Belinda Willard

A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β(2)GPI, and activate endothelial cells (ECs) in a β(2)GPI-dependent manner after binding of β(2)GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β(2)GPI Ab-mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88-dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β(2)GPI Ab-mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β(2)GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β(2)GPI-associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.

Evaluation of Sperm Proteins in Infertile Men: A Proteomic Approach

In this study, the sperm protein profile was compared between fertile and infertile men using 2-dimensional gel electrophoresis, liquid chromatography mass spectrometer analysis, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. Four unique proteins, semenogelin II precursor, prolactin-induced protein, clusterin isoform 1, and prostate-specific antigen isoform 1 preproprotein, were predominantly present in the semen of healthy men; however, semenogelin II precursor and clusterin isoform 1 were not seen in the semen of infertile men, suggesting unique differences in the spermatozoa protein profiles of fertile and infertile men.

Target-Selective Protein S-Nitrosylation by Sequence Motif Recognition

S-nitrosylation is a ubiquitous protein modification emerging as a principal mechanism of nitric oxide (NO)-mediated signal transduction and cell function. S-nitrosylases can use NO synthase (NOS)-derived NO to modify selected cysteines in target proteins. Despite proteomic identification of over a thousand S-nitrosylated proteins, few S-nitrosylases have been identified. Moreover, mechanisms underlying site-selective S-nitrosylation and the potential role of specific sequence motifs remain largely unknown. Here, we describe a stimulus-inducible, heterotrimeric S-nitrosylase complex consisting of inducible NOS (iNOS), S100A8, and S100A9. S100A9 exhibits transnitrosylase activity, shuttling NO from iNOS to the target protein, whereas S100A8 and S100A9 coordinately direct site selection. A family of proteins S-nitrosylated by iNOS-S100A8/A9 were revealed by proteomic analysis. A conserved I/L-X-C-X2-D/E motif was necessary and sufficient for iNOS-S100A8/A9-mediated S-nitrosylation. These results reveal an elusive parallel between protein S-nitrosylation and phosphorylation, namely, stimulus-dependent posttranslational modification of selected targets by primary sequence motif recognition.

Ceramide as a Mediator of Non-Alcoholic Fatty Liver Disease and Associated Atherosclerosis

Cardiovascular disease (CVD) is a serious comorbidity in nonalcoholic fatty liver disease (NAFLD). Since plasma ceramides are increased in NAFLD and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD, the role of ceramides in dyslipidemia was assessed using LDLR-/- mice, a diet-induced model of NAFLD and atherosclerosis. Mice were fed a standard or Western diet (WD), with or without myriocin, an inhibitor of ceramide synthesis. Hepatic and plasma ceramides were profiled and lipid and lipoprotein kinetics were quantified. Hepatic and intestinal expression of genes and proteins involved in insulin, lipid and lipoprotein metabolism were also determined. WD caused hepatic oxidative stress, inflammation, apoptosis, increased hepatic long-chain ceramides associated with apoptosis (C16 and C18) and decreased very-long-chain ceramide C24 involved in insulin signaling. The plasma ratio of ApoB/ApoA1 (proteins of VLDL/LDL and HDL) was increased 2-fold due to increased ApoB production. Myriocin reduced hepatic and plasma ceramides and sphingomyelin, and decreased atherosclerosis, hepatic steatosis, fibrosis, and apoptosis without any effect on oxidative stress. These changes were associated with decreased lipogenesis, ApoB production and increased HDL turnover. Thus, modulation of ceramide synthesis may lead to the development of novel strategies for the treatment of both NAFLD and its associated atherosclerosis.

Proteomic Analysis of Resistance Mediated by Rcm 2.0 and Rcm 5.1, Two Loci Controlling Resistance to Bacterial Canker of Tomato

Two quantitative trait loci from Lycopersicon hirsutum, Rcm 2.0 and Rcm 5.1, control resistance to Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato. Lines containing Rcm 2.0 and Rcm 5.1 and a susceptible control line were compared at 72 and 144 h postinoculation, using 2-dimensional gel electrophoresis to identify proteins regulated in response to C. michiganensis subsp. michiganensis infection. A total of 47 proteins were subjected to tandem mass spectrometry. Database queries with resulting spectra identified tomato genes for 26 proteins. The remaining 21 proteins were either identified in other species or possessed no homology to known proteins. Spectra were interpreted to deduce peptide amino acid sequences that were then used to query publicly available data. This approach identified tomato genes or expressed sequence tags for 44 of the proteins analyzed. Three superoxide dismutase (SOD) enzymes were differentially regulated among genotypes, and patterns of hydrogen peroxide accumulation were genotype- and tissue-specific, indicating a role for oxidative stress in response to C. michiganensis subsp. michiganensis. Steady-state mRNA and protein levels for SOD, thioredoxin M-type, S-adenosylhomocysteine hydrolase, and pathogenesis-related proteins demonstrated similar patterns of differential regulation. Lines containing Rcm 2.0 and Rcm 5.1 accumulate different proteins and steady-state mRNAs in response to inoculation, suggesting that the two loci may confer resistance through distinct mechanisms.

Proteomic analysis of human spermatozoa proteins with oxidative stress

BackgroundOxidative stress plays a key role in the etiology of male infertility. Significant alterations in the sperm proteome are associated with poor semen quality. The aim of the present study was to examine if elevated levels of reactive oxygen species cause an alteration in the proteomic profile of spermatozoa.MethodsThis prospective study consisted of 52 subjects: 32 infertile men and 20 normal donors. Seminal ejaculates were classified as ROS+ or ROS- and evaluated for their proteomic profile. Samples were pooled and subjected to LC-MS/MS analysis through in-solution digestion of proteins for peptide characterization. The expression profile of proteins present in human spermatozoa was examined using proteomic and bioinformatic analysis to elucidate the regulatory pathways of oxidative stress.ResultsOf the 74 proteins identified, 10 proteins with a 2-fold difference were overexpressed and 5 were underexpressed in the ROS+ group; energy metabolism and regulation, carbohydrate metabolic processes such as gluconeogenesis and glycolysis, protein modifications and oxidative stress regulation were some of the metabolic processes affected in ROS+ group.ConclusionsWe have identified proteins involved in a variety of functions associated with response and management of oxidative stress. In the present study we focused on proteins that showed a high degree of differential expression and thus, have a greater impact on the fertilizing potential of the spermatozoa. While proteomic analyses identified the potential biomarkers, further studies through Western Blot are necessary to validate the biomarker status of the proteins in pathological conditions.

Measuring protein synthesis using metabolic 2H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O.

REH2 RNA helicase in kinetoplastid mitochondria: ribonucleoprotein complexes and essential motifs for unwinding and guide RNA (gRNA) binding.

Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3′-terminal maturation, RNA editing, turnover, and translation; however, these processes remain poorly studied. Core editing complexes and their U-insertion/deletion activities are relatively well characterized, and a battery of ancillary factors has recently emerged. This study characterized a novel DExH-box RNA helicase, termed here REH2 (RNA editing associated helicase 2), in unique ribonucleoprotein complexes that exhibit unwinding and guide RNA binding activities, both of which required a double-stranded RNA-binding domain (dsRBD) and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression.

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. DOI: http://dx.doi.org/10.7554/eLife.10067.001

Proteomic analysis of seminal fluid from men exhibiting oxidative stress

BackgroundSeminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men.MethodsThis prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins.ResultsA total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactin-induced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes.ConclusionsWe have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility.