied compared with the IL-6 rs1800795.The common n cancer , breast n cancer ,29, neurn cancer  and heman cancer . To the patients . Liao etpatients  showed aulations , while tIL-6R genetic variant. In multiple myeloma patients the minor rs8192284 C allele showed association with lower overall survival [sIL-6R and IL-6R genetic variants may display variable clinical effects depending on tumor type, tumor stage, concomitant treatments, host related features involving the immune system and the fine tuning of other cytokines.Less information is available on the clinical impact of the rs8192284 survival , but in survival . Accordisurvival , it is lClinical data on the activation of the IL-6/IL-6R system should be considered beyond the possible prognostic role. In fact, the effects of IL-6/IL-6R may contribute to explain different sensitivity and clinical outcomes of patients treated with novel target therapies. At the same time, IL-6/IL-6R analyses could offer the opportunity of developing an alternative therapeutic strategy. In patients with metastatic renal cell cancer, high IL-6 serum levels were predictive of improved progression-free survival from the multi-kinase inhibitor Pazopanib compared with placebo . In expeLimitations of this study are its retrospective nature and the lack of a concomitant analysis of the cytokines circulating levels. Therefore, additional studies are needed for confirming the prognostic role of IL-6 analyses, and for corroborating the hypothesis that subjects with elevated baseline IL-6 levels and/or an IL-6 enhancing genetic profile may represent the target population for evaluating the effects of the anti-IL-6 MoAbs in cancer patients.HR: Hazard ratio; CI: Confidence interval; ECOG PS: Eastern Cooperative Oncology Group Performance Status; RR: Response rate; CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease; Mets: metastasis; LA/LR: Locally advanced/local relapse.The authors declared that they have no competing interests.AR, EC, EG, MM and FG conceived and designed the experiments. VC, DS, GT, BV, GF and FG provided the samples. AR, EC, EG, MM and FG analyzed and interpreted the data. AR, EG, MM and FG contributed in the writing of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/357/prepub


These results suggest that Sch A and Sch B may act differentially on activating GST/ depleting cellular GSH and inducing an antioxidant response involved in their anti-inflammatory actions.Schisandrin A (Sch A) and schisandrin B (Sch B) are active components of Schisandrae Fructus. We compared the biochemical mechanism underlying the anti-inflammatory action of Sch A and Sch B, using cultured lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and concanavalin (ConA)-stimulated mouse splenocytes. Pre-incubation with Sch A or Sch B produced an anti-inflammatory action in LPS-stimulated RAW264.7 cells, as evidenced by the inhibition of the pro-inflammatory c-Jun N-terminal kinases/p38 kinase/nuclear factor-κB signaling pathway as well as the suppression of various pro-inflammatory cytokines and effectors, with the extent of inhibition by Sch A being more pronounced. The greater activity of Sch A in anti-inflammatory response was associated with a greater decrease in cellular reduced glutathione (GSH) level and a greater increase in glutathione S-transferase activity than corresponding changes produced by Sch B. However, upon incubation, only Sch B resulted in the activation of the nuclear factor (erythroid-derived 2)-like factor 2 and the induction of a significant increase in the expression of thioredoxin (TRX) in RAW264.7 cells. The Sch B-induced increase in TRX expression was associated with the suppression of pro-inflammatory cytokines and effectors in LPS-stimulated macrophages. Studies in a mouse model of inflammation (carrageenan-induced paw edema) indicated that while long-term treatment with either Sch A or Sch B suppressed the extent of paw edema, only acute treatment with Sch A produced a significant degree of inhibition on the inflammatory response. Although only Sch A decreased the cellular GSH level and suppressed the release of pro-inflammatory cytokines and cell proliferation in ConA-simulated splenocytes  The inflammatory response, as