AS) whose catalytic activity is specifically and rapidly inhibited by a small molecule. Inhibition of CskAS during TCR stimulation led to stronger and more prolonged TCR signaling and to increased proliferation. Inhibition of CskAS enhanced activation by weak but strictly cognate agonists. Titration of Csk inhibition revealed that a very small increase in SFK activity was sufficient to potentiate T cell responses to weak agonists. Csk plays an important role, not only in basal signaling, but also in setting the TCR signaling threshold and affinity recognition.The C-terminal Src kinase (Csk), the primary negative regulator of Src-family kinases (SFK), plays a crucial role in controlling basal and inducible receptor signaling. To investigate how Csk activity regulates T cell antigen receptor (TCR) signaling, we utilized a mouse expressing mutated Csk . Antigen recognition causes the TCRs to transmit signals to the inside of the T cell that trigger an immune response. The degree to which the TCRs are active, and the length of time that they transmit signals regulates the size of the immune response. Therefore, developing new drugs that manipulate the activity of TCRs could be useful to treat many diseases.An enzyme called Csk inhibits the activities of a small family of proteins involved in a variety of different processes. One protein that Csk targets is called Lck and is required for the activation of immune responses in T cells. However, it is not clear whether Csk interacting with this protein stops the release of signals from TCRs, or whether it alters the level to which TCRs need to be activated before they transmit the signals.Manz, Tan et al. studied T cells from mice that had a mutant form of Csk that is inhibited by a drug called 3-iodo-benzyl-PP1 (3-IB-PP1), but otherwise works normally. When these cells detected an antigen in the presence of the drug, its TCRs were more highly activated and transmitted signals for a longer period of time than cells not exposed to the drug. The drug especially enhanced immune responses to very weak antigens, ones that might not activate T cells under normal circumstances.Manz, Tan et al.'s findings confirm that Csk plays a negative role in the activation of T cells and suggest that Csk may be a useful target for drug therapies that aim to fine-tune immune responses. The next challenge is to find a drug that can inhibit normal Csk enzymes and test this in mice and other animals.DOI:http://dx.doi.org/10.7554/eLife.08088.002 SFKs are ubiquitous regulators of basal and inducible receptor signaling. The seven family members are expressed in various combinations in different cell types, have unique substrate specificity and are differentially regulated by phosphatases and localization . HoweverIn T cells, the strength and duration of T cell antigen receptor (TCR) signaling in response to antigen stimulation dictates the magnitude and quality of primary and secondary immune responses . The TCRAlthough the molecular regulation of Lck is understood, its localization and changes in activity during TCR signaling are still unclear . Lck is Csk is a cytoplasmic protein, yet its predominant role is to phosphorylate the inhibitory tyrosine of SFKs that are localized to membranes . Hence, The precise role of Csk in TCR signaling has also been difficult to study by genetic inactivation . GermlinAS) in the absence of endogenous Csk . Studies in thymocytes in this model system support the notion that the combined actions of CD45 and Csk establish the basal activity of SFK  by titrating anti-CD3ε antibody in the absence or presence of a high dose of the CskAS inhibitor, 3-IB-PP1, and examined proximal TCR signaling tyrosine phosphorylation events. As previously shown, inhibition of CskAS alone induced strong activation of Lck, as well as Fyn, which was indicated by the hyperphosphorylation of their activation loop tyrosines and the reduced phosphorylation of their inhibitory tyrosines , likely due to spurious activation of a few cells  (Short-term Csk (<1 μM) , as demoAS;OTI CD8+ T cells were stimulated by APCs preloaded with peptides of different agonist potency during strong CskAS inhibition (5 μM), we observed marked augmentation of CD69 expression in responses to weak  agonists, contrasting with only slight enhancement of the responses to strong  agonists . To test the extent of Csk inhibition necessary to boost weak agonist signaling, we titrated 3-IB-PP1 and assayed responses to weak agonists Q4H7 and G4 . SurprisOur results raise the question of how Csk activity controls SFK activity and specifically the TCR activation threshold. Since a substantial amount of active Lck is present in the basal state and does not change with TCR ligation, it has been hypothesized that the pre-existing pool of active Lck is responsible for the highly sensitive and rapid initiation of TCR signaling in response to ligand engagement . Hence, Our data supports the model that the size of the active pool of Lck is especially critical for affinity discrimination of different agonistic peptides. Strong agonists have a long half-life of pMHC-TCR and have sufficient time to recruit active Lck and initiate downstream events, while weak agonists with short half-lives are most sensitive to the availability of active Lck before the pMHC-TCR complex falls apart. The different amounts of Lck loaded on the CD4 and CD8 co-receptors can explain the different half-life range of positively and negatively selecting ligands for CD4 and CD8 cells ; howeverAS inhibition at 200 nM 3-IB-PP1, led to relatively large pY394 changes in the setting of small changes in pY505. Since Y394 phosphorylation is the result of trans-autophosphorylation, it seems likely that a small reduction in pY505 induced by low levels of Csk inhibition, could lead to autoactivation of a large pool of Lck. Such a larger pool of active Lck could then show strong synergy with the TCR stimulus and is sufficient to markedly enhance the response to very weak agonists. The synergy we observed between TCR stimulus and Csk inhibition, strikingly, had its greatest impact on the activation of downstream signaling molecules. This is most likely due to positive feedback/signal amplification along the pathway. Eventually, the integration of stronger signaling over hours led to profound differences in the fraction of cells upregulating CD69 or proliferating. On the other hand, this minimal change in Lck activity highlights how tightly it must be regulated in both the basal and inducible states by kinases and phosphatases and their respective adaptors. It is possible that, although small changes in Lck activity greatly sensitize the TCR to weak agonists, such small changes do not as effectively engage negative feedback mechanisms.The ability to acutely titrate the activity of Csk, and consequently Lck, allowed us to assess how graded increases of Lck activity affects TCR signaling without concerns about basal level adaptation and long-term compensation that occurs when using genetic manipulation of Csk amounts or function. Surprisingly, even very small changes in Lck activity, caused by CskLoss of Csk from the cell membrane, or biochemically defined lipid raft fractions has been proposed to lead to TCR signal initiation via activation of Lck . This moCsk has also been proposed to regulate TCR signal downregulation by returning to the plasma membrane after signal initiation. It has been challenging to study this role using genetic ablation approaches since Csk deficiency affects TCR basal state signaling. Using acute inhibition of Csk concurrently with TCR stimulation, we were able to follow TCR signal downregulation in the absence of Csk activity and observed that it is delayed but not abolished. Our data suggest that other molecules are involved in signal termination more prominently than Csk.New insights into how Csk is regulated will enable the development of strategies to manipulate Csk activity and hence the sensitivity of T cells to various activation stimuli. Such manipulation of Csk activity by small molecule inhibitors could be useful therapeutically, for example in pathologic settings where suppression  or augmentation  of T cell responses to antigen is desirable.Mice used for these studies were 6–12 weeks of age. All mice were housed in a specific pathogen-free facility at UCSF according to the University Animal Care Committee and National Institutes of Health (NIH) guidelines.3-IB-PP1 has been described .CD4 PerCP-Cy5.5 (550954), TCR Vα2 PE (553289) and Lck-pY505 (BD Biosciences 612390) are from BD Biosciences ; p44/42 MAPK pThr202/Tyr204 (4377), Src 416 (2101), ZAP-70-pY319 (2701) are from Cell Signaling ; LAT-pY132 (44-224), PLC-γ1-pY783(44-696G) and CFSE (C34554) are from Life Technologies ; actin ; GAPDH ; Goat anti-armenian hamster IgG(H+L) (127-005-160) and donkey anti-rabbit IgG Ab conjugated to APC (711-136-152) are from Jackson Immunoresearch ; Horseradish peroxidase (HRP)-conjugated goat antibody α-rabbit IgG (H+L) (4050-05), α-mouse IgG (H+L) (1031-50), Alexa647-conjugated α-mouse IgG (H+L) (1010-31) are from Southern Biotech ; I-A(b) tetramers loaded with human class II-associated invariant chain peptide 103–117 or chicken OVA peptide 328–337 are from NIH tetramer core facility.Stained cells were analyzed on a BD Fortessa . Data analysis was performed using FlowJo software  and GraphPad Prism  and ImageLab .Before stimulations, cells were serum-starved at 37°C for at least 20 min. Stimulations were performed in serum-free RPMI at 37°C. CD3ε crosslinking was induced by addition of anti-CD3ε followed by goat anti-armenian hamster IgG(H+L) to a final concentration of 50 μg/ml. For bead-based stimulations, 4.5 μm styrene beads  were coated overnight at 4°C or for 1 hr at room temperature with p-MHC tetramers or αCD3 (2c11) in PBS under continuous rotation at 4°C. Biotinylated p-MHC for CD8 stimulation were precoated with 5 μg/ml streptavidin. Beads were then saturated with 1% BSA in PBS under continuous rotation for 2 hr at room temperature, and washed with serum-free RPMI before use. Ice-cold rested cells and beads were span together and signaling was initiated by transfer to 37°C. Intracellular phospho-ERK was performed as previously described .Immunoblotting was performed as previously described .