Despite efforts in the last decade, signaling aberrations associated with obesity remain poorly understood. To dissect molecular mechanisms that define this complex metabolic disorder, we carried out global phosphoproteomic analysis of white adipose tissue (WAT) from mice fed on low-fat diet (LFD) and high-fat diet (HFD). We quantified phosphorylation levels on 7696 peptides, and found significant differential phosphorylation levels in 282 phosphosites from 191 proteins, including various insulin-responsive proteins and metabolic enzymes involved in lipid homeostasis in response to high-fat feeding. Kinase-substrate prediction and integrated network analysis of the altered phosphoproteins revealed underlying signaling modulations during HFD-induced obesity, and suggested deregulation of lipogenic and lipolytic pathways. Mutation of the differentially-regulated novel phosphosite on cytoplasmic acetyl-coA forming enzyme ACSS2 (S263A) upon HFD-induced obesity led to accumulation of serum triglycerides and reduced insulin-responsive AKT phosphorylation as compared to wild type ACSS2, thus highlighting its role in obesity. Altogether, our study presents a comprehensive map of adipose tissue phosphoproteome in obesity and reveals many previously unknown candidate phosphorylation sites for future functional investigation. Obesity, characterized by excess fat accumulation, is an epidemic and complex metabolic disorder caused by both life style and genetic variation256White adipose tissue (WAT) is the predominant site for storage of fat with adipocytes representing the majority cell type within this tissue. Adipocytes synthesize and store triglycerides during feeding, and upon fasting they hydrolyze and release triglycerides as FFA and glycerol1011131417in vivo quantitative phosphoproteome profiling of WAT from mice fed on high-fat diet (HFD) or low-fat diet (LFD). Through comprehensive analysis of the modulated phosphoproteins, we extracted site-specific dephosphorylation events on several key enzymes involved in the lipogenic and lipolytic pathways, reflective of metabolic imbalance in lipid homeostasis during obesity. In particular, we observed phosphorylation changes on acetyl-coenzymeA synthetase (ACSS2), a key enzyme involved in lipid metabolism and energy generation. As a validation of our approach we show that the phosphorylation status on a serine residue (S263) of ACSS2 plays crucial roles in triglyceride synthesis.To obtain an in-depth molecular perspective of altered events in adipocytes during obesity, we performed label-free We adopted a label-free quantitative mass spectrometry (MS)-based phosphoproteomic strategy to identify and characterize high-fat diet (HFD)-induced phosphorylation changes in mouse epididymal WAT . To esta19To gain functional insights into processes that were regulated by HFD-responsive phosphoproteins in WAT, we performed GO enrichment  for phosphoproteins with increased and decreased phosphorylation levels separately . The resModulation of signaling response relies upon intricate wiring of protein networks and activity status of the kinases involved. As changes in phosphorylation landscape are reflective of alterations in kinase activity, we used the phosphoproteomic data to systematically identify deregulated kinases in WAT upon HFD, mainly aiming to identify kinases that regulate uncharacterized phosphorylation events in metabolic pathways.2+/calmodulin-dependent protein kinase-like (CAMK-like) family of kinases, including the AMPK-related salt-inducible kinases (SIKs), sucrose non-fermenting-related kinase (SNRK), STE20-related proline alanine rich kinase (SPAK) and maternal embryonic leucine zipper kinase (MELK). Among these, SIK2 and SIK3 were significantly deregulated with reduced phosphorylation on multiple sites in our dataset. These kinases, regarded as gatekeepers of hepatic gluconeogenesis, are also known to regulate triglyceride level30First, we predicted kinase-substrate relationships for all the upregulated and downregulated phosphosites based on short linear motifs and protein-protein interactions as implemented in iGPS and NetworKIN computational approaches2324Substrate-based kinase prediction also revealed that the activities of certain kinases belonging to CDK subfamily , p38 MAP kinase, and casein kinase may be enhanced in response to HFD. In fact, a close homolog of predicted CDK18, PCTAIRE-1, was identified as a negative regulator of insulin-induced glucose transport in an RNA interference-based screening in adipocytesTo explore the functional connectivity among the regulated phosphoproteins, we constructed an integrated protein-protein interaction network by combining interactions from STRING, Reactome Functional Interaction (Reactome FI) and GeneGo Metacore35Functional enrichment analysis of the network revealed components of the insulin signaling pathway suggesting that the known proteins are successfully mapped onto the network. Densely connected cluster of proteins within the network were analysed using ClusterONE, and a total of six clusters with a p value < 0.05 and one cluster with p value < 0.1 were identifiedThe most striking feature in the WAT phosphoproteome signature, also evident from our network (cluster yellow and blue), was the selective enrichment of metabolic enzymes in the downregulated phosphoproteins. Thus, we focused on such dephosphorylation events associated with metabolic pathways, particularly lipid metabolism. The fold changes associated with the differentially regulated unique phosphopeptides from these metabolic enzymes are shown in 40Upstream of ACACA, we observed downregulated phosphosite at serine 293 of pyruvate dehydrogenase E1 component subunit alpha (PDHA1) with HFD. Dephosphorylation of this site increases the enzyme activityFurthermore, downstream of ACACA, the phosphosite abundance on fatty acid synthase (FASN) that acts as the second major enzyme of fatty acid biosynthesis was significantly altered. This specific site, threonine 976, has also been previously reported in brown adipose tissuesKey proteins involved in the lipolytic pathway were also observed with decreased phosphorylation. Indeed, we identified dephosphorylation of the rate-limiting hormone-sensitive lipase (HSL) at the major activity regulatory site serine 964 (serine 660 in rat) that is known to be phosphorylated by PKATaken together, our analysis shows a shift in the balance between lipogenesis and lipolysis pathways in WAT as a result of changes in the kinase signaling mechanisms during high-fat feeding.in vitro insulin resistance model with high lipid content  or 1 μM Dexamethasome (Dex) for 16 hours. Insulin signaling was monitored by challenging the adipocytes with acute insulin or metformin. While CI and Dex both led to impairment of insulin signaling as observed by decreased AKT phosphorylation status , Dex cou content . Since m content . We obse content . We furt content . ACSS2 Sin vivo during obesity. Our atlas revealed several uncharacterized candidate sites in the context of obesity and hence is a valuable resource for elucidating molecular events accompanying HFD-induced adipocyte dysfunction, obesity and insulin resistance.In this study, we provide a system-wide quantitative atlas of phosphorylation changes in WAT in response to HFD-induced obesity using label-free phosphoproteomics. In addition to reflecting known signaling events implicated in obesity, such as aberrant insulin signaling and associated insulin resistance, the snapshot of the adipocyte phosphoproteome points to intricate rewiring of metabolic network in a phosphorylation-dependent manner. Our dataset uncovered an array of phosphorylation changes on several key enzymes involved in lipid and glucose homeostasis, implying that these events may underlie important functional consequences in adipocyte reprogramming during obesity or insulin resistance. Though other large-scale proteomic studies have been previously performed in adipose tissues1016Metabolic disorders are characterized by dysregulated insulin response, impaired glucose homeostasis and insulin resistance. Insulin resistance is manifested by aberrant trafficking of insulin-responsive glucose transporter GLUT4 (known as SLC2A4) to the plasma membrane, and this translocation is dependent on the phosphorylation of TBC1D451Despite the wealth of data that can be acquired from a typical phosphoproteome experiment, the elucidation of the overall functional consequences of phosphorylation events is quite challenging. The phosphoproteome landscape as such is reflective of modulated kinase signaling events and linking the altered kinase signaling events in the context of protein or metabolic networks can therefore reveal activated or suppressed molecular events during obesity. Using our dataset, we predicted several upstream kinases that are likely affected during high-fat feeding. Apart from the well-documented AKT and AMPK-family of kinases, we predicted several others including MRCKα or PAK 1/2 which are not clearly understood in the context of obesity. These kinases function as a part of the CDC42 signaling, and a role for CDC42 in the insulin signaling cascade and glucose translocation has been previously suggestedTo respond to various nutritional statuses, cycles of lipogenesis and lipolysis are regulated to maintain energy homeostasis. The balance between these processes controls flux of fatty acid and ectopic lipid accumulation; however, there have been contradictory observations with respect to their status in the adipose tissue6061In summary, this study expands our understanding of the molecular mechanisms modulated during obesity by providing an in-depth systems level analysis of the altered phosphorylation profile. Our global phosphoproteome profile stresses on a phosphorylation-dependent modulation of metabolic output, and also unveils several uncharacterized phosphosites that may play important regulatory roles in glucose and lipid metabolic pathways. We believe that an extensive phosphoproteomic interrogation of various tissues in different metabolic milieu will provide a holistic view of the molecular aspects that underlie complex metabolic disorders like obesity, and reveal context-dependent or tissue-specific regulation of proteins maintaining energy homeostasis.Insulin levels were measured using Rat/Mouse Insulin ELISA kit (Merck). Triglycerides and FFAs were measured by Triglyceride Quantification Colorimetric/Fluorometric Kit (Biovision). pLenti-GFP-Zeo was purchased from Addgene. Mutants were generated by using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). AKT, pAKT, pAMPK, and pACC antibodies were obtained from Cell Signaling Technology.http://grants1.nih.gov/grants/olaw/references/phspol.htm, accessed 1 January 2012) were followed.Mice were kept on a 12-hour light-dark cycle and fed on either a standard rodent chow , or a high-fat diet . HFD was initiated at 2 months of age for 4 months. Body weights were monitored every other week. All procedures involving animal experimentation were approved by the Institutional Animal Care and Use Committee (IACUC) of A*STAR. Principles of laboratory animal care  for 16 hr.3T3L1 pre-adipocyte cell line was obtained from the American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% calf serum. 