First Identification of a Large Set of Serine Hydrolases by Activity-Based Protein Profiling in Dibutyl Phthalate-Exposed Zebrafish Larvae

Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposure to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120 h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120 h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches, such as ABPP, can, therefore, be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology

Comprehensive Analysis of Transcript and Protein Relative Abundance During Blood Stages of Plasmodium falciparum Infection

International audiencePlasmodium falciparum is the main causative agent of human malaria. During the intraerythrocytic development cycle, the P. falciparum morphology changes dramatically from circulating young rings to sequestered mature trophozoites and schizonts. Sequestered forms contribute to the pathophysiology of severe malaria as the infected erythrocytes obstruct the microvascular flow in deep organs and induce local inflammation. However, the sequestration mechanism limits the access to the corresponding parasitic form in the clinical samples from patients infected with P. falciparum. To complement this deficiency, we aimed to evaluate the relevance of mRNA study as a proxy of protein expression in sequestered parasites. To do so, we conducted a proteotranscriptomic analysis using five independent P. falciparum laboratory strain samples. RNA sequencing was performed, and the mRNA expression level was assessed on circulating ring-stage parasites. The level of protein expression were measured by LC-MS/MS on the corresponding sequestered mature forms after 18-24 h of maturation. Overall, our results showed a strong transcriptome/transcriptome and a very strong proteome/proteome correlation between samples. Moreover, positive correlations of mRNA and protein expression levels were found between ring-stage transcriptomes and mature form proteomes. However, twice more transcripts were identified at the ring stage than proteins at the mature trophozoite stage. A high level of transcript expression did not guarantee the detection of the corresponding protein. Finally, we pointed out discrepancies at the individual gene level. Taken together, our results show that transcript and protein expressions are overall correlated. However, mRNA abundance is not a perfect proxy of protein expression at the individual level. Importantly, our study shows limitations of the "blind" use of RNA-seq and the importance of multiomics approaches for P. falciparum blood stage study in clinical samples

Proteomic Landscape of Neutrophils in Sickle Cell Anemia: An Unexpected Autoimmune Profile

Meeting: 60th Annual Meeting of the American-Society-of-Hematology (ASH)Location: San Diego, CADate: DEC 01-04, 2018Sponsor: Amer Soc HematolInternational audienceAbstract Although sickle cell disease (SCD) is a red cell disorder, many cell types, including endothelial cells and polymorphonuclear neutrophils (PMNs), contribute to its pathophysiology. In particular, activated PMNs have been implicated to play an important role in the initiation and propagation of vaso-occlusive events in SCD. Activated PMNs engage in a complex process of abnormal interactions with activated endothelial cells, platelets and circulating erythrocytes contributing to endothelial injury and decreased blood flow. In the present study, global proteomic analysis was performed using label-free mass spectrometry of PMNs from 4 SCD patients (SS) in steady state and from 4 control subjects (AA). We identified a total of 4,534 proteins both in AA and SS PMNs with 3,080 of these proteins identified in at least three samples for each condition. 50 proteins were significantly over-expressed in SS PMNs compared to AA PMNs (ratio > 1.4). STRING employed to monitor potential interaction between the overexpressed proteins showed that the main interactive clusters consist of STAT1 and STAT2, OAS 1, 2 and 3, and many Interferon Signaling Proteins i.e. IFIT1, IFIT2, IFIT 3, ISG15, ISG20, GBP2, IFI35, MX1 and MX2, TLR8 proteins (Fig. 1). This finding implies a strong activation of the type I interferon (IFN) signaling pathway in the SS PMNs (between 10 and 100-fold increase in SS vs AA). In addition, 33 proteins showed significantly lower expression in SS PMNs compared to AA PMNs. Among these were L-selectin (CD62L) and IL-17 receptor A (IL17RA) (p = 0.01). These findings are consistent with previously described phenotypes of aged neutrophils and acute inflammatory responses in SCD. Similar proteomic analysis performed on PMNs from SS patients treated with hydroxycarbamide (HC, n=4) showed that 14 proteins had significantly lower expression compared to untreated-SS patients (ratio <0.7). Interestingly, HC restored a normal expression pattern for most of the interferon signaling proteins. Type I IFNs represent the major effector cytokines of the host immune response against viruses and other intracellular pathogens. These cytokines are produced via activation of STAT1 and of pattern recognition receptors, including the Toll-like receptor signaling network. To determine if type I IFN-α could be detected at the protein level in the plasma of SS patients, we used the novel digital-ELISA technology (SIMOA, Quanterix) developed by Wilson et al (J Lab Autom, 2016). Interestingly, we found an increased level of INFα in plasma from SS patients compared to AA (n=32) (p<0.001) and it is noteworthy that while 50% of SS patients have similar level of INFα compare to AA individuals the other 50% exhibit 10 to 1,000-fold increased levels (Fig. 2). In summary, our novel proteomic analysis documents a high level expression of interferon signaling proteins, STAT1 and TLR8 in the proteome of neutrophils from SS patients and strongly suggests autoimmune or auto-inflammatory phenomena at basal state in SCD. Our results provide strong support for an important role for the innate immune system in the pathophysiology of SCD. Future studies will help determine the relationship between the plasmatic level of IFN-α and clinical complications and will establish if interferon signaling proteins and IFN-α could represent new therapeutic targets in SCD. Disclosures Hermand-Tournamille: Imara: Research Funding. Le Va Kim:Imara: Research Funding. Koehl:Imara: Research Funding