+ or CD8+ T cells were obtained by negative selection according to manufacturer's protocol (STEMCELL Technologies 19852) or with an in-house procedure as follows: The following biotinylated antibodies were combined and dialyzed in 1× PBS with Slide-a-lyzer 10,000 Molecular Weight cutoff dialysis cassette , then filter sterilized: biotin anti-CD8a (clone 53-6.7) or biotin anti-CD4 (clone GK1.5), biotin anti-CD11b (clone M1/70), biotin anti-CD11c (clone N418), biotin anti-CD19 (clone 1D3) from were Tonbo Biosciences ; biotin anti-CD24 (clone M1/69), biotin anti-CD45R (B220) (clone RA3-6B2), biotin anti-CD49b (clone DX5), biotin anti-TER119 (clone TER-119) were from Biolegend . ACK-lysed splenocytes and lymphocytes were incubated at room temperature with the antibody cocktail for 10 min in PBS with 1% FBS, 2 mM EDTA and 5% normal rat serum at 108 cells/ml. Cells were then washed once with PBS with 1% FBS, 2 mM EDTA, resuspended at 0.85 ml per 108 cells, and mixed with 0.15 ml anti-biotin MACSiBead per 108 cells . After 5 min at room temperature, cell-bead mix was placed in separation magnet for 5 min and the unbound cells were collected.Enriched populations of pan T cells were obtained by negative selection according to manufacturer's protocol . Enriched populations of CD4+ or CD8+ T cells were resuspended in PBS, labeled for 4 min at room temperature with 2 μM CFSE, and quenched with 100% FBS. Labeled CD4+ T cells were stimulated by plate-coated OVA-MHC or α-CD3 (2c11) for 72 hr. At indicated timepoint, DAPI were analyzed for CFSE dilution.Purified CD4+ OTI T cells were stimulated for indicated time by Mitomycin C-treated Calpha−/− or BoyJ splenocytes, preloaded for 1 hr with indicated peptide and washed of excess peptide. At indicated time, cells were fixed and stained for CD69, TCRValpha2 and CD45.1, and analyzed by flow cytometry.Purified CD8 review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled “Small molecule inhibition of Csk alters affinity recognition by T cells” for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:The authors use the Csk-AS model to investigate the impact of acute Csk inhibition on both CD3 Ab and MHC-peptide-induced biochemical signaling pathways and downstream effects on activation marker expression, TCR downregulation and proliferation. The major conclusions that Csk 1) regulates the basal activity of Lck (and Fyn) and 2) influences the extent of T cell activation in response to low-dose / low affinity Ags are supported by the data.Furthermore, Csk inhibition only weakly prolongs the duration of TCR-induced signaling – suggesting that other inhibitory pathways are required for signal termination. Overall, this elegant study establishes that Csk controls the size of the active pool of Lck and thus contributes to set the TCR signaling threshold and thus enforces TCR affinity discrimination in peripheral T cells.Essential revisions:1) Using CskAS expressing OT1 T cells, the authors made the puzzling finding that the phosphorylation of the inhibitory tail pY505, the direct target of Csk, did not change in response to 3-IB-PP1 treatment. In contrast, a 3-4 fold increase in Y394. This observation is inconsistent with most present model(s) of Lck regulation. How do the authors account for this intriguing observation?doi:10.1038/icb.2014.100) suggests that the pool of active Lck is smaller than reported and that stimulation of T cells by physiological TCR and CD4 ligation does increase the number of active Lck molecules. This work needs to be discussed.2) Results, first paragraph. The recent work of Ballek et al. (3) In  1) Using CskAS expressing OT1 T cells, the authors made the puzzling finding that the phosphorylation of the inhibitory tail pY505, the direct target of Csk, did not change in response to 3-IB-PP1 treatment. In contrast, a 3-4 fold increase in Y394. This observation is inconsistent with most present model(s) of Lck regulation. How do the authors account for this intriguing observation?The reviewers point out the puzzling observation that phosphorylation of the direct target of Csk, Lck Y505, does not change much upon Csk inhibition, whereas phosphorylation of the activating site Y394 on Lck goes up dramatically.AS mice have been examined and they all respond with Src family kinase (SFK) activation after Csk inhibition, however the phosphorylation status of the inhibitory tyrosines differs substantially. In thymocytes (doi:10.1038/ni.2772) only partial loss is observed at high drug doses, even after prolonged treatment, while in bone marrow derived macrophages, the inhibitory tyrosines of the SFKs are rapidly and almost completely de-phosphorylated . Moreover, similar more complete dephosphorylation of the inhibitory site is observed in B cells (unpublished). Likewise, in Jurkat T cells expressing a membrane targeted CskAS construct that functions a dominant inhibitory allele, inhibition of Csk led to near complete dephosphorylation of the Lck inhibitory site . We conclude that the SFKs are differentially regulated in different cell types, perhaps via differential sequestration from CD45, the contribution of other phosphatases such as CD148, or pools that are prone to autophosphorylation of the activating site pY394. To address the difference between CD4+ and CD8+ T cells, we immunoblotted lysates from equivalently stimulated cells side by side  Results, first paragraph. The recent work of Ballek et al. (doi:10.1038/icb.2014.100) suggests that the pool of active Lck is smaller than reported and that stimulation of T cells by physiological TCR and CD4 ligation does increase the number of active Lck molecules. This work needs to be discussed.The reviewers point out a recent estimate of active Lck in T cells  that is much lower than a previous one  with which our data is consistent.Our study was not designed to answer the question of how much Lck is active in the basal state, which was the focus of both of the above studies, and they approached it with different experimental techniques. By acute Csk inhibition, we were able to activate significantly more Lck molecules and based on the plateau level, we inferred an estimate of the basally active fraction. Although it is consistent with Nika et al.'s results, it is possible that there remains a pool of Lck that cannot be activated acutely by Csk inhibition, due to sequestration away from Csk, proximity to CD45 or another mechanism. Therefore our approach is not suited to quantitatively answer this question. This distinction has been noted in the revised text.3) In– these are (to me at least) a little confusing. Any effect of Csk inhibition on levels of Lck pY394 would be secondary to direct effects on pY505. It is possible that to see changes in pY505 following Csk inhibition requires a dephosphorylation event by CD45, but it seems a bit odd that large changes in pY394, as seen with high doses of 3-IB-PP1, should be apparent without any change in pY505? Given the results inappear to give quite different results as regards the effects of the drug on basal pY505 in CD4  and OT-1  cells, this should be discussed. Is this a “real” effect – i.e., is pY505 regulated differently is CD4 and CD8 T cells?+ and CD8+ T cells.The reviewers point out the surprising activation of pY394 with very subtle changes in pY505, which was addressed in point 1, and the apparent discrepancy between CD4+ and CD8+ T cells, we compared them side by side, as seen in the new + T cells having slightly more Lck and a larger fraction of it is phosphorylated at Y505. It is important to note that most of the data with CD8+ T cells was performed with much lower doses of the Csk inhibitor where changes at the Lck phosphorylation sites were relatively small. On the other hand, the CD4+ T cell experiments used high drug doses (5 or 10 µM) where greater change was observed.To quantitatively address any differences between CD4The mechanism behind the relatively small changes in pY505 and larger changes in pY394 is unknown. As mentioned in our response to point 1, we hypothesize that the trans-autocatalytic nature of Lck may account for dramatic changes in the activation site. This would allow a small number of Lck molecules that are initially activated through Csk inhibition to rapidly phosphorylate the active site of other Lck molecules and amplify the overall level of activation. This explanation has been added to the text.