an integral part of innate immunity, enables the removal of pathogens and the clearance of injured cells by means of a complex interplay involving resident immune cells (resident macrophages) and patrolling immune cells  via a series of biochemical events . In brieDespite the fact that the inflammatory response is beneficial to the host, a chronic and low-grade inflammation was found to be associated with the pathogenesis of a number of diseases, such as inflammatory bowel disease , cardiovSchisandra sphenanthera and Schisandra chinensis. Schisandrin A (Sch A) and schisandrin B (Sch B) are the most abundant active dibenzocyclooctadiene lignans in the fruits of Schisandra sphenanthera and Schisandra chinensis, respectively , a traditional Chinese herb clinically prescribed for the treatment of hepatitis, contains two species, namely the dried fruits of ectively . While tof Sch A . Sch B wof Sch A –13. The of Sch A  and concof Sch A . HoweverGiven the differential abilities of Sch A and Sch B in triggering the redox signaling pathway , it remaDulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), mouse TNF-, IL-1β, IL-2 ELISA kit, Lipofectamine LTX & PLUS reagent, Lipofectamin RNAiMAX reagent, BLOCK-iTTM Fluorescent Oligo, PureLink RNA Mini Kit, SuperScript VILO Master Mix, customized TaqMan array plates and TaqMan universal PCR Master Mix were obtained from Life Technologies . Histopaque-1083, RPMI 1640 medium, sodium pyruvate, LPS, proteinase inhibitor cocktails and phosphatase inhibitor cocktails were purchased from Sigma-Aldrich Co . Sch A and Sch B were prepared as previously described . All oth2. RAW264.7 cells used for the experiments were seeded at a density of 1.25×105 cell/mL on a 12 or 96-well culture plate and were allowed to grow to 60–80% confluence within 24 h prior to incubation with the test compounds.A murine RAW264.7 macrophage cell line was purchased from American Type Culture Collection . RAW264.7 cells were cultured in a monolayer using DMEM supplemented with 10% FBS, 100 units/mL penicillin, 0.1 mg/mL streptomycin. RAW 264.7 and isolated peritoneal macrophages were kept at 37°C in a humidified atmosphere of air and 5% COad libitum in the Animal and Plant Care Facility at the Hong Kong University of Science and Technology (HKUST). All experimental protocols were approved by the University Committee on Research Practice at the HKUST on 7 April 2013 (reference number: SC019).Adult Imprinting Control Region (ICR) mice  were maintained under a 12-h dark/light cycle at about 22°C, and allowed food and water 6 viable cells/mL. The viability of isolated splenocytes was determined by Trypan Blue exclusion.Adult female ICR mice were sacrificed by cardiac excision under ketamine chloride-induced anesthesia. Splenic tissue obtained from mice was teased with a plastic syringe in a culture dish (60 mm) containing 10 mL of RPMI-1640 medium, and it was gently strained through a 200 mesh stainless steel sieve to remove clumps to produce a cell suspension. The cell suspension was washed 3 times with RMPI-1640 medium by centrifugation and re-suspension, and the cells were finally resuspended in RPMI-1640 medium supplemented with 10% FBS at a concentration of 5 × 10For the investigation of the direct anti-inflammatory activities of Sch A and Sch B, macrophages were activated by LPS immediately following Sch A/Sch B incubation . In brieFor the investigation of indirect anti-inflammatory activities of Sch A and Sch B, Sch A/Sch B-incubated macrophages were activated by LPS after an additional incubation for 16 h in tested compound-free medium for the induction of the antioxidant response . RAW264.To delineate the involvement of various components in the anti-inflammatory actions of Sch A and Sch B, specific enzyme inhibitors and antioxidants were used. Quinidine , N-acetyl cysteine , Trolox  and 1-aminobenzotriazole  were co-incubated with Sch A/Sch B in the LPS-activated macrophages.5-UUAAGUGGCCCAAGUCUUGCUCCA-3; antisense, 5-UGGAGCAAGACU UGGGCCACUUAAA-3 for Nrf2 siRNA . Cells were transfected with Nrf2 siRNA (50 nmol/L) with Lipofectamin RNAiMAX transfection reagent according to the procedures modified by Chiu et al.  for 1.15 h at 100 V. Membranes were blocked for 