3T3L1 pre-adipocytes were differentiated into 3T3L1 adipocytes as previously describedStatistical analyses were performed by Graphpad Prism 5.0. Differences between the two groups of mice that were fed on HFD or LFD were calculated using an unpaired Student’s t-test (two-tailed). P value <  0.05 was considered statistically significant.TM C18-SD Cartridge (3 M Empore) and the amount of peptide obtained was measured using Nanodrop (ThermoScientific).Epididymal white adipose tissues (WAT) of the HFD-fed or LFD-fed mice (1.3 mg) was lysed in 9 M urea buffer consists of 20 mM HEPES (pH 8), 9 M urea, 1 mM activated sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM ß-glycerophosphate, yielding 1.3 mg protein. Reduction of the protein was carried out by the addition of 1/10 volume of 55 mM DTT to the cleared cell supernatant and incubation for 30 minutes at room temperature. This was followed by protein alkylation performed by the addition of the same volume of 120 mM iodoacetamide and incubation for 30 min at room temperature in the dark. The samples were then diluted using 20 mM Hepes (pH8) to a final concentration of 6 M urea before the addition of LysC (Wako 129-02451) with a LysC to protein ratio of 1:100 and incubation at 37 °C overnight. The samples were further diluted to a final concentration of ~1 M urea using 20 mM Hepes (pH8). Sequencing-grade trypsin (Promega) was added with a trypsin to protein ratio of 1:50 (w/w) and digestion was carried out for 4 hours at 37 °C, The resultant peptide solution was cleaned up using an Emporeet al. with slight modificationsIMAC beads were prepared as described by Ficarro 650 μg tryptic peptides were reconstituted in 650 μL 50% acetonitrile/0.1% trifluoroacetic acid, before being diluted 1:1with 100% acetonitrile/0.1% trifluoroacetic acid. Phosphopeptide enrichment was carried out by incubating the tryptic peptides with 10 μL IMAC beads for 30 minutes with end-over-end rotation. The beads were then loaded onto a self-packed C18 StageTip6, and a maximum injection time of 10 ms. The twenty most intense peptide ions in each survey MS scan with an intensity threshold of 10 k, underfill ratio of 1% and a charge state ≥2 were isolated in succession with an isolation window of 2 Th to a target value of 50 k, maximum injection time of 50 ms and fragmented by high energy collision dissociation using a normalized collision energy of 25% in the high energy collision cell. The MS/MS was acquired with a resolution of 17.5 k and a starting m/z of 100. A dynamic exclusion with exclusion duration of 15 s was applied.Reconstituted peptides were analysed on an EASY-nLC 1000  coupled to a Q-Exactive (Thermo Fisher Scientific). Peptides were first trapped onto a C18 pre-column and then separated on a 50 cm analytical column  at 50 °C. A 245 min gradient ranging from 0 to 40% acetonitrile/0.1% formic acid was used. This was followed by a 10 min gradient ranging from 40 to 80% acetonitrile/0.1% formic acid and remained at 80% acetonitrile/0.1% formic acid for 10 min. Survey full scan MS spectra (m/z 310–2000) were acquired with a resolution of 70k, an AGC target of 3 × 10Database search was performed with X!Tandem203PO4 (97.97690); decoy site localization mass 79.966331. Scoring was performed for all peptides with initial PeptideProphet probability 0.5 and above in charge state 2+ and 3+.To perform semi-quantitation by spectral counting at individual phosphorylation sites, site localization algorithm LuciPHOrFor the peptides with false site localization error rate (FLR) below 1%, the number of MS/MS spectra supporting phosphorylation on each candidate residue (S/T/Y) was counted to represent the “spectral counts” of each site. Spectral count-based tool QSPEC was used to perform differential expression analysis for each phosphosite, controlling the FDR at 10%For analysing the functional enrichment across the phosphoproteins, the significantly altered phosphopeptides with upregulated or downregulated sites upon high-fat feeding were categorized based on GO Biological Process using BINGO functional annotation tool as implemented in Cytoscape. The GO terms for each of the HFD- and LFD- fed groups were filtered to retain only those categories that were significantly enriched with p value < 0.005 and contained at least three representing genes in either of the analysed groups. The filtered p values were subjected to hierarchical clustering based on Euclidean distance and average linkage, and visualized as a heat map. Pathway annotation for metabolic enzymes was retrieved from Reactome pathway database.−4 were considered significantly altered. For those kinases that were predicted to be significantly enriched both in the upregulated and downregulated substrate groups, kinase-substrate enrichment analysis based on average abundances (log2 fold change) of phosphorylated substrates was additionally performed to assign the activity of the kinase to either the HFD- or LFD-fed groups.To predict potential kinase-substrate relationships and thus the kinase activity in the WAT of HFD- and LFD-fed mice groups, both iGPS (version 1.0) and NetworKIN (version 3.0) algorithm were used2324Protein-protein interactions as implemented in STRINGFor metabolic network mapping, the substrates, products and pathways associated with each enzyme was manually obtained from KEGG and the schema was constructed using components of the pathway map corresponding to ‘Fatty acid biosynthesis’, ‘Glycerolipid metabolism’ and ‘Citrate cycle’. Known regulations such as upstream kinases and functional consequences were obtained through literature. Finally, all the information was visualized in a schematic manner.http://proteomecentral.proteomexchange.org/) via the PRIDE partner repository with the dataset identifier PXD002927.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium .


Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits. Phosphorylation is the process by which a phosphate group is added to a protein. It leads to either activation or deactivation of a great number of proteins and represents a major building block for network regulation Numerous studies designed to understand phosphorylation-mediated regulatory mechanisms have been reported recently. Early models employed Michaelis-Menten kinetics of the simplest phosphorylation reaction The classical models assume that it is possible to ignore the concentrations of the Michaelis complexes in those cases where the total concentration of protein substrate significantly exceeds the concentrations of the kinase and the phosphatase. This approach was used as a basis in many biochemical networks with phosphorylation-dephosphorylation reactions The proportion of maximally phosphorylated substrate as a function of the kinase and phosphatase activities was recently determined to show that steeper switch-like regulation is due to increasing of number of phosphorylation sites Phosphorylation plays a critical role in the regulation of the immune system. However, there is a clear gap in the mechanistic understanding of the role of multisite phosphorylation in this process. Phosphorylation governs protein signaling via Signal Transducers and Activators of Transcription (STAT) proteins The STAT proteins are critical for many fundamental cellular processes such as proliferation, differentiation, cell growth and survival The STAT proteins are activated by phosphorylation of their C-terminal transactivation domain (CTD) by Januse Kinases (JAKs) at Tyr701 for STAT1 in response to type II interferons Interferon Regulatory Factor 5 (IRF-5) is a latent transcription factor involved in autoimmunity Several models for STAT3 and IRF-5 phosphorylation as part of larger models have been published recently. A classical approach for the phosphorylation of STAT3 by JAK has been employed in The cells that differentiate in the thymus and are involved in cell mediated immunity are known as T cells. They circulate in the lymphoid organs and the blood in the form of naive T cells, which have not been in contact with antigens yet. After the interaction with the antigen the naive CD4+ T cells are activated and can differentiate into the specific T cell phenotypes, namely T helper 1 (Th1), Th17 and regulatory T cells (Tregs). Each of these phenotypes has its own function in the regulation of the immune response and a specific cytokine signature. Th1 and Th17 cells play a critical role in the regulation of the activity of the immune response and inflammation. Tregs are known for their anti-inflammatory properties and for maintaining the immune tolerance. Th1 cells are defined by expressing IFN-γ, Th17 cells by IL-17 and Tregs by IL-10 In this study a new model for multisite phosphorylation has been developed. The model has been compared with previously reported models In this study, we developed a new mathematical model for multisite phosphorylation signaling. The model predicts probabilities for a protein to be phosphorylated at various phosphorylation sites as a function of the kinase activity. The newly developed and previously reported models were compared with the experimental data for the transcriptional regulation of the STAT and IRF-5 proteins.It has been shown that IRF-5 contributes to the polarization and plasticity of macrophages p . However the model predictions differ when the corresponding concentrations are similar.p  and highT kinase, which leads to the ultrasensitivity that is characterized by the concentrations of STAT3p being more sensitive to change in stimulus than would be expected from a Michaelis-Menten response The figures show that the system may operate in a switch-like manner with an increasing concentration of JAKp activity depends on the ratio of the total STAT3 and SHP-1 concentrations. This prediction differs from the previous study T is significantly smaller than STAT3T. However, our model offers significantly different predictions for comparable or higher phosphatase concentrations than STATs, consistent with the T cell phenotype dependence on intracellular phosphorylation signaling summarized on T and SHP-1T concentrations are comparable, the phosphorylated STAT3 species (STAT3p) increase as a function of the ratio of the forward phosphorylation reaction rate, Our calculations suggest that if the STAT3n rate,  . The intn rate,  .T and SHP-1T are significantly smaller than that of STAT3T. While these situations can occur in nature, most living cells exhibit comparable concentrations of enzymes and their substrates. Therefore, the physiological range of applications considered in Our analysis suggests that the STAT3 phosphorylation system with switch-like characteristics depends on the parameters of the model. The approach proposed by Goldbeter and Koshland We next investigated multisite phosphorylation reactions in other inflammatory signalling pathways and studied the activation of IRF-5 as an example. IRF-5 is phosphorylated by the TBK-1 kinase and dephosphorylated by Alkaline Phosphatase (AP)  [36], [3We compared our model predictions with the previously reported method of Goldbeter and Koshland Our analysis shows that alterations of phosphorylation rates and total IRF-5 to AP ratios do not have any impact on the model in Our results suggest that the selective activity of the multisite phosphoprotein-mediated response is regulated by the ratio of the total amounts of the protein to phosphatase and by the relative rates of the phosphorylation-dephosphorylation reactions. These effects have not been observed in previously reported mathematical models of phosphorylation In this study we propose a new model for multisite phosphorylation with applications to intracellular signaling in the immune system. The model extends previous models for activation of proteins by single- The proposed model has been applied to the STAT3 signaling circuit and compared with one of the previously published models We show here that IRF-5 is activated in a switch-like manner , which lThis work introduces universal mechanisms for single and multisite phosphorylation and proposes an accurate model for phosphorylation-mediated regulation. The analysis reveals the physiological conditions under which the model coincides and differs from the classical models. This approach can have applications in a variety of molecular systems where the information is transmitted through a phosphorylation mechanism. The predictions of the model applied to the STAT3 and IRF-5 regulatory circuits has a broad impact to Systems Immunology and may increase our understanding of the mechanisms of inflammatory diseases.Here we consider a general mechanism of phosphorylation of protein The reactions can be represented as follows:We introduce the following notation: The conservation equations for the elements involved in the above reactions are as follows:The steady-state solutions of The rate of change of From The non-dimensional form of the According to the above notation, We can therefore rewrite the law of mass conservation for In In general, conditions Substituting The range of It can be shown that For any real positive values of the parameter We find the real root of Since the domain is To find the roots of Thus there is a system of equations for To simplify the above system of equations, we use the following parameters:Solving the system described by Thus, a steady-state solution for From Since In this section we consider a system with In this case, the normalized parameters are written as follows:When STAT3 can form a dimer and be activated when it is phosphorylated at one site by JAK and dephosphorylated by SHP-1. Thus, Here we consider phosphorylation of Interferon regulatory factor 5 (IRF5). Our model assumes that the molecule contains 6 independent phosphorylation sites. IRF-5 can be phosphorylated by TBK-1 kinase and dephosphorylated by Alkaline Phosphatase. In this case we use