A receptors. Photolabeling also indicates that pentobarbital binds to some of the same sites where propofol and etomidate act. Quantitative allosteric co-agonist models for propofol and etomidate account for modulatory and agonist effects in GABAA receptors and have proven valuable in establishing drug site characteristics and for functional analysis of mutants. We therefore sought to establish an allosteric co-agonist model for pentobarbital activation and modulation of α1β3γ2L receptors, using a novel approach to first correct pentobarbital activation data for inhibitory effects in the same concentration range.Pentobarbital, like propofol and etomidate, produces important general anesthetic effects through GABAA receptors and two-microelectrode voltage-clamp, we quantified modulation of GABA responses by a low pentobarbital concentration and direct effects of high pentobarbital concentrations, the latter displaying mixed agonist and inhibitory effects. We then isolated and quantified pentobarbital inhibition in activated receptors using a novel single-sweep “notch” approach, and used these results to correct steady-state direct activation for inhibition.Using oocyte-expressed α1β3γ2L GABA50s near 1 mM, while co-agonist model parameters suggest two allosteric pentobarbital agonist sites characterized by KPB ≈ 5 mM and high efficacy. The results also indicate that pentobarbital may be a more efficacious agonist than GABA.Combining results for GABA modulation and corrected direct activation, we estimated receptor open probability and optimized parameters for a Monod-Wyman-Changeux allosteric co-agonist model. Inhibition by pentobarbital was consistent with two sites with ICA receptor mutations in putative pentobarbital binding sites.Our novel approach to quantifying both inhibitory and co-agonist effects of pentobarbital provides a basis for future structure-function analyses of GABA A) receptors  and [PB] was fitted by non-linear least squares (Origin 6.1) with Allosteric co-agonist modeling was performed as previously described ,17. Brie0 in 0-1. Initially, L0 was set at 25,000, based on previous estimates for α1β2γ2L GABAA receptors  > 3 mM, the range where maximal agonism, but also maximal inhibition, is observed. The high efficacy of PB agonism evident in the corrected data also increases uncertainty in co-agonist model parameters for PB binding and efficacy, because model sensitivity drops as the fraction of activated receptors approaches 1.0. The mathematical approach we took to correcting PB-dependent activation data for PB inhibition presumes that agonist and antagonist mechanisms are independent. Our approach also presumes that PB inhibition is independent of receptor open probability. This is a feature that was also included in the optimal model identified by Serafini et al , but nottivation . IndepenA receptors expressed in Xenopus oocytes, and used these results to correct steady-state activation data elicited with high PB concentrations. Corrected results for PB activation combined with PB modulation of GABA responses enabled fitting with an allosteric co-agonist model. Our approach provides independent characterization of the stoichiometries, affinities, and efficacies of PB sites mediating both positive modulation and inhibition. These results agree with estimates from some earlier kinetic studies based on patch-clamp techniques. Our new experimental-analytical approach is expected to be useful in studies of the co-agonist vs. inhibitory effects of PB in receptors with mutations in putative PB binding sites.We developed a novel experimental approach to electrophysiologically isolate and quantify PB inhibition of GABA


The invasive blood-stage malaria parasite – the merozoite – induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. The blood stage malaria parasite, the merozoite, achieves a remarkable feat in terms of cell biology. In less than two minutes it manages to successfully invade a target erythrocyte, a cell that is not only non-phagocytic, but whose membrane architecture also represents one of the most strong, resilient and flexible cellular structures knownErythrocyte invasion is a stepwise process that begins when a merozoite forms an initial low-affinity attachment to a red blood cell via any point on its surface. Attachment is associated with erythrocyte membrane deformation and is followed by reorientation, where the merozoite brings its apical tip into contact with the target host cell5Toxoplasma gondii7T. gondii tachyzoite invasion of fibroblasts1112Plasmodium berghei, sporozoite invasion of hepatocytesT. gondii and Plasmodium invasion proteins 20242627P. falciparum merozoitesHere, we exploit the ability to achieve synchronised erythrocyte invasion using free and viable P. falciparum merozoite invasion requires host cell ATP, erythrocytes were pre-treated in ATP depletion medium containing 6 mM iodoacetamide, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, and 10 mM inosine, which together accelerate ATP consumption and block its production through glycolysisTo determine if P. falciparum invasion and growth, while also enabling the incorporation of fluorescent markers . Under all other conditions invasion rates were unaffected. Of note, all resealed erythrocyte populations contained a mixture of normal and mildly echinocytic erythrocytes, with the exception of erythrocytes resealed in the presence of 10 mM AMP-PNP, which all appeared reduced in size and severely echinocytic , no invasion inhibition was achieved when merozoites were pre-treated with blebbistatin . Given t3739Therefore, to more specifically target erythrocyte actin, erythrocytes were resealed in the presence of increasing concentrations of phalloidin and fluorescent dextran then subjected to merozoite invasion. Phalloidin only enters erythrocytes upon their permeabilizationHaving argued against a requirement for host cell ATP-dependent actin and myosin activity for malaria parasite entry, but observing some evidence for cytoskeletal remodelling (in particular adducin), we next sought to test whether early events in merozoite invasion might stimulate erythrocyte-mediated changes in host surface or cytoskeletal protein organisation. The centrality of phosphorylation in regulating erythrocyte membrane and cytoskeletal protein associations is well established332PO4) suggested that, relative to non-invaded controls, erythrocytes incubated with merozoites for 2 min exhibited a marked increase in general protein phosphorylation, with a particularly dramatic response seen for of high molecular weight proteins  under three different conditions, either in the presence of heparin, which inhibits firm merozoite attachment and reorientation45P. falciparum peptides collected can be found in Median Peptide fold-change values for high confidence peptides (FDR ≤1%) were produced by comparing the abundance of individual peptides in samples containing erythrocytes and merozoites (numerator) versus erythrocytes alone (denominator) for each of the three conditions tested. In order to directly test the effect of merozoites on erythrocytes, each control sample (denominator) was subjected to the same conditions, including the same drug treatment, as its corresponding invasion sample (numerator). Across the four experiments under all three conditions, most peptide median fold-change values were low and clustered together, while a small number of high fold-change and negative fold-change peptides were detected . The sumThe potentially transient nature of host protein phospho-modification allied to the small proportion of parasites undergoing active invasion within invasion assays provides significant challenges to the identification of sites of specific protein phosphorylation associated with merozoite attachment and/or invasion. The robust regression and outlier removal (ROUT) method was used to identify peptides with fold-change values that likely represented clear responses to merozoite presence, as opposed to those that likely represented natural variation or noise in the data. Outlier peptides with high median fold-change values, representing an increase in phosphorylation in response to merozoites, were detected in all experiments and across all conditions . A total5 merozoites/μL , yielding a merozoite to erythrocyte ratio in these invasion proteomics assays of between approximately 5:1 and 7:1. In contrast, the rate of erythrocytes invaded in these rapid 1.5 min assays ranged from around 2–4%, such that even in uninhibited invasion samples the vast majority of erythrocytes will have experienced merozoites transiently interacting or binding rather than active invasion events. As a result, phosphorylation events specific to invasion itself (downstream of erythrocyte interactions that occur under heparin treatment) are likely masked in a proteomic assay by the predominant host-parasite interaction occurring in the samples, that of transient and weak merozoite binding.The lack of outlier peptide segregation into the different invasion assay conditions is likely explained by experimental conditions. In order to achieve high invasion rates, invasion assays involved applying an excess of merozoites to erythrocytes compared to normal culture conditionsbone fide target of phosphorylation during invasion  were detected in only one of the four proteomics repeats. This variability may be due to differences in merozoite numbers, variability between donor erythrocyte samples, efficiency of phospho-enrichment or chance differences in phospho-peptides detected by the mass spectrometer between experiments, with the abundance of both haemoglobin and merozoite peptides present within the sample likely limiting the depth of sequencing attained. Thus, to increase confidence in assignment of real changes, we took a conservative approach and limited analysis to outlier phospho-peptides that appeared in at least two of the four proteomics experiments . Where minvasion .2 (fold-change) values for beta-spectrin peptides corresponding to p-S1301/Y1302 across all three conditions ranged from 3.0 to 6.5 , indicating a large increase in abundance in response to merozoites, whereas the two other detected phosphorylation sites in the protein (S36 and S2015) displayed minimal changes and were never detected as outliers  values  values , and pho) values .The remaining shortlisted phospho-peptides from glycophorin C, eukaryotic translation