1 h with 5% non-fat milk in Tris-buffered saline with Tween-20  at room temperature and subsequently probed with anti-JNK (1:1000), anti-phospho JNK (1:1000), anti-p38 (1:1000), anti-phospho p38 (1:1000), anti-COX2 (1: 500), anti-iNOS (1:1000), anti-Nrf2 (1:1000) or TRX (1:500) overnight. The membranes were rinsed with TBS-T and incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000). Following the incubation, the membranes were rinsed with TBS-T, and bound antibodies were detected by using enhanced chemiluminescence, following the manufacturer’s instructions . The membranes were stripped and probed for anti-β-actin (1:5000) or anti-lamin B1 (1:1000) as loading controls. The molecular weight of the protein of interest was estimated using Blue Protein Standard marker, Broad Range . Individual band densities were quantified by Quantiscan.The levels of TNF-α, IL-1β and IL-6, IL-2 in culture media or mouse paw tissue were measured using EILSA Kits . The level of PGE2 in the culture medium from LPS-activated macrophages was measured by ELISA using an assay kit purchased from GE healthcare . The levels of T helper cell 1 (Th1), T helper cell 2 (Th2) and T helper cell 17 (Th17) in the culture medium from the Con A-activated lymphocytes isolated from Sch A/Sch Btreated mice were measured by using mouse Th1/Th2/Th17 Cytokine Muti-analyte ELISArray Kits purchased from Qiagen Inc. . All ELISAs were performed according to manufacturer’s instructions.Nrf2-luciferase reporter, which consists of an Nrf2 responsive firefly luciferase construct and a constitutively expressing Renilla luciferase construct at a ratio of 40:1, was purchased from SABiosciences . NF-κB-luciferase reporter, which consists of an NF-κB responsive firefly luciferase construct and a constitutively expressing Renilla luciferase construct at a ratio of 40:1, was purchased from SABiosciences. In brief, RAW264.7 cells were grown to 70–80% confluence in a 24-well cell culture dish and transfected with Nrf2-luciferase or NF-κB-luciferase reporter (0.5 μg/well) using Lipofectamine LTX & PLUS reagent according to the manufacturer's protocol. At twenty-four hours post-transfection, the transfected cells were incubated with Sch B as indicated previously. The incubated cells were washed with phosphate-buffered saline (PBS) and lysed with the buffer provided by the dual-luciferase reporter assay kit [Promega ]. Luciferase activities in the cell lysates were then measured using a Victor V3 Multi-label Counter. Renilla luciferase expression was used to normalize the various transfection efficiencies in the samples.et al.  using a tissue homogenizer ULTRA-TURRAX T25  at 95,000 rpm and the cytosolic fraction was obtained by centrifugation at 10,000 ×g at room temperature for 30 min. The lymphocyte-rich opaque layer was collected and washed 3 times with PBS. The isolated lymphocytes were then lysed with 3% 5-sulfosalicylic acid. GSH levels of the samples were then measured.Female ICR mice were randomly divided into groups of 5–6 animals each. Mice were given a single oral dose of Sch A (1 mmol/kg), Sch B (1 mmol/kg) and Sch A+Sch B (0.5 mmol/kg each) while control mice received the vehicle (oil) only. One hour after the Sch A/Sch B treatment, heparinized blood samples were drawn from ketamine-anesthetized mice by cardiac puncture. Mice were then sacrificed by cardiac excision under ketamine chloride-induced anesthesia. Lymphocytes were isolated by centrifugation in Histopaque 1083 at 400 ×6 cell/mL) was activated with Con A  for the assessment of Th1/Th2/Th17 cytokine profiles or with Con A  for the examination of cell proliferation.Female ICR mice were randomly divided into different groups of 5–6 animals each. Mice were given Sch A (0.25 nmol/kg/d × 5d), Sch B (0.25 nmol/kg/d × 5d) and Sch A+Sch B (0.125 nmol each/kg/d × 5d) administered orally while control mice received the vehicle (oil) only. One hour after the last dosing, mice were then sacrificed by cardiac excision under ketamine chloride-induced anesthesia. Lymphocytes were isolated using the method described above. The cell suspension .t-test or one-way Analysis of Variance (ANOVA). Post-hoc multiple comparisons were performed using TUKEY. P values < 0.05 were regarded as statistically significant. All statistical analyses were performed using GraphPad Prism 3, SPSS 17.0 or Microsoft Office Excel 2013.Data were analyzed by Incubation with Sch A or Sch B (25 and 50 μM) increased cellular GST activity in RAW264.7 macrophages, with the degree of stimulation caused by Sch A being greater  . The co-LPS activated the pro-inflammatory signaling cascade, as evidenced by increases in the expression of JNK1 (8.2 fold), JNK2 (11.7 fold), p38 (33.9 fold) and NF-κB 58%) . The act8% . The To investigate the possible involvement of GST in the anti-inflammatory action produced by Sch A or Sch B incubation, the effects of quinidine  on Sch A/Sch B-pre-incubated and LPS-activated macrophages were examined . QuinidiIncubation with Sch B (25 and 50 μM), but not Sch A, activated Nrf2, as indicated by an increase in nuclear translocation of Nrf2 (18 and 85%) . Only ScIncubation with Sch A, Sch B or Sch A+Sch B did not produce detectable changes in the pro-inflammatory signaling cascade, as assessed by the activation of JNK1, JNK2, p38 and NF-κB, at 16 h-post exposure in LPS-activated macrophages (data not shown). Only incubation with Sch B (but not Sch A and Sch A+Sch B) produced an anti-inflammatory action in LPS-activated macrophages. While the incubation with Sch B at 25 μM differentially attenuated the tested inflammatory parameters, the incubation with Sch B at 50 μM consistently suppressed all tested pro-inflammatory cytokines   and inflEarlier studies in our laboratory have demonstrated that Sch B can induce an Nrf2-driven antioxidant response via the CYP-catalyzed production of signaling ROS in various cell lines. In this regard, we investigated the involvement of CYP in the anti-inflammatory action of Sch B using a CYP inhibitor . ABT a . The traIncubation with Sch A concentration-dependently decreased cellular GSH levels in isolated mouse splenocytes  . IncubatTo extend the results obtained from cell-based studies, the anti-inflammatory actions of Sch A and Sch B were examined in a mouse model of inflammation—namely, carrageenan-induced paw edema. Carrageenan injection  into the paws of mice caused edema (by 104%), when compared with vehicle-injected mice. Treatment with a single bolus dose of Sch A (1 mmol/kg), but not Sch B (1 mmol/kg) or Sch A+Sch B (0.5 mmol/kg each), significantly suppressed paw edema in carrageenan-injected mice, with the degree of inhibition being 31% . The supin vitro splenocyte study, an ex vivo model was adopted to investigate the effect of Sch A/Sch B pretreatment on Con A-stimulated splenocyte proliferation in mice. Long-term treatment with Sch A, Sch B and Sch A+Sch B did not produce any detectable change in cellular GSH levels in splenocytes isolated from pretreated mice. However, long-term treatment with Sch A, Sch B and Sch A+Sch B did inhibit the Con A-activated splenocyte proliferation  (To confirm the results obtained from the ctively) . The inhctively) .The LPS-activated signal transduction in macrophages, which involves a cascade of the Toll-like receptor 4/JNK/P38/NF-κB pathway, is commonly investigated in cell models for assessing anti-inflammatory activities of a variety of candidate compounds –24. Consin vivo. Given that the maximum achievable blood concentration of Sch B in mice is around 10 μM (unpublished data) which is lower than the effective concentrations of Sch B (25–50 μM) in in vitro assays, the immune cells may interact synergistically in in vivo condition in response to Sch B and thereby produce the anti-inflammatory action.Accumulating experimental evidence has demonstrated the involvement of an Nrf2-driven antioxidant response in the suppression of inflammation. Consistent with our previous finding in cultured murine hepatocytes and cardiomyocytes ,13, the The ability of Sch B to induce an antioxidant response is likely mediated by the CYP-catalyzed metabolism of Sch B, with the production of signaling ROS resulting in activation of Nrf2 ,13. Thisex vivo study that levels of Th1 cytokines  were suppressed in Con A-activated splenocytes isolated from Sch A-pretreated mice. Similar to the results obtained from the study in macrophages, the Sch A-induced GSH depletion seems to be associated with immunosuppression in Con A-activated splenocytes. In this regard, it has been reported that the proliferation of cultured murine lymphocytes is dependent on the availability of cellular GSH [Con A, a lectin isolated from jack-beans, is a T lymphocyte-specific antigen that preferentially triggers the differentiation and proliferation of immature lymphocytes into T lymphocytes . In thisular GSH . In addiin vitro or in in vivo assay systems. The possibility of synergism produced by Sch A and Sch B in combination may possibly be revealed in other experimental models of inflammation.Given that the differential biochemical mechanism underlying the anti-inflammatory action of Sch A (mainly involving GST activation/ GSH depletion) and Sch B (mainly via the induction of an antioxidant response), whether or not a synergistic effect can be produced by the combined treatment with Sch A and Sch B, is of considerable pharmacological interest. Unexpectedly, under the present experimental conditions, no synergistic anti-inflammatory effects were observed for Sch A and Sch B in either in vitro and in vivo  during the incubation with Sch A or Sch B. After one h co-incubation, GST activity was measured . After 6 h, the GSH level was also measured . Data are expressed as % non-quinidine control by normalizing relative to non-quinidine-incubated cells (left panels). Value given are means ± SEM, with n = 3–5. * Significantly different from the non-quinidine control; # significantly different from the quinidine control. After 6 h co-incubation, RAW264.7 macrophages were stimulated with LPS (1 μg/mL) for 18 h and 24 h, for the measurement of iNOS and NO levels, respectively. Levels of iNOS and NO were measured as described in (TIF)Click here for additional data file.S2 FigRAW264.7 macrophages were co-incubated with NAC (5 mM) during the 6-h incubation with Sch A or Sch B. GSH levels were measured (left panel). Data are expressed as % non-NAC control by normalizing relative to non-NAC incubated control cells. Value given are means ± SEM, with n = 3–5. * Significantly different from the non-NAC control; # significantly different from the NAC control. After the 6-h co-incubation, RAW264.7 macrophages were challenged with LPS (1 μg/mL) for 18 or 24 h, for the measurement of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels, respectively. Levels of iNOS and NO were measured as described in (TIF)Click here for additional data file.S3 FigRAW264.7 macrophages were co-incubated with Trolox (500 μM) during the 6-h incubation with Sch A or Sch B. GSH levels were measured (left panel). Data are expressed as % non-Trolox control by normalizing relative to non-Trolox incubated control cells. Value given are means ± SEM, with n = 3–5. * Significantly different from the non-Trolox control; # significantly different from the Trolox control. After the 6-h co-incubation, RAW264.7 macrophages were challenged with LPS (1 μg/mL) for 18 or 24 h, for the measurement of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) levels, respectively. Levels of iNOS and NO were measured as described in (TIF)Click here for additional data file.S4 FigRAW264.7 macrophages were transfected with Nrf2 luciferase reporter, as described in Materials and Methods. The transfected macrophages were co-incubated with ABT (10 mM) during the incubation with Sch A or Sch B for 6 h. At 16 h post- exposure, the luciferase activities in the cell lysates were measured. Nrf2 reporter activities were expressed as % control, by normalizing relative to non-ABT controls . At 16 h post-exposure, the level of thioredoxin (TRX) was also measured, as described in (TIF)Click here for additional data file.S5 FigRAW264.7 macrophages were co-transfected with Nrf2 luciferase reporter as well as siRNA of Nrf2, as described in Materials and Methods. The transfected macrophages were incubated with Sch B (50 μM) for 6 h. Followingt 16 h of incubation, the luciferase activities in the cell lysates were measured. The Nrf2 reporter activity was expressed as % control, by normalizing relative to the value of the non-siRNA control . Following 16 h of incubation, the level of thioredoxin (TRX) was measured, as described in (TIF)Click here for additional data file.