Several recent randomized clinical trials have preliminarily demonstrated that initial targeted therapy with combined BRAF and MEK inhibition is more effective in metastatic melanoma (MM) than single agent. To guide therapeutic decisions, we did a comprehensive network meta-analysis to identify evidence to robustly support whether combined BRAF and MEK inhibition is the best initial targeted therapeutic strategy for patients with MM.The databases of PubMed and trial registries were researched for randomized clinical trials of targeted therapy. Data of outcome were extracted on progression-free survival (PFS), objective response rate (ORR), and overall survival (OS). Network meta-analysis using a Bayesian statistical model was performed to evaluate relative hazard ratio (HR) for PFS and OS, odds ratio (OR) for ORR.P < 0.0001) or MEK inhibition alone . Combined BRAF-MEK inhibition also improved the OS over BRAF inhibition  or MEK inhibition alone . The ORR was superior in combined BRAF and MEK inhibition comparing with BRAF inhibition  or MEK inhibition alone .Finally, 16 eligible trials comprising 5976 participants were included in this meta-analysis. PFS were significantly prolonged in patients who received combined BRAF-MEK inhibition compared with those who received BRAF inhibition (HR: 0.58, 95%CI: 0.51-0.67, This study indicates that concurrent inhibition of BRAF and MEK improved the most effective therapeutic modality as compared as single BRAF or MEK inhibition for patients with MM. Metastatic melanoma (MM) used to be a fatal disease with an average survival of 7 months after diagnosis, since treatment options were limited. The discovery of driver oncogenic mutations of BRAF  opens a new era in targeted therapy for MM. Indeed, the potent and specific BRAF  inhibition, as compared with chemotherapy, have provided considerable clinical benefits including progression-free survival (PFS), overall survival (OS) and objective response rate (ORR) in patients with MM , 2. HoweConcurrent targeting BRAF and MEK has been considered the possibility to enhance tumor growth inhibition, delay acquired resistance, and abrogate paradoxical activation of the MAPK pathway in preclinical models of melanoma . RecentlWe identified 451 relevant references for review title and abstract. After initial screening, we retrieved the full text of 32 potentially eligible clinical trials for detailed assessment. Of these, 22 randomized controlled trials were evaluated in more detail, and 18 randomized controlled trials with phase II or III were found that met the eligibility criteria for this study. Finally, 16 eligible trials reporting randomized controlled trials were included for meta-analysis, with a total of 5976 patients randomized to receive two of the six treatment strategies , 13-21. P < 0.0001) or MEK inhibition alone , respectively. The network graph and forest plot of traditional pair-wise direct comparison were drawn to graphically display the results of the available direct comparisons between treatments. Comparing results from traditional pairwise meta-analysis .Data on PFS were available in fifteen studies, and HR values were explicitly reported in those studies. We summarized the results of our random-effects network meta-analysis for PFS in Figure s Figure  and netwP < 0.0001) or MEK inhibition alone , respectively. Whereas, these results demonstrated that single BRAF inhibition had a statistically significantly longer in OS than MEK inhibition alone , and combined BRAF inhibition and chemotherapy . This finding strengthened the results of the direct comparisons , followed by BRAF inhibition alone , and combined MEK-chemotherapy . Furthermore, the ORR was superior in patients who received combined BRAF-MEK inhibition compared with those who received BRAF inhibition  or MEK inhibition alone . The single BRAF inhibition yielded better response rate than MEK inhibition alone . Values of surface under the cumulative ranking probability curve . In the Figure Despite major advancements in targeted therapy for MM, however, most patients relapse and show progressive disease after 7 months with treatment of BRAF or MEK inhibition alone. The crucial issue is how to combine targeted inhibition to maximize survival for patients with MM  and to aP < 0.0001) or MEK inhibition alone , respectively. Combined BRAF-MEK inhibition also improved the OS over BRAF inhibition  or MEK inhibition alone . The similar benefit has been found in the ORR, which was superior in combined BRAF and MEK inhibition compared with BRAF inhibition  or MEK  inhibition alone. These data provide clear evidence for the benefit of BRAF-MEK inhibition combination therapy over BRAF or MEK inhibition alone in prolonging survival and higher response rate. This promising result of combined BRAF and MEK inhibition will allow physicians to select this concurrent inhibition as the preferred therapeutic strategy for patients with MM. We also believe this meta-analysis is the largest and most comprehensive study of initial targeted therapy for MM so far, and provides the highest level of evidence for patients with MM.Different measures of survival can be combined in a single analysis on the HR scale, avoiding potential selection bias and loss of information due to only including studies with the same measure or doing separate analyses for different measures . NetworkThe survival benefit of combined BRAF and MEK inhibition, not only patients with MM but also those with operable BRAF mutation-positive melanoma has been reported previously . FurtherP < 0.0001), OS , and higher OSS  than those in MEK inhibition alone.Prospective trials directly comparing single BRAF inhibition to single MEK inhibition are lacking. This meta-analysis is the first to assess the PFS, OS, and OSS between single BRAF and MEK inhibition, and fills a crucial knowledge gap of MAPK pathway. Our present results demonstrated that the single BRAF inhibition had a statistically significantly longer in PFS  and MEK , trial and melanoma were used to search relevant studies according to our previous MAPK therapy review [We searched database of PubMed, the Cochrane Collaboration Central Register of Controlled Clinical Trials, Cochrane Systematic Reviews, and y review .First, the titles and abstracts of study reports have been identified by the search strategies for eligibility, and then full-text versions of all eligible studies were obtained for data synthesis. All randomized controlled trials that compared at least two arms of different treatment regimens involving targeted therapy were obtained. We required trials to include data for hazard ratio (HR) for PFS and OS, and conformed to the convention of reporting HR showing benefit of experimental drug versus control . The ORR defined as complete (CR) or partial response (PR) was according to Response Evaluation Criteria in Solid Tumors (RECIST).Clinical Trials.gov number, randomized phase and treatments of experimental and control group were recorded. Primary and secondary endpoints were also recorded. The primary end point was PFS. Secondary end points included OS and ORR, which were measured according to the Response Evaluation Criteria in Solid Tumors (RECIST) [Three investigators  independently reviewed the full article of eligible trials and extracted information into an electronic database. From each eligible trial, the first author, year of publication, sample size, BRAF mutation, (RECIST) . The rep(RECIST) . When HR(RECIST) . We extrP value from the 95% confidence interval has been evaluated according to the method described by Altman DG [The traditional pair-wise meta-analysis has been performed by Stata 12  for PFS, OS and ORR, respectively. For network meta-analysis, the model applied to analyze the HR of PFS and OS was a Bayesian consistency model as described in Woods et al. , with 24ltman DG . We did ltman DG .


Here we study the functional and biophysical characteristics of a PLB mutant associated with human dilated cardiomyopathy (DCM), with a deletion of arginine at position 14 (PLBR14Δ). In agreement with recent findings, we find that PLBR14Δ has a reduced inhibitory effect on SERCA compared to wild type PLB (PLBWT) when reconstituted into lipid membranes. The mutation also leads to a large reduction in the protein kinase A-catalysed phosphorylation of Ser-16 in the cytoplasmic domain of PLBR14Δ. Measurements on SERCA co-reconstituted with an equimolar mixture of PLBWT and PLBR14Δ  indicates that the loss-of-function mutation has a dominant effect on PLBWT functionality and phosphorylation capacity, suggesting that mixed PLBWT/PLBR14Δ pentamers are formed that have characteristics typical of the mutant protein. Structural and biophysical analysis of PLBR14Δ indicates that the mutation perturbs slightly the helical structure of the PLB cytoplasmic domain and reduces its affinity for the phospholipid bilayer surface, thereby altering the orientation of the cytoplasmic domain relative to the wild-type protein. These results indicate that the structure and function consequences of the R14 deletion have profound effects on the regulation of SERCA which may contribute to the aetiology of DCM.Phospholamban (PLB) is a pentameric protein that plays an important role in regulating cardiac contractility via a reversible inhibitory association with the sarcoplasmic reticulum Ca Phospholamban (PLB) is a 52 amino acid single-pass transmembrane protein located in the sarcoplasmic reticulum (SR) membrane, where it exists in dynamic equilibrium between monomeric and pentameric forms Cardiac contractility is determined by tight regulation of calcium transients within cardiac myocytes, with the force of contraction and rates of relaxation in part determined by the calcium-pumping action of the sarcoplasmic reticulum Ca2+], the monomeric form of PLB interacts with SERCA reducing the calcium affinity of the enzyme and reducing the rate of calcium uptake by the sarcoplasmic reticulum (SR) 2+] or by