Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Chronic myeloid leukaemia (CML) arises after transformation of a haemopoietic stem cell (HSC) by the protein-tyrosine kinase BCR-ABL. Direct inhibition of BCR-ABL kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSCs), which maintain CML. LSCs are independent of BCR-ABL for survival, providing a rationale for identifying and targeting kinase-independent pathways. Here we show--using proteomics, transcriptomics and network analyses--that in human LSCs, aberrantly expressed proteins, in both imatinib-responder and non-responder patients, are modulated in concert with p53 (also known as TP53) and c-MYC regulation. Perturbation of both p53 and c-MYC, and not BCR-ABL itself, leads to synergistic cell kill, differentiation, and near elimination of transplantable human LSCs in mice, while sparing normal HSCs. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSCs can be eradicated.

Preexisting hypertension and pregnancy-induced hypertension reveal molecular differences in placental proteome in rodents.

Preexisting or new onset of hypertension affects pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. In certain cases, it also leads to long-term maternal cardiovascular complications. The placenta is a key player in the pathogenesis of complicated hypertensive pregnancies, however the pathomechanisms leading to an abnormal placenta are poorly understood. In this study, we compared the placental proteome of two pregnant hypertensive models with their corresponding normotensive controls: a preexisting hypertension pregnancy model (stroke-prone spontaneously hypertensive rats; SHRSP) versus Wistar-Kyoto and the transgenic RAS activated gestational hypertension model (transgenic for human angiotensinogen Sprague-Dawley rats; SD-PE) versus Sprague-Dawley rats, respectively. Label-free proteomics using nano LC-MS/MS was performed for identification and quantification of proteins. Between the two models, we found widespread differences in the expression of placental proteins including those related to hypertension, inflammation, and trophoblast invasion, whereas pathways such as regulation of serine endopeptidase activity, tissue injury response, coagulation, and complement activation were enriched in both models. We present for the first time the placental proteome of SHRSP and SD-PE and provide insight into the molecular make-up of models of hypertensive pregnancy. Our study informs future research into specific preeclampsia and chronic hypertension pregnancy mechanisms and translation of rodent data to the clinic.

Utilizing proteomics to understand and define hypertension: where are we and where do we go?

INTRODUCTION: Hypertension is a complex and multifactorial cardiovascular disorder. With different mechanisms contributing to a different extent to an individual's blood pressure, the discovery of novel pathogenetic principles of hypertension is challenging. However, there is an urgent and unmet clinical need to improve prevention, detection, and therapy of hypertension in order to reduce the global burden associated with hypertension-related cardiovascular diseases. Areas covered: Proteomic techniques have been applied in reductionist experimental models including angiotensin II infusion models in rodents and the spontaneously hypertensive rat in order to unravel mechanisms involved in blood pressure control and end organ damage. In humans proteomic studies mainly focus on prediction and detection of organ damage, particularly of heart failure and renal disease. While there are only few proteomic studies specifically addressing human primary hypertension, there are more data available in hypertensive disorders in pregnancy, such as preeclampsia. We will review these studies and discuss implications of proteomics on precision medicine approaches. Expert commentary: Despite the potential of proteomic studies in hypertension there has been moderate progress in this area of research. Standardized large-scale studies are required in order to make best use of the potential that proteomics offers in hypertension and other cardiovascular diseases.

Urinary biomarkers for renal tract malformations.

INTRODUCTION: Renal tract malformations (RTMs) are congenital anomalies of the kidneys and urinary tract, which are the major cause of end-stage renal disease in children. Using immunoassay-based approaches (ELISA, western blot), individual urinary proteins including transforming growth factor ß, tumor necrosis factor and monocyte attractant proteins 1 were found to be associated to RTMs. However, only mass spectrometry (MS) based methods leading to the identification of panels of protein-based markers composed of fragments of the extracellular matrix allowed the prediction of progression of RTMs and its complications. Areas covered: In this review, we summarized relevant studies identified in "Pubmed" using the keywords "urinary biomarkers" and "proteomics" and "renal tract malformations" or "hydronephrosis" or "ureteropelvic junction obstruction" or "posterior urethral valves" or "vesicoureteral reflux". These publications represent studies on potential protein-based biomarkers, either individually or combined in panels, of RTMs in human and animal models. Expert commentary: Successful use in the clinic of these protein-based biomarkers will need to involve larger scale studies to reach sufficient power. Improved performance will potentially come from combining immunoassay- and MS-based markers.

An assessment of peptide enrichment methods employing mTRAQ quantification approaches.

