Evaluating changes in firefighter urinary metabolomes after structural fires: an untargeted, high resolution approach.

Firefighters have elevated rates of urinary tract cancers and other adverse health outcomes, which may be attributable to environmental occupational exposures. Untargeted metabolomics was applied to characterize this suite of environmental exposures and biological changes in response to occupational firefighting. 200 urine samples from 100 firefighters collected at baseline and two to four