protein kinase A catalysed phosphorylation of PLB at Ser16 and/or Thr17 in response to β-adrenergic stimulation Under conditions of low   =  ΔA340/6.22 mM−1cm−1. Initial rates of phosphorylation were calculated by linear regression analysis of the A340 values over the first 20 min after initiation of the reaction.Phosphorylation was carried out by an adaptation of a previous method, using 0.15 mg each of PLB and R14Δ solubilised in 0.3 ml of phosphorylation buffer . Equal volume aliquots of each protein or the mixture were applied to two PVDF membranes; these were then processed simultaneously using the same solutions differing only at the primary Ab step.Ca values and phosphorylation rates are presented as means ± SEM. Multiple group comparisons were performed using one-way ANOVA with Tukey's post hoc test (Minitab 17). P<0.05 was considered significant in all analyses.KFar-UV circular dichroism measurements were carried out on a Jasco J-180 spectropolarimeter. Circular dichroism (CD) spectra were recorded from 250 to 195 nm using a 0.1 cm cuvette, a scan rate of 20 nm/min and an average of 4 accumulations per sample. All peptides were prepared to 100 µM in 10 mM phosphate, pH 7. Measurements were made using a stepwise titration with trifluoroethanol (TFE) to generate measurements up to 40% TFE. Controls were carried out by titrating TFE into buffer minus the peptides and deducting the values obtained from the experimental values.23WT or NAcPLB23R14Δ was measured using the ultrasensitive iTC200 MicroCalorimeter . Reaction cell volume and total injection volume were 200 µl and 40 µl respectively. Experiments were performed at 25°C, at a power reference setting of 6 µcal/sec with stirring at 800 rpm. Data analysis was carried out using the Origin v.7 software  and fitted using the ‘one set of sites’ model. The reaction cell contained a 100 µM solution of peptide in 10 mM Tris; 1 mM EDTA, 30 mM NaCl; pH 7.4. Lipid SUV's comprising 10 mM 2∶1 L-α-dimyristoylphosphatidylcholine/L-α-phosphattidylglycerol (DMPC/DOPG) in the same buffer, were prepared using a probe sonicator and sonicated at 50% duty cycle for 2×30 s and injected via the syringe. Titrations were carried out at intervals up to 2 minutes in 1 or 2 µl aliquots following an initial 0.5 µl discard aliquot. Each injection generates a heat of reaction, determined by integration of the individual peaks from the heat flow trace.Heat flow resulting from binding of lipid small unilamellar vesicle (SUV's), to NAcPLB15N labelled full-length PLBWT or PLBR14Δ prepared recombinantly were reconstituted with 4∶1 DOPC/DOPE (L-α-dioleoylphosphatidylethanolamine) with a total of 50 mg of lipid. Samples were solubilised in up to 2 ml 50/50 chloroform: acidified methanol (1% acetic acid), before transferring to ∼ 25 glass plates ), in a series 20 µl repeated aliquots, which were allowed to dry between additions. The plates were then transferred to a desiccating cabinet for several days and allowed to dry. The samples were hydrated either by applying a 10 µl aliquot of MilliQ grade water and transferring to a humidifier at 37°C or in the case of PLBR14Δ by omitting the addition of water and transferring directly to the humidifier. The samples were again left for several days until fully hydrated before stacking and wrapping in cling film ready for analysis by solid state NMR. The stacked plates were inserted into a Bruker double resonance flat coil probehead equipped with coil of dimensions 9×9×3 mm and 15N NMR spectra were acquired on a Bruker Avance 400 NMR spectrometer operating at a frequency of 400.13 MHz for protons. Spectra were obtained with Hartmann-Hahn cross polarization at a contact time of 1 ms and proton field of 63 kHz, and recycle delay of 1 s. Each spectrum was the result of accumulating between 300,000 and 400,000 transients, and so the experimental temperature was reduced to −10°C to avoid sample dehydration over the lengthy acquisition times. 31P spectra of the lipid headgroups were obtained under the same conditions before and after the 15N measurements to confirm the macroscopic alignment of the membrane sample.Uniformly 1H-13C spectra at 4°C and a MAS frequency of 8 kHz. Spectra were acquired using the States-TPPI method to obtain phase-sensitive quadrature detection in the indirect dimension and 64 increments were collected in t1.Chain perdeuterated DMPC membranes containing PLBWT or PLBR14Δ (at a lipid:protein molar ratio of 20∶1) were rehydrated in 10 mM phosphate, pH 7 and centrifuged at 13000 rpm in a bench top microfuge. Pellets were transferred to a 4 mm diameter zirconia magic angle spinning (MAS) rotor. NMR measurements were carried out on a Bruker Avance 400 spectrometer equipped with a 4 mm triple resonance magic-angle spinning (MAS) probehead. The refocused INEPT (rINEPT) experiment was used to obtain two-dimensional Immunoblotting analysis of N-flag tagged PLB suggested that the R14Δ mutation may partially destabilise the pentamer form of the peptide generating different sized oligomers Studies were carried out to determine the regulatory effect of recombinant PLBWT, PLBR14Δ and an equimolar mixture of the two  on SERCA when incorporated with the enzyme in DOPC membranes. Calcium-dependent ATPase activity measurements  confirm 1-26), which is highly homologous to PLB, generated no reaction with either PLB Ab (data not shown). It should be noted that the R14Δ mutation occurs within the epitope region for A1 (L7 – S16), and PS16 (R9 – S16 (PO4)), and could therefore negatively impact on their ability to react to the mutant form of PLB. The PS16 Ab is more likely to be affected since this is much more specific than A1, a more promiscuous Ab capable of reacting with multiple forms of PLB. This may in part account for the minimal reaction of PS16 with PKA treated PLBR14Δ. Qualitative inspection of the PS16 blot for the mixed PLBWT and PLBR14Δ sample in PLBR14Δ was next tested for its ability to undergo PKA-catalysed phosphorylation when incorporated alone into DOPC membranes and whether it interferes with phosphorylation of PLBWT in a 1∶1 mixture. The reaction was carried out with protein at a concentration of ∼40 µM and the presence of phosphorylated protein was confirmed by antibody (Ab) dot blot . Ab A1 r340 (−1) than the phosphorylation of PLBWT , as reflected in the dot blotting data representing the end-point of the phosphorylation reaction. In an equimolar PLBR14Δ/PLBWT mixture the overall rate of phosphorylation of PLBWT (30±4 µM h−1) is not significantly different from PLBWT alone (p>0.05), also in agreement with the dot blotting data. Together these results indicate that the R14Δ mutation impedes PKA-dependent phosphorylation of PLBWT, as reported in earlier work While Ab dot blot analysis is a useful technique for detecting and confirming the presence of phosphorylated protein, it tells us nothing about the rate or efficiency of phosphorylation. A second measure of phosphorylation was used, therefore, in which the production of ADP during the PKA-catalysed reaction is linked to oxidation of NADH using the coupled enzyme assay, and monitored by observing the absorbance at A340 . This al340  [37], an1-23WT and PLB1-22R14Δ) were analysed for differences in overall secondary structure using CD spectroscopy and titrating the peptides with TFE to stabilise any intrinsic helical structure. The spectra are consistent with a shift from an unstructured state in 0% TFE . Under these conditions the 15N spectra are consistent with the proteins having a unique orientation in the magnetic field, and despite the low signal to noise some differences between the spectra of wild-type and mutant proteins could be discerned. The spectrum of PLBWT exhibits a line shape with enhanced intensity around 60–120 ppm and 200 ppm and very little signal from 120–200 ppm. This line shape is consistent with helical domains oriented approximately perpendicular and parallel to the bilayer normal, respectively. A theoretical 15N spectrum was simulated from the atomic coordinates for PLBWT pentamers in the “pin-wheel” structure of the T-state  PLBWT adopts a structure reminiscent of the T-state. The spectrum of PLBR14Δ covers the 100–200 ppm range with a maximum around 200 ppm, suggesting that there is overall much more orientational disorder in the mutant  and the other in which the cytoplasmic domain is dynamically disordered and dissociated from the membrane surface (“R-state”) 1-23WT and PLB1-22R14Δ) for phospholipid membrane surfaces. Previous work demonstrated that SUV's of 2∶1 DMPC/DOPG are a useful model system to compare the membrane affinities of amphipathic peptides, giving measureable enthalpic changes for PLB1-23WT (23R14Δ do not give rise to measureable enthalpic changes (23R14Δ are substantially weaker than for PLBWT.The ordered T-state of PLB may depend in part on the amphipathic cytoplasmic helix being stabilised by electrostatic interactions with the membrane surface, i.e., the phospholipid headgroups. If PLBR14Δ is predisposed toward the more disordered state as suggested by the NMR measurements, this may arise from a weakening of the stabilising membrane interactions resulting from the loss of the cationic arginine residue. Isothermal titration calorimetry was used therefore to determine the relative affinities of the PLBWT and PLBR14Δ cytoplasmic domains (represented by PLBLB1-23WT . Here, t changes , indicatm of SERCA for calcium, but it has been variably shown to decrease or increase Vmax or to have no effect We have used a variety of techniques to investigate the functional and conformational properties of PLBWT and the mutant PLBR14Δ lacking R14 in the cytoplasmic domain. It is generally accepted that PLBWT reduces the KThe diminished capacity of PLBR14Δ to inhibit SERCA may arise from changes to the oligomeric status of the PLB mutant. It is widely recognised that PLB is more inhibitory as a monomer 1-23R14Δ interacts more weakly with membranes than does PLB1-23WT. The effects of the membrane environment on PLB structure and function are well established, with membranes representative of SR favouring a non-extended PLB conformation with an T-type structure The loss of function of PLBR14Δ may arise from the preference of the cytoplasmic domain to adopt a structurally disordered, membrane-disordered state akin to the R-state observed for the wild-type protein. The PLBWT cytoplasmic domain can undergo a shift from a folded, membrane-associated T-state to an unfolded, excited, membrane free R-state The phosphorylation measurements reported here  agree wi


Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation. Mitochondria are the powerhouses of the eukaryotic cell, hosting the production of energy in the form of ATP by oxidative phosphorylation of ADP. They possess their own genome (mt-DNA), which codes in humans for 13 of the respiratory chain subunits, 22 tRNAs and 2 rRNAs. More than one thousand proteins, encoded by the nuclear genome, synthesized in the cytosol and subsequently imported into mitochondria, are also required for mitochondrial biogenesis and functioning WARS2) are affected by pathology-related mutations17891011158In 2007, it was shown for the first time that a mt-aaRS is implicated in a disease when mutations were found in the DARS2 gene within a cohort of 78 patients belonging to 58 families141516171817Human mt-AspRS is the most prominent case with the largest number of reported mutations 60). These are spread throughout the 0. These in vitro analyses of an initial set of human mt-AspRS mutants revealed that only some of them had lower aminoacylation activities22Initial in vitro assays had shown that R263Q is the only mutation of these six that significantly impairs the aminoacylation property of recombinant proteinWe recently determined the X-ray structure of human mt-AspRSE. coli for in vitro biophysical analysis. Proteins were expressed without the first 40 N-terminal amino-acids, corresponding to the putative MTS, and with a His-tag appended at the C-terminus. After purification as initially reported for the WT enzymein cellulo solubility analysis as full-length sequences flanked by a C-terminal Flag®-tag for immunodetection. In this case, the MTS was naturally cleaved after import into mitochondria.In the present study, wild-type (WT) mt-AspRS and six mutants  were proin vitro and in cellulo experiments, the mutants and the WT synthetase were prepared in parallel and treated in the same way for strictly comparative biochemical and biophysical analyses of solubility (by protein quantification), thermal stability , 2D and 3D structure in solution . The stabilizing effect of a synthetic inhibitor derived from the aspartyl-adenylate (Asp-AMS), which binds to the active site of the enzymeFor both h = 10 ± 1 nm, except Q184K whose size was slightly greater  . The Tm The structural stability of WT and mutant mt-AspRSs was further characterized by recording SRCD spectra as a function of the temperature to monitor the loss of secondary structure elements during unfolding. CD spectra are displayed in WT and mutants of mt-AspRS were expressed in BHK21 cells and their abundance in soluble and insoluble fraction in either whole cell extracts or enriched mitochondria was determined by western blot . Histogrin vitroMutants Q184K and R263Q, showing respectively the highest impact on protein solubility in cellular extracts (either whole cells or enriched mitochondria) and the highest impact on aminoacylation E. coli, the C-terminal tail encompasses only ten residues and is thus slightly shorter. The last five residues are also not visible in the crystal structure of the free enzyme (PDBid: 1C0A), confirming the flexible nature of C-terminal regions in bacterial-type AspRSs.The major component of the three protein samples eluting as peak 3 consisted of mt-AspRS dimers with an Rg of 40 ± 1 Å, slightly larger than the Rg (36 Å) calculated from the crystal structure (PDBid: 4AH6), which does not include the C-terminal tails. These tails were not visible by X-ray crystallography probably because they do not adopt a fixed position in the crystal packing. In solution, however, their presence was revealed by the maximal interatomic distance (dmax = 150–155 Å) in the distance distribution function p(r) of the dimer . Accordiab initio bead modeling did not converge to a satisfactory representation of these entities, the similarity of their SAXS profiles clearly indicated that they were not random aggregates. According to their radii of gyration  and their Porod molecular volumes, these large complexes correspond to hexamers of dimers in peak 1 and dimers of dimers in peak 2 , in the catalytic domain at the dimer interface  or in the C-terminal extension  involved in subunit dimerization within bacterial-type AspRSs.Previous studies on human mt-AspRS had shown that mutations associated with the LBSL pathology have various impacts on the molecular and cellular properties of the enzyme, but reduce only marginally the catalytic activity of mt-AspRS and are thus not always deleterious to the housekeeping function222324It is widely believed that mutation of strictly conserved residues would be detrimental to the architecture or activity . Therefore we investigated the amino acid conservation of relevant residues in structural sequence alignments made of 180 sequences of bacterial-type AspRSs . We found that none of them are strictly conserved , indicatThe particle diameters determined by DLS showed that the tested mutants were dimers like the WT mt-AspRS, but that mutant Q184K was of slightly greater size. SAXS data confirmed that mutants Q184K and R263Q, like the WT, are predominantly composed of dimers . The simSAXS analysis further confirmed the floppy nature of the C-terminal tails in the mt-AspRS dimer that were not visible in the X-ray structure. These extensions pointing into the solvent  and 6A mAll enzymes, WT and mutant mt-AspRSs, sharing the same structure were subjected to thermal denaturation in order to detect differences in stability. Three approaches were used to assess the propensity to aggregate (recorded by DLS), to unfold  and to expose hydrophobic regions to the solvent upon unfolding (monitored by DSF). Interestingly, every mutant behaved in a different manner in response to thermal denaturation. This response varied from one analytical method to the other as summarized in Our previous analyses of human WT mt-AspRS had revealed that the synthetic analog of the aminoacylation intermediate aspartyl-adenylate, Asp-AMS, binds cooperatively29E. coli were soluble in vitro up to at least 10 mg/ml in standard solvent conditions. The situation was different in BHK cells grown at 37 °C. As shown in The differential thermal stability and effect of Asp-AMS led us to examine the stability of the mutants in the mitochondria of mammalian cells. All recombinant proteins produced in a priori possess a mixture of homo- and hetero-dimers. Here we examined homodimers composed of either two WT or two mutated monomers, which represent two-thirds of the possible combinations  can be summarized as follows:Up to now LBSL is the neurodegenerative disease correlated with the greatest number of mutations in a human mitochondrial aminoacyl-tRNA synthetase, and with the largest cohort of diagnosed patients. All investigations performed thus far on the possible impacts of LBSL-causing mutations on molecular, cellular, and in the present contribution on biophysical properties of the mt-AspRS, highlight that each mutation has an individual signature (as illustrated in 3233TM2(DE3) was purchased from Merck Millipore, Anti-Flag® antibody and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were from Sigma, anti-SOD2 (human) and anti-prohibitin (human) antibodies were from Abcam®, goat anti-rabbit secondary antibody conjugated to horseradish peroxidase [HRP] was from BioRad, chemiluminescent detection kit from Pierce (Thermo Scientific), aminoacyl-adenylate analog 5′-O-[N-(L-aspartyl)sulfamoyl]adenosine (Asp-AMS) from Integrated DNA Technologies (Belgium), and SYPRO OrangeTM from Molecular Probes Invitrogen. Mini-Protean® TGX Precast polyacrylamid gels, Trans-Blot Turbo system and SEC protein standard  were from BioRad. ArrestTM protease inhibitor cocktail and polyethylenimine  were purchased from GBiosciences and Polysciences, respectively.Baby Hamster Kidney cells strain 21 (BHK21) (ATCC # CRL-12072) and modified Vaccinia Ankara strain (MVA-EM24) were generous gifts from Robert Drillien . Bacterial strain RosettaTM2(DE3) as previously described23The genes for wild-type mt-AspRS and those carrying the mutations 172C > G (R58G), 406A > T (T136S), 550C > A (Q184K), 788G > A R263Q), 1837C > T (L613F) and 1876T > A (626Q) were cloned in bacterial strain Rosetta3Q, 1837CTM and Malvern ZetasizerTM NanoS light scattering instruments, respectively−3 Pa.s. Particle masses determined by SLS were corrected for refractive index concentration dependence dn/dc 0.185 ml/g. The behavior of samples subjected to a temperature gradient from 20 °C to 60 °C was monitored by DLS in the Zetasizer. A shift in the autocorrelation function towards greater times was indicative of the onset of aggregation.Samples containing 2 mg/ml mt-AspRS in standard buffer  were ultracentrifuged for 1 h at 45 000 rpm or 100 000 × g. The supernatant was transferred into 2 μl or 20 μl quartz cells for parallel analyzes at 20 °C in Wyatt Technology NanostarTM 5000X stock solution diluted 20-fold in dimethylsulfoxide) in 0.2 ml transparent plastic qPCR tubes. Fluorescence variation was monitored along a temperature gradient from 20 to 90 °C using an Agilent Stratagene Mx3005P instrument. The inflection point of the fluorescence intensity versus temperature plot was considered to be the transition mid-point (Tm).Twenty μl of mt-AspRS solution containing 2 mg/ml were mixed with 0.5 μl dye  glycerol, 0.1 mM EDTA and 1 mM DTT, were separated by SEC and analyzed by SAXS online. Thus, 45 μl protein solution was loaded onto an Agilent Bio SEC-3 column  installed on an Agilent HPLC system and maintained at 15 °C. Proteins were eluted at a flow rate of 0.2 ml/min with a mobile phase containing 100 mM HEPES-Na pH 7.5, 250 mM NaCl, 5% (v/v) glycerol and 1 mM TCEP. The eluate was analyzed by SAXS in a continuous flow capillary cell with a frame duration of 1000 ms at intervals of 500 ms. Data processing, analysis and modeling step were done with PRIMUS and other programs of the ATSAS suiteg) were derived from Guinier approximation and used to estimate the molecular Porod volume. Rg was also calculated from the entire scattering pattern using the indirect transform package GNOMRadii of gyration  (Consensus CDS Gene CCDS1311.1). Constructs were introduced in BHK21 using a protocol adapted from the transfection-infection approach6 cells were harvested, washed with PBS.WT and mutants constructs were generated by directed mutagenesis from pBCJ749.77 plasmid, derived from described pBCJ739.14TM 5G machine , in 1.5 ml of an isotonic lysis buffer  containing a protease inhibitor cocktail. Mechanically lysed cells were centrifuged 10 min at 400 g (4 °C). The pellet (nuclei and cell debris) was discarded, and the supernatant was centrifuged 10 min at 12 000 g (4 °C) to pellet mitochondria. Whole cells and mitochondria were sonicated (6 × 10 s) on ice in 160 μl of 10 mM Tris-HCl pH 7.5 containing a protease inhibitor cocktail. The lysates were then ultracentrifuged 30 min at 125 000 g (4 °C) and the soluble fractions (supernatants) were separated from the insoluble fractions (pellets). An aliquot of each soluble fraction (16 μl) was withdrawn and added to 4 μl of Laemmli denaturing buffer, heated to 95 °C for 10 min and loaded on a 10% (m/v) polyacrylamide gel. Pellets were solubilized in 160 μl of 10 mM Tris-HCl pH 7.5 to which 40 μl Laemmli buffer was added before heating and 20 μl of this mixture was analyzed on a gel. Proteins were blotted onto a PVDF membrane and recombinant proteins were detected with an antibody specific for the Flag® tag peptide. Chemiluminescent detection was carried out using the Pierce Detection Kit according to manufacturer’s instructions. Exposed films were digitized using Epson Perfection 3490 Photo scanner and band intensities were quantified on images using ImageJ softwareA flowchart of the experimental procedure to investigate soluble/insoluble state of the proteins is given in Mt-AspRS in each sample distributed in either soluble or insoluble fraction, the total corresponding to 100%. The blots of these fractions were developed simultaneously in the same bath to allow for strict comparison of protein amounts and determination of relative distribution in both fractions. In that way, the result was independent of expression. The loading controls, SOD2 or prohibitin respectively for soluble or insoluble fractions, were used to adjust the amount of mt-AspRS in every lane of a given blot. Resulting mt-AspRS intensities were converted to percentages of protein amount (WT or mutants) in soluble and insoluble fractions.Mean values and standard deviations were derived from three independent experiments. Histograms were normalized with regard to WT mt-AspRS to compare the relative abundance of the mutated proteins in whole cell extracts and enriched mitochondria.How to cite this article: Sauter, C. et al. Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures. Sci. Rep.5, 17332; doi: 10.1038/srep17332 (2015).


