The plasma level of cystatin C is a better marker than plasma creatinine for successful aging. It has been assumed that the advantage of cystatin C is not only due to it being a better marker for glomerular filtration rate (GFR) than creatinine, but also because an inflammatory state of a patient induces a raised cystatin C level. However, the observations of an association between cystatin C level and inflammation stem from large cohort studies. The present work concerns the cystatin C levels and degree of inflammation in longitudinal studies of individual subjects without infl ammation, who undergo elective surgery.Cystatin C, creatinine, and the inflammatory markers CRP, serum amyloid A (SAA), haptoglobin and orosomucoid were measured in plasma samples from 35 patients the day before elective surgery and subsequently during seven consecutive daysTwenty patients had CRP-levels below 1 mg/L before surgery and low levels of the additional inflammatory markers. Surgery caused marked inflammation with high peak values of CRP and SAA on the second day after the operation. The cystatin C level did not change significantly during the observation period and did not correlate significantly with the level of any of the four inflammatory markers. The creatinine level was significantly reduced on the first postoperative day but reached the preoperative level towards the end of the observation period.The inflammatory status of a patient does not influence the role of cystatin C as a marker of successful aging, nor of GFR. Increased levels of cystatin C have been shown to be associated with less successful aging than normal levels of cystatin C and cystatin C has been described to have a better diagnostic performance than creatinine to predict successful aging –6. It isThirty-five patients scheduled for elective surgery, with normal body temperatures and able to provide written informed consent, without the need for a proxy respondent, were considered for inclusion in the study. The study complied with the Declaration of Helsinki as revised in 1983. Tissue trauma during surgical procedures varied from minor to medium.Venous blood samples were collected in EDTA-containing tubes on the day before surgery (Day 0) and daily postoperatively for seven days (Day 1 to 7).Plasma CRP, orosomucoid  and haptoglobin were determined by immunoturbidimetry using a Roche-Hitachi cobas 6000 analysis platform and with a calibrator based upon the international calibrator CRM 470 . Referenp-values < 0.05 were considered significant.All statistical procedures were performed using SPSS release 15.0.0 . To limit the problem of multiple comparisons, the time course of the levels of each inflammatory marker , as well as of creatinine and cystatin C, were evaluated by two distinct statistical tests only: (i) Differences between preoperative levels and the first  or second (haptoglobin and orosomucoid) postoperative day were tested using Wilcoxon signed rank test; and (ii) for CRP, SAA, creatinine and cystatin C, log-linear trends in the changes in levels on postoperative day 1 through 7 were tested overall, using a linear mixed model with the logarithm of the marker day 1 through 7 as dependent variable, day as a fixed continuous covariate and with no random effect but a first-order autoregressive model for the residuals. To assess the correlation between infl ammatory markers and creatinine and cystatin C, the mixed models with inflammatory markers as dependent variables were extended by including the natural logarithm of creatinine and cystatin C, respectively, as fixed covariates. Due to missing data at several time points, mixed models were preferred to methods requiring complete data at all time points. The presence of systemic inflammation in 35 patients scheduled for elective surgery was investigated by analysis of the plasma levels of four commonly used markers for inflammation: CRP, SAA, haptoglobin and orosomucoid. The preoperative CRP levels in 20 of the patients were below 1 mg/L, which is below the median CRP level in the third decade of a general adult population  indicatip < 0.001 and p < 0.001, respectively) and reached maximal values on the second postoperative day with median levels corresponding to 46 and 32 times the preoperative levels of CRP and SAA, respectively , and remained stable during day 1–7 (p = 0.49). Creatinine decreased from 81–65 µmol/L in median on the first postoperative day (p = 0.009), but tended to increase again during day 1–7 (p = 0.19). The median values and the corresponding 1 st and 3 rd quartiles of the CRP, SAA, haptoglobin, orosomucoid, creatinine and cystatin C levels are given in In contrast to the levels of the infl ammatory markers, the cystatin C level did not change significantly on the first postoperative day . The creatinine level displayed a significant correlation with the SAA level (p = 0.019), but not with the levels of any of the other infl ammatory markers .The cystatin C level showed no statistically significant correlation with the levels of any of the inflammatory markers (SAA: It has been suggested that cystatin C is a better marker than creatinine for survival success not only because it is a better marker for GFR but also because the presence of an inflammatory reaction in an individual produces a raised cystatin C level ,10–13. TAlthough the results of the present study might seem to be in confl ict with the previous observations of significant correlations between the plasma levels of CRP and cystatin C in cohort studies ,10–13, wThe present study indicates that the advantage of cystatin C compared to creatinine as a marker of successful aging is not due to the fact that an increased level of cystatin C signals the presence of infl ammation in addition to a decrease in GFR, as previously suggested ,10–13. T


The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity. In brain synapses, the release of neurotransmitter is restricted to a specialized region of the presynaptic plasma membrane, the active zone, which is characterized by the presence of a dense proteinaceous matrix, called cytomatrix at the active zone (CAZ). The CAZ forms a distinct scaffold for multiple membrane trafficking processes occurring during neurotransmitter release Here, we have identified the small ubiquitous adaptor protein 14-3-3 as a novel interaction partner for Bassoon. The interaction critically depends on phosphorylation of the 14-3-3-binding motif of Bassoon. 14-3-3s are dimeric, highly abundant proteins with multiple cellular functions including regulation of signal transduction, cell survival and differentiation. They usually bind to phosphoserine-based motifs in their target proteins and often control the incorporation into multiprotein complexes and/or the subcellular localization of their binding partners The following primary antibodies were used for Western blots: rabbit antibodies against pan 14-3-3 , 14-3-3η , Bassoon sap7f  in GFP-Bsn expression vector and the mutation was confirmed by sequencing. To generate His-Bsn11 (Bsn aa 2714–2867) the fragment was cloned via EcoRI/BamHI into the pET32a vector. 14-3-3η, 14-3-3β, 14-3-3γ and 14-3-3ε were generated using PCR on a rat brain Matchmaker cDNA library  as template with extended primers to add EcoRI and XhoI restriction sites at the 5′ and 3′ ends of the fragments, respectively. The introduced restriction sites were used for cloning into pmRFP-C2 The Bassoon fragments Bsn28 (aa 2715–3013) and GFP-Bsn (aa 95–3938) were described previously For a cDNA library screening, the Matchmaker Two-Hybrid System 3  was used with a pACT2 rat brain Matchmaker cDNA library  as prey and Bassoon fragment Bsn28 (aa 2715–3013 of rat Bassoon) as bait. Transformation and selection was performed according to the manufacturer's protocols.Transfection of COS-7 cells  grown on glass coverslips coated with Poly-D-Lysine (Sigma-Aldrich) was performed using the transfection reagent Polyfect (Qiagen) according to the manufacturer's protocol. After 24 h, cells were fixed in 4% paraformaldehyde and 4% sucrose in PBS, pH 7.4, for 5 min at room temperature and embedded in Mowiol 4–88. Images were taken with an upright microscope  equipped with a camera  or Spot RT-KE; Diagnostic Instruments, Inc.) and MetaVue software . Constructs and procedure of the mitotargeting assay were described previously 2 were mixed with 4 µg DNA. Then, 50 µl of 140 mM NaCl, 50 mM HEPES, and 1.5 mM Na2PO4, pH 7.05, were added and, after 1 min, applied to cells in culture. The cells were incubated for 4 to 6 h at 37°C in 5% CO2 atmosphere before exchanging growth media. Cells were lysed in 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.5% Triton-X-100, for pull downs, or 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 10% glycerol, for co-immunopreciptations, containing Complete protease inhibitors (Roche) and PhosStop (Roche) for 10 min at 4°C. Insoluble material was removed by centrifugation. For dephosphorylation of proteins 10 units of calf intestine alkaline phosphatase (Fermentas) were added to the cell lysates and incubated for 1 h at 37°C. For pull downs 25 µg purified GST or 50 µg purified GST-14-3-3h were coupled to glutathion sepharose Fast Flow (Amersham) and incubated with cell lysates overnight at 4°C. After washing with lysis buffer bound material was eluted by incubation with 10 mM glutathione, 50 mM Tris-HCl, pH 7.5, for 30 min at RT. Co-immunoprecipitations were done using MicroMACS anti-GFP MicroBeads and MicroColumns (Miltenyi Biotec) according to the manufacturer's protocol but using the lysis buffer in all washing stepsHEK293T  were grown in 6-well plates and transfected using the calcium phosphate method. In brief, 150 µl of 0.5 M CaCl2 and 1 mM ATP (Sigma-Aldrich) was added and incubated at 30°C for 30 min. The reaction was stopped by addition of 10 mM EDTA. Protein concentrations were determined by amidoblack assay, the samples were adjusted by adding of 1×loading buffer to equal protein concentrations and loaded on SDS polyacrylamide gels for subsequent Western blotting. All experiments involving animals were carried out in accordance with the European Committees Council Directive (86/609/EEC) and approved by the local animal care committee .Bassoon mutant mice  RIPL; Stratagene) and purified following the manufacturer's protocol. 5 µmol of His-fusion proteins were incubated with 0 to 40 mU 90-kDa ribosomal S6 protein kinase (RSK) protein  in 50 mM Tris-HCl, pH 7.5, 10 mM MgClLive imaging was performed at 37°C using an inverted microscope  in a heated imaging chamber TC-344B (Warner Instruments) and an EMCCD camera  controlled by MetaMorph Imaging  and VisiView (Visitron Systems GmbH) software. The FRAP laser DL-473 (Rapp Optoelectronics) was driven by the FRAP targeting device Visifrap 2D (Visitron Systems GmbH). Videos were taken prior to bleaching 10 s , then for fluorescence bleaching a laser pulse of ms was applied and the recovery was monitored for 25 s  and additional 300 s . For image analyses ImageJ For vesicle mobility experiments video streams of 25-s recorded at 4 Hz with an exposure time of 250 ms were analyzed using MetaMorph software. To determine velocities and running distances of vesicles carrying GFP-Bsn or GFP-Bsn14-3-3BM, traces of mobile particles were visualized on kymographs of axonal segments. Traces showing processive movement (without stops and changes in velocities or movement directions) were analyzed. Statistical analysis of all data was done with the software GraphPad Prism 5 (GraphPad Software).S2845A), in which the critical serine residue S2845 was changed to alanine, were expressed in HEK 293T cells. The expression of the Bassoon fragments in mammalian cells was chosen to allow the phosphorylation of the binding motif, which was previously reported to be a necessary prerequisite for 14-3-3 binding to almost all reported targets S2845A showed no binding to GST-14-3-3h confirming that the intact S2845-containing motif was required for interaction of Bassoon with 14-3-3η  screen of a rat brain cDNA library with the cDNA fragment Bsn28 covering amino acid residues 2715 – 3013 of rat Bassoon as a bait . One int 14-3-3η . To test 14-3-3η . To mimi 14-3-3η , which i2 and 1 mM ATP at 30°C for 30 min leads to a shift of the Bassoon bands to higher molecular weights suggesting a change in the charge and conformation of Bassoon due to increased phosphorylation. Further blot membranes prepared in parallel were incubated with purified recombinant GST or a GST-14-3-3η fusion protein. Immunodetection with α-GST antibodies showed the binding of GST-14-3-3η at the exact location of the wild-type Bassoon band in lysates from wild type animals but not in lysates from BsnΔEx4/5 mice indicating the interaction of 14-3-3 with Bassoon  and Bassoon mutant mice  and an mRFP fusion protein of 14-3-3η (mRFP-14-3-3η) results in a clear enrichment of mRFP-14-3-3η at mitochondria carrying mitoGFP-Bsn28 (S2845A) or with mitochondria-targeted GFP (mitoGFP), mRFP-14-3-3η showed a diffuse cellular localization  by introducing the point mutation S2845A, which disrupted the binding of Bassoon to 14-3-3 proteins in vitro, into GFP-Bsn. In accordance with previous observations, both GFP-fusion proteins were localized at synapses in a similar way as the endogenous Bassoon protein. In addition, GFP-Bsn was localized in ectopic cytoplasmic clusters formed by mis-targeted over-expressed protein in the transfected neurons S2845A . This demonstrates that Bassoon can sequester 14-3-3η in neurons and that this is crucially dependent on presence of S2845 residue in Bassoon. Since the GFP-BsnS2845A construct did not recruit 14-3-3η we assume that the described binding motif represents the only interaction interface for 14-3-3 in Bassoon.Next we investigated, whether Bassoon interacts with 14-3-3η in neurons. To this end primary hippocampal neurons were co-transfected with mRFP-14-3-3η and a GFP-tagged construct comprising the amino acids 95 to 3938 of rat Bassoon (GFP-Bsn). This construct behaves in many respects like wild-type Bassoon when expressed in primary neurons s S2845A . We analS2845A. The α-pS2845 Bsn antibody recognized GFP-Bsn28 but failed to recognize its S2845A mutant or dephosphorylated GFP-Bsn28 in cell lysates treated with alkaline phosphatase . To test the specificity of this antibody for Bassoon, we tested it on brain extracts (membrane enriched P2 fractions) from wild-type and BsnΔEx4/5-mutant mice. The α-pS2845 Bsn antibody reliably recognized a band with a molecular weight of ∼450 kDa corresponding to Bassoon in hyperphosphorylated extracts from wild-type mice. No corresponding immunoreactivity was observed in the extracts from mutant mice, confirming the specificity of this antibody for Bassoon . To testsphatase . The effTo understand the physiological context under which the phosphorylation of Bassoon on S2845 occurs, it is important to identify the protein kinases driving this modification. To address this question we used in silico predictions of the online services NetPhosK S2845A) in cultured hippocampal neurons. Both constructs showed an identical general cellular distribution and degree of co-localization with synaptic markers . To check, whether the 14-3-3 interaction influences the dynamic properties of synaptic Bassoon we applied a FRAP assay . The quantitative analysis of the fluorescence intensities of GFP-BsnS2845A puncta showed no significant difference compared to GFP-Bsn puncta  indicating that the decreased recovery cannot be explained by differences in the expression levels of both constructs.In many cases 14-3-3 induces a spatial redistribution of its binding partners AP assay . The lonS2845A contributes to observed decreased recovery we analyzed the mobility properties of fluorescently-labeled vesicles in neurons transfected with wt and mutant constructs. We measured the vesicle velocities, the travelled distances of the vesicles and the amount of moving vesicles in relation to stable puncta. The velocity of vesicle movement and the travelled distances were analyzed by creating kymographs of axonal segments of transfected neurons imaged for 25 s at the rate of 4 Hz. Continuous vesicle traces without stop or change of direction were counted as single events and measured for their velocity and distance. Although there was a slight tendency for an increased travelling distance of the GFP-BsnS2845A the statistical analysis did not reveal significant difference between velocities  or travelling distances . The results imply that the disruption of the 14-3-3 interaction site on Bassoon does not significantly influence the transport properties of the Bassoon transporting vesicles. The relative mobility of these vesicles was analyzed by counting the moving and stable GFP-Bassoon and GFP-BsnS2845A puncta in the videos during a time period of 25 s (100 frames). The relative percentage of immobile puncta of GFP-Bsn did not significantly differ from GFP-BsnS2845A . Taken together, the lower recovery rate of GFP-BsnS2845A cannot be explained by a defect in the supply of Bassoon from the mobile pools to the synapse. We therefore propose that the S2845 mutation in Bassoon leads to a stronger anchoring to the presynaptic cytomatrix (or to a lower rate of dissociation from it), what eventually leads to the observed lower exchange rate.Bassoon and its paralogue Piccolo are transported into the axon on membranous organelles The integrity of the CAZ is essential for properly controlled release of neurotransmitter from presynaptic terminals Protein phosphorylation is a fast and reversible way to modulate protein function and was recognized to induce rearrangements of numerous protein complexes in processes of synaptic plasticity 2+/ATP upon incubation at 30°C or in lysates of cells treated with the protein phosphatase inhibitor okadaic acid (data not shown) demonstrating that this epitope can be phosphorylated by endogenous kinases. Interestingly, okadaic acid – induced phosphorylation of S2845 was reverted by pretreatment of cells with the specific RSK family inhibitor BI-D1870 (data not shown), further supporting role of RSKs in the phosphorylation of Bassoon at this residue.Our in vitro experiments showed that binding of 14-3-3 to Bassoon critically depends on the phosphorylation of Bassoon S2845. Multiple unbiased proteomic studies identified the phsophorylation of Bassoon S2845 The α-pS2845 Bsn antibody was instrumental for identification of the RSK family kinases to phosphorylate the S2845 of Bassoon in in vitro phosphorylation assays. RSK1 and 3 were about twice as potent compared to RSK2 and 4 in vitro. All RSKs genes are expressed in the nervous system, having restricted but overlapping developmental and regional expression patterns S2845A suggesting that 14-3-3 binding favors dissociation of Bassoon from its presynaptic anchor. It is also possible 14-3-3 associates with free Bassoon and interferes with its association. In either scenario phosphorylation of S2845 and binding of 14-3-3 decreases attachment of Bassoon to the presynaptic CAZ. The complex and tightly interwoven character of this presynaptic protein meshwork is caused by diverse interactions between the single CAZ constituents 14-3-3 proteins frequently function as dimers 1) to induce a conformational change of target protein by interacting with two interaction sites on the same protein and clamping it, 2) to stabilize protein complex formation by bridging two 14-3-3 interaction partners, or 3) to inhibit protein-protein interactions by competing for binding sites During synaptogenesis membrane-associated Bassoon is transported on Piccolo-Bassoon transport vesicles from the cell bodies to the distal axons to be inserted into nascent synapses


CRIM1 , ZNF71 , TLN1 , and SYNPO2 . Regarding the association of specific IgE to house dust mites, it was observed that intergenic SNPs nearby to OPRK1 and LOC730217 might be associated with Dermatophagoides pteronyssinus (D.p.) and Dermatophagoides farinae (D.f.) in asthmatics, respectively. In further pathway analysis, the phosphatidylinositol signaling system and adherens junction pathways were estimated to play a role in the regulation of total IgE levels in asthma. Although functional evaluations and replications of these results in other populations are needed, this GWAS of serum IgE in asthmatics could facilitate improved understanding of the role of the newly identified genetic variants in asthma and its related phenotypes.Immunoglobulin E (IgE) is one of the central players in asthma and allergic diseases. Although the serum IgE level, a useful endophenotype, is generally increased in patients with asthma, genetic factors influencing IgE regulation in asthma are still not fully understood. To identify the genetic variations associated with total serum and mite-specific IgEs in asthmatics, a genome-wide association study (GWAS) of 657,366 single nucleotide polymorphisms (SNPs) was performed in 877 Korean asthmatics. This study found that several new genes might be associated with total IgE in asthmatics, such as  CTLA4 and C11orf30-LRRC32 regions, have been shown to be associated with total serum IgE levels in patients with asthma FCER1A as a novel susceptibility locus. Furthermore, two other recent GWASs have found additional genes (such as IL13 and HLA-DQB1) that are associated with the total serum IgE Asthma, a chronic inflammatory respiratory disease, is characterized by bronchial hyperresponsiveness. Asthma and its related illnesses are complex diseases resulting from interactions among multiple genetic factors as well as environmental components interleukin 4 (IL4) and IL13, which stimulate B cells to produce IgE, genetic variants have been determined to be significantly associated with serum IgE in childhood asthma and atopy DPP10 and the C11orf30-LRRC32 region, may affect IgE levels in asthmatics IgE is a class of antibody that plays an essential role in immediate hypersensitivity response, which is the hallmark of allergic diseases including asthma Dermatophagoides pteronyssinus (D.p.) Dermatophagoides farinae (D.f.), are generally considered among the most implicated asthma triggers associated with blood allergen-specific IgEs It has been reported that asthma severity is positively correlated with serum concentration of both total IgE and specific IgE to 1) and/or airway hyperreactivity to <10 mg/mL of PC20 methacholine. Twenty-four common inhalant allergens, including dust mites (D.p. and D.f.), car fur, dog fur, cockroaches, grass, tree, pollens, ragweed, and the aspergillus species , were used for the skin-prick test. Atopy was defined as having a wheal reaction to an allergen that was equal to or greater than that to histamine (1 mg/ml), or that was 3 mm in diameter.A total of 877 asthma patients were recruited from Soonchunhyang University, Chungbuk National University, Chonnam National University, Seoul National University, and Chung-Ang University in Korea. All subjects provided written informed consent. In the case of children patients, their parents provided written informed consent before the study commenced. The study protocols were approved by an Institutional Review Board of the Soonchunhyang University Bucheon Hospital, a member of the National Biobank of Korea (IRB No. SCHBC_IRB_05_02). All patients met the criteria for clinical symptoms in accordance with the definition of asthma in the Global Initiative for Asthma (GINA) guidelines. All patients had airway reversibility, as documented by a positive bronchodilator response of >15% increase in forced expiratory volume in one second . UniCAP-specific IgE calibrators were used for the determination of specific IgE antibodies. Specific IgE (D.p and D.f.) was classified as 0-6, based on the semi-quantitatively expressed concentrations . For UniP-value <0.001. SNPs in chromosome X of males were excluded from HWE analysis. Although there were significantly deviated SNPs from HWE (as measured by an asymptotic test) in asthmatics, these SNPs were included in the association analysis in order not to miss any SNPs underlying both asthma and total or specific IgEs . X-chromosomal markers were also included in the association study. For the additive genetic model among males, genotypes of X chromosome were coded as “0”  or “2” . A total of 70,601 SNPs were excluded, and 442,098 SNP markers were examined for further association analysis.Genome-wide SNP genotyping of asthma subjects was performed using approximately 200 ng of genomic DNA from the subjects’ peripheral blood lymphocytes on Illumina’s Human660W-Quad BeadChip® , according to the manufacturer’s protocol. Quality controls of call rate (>98%) and high minor allele frequency (MAF >0.05) were measured. In addition, visual inspection of the genotype cluster image was performed for the SNPs with deviation from Hardy-Weinberg equilibrium (HWE) and with an association 2 statistics by 0.456) was computed as 1.003, indicating no significant deviation of the population stratification. For genome-wide evaluation, associations of genotype distributions were calculated using linear regression analysis for total IgE  association and logistic analysis for specific IgE association, adjusted for age , sex , and smoking status  as covariates, using HelixTree® software . Statistical power analysis of linear regression for total IgE was obtained from a two-tailed t-test; case-control analysis for specific IgE was obtained from the CaTS power calculator All subjects were of Korean ethnicity. Using the principal component analysis (PCA) for population stratification http://vortex.cs.wayne.edu/projects.htm) is represented by the corrected gamma P-value, as the P-value provided by an impact analysis calculated as the sum of the statistically significant number of genes showing biological meanings in the given pathway Statistical test used in the Pathway Express . First, we tested association with total serum IgE levels in asthmatics using linear regression analysis corrected for age, gender, and smoking status as covariates. In the case of genetic homogeneity of the study subjects, the measured genomic control inflation factor was λ = 1.003, indicating no inflation of the type I error. The quantile-quantile (Q–Q) plot for the association test with total IgE showed that distributions between observed and expected eme tail .P<1.0×10−5, CRIM1 , ZNF71 , TLN1 , and SYNPO2 , as susceptibility markers for total IgE levels in asthmatics (SYNPO2 in the chromosome 4q26 (P = 8.36×10−6 for rs1472066 to P = 0.0056 for rs1021377) when compared to other top signals , top signals  were detected as the most significant polymorphisms associated with the specific IgEs of D.p. and D.f. in asthmatics, respectively.In further association analyses with specific IgE to house dust mites , along whttp://vortex.cs.wayne.edu/projects.htm). Among the significantly associated SNPs with total IgE (P<0.001), only the SNPs in the gene regions were analyzed. The phosphatidylinositol signaling system (P = 2.89×10−18) and adherens junction (P = 1.57×10−13) pathways were estimated to play a role in the regulation of total IgE levels in asthmatics  epithelial cells, and (4) lung function. Although IgE and its binding to FcεRI (high-affinity Fc receptor for IgE) regulates the activation of mast cells and basophils mainly in the first and second mechanisms, IgE is associated with a more complicated network of proteins IgE is considered an important target in the treatment of asthma. Furthermore, anti-IgE therapy, i.e., using a humanized antibody drug of Omalizumab against IgE, has been demonstrated to be efficacious for the management of asthma as well as allergic diseases CRIM1, ZNF71, TLN1, and SYNPO2. Recently, SYNPO2 has been reported to be significantly associated with airway hyperresponsiveness in patients with asthma http://www.umd.be/HSF/) TLN1, rs1472066 and rs1038770 of SYNPO2 were predicted as potential branch point (BP) sites for alternative splicing , which is a limitation of this study. According to the power calculation, this failure to reach a genome-wide signal might be due to an underpowered study  for total serum IgE levels in asthma patients. Among the several well-known IgE-influencing genes  CD28 SNPs and total IgE in asthmatics . Serum total IgE levels have been elucidated to be correlated with CD28, an important regulator of T-cell activation and subsequent IgE production C11orf30-LRRC32 region were matched with those of this GWAS), and a diversity of clinical phenotypes among asthma patients.Recent results from GWASs on total serum IgE levels have identified associated genes, including NPSR1 and IRAK3 were also observed to be associated with total IgE among asthmatics in this study  have been found to be associated with increased serum IgE levels or asthma, and have been functionally evaluated to be distinctively distributed between protein isoforms in bronchial biopsies from healthy and asthmatic subjects NPSR1 and its genetic variations with total IgE level and/or asthma have also been reported IRAK3 rs1821777, which showed a nominal signal (P = 0.03) and in Sardinians (P = 0.001) of Italy IRAK3 SNPs  and in a meta-analysis of Spanish and Sardinian populations (P = 0.013) IRAK3 with total IgE in asthmatics . The result showed that the phosphatidylinositol signaling system (P = 2.89×10−18), as supported by previous strong evidence P = 1.57×10−13) pathways might play a role in the regulation of total IgE levels in asthmatics  were shown to have functional connectivity , and specific IgE (D.f.). The observed P-value (y-axis) is compared with the expected P-value  for (A) total IgE, (B) specific IgE (D.p.), and (C) specific IgE (D.f.).(DOC)Click here for additional data file.Figure S2Regional association plot and LD of CRIM1 for the total IgE in asthmatics. GWAS associations (−log10P) of SNPs across approximately 310 kb region around CRIM1 in the chromosome 2p21 are shown. Relatively strong associations are shown as large black circles; relatively less significances as small gray diamonds.(DOC)Click here for additional data file.Figure S3Regional association plot and LD of ZNF71 for the total IgE in asthmatics. GWAS associations (−log10P) of SNPs across approximately 245 kb region around ZNF71 in the chromosome 19q13.4 are shown. Relatively strong associations are shown as large black circles; relatively less significances as small gray diamonds.(DOC)Click here for additional data file.Figure S4Regional association plot and LD of TLN1 for the total IgE in asthmatics. GWAS associations (−log10P) of SNPs across approximately 126 kb region around TLN1 in the chromosome 9p13 are shown. Relatively strong associations are shown as large black circles; relatively less significances as small gray diamonds.(DOC)Click here for additional data file.Figure S5Manhattan plots of specific IgE against house dust mites (D.p. and D.f.). Associations for the specific IgE against (A) D.p. and (B) D.f. in asthma patients are obtained by correcting for age, gender, and smoking status as covariates.(DOC)Click here for additional data file.Figure S6In silico analysis of top 6 SNPs in intron regions. (A) Potential branch point (BP) sites are predicted by the change between major allele and minor allele using the Human Splicing Finder (http://www.umd.be/HSF/). (B) A schematic plot of alternative splicing by the predicted additional BP site. The picture of consensus sequence is modified from Desmet et al. Nucleic Acids Research 37(9): e67, 2009.(DOC)Click here for additional data file.Table S1Classification of specific IgE.(DOC)Click here for additional data file.Table S2Top 100 SNPs associated with total IgE of asthmatics in the GWAS.(DOC)Click here for additional data file.Table S3Top 100 SNPs associated with specific IgE for D.p. in the GWAS.(DOC)Click here for additional data file.Table S4Top 100 SNPs associated with specific IgE for D.f. in the GWAS.(DOC)Click here for additional data file.Table S5Pathway analysis for significantly associated SNPs (P<0.001), only in the gene regions from the results of GWAS, on total IgE in asthmatics.(DOC)Click here for additional data file.Table S6Statistical power for the analysis in each subgroup of study subjects.(DOC)Click here for additional data file.Table S7Genetic associations from this GWAS compared to the previously identified SNPs from other GWASs.(DOC)Click here for additional data file.Table S8SNPs of other potential genes (but not well-known IgE-influencing genes) associated with total IgE in asthmatics (P<0.01).(DOC)Click here for additional data file.Table S9Genes based clustering from GRAIL.(DOC)Click here for additional data file.


Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus. Plasmodium sp. More than 100 species that can infect vertebrates have been identified in nature and Plasmodium falciparum is the most lethal among them Malaria, one of the most severe parasitic diseases, is caused by The life cycle of the malaria parasite is extremely complex and is characterized by an asexual phase, which occurs inside the vertebrate host, and a sexual phase that develops in the mosquito vector Plasmodium falciparum (PfPKA), which is also involved in parasite invasion Plasmodium sp.The mechanism of erythrocyte invasion by merozoites has been studied by several groups and it is characterized as a multi-step process involving host erythrocyte membrane involution and deformation, leading to invagination and cell swallowing Aedes aegypti contaminated with Plasmodium gallinaceum by blocking accumulation of sporozoites in mosquito salivary glands without affecting vector survival P. falciparum entry is scarce. In this report, we describe studies aimed at identifying the molecular mechanisms induced by Ang II that are involved in the modulation of the P. falciparum erythrocytic cycle.A possible candidate is angiotensin II (Ang II), a peptide that belongs to the renin-angiotensin system (RAS), which has confirmed proinflammatory effects and antiplasmodial activity when injected in the hemolymph of P. falciparum erythrocytic cycle, increasing concentrations of the peptide (10−12–10−6 M) were assayed in vitro against a synchronized culture in the schizont form of the W2 strain of P. falciparum maintaining 5% hematocrit and 2% parasitemia. Twenty-four hours after treatment, Ang II was able to reduce parasite invasion in a dose-dependent manner with the maximum effect observed at 10−8 M, promoting 47% of inhibition (−8 M Ang II was not changed by different inoculum.To assess the effect of Ang II in the hibition . The sch−8 M Ang II for 4 consecutive days (2 complete parasite life cycles) with an initial inoculum of 0.5%. The inhibitory effect of Ang II was maintained throughout the experiment (−8 M) for 4 consecutive days, as described before. The culture was then split and maintained for 11 days without Ang II. Parasitemia was then evaluated. The pre-incubation treatment decreased parasitemia compared with the control , the positive control used. In agreement with our results, AT1 receptor isoforms with molecular weight ranging from 60 to 73 kDa have been observed in the kidneys of the Sprague-Dawley rat, desert rodent Meriones crassus and human ovarian carcinoma cells To verify the presence of Ang II receptors on the surface of erythrocytes, membrane fractions were subjected to immunoblotting analysis. Rat renal cortex homogenate was used as positive control because it is well known that this preparation has AT−6 M losartan, 10−7 M PD123319 or 10−7 M A779, antagonists of AT1, AT2 and MAS receptors, respectively. These antagonists were added 30 min before the addition of Ang II and maintained during the course of the reaction with Ang II. The inhibitory effect of Ang II on parasite invasion was partially abolished when PD123319 was added and completely abolished in the presence of A779. The addition of PD123319 or A779 without Ang II did not change the parasite invasion . The supernatant was collected and analyzed by mass spectroscopy as described in the −5 M Ang II. Ang II is metabolized under both conditions (−12–10−6 M) were added to the invasion assay, parasite invasion was reduced in a dose-dependent manner with the maximum effect observed at a concentration of 10−8 M, achieving 41% of inhibition . When both Ang II and Ang-(1–7) were assayed concomitantly against the P. falciparum culture, the same level of inhibition was observed, demonstrating a non-additive effect of both peptides (−7 M), a specific antagonist of MAS receptor, known to transduce Ang-(1–7) signaling, completely reversed the effect of Ang-(1–7). PD123319 (10−7 M) was also able to partially reduce the inhibitory effect of Ang-(1–7) on the parasite infection. On the other hand, losartan (10−6 M) did not change the inhibitory effect of Ang-(1–7)  . Thus, tng-(1–7)  and 5C.−12 M) (−8 M. Ang IV has a more potent inhibitory effect compared with Ang-(1–7) and Ang II  . Ang II d Ang II  and its losartan .P. falciparun also expresses PKA (PfPKA) we measured the effect of Ang II or Ang-(1–7) on erythrocyte PKA activity using the membrane preparation (−8 M Ang II or 10−8 M Ang-(1–7) inhibited the basal PKA activity and this effect was reversed by 10−7 M A779  increased the level of infection , a well-known protein kinase C (PKC) activator, did not change the level of infection.It is known that erythrocyte Gs protein is a crucial player during the malaria blood cycle paration . In this7 M A779 . In addinfection . On the on assay . 10−12 MP. falciparum blood stage Plasmodium sp. have been well described, the physiological aspects involving host components in this process are still poorly understood. Our results provide the first evidence of the important role of RAS components in erythrocyte infection by P. falciparum. Here we show that Ang II, through its carboxypeptidase-mediated metabolic product, Ang-(1–7), decreases infection of new erythrocytes during development of the P. falciparum blood stage. This effect of Ang-(1–7) is specific and probably involves a MAS-mediated PKA inhibition.The invasive stages of the malaria parasite are considered attractive targets for the development of antimalarial drugs and vaccines, especially the merozoite invasion of erythrocytes during the P. falciparum? Recently, local tissue generation of Ang II has been described; this peptide could have paracrine or autocrine effects P. falciparum.The first question that arises from our results is: What is the source of Ang II involved in the modulation of the erythrocyte cycle of P. falciparum blood stage are mediated by Ang-(1–7) reveals a new role for the (ACE2)/Ang-(1–7)/MAS receptor axis. This idea is strengthened by the observation that concentrations of Ang-(1–7) that inhibit parasite invasion (10−12–10−10 M) are compatible with the physiological serum concentration of Ang-(1–7) The observation that the effects of Ang II on Aedes aegypti infected with Plasmodium gallinaceum. However, the mechanism involved in antiplasmodial activity in the human host has not yet been determined. In the present study, we showed that the antiplasmodial effect of Ang II on the P. falciparum erythrocytic cycle is mediated by its conversion into Ang-(1–7).It is well known that Ang II promotes endothelial dysfunction P. falciparum erythrocytic cycle is what is the molecular mechanism involved in this process? The possibility of explaining our results based on the effect of Ang II on the plasma membrane structure, as shown by Maciel et al. P. falciparum erythrocytic cycle were completely abolished by specific receptor antagonists, indicating a correlation between the structure and function of angiotensin peptides in the effect observed in the present work.An important question regarding the effect of angiotensin peptides on the 1, AT2 and MAS (or AT(1–7)), in erythrocyte membranes, and their selective blockage by specific antagonists revealed important clues about their involvement in parasite invasion. The observation that Ang II and Ang-(1–7) effects are blocked by A779 and not changed by losartan show the involvement of the MAS receptor in the modulation of the P. falciparum erythrocytic cycle. This agrees with the observation that Ang II is metabolized to Ang-(1–7) in non-infected or even in infected cultures in which Ang-(1–7) formation is reduced. In addition, the effect of these peptides was partially abolished by the AT2 receptor antagonist, PD123319. These results indicate that MAS and AT2 receptors could be working in a coordinated manner to mediate the effect of Ang-(1–7). Similar results have been obtained in other systems such as in the modulation of blood pressure where the effect of Ang-(1–7) depends on the interaction of both receptors 4) Here, we clearly show the presence of angiotensin receptors, AT1, AT2 and MAS belong to the GPCR family. MAS receptor was recently cloned, but the signaling pathways activated by this receptor are not well established P. falciparum erythrocytic cycle. In agreement with the role of PKA in the P. falciparum erythrocytic cycle, it was observed that PKA inhibitor was also able to inhibit the infection, whereas the permeable analogue of cAMP increases it. This effect is specific for PKA because the activation of PKC by PMA did not change the P. falciparum erythrocytic cycle. These results indicate that the inhibitory effect of Ang-(1–7) on the P. falciparum erythrocytic cycle is mediated by the inhibition of PKA. P. falciparum PKA (PfPKA) has been recognized to have an important role in the development of blood-stage parasites with highest activity in schizonts P. falciparum erythrocytic cycle by modulating erythrocyte PKA activity. In addition, the α subunit of the Gs protein (Gαs) was found in detergent-resistant rafts of erythrocyte membrane Angiotensin peptide receptors ATP. falciparum blood stage. These results open new avenues for the development of new antimalarial drugs.Together, the results obtained here reveal a potential role of RAS in the Collection of human blood samples for this study was conducted according to the protocols approved by the Research Ethics Committee of the Hospital Universitário Clementino Fraga Filho from Federal University of Rio de Janeiro (Permit Number 074/10). All patients provided written informed consent for the collection of samples and subsequent use. The use of this material follows long-standing protocols and has not been associated with any adverse or other unforeseen events and no data of relevance to specific patients has been generated. Also, this study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Ethics Committee of Federal University of Rio de Janeiro (Permit Number IBCCF098).N-2-Hydroxyethylpiperazine N-2-ethanesulfonic acid (HEPES), (tris-hydroxymethyl)-aminomethane (Tris), glucose, sodium bicarbonate, hypoxanthine, adenosine triphosphate , magnesium chloride, ethylenediaminetetraacetic acid (EDTA), histone, protein kinase A inhibitor peptide (PKAi), PD123319, Ang II , Ang IV  and Ang-(1–7)  were purchased from Sigma Aldrich Co. . Percoll was purchased from GE Healthcare . [32Pi]Pi was obtained from the Brazilian Institute of Energetic and Nuclear Research, São Paulo, Brazil. [γ-32Pi]ATP was synthesized according to the procedures described by Maia et al. 1 receptor selective antagonist, losartan, was obtained from Medley S.A.  and A779, the MAS receptor blocker, was kindly provided by Dr Robson Augusto Souza dos Santos . All other reagents were of the highest purity available.P. falciparum W2 strain, characterized as chloroquine resistant and mefloquine sensitive Erythrocytic asexual stages of Parasite mature forms were collected using a Percoll/sorbitol gradient as described in standard protocols m/z = 904.47), angiotensin I (m/z = 1296.69), Glu1-fibrinopeptide B (m/z = 1570.68), and adrenocorticotropic hormone (18–39) (m/z = 2465.20). The spectrum files (T2D) were generated from the raw (or native) mass spectrometry data using Data Explorer Software v.4.8 (Applied Biosystems). To monitor angiotensin metabolism, the areas of the peaks for different angiotensin forms  present in the same spectrum, masses 1046.19, 774.92 and 899.02 Da, respectively, were obtained and their summation was arbitrarily assigned as 100%, making it possible to observe the relative changes of each form as a function of time.C18 ZipTip micropipette tips were used for desalting angiotensin peptides in the medium. The tips were first activated with acetonitrile and equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) in water. The samples were aspirated and dispensed for eight cycles and the tips were washed with 0.1% (v/v) TFA in water three times. The peptides retained on the tips were eluted using 1.5 µl of a mixture of 50% acetonitrile and 0.1% (v/v) TFA in water. Then, 0.3 µl of this eluate was immediately spotted on the ABI 192-target MALDI plate  by cocrystallization with 0.3 µl of an α-cyano-4-hydroxycinnamic acid matrix  TFA in water). Raw data for angiotensin forms were obtained on the 4700 Proteomics Analyzer . Mass spectrometry data were acquired in positive and reflectron mode, mass range 600–1100 Da, using a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser with a 200-Hz repetition rate. Typically, 1600 shots were accumulated for spectra. External calibration was performed using a mixture of four peptides: des-Arg1-bradykinin , and cells were lysed by freezing in liquid nitrogen and thawing naturally at room temperature. The resulting fluffy pellet was re-suspended and homogenized in 5 mM Hepes–1 mM EDTA buffer (pH 7.4), and hemoglobin was washed out by centrifugation at 10,409×g, for 17 min, at 4°C. A second wash was carried out using 5 mM Hepes buffer (pH 7.4) and then a final wash with a buffer containing 10 mM Tris, 130 mM KCl, 0.5 mM MgCl2 and 50 mM CaCl2. The clarified preparation was re-suspended in a minimal amount of the same buffer. The final protein concentration in this enriched membrane fraction was determined by the Folin phenol method Erythrocyte membranes were prepared from fresh human A3VO4, 5 mM Na4P2O7 and 1× protease inhibitor cocktail, Roche, IN, USA). Aliquots containing 50, 150 or 200 µg protein were re-suspended in SDS-PAGE loading buffer, resolved on SDS 9% acrylamide gels and transferred onto Immobilon-P membranes . After blocking with 5% non-fat dry milk/Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, membranes were probed overnight, at 4°C, with primary specific antibodies, followed by horseradish peroxidase-labeled secondary antibodies (Amersham Biosciences) and visualized with ECL®-plus reagent . Rabbit polyclonal anti-human MAS1 was purchased from Abcam ; mouse monoclonal anti-human AT1 and rabbit polyclonal anti-human AT2 were obtained from Santa Cruz Biotechnology, Inc. .The membrane preparation was re-suspended and lysed on ice for 20 min in Ripa buffer  freshly supplemented with phosphatase and protease inhibitors , using histone as substrate. The composition of the reaction medium was 4 mM MgCl2, 20 mM HEPES–Tris (pH 7.0), 1.5 mg/ml histone and 0.7 mg/ml protein. After 10 min, the reaction was stopped with 40% trichloroacetic acid (TCA) and the sample was immediately placed on ice. An aliquot (0.1 ml) was filtered through a Millipore filter (0.45 µm pore size) and washed with ice-cold 20% TCA solution and 0.1 M phosphate buffer (pH 7.0). The radioactivity was quantified using a liquid scintillation counter. The specific PKA activity was calculated from the difference between the activity in the absence and in the presence of 10−8 M PKAi The PKA activity of isolated erythrocyte membranes was measured by protein kinase inhibitor-sensitive incorporation of [t-test. Statistical analysis was performed using absolute values.Each experiment was performed in independent cell suspensions. The data were analyzed by two-way analysis of variance (ANOVA), considering the treatments as factors. The significance of the differences was verified by the Bonferroni


Background: Protein oligomers may be toxic, but are unstable, difficult to study, and challenging therapeutic targets.Results: Oligomers can be stabilized and used to generate and isolate antibodies with high selectivity for amyloid oligomers.Conclusion: Oligomer stabilization is a powerful strategy to study and target amyloid oligomers.Significance: We describe methods to generate oligomer-specific antibodies for molecular characterization, diagnosis and treatment of amyloid disorders. The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers . These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers. A major challenge in future healthcare is managing the growing number of patients affected by protein conformational disorders. Protein misfolding is implicated in the pathogenesis of neurodegenerative pathologies like Alzheimer, Huntington, and Parkinson diseases, the spongiform encephalopathies, age-related diabetes mellitus, cystatin C amyloidosis with brain hemorrhage, and a number of other conditions characterized by tissue amyloid deposition. To date, at least 27 different amyloidogenic proteins involved in human disease have been identified .The molecular pathophysiological process in the amyloid disorders usually involves the transformation of a soluble functional monomeric protein into potentially toxic aggregates/oligomers and insoluble amyloid fibrils , 3. SeveWe have previously shown that a stabilization of the monomeric form of cystatin C by insertion of a disulfide bridge will stop its transition into amyloid fibrils by preventing domain swapping of the monomer . In the 384 microplate absorbance reader was from Molecular Devices . Ultrafiltration devices  were from A-filter . Microtiter plates  and polyolefin sealing tapes were from Nunc . Polypropylene tubes, 0.5 and 1.5 ml, were purchased from Applied Biosystems and VWR , respectively. Nitrocellulose membranes were obtained from Ancos . NuPAGE polyacrylamide gels, sample loading buffer, Mark12-unstained standards, phosphate-buffered saline , and polyclonal rabbit anti-oligomer antibodies (A11) were obtained from Invitrogen. Silver staining kit, ChemiDoc XRS+ system, and ImageLab software  were purchased from Bio-Rad. Dried nonfat milk was from Semper . Polyclonal rabbit anti-human cystatin C antibodies, horseradish peroxidase (HRP)-labeled polyclonal swine antibodies against rabbit immunoglobulins and HRP-labeled polyclonal rabbit antibodies against mouse immunoglobulins were from Dako . Immobilon Western enhanced chemiluminescence (ECL) substrate was from Millipore . Papain and legumain substrates were acquired from Bachem Feinchemikalien . Agarose (SeaKem LE) was purchased from BioWhittaker Molecular Applications . Coomassie Brilliant Blue R-250 was from VWR. Brij-35 was obtained from Kebo . Guanidine hydrochloride  was from Sigma. All other reagents were of at least reagent grade and, unless specified, obtained from Sigma.Chromatography columns and resins, gel filtration protein standards, and the ÄKTA FPLC system were obtained from GE Healthcare. Analytical gel filtrations were performed on Superdex 75 PC 3.2/30 or Superdex 200 PC 3.2/30 columns in a HPLC system, including a 600e controller and a 2996 Photodiode Array detector from Waters . Fluorimeter (Fluoroskan Ascent), spin desalt columns, dialysis cassettes ),Escherichia coli extracts using ion-exchange and size exclusion chromatography (SEC), subsequently stored as lyophilized powder at room temperature, and reconstituted in appropriate buffers just before use. The L68Q cystatin C variant was purified from solubilized E. coli inclusion bodies as previously described  of 0.83  of 1.35 was assumed for purified rabbit immunoglobulins (Protein concentrations of recombinant cystatin C variants were measured by UV absorption spectroscopy at 280 nm in a NanoDrop 2000 spectrophotometer (Fisher Scientific) using a mass extinction coefficient . Portions of 10 μl of the mixtures were distributed into the 25-μl wells of 384-well microtiter plates and either of the reducing agents dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine was added to each well to final concentrations  ranging from 0 to 150 μm (i.e. up to 10-fold molar excess of the reducing agents). The plates were sealed with sealing tapes and incubated at 37 or 40 °C. For each time point  the solutions were analyzed by agarose gel electrophoresis, as described previously (etc.) were established in 10.4-μl assay volumes by increasing the cystatin C concentration to 3 mg/ml under the optimal dimer-inducing conditions, i.e. equimolar DTT and 1 m GdnHCl, and decreasing the incubation temperature and time, with the results followed by SEC. The incubation mixtures were transferred to 0.5-ml tubes and centrifuged at 10,000 × g for 10 min and 5 μl were analyzed in a HPLC system using a Superdex 75 PC 3.2/30 column run in 150 mm ammonium bicarbonate, pH 7.8, with a flow rate of 0.1 ml/min and the eluate monitored by its absorption at 280 nm. In scale-up experiments of oligomer-inducing mixtures, using the optimized conditions, 0.5-ml incubation volumes showed identical chromatograms compared with 10.4-μl volumes.Lyophilized stab1 cystatin C was dissolved in PBS and aliquots of the stock solution (1 mg/ml) were added to solutions of GdnHCl in PBS, giving final concentrations of GdnHCl varying from 0 to 1.0 eviously , with upeviously . The optm stock solution, giving final concentrations of GdnHCl of 1 m. To this mixture was added freshly dissolved DTT in PBS at 0.9 mg/ml (6.25 mm) giving final concentrations of DTT and cystatin C of 225 μm (corresponding to a cystatin C concentration of 3 mg/ml), and the tubes were kept at room temperature for 2 h. After centrifugation at 10,000 × g for 10 min, 0.5-ml samples were injected in an ÄKTA FPLC system running a Superdex 75 GL 10/300 column in 150 mm ammonium bicarbonate, pH 7.8, with a flow rate of 0.5 ml/min and the eluate was monitored by its absorption at 280 nm. Fractions of 0.5 ml were collected and the tubes stored open to ambient air overnight at 4 °C to allow re-formation of disulfide bonds by spontaneous oxidation. Fractions were analyzed by nonreducing silver-stained SDS-PAGE as described below, pooled, and concentrated to 1–3 mg/ml in ultrafiltration devices . The protein concentration was measured, samples were aliquoted and either lyophilized or stored at 4 or −20 °C.Lyophilized stab1 cystatin C was reconstituted in 0.5 ml of PBS in 1.5-ml tubes, the concentration was measured in 1.5-μl droplets using a NanoDrop 2000 spectrophotometer, and concentrations were adjusted before the addition of GdnHCl from a 8 Purified monomers/oligomers were analyzed by SDS-PAGE using pre-cast NuPAGE BisTris gradient gels (4–12%). Samples were boiled, without the addition of reducing agents, in 2% SDS loading buffer according to the manufacturer and the gels run with the MES buffer system, at 200 V for 35 min. The gels were either stained with Coomassie or silver-stained as described by the manufacturer. The gels were digitized in a ChemiDoc system and analyzed using the included ImageLab software.m ammonium bicarbonate, pH 7.8 (nonreducing buffer), were mixed 1:1 with the matrix 2,6-dihydroxyacetophenone and 1 μl was applied to the ground steel plate before analysis by a positive method operating in reflectron mode and a 25 kV acceleration voltage. The analyses were calibrated using external calibrants  and re-calibrated using wt cystatin C .Isolated monomers and oligomers were analyzed by mass spectrometry using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) ultrafleXtreme mass spectrometer . Samples at 1 mg/ml in 150 mg for 10 min and analyzed in a HPLC system using a Superdex 200 PC 3.2/30 column equilibrated in 150 mm ammonium bicarbonate, pH 7.8, with a flow rate of 0.1 ml/min and the eluate was monitored by its absorption at 280 nm. The column was calibrated with protein standards . A calibration curve was constructed from calibrator protein elution volume peaks and the mass of each cystatin C oligomer calculated from its elution volume.Eight-μl samples of purified monomers/oligomers were centrifuged at 10,000 × m ammonium bicarbonate, pH 7.8, and 5 μl were applied to glow-discharged carbon-coated copper grids (400 mesh) and after 1 min of adsorption the excess liquid was removed with filter paper. The samples were negatively stained with 5 μl of 2% (w/v) aqueous uranyl acetate for 30 s before the staining solution was removed with filter paper. The samples were examined with a Technai G2 Spirit electron microscope , operating at an excitation voltage of 80 kV and equipped with an Eagle 16 megapixel CCD camera. Electron micrographs were analyzed using the software ImageJ (Protein samples were diluted 1:10 or 1:40 in 150 me ImageJ .2O. The sample solutions (5 μl) were deposited on a freshly cleaved mica surface and dried before visualization. The intermittent (air) contact mode imaging was carried out with a silicon nitride cantilever. The images were analyzed using the software package Gwyddion  (Atomic force microscopy (AFM) measurements were carried out using the NanoWizard II system . Oligomer samples were prepared as described above and diluted 1:2000 using double-distilled Hon 2.30)  and the m sodium phosphate, pH 6.5, 4 μm DTT, 4 mm EDTA). Five-μl samples of purified stab1 monomers/oligomers or monomeric wt cystatin C at equal mass concentrations in 150 mm ammonium bicarbonate, pH 7.8, were added to the papain solution and the mixtures preincubated for 10 min at 37 °C. Twenty microliters of the synthetic peptide substrate Z-Phe-Arg-NHMec (from a 200 μm stock solution in 0.01% (w/v) Brij-35) were added and fluorescence was measured every 30 s at excitation and emission wavelengths of 355 and 460 nm, respectively. All measurements were carried out in duplicates. Full enzymatic activity (A) was defined as the mean of fluorescence values obtained after a 20-min substrate development in mixtures without the addition of monomeric, or oligomeric, cystatin C. Essentially the same protocol was employed in legumain inhibition assays using the fluorogenic substrate Z-Ala-Ala-Asn-AMC at 20 μm and assay buffer composed of 100 mm sodium citrate, pH 5.8, 4 μm DTT, 1 mm EDTA, and 0.1% (w/v) CHAPS .The inhibitory capacity of purified and stabilized cystatin C oligomers was measured using the cysteine proteases papain and legumain. When setting up the assays , enzymesactivity A was defm sodium acetate, pH 4.0, 100 mm NaCl, and the protein concentration adjusted to 1 mg/ml. Purified stab1 cystatin C monomers and oligomers were subjected to buffer exchange into the sodium acetate buffer using spin columns (0.5 ml) with desalting resins according to the manufacturer's instructions and the protein concentration was adjusted to 1 mg/ml. Ten-μl samples were distributed into the wells of 384-well microtiter plates and the plates placed in a thermoshaker operating at 48 °C and at a speed of 900 rpm in orbits of 2 mm. HMW-oligomers/fibrils were visualized by electron microscopy as described above, or nonreducing SDS-PAGE using silver-stained NuPAGE gels as described above, but without boiling samples and with a low (0.1% (w/v)) SDS sample buffer, according to Ref. Wild type cystatin C was reconstituted in 50 mm Tris, pH 7.4, 150 mm NaCl) at −20 °C. The specific reactivity of antisera and immunoglobulin fractions against monomeric, dimeric, and HMW-oligomeric cystatin C was tested by Ouchterlony radial immunodiffusion as described below.Immunizations were performed according to the ethical requirements and in-house protocols at Dako Denmark A/S. Prepared and isolated as described above, the stabilized dimers and HMW-oligomers, respectively, were used as immunogens in two groups of rabbits. Each group received initial and repeated booster injections of 0.1 mg of dimeric or 0.05 mg of HMW-oligomeric cystatin C. The immunoglobulin fractions of the antisera were isolated using standard ammonium sulfate precipitation and protein A-Sepharose fractionation, according to the instructions of the manufacturer. The immunoglobulins were stored in neutral buffer , dialyzed against PBS, and stored at −20 °C.To remove monomer-binding antibodies from the oligomer-reactive immunoglobulin fractions, a two-step purification procedure, illustrated in The Ouchterlony procedure was used for in-gel immunodiffusion assays .i.e. stabilized monomers and dimers, at equal final concentrations) were mixed in 10-μl volumes in wells of 384-well plates and allowed to react at room temperature for 30 min. Ten-μl samples were applied to the gel and electrophoresed at 250 V for 30 min at 4 °C. Gels were stained with Coomassie and band patterns were digitized by optical scanning in an EPSON 1680 Pro flatbed scanner. Protein band patterns were subjected to densitometric analyses using ImageJ software.Agarose gel electrophoresis was preformed as described . AntibodThe dot blot procedures were performed according to the protocol from the manufacturer of the A11 antibodies. Briefly, 5-μl protein samples were applied to nitrocellulose membranes and allowed to dry. For A11 dot blots, the sample concentration was 1 mg/ml, and for cystatin C blots the samples were first diluted 1:25. The membranes were blocked in a solution containing 10% (w/v) milk proteins and probed with A11, polyclonal rabbit anti-human cystatin C antibodies (Dako) or the novel oligomer-specific antibodies. After incubation with HRP-labeled polyclonal swine antibodies against rabbit immunoglobulins, membranes were developed with ECL substrate and digitized in a ChemiDoc detection system.m ammonium bicarbonate, pH 7.8) was 0.3 ml/min. The cystatin C concentration in the chromatography fractions containing monomeric or dimeric cystatin C was measured using the ELISA system as previously described  introduced at strategic positions by site-directed mutagenesis, forming a disulfide link between the adjacent β2- and β3-strands, which (in wt cystatin C) separate during domain swapping and dimer formation  intact, and only reduce the additional (solvent-exposed) disulfide bridge to enable domain swapping of the monomeric protein. Indeed, after screening a number of different conditions, it was evident that when we used neutral buffers, pH 7.4, mild denaturation (0.5 or 1.0 m GdnHCl) and the addition of exactly 1:1 molar equivalents of DTT, we observed dimer formation without precipitation, as evident from agarose gel electrophoresis .We employed a miniaturized escribed  to find ormation . In the teines L4C/G69C inA). The yield of some oligomers was low, but, after ultrafiltration and re-chromatography, high enough to permit their characterization. Fractionation of the incubation mixtures by gel filtration in ammonium bicarbonate buffers enabled not only isolation of oligomers, but also removal of salts and oxidized DTT. All of the oligomers were SDS-stable (B), but DTT-sensitive, in SDS-PAGE (data not shown), indicating that intermolecular disulfide bridges stabilized them, thus supporting a model of domain swapping in the formation of oligomers  was utilized to establish the optimal protocol for the induction of not only dimers, but also trimers and higher-order oligomers. This was accomplished by increasing the protein concentration, still retaining the 1:1 molar equivalents of DTT, and decreasing the temperature and incubation time to minimize protein loss by precipitation. By incubating mixtures of GdnHCl and DTT-treated stab1 cystatin C at 3 mg/ml in 0.5-ml volumes at room temperature, we were able to obtain and purify a number of cystatin C oligomers using preparative gel filtration A. The yiS-stable B, but DTligomers , 21. ConB). Tetramers were obtained in some preparative runs and observed in some decamer fractions (C). The HMW-oligomers migrated in SDS-PAGE as a diffuse band with a molecular mass of >200 kDa, similar to the annular oligomers formed from wt and L68Q cystatin C, as previously described . The decamer sample contained slightly smaller circular objects, with a diameter of 9–13 nm (B). A significant number of both the HMW-oligomeric and decameric objects displayed central cavities. The isolated HMW-oligomers and decamers were further characterized by AFM and, as shown in C and D, the cross-sectional analysis showed a height of 1.2–1.4 nm for both objects. Crystallographic data for chicken cystatin and stab1 cystatin C indicate that this corresponds to monomolecular layers in both objects . The retained inhibitory capacity toward legumain was also shown for the stabilized trimer (99%) and was only partially lost in the decamer and HMW-oligomer fractions . In contrast, the dimer and the other oligomers showed complete loss of inhibitory capacity toward papain . Fig. 4B shows structural models explaining the loss of the papain inhibitory activity, whereas the legumain inhibitory activity is preserved.To investigate the conformational changes induced during cystatin C oligomerization, we tested the inhibitory capacity of the stabilized oligomers toward the two cysteine proteases papain and legumain, both of which are inhibited by monomeric cystatin C . The staFig. 4Amyloid oligomers might display unique epitopes common to all oligomers, but not present in monomers, and oligomers formed from a number of different amyloidogenic proteins can be detected with use of the A11 antibody . As showA, during the lag phase of fibril formation, already after 1 h of incubation at 48 °C in mildly denaturing buffers, pH 4.0, both wt and L68Q cystatin C form ring-shaped oligomers detectable by SDS-PAGE or electron microscopy. Prolonged incubation results in fibril formation, as shown for wt cystatin C after 3 weeks of incubation . When isolated stabilized cystatin C monomers, dimers, trimers, and decamers were incubated under these conditions, no formation of HMW-oligomers  or fibrils could be found. In contrast, nonstabilized monomeric wt cystatin C formed both HMW-oligomers and fibrils (A-C). This supports the notion that domain swapping, propagated in an open-ended fashion, is a prerequisite for formation of, not only fibrils . They also precipitated stabilized HMW-oligomers (data not shown). No difference could be observed between antibodies obtained by immunizing with stabilized dimers and those obtained by immunizing with stabilized HMW-oligomers. The cerebral hemorrhage-causing L68Q variant of cystatin C spontaneously forms dimers (A). The antibodies reacted strongly with HMW-oligomers, decamers, and trimers, and weakly with dimers, but not at all with monomers, in immunoblotting experiments (B).The specific antibodies from oligomer-immunized rabbits precipitated stabilized dimers, but not monomers, in double radial immunodiffusion A. They as dimers  and the s dimers A. The aneriments B.To assess the reactivity of the oligomer-specific antibodies in free solutions of dimeric and monomeric cystatin C, the following system was used. Mixtures of antibodies, stabilized monomers, and stabilized dimers were prepared and electrophoresed in agarose gels under native conditions. The results showed that dimers were the preferred antigen, although a minor monomer reactivity could be detected. As shown in g for 10 min, the mixtures were run in the SEC-ELISA system. A significant and dose-dependent decrease in cystatin C immunoreactivity in the fractions corresponding to dimers was observed, whereas monomeric cystatin C was less affected . A concomitant increase in cystatin C immunoreactivity was also observed in the void volume of the column (SEC exclusion >100 kDa).To test if the oligomer-specific antibodies would selectively target oligomers also in complex biological systems, we developed a sensitive system for the detection of monomers and dimers in blood plasma using gel filtration followed by immunodetection (SEC-ELISA), as described under “Experimental Procedures.” The oligomer-specific antibodies were, in increasing concentrations, added to equal aliquots of plasma containing 0.9 mg/liter of monomeric and 1.7 mg/liter of dimeric cystatin C and, after a 2-h incubation at room temperature and centrifugation at 10,000 × affected . Using aectively A. A concProtein misfolding underlies neurodegeneration and chronic diseases commonly affecting the aging population. An important example is Alzheimer disease, which is characterized by progressive cognitive decline and cerebral amyloid deposition, and is expected to affect more than 100 million individuals by 2050 . No dise2-microglobulin, ribonuclease A, T7 endonuclease I, antithrombin, the prion protein, and amyloid β , a mutation in the cystatin C gene produces a protein variant, L68Q cystatin C, which rapidly forms oligomers and amyloid deposition in the brain arteries, resulting in brain hemorrhage and death in early adult life . L68Q cyin vitro , 21. Theswapping , 21. Theswapping . By seleetramers . But foretramers . In addietramers . Furtherin vivo in different amyloid disorders. We have observed that the set is influenced by the incubation conditions. For example, the incubation conditions used for crystallization of monomeric cystatin C resulted in a population of nearly 100% cystatin C dimers  produced in our study are relevant to the set of oligomers present C dimers . The preC dimers , might rThe homogeneity and stability of the oligomers might allow studies of their detailed structures using x-ray diffraction. This would be of particular interest for the decamers, as their gross structure, as defined by EM and AFM, is similar to those of oligomers described for other amyloidogenic proteins , 40. It B, the two inhibitory sites (for papain or legumain) of monomeric cystatin C are well separated. Our previous structure-function studies of dimeric cystatin C . These results strongly suggest that the isolated oligomers are not random aggregates, but instead highly ordered, domain-swapped assemblies of monomeric cystatin C with the two disulfide bridges of native cystatin C intact in the subunits of the oligomers. The stability of the oligomers is most likely induced by novel intermolecular Cys47–Cys69 bonds, stabilizing the swapped domains.The stoichiometry of the reduction procedure used in the present study indicated that only the introduced disulfide bridge, preventing domain swapping in stab1 cystatin C, was reduced in the process, leaving the other two intramolecular disulfide bridges of native cystatin C intact . FurtherFig. 4statin C , 24 havestatin C . The resC). Therefore, at least for cystatin C, domain swapping seems to be required for all steps in the production of HMW-oligomers. This means that a substance, preventing (propagated) domain swapping, potentially would suppress the formation of larger oligomers from smaller ones and from monomeric cystatin C. It has previously been shown that formation of amyloid fibrils from monomeric wt cystatin C requires domain swapping . Such a mechanism, together with the monomolecular layer organization of subunits observed in AFM, might explain the toroidal (doughnut-shaped) morphology of the structures observed in EM and AFM. Similar morphologies have been described for the pore-like oligomeric assemblies described by others (The evident stability of the cystatin C oligomers also indicates that free cysteine thiols are absent, suggesting that all oligomers are closed by domain swapping head-to-tail D. Such ay others –41 and aThe blood of patients with HCCAA contains dimers of L68Q cystatin C and perhaps larger oligomers . The ava


Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton’s tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of  Recently, several high-throughput studies designed to analyze the global properties of phosphorylation networks in various model organisms have been reported . Though  (MS/MS) . Togethein vivo. This strategy, which we have dubbed ‘CEASAR’ because it provides a general framework for Connecting Enzymes And Substrates at Amino acid Resolution, was used to construct a high-resolution map of human phosphorylation networks that connects kinases to specific phosphorylation sites on their downstream substrates. In addition to in vivo phosphosites, such a map requires two key elements: (1) an activity-based phosphorylation network based on direct KSRs and (2) information about the consensus phosphorylation motif of each kinase in the network. To this end, we first employed human protein microarrays to experimentally determine substrates for 289 unique human kinases  for each of the 24 046 KSRs. Using a P-value of 0.05 as a threshold, we predicted 3656 refined KSRs (refKSRs)—involving 255 unique kinases and 742 substrate proteins—that were most likely to be physiologically relevant . For this analysis, we hypothesized that a kinase and its physiologically relevant substrate(s) are more likely to share similar tissue expression patterns, localize to the same subcellular compartment, and/or physically interact with one another, either directly or indirectly . We thenrelevant .in vivo—based mainly on global MS/MS analysis—was significantly improved in the refKSR list, increasing from 66% (1291/1967) in the rawKSR data set to 77% (567/741) in the refKSR data set (P=7.87 × 10−16). Second, cross-validation analysis revealed that the Bayesian approach increases the recovery rate of known KSRs over five-fold (P=1.37 × 10−15). Third, the enriched functions of the identified substrates for individual kinases showed better agreement with the known functions of their upstream kinases. Specifically, known functions were recovered for 53 kinases based on the enriched functions of their respective substrates, a 2.4-fold improvement over the rawKSR data set (P=2.1 × 10−11). Taken together, these findings suggest that the refKSR set, as compared with the rawKSR set, is significantly improved with regard to its physiological relevance.Three lines of computational evidence suggest that the Bayesian analysis improves the fidelity of our refKSR data set . First, To experimentally evaluate the refKSRs, we randomly selected 243 KSRs, involving 75 kinases and 136 substrates, and tested their relationships in transfected cells . Of the 2+ signaling pathways . These data suggest that many kinases may control protein stability, either directly or indirectly. To determine whether the observed changes in protein stability were due to direct phosphorylation of the substrate by its cognate kinase, a second round of validation experiments were conducted. During these experiments, we chose to focus on the action of endogenous kinases. Therefore, we selected 19 KSRs in which the kinases were restricted to protein kinase C (PKC), PKA, protein kinase B (PKB/Akt), and extracellular signal-regulated kinase 1 (Erk1/MAPK3). These kinases were chosen because (1) their endogenous activity can be both induced and inhibited pharmacologically and (2) the phosphorylation of their substrates can be readily detected using commercially available antibodies. For instance, in the first round of validation experiments, we observed that DDEFL1/ASAP3, a GTPase activating protein involved in cell differentiation and migration , underwepathways .In another case, co-expression of PKA caused a reduction in the levels of holocarboxylase synthetase (HLCS), an essential biotin ligase involved in chromatin remodeling and several metabolic processes , top. ClOverall, 15 of the 19 KSRs tested (79%) were confirmed in the second round validation, suggesting that in most cases the observed differences in substrate protein levels reflected direct phosphorylation by their corresponding kinase . Taken tMotif discovery based on Microarray and MS/MS), to systematically identify phosphorylation motifs  centered about these phosphosites were then binned into groups according to the identified KSRs. Though it is possible that some of the sequences within a given group contained phosphorylation sites that are recognized by several kinases, we assumed that, among all of the 15-mers for a particular kinase, those sequences recognized by the kinase-of-interest carry a statistically enriched consensus motif. Therefore, M3 is designed to utilize an iterative approach to identify statistically enriched consensus motifs within each group   are then compared with this PWM to identify the top-matched sequence. This sequence is then added to the seed sequences and the PWM is updated (Step 3). The entire process is repeated until the best score of the remaining sequences is below a cutoff, which is determined based on the distribution of matching scores for random sequences, or until the number of seed sequences is equal to the number of substrates of the kinase determined during the phosphorylation assay (Step 4). In the case of dual-specificity kinases, we separately considered motifs that contained pS/T or pY sites. Using this approach, we identified 300 consensus motifs for 284 human kinases, representing 55% of the human kinome  (in vitro phosphorylation reactions using purified wild-type (WT) and mutated substrate proteins. As illustrated in To experimentally evaluate the fidelity of these newly identified connections, we examined what effect mutation of the predicted phospho-acceptor site had on the substrates in the presence of kinase for three selected KSRs see . For exaS688A) . SimilarS688A) . To evalS688A) , suggestIgH locus . Moreover, when combined with information derived from our high-resolution phosphorylation map, this type of analysis can offer insights into the physiological consequences of the specific phosphorylation events underlying these connections. For instance, Btk is known to play an important role during BCR signaling while its binding partner, ARID3A, has been implicated in the transcriptional regulation of the gH locus . Recent gH locus . HowevergH locus . Based oTo test this hypothesis, we first validated the Btk→PKA and PKA→ARID3A KSRs in HeLa cells using the cell-based assays described above. In the case of PKA→ARID3A, either ectopic expression of PKA or activation of endogenous PKA led to increases in both ARID3A