Less understood is the role of electrostatic repulsion between groups of like charge in destabilizing structure or directing molecular recognition. To study this, we apply a “charge crowding” approach, where repulsive interactions between multiple aspartate side chains are found to destabilize the homotrimer states in triple helical peptide system and can be utilized to promote the formation of heterotrimers. Neutralizing surface charge by increasing salt concentration or decreasing pH can enhance homotrimer stability, confirming the role of charge crowding on the destabilization of homotrimers via electrostatic repulsion. Charge crowding may be used in conjunction with other approaches to create specific collagen heterotrimers.Charge-pair interactions between acidic and basic residues on the surface of collagen can promote stability as well as control specificity of molecular recognition. Heterotrimeric collagen peptides have been engineered  Collagen is the most abundant protein in the human body, accounting for approximately one-third of protein mass . Collagede novo α-helical coiled coils shows that peptide systems assemble using electrostatic forces, both to encourage specific association of the desired state and prevent competing states. Favorable charge-pair interactions serve to increase specificity and stability by encouraging certain conformations and aiding in the stabilization process [Early work on the protein design focused on surface electrostatic interaction for coiled-coil and globular proteins. Extensive study of  process ,8,9. Eng process ,11,12. SAlthough sophisticated designs of α-helical coiled-coils have been achieved, little attention was given to computational design of the fibrous protein collagen until recently. Utilizing the surface electrostatic interactions, charged heterotrimers have been designed by forming a network of electrostatics on the surface of triple helix ,18,19,20N- and C-terminal peptides significantly destabilizes the triple helical structure, whereas charged amino acids in the middle of the sequence can disrupt the formation of triple helical structure. Salt and pH titrations confirm electrostatic repulsions prevent triple helix formation. The resulting charge crowding promotes the formation of binary collagen heterotrimers. We believe that charge crowding can be used in conjunction with other methods to design specific heterotrimers by destabilizing competing states and favoring targeted state. To isolate the impact of repulsive interactions, three collagen peptides were designed to include acidic triplets either flanking or in between Pro-Hyp-Gly sequence repeats. Our result suggests that charge crowding on 3 located on the C- or N-termini or in the middle of (Pro-Hyp-Gly)7, peptides A, C and B respectively. Assuming the POG-rich region specifies the alignment of the strands in a homotrimer, this would place the six negative charges per chain within 10–15 Å of each other, resulting in a total net charge of -18 within a local volume of approximately 1,500 Å3. It is hypothesized that this degree of charge crowding would prevent local folding of the charged regions and destabilize the global fold of homotrimers. Furthermore, in mixtures of multiple peptide types, these domains could facilitate the formation of heterotrimers where staggered placement of charge domains might relieve repulsive interactions.Previous studies applied the principles of positive and negative design to create a heterospecific trimer; repulsive forces were penalized while attractive interactions were rewarded ,19,20,21A:7(Asp-Asp-Gly)3-NH2Ac-(Pro-Hyp-Gly)C-terminalB:3(Asp-Asp-Gly)3(Pro-Hyp-Gly)4-NH2Ac-(Pro-Hyp-Gly)MiddleC:3 (Pro-Hyp-Gly)7-NH2Ac-(Asp-Asp-Gly)N-terminal10 whose melting temperature is approximately 68 °C [7 which lacks the N or C-terminal charged domains is also significantly more stable with a previously reported mT around 40 °C [Peptides A, B, and C were dissolved in 10 mM pH7.0 phosphate buffer as described in the experimental section. Peptides A and C formed structured homotrimers in solution at 4 °C, showing the characteristic Circular Dichroism (CD) spectrum of a triple helix with a positive band at 225 nm . Peptidely 68 °C ,22,23.  