AD) brain. Previous studies showed that glucose deprivation increases amyloidogenesis via a BACE‐1‐dependent mechanism. However, no data are available on the effect that this condition may have on tau phosphorylation. In this study, we exposed neuronal cells to a glucose‐free medium and investigated the effect on tau phosphorylation. Compared with controls, cells incubated in the absence of glucose had a significant increase in tau phosphorylation at epitopes Ser202/Thr205 and Ser404, which was associated with a selective activation of the P38 mitogen‐activated protein kinase. Pharmacological inhibition of this kinase prevented the increase in tau phosphorylation, while fluorescence studies revealed its co‐localization with phosphorylated tau. The activation of P38 was secondary to the action of the apoptosis signal‐regulating kinase 1, as its down‐regulation prevented it. Finally, glucose deprivation induced cell apoptosis, which was associated with a significant increase in both caspase 3 and caspase 12 active forms. Taken together, our studies reveal a new mechanism whereby glucose deprivation can modulate AD pathogenesis by influencing tau phosphorylation and suggest that this pathway may be a new therapeutic target for AD.Alterations of glucose metabolism have been observed in Alzheimer's disease ( ADAlzheimer's diseaseNFTneurofibrillary tanglesCNScentral nervous systemAβamyloid betaAPPamyloid beta precursor proteinBACE‐1beta secretase‐1GSK3glycogen synthase kinase 3CDK5cyclin kinase 5MAPKmitogen‐activated protein kinaseASK1apoptosis signal‐regulating kinase 1DMEMDulbecco's modified Eagle's mediumet al., et al., Alzheimer's disease (AD) is an age‐related neurodegenerative disorder with dementia characterized by progressive accumulation of amyloid beta (Aβ) peptides and abnormal aggregates of the microtubule‐associated tau protein, also known as neurofibrillary tangles (NFTs) , as under physiological conditions neuronal cells are entirely dependent on a continuous supply of glucose for their correct functioning. The CNS is particularly vulnerable to hypoglycemic damage, and changes in glucose metabolism have been observed in a variety of neurodegenerative conditions associated with dementia , BACE‐1 and Aβ levels become elevated, suggesting that energy deprivation may be amyloidogenic However, to the best of our knowledge no data are available on the effect that a dysregulation of glucose levels may have on tau and its phosphorylation state. To address this important biological question in the current study, we exposed neuronal cells to a glucose‐free medium and investigated the effect on tau phosphorylation and the potential mechanisms involved in this biological effect. Under this experimental condition, we found that, compared with controls, glucose‐deprived cells had significant increase in tau phosphorylation at specific epitopes, which was dependent on the activation of the P38 mitogen‐activated protein kinase (MAPK) pathway.N2A cells were incubated for 24 h in either DMEM with glucose, or DMEM without glucose. Compared with controls, no changes in levels of total tau were observed in cells that were exposed to medium without glucose Fig. A. HoweveTo study the mechanism underlying the increase in tau phosphorylation after glucose deprivation, we next examined some of the kinases and phosphatases which are considered major regulators of this post‐translational modification. As shown in Figure m) , next we investigated the involvement of this kinase under our experimental conditions. To this end, we down‐regulated ASK1 protein levels using a siRNA approach and found that the reduced availability of this kinase in glucose‐deprived cells was sufficient to prevent the phosphorylation/activation of P38 MAPK , all implicated in tau phosphorylation when activated by dual phosphorylation at Thr180 and Tyr182. Several studies have shown that P38 active form  is associated with neuritic Aβ‐amyloid plaques and tau NFT in postmortem brains of AD patients and that P38 activation occurs at very early stage of the disease  of the in vitro experimental evidence that in response to a condition of glucose deprivation stress, neuronal cells manifest an increase in tau phosphorylation and apoptotic response which were mediated by the activation of the P38 MAPK pathway via the participation of the ASK1 kinase.In summary, our work using a model of energy deficiency provides the first Considering that impaired glucose utilization is a well‐known AD risk factor, the targeting of this kinase could afford a new therapeutic opportunity for developing preventative and disease‐modifying therapies for AD.−1 penicillin, 100 μg mL−1 streptomycin, and 400 μg mL−1 G418 (Gibco), at 37 °C in the presence of 5% CO2, as previously described  neuronal cells stably expressing human APP carrying the K670 N, M671 L Swedish mutation (APPswe), were grown in Dulbecco's modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum, 100 U mLm Tris base, 750 mm NaCl, 5% NP‐40, 25 mm EDTA, 2.5% sodium deoxycholate, 0.5% SDS, and an EDTA‐free protease and phosphatase inhibitors cocktail tablet . Samples were centrifuged at 15.7 g for 20 min at 4 °C, and supernatants were used for immunoblot analysis, as previously described  prior incubation with the glucose‐free medium (DMEM) and then treated with the same inhibitor for additional 24 h, after which cells pellets were harvested in lytic buffer for biochemical analyses. In another set of experiment, before the incubation with the glucose‐deprived medium, cells were treated overnight with small interfering (si) RNA for ASK1 .To test the effect of a P38 MAPK inhibitor on tau phosphorylation, N2A‐APPswe cells were grown to 70% confluence, then incubated for 1 h with the P38 MAPK inhibitor  . Briefly, cells were fixed with a cross‐linking fixative such as 4% methanol‐free formaldehyde and then permeabilized by suspending them in 0.2% Triton X‐100 solution in PBS for 5 min. Cells were then incubated with the terminal deoxynucleotidyl transferase recombinant enzyme (rTdT) for 60 min at 37 °C. The 3′ OH‐termini of the DSBs serve as primers and become labeled in this procedure with fluorescin‐12‐dUTP when incubated in the reaction catalyzed by rTdT. Positive controls were prepared using DNase I. Concurrent staining of DNA with propidium iodide was applied. Coverslips were mounted using VECTASHIELD mounting medium (Vector Laboratories) and analyzed with the confocal microscope described above.anova followed by Bonferroni's multiple comparison tests were performed using graphpad prism 5.0 . All data are presented as mean ± S.E.M. Significance was set at P < 0.05.One‐way E.L. and P.D. designed the study. E.L. executed all the experiments. E.L. and D.P. interpreted the data and wrote the manuscript. Both authors read and approved the final version of the manuscript.No funding information provided.The authors declare no competing interest to disclose.


PKs transfer a phosphate from ATP to the side-chain hydroxyl group of a serine, threonine or tyrosine residue of a substrate protein. This in turn can alter that protein's function; modulating fundamental cellular processes including, metabolism, transcription, growth, division, differentiation, motility and survival. PKs are subdivided into families based on homology. One such group are the stress-activated kinases, which as the name suggests, are activated in response to cellular stresses such as toxins, cytokines, mechanical deformation and osmotic stress. Members include the p38 MAPK family, which is composed of α, β, γ and δ, isoforms which are encoded by separate genes. These kinases transduce extracellular signals and coordinate the cellular responses needed for adaptation and survival. However, in cardiovascular and other disease states, these same systems can trigger maladaptive responses that aggravate, rather than alleviate, the disease. This situation is analogous to adrenergic, angiotensin and aldosterone signalling in heart failure, where inhibition is beneficial despite the importance of these hormones to homeostasis. The question is whether similar benefits could accrue from p38 inhibition? In this review, we will discuss the structure and function of p38, the history of p38 inhibitors and their use in preclinical studies. Finally, we will summarize the results of recent cardiovascular clinical trials with p38 inhibitors. GlaxoSmithKline has the most active programme with a number of phase 1 trials suggesting a potential benefit in patients with early (Cheriyan −1), increase in serum creatinine at 12 weeks in the losmapimod groups. Study drug discontinuation was twice as frequent among patients receiving losmapimod than among those receiving placebo. This observation was of borderline significance and the cause was unknown. Serious adverse events were very similar across groups.In terms of safety, there was no statistically significant increase in liver enzymes, but alanine transaminase elevations three times above the upper limit of normal were more common in the losmapimod groups. In addition, there was a statistically significant, but very small  did not differ by treatment group. However, in an MRI substudy (92 patients with paired MRI scans) losmapimod was associated with significant improvements in ejection fraction and left ventricular end-diastolic and end-systolic volumes, and also a significant reduction in discharge brain natriuretic peptide in the main cohort. Collectively, these signals hint of a benefit with losmapimod and p38 inhibition in the setting of myocardial infarction despite the fact that SOLSTICE failed to meet its primary efficacy end points. Moreover, this suggestion of benefit was observed despite a losmapimod oral dosing regime that probably only achieved a maximum of 50% inhibition of p38 at the time of peak effect which occurred 4 h after oral dosing  will involve approximately 26 000 patients worldwide with myocardial infarction and have a study design similar to SOLSTICE, but without the 15 mg loading group (i.e. losmapimod 7.5 mg twice per day vs. placebo). The primary efficacy end point is a composite of major adverse cardiovascular events comprising cardiovascular death, reinfarction or recurrent myocardial ischaemia requiring urgent revascularization at 12 weeks. The main inclusion criterion is type 1 myocardial infarction (an atherosclerotic plaque rupture event) and will include presentation electrocardiograms with both ST and no ST elevation. Study medication will be continued for 12 weeks and follow-up for 24 weeks. It is anticipated that the study will be completed in December 2018.et al., et al., The p38 MAPK pathway is integrated with multiple other signalling pathways at different levels. For example, p38α has been shown to phosphorylate glycogen synthase kinase 3 and the Wnt signalling pathways (Bikkavilli Global inhibition of p38α can lead to detrimental effects to the homeostasis of the organism. Pharmacological inhibition of p38 has been achieved, but the results of the preclinical trials, although encouraging, have not translated to clinical trials with a similar level of success. Therefore, a more selective targeted inhibition of the kinase that could ameliorate the detrimental consequence of activation of the kinase while maintaining the essential functions of the kinase would be highly desirable. The recent studies indicating the TAB1-induced activation of the kinase that mediates myocardial damage would be an eligible target for inhibition and that would allow the circumstance-specific mode of activation to be exploited.