initiation factor 4B (EIF4B) and ankyrin, although outliers in at least two experiments, were also detected as non-outliers in other experiments . In addiHaving demonstrated that erythrocytes respond to the presence of merozoites through phosphorylation, we wanted to exclude the possibility that this was simply due to mechanical stimulation by small particles. This question is particularly relevant given that these merozoite invasion assays are carried out in shaking conditions and noting that one of our top outlier phospho-peptides was from PIEZO1, a mechano-sensitive ion channel. To test whether mechanical force applied by merozoite-sized bodies could stimulate the phosphorylation events detected in invasion assays, we incubated erythrocytes under invasion-assay conditions with fluorescent microspheres of 1μm diameter. Erythrocytes were incubated with shaking alone or in the presence of beads pre-coated with wheat germ agglutinin (WGA), or BlockAid, a commercially available blocking solution. This experiment was repeated twice and the two repeats were processed for quantitative phospho-proteomics and analysed as for merozoite invasion samples. Compared to the range of median fold-change values obtained from our invasion assay proteomics , the disShortlisted outlier peptides identified in invasion quantitative-proteomic assays were either absent among the phospho-peptides from the bead experiments, or present as non-outlier peptides , indicatP. falciparum invasion by flow cytometry, we demonstrate that a reduction in erythrocyte ATP indeed leads to a severe defect in merozoite invasion. Although the precise role of ATP in this context remains unclear, given the many morphological, biophysical and biochemical repercussions of ATP depletion, along with potential off-target effects of the treatments used to interfere with ATP, these results nonetheless support a model in which the red blood cell is co-opted to actively participate in its own infection by the merozoite.The proposition that merozoites hijack host pathways of membrane and cytoskeletal remodelling to invade requires, at its base, an erythrocyte capable of carrying out active processes. The possibility that red blood cell ATP is required for merozoite entry was first suggested over two decades ago, however to date no studies have been able to determine whether host ATP was a requirement of invasion itself or downstream growth up to ~20 hours of intracellular development1518P. falciparum merozoite entry as observed in other apicomplexan modes of invasionBased on our combined imaging and invasion-inhibition analyses, which were unable to demonstrate either the presence of or a reliance on erythrocyte actin filament turnover during An alternative host-cell involvement during merozoite entry might be the localized destabilization of the erythrocyte cytoskeleton, which would likely increase the potential for membrane wrapping, thereby leading to a lower parasite energy requirement for invasionPhosphorylation provides a key mechanism by which the erythrocyte is able to regulate the affinity of interactions between membrane and cytoskeletal proteins in order to remodel its structure in response to stimuli. Here, we clearly demonstrate that a large number of red blood cell proteins are phosphorylated in the presence of merozoites, with beta-spectrin and PIEZO1 consistently identified as targets of phosphorylation across biological and technical replicates. The fact that these increases in erythrocyte protein phosphorylation were detected not only in samples where parasites can enter host cells, but also in the presence of the invasion inhibitors R1 and heparin, suggests that erythrocytes respond to early interactions with merozoites. R1 treatment stalls invasion at reorientationP. falciparum invade using different sets of initial receptor-ligand interactions   does not inhibit invasion, genetic approaches have indicated that the P. falciparum CKII homologue is likely essential for blood stage survival and its activity has been implicated in merozoite invasion 56575859P. falciparum CKII in the future 58Is an erythrocyte kinase recruited to prime the host cell for invasion? Since erythrocyte protein phosphorylation was detected prior to the point at which merozoites begin to secrete effectors into the target erythrocyte, it follows that a host cell kinase must be mediating these events. The kinases responsible for phosphorylating the outlier sites identifies through proteomics are unknown. Kinase prediction using NetPhosK is suggestive of a role for CKII and Ca(CAMKII) . Indeed,P. falciparum infected erythrocytesIt is possible that the observed phosphorylation events may represent responses to merozoite contact or secreted factors with little or no functional consequence, or serve as markers of other invasion-related events in the host cell. More attractively, phosphorylation may function to alter the properties of the cytoskeleton and potentially ‘prime’ the erythrocyte to be amenable to downstream invasion steps, however the consequences of each phosphorylation event identified in this study are currently unknown. Thus far, the only report of phosphorylation at beta-spectrin S1301 was in Plasmodium merozoite. With future development of phospho-specific antibodies to each target protein and development of live imaging probes, we anticipate that this study will inform future efforts focused toward deciphering the potential for host cell signalling and kinase involvement in invasion and to understand the precise membrane and cytoskeletal protein remodelling events that underscore red blood cell responses to malaria parasite invasion.In summary, we present the first quantitative phospho-proteomic dataset addressing erythrocyte protein modification in response to stimulus by the early events of blood stage malaria parasite entry. We can exclude a significant contribution of host cell actin dynamics during the invasion process whilst demonstrating a host kinase response to the earliest membrane contact with the merozoite through the phosphorylation of prominent erythrocyte membrane and cytoskeletal proteins. It is now clear from this work that the erythrocyte is capable of and indeed does respond actively to attachment and invasion of the P. falciparum strains D10 M3′ and D10-PHG3 and either 0.4% (w/v) Albumax II (GIBCO) or a combination of 0.2% (w/v) Albumax II and 5% (v/v) heat inactivated human serum from O+ donors (complete culture medium)   and an Australian Red Cross Blood Service Agreement (11-09VIC-01)). Cultures were synchronised as previously described using a combination of sorbitol (Sigma) synchronizationUnless otherwise specified, free merozoites were isolated and used in standard erythrocyte invasion assays according to published protocols® 488 , goat anti-rabbit Alexa Fluor® 488 (A-11008), goat anti rabbit Alexa Fluor® 594 (A-11037) or goat anti-rat Alexa Fluor® 594 (A-11007). Phalloidin Alexa Fluor® 594 1:50  was used to label erythrocyte F-actin. Samples were mounted in Vectashield  containing 0.1 ng/μL 4′,6-diamidino- 2-phenylindole  and widefield deconvolution imaging performed using a DeltaVision Elite equipped with a TrueLight illumination system, Photometrics CoolSnap HQ2 camera, 100 × 1.40 NA objective and SoftWoRx v6 software.Imaging of invasion followed established methodsWhere indicated, erythrocytes were pre-treated with the actin and myosin inhibitors cytochalasin D , blebbistatin  and 2,3-Butanedione monoxime  prior to merozoite invasion for 1 h at 37 °C, and washed two times. Filtered merozoites were then added to treated erythrocytes and invasion assays completed as publishedTo deplete erythrocytes of ATP prior to invasion, erythrocytes were washed three times in PBS and incubated at 8% haematocrit in either PBS alone or depletion media containing 6 mM iodoacetamide  and 10 mM inosine  in PBS for between 10 min and 3 h at 37 °C in a humidifying oven on rotation. Treated cells were then washed three times in incomplete culture medium and used in merozoite invasion assays.Quantitative proteomic experiments were performed using lysates of erythrocytes undergoing active merozoite invasion or in the presence of either 40 IU/mL heparin (Pfizer) or 100 μg/mL R1 peptide (GL Biochem), where invasion was allowed to continue for 1.5 min to enrich for mid-invasion events. As such, invasion rates obtained tended to be lower than for standard flow cytometry based assays (above), which allow merozoite-erythrocyte interaction for 30–40 mins. For assays where D10-PHG parasites were used, a proportion of the sample was used for evaluation of invasion rate by flow cytometry. The remaining sample was then rapidly centrifuged , supernatant containing media and unbound merozoites removed and pellets snap frozen on dry ice to stop invasion.+ platelet apheresis blood waste  from healthy donors with informed consent for research use in accordance with the Declaration of Helsinki and approval from the Local Research Ethics Committee (REC 12/SW/0199). Fluospheres of 1 μm diameter  (0.67% solution) were incubated with BlockAid solution  or wheat germ agglutinin  at 50μg/mL for 1 h with rotation at room temperature. Beads were washed once in complete culture media, resuspended in complete culture media at a concentration of 3.6 × 106/μL and sonicated for 1 min in a sonicating water bath. A concentration of 3.6 × 106/μL microbeads was selected to mimic standard merozoite invasion assay conditions, which typically involve incubation of erythrocytes with filtered merozoites at between 3 × 106/μL and 4 × 106/μL . Assays were performed as described for merozoite invasion assays.For control microbead assays, erythrocytes were isolated from O+ erythrocytes supplied in equal amounts from two different donors were washed three times in incomplete culture medium, and 100 μL of packed erythrocytes (pRBCs) used per assay. To lyse, pRBCs were resuspended in 100 μL of lysis buffer at 4 °C  and incubated on ice, with periodical agitation, for 10 min. To reseal, 20 μL of resealing buffer  was added to the lysing erythrocytes and each sample incubated at 37 °C on rotation for 1 h in a humidifying oven. Samples were washed three times in incomplete culture medium. For resealing of membrane impermeable compounds into erythrocytes, the desired compounds were included in the lysis buffer. To label resealed erythrocytes, dextran-Alexa Fluor® 647 10,000 MW  was