Quantitative analysis of a phosphoproteome readily altered by the protein kinase CK2 inhibitor quinalizarin in HEK-293T cells.

CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50µM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.

The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker.

AIMS/HYPOTHESIS: Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. METHODS: Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. RESULTS: Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. CONCLUSIONS/INTERPRETATION: We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.

Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression.

Myeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC(+)BAP(+) MCCs compared with those in OC(-)BAP(-) cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC(+)BAP(+) MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cells.

No peptide left behind: the "out of range" recovery in IPG-IEF fractionation.

IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.

Improvements to polar 2-D electrophoresis for proteomic applications.

Recently, we reported a new way of performing 2-DE, called P-dimensional electrophoresis (2-PE). In this approach, the second dimension is achieved in a radial gel which can accommodate up to six 7 cm long IPG strips simultaneously, improving reproducibility and throughput power in respect to 2-DE. Nevertheless, 2-PE was up to now limited to the use of only short strips because of technical difficulties. Here, we describe how to load longer strips (e.g., 18-24 cm) on 2-PE and report some representative images for a qualitative assessment.

Myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.

Skin fibroblasts as a tool for identifying the risk of nephropathy in the type 1 diabetic population.

Human fibroblasts in culture have been employed as an in vitro system to investigate some pathophysiological mechanisms of diabetes mellitus also associated with the development of diabetic nephropathy. In fact, there is increasing evidence that genetic factors either convey the risk of, or protect from, diabetic nephropathy and that the expression profiles and/or the behaviour of the cultured skin fibroblasts from type 1 diabetic patients could reflect these genetic influences. On the other hand, alterations could be attributable not only to changes in DNA sequence, but also to epigenetic factors. Our aim is to make a critical overview of the studies involving primary cultures of skin fibroblasts as tools to investigate the pathophysiology of diabetic nephropathy performed until now in this area. Cultured skin fibroblasts could be useful not only for the identification of patients at risk of developing diabetic renal disease, but also for a better understanding of the complex multifactorial mechanisms leading to the long-term complications in diabetes.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

Operator- and software-related post-experimental variability and source of error in 2-DE analysis.

In the field of proteomics, several approaches have been developed for separating proteins and analyzing their differential relative abundance. One of the oldest, yet still widely used, is 2-DE. Despite the continuous advance of new methods, which are less demanding from a technical standpoint, 2-DE is still compelling and has a lot of potential for improvement. The overall variability which affects 2-DE includes biological, experimental, and post-experimental (software-related) variance. It is important to highlight how much of the total variability of this technique is due to post-experimental variability, which, so far, has been largely neglected. In this short review, we have focused on this topic and explained that post-experimental variability and source of error can be further divided into those which are software-dependent and those which are operator-dependent. We discuss these issues in detail, offering suggestions for reducing errors that may affect the quality of results, summarizing the advantages and drawbacks of each approach.

Effects of chronic metabolic acidosis on splanchnic protein turnover and oxygen consumption in human beings.

BACKGROUND & AIMS: Although metabolic acidosis stimulates protein catabolism, its effects on splanchnic protein turnover and energy expenditure have not been measured in human beings. We investigated the effects of chronic metabolic acidosis (CMA) on splanchnic protein dynamics and oxygen consumption in human beings by using a leucine tracer and mass-balance techniques. METHODS: Five subjects were studied after 6 days of HCl-, CaCl(2)-, and NH(4)Cl-induced acidosis; 8 subjects served as controls. Blood samples were collected from the radial artery and the hepatic veins. Measurements were performed on plasma and whole-blood samples. RESULTS: Based on plasma measurements, subjects who had undergone CMA had lower rates of splanchnic proteolysis (-35%) and protein synthesis (-50%; P < .05) than controls, as well as a negative leucine kinetic balance (-6.81 +/- 2.48 micromol/kg/min/1.73 m(2) body surface [BS](-1)), compared with the neutral balance in control plasma samples (0.76 +/- 2.11 micromol/kg/min/1.73; P < .05 between groups). Based on measurements from whole blood, splanchnic proteolysis and protein synthesis did not differ significantly between CMA and control samples, and the net leucine kinetic balance was neutral in both groups (CMA, -0.69 +/- 1.57; controls, -0.74 +/- 3.45 micromol/kg/min/1.73). In CMA whole-blood measurements, splanchnic oxygen consumption (44.8 +/- 4.3 mL/min/1.73 m(2) BS) was slightly lower than in controls (57.5 +/- 8.4 mL/min/1.73 m(2) BS; P = NS). Splanchnic protein synthesis correlated with oxygen consumption (r = 0.82; P < .001). CONCLUSIONS: CMA reduces splanchnic protein turnover and results in a negative leucine balance--an effect that apparently is offset by the contribution of blood cells to organ leucine (and protein) dynamics. Protein synthesis is a major contributor (about 67%) to energy expenditure in splanchnic organs.