protein levels and the extent of PKA-mediated phosphorylation on ARID3A . Based oin vitro, as predicted by the high-resolution map (Y331F) completely abolishes the ability of Btk to enhance PKA-mediated phosphorylation of ARID3A  . The obs2+ release from intracellular stores in a dose-dependent manner following BCR activation, leading to a pronounced reduction in the extent of Ca2+ influx =34.4% and the recovery rate is 758/1156=65.6%. That being said, the possibility exists that the sites predicted to be phosphorylated on a given substrate are not necessarily the sites targeted by the kinase-of-interest during the protein microarray experiments. For instance, the true target site(s) may not yet be identified by MS/MS. Nevertheless, based on our validation experiments, each of the 5 kinase-phosphosite interactions examined were confirmed by protein mutagenesis experiments  were associated with changes in protein stability. We believe that one explanation for this observation may lie, in part, in the fact that a large proportion of the substrates in our refKSR data set are composed of TFs and their co-regulators. Indeed, phosphorylation-dependent degradation/stabilization of TFs is well documented and may be a common mode of regulation for this class of proteins . In supphttp://phosphonetworks.org, will serve as a valuable resource for the research community in several ways. For instance, aside from generating more human KSRs and more phosphorylation motifs than all previous studies combined, this study also provides a blue print for mapping kinase-dependent connections and serves as a foundation for the development of new tools, such as genetically targetable kinase activity reporters and phosphorylation site-specific antibodies, that promise to offer important insights into the regulation of kinases and their downstream substrates. Likewise, the large number of human KSRs identified in these studies can be used in conjunction with KSR data sets from other species  to explore the extent to which kinase-dependent signaling networks are conserved across species . Each clone was verified by restriction digestion. Each kinase was expressed as a GST fusion in the budding yeast, eviously .in vitro substrates for each kinase, a protocol similar to that described by Human protein microarrays were generated as described previously . Each miin vivo, we used a naïve Bayesian approach (phospho.elm.eu.org). We also constructed an artificial data set as a negative data set that contains 10 000 protein pairs where no kinases were included in the set. The relative weights for these three features were learned from the known data sets and applied to each of the rawKSRs.To predict KSRs that are likely to occur approach  to integTo predict consensus phosphorylation motifs for the kinases in our collection, we integrated the following data sources: the rawKSRs determined by protein microarray, the phosphorylation sites determined by MS/MS, and the phosphorylation sites with known upstream kinases obtained from the literature. Each site was mapped to the protein sequences of substrates identified in the rawKSR data set and subject to an iterative process by the M3 algorithm.List of rawKSRList of refKSRSummary of the first-stage KSR validation resultsList of comKSR


In contrast by altering the scaffold of the promiscuous CK2 inhibitor TBI a new class of HIPK2 inhibitors has been generated. One of these, TBID, displays toward HIPK2 unprecedented efficacy (IC50 = 0.33 µM) and selectivity (Gini coefficient 0.592 out of a panel of 76 kinases). The two other members of the HIPK family, HIPK1 and HIPK3, are also inhibited by TBID albeit less efficiently than HIPK2. The mode of action of TBID is competitive with respect to ATP, consistent with modelling. We also provide evidence that TBID is cell permeable by showing that HIPK2 activity is reduced in cells treated with TBID, although with an IC50 two orders of magnitude higher (about 50 µM) than in vitro.Homeodomain-interacting protein kinase 2 (HIPK2) is a Ser/Thr kinase controlling cell proliferation and survival, whose investigation has been hampered by the lack of specific inhibitors able to dissect its cellular functions. SB203580, a p38 MAP kinase inhibitor, has been used as a tool to inhibit HIPK2 in cells, but here we show that its efficacy as HIPK2 inhibitor is negligible (IC The CMGC group of the human kinome is split into several branches, one of which, also including DYRKs and CLKs, gives rise to a sub-branch composed by so called “homeodomain-interacting protein kinases” (HIPKs). Four HIPKs are present in human, with HIPK2 attracting special attention for its role as a regulator of growth and apoptosis in various types of cells These studies have been hampered by the lack of selective inhibitors of HIPK2. Small cell permeable inhibitors of protein kinases have become invaluable reagents for dissecting signaling pathways mediated by each of them. In recent years a huge repertoire of compounds purported to be “specific” toward a large number of protein kinases have become available. Since however the human kinome is composed by some 500 members the issue of selectivity is critical and only in a limited number of cases inhibitors have been shown to display a really narrow selectivity window hitting only few and in very rare cases one individual protein kinases In the case of HIPK2 the rational design of specific inhibitors has never been reported, the only HIPK2 inhibitor mentioned in the literature being SB203580, a compound firstly employed as HIPK2 inhibitor In the course of our studies aimed at the identification and development of compounds able to inhibit CK2, a highly pleiotropic kinase These observations prompted us to design modifications of the tetrabromo-benzimidazole scaffold aimed at decreasing the efficacy toward CK2 and other kinases drastically inhibited by TBI and TBB, while maintaining or eventually improving that toward HIPK2.H-imidazol-2-yl)isoindoline-1,3-dione (TBID) which is able to inhibit HIPK-2 with a selectivity much higher than that of TBI, not to say of SB203580, whose ability to inhibit HIPK2 is in our hands negligible. These properties in conjunction with cell permeability, make TBID the first choice inhibitor of HIPK2 presently available for both in vitro and in cell studies.Here we describe the properties of one of these derivatives, 4,5,6,7-tetrabromo-2-, and the synthetic peptide substrate NKRRRSPTPPE HIPK2 (63 ng) was preincubated at 37°C for 10 minutes either in the absence or in the presence of increasing amounts of each inhibitor in a final volume of 20 µl of solution containing 50 mM Tris-HCl pH 7.5, 0.1% (v/v) 2-mercaptoethanol, 0.1 mM EGTA, 10 mM magnesium acetate. The reaction was started by addition of 5 µl of a reaction mixture containing 20 µM ATP (500–1000 c.p.m./pmol).PIM1, CK2, CK1 phosphorylation assays were performed following similar procedures used for HIPK2 except that preincubation was omitted. In detail, PIM1 activity was determined at 100 µM ATP concentration and in the presence of 30 µM synthetic peptide substrate RKRRQTSMTD. CK2 and CK1 activities were assayed in a buffer containing 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 12 mM MgClConditions for the activity assays of all other protein kinases tested in selectivity experiments are as described or referenced in Initial velocities were determined at each of the ATP concentration tested, at 1 mM NKRRRSPTPPE peptide concentration. Km values were calculated either in the absence or in the presence of increasing concentrations of inhibitor, from Lineweaver-Burk double-reciprocal plots of the data. Inhibition constants were then calculated by linear regression analysis of Km/Vmax versus inhibitor concentration plots.Lorenz curves were derived from the selectivity data. Gini coefficients and hit rates (expressing the percent of kinases inhibited >50% by a given compound) were calculated as described in rms of conjugate gradient was <0.05 kcal mol−1 Å−1 keeping all the heavy atoms fixed Human HIPK2 protein kinase was built using an homology modeling approach implemented into Molecular Operating Environment (MOE) After the calibration phase, all compound structures were docked directly into the ATP binding site of the human HIPK2 model, by using the docking tool part of the GOLD suite 2, at 37°C. Treatments with TBID were performed in the same medium but with 1% fetal calf serum; control cells were treated with the solvent (DMSO). Total cell lysates were prepared as in HepG2 cells (human hepatocellular carcinoma) were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 unit/ml penicillin and 100 µg/ml streptomycin; CEM cells (human T lymphoblastoid cells) were cultured n RPMI-1640 (Sigma) with the same supplements. Cells were cultured in an atmosphere containing 5% COEndogenous HIPK2 activity was evaluated by measuring the phosphorylation level of its target site Ser46 of p53: to this purpose, CEM cells were treated for 6 h as indicated, then lysed. 10 µg of total proteins were loaded on 11% SDS-PAGE, blotted on Immobilon-P membranes (Millipore), and analyzed by western blot (WB) using an anti-phospho Ser46 p53 antibody (BD Biosciences); chemiluminescence signals were acquired with a Kodak 4000MM Pro Image Station. Bands were quantified by Carestream Molecular Imaging Software (Kodak) and the obtained values were normalized to total p53 signal with a Cell Signaling Technology antibody; anti-actin (Sigma) was used as loading control.Alternatively, HIPK2 was immunoprecipitated with 2.5 µl anti-HIPK2 (Epitomics) from 350 µg of total lysate proteins deriving from HepG2 cells either treated or not with TBID following a protocol elsewhere described 5 cells/100 µl were incubated in a 96-well plate and treated for 6 h as indicated. 1 h before the end of the incubation, 10 µl of MTT solution (5 mg/ml in PBS) was added to each well. Incubations were stopped by addition of 20 µl of lysis solution at pH 4.7, as described elsewhere Cell viability was evaluated by means of MTT -3,5-diphenyltriazolium bromide) reagent; 1050 values with either HIPK and CK2 are nearly identical isoindoline-1,3-dione , The synthesis of TBID (5a) and analogs 5b-5i was carried out following a published general synthesis protocol The structures of two commonly used CK2 inhibitors, TBB and TBI are shown in 5a) inhibits HIPK2 with the same efficiency as TBI, while displaying toward CK2 a more than10-fold higher IC50 value.As shown in 5b-5i were less potent HIPK2 inhibitors  was only slightly higher than that with TBID, the latter however being much more selective as also highlighted by the observation that the number of kinases inhibited >90% by either 10 µM TBID or TBI in the same panel is 1 was developed to inhibit different classes of protein kinases and its efficacy to inhibit HIPK2 activity is questionable, as clearly shown here and elsewhere File S1Table S1, Selectivity profiles of TBID on a 125 kinase panel. These are expressed in % activity of the enzyme. Figure S1, Synthesis of 4,5,6,7-tetrabromo-2-(1H-imidazol-2-yl)isoindoline-1,3-diones. Reagents and conditions: (i) acetic acid, reflux, 1–3 h, 11–64%. For synthesis of 5a, instead of a free heterocyclic base the 2-aminoimidazolium sulfate was used in the presence of DBU.(DOC)Click here for additional data file.