The carbonyl oxygen of Thr113 is one of the residues that coordinate K+ in the active site. Even though the side chain of Thr113 does not participate in binding K+, the strict co-evolution between position 117 and 113 suggests that T113 may be the result of the evolutionary pressure to maintain the selectivity of pyruvate kinase activity for K+. Thus, we explored if the replacement of Thr113 by Leu alters the characteristics of the K+ binding site. We found that the polarity of the residue 113 is central in the partition of K+ into its site and that the substitution of Thr for Leu changes the ion selectivity for the monovalent cation with minor changes in the binding of the substrates. Therefore, Thr113 is instrumental in the selectivity of pyruvate kinase for K+.In a previous phylogenetic study of the family of pyruvate kinase, we found one cluster with Glu117 and another with Lys117. Those sequences with Glu117 have Thr113 and are K WT-RMPK+ in catalysis has not totally been understood, even when it has been extensively studied [+ changes the kinetic mechanism of WT-RMPK, inducing the closure of the active site and promoting a rearrangement of the residues involved in the binding of ADP-Mg [+ lies in a site formed by the carbonyl oxygen of T113, Oγ of S76, Oδ1 of N74 and Oδ1 of D112 [Up to now, the role of K studied ,4,5,6,7.f ADP-Mg . The cry of D112 ,10 . However, as more enzymes were characterized, it became apparent that the activity of a substantial number of PKs was K+-independent. To explore the molecular basis of this behavior, Laughlin and Reed [+-independent bacterial enzymes. The authors found that E117 of WT-RMPK, which is close to the K+ binding site, is replaced by Lys in the bacterial enzymes; they constructed the E117K mutant of the rabbit enzyme and found that the mutant was >200-fold more active than the wild-type in the absence of K+ and that the activity was not stimulated by monovalent cations.For a long time, it was considered that the dependence of Kand Reed  compared+-independent activities, an extensive phylogenetic study of the PK family was performed [+-dependent activity, whereas those with K117 are K+-independent PKs. Ninety seven percent of the PKs that have E117 also have T113, and 97% of the enzymes that have K117 have L113. Laughlin and Reed [+ in WT-RMPK with that of the two bacterial PKs that do not require K+ for activity; they found a strict conservation of the residues contributing the K+ binding pocket in the three enzymes, except for the substitution of Leu by Thr at residue 113 in the PKs of the bacterial enzymes that do not require monovalent cations for activity. The authors considered that this was a conservative substitution, because the main-chain carbonyl oxygen of T113, and not the O in its side chain, is the K+ coordinating moiety  and ADP-Mg diminished independently of the size or dehydration energy.The strict co-evolution of the amino acids in positions 117 and 113 in the two clusters of the PK phylogenetic tree  suggests+, NH4+, Rb+ and Cs+  [mK or 0.5K for all cations studied, except for Cs+, were higher in the mutant than in the WT-RMPK; however, the relative increase in mK was different for each cation, indicating that T113 may participate in the selectivity of PK for a monovalent cation.We measured and compared the activation of WT-RMPK and T113L-RMPK by different concentrations of K and Cs+  in media and Cs+ A; in T11K+ > Cs+ B. The ex± 69 mM) . The kin+, NH4+, Rb+ and Cs+ have been documented [+ has the highest energy barrier of dehydration. Thus, to examine if there is a correlation between the energy of dehydration of the monovalent cations and their partition into the WT-RMPK and T113L-RMPK, the ratio of the mK or 0.5K of the mutant and the wild-type enzymes with K+, NH4+, Rb+ and Cs+ were plotted against the heats of dehydration of the respective cations , whereas with Na+, up to 100 mM, there is only a low non-saturating activation (2.5 µmol/min·mg) (data not shown). On the other hand, for comparative purposes, the effect of NH4+ was included in the data, particularly because it is a good activator of PK, albeit that it does not have the physicochemical properties of the alkali metal ions.Accordingly, we asked how the change of Thr by Leu could affect the selectivity for the monovalent cations. In search of an explanation, we examined the crystal structure of PK. The data show that the side chain of T113 faces the outside of the active site ,10 and tcumented ,18; and  cations C. It was+, NH4+, Rb+ and Cs+ on the kinetics of WT-RMPK and T113L-RMPK in media with 20% DMSO. This is because it has been well documented that the transfer of relatively polar ligands into relatively hydrophobic protein binding sites is favored by including DMSO in the media [+, NH4+, Rb+ or Cs+ on the activity of WT-RMPK (3− and ADP-Mg that were 10-times above their mKapp)( or 0.5Kapp)( for PEP3− (3− and ADP-Mg that were 10-times above their mKapp)( or 0.5Kapp)/appK(DMSO) ratios of K+, NH4+, Rb+ or Cs+ against their −∆Hhyd in WT-RMPK and T113L-RMPK, respectively. These ratios indicate that the affinities for K+, NH4+, Rb+ and Cs+ increase ~8-fold when the enzyme is transferred from 100% water to a medium with 20% DMSO. These K(water)/appK(DMSO) ratios do not correlate with their dehydration energy /appK(DMSO) ratios were clearly dependent on the dehydration energy; the smaller the cation, the higher the cost to dehydrate it and transfer it into its hydrophobic binding site  in WT-RMPK to those of T113L-RMPK for K+, NH4+, Rb+ and Cs+ were 4.6, 1.6, 1.5 and 0.7 in 100% water and 1.8, 1.5, 1.5 and 1.8 in 20% DMSO, respectively. These values represent the extent of preference of WT-RMPK for a monovalent cation compared with T113L-RMPK, the larger the ratio, the more the discrimination of T113L-RMPK for a given monovalent cation. The ratios of specificity constants for each monovalent cation for WT-RMPK and T113L-RMPK in 100% water and in 20% DMSO versus their dehydration energy are in + (with the highest dehydration energy) than T113L-RMPK, whereas the T113L mutant is 1.4-fold more specific for Cs+ (with the lowest dehydration energy) than WT-RMPK. In addition, Rb+ and NH4+ (with the intermediate dehydration energies) are about 1.5-fold more specific for WT-RMPK than for T113L-RMPK, either in 100% water or 20% DMSO. In contrast, when DMSO exerts its effect in partitioning monovalent cations into their binding site, WT-RMPK is about 1.8-fold more specific for K+ and Cs+, respectively, than for T113L-RMPK. These data indicate that T113L-RMPK discriminates K+ in 100% water; and that when the partition of the cation into its binding site is favored by DMSO, the discrimination is almost abolished. Therefore, the substitution of Thr by Leu is instrumental in the discrimination of T113L-RMPK for K+.The specificity constant, KT113L/KWT of PEP3−  to a similar extent in both water medium or medium with 20% DMSO; i.e., the ratios KT113L/KWT of the affinity for PEP3− varied from 0.8- to 2.5-fold and those for ADP-Mg varied from 1.2- to 3.3-fold. This effect on the affinities for both substrates did not correlate with the heats of dehydration or the size of the respective monovalent cations. Therefore, the binding of the substrates into T113L-RMPK is independent of the restriction imposed to the monovalent cation binding site.In order to explore if the substitution of Thr113 by Leu113 affects the binding of the substrates, we compared the ratios of PEP3− A in the  and Cs+ B. The ex+-dependent PKs shows that residues K114 and T120 covariate 89% and 81%, respectively, with E117 [+-independent PKs, Q114 and  120 covariate 82% and 93%, respectively with K117. Therefore mutants K114Q and T120L of RMPK were also constructed and studied. Mutant K114Q preserved ion selectivity and kinetic constants for monovalent cations similar to those exhibited by WT-RMPK. Only minor changes were found in the kinetic parameters for the substrates. On the other hand, mutant T120L was poorly expressed, unstable and did not exhibit any activity in the free salt form. Albeit having K+ or NH4+, maxV of the T120L mutant was 400-fold lower than that of WT-RMPK, with similar affinities for the substrates (data not shown). Residue 120 is part of the hinge that closes and opens the lid (domain B) over the active site (domain A). In the open conformation, Oγ of Thr120 forms hydrogen bonds with main-chain NH of Lys206 and with a water molecule. In the closed conformation, none of these interactions are present. Lys206 moves 10.4 Å when the lid is closed to coordinate the ribose of the nucleotide in the active site. Therefore, it is likely that the movement of the lid is altered in the mutant, T120L.In addition to the co-evolution of T113 with E117, the phylogenetic analysis of KE. coli strain devoid of PK (PB25) [The T113L-RMPK was constructed using the WT-RMPK plasmid by means of polymerase chain reaction (PCR) using the Expand High Fidelity PCR System . The mutagenic oligonucleotides were 5'-GCTCTGGACCTGAAGGGACC-3' and 5'-GGTCCCTTCAGGTCCAGAGC-3', and the external oligonucleotides were 5'-TAATCATCCGGCTCGTATAATGTG-3' and 5'-GGCTGAAAATCTTCTCTCATCCGC-3'. The mutation was introduced as follows: 5 min at 94 °C, 30 cycles for 3 min at 94 °C, 1 min at 67 °C and 1 min at 72 °C; and the extension incubation for 10 min at 72 °C. The PCR product was cloned in the pTrc99A vector after digestion with EcoRI and sequenced completely. The gene with the appropriate mutation was introduced by transformation into an K (PB25) .d-galactopyranoside at an A600 of 0.6. The enzyme was purified as in [LB medium containing 100 µg/mL ampicillin, 50 µg/mL kanamycin and 30 µg/mL chloramphenicol was inoculated with PB25-T113L. Expression was induced with 1 mM isopropyl 1-thio-β-ed as in . The T114+, Na+ and K+ in reaction mixtures were below the detection limit (10 µM), as shown elsewhere [WT-RMPK and LDH were obtained as ammonium sulfate suspensions from Roche Applied Science. Ammonium sulfate-free enzymes were obtained as in . The cyclsewhere . The forlsewhere .w/v) DMSO, contained 25 mM HEPES-tetramethylammonium hydroxide, pH 7.4, and the concentrations of cations  indicated under the “Results and Discussion”. The ADP-Mg complexes and free Mg2+ concentrations were calculated using the software, CHELATOR [K value of 6.3 [w/v) are very similar  [3− in media with 20% DMSO were calculated as described above. For the experiments carried out in 100% water with saturating concentrations of the substrates, the fixed concentrations of PEP3− for WT-RMPK were 0.46 mM for all monovalent cations and for T113L-RMPK were 1.19, 0.46, 0.64 and 0.34 mM, with K+, NH4+, Rb+ and Cs+, respectively. The fixed concentrations for the ADP-Mg complex for WT-RMPK were 3.7 mM for K+ and Rb+ and 2.42 and 2.93 mM for NH4+ and Cs+, respectively. For T113L-RMPK, ADP-Mg concentrations were 12.1, 2.96, 7.26 and 10.28 mM for K+, NH4+, Rb+ and Cs+, respectively. For the experiments carried out in 20% DMSO with saturating concentrations of the substrates, the fixed concentrations of PEP3− were 0.46 mM for all monovalent cations and for T113L-RMPK were 0.23, 0.11, 0.129 and 0.23 mM for K+, NH4+, Rb+ and Cs+, respectively. The fixed concentrations for the ADP-Mg complex for WT-RMPK were 1.06, 0.73, 0.86 and 0.46 mM for K+, Rb+, NH4+ and Cs+, respectively, and for T113L-RMPK were 2.93, 1.32, 1.65 and 0.99 mM for K+, NH4+, Rb+ and Cs+, respectively. HEPES was added to achieve a constant ionic strength of 0.223 M using the software, Maxchelator (http://maxchelator.stanford.edu), when different ligand concentrations  were used. It was checked that the concentration of LDH added to the reaction mixture  sufficed to trap all of the formed pyruvate, i.e., the specific activity was not increased by a 5-fold increase in the concentration of LDH. It is noted that divalent metal ions are essential for phospho transfer during the catalytic cycle of PKs and that Mg2+ is considered to be the physiologically relevant divalent metal ion. Therefore, all of the experiments were carried out in the presence of a non-inhibitory free Mg2+ concentration (2.96 mM). Activity was initiated with 0.1 µg/mL of WT-RMPK or T113L-RMPK. The temperature was 25 °C. Kinetic data were fitted either to the Michaelis–Menten equation (v = Vmax ∙ S/mK + S) or to the Hill equation (v = Vmax ∙ nS/0.5nK + nS) using Origin version 7.0. The standard deviations from two experiments are shown in the tables of Supplemental Data. We computed the standard deviations of the ratios shown in The reaction mixtures, either in conventional aqueous media (100% water) or composed by 20% (CHELATOR . Ionizede of 6.3 . Since dctively) , the con+-dependent PKs [+ is coordinated by its carbonyl oxygen and not by the oxygen of its side chain, which, in fact, is oriented toward the outside of the monovalent binding site. We found that the polarity of the residue 113 is determinant in the partition of K+ into its site. We also found that the substitution of Thr by Leu into the active site of PK locally altered the binding site for the monovalent cation with minor changes in the binding site for PEP3− and ADP-Mg. Therefore, T113 was conserved, because it participates in the selectivity for K+ in the K+-dependent family of PK.The goal of this work was to understand why T113 is a conserved residue in the family of the Kdent PKs . During


