Isabelle Kelly

Leishmania Exosomes Modulate Innate and Adaptive Immune Responses through Effects on Monocytes and Dendritic Cells

We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ–producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100−/− L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100−/− leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100−/− exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

Molecular basis of film formation from a soybean protein: comparison between the conformation of glycinin in aqueous solution and in films

Fourier transform infrared spectroscopy has been used to investigate the conformational changes of glycinin. a major storage protein of soybean seeds, upon film-forming. The results show that the secondary structure of glycinin is mainly composed of a beta-sheet (48%) and unordered (49%) structures. The amide I band of glycinin in film-forming conditions, i.e. in alkaline media and in the presence of plasticizing agent, reveals the conversion of 18% of the secondary structure of the protein from the beta-sheet (6%) and random coil (12%) to the alpha-helical conformation due to the helicogenic effect of the ethylene glycol used as the plasticizing agent. Conformational changes also occur upon the film-forming process leading to the formation of intermolecular hydrogen-bonded beta-sheet structures. Results obtained from other plant families indicate that, whatever the origin and conformation of protein, formation of films leads to the appearance of intermolecular hydrogen-bonded beta-sheet structures, suggesting that this type of structure might be essential for the network formation in films. Thus, it is hypothesized that, in the film state, intermolecular hydrogen bonding between segments of beta-sheet may act as junction zones in the film network. This study reveals for the first time that there is a close relationship between the conformation of proteins and the mechanical properties of films.

Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress

Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.

Immunization with Live and Dead Chlamydia muridarum Induces Different Levels of Protective Immunity in a Murine Genital Tract Model: Correlation with MHC Class II Peptide Presentation and Multifunctional Th1 Cells

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ+–producing CD4+ T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4+ T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.

An SNP in an ultraconserved regulatory element affects Dlx5/Dlx6 regulation in the forebrain

Dlx homeobox genes play a crucial role in the migration and differentiation of the subpallial precursor cells that give rise to various subtypes of γ-aminobutyric acid (GABA)-expressing neurons of the forebrain, including local-circuit cortical interneurons. Aberrant development of GABAergic interneurons has been linked to several neurodevelopmental disorders, including epilepsy, schizophrenia, Rett syndrome and autism. Here, we report in mice that a single-nucleotide polymorphism (SNP) found in an autistic proband falls within a functional protein binding site in an ultraconserved cis-regulatory element. This element, I56i, is involved in regulating Dlx5/Dlx6 homeobox gene expression in the developing forebrain. We show that the SNP results in reduced I56i activity, predominantly in the medial and caudal ganglionic eminences and in streams of neurons tangentially migrating to the cortex. Reduced activity is also observed in GABAergic interneurons of the adult somatosensory cortex. The SNP affects the affinity of Dlx proteins for their binding site in vitro and reduces the transcriptional activation of the enhancer by Dlx proteins. Affinity purification using I56i sequences led to the identification of a novel regulator of Dlx gene expression, general transcription factor 2 I (Gtf2i), which is among the genes most often deleted in Williams-Beuren syndrome, a neurodevelopmental disorder. This study illustrates the clear functional consequences of a single nucleotide variation in an ultraconserved non-coding sequence in the context of developmental abnormalities associated with disease.

Interaction between β-purothionin and dimyristoylphosphatidylglycerol: A 31P-NMR and infrared spectroscopic study

Abstract The interaction of β -purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of dimyristoylphosphatidylglycerol (DMPG) was investigated by 31 P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of β -purothionin. The results indicate that β -purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that β -purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by β -purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of β -purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24°C to ∼33°C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20°C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of β -purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the α -helices and the β -sheet of β -purothionin have tilt angles of ∼60° and 30°, respectively, relative to the normal of the ATR crystal.

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.

Evaluation of iTRAQ and SWATH-MS for the Quantification of Proteins Associated with Insulin Resistance in Human Duodenal Biopsy Samples.

Insulin resistance (IR) is associated with increased production of triglyceride-rich lipoproteins of intestinal origin. In order to assess whether insulin resistance affects the proteins involved in lipid metabolism, we used two mass spectrometry based quantitative proteomics techniques to compare the intestinal proteome of 14 IR patients to that of 15 insulin sensitive (IS) control patients matched for age and waist circumference. A total of 3886 proteins were identified by the iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry approach and 2290 by the SWATH-MS strategy (Serial Window Acquisition of Theoretical Spectra). Using these two methods, 208 common proteins were identified with a confidence corresponding to FDR < 1%, and quantified with p-value < 0.05. The quantification of those 208 proteins has a Pearson correlation coefficient (r2) of 0.728 across the two techniques. Gene Ontology analyses of the differentially expressed proteins revealed that annotations related to lipid metabolic process and oxidation reduction process are overly represented in the set of under-expressed proteins in IR subjects. Furthermore, both methods quantified proteins of relevance to IR. These data also showed that SWATH-MS is a promising and compelling alternative to iTRAQ for protein quantitation of complex mixtures.

Key intestinal genes involved in lipoprotein metabolism are downregulated in dyslipidemic men with insulin resistance

Insulin resistance (IR) is associated with elevated plasma levels of triglyceride-rich lipoproteins (TRLs) of intestinal origin. However, the mechanisms underlying the overaccumulation of apolipoprotein (apo)B-48-containing TRLs in individuals with IR are not yet fully understood. This study examined the relationships between apoB-48-containing TRL kinetics and the expression of key intestinal genes and proteins involved in lipid/lipoprotein metabolism in 14 obese nondiabetic men with IR compared with 10 insulin-sensitive (IS) men matched for waist circumference. The in vivo kinetics of TRL apoB-48 were assessed using a primed-constant infusion of L-[5,5,5-D3]leucine for 12 h with the participants in a constantly fed state. The expression of key intestinal genes and proteins involved in lipid/lipoprotein metabolism was assessed by performing real-time PCR quantification and LC-MS/MS on duodenal biopsy specimens. The TRL apoB-48 pool size and production rate were 102% (P < 0.0001) and 87% (P = 0.01) greater, respectively, in the men with IR versus the IS men. On the other hand, intestinal mRNA levels of sterol regulatory element binding factor-2, hepatocyte nuclear factor-4α, and microsomal triglyceride transfer protein were significantly lower in the men with IR than in the IS men. These data indicate that IR is associated with intestinal overproduction of lipoproteins and significant downregulation of key intestinal genes involved in lipid/lipoprotein metabolism.

Effect of sitagliptin therapy on triglyceride-rich lipoprotein kinetics in patients with type 2 diabetes

To investigate the effects of sitagliptin therapy on the kinetics of triglyceride‐rich lipoprotein (TRL) apolipoprotein (apo)B‐48, VLDL apoB‐100, apoE and apoC‐III in patients with type 2 diabetes.