Test Groups, Not Individuals: A Review of the Pooling Approaches for SARS-CoV-2 Diagnosis.

Massive molecular testing for SARS-CoV-2 diagnosis is mandatory to manage the spread of COVID-19. Diagnostic screening should be performed at a mass scale, extended to the asymptomatic population, and repeated over time. An accurate diagnostic pipeline for SARS-CoV-2 that could massively increase the laboratory efficiency, while being sustainable in terms of time and costs, should be based on a pooling strategy. In the past few months, researchers from different disciplines had this same idea: test groups, not individuals. This critical review intends to highlight both the general consents-even if the results from different publications have been obtained with different protocols-and the points of disagreement that are creating some interpretative/comprehension difficulties. Different pooling schemes and technical aspects associated to the type of pooling adopted are described and discussed. We hope that this review can consolidate information to support researchers in designing optimized COVID-19 testing protocols based on pooling.

The effects of rosiglitazone and high glucose on protein expression in endothelial cells.

Rosiglitazone is a thiazolidinedione used to treat insulin resistance in diabetes. Although thiazolidinediones may also exert cardiovascular effects, contrasting results were reported. Favorable effects were shown for pioglitazone, whereas adverse reactions were suspected for rosiglitazone. Therefore, a reassessment of the molecular effects of rosiglitazone on vascular cells is required. We tested the effects of rosiglitazone on the proteome of human endothelial cells grown under either normal or high glucose levels. Protein profiles were analyzed in both membrane and cytosolic fractions. About 150 cytosolic proteins, and approximately 100 membrane proteins, were detected. Two-thirds of the proteins significantly altered by high glucose were also modulated by rosiglitazone in an antagonistic way. Half of these proteins are involved in apoptosis. Using an independent assay of apoptosis based on nucleosome quantification, an approximately 20% stimulation by high versus normal glucose was shown (p < 0.05). Conversely, rosiglitazone reduced apoptosis by approximately 30-50% in cells exposed to either glucose conditions (p < 0.001). In addition, rosiglitazone differently modulated cytoskeleton and energy metabolism-related proteins. Our data show novel, potential sites of action of rosiglitazone through protein expression of endothelial cells. These mechanisms may foster new investigations on the overall vascular effects of this compound, and help to discriminate between desired and adverse effects.

Proteomic analysis of clonal interstitial aortic valve cells acquiring a pro-calcific profile.

Calcific degeneration represents the most frequent aortic valve disease observed in industrialized countries. Our aim is to study modifications in the cytosolic and membrane protein profile of aortic interstitial valve cells (VIC) acquiring a pro-calcific phenotype. We studied a clonal population of bovine VIC that expresses bone-related proteins (such as alkaline phosphatase [ALP]) and calcifies a collagen matrix in response to endotoxin (LPS) treatment. A proteomic analysis was performed on proteins extracted from cells treated for 12 days with LPS (100 ng/mL) versus control. We identified 34 unique cytosolic and 10 unique membrane-associated proteins showing significant changes after treatment. These proteins are involved in several cellular functions, such as chaperone-mediated protein folding, protein metabolism and transport, cell redox/nitric oxide homeostasis, and cytoskeletal organization. Reduced expression of proteins involved in NOS bioactivity (such as DDAH-1 and -2) suggested a role for the l-arginine/ADMA ratio in controlling VIC phenotypic profile. In accordance with this hypothesis, we observed that exposure of clonal cells to l-arginine prevented LPS-induced ALP expression and collagen calcification. In conclusion, we identified several proteins involved in structural, metabolic, and signaling functions that are significantly altered in aortic VIC acquiring a pro-calcific profile, thus giving new insights into the pathogenesis of aortic valve degeneration.

The inter- and intra-operator variability in manual spot segmentation and its effect on spot quantitation in two-dimensional electrophoresis analysis.

Separation of complex mixtures of proteins by 2-DE is a fundamental component of current proteomic technology. Quantitative analysis of the images generated by digitization of such gels is critical for identifying alterations in protein expression within a given biological system. Software packages are designed for this purpose. The accurate definition of protein spot boundaries, using a suitable method of image segmentation, is a key requirement for image analysis. It is often necessary for operators to intervene manually to correct mistakes in spot segmentation; therefore operator subjectivity and differences in ability can weaken the analysis. We estimated the error in spot quantification after manual spot segmentation, which was performed by different operators, using two different software packages. Our results clearly show that this operation was associated with significant inter- and intra-variability and an overestimation of subsequent spot intensity, especially when spots were weak. For comparative studies, we suggest separately analysing spots which have been manually segmented by imposing a requirement for at least a threefold difference in spot intensity in addition to use of statistical tests.