This regulation is dependent on the DNA-binding transcription factor ETS-1 which recruits EMSY and the histone demethylase KDM5B to the miR-31 promoter, thus repressing its transcription.miR-31 is well known as an anti-metastatic microRNA (miRNA) in the context of breast cancer. However, to date the mechanism of regulation of this miRNA has yet to be elucidated. The recent publication by Viré The online version of this article (doi:10.1186/s13058-014-0467-x) contains supplementary material, which is available to authorized users. Moreoved in vivo.EMSY, a putative oncogene involved in gene regulation, is known to be amplified in approximately 13% of sporadic breast cancers . HoweverThe publication from the Kouzarides group reports that the oncogene EMSY targets miR-31 through co-operation with the transcription factor ETS-1 and the histone lysine demethylase KDM5B, resulting in transcriptional repression of the miRNA and numerous phenotypic effects . However, as EMSY contains no DNA-binding domain another factor was necessary for promoter recruitment. Bioinformatic analysis of the miR-31 promoter revealed binding sites for ETS-1, ETV4/PEA3 and GATA1 with site-directed mutagenesis of any of the ETS-1 binding sites in the promoter reducing promoter activation. ETS-1 was subsequently confirmed to bind to the miR-31 promoter. Furthermore, this transcription factor was then shown to recruit the histone H3K4me3 demethylase KDM5B to the miR-31 promotor, thus inhibiting transcription of miR-31 through the demethylation of Lys4 on histone 3.This article is the first to mechanistically anchor an oncogenic role for EMSY and demonstrate a direct mechanism of regulation of miR-31, an important metastasis-associated miRNA, in breast cancer. Numerous mechanisms of miRNA regulation exist, including epigenetic silencing via DNA methylation or histone modification , direct in vitro and in vivo studies provides a convincing argument for the classification as EMSY as an oncogene, as well as the direct regulation of miR-31 by this oncogene. However, the story is undoubtedly much more complex, as EMSY may regulate a number of different miRNAs/genes. Nor is this the only potential mechanism of direct regulation of miR-31. Notably, as EMSY amplification is only seen in approximately 13% of sporadic breast cancers, it is imperative to also study the regulation of this miRNA in an EMSY non-amplified context. Moreover, given that there were only 45 samples with EMSY amplification and corresponding miRNA data in the cohort studied, expanding the analysis into larger cohorts would provide additional evidence as to the relevance of this discovery in breast cancer patients.The evidence presented from both Viré and colleagues have elegantly documented a novel role for EMSY in breast cancer. However, they have also presented the case for a second scenario, that in which the oncogenic properties of this gene may be functionally propagated by potential oncomiRs. Indeed, EMSY depletion in cell lines led to the upregulation of 29 miRNAs and 6 miRNAs were found to be overexpressed in breast cancer patients with amplification of the gene. The mechanism of targeting of these miRNAs by EMSY, either directly or indirectly, has yet to be investigated.To date, the focus of the majority of miRNA publications has been to evaluate gene targets observed to affect a functional response. This study highlights a need to fully elucidate the mechanisms of regulation of the miRNAs themselves, not just their targets. This may lead to the discovery of a myriad of feedback loops similar to that between the miR-200 family of miRNAs and the transcriptional repressors ZEB1/SIP1 which functions to regulate epithelial-mesenchymal transition (EMT) . As miRN


Oryza sativa) MTP8.1 (OsMTP8.1) is a tonoplast-localized manganese transporter of the cation diffusion facilitator family. Here we present a structure-function analysis of OsMTP8.1 based on the site-directed and random mutagenesis and complementation assays in manganese hypersensitive yeast, in combination with three-dimensional (3D) structure modeling based on the crystal structure of the Escherichia coli CDF family member, EcYiiP. Two metal-binding sites are conserved in OsMTP8.1 with EcYiiP, one is between transmembrane helices TM2 and TM5, the other is the cytoplasmic C-terminus. In addition to these two metal-binding sites, there may exist other Mn-binding sites such as that at the very end of the CTD. Two residues (R167 and L296) may play an important role for the hinge-like movement of CTDs. Several mutations such as E357A and V374D may affect dimer formation, and S132A may induce a conformational change, resulting in a loss of transport function or modification in metal selectivity. The N-terminus of OsMTP8.1 was not functional for Mn transport activity, and the real function of NTD remains to be investigated in the future. The findings of the present study illustrate the structure-function relationship of OsMTP8.1 in Mn transport activity, which may also be applied to other plant Mn-CDF proteins.Rice ( Manganese (Mn) is an essential micronutrient for plant growth and development. In addition to being a cofactor in many enzymatic processes involved in glucose metabolism and energy production, it also acts as a cofactor of MnSOD in the mitochondrion and as part of the water-splitting complex in PSII in the chloroplast1Oryza sativa L.) have been identified. Members of the transporter gene family include natural resistance-associated macrophage protein (NRAMP)456810OsNramp5 and OsMTP9OsNramp5 is an influx transporter that has been localized to the distal side of the exodermis and endodermisOsMTP9 is an efflux transporter that has been localized to the proximal side of both cell layersOsYSL2 and OsYSL6 have been implicated in Mn homeostasis. OsYSL2 is a metal-nicotianamine transporter that is required for long-distance transport of iron and MnOsYSL6 translocates Mn from the apoplast to the symplast when plants are exposed to high levels of MnOsMTP8.1 that was isolated from a rice shoot cDNA library conferred Mn tolerance in Saccharomyces cerevisiae, and its expression was found to enhance tolerance and accumulation of Mn, but not other heavy metals. OsMTP8.1 has been localized to the tonoplast and transports cytosolic Mn2+ into the vacuole. Disruption of OsMTP8.1 results in decreased chlorophyll content in the youngest leaf blades. Rice mtp8.1 Tos-17 insertion mutants and transgenic OsMTP8.1 RNAi knockdown lines are hypersensitive to Mn and accumulate less Mn in shoots and roots than wild-type plantsRecently, some of the genes responsible for Mn uptake and homeostasis in rice  are grouped into two bundles with four (TM1–TM2–TM4–TM5) and two (TM3–TM6) helices. When Zn2+ binds to site C, the C-terminus moves the TM3-TM6 pair, causing reorientation of TM5, which allosterically changes the geometry of the active Zn2+-binding site (site A), and thereby accomplishes transmembrane transport of Zn2+.MTPs belong to the cation diffusion facilitator (CDF) transporter family and are important for the maintenance of cation homeostasis in bacteria, yeast, plants, and mammals1415et al. studied the structural basis of Zn2+ selectivity and transport activity in AtMTP1AtMTP1 with EcYiiP. They also found that the N-terminus of AtMTP1 is very important for metal selectivity. They suggested that the N-terminal domain, the His-rich loop, and the leucine zipper motif in TM6 may contribute to the high Zn2+ selectivity in AtMTP1. The His-rich loop of AtMTP1 acts as a sensor of cytosolic Zn to maintain an essential level of Zn in the cytosolOsMTP8.1 differs from that of AtMTP1; for example, OsMTP8.1 does not have the His-rich loop between TM4 and TM5, and no leucine zipper motif exists within TM6. The sequence of plant Mn-CDF members are highly similar and differ from EcYiiP and AtMTP1 only in terms of structure-function relationships.By using site-directed mutagenesis, Kawachi OsMTP8.1.In the present study, we are used site-directed and random mutagenesis to investigate the structural basis of Mn transport activity in a plant Mn-CDF member, OsMTP8.1 (Os03g0226400) was RT-PCR amplified using sequence information derived from the Rice Annotation Project of NCBI (http://www.ncbi.nlm.nih.gov). OsMTP8.1 encodes a putative protein of 396 amino acids in length. Phylogenetic analysis of OsMTP8.1 indicated that it belongs to the Mn clade of the CDF familyOsMTP8.1 was assessed by complementation assay using a number of yeast mutants that were deficient in various metal transporters. OsMTP8.1 was expressed in S. cerevisiae yeast mutants lacking the endogenous metal transporters for Zn (zrc1), Co (cot1), Cu (cup2), Cd (ycf1), Ni (smf1), and Mn (pmr1). The results showed that a pmr1 strain but not other mutants were restored after transformation with OsMTP8.1  domains, an N-terminal domain, and a C-terminal domain. To study the function of the N- and C-terminal domains of OsMTP8.1, we construct several deletion mutants. For the N-terminal domain, Mn tolerance conferred by these deletion mutants was equivalent to that conferred by wild-type OsMTP8.1 . The N-terminal domain of OsMTP8.1 was much longer than EcYiiP  and two residues from TM5 (His153 and Asp157), two cytoplasmic binding sites, sites B and C. Site B is localized to the intracellular loop that connects TM2 and TM3 (IL1), in which Zn2+ is coordinated with one water molecule and three residues Asp68, His71 and His75, and site C in the C-terminal domain that hosts two Zn2+ ions bound in a binuclear cluster by Asp285, His232, His248, His283, and His261 from the neighboring subunit of EcYiiP. This site hosts four Zn2+ ions that mediate a tight interaction between CTDs and play an important role in stabilizing the dimer19Biochemical studies and X-ray structural analysis of OsMTP8.1 consists of D135 and D139 from TM2 and D244 and D248 from TM5  of from TM5 . The seqEcYiiP and OsMTP8.1 in site B  between TM2 and TM3. This site is also not conserved within other CDF family members such as MmCDF3 from Maricaulis maris MCS10AtMTP1EcYiiP protomersOsMTP8.1, IL1 contains critical residues  for transport function   of nd D377) . EcYiiP and L376 . Yeast cOsMTP8.1 . The seqm EcYiiP , and mutEcYiiP consist of two independent bundles; TM1, TM2, TM4, and TM5 form a four-helix bundle, whereas TM3 and TM6 compose another bundle. The orientation of the TM3–TM6 bundle is stabilized by four interlocking salt bridges formed by K77 of TM3 and D207, which are located in IL3. The charge interlock also plays an important role in stabilizing dimer associations and is essential for Zn transportOsMTP8.1, two residues (R167 from TM3 and L296 from TM6) near the membrane surface play critical roles in Mn transport  according to the manufacturer’s instructionsNotI site of the pFL61 vector. The primers used and the mutations induced are listed in pmr1) or Zn-sensitive mutant Y00829 (zrc1) and its parental strain BY4741 for Mn and Zn complementation analyses. Yeast transformation was performed by using the lithium acetate/PEG transformation methodOsMTP8.1, or mutated OsMTP8.1 variants were pre-cultured in SD-Ura liquid medium at 30 °C for 16 h. Pre-cultured cells were diluted to an OD600 nm of 1.0, and 10-μL aliquots were spotted onto SD-Ura plates containing various concentrations of metals as indicated elsewhereThe mutants and the wild-type yeast strains used in this research were obtained from the Euroscarf collectionOsMTP8.1 cDNA fragment without a stop codon was amplified and was cloned into the BamHI and KpnI sites of pXZP008MluI site, and cloned into the yeast expression vector pFL61-GFP. The construct was transformed into pmr1 and GFP signal was observed by confocal laser scanning microscopy.For the subcellular localization in rice protoplasts, the Arabidopsis thaliana AtMTP1 (GenBank Acc. No. At2g46800), AtMTP8 (GenBank Accession Number At3g58060) and AtMTP11 (GenBank Acc. No. At2g39450), Cucumis sativus CsMTP8 (GenBank Acc. No. AFJ24703), Hordeum vulgare HvMTP8.1 (GenBank Acc. No. AFP33387), PtMTP11.1 (Phytozome: POPTR_0010s21810) and PtMTP11.2 (Phytozome: POPTR_0008s04940) from Populus trichocarpa, OsMTP9 (Phytozome: LOC_Os01g03914) and OsMTP8.1 (Phytozome: LOC_Os03g12530) from Oryza sativa, and E. coli K12 YiiP (P69380) were performed using multalinhttp://swissmodel.expasy.org/)383940EcYiiP (PDB ID 3H90). Images were generated by using PyMOL 1.6.x.Multiple sequence alignments of TtCzrB (GenBank Acc. No. AJ307316.1) from Thermus thermophilus, How to cite this article: Chen, X. et al. A mutagenic study identifying critical residues for the structure and function of rice manganese transporter OsMTP8.1. Sci. Rep.6, 32073; doi: 10.1038/srep32073 (2016).