Impact of Fetal Growth Restriction on the Neonatal Microglial Proteome in the Rat

Microglial activation is a key modulator of brain vulnerability in response to intra-uterine growth restriction (IUGR). However, the consequences of IUGR on microglial development and the microglial proteome are still unknown. We used a model of IUGR induced by a gestational low-protein diet (LPD) in rats. Microglia, isolated from control and growth-restricted animals at P1 and P4, showed significant changes in the proteome between the two groups. The expression of protein sets associated with fetal growth, inflammation, and the immune response were significantly enriched in LPD microglia at P1 and P4. Interestingly, upregulation of protein sets associated with the oxidative stress response and reactive oxygen species production was observed at P4 but not P1. During development, inflammation-associated proteins were upregulated between P1 and P4 in both control and LPD microglia. By contrast, proteins associated with DNA repair and senescence pathways were upregulated in only LPD microglia. Similarly, protein sets involved in protein retrograde transport were significantly downregulated in only LPD microglia. Overall, these data demonstrate significant and multiple effects of LPD-induced IUGR on the developmental program of microglial cells, leading to an abnormal proteome within the first postnatal days

Infected erythrocytes and plasma proteomics reveal a specific protein signature of severe malaria

Abstract Cerebral malaria (CM), the most lethal complication of Plasmodium falciparum severe malaria (SM), remains fatal for 15–25% of affected children despite the availability of treatment. P. falciparum infects and multiplies in erythrocytes, contributing to anemia, parasite sequestration, and inflammation. An unbiased proteomic assessment of infected erythrocytes and plasma samples from 24 Beninese children was performed to study the complex mechanisms underlying CM. A significant down-regulation of proteins from the ubiquitin–proteasome pathway and an up-regulation of the erythroid precursor marker transferrin receptor protein 1 (TFRC) were associated with infected erythrocytes from CM patients. At the plasma level, the samples clustered according to clinical presentation. Significantly, increased levels of the 20S proteasome components were associated with SM. Targeted quantification assays confirmed these findings on a larger cohort (n = 340). These findings suggest that parasites causing CM preferentially infect reticulocytes or erythroblasts and alter their maturation. Importantly, the host plasma proteome serves as a specific signature of SM and presents a remarkable opportunity for developing innovative diagnostic and prognostic biomarkers

Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis

International audienceHereditary xerocytosis is a dominant red cell membrane disorder characterized by an increased leak of potassium from the inside to outside the red blood cell membrane, associated with loss of water leading to red cell dehydration and chronic hemolysis. 90% of cases are related to heterozygous gain of function mutations in PIEZO1, encoding a mechanotransductor that translates a mechanical stimulus into a biological signaling. Data are still required to understand better PIEZO1-HX pathophysiology. Recent studies identified proteomics as an accurate and high-input tool to study erythroid progenitors and circulating red cell physiology. Here, we isolated red blood cells from 5 controls and 5 HX patients carrying an identified and pathogenic PIEZO1 mutation and performed a comparative deep proteomic analysis. A total of 603 proteins were identified among which 56 were differentially expressed (40 over expressed and 16 under expressed) between controls and HX with a homogenous expression profile within each group. We observed relevant modifications in the protein expression profile related to PIEZO1 mutations, identifying two main “knots”. The first contained both proteins of the chaperonin containing TCP1 complex involved in the assembly of unfolded proteins, and proteins involved in translation. The second contained proteins involved in ubiquitination. Deregulation of proteins involved in protein biosynthesis was also observed in in vitro -produced reticulocytes after Yoda1 exposure. Thus, our work identifies significant changes in the protein content of PIEZO1-HX erythrocytes, revealing a “PIEZO1 signature” and identifying potentially targetable pathways in this disease characterized by a heterogeneous clinical expression and contra-indication of splenectomy

Proteome analysis of formalin‐fixed paraffin‐embedded colorectal adenomas reveals the heterogeneous nature of traditional serrated adenomas compared to other colorectal adenomas: Proteomic characterization of colorectal adenomas using FFPE samples

International audienceTraditional serrated adenoma (TSA) remains the least understood of all the colorectal adenomas although these lesions have been associated with a significant cancer risk, twice that of the conventional adenoma (CAD) and of the sessile serrated adenoma (SSA/P). This study was performed to investigate the proteomic profiles of the different colorectal adenomas to better understand the pathogenesis of TSA. We performed a global quantitative proteome analysis using the label-free quantification (LFQ) method on 44 colorectal adenomas (12 TSAs, 15 CADs, 17 SSA/Ps) and 17 normal colonic mucosa samples, archived as formalin-fixed paraffin-embedded blocks. Unsupervised consensus hierarchical clustering applied to the whole proteomic profile of the 44 colorectal adenomas identified four subtypes: C1 and C2 were well-individualized clusters composed of all the CADs (15/15) and most of the SSA/Ps (13/17), respectively. This is consistent with the fact that CADs and SSA/Ps are homogeneous and distinct colorectal adenoma entities. In contrast, TSAs were subdivided into C3 and C4 clusters, consistent with the more heterogeneous entity of TSA at the morphological and molecular levels. Comparison of the proteome expression profile between the adenoma subtypes and normal colonic mucosa further confirmed the heterogeneous nature of TSAs which overlapped either on CADs or SSA/Ps, while CADs and SSAs formed homogeneous and distinct entities. Furthermore, we identified LEFTY1 a new potential marker for TSAs that may be relevant for the pathogenesis of TSA. LEFTY1 is an inhibitor of the Nodal/TGFβ pathway, which we found to be one of the most overexpressed proteins specifically in TSAs. This finding was confirmed by immunohistochemistry. Our study confirms that CADs and SSA/Ps form homogeneous and distinct colorectal adenoma entities, while TSAs are a heterogeneous entity and may arise from either SSA/Ps or from normal mucosa evolving through a process related to the conventional adenoma pathway. This article is protected by copyright. All rights reserved

Skyline output and calibration range for GHTVDSELTTEDPVIQKK.

<p>Retention time is displayed on x axis and intensity for each ion or fragment is displayed on y axis. Each parent ion and fragment is shown in a different colour. AUC corresponds to the integration of signal under the curve (A). Regression line is obtained using AUC from each concentration point.</p

Geographic origin of the samples used for genomic studies of <i>PFI1785w</i>.

<p>Geographic origin of the samples used for genomic studies of <i>PFI1785w</i>.</p