The human plasma peptidome has potential in biomarker discovery not least because the plasma proteome is a challenging matrix due to its complexity and dynamic range. However, methods to significantly reduce the amount of protein present in plasma while retaining the less abundant peptides present in plasma samples has been a major issue. Here, we present a novel strategy which has been employed to assess the effectiveness of removing interfering proteins while retaining peptides of interest. To monitor peptide retention, a spiked in digested protein, in this case a synthetic QconCAT protein, was employed. This enabled a variety of target analytes (peptides) to be monitored for their retention in liquid phase, providing a broader picture of peptide loss from each method assessed. The incorporation of mTRAQ labeling allowed the presence of each peptide to be monitored, and accurate peptide losses to be determined in a Selected Reaction Monitoring (SRM) assay, thus, enabling an objective semiquantitative conclusion to be drawn regarding the suitability of each method for protein removal and peptide retention. We also assessed a range of methods for retaining nontryptic peptides in a plasma peptidomics workflow. From these data, we determined an optimal workflow for removing intact protein, while retaining peptides for MS-based analyses.

Identification of the phosphorylation sites on the E3 ubiquitin ligase Pellino that are critical for activation by IRAK1 and IRAK4.

The E3 ubiquitin ligase Pellino can be activated by phosphorylation in vitro, catalyzed by IL-1 receptor-associated kinase 1 (IRAK1) or IRAK4. Here, we show that phosphorylation enhances the E3 ligase activity of Pellino 1 similarly with any of several E2-conjugating enzymes (Ubc13-Uev1a, UbcH4, or UbcH5a/5b) and identify 7 amino acid residues in Pellino 1 whose phosphorylation is critical for activation. Five of these sites are clustered between residues 76 and 86 (Ser-76, Ser-78, Thr-80, Ser-82, and Thr-86) and decorate a region of antiparallel beta-sheet, termed the "wing," which is an appendage of the forkhead-associated domain that is thought to interact with IRAK1. The other 2 sites are located at Thr-288 and Ser-293, just N-terminal to the RING-like domain that carries the E3 ligase activity. Unusually, the full activation of Pellino 1 can be achieved by phosphorylating any one of several different sites (Ser-76, Thr-86, Thr-288, or Ser-293) or a combination of other sites (Ser-78, Thr-80, and Ser-82). These observations imply that dephosphorylation of multiple sites is required to inactivate Pellino 1, which could be a device for prolonging Pellino's E3 ubiquitin ligase activity in vivo.

Identifying molecules as biosignatures with assembly theory and mass spectrometry.

The search for alien life is hard because we do not know what signatures are unique to life. We show why complex molecules found in high abundance are universal biosignatures and demonstrate the first intrinsic experimentally tractable measure of molecular complexity, called the molecular assembly index (MA). To do this we calculate the complexity of several million molecules and validate that their complexity can be experimentally determined by mass spectrometry. This approach allows us to identify molecular biosignatures from a set of diverse samples from around the world, outer space, and the laboratory, demonstrating it is possible to build a life detection experiment based on MA that could be deployed to extraterrestrial locations, and used as a complexity scale to quantify constraints needed to direct prebiotically plausible processes in the laboratory. Such an approach is vital for finding life elsewhere in the universe or creating de-novo life in the lab.

Polarized Trout Epithelial Cells Regulate Transepithelial Electrical Resistance, Gene Expression, and the Phosphoproteome in Response to Viral Infection.

The burden of disease is a major challenge in aquaculture production. The fish gill characterized with a large surface area and short route to the bloodstream is a major environmental interface and a significant portal of entry for pathogens. To investigate gill responses to viral infection the salmonid gill cell line RTgill-W1 was stimulated with synthetic dsRNA and the salmonid alphavirus subtype 2 (SAV-2). Epithelial integrity in polarized cells can be measured as transepithelial electrical resistance (TEER) which is defined as the electrical resistance across a cell monolayer. TEER is a widely accepted quantitative measure of cellular integrity of a cell monolayer. TEER increased immediately after stimulation with the synthetic dsRNA, polyinosinic:polycytidylic acid (poly(I:C)). In parallel, tight junction and gene expression of innate immune activation markers was modulated in response to poly(I:C). The SAV-2 virus was found to replicate at a low level in RTgill-W1 cells where TEER was disturbed at an early stage of infection, however, gene expression related to tight junction regulation was not modulated. A strong poly(I:C)-driven antiviral response was observed including increases of Rig-like receptors (RLRs) and interferon stimulating genes (ISGs) mRNAs. At the level of signal transduction, poly(I:C) stimulation was accompanied by the phosphorylation of 671 proteins, of which 390 were activated solely in response to the presence of poly(I:C). According to motif analysis, kinases in this group included MAPKs, Ca2+/calmodulin-dependent kinase (CaMK) and cAMP-dependent protein kinase (PKA), all reported to be activated in response to viral infection in mammals. Results also highlighted an activation of the cytoskeletal organization that could be mediated by members of the integrin family. While further work is needed to validate these results, our data indicate that salmonid gill epithelia has the ability to mount a significant response to viral infection which might be important in disease progression. In vitro cell culture can facilitate both a deeper understanding of the anti-viral response in fish and open novel therapeutic avenues for fish health management in aquaculture.