included in lysis buffer resulting in a final concentration of 125 μg/mL. Phalloidin (P3457) was supplied by ThermoFisher. For ATP studies, various concentrations of either ATP  or AMP-PNP triphosphate tetralithium salt hydrate, A2647, Sigma), were included in both the lysis and resealing buffers, with ATP or analogue buffer concentrations ranging from 1 mM to 10 mM in equal amounts of solvent (DDW).O3VO4, 2 mM PMSF, 1% (v/v) protease inhibitor cocktail set V (Merck Millipore) and 1% (v/v) phosphatase inhibitor cocktail set IV (Merck Millipore). Total protein concentration of lysates was quantified using a DC Protein Assay (BioRad) and 100 μg of each sample labelled using TMTsixplex reagents according to manufacturer’s instructions (Thermo Scientific). The labelled samples were pooled and then subjected to phospho-peptide enrichment using a TiO2-based enrichment kit, according to the manufacturer’s instructions (Pierce). The phospho-enriched sample was then analysed by RP nano-LC MSMS using an LTQ-Orbitrap Velos mass spectrometer.Samples were lysed for 10 min on ice in lysis buffer containing 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% (v/v) Nonidet P-40, 10 mM NaEach phospho-enriched sample was fractionated using an Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). In brief, peptides in 1% (v/v) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific). After washing with 0.5% (v/v) acetonitrile 0.1% (v/v) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Thermo Scientific) over a 150 min organic gradient, using seven gradient segments  with a flow rate of 300 nL/min. Solvent A was 0.1% (v/v) formic acid and Solvent B was aqueous 80% (v/v) acetonitrile in 0.1% (v/v) formic acid. Peptides were ionized by nano-electrospray ionization at 2.1 kV and a capillary temperature of 250 °C.Tandem mass spectra were acquired using an LTQ- Orbitrap Velos mass spectrometer controlled by Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode. The Orbitrap was set to analyse the survey scans at 60,000 resolution (at m/z 400) in the mass range m/z 300 to 1800 and the top 10 multiply charged ions in each duty cycle selected for MS/MS fragmentation using higher-energy collisional dissociation (HCD) with a normalised collision energy of 45%, activation time of 0.1 ms and at a resolution of 7500 within the Orbitrap. Charge state filtering, where unassigned precursor ions were not selected for fragmentation, and dynamic exclusion  was used.P. falciparum 3D7 databases using the SEQUEST algorithm. Peptide precursor mass tolerance was set at 10 ppm, and MS/MS tolerance was set at 20 mmu. Search criteria included carbamidomethylation of cysteine (+57.0214) and the addition of the TMT 6Plex mass tag (+229.163) to peptide N-termini and lysine as fixed modifications and oxidation of methionine (+15.9949) and phosphorylation of serine, threonine and tyrosine (79.966Da) as variable modifications. Searches were performed with full tryptic digestion and a maximum of 1 missed cleavage was allowed. The reverse database search option was enabled and all peptide data was filtered to satisfy false discovery rate (FDR) of 5%. The Proteome Discoverer software generates a reverse “decoy” database from the same protein database used for the search and any peptides passing the initial filtering parameters that were derived from this decoy database are defined as false positive identifications. The minimum cross-correlation factor (Xcorr) filter was readjusted for each individual charge state separately to optimally meet the predetermined target FDR of 5% based on the number of random false positive matches from the reverse decoy database. Thus each data set has its own passing parameters. Quantitation was done using a peak integration window tolerance of 0.0075 Da with the integration method set as the most confident centroid.The raw data files were processed using Proteome Discoverer software v1.2 (Thermo Scientific) and searched against a combined database consisting of the UniProt Human and Quantitative peptide fold-change data was manually curated using Excel v14.4.1 to select peptides of human origin with a high confidence measure (FDR ≤ 1%). Peptides derived from known serum proteins, based on localisation data from the Uniprot database, were excluded from further analysis. Where multiple spectra were obtained for the same peptide within an experiment, the median fold-change of the reads was taken. Peptide medians were then transformed to log base 2 and median centered to adjust for loading, a linear transformation commonly used in microarray analysesThe confidence of phospho-site localisations within individual outlier peptides was assessed using Scaffold v4 (Proteome Software) to calculate the SEQUEST delta Cn score, which is a measure of the difference in the top two peptide sequence correlation scores (Xcorr), where a score of > 0.1 is considered a confident modified site assignment6869P. falciparum origin was filtered using Excel v14.4.1 to exclude medium confidence peptides (1% ≤ FDR ≤ 5%) and retain only high confidence peptides (FDR ≤ 1%).Data collected for peptides of How to cite this article: Zuccala, E. S. et al. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton. Sci. Rep.6, 19766; doi: 10.1038/srep19766 (2016).


Retromer is a protein assembly that orchestrates the sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma-membrane transport pathways. Here, we have employed quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein . Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo-specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma-membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo-specific functions for retromer-interacting proteins. Further analysis of the retromer–VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to mistrafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall, these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex. On arriving in the endosomal network, internalized transmembrane protein cargos have two principal fates – either they are sorted from the limiting membrane of the endosome into intraluminal vesicles for delivery to the lysosome for degradation or, alternatively, they are exported from the endosome for recycling to the plasma membrane or to the trans-Golgi network (TGN) . AlthougPioneering studies in yeast led to the identification of a pentameric protein complex termed ‘retromer’ that was essential for the recycling of the Vps10 sorting receptor from endosomes to the TGN . In yeasThe CSC is an ancient protein assembly that evolved before the last common eukaryotic ancestor and has been highly conserved throughout eukaryotic evolution . UnsurprThe diversification of the roles of retromer has raised the fundamental question of how the common CSC is able to sort such a wide array of cargo proteins into distinct endosome-to-TGN and endosome-to-plasma-membrane transport pathways . PotentiTo further explore the role of the CSC as an endosomal scaffold and provide additional insight into the functional heterogeneity of retromer, we here describe the employment of stable isotope labeling with amino acids in cell culture (SILAC)-based labeling and high-resolution mass spectrometry to quantify the interactome of human VPS35. In addition to established interactors, including all components of the WASH complex, this has identified a number of new retromer accessory proteins that we have functionally validated in the context of retromer-mediated endosome-to-plasma-membrane recycling. These include ANKRD50, a multiple ankyrin-repeat domain containing protein with no described function; SDCCAG3, an endosome-associated protein implicated in cytokinesis , and VARsupplementary material Table S1), 63 proteins were considered to comprise the VPS35 interactome after filtering of the data by two criteria – protein quantification from two or more peptides and an enrichment of >2.5-fold over control. Gene ontology analysis revealed that the majority of these interactors had roles in ‘establishment of localization’, ‘localization’ and ‘transport’ . Of these, we selected the top three proteins, each with high levels of enrichment and numerous quantified peptides, for further validation and functional analysis. These proteins were VARP , ANKRD50  and SDCCAG3 . In addition, we further characterized the role of RME-8 , a protein previously implicated in retromer-mediated sorting . All of these data are indicative of endosomal missorting leading to an enhanced degradation for these SNX27–retromer cargos under VARP-suppressed conditions.In contrast to the subtle effects on GLUT1, subsequent examination of the additional SNX27–retromer cargos MCT1, CD97 and TRAILR1 revealed a functional link between retromer and VARP. Suppression of VARP and, as published previously, VPS35  led to asupplementary material Fig. S1Aiii). Thus, of those SNX27–retromer cargos examined in the present study, only GLUT1 appears to require association with FAM21 and the actin polymerizing WASH complex for optimal endosomal sorting and transport.To further probe the molecular heterogeneity in SNX27–retromer-mediated endosome-to-plasma-membrane sorting, we performed parallel assays under conditions where additional retromer accessory proteins were suppressed. It was notable that disruption of the WASH complex through FAM21 depletion had no or only minor effects on MCT1, CD97 and TRAILR1 trafficking .For RME-8 and SDCCAG3, RNAi-mediated suppression produced minor effects on CD97 surface levels  and the supplementary material Fig. S1Avii).Finally, and as briefly discussed previously, suppression of ANKRD50 expression generally phenocopied a loss of SNX27–retromer. From flow cytometric analysis, suppression of ANKRD50 led to an ∼35% decrease in the surface levels of CD97 , alongsiGiven the strong functional relationship between retromer and VARP in the endosomal sorting of MCT1, CD97 and TRAILR1, we analyzed the binding of VARP to the retromer complex in more detail. VARP comprises an N-terminal VPS9 domain and two C-terminal ankyrin repeat regions, ANKR1 and ANKR2 . Co-immuWe were intrigued to observe that endogenous VARP protein levels were consistently decreased by ∼50% in cells treated with siRNA against VPS35 . That thIn light of the evidence that the retromer CSC functions as a scaffold for aiding the endosomal association of various accessory proteins including the WASH complex, we