compared to any homotrimer states  were evaluated. Only 2A:C, A:2C, 2A:B, and A:B:C showed cooperative unfolding upon melting B,C. Hower states D,E indicTo resolve whether A:B:C is formed, we performed NMR HSQC measurements on seven different mixtures of A, B, C peptides . Residues in different species would experience dissimilar chemical environments, giving rise to distinct resonances. Total overlap is observed between spectra of the A:B mixture and the combined spectra of A and B, consistent with a lack of interaction between A and B measured by CD. B:C mixtures also did not show any evidence of heterospecific interactions C. Howeve7 (Tm = 36 °C) [To confirm the effect of electrostatic repulsion via charge crowding, the structure and stability of peptides A, B, and C were measured under a series of salt concentrations ranging from 0.0 to 1.0 M NaCl. The stability and folding of peptide A and C increased with salt concentration A,C. Stab= 36 °C)  alone, wa of the aspartate carboxylate side chain (~4). Side chains should be neutral at this pH. Lowering pH promoted triple helix formation for peptide A and C , well below the pK A and C A,C. Stab A and C D,F. At lN- and C-termini were uncapped. It has been show that uncapped ends have minor effects on the stability of collagen model peptides near neutral pH [2PO4) buffer. The peptides were synthesized using solid-phase FMOC chemistry at the Tufts University Core Facility, Boston, MA, USA. utral pH . Peptide7(Asp-Asp-Gly)3-NH2A: Ac-(Pro-Hyp-Gly)3(Asp-Asp-Gly)3(Pro-Hyp-Gly)4-NH2B: Ac-(Pro-Hyp-Gly)3 (Pro-Hyp-Gly)7-NH2C: Ac-(Asp-Asp-Gly)The sequences of the characterized peptides are listed below:214 = 2,200 M−1cm−1. After preparing mixtures at room temperature, they were heated to 80 °C for 30 min and stored at 4 °C for at least 48 h. To assess the effect of ionic effect, peptides were allowed to fold in the presence of salt at a series of concentrations: 0.0, 0.01, 0.1, 0.5, and 1 M NaCl. Low pH studies were carried out in 0.1 mM pH2.5 phosphate citrate buffer. All peptide solutions were prepared in 10 mM pH7.0 phosphate buffer. Peptide concentrations in solution were estimated by obtaining the absorbance at 214 nm using εCD measurements were conducted using the Aviv model 420SF spectrophotometer equipped with a Peltier temperature controller. For wavelength spectra, measurements were made at every 0.5 nm step with an averaging time of 10 s at each wavelength. Wavelength scans were conducted from 190 to 260 nm at 5 °C. Observed ellipticity was converted to molar ellipticity by dividing raw values by the peptide concentration, number of residues, and cell path length. For temperature induced denaturation, ellipticity was measured at 225 nm for peptides using a total peptide concentration of 0.2 mM. CD melt were smoothed using the Savitsky-Golay algorithm with nineteen points and a third-order polynomial [The three peptides A, B, and C were mixed to get seven different combinations: A, B, C, A:B, A:C, B:C, and A:B:C. Final concentrations were 5.0 mM in 10% D2O with 135 mM NaCl, 10 mM Tris, and 2.3% Glycerol at pH8.0. 1H−13C single-bond correlation spectra were recorded at 7 °C on a Bruker Biospin 600 MHz spectrometer equipped with cryoprobe using the echo/anti-echo-TPPI gradient selection with decoupling during acquisition, phase-sensitive pulse program (hsqcetgp). Spectral widths were 8,389 Hz for 1 H and 13,979 Hz for 13 C.In this study, charge crowding of negative amino acids has been rationally designed to control stability and heterospecificity of collagen-like triple helix assembly. Using a simple model, we showed the effect of electrostatic repulsion on the stability and folding of triple-helical peptide. In past stability and specificity of three sequences were generated using electrostatic attractive interactions via favorable salt bridges. Understandably, natural collagens avoid charge crowding. While favorable charge interactions are common, the presence of adjacent like-charge repulsions is rare. In a survey of the triple-helix forming regions of various human collagens—COL1A1, COL3, and COL5A1—the number of adjacent like-charge residues is limited to two or at most three . Often,