The IRAK-catalysed activation of the E3 ligase function of Pellino isoforms induces the Lys63-linked polyubiquitination of IRAK1.

The protein kinases IRAK [IL-1 (interleukin 1) receptor-associated kinase] 1 and 4 play key roles in a signalling pathway by which bacterial infection or IL-1 trigger the production of inflammatory mediators. In the present study, we demonstrate that IRAK1 and IRAK4 phosphorylate Pellino isoforms in vitro and that phosphorylation greatly enhances Pellino's E3 ubiquitin ligase activity. We show that, in vitro, Pellino 1 can combine with the E2 conjugating complex Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) to catalyse the formation of K63-pUb (Lys63-linked polyubiquitin) chains, with UbcH3 to catalyse the formation of K48-pUb chains and with UbcH4, UbcH5a or UbcH5b to catalyse the formation of pUb-chains linked mainly via Lys11 and Lys48 of ubiquitin. In IRAK1-/- cells, the co-transfection of DNA encoding wild-type IRAK1 and Pellino 2, but not inactive mutants of these proteins, induces the formation of K63-pUb-IRAK1 and its interaction with the NEMO [NF-kappaB (nuclear factor kappaB) essential modifier] regulatory subunit of the IKK (inhibitor of NF-kappaB kinase) complex, a K63-pUb-binding protein. These studies suggest that Pellino isoforms may be the E3 ubiquitin ligases that mediate the IL-1-stimulated formation of K63-pUb-IRAK1 in cells, which may contribute to the activation of IKKbeta and the transcription factor NF-kappaB, as well as other signalling pathways dependent on IRAK1/4.

Comparison of Urine and Plasma Peptidome Indicates Selectivity in Renal Peptide Handling.

PURPOSE: Urine is considered to be produced predominantly as a result of plasma filtration in the kidney. However, the origin of the native peptides present in urine has never been investigated in detail. Therefore, the authors aimed to obtain a first insight into the origin of urinary peptides based on a side-by-side comprehensive analysis of the plasma and urine peptidome. METHODS: Twenty-two matched urine and plasma samples are analyzed for their peptidome using capillary electrophoresis coupled to mass spectrometry (CE-MS; for relative quantification) and CE or LC coupled to tandem mass spectrometry (CE- or LC-MS/MS; for peptide identification). The overlap and association of abundance of the different peptides present in these two body fluids are evaluated. RESULTS: The authors are able to identify 561 plasma and 1461 urinary endogenous peptides. Only 90 peptides are detectable in both urine and plasma. No significant correlation is found when comparing the abundance of these common peptides, with the exception of collagen fragments. This observation is also supported when comparing published peptidome data from both plasma and urine. CONCLUSIONS AND CLINICAL RELEVANCE: Most of the plasma peptides are not detectable in urine, possibly due to tubular reabsorption. The majority of urinary peptides may in fact originate in the kidney. The notable exception is collagen fragments, which indicates potential selective exclusion of these peptides from tubular reabsorption. Experimental verification of this hypothesis is warranted.

Dopa-responsive dystonia (Segawa syndrome).

A 12 year old boy with gradually worsening global developmental delay was diagnosed and managed as quadriplegic cerebral palsy since child-hood. Subsequent evaluation revealed marked dystonia over spasticity leading to suspicion of Segawa syndrome. Dramatic improvement in clinical condition followed after therapy with low dose L-Dopa.

Development of a MALDI MS-based platform for early detection of acute kidney injury.

PURPOSE: Septic acute kidney injury (AKI) is associated with poor outcome. This can partly be attributed to delayed diagnosis and incomplete understanding of the underlying pathophysiology. Our aim was to develop an early predictive test for AKI based on the analysis of urinary peptide biomarkers by MALDI-MS. EXPERIMENTAL DESIGN: Urine samples from 95 patients with sepsis were analyzed by MALDI-MS. Marker search and multimarker model establishment were performed using the peptide profiles from 17 patients with existing or within the next 5 days developing AKI and 17 with no change in renal function. Replicates of urine sample pools from the AKI and non-AKI patient groups and normal controls were also included to select the analytically most robust AKI markers. RESULTS: Thirty-nine urinary peptides were selected by cross-validated variable selection to generate a support vector machine multidimensional AKI classifier. Prognostic performance of the AKI classifier on an independent validation set including the remaining 61 patients of the study population (17 controls and 44 cases) was good with an area under the receiver operating characteristics curve of 0.82 and a sensitivity and specificity of 86% and 76%, respectively. CONCLUSION AND CLINICAL RELEVANCE: A urinary peptide marker model detects onset of AKI with acceptable accuracy in septic patients. Such a platform can eventually be transferred to the clinic as fast MALDI-MS test format.