examined whether this was also the case for the endosomal association of VARP. However, and entirely consistent with the ability of VARP to bind to endosome-associated Rab32–GTP and Rab38–GTP , RNAi-meTo further validate the functional significance of the observed interaction between retromer and VARP, we turned to analyzing the established role of VARP in melanosome biogenesis. Here, the Rab32/Rab38 and VAMP7 binding activities of VARP, but not its Rab21 guanine nucleotide exchange factor (GEF) activity , are reqHere, we have employed quantitative SILAC proteomics to define the interactome of VPS35. Besides detecting most established retromer interactors, which validates our proteomic approach, we identified a number of new interactors, three of which – ANKRD50, VARP and SDCCAG3 – we have characterized in more detail. In addition, we have further clarified the functional significance of the previously identified retromer interactors RME-8 and the WASH component FAM21. For the functional analysis, we focused on the emerging role of SNX27–retromer as a major regulator of endosome-to-plasma-membrane recycling . We did Interestingly, of the new interactors, or any of the established interactors, none appeared to be fully essential for core SNX27–retromer function, as their efficient knockdown resulted in only partial or cargo-specific phenotypes. The exception was ANKRD50, a 220-kDa protein that contains 19 ankyrin repeats flanked by two termini with no homology to known functional domains. Nothing is known about this protein but our data has established that it is essential for SNX27–retromer function, as its knockdown strikingly phenocopies the loss of this sorting complex across multiple cargos. Precisely what role ANKRD50 plays in SNX27–retromer function remains to be described, and experiments are currently underway to elucidate this.Our analysis has also established that the WASH complex is not essential for the recycling of all SNX27–retromer cargos. Its suppression does not affect MCT1 sorting and has only minor effects on the sorting of CD97 and TRAILR1. Thus, not all SNX27–retromer cargos appear to require actin polymerization by the WASH complex for efficient sorting. Moreover, it is interesting to note that VARP suppression affected all of those cargos  that were not affected by the suppression of WASH complex function. VARP therefore appears to be essential for the sorting of these non-WASH-dependent cargos. The molecular reason(s) behind the differences in the itineraries of these cargos certainly warrants further investigation.For several reasons, we focused our study on the newly identified retromer interactor VARP. VARP has emerged as a regulator of late endosomal dynamics through its effect on VAMP7 . It playBesides this specific role in melanosome biogenesis, the identification of VARP as a retromer accessory protein provides a further molecular link bridging retromer with the timing of Rab GTPase switches. Direct binding of VPS29 to the proposed Rab7 GTPase-activating protein TBC1D5 has been implicated in the control of retromer targeting to the late endosome through modifying the availability of Rab7–GTP for binding to VPS35 . TBC1D5 Finally, the described role of VARP in controlling the fusogenic state of VAMP7 and limiting late-endosome–lysosome fusion , is of iDuring the review of our paper, David Owen and colleagues also identified the interaction between retromer and VARP . ImportaAntibodies used in the study were against the following proteins; SNX1 , WASH1 and FAM21 . The Cytoscape plug-in Enrichment Map was used to display overlap between gene ontology terms within Cytoscape (version 2.8.2). Node size was mapped to the number of genes within a Gene Ontology category and node color was mapped to the t-test. For immunoprecipitation experiments, the respective cell lines stably transduced with the desired GFP-tagged constructs were lysed in Tris-based immunoprecipitation buffer , and GFP was precipitated with GFP-trap beads (Chromotek). Western blots were performed using standard procedures. Detection was carried out on a Li-Cor Odyssey Infrared scanning system using fluorescently labeled secondary antibodies. Melan-a cell culture, siRNA transfection, immunostaining of melanogenic enzymes (tyrosinase and Tyrp1) and bafilomycin treatment were performed as described previously (HeLa and RPE1 cells were maintained in DMEM (Gibco-Invitrogen) plus 10% (v/v) fetal calf serum (Sigma-Aldrich) and penicillin-streptomycin (PAA). For immunofluorescence analysis, plasmids containing the stated constructs were transfected using Lipofectamine LTX reagent (Invitrogen). At 48 h after transfection, cells were fixed in 0.1 M phosphate buffer containing 4% (w/v) paraformaldehyde for 10 min on ice and permeabilized with 0.1% (v/v) Triton X-100 for 5 min. Thereafter, cells were incubated with 0.5% (w/v) bovine serum albumin for 30 min, followed by incubation with the indicated primary antibodies and subsequent incubation with secondary antibodies (Molecular Probes). For nuclear staining, DAPI was used. Images were recorded on a Leica SPE or a Leica AOBS-SP2 confocal microscope. Following acquisition, images were analyzed with the Volocity software package (Perkin Elmer); to filter noise, thresholds were applied uniformly across conditions. After setting of the thresholds, the percentage of colocalization and the Pearson's correlation between the respective channels were quantified with the colocalization tool of the Volocity software. Each colocalization analysis was based on the quantification of ≥150 images. Statistical significance was determined by using an unpaired Student's eviously .For VPS35 suppression, the ON-TARGETplus human SMARTpool (Dharmacon) was used, except when suppressed in cells stably expressing a GFP–VPS35 siRNA-resistant construct, in which case only oligo 3 from the SMARTpool was used. RME-8, ANKRD50 and FAM21 were suppressed using ON-TARGETplus human SMARTpools (Dharmacon). VARP was suppressed using the oligo 5′-AAGAUGUGUCACCCUCUCUGCTT-3′. For GLUT1 immunofluorescence localization experiments, SDCCAG3 was suppressed using the oligo 5′-AAUUCUAAGCUGAGAAGAATT-3′, and for examination of surface levels of GLUT1 and MCT1 a second oligo was also used (5′-UACGACGCACUGAAAGAUGAATT-3′). Mouse VPS35 was suppressed using the following oligos : 5′-GGUGUAAAUGUGGAACGUU-3′ (site 1) and 5′-CCUGUAGAAGACCCUGAU C-3′ (site 2).For the quantification of protein surface levels, cells were surface biotinylated with a commercially available kit (Pierce/Thermo) according to the manufacturer's instructions at 72 h post transfection. Cells were lysed in PBS with 2% Triton X-100, lysates were pooled and streptavidin–agarose  was used to capture biotinylated membrane proteins. After capture, biotinylated proteins were washed extensively in 1.2 M NaCl containing PBS with 1% Triton X-100 to remove cytoskeletal proteins and contaminants from the transmembrane proteins, followed by elution of the proteins by boiling in dithiothreitol-containing sample buffer.Flow cytometric detection of TRAILR1 and CD97 was performed as described previously . Antibodt = 0.To measure the degradation of surface proteins, HeLa cells were transfected with the requisite siRNA. At 24 h post transfection, before the effects of the siRNA were fully established, surface proteins were biotinylated with sulpho-NHS-biotin (Pierce/Thermo). Cells were lysed at 0, 12 and 24 h after biotinylation , and biotinylated proteins were captured with streptavidin–agarose  and detected by quantitative western blotting on an Odyssey scanner. Degradation was quantified as the fluorescence signal remaining after 12 and 24 h as a percentage of the signal intensity at VPS35 was subcloned into pXLG3. The shRNA viral construct against the 3′UTR of VPS35 was purchased from Sigma (TRCN0000337019). siRNA-resistant VPS35 was generated by introducing 6 silent base mis-matches  into the open reading frame, conferring resistance to siRNA VPS35-3 described above.t = 0 (degradation assays) for each condition. The mean and standard error of the mean over the indicated number of independent experiments were calculated, followed by performing an unpaired Student's t-test to determine statistical significance. Colocalization data from at least three independent experiments was averaged across individual experiments and standard deviation was calculated across the three experiments. An unpaired Student's t-test was then used to analyze statistical significance. For all statistical tests, a P-value of <0.05 was considered significant and is indicated by an asterisk.All quantified western blot and confocal colocalization data are shown as the mean of the indicated number of independent experiments. The raw data from the quantitative western blotting were first normalized to percentage of control (comparative western blots) or to percentage of


