Dataset Information

PROTEOLYTIC SIGNATURES OF THROMBIN-DERIVED PEPTIDES by LC-MS/MS

ABSTRACT: We hypothesized that the peptide profile could reflect the action of different proteases present in wounds. However, the complexity of such peptide systems, and lack of efficient analytic tools, until recently has hampered their clinical exploitation and use. In this project, we set out to define cleaved thrombin fragments generated by both endogenous and bacterial proteases.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

DISEASE(S): Wounds And Injuries

SUBMITTER: Sunil Adav

LAB HEAD: Prof. Sze S K

PROVIDER: PXD006992 | Pride | 2017-10-20

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	170227_SunilRathi_LKC_ALYSA3h_A.raw	Raw
	170227_SunilRathi_LKC_ALYSA3h_B.raw	Raw
	170227_SunilRathi_LKC_ALYSA6h_A.raw	Raw
	170227_SunilRathi_LKC_ALYSA6h_B.raw	Raw
	170227_SunilRathi_LKC_ALYSB3h_A.raw	Raw

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Publications

Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

Saravanan Rathi R Adav Sunil S SS Choong Yeu Khai YK van der Plas Mariena J A MJA Petrlova Jitka J Kjellström Sven S Sze Siu Kwan SK Schmidtchen Artur A

Scientific reports 20171013 1

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a stra ...[more]

PMID: 29030565

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Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Thrombin (2 U/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments, Control Monosome, Control Polysome, Thrombin Monosome, and Thrombin Polysome. Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (thrombin polysome/thrombin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. TSAA data suggests that JunB is translationally regulated by thrombin stimulation. Immunocytochemistry, western blotting and RT-PCR were used to verify the results of TSAA. Polysome Profiling. HUVECs were stimulated with thrombin (2 U/mL) for 2 hours. Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was removed and cells were carefully resuspended in 375 microL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice. 125 microL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting. The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 degrees C) to remove nuclei and cellular debris. The supernatant was transferred to a new tube containing 50 microL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 microL 5M NaCl. This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients were spun at 43,700 rpm for 90 minutes (4 degrees C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles. Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer. Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen). These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters. Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters. To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter. Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array. Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1. Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter. The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction: TS=P/M. The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls. In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1. Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated. A measure of transcriptional activity was also determined from the signal intensities of the array elements. Summing the normalized signal intensities from the M and P fractions of the experimental cells and dividing that value by the sum of the normalized signal intensities from the control fractions calculated the Transcription Index (TCI). Therefore, TCI = (Me + Pe)/(Mc + Pc). If transcript levels for a specific gene do not change, the TCI value for that gene will be 1. Genes that are transcribed upon stimulation of the cells will have a TCI value greater than 1, while genes that are not transcribed or transcription is down-regulated upon stimulation will have a TCI value less than 1. Arbitrary values of 2 and 0.5, a doubling or halving of the amount of mRNA for that element, respectively, were used to categorize individual array elements as being transcriptionally regulated.

Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-RÂ´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Experiment Overall Design: HUVEC in passage 1 were cultured in serum-free EC medium until cells reached confluency. HUVEC were stimulated by 100 nM LTD4 (n = 4) or 10 nM thrombin (n = 4) or both agonists concomitantly (n = 3). The immediate-early gene programs of these experimental groups were identified at 60 minutes. In addition, one HUVEC preparation was stimulated with 100 nM LTD4 for 6 h and 24 h.