SDS-PAGE and two-dimensional maps in a radial gel format.

A novel method for performing 2-D map analysis is here reported, consisting in a modification of the second dimension run, which is performed not in a conventional square- or rectangular-size gel, but in a radial surface. This has the advantage of permitting resolution of closely adjacent bands, representing strings of isoforms of similar or identical mass but of closely spaced isoelectric points. When used in a mono-dimensional, SDS-PAGE format, this system allows the simultaneous running of 62 sample tracks. Examples are given of separation of plasma and urinary proteins.

Delta2D and Proteomweaver: Performance evaluation of two different approaches for 2-DE analysis.

2-DE is a fundamental technology used in proteomics research. However, despite its high capacity to simultaneously separate several proteins for subsequent identification and quantitative comparison studies, a drawback for this technique is its limited reproducibility, especially when comparing data from different laboratories. 2-DE-related variability can be broadly divided into two categories: experimental and post-experimental. Experimental variability depends on physical and chemical parameters, whereas post-experimental variability arises when gels are analyzed by different software packages, particularly when different workflows are followed. In this paper, we compared the analysis performance of two software packages, Delta2D and Proteomweaver, using both standard and experimental gel images. Using standard gel images, the false negative spot count was 50% lower, the false positive count was 77% lower, the true positive count was 19% higher and spot matching was 4% higher in Delta2D when compared to Proteomeweaver. Using experimental gel images, we found that the total amount of time taken to complete the analysis with Delta2D was 30% that of the time needed with Proteomweaver and required fewer user interventions. The differences between ease of use and workflow strategy of these programs is discussed.

Improved instrumentation for large-size two-dimensional protein maps.

Novel instrumentation for performing large-size (>25 cm) 2-D maps is reported here. To perform the first dimension, we developed a power supply that can deliver a voltage of up to 15,000 V and allows regulation of current (up to 200 µA) onto each individual focusing IPG strip. The IEF strip tray can accommodate up to 12 IPG strips and the electrodes slide on a ruler, thus permitting running strips of any length up to 45 cm. In addition, this apparatus also includes a second power supply that allows the performance of electrophoresis at high amperage (400 mA) and a Peltier system that allows a 10-80°C temperature control.

Effect of liver cirrhosis on phenylalanine and tyrosine metabolism.

PURPOSE OF REVIEW: Phenylalanine conversion to tyrosine (i.e., 'hydroxylation') is the first irreversible step in phenylalanine catabolism and a source of circulating tyrosine. The purpose of the present review is both to examine hydroxylation from a biochemical standpoint and to report data measured in vivo under physiological conditions, as well as in liver and kidney disease. RECENT FINDINGS: The simultaneous infusion of phenylalanine and tyrosine tracers in humans allows us to determine the hydroxylation rate in vivo. Hydroxylation accounts for a minor ( approximately 10-20%) although significant portion of tyrosine flux. The liver and the kidney are the key organs accounting for virtually the whole-body hydroxylation rates. It is regulated by substrate availability, being acutely stimulated by mixed meal ingestion and by dietary adaptation to high phenylalanine intakes. Theoretically, it may be impaired in advanced liver and kidney disease. Nevertheless, in compensated liver cirrhosis, hydroxylation as well as tyrosine flux are not decreased but rather increased. Only in end stage liver disease hydroxylation may be impaired and is corrected by transplantation. Hydroxylation is also reduced in end stage renal disease. SUMMARY: Phenylalanine hydroxylation in vivo appears to represent a regulatory step of phenylalanine disposal and tyrosine production under acute and/or extreme conditions.

Informed sequential pooling approach to detect SARS-CoV-2 infection.

The alarming spread of the pandemic coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus requires several measures to reduce the risk of contagion. Every successful strategy in controlling the SARS-CoV-2 infection depends on timely diagnosis, which should include testing of asymptomatic carriers. Consequently, increasing the throughput for clinical laboratories for the purposes of conducting large-scale diagnostic testing is urgently needed. Here we support the hypothesis that standard diagnostic protocol for SARS-CoV-2 virus could be conveniently applied to pooled samples obtained from different subjects. We suggest that a two-step sequential pooling procedure could identify positive subjects, ensuring at the same time significant benefits of cost and time. The simulation data presented herein were used to assess the efficiency, in terms of number of required tests, both for random assignment of the subjects to the pools and for situations in which epidemiological and clinical data are used to create "informed" pools. Different scenarios were simulated to measure the effect of different pool sizes and different values for virus frequency. Our results allow for a customization of the pooling strategy according to the specific characteristics of the cohort being tested.

l-Arginine prevents inflammatory and pro-calcific differentiation of interstitial aortic valve cells.

BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.