P = 0.017). Participants lacking strongly inhibitory antibodies with albuminuria ≥1 g/g creatinine had a significantly increased primary event hazard ratio, 3.41 – 95% confidence intervals  compared to those lacking strongly inhibitory antibodies with lower baseline albuminuria ( <1 g/g creatinine). These results suggest that anti-endothelial cell antibodies interact significantly with albuminuria in predicting the composite endpoint of death, ESRD, or substantial decline in renal function in older, adult type 2 diabetic nephropathy.Albuminuria is a strong predictor of diabetic nephropathy chronic kidney disease outcomes. Yet, therapeutic albuminuria-lowering has not consistently translated into a reduction in clinical events suggesting the involvement of additional pathogenic factors. Our hypothesis is that anti-endothelial cell autoantibodies play a role in development and progression in diabetic nephropathy. We determined anti-endothelial cell antibody (AECA) bioactivity in protein A-elutes of baseline plasma in 305 participants in the VA NEPHRON-D study, a randomized trial of angiotensin receptor blocker (ARB) or dual ARB plus angiotensin-converting enzyme inhibitor therapy in type 2 diabetes with proteinuric nephropathy. Thirty-eight percent (117/305) of participants had significantly reduced endothelial cell survival ( ≤80%) in the IgG fraction of plasma. A VA NEPHRON-D primary endpoint  was experienced by 58 individuals. In adjusted Cox regression analysis, there was a significant interaction effect of baseline anti-endothelial cell-mediated cell survival and albuminuria on the hazard rate (HR) for primary composite endpoint ( Renin–ahropathy . The lachropathy  or in thhropathy  has refoin vitro  . Increasin vitro , we hypoin vitro , a rando2  rate 30–89.9 ml/min/1.73 m2 . IndividOvernight fast blood draws were performed at each site. EDTA plasma was aliquoted and shipped frozen to a central repository and stored at −80°C. Archived, coded frozen EDTA plasma was shipped to the laboratory of Dr. Zimering  where EC bioassays were performed. The samples were kept frozen (−40°C) prior to assay for protein A-eluted EC bioactivity. EC bioactivity in the protein A eluates of plasma was previously shown to be stable for 5 years or longer at −20°C .Comorbidity was determined using chart review and patient history; race and ethnicity were by self-report. Cardiovascular disease is defined as the prior occurrence of any one of the following at the baseline study evaluation visit . Standard laboratory and clinical measures were determined at individual study sites. Creatinine value for the primary endpoint was measured in a central lab using an IDMS traceable creatinine assay. Estimated GFR was calculated using the Modification of Diet in Renal Disease (MDRD) formula for IDMS traceable creatinine. Urinary albumin excretion rate was determined in a random sample in the vast majority of participants.Protein A chromatography was carried out as previously described , 15. FouProtein concentrations were determined by a bicinchoninic acid protein assay kit .Bovine pulmonary artery (BPA) ECs  were maintained at 37°C in 5% CO2/95% air in Medium 199 plus 10% fetal calf serum. BPA cells were passaged continuously and used between passages 4 and 10. BPA EC  were incubated (at 37°C× 48 h) with (30 μg/mL) of the protein A eluate of plasma. BPA ECs mitogenicity is exquisitely sensitive to low concentrations of basic fibroblast growth factor (bFGF) . We chos3 cells/well in Medium 199 plus 10% fetal calf serum in 96-well plates. After 1 or 2 days incubation for cells to reach 60–80% confluency, test fractions (30 μg/mL of protein A eluates of plasma) were added to wells in quadruplicate. After 48 h incubation in the presence of test fractions, cells were washed with PBS and processed for the colorimetric estimation of cell number, i.e., cell-associated acid phosphatase activity, as previously described (n = 2–4 assays in each patient).Endothelial cell number assays were carried out as previously reported . Conflueescribed . There wescribed . Growth-escribed . Each pot-test or Chi-square test was used for the comparisons between antibody groups shown in Table Student’s P-values for all outcomes were two-sided; values <0.05 were considered statistically significant. Statistical analyses were performed using SAS, version 9.3.n = 305) did not differ significantly in any of the mean baseline risk factor levels shown in Table 2. We observed 38% (117/305) strongly inhibitory EC activity  in the immunoglobulin G (IgG) fraction of plasma. Table 1c), LDL cholesterol, systolic or diastolic blood pressure, smoking, or medication use. We observed that participants with less inhibitory AECA were younger. Individuals with strongly inhibitory AECA were more likely to have diabetic retinopathy, but less likely to have a history of a prior macrovascular event. There was a trend of a significant association between strongly inhibitory AECA and baseline atrial fibrillation history.All of the 305 individuals in this analysis were men. The study population (rved 38% 17/305 stP = 0.017) (Table P < 0.001); whereas participants with strongly inhibitory antibodies and higher baseline albuminuria ( ≥1 g/g creatinine) or those with strongly inhibitory antibodies and lower baseline albuminuria ( <1 g/g creatinine) had a non-significant hazard for primary endpoint occurrence HR, 1.44  and HR, 1.53 , respectively  for primary composite endpoint occurrence (7) Table . CompareP = 0.312), baseline eGFR ( <60 vs. ≥60 mL/min/1.73 m2) (P = 0.580), prior macrovascular event (P = 0.388) or age (P = 0.078) , 1.37–3.69; P = 0.08 compared to lower albuminuria subgroups)  Table . In the s) Table . Primarys) Table .The present data are the first to suggest a substantially increased prevalence (38%) of strongly inhibitory plasma anti-EC autoantibodies in proteinuric adult type 2 diabetic nephropathy. These findings are consistent with prior reports of an association between strongly inhibitory EC autoantibodies and proteinuric type 2 diabetic nephropathy , 18. TakSeveral of the known properties of anti-EC antibodies suggest their possible involvement in mediating one or more pathological findings  reported in renal biopsy specimens in human diabetic nephropathy . For exaAngiotensin II increases bFGF and transforming growth factor beta (TGF-β) expression in vascular smooth muscle cells ; bFGF an2) than in the baseline VA-NEPHRON-D, participants were 2.5 times more likely to have had (low titer) weakly inhibitory and (high titer) moderately stimulatory (FGF-like) EC plasma antibodies  than AECA ≤80%, i.e., strongly inhibitory anti-EC antibodies at both low and high titers tested . Growth stimulatory antibodies having properties resembling those of bFGF stimulated proliferation in both ECs and in fibroblasts  may be a marker involved in mediating the increased hazard for primary endpoint in patients having higher albuminuria. Prior work in older adult type 2 DM  and in croblasts . They maroblasts .The albuminuria × anti-EC antibody-mediated cell survival interaction demonstrated here suggests that higher albuminuria is necessary, but may not be sufficient to explain a substantial degree of the variability in renal composite endpoint occurrence in an older, high-risk diabetic nephropathy population. Endothelial damage and inflammation have been identified as possible markers of residual high risk in subsets of diabetic nephropathy . Basic Fp < 0.001, n = 9 patients). The latter observation is consistent with a possible role for higher titer, strongly inhibitory anti-EC antibodies in modulating HBGF bioavailability in the renal interstitium.Among several limitations of our study, one is that the findings may only reflect the experience of middle-aged or older men with type 2 DM. More study is needed in additional and larger diabetic populations to confirm a role for AECA-mediated cell survival/albuminuria interaction effect on primary endpoint occurrence. Non-diabetic renal disease may have coexisted in a small subset of adult type 2 diabetes and possibly contributed to the observed effect of AECA-mediated cell survival/albuminuria interaction on the overall rate of primary end point occurrence. An increased prevalence of anti-EC antibodies was reported in advanced non-diabetic CKD compared to a normal population  and simiIn summary, the current findings demonstrate involvement of a novel, putative pathogenic factor, i.e., anti-EC autoantibodies, as a significant modulator of the occurrence of the composite endpoint of renal function decline, ESRD, or death in adult proteinuric, type 2 DM. Much more study is needed to determine whether the albuminuria/AECA interaction implies a dual role for strongly inhibitory and/or stimulatory antibodies associated with less inhibitory antibodies in providing a brake for or accelerating growth factor-driven cellular processes underlying declining renal function (or premature death) in adult type 2 DM nephropathy.Dr. Nicholas Emanuele received honoraria as member of the Speaker’s bureau for Merck and Co., Inc. The other co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The expression of miR-26a in the liver was induced in a TGR5-dependent manner after feeding the mice with a bile acid diet. TGR5 activation strongly increased the expression of miR-26a in macrophages, including the Kupffer cells in the liver. We further demonstrated that JNK pathway was required for miR-26a induction by TGR5 activation. Interestingly, we located the TGR5-responsive DNA element to a proximal region of miR-26’s promoter, which was independent of the transcription of its host genes. These results unravel a new mechanism by which bile acid receptor TGR5 activates a miRNA gene expression.GPBAR1/TGR5 is a G protein–coupled receptor of bile acids. TGR5 is known to regulate the BA homeostasis and energy metabolism. Recent studies highlight an important role of TGR5 in alleviating obesity and improving glucose regulation, however, the mechanism of which is still unclear. Here we report that TGR5 is involved in mediating the anti-obesity and anti-hyperglycemia effect of a natural compound, oleanolic acid. By comparing the miRNA profiles between wild type and TGR5 Recent studies have highlighted bile acid (BA) signaling as a new therapeutic target for the treatment of metabolic disorders, including obesity and type 2 diabetes (T2D) . Two majObesity and its major co-morbidity, T2D, have reached an alarming epidemic prevalence without an effective treatment available, thus highlighting an urgent medical need for new treatment strategies . RecentlHere we show that TGR5 is the receptor that partially mediates the anti-obesity and anti-hyperglycemia effect of a natural compound, oleanolic acid (OA). TGR5 activation increases the expression of miR-26a in macrophages through a JNK-dependent pathway. This study thus provides a potential link between bile acid receptor TGR5 and miR-26a expression.BMS-345541, SP600125, PD98059, and H89 were purchased from Calbiochem . Oleanolic acid (OA), Lipofectamine 2000 and Lipofectamine LTX as well as other reagents for cell transfection were obtained as we described previously .-/- mice in C57BL/6 background were described previously [2. All euthanasia procedures will follow the recommendation of the Panel on Euthanasia of the American Veterinary Medical Association.C57BL/6 wild type (WT), c-Jun-N-terminal kinase 1 (JNK1) knockout and TGR5eviously . Animal When animal feeding was terminated, all mice were euthanized. Liver tissues were fixed in 10% formalin, dehydrated and embedded in paraffin. Hematoxylin and eosin staining was performed for standard histological examination by Pathology Core at City of Hope.Isolation and cultivation of both Kupffer cells and Peritoneal macrophages were described in detail previously .Blood glucose levels were determined using a portable glucose meter (Abbot Laboratories). For glucose tolerance test (GTT), mice were fasted for 16 hours and then injected intraperitoneally with D-glucose (2 g/kg body weight). Blood glucose levels were then determined immediately before glucose injection or 15, 30, 60 and 120 minutes after injection.The RNAs preparation and quantification by real-time polymerase chain reaction (RT-PCR) were described in detail previously . Primer Transient transfection was performed in a 96-well plate. Briefly, RAW264.7 cells were transfected with plasmids pGL3-TK-miR-26a-Luc using Lipofectamine LTX reagent according to the manufacturer’s instructions. For mIR-26a promoter analysis, different deletions of miR-26a promoter DNA sequences were cloned into pGL3-TK-Luc plasmids. To normalize the transfection efficiency, cells were co-transfected with pGL3-TK plasmid. Cells were treated with either DMSO or OA (10 μM) respectively for 24 hours. Luciferase activity was assessed using the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions. At least three replicates of each transfection were performed.P < 0.05, **P < 0.01, *** P < 0.001) unless otherwise indicated.Results of experiments are presented as mean ± SEM. Two-tailed Student's t test was used to determine the significant differences between data groups  and TGR5-/- mice of 8 weeks of age were fed with HFD or the control regular chow diet. After 8 weeks, the mice fed with HFD were then randomly divided into two groups. One group were kept feeding with HFD. The other group was switched to HFD+OA for additional 8 weeks. Mouse body weights were monitored during the entire process. In both WT and TGR5-/- mice, HFD significantly increased mouse BW. As expected, OA treatment starting at 8th week significantly reduced the mouse BW compared to that of HFD control mice , which can be activated by TGR5 as we previously described . Thereforeatment . These rmiR-26 family has two members, miR-26a and miR-26b. In both the human and mouse genomes, two loci align with miR-26a—miR-26a-1 and miR-26a-2—which are located in intronic regions of CTDSPL and CTDSP2, respectively  to alleviate obesity-induced glucose intolerance . HoweverCTDSP1 . HoweverIn summary, the present work demonstrates that membrane-bound BA receptor TGR5 activates miR-26a expression through a JNK-dependent pathway. Our results thus provide a novel link between TGR5 and miRNA expression. These findings also suggest that activation of TGR5 by small molecules could be a novel approach to modulate the expression of miR-26a for the treatment of metabolic diseases.S1 Fig(A) Genomic location of miR-26a family members . The promoters of miR-26a family members are shown. (B) RT-PCR strategy for detection of pri- and mature miR-26a.(TIF)Click here for additional data file.S2 FigAt the end of the feeding, mice were euthanized and the white adipose tissues from either WT or TGR5-/- mice were weighted and compared.(TIF)Click here for additional data file.S3 Fig-/- mice were subjected to H&E staining.At the end of the feeding, mice were euthanized and the liver tissue sections from either WT or TGR5(TIF)Click here for additional data file.S4 FigWT mice were fed with regular chow diet (Re) or 1% CA for 3 days. WT mice were orally gavaged with vehicle (Veh) or GW4064  once every day for 3 days. MiR26a expression in the liver was determined by QRT-PCR.(TIF)Click here for additional data file.S5 Fig-/- (TGR5) mice were i.p injected with DY284 (30 mg/kg). Two days later, livers RNAs were prepared and used for measuring miR-26a levels by QRT-PCR.Wild type (WT) and TGR5(TIF)Click here for additional data file.S6 FigSK-MEL-28 and A375 melanoma cell lines were treated with OA (10 M) for 24h. The expression of miR-26a was measured by QRT-PCR.(TIF)Click here for additional data file.


It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. Atherothrombosis, which is triggered by activated human platelets and caused high mortality or disturbance of daily life, is a major health concern to contribute cerebrovascular events and cardiovascular diseases ,2. ActivType 2 diabetes mellitus (DM) is currently considered a major health problem not only in developed countries but also in developing ones . Even inHeat shock proteins (HSPs), which could be expressed by a variety of biological stresses in mammalian cells, generally act as molecular chaperones that protect unfolded proteins from aggregation . Heat shIn the present study, we conducted the investigation into the relationship between the phosphorylation of HSP27 and TRAP-stimulated platelet activation in the patients of type 2 DM. We herein show that TRAP-induced activation of Akt in addition to p38 MAP kinase is involved in the release of phosphorylated-HSP27 from human platelets, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.n = 47) are presented in The characteristics of the subjects  using the laser scattering system are shown in Human HSP27 is phosphorylated at three residues of serine  ,13. In oIt has recently been reported that the 3-phosphoinositide-dependent protein kinase-1 (PDK1)-regulating phosphorylation of Akt is involved in thrombin-induced platelet activation . We haveR2 = 0.383, p = 0.005)  were significantly correlated with those of the HSP27 phosphorylation (Ser-78) (= 0.005) .R2 = 0.589, p < 0.001)  .R2 = 0.399, p = 0.004, n = 16)  .R2 = 0.137, p = 0.010, n = 47) (PDGF-AB that is secreted accompanied with platelet activation promotes the progress of atherosclerosis as a mitogenic growth factor . We have n = 47) .R2 = 0.129, p = 0.013; R2 = 0.257, p < 0.001; R2 = 0.327, p < 0.001, respectively, n = 47)   of platelet aggregation including transmittance and the size of aggregates against the levels of released phosphorylated-HSP27 induced by 10 µM TRAP in the platelets of type 2 DM patients. The levels of released phosphorylated-HSP27 were significant to AUC of light transmittance A. In add n = 47) B,C. Howe n = 47) D.We further examined the effect of deguelin, an inhibitor of Akt , on the We have recently shown that in type 2 DM patients the release of HSP27 from platelets is accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation . ThrombiWe next investigated whether the phosphorylated-HSP27 induced by TRAP is released from the platelets of type 2 DM patients. We measured the release of phosphorylated-HSP27 under the stimulation of 10 µM TRAP and analyzed the relationship between the individual levels of extracellular phosphorylated-HSP27 and intracellular TRAP-induced phosphorylation of HSP27 in the platelets. The levels of released phosphorylated-HSP27 stimulated by TRAP were directly proportional to the levels of intracellular phosphorylated-HSP27. Therefore, our findings suggest that the TRAP-induced phosphorylation of HSP27 elicits the release of HSP27 from the TRAP-activated platelets into circulation.p = 0.058) but looks inversely proportional. Taking our findings into account, it is probable that the phosphorylated-HSP27 release reflects the hyper-aggregation of platelets in the pathological state of type 2 DM. Evidence is accumulating that HSP27 not only intracellularly acts as a molecular chaperone but also extracellularly affects the behavior of the surrounding environment [We demonstrated here that there is a close correlation between the individual levels of the TRAP-induced release of phosphorylated-HSP27 and the secretion of PDGF-AB, a mitogenic mediator promoting atherosclerosis , from thironment . It has ironment , whereasironment . As for ironment . It is wironment ,2. In adironment . We haveironment . Based oironment . In addiironment . Taking TRAP  was purchased from Bachem AG . Phospho-specific HSP27 (Ser-78) antibodies were purchased from Stressgen Biotechnologies . Phospho-specific Akt (Ser-473 and Thr-308) antibodies, phospho-specific p44/p42 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies and phospho-specific HSP27 (Ser-82) antibodies were purchased from Cell Signaling Technology, Inc. . GAPDH antibodies and deguelin were purchased from Santa Cruz Biotechnology, Inc. . The PDGF-AB ELISA kit was purchased from R&D . The phosphorylated-HSP27 ELISA kit was purchased from Enzo Life Science, Inc. . Other materials and chemicals were obtained from commercial sources. Deguelin was dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the detection of protein expression by a Western blot analysis.The inclusion criteria for the study were the presence of type 2 DM according to the criteria of the World Health Organization. We excluded the patients who were complicated with a malignancy, infectious diseases including hepatitis B and hepatitis C, or autoimmune disorders. All participants were advised to avoid sleep deprivation or blood donation. The study was approved by the committee of the conduct of human research at National Center for Geriatrics and Gerontology , and at Gifu University Graduate School of Medicine . Written informed consent was obtained from all of the patients and healthy donors.g for 12 min at room temperature. Platelet-poor plasma (PPP) was prepared from the residual blood by centrifugation at 1400× g for 5 min.Ten ml of blood was drawn from the vein between 8:00 and 9:00 after at least 15 min of bed rest to preserve steady state conditions. Sodium citrate (14 µM) was added to the blood immediately as an anticoagulant, and platelet-rich plasma (PRP) was obtained by centrifugation at 155× g at 4 °C for 2 min. The supernatant was collected and stored at −80 °C. The pellet was washed twice with PBS, and then lysed immediately by boiling in a lysis buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol for a Western blot analysis.Platelet aggregation was measured using an aggregometer  with a laser-scattering system as described previously ,8. In brA Western blot analysis was performed as described previously . In brieThe levels of PDGF-AB and phosphorylated-HSP27 in the supernatant of the conditioned mixture after platelet aggregation were determined using ELISA kits for PDGF-AB and phosphorylated-HSP27, respectively, according to manufacture’s instructions.The data were presented as the mean ± SD. The statistical significance of the correlation between two variables, linear regression analysis was adopted using SPSS ver. 19.0  as the software. A probability of less than 5% was considered to be statistically significant.In conclusion, our present findings strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the acceleration of platelet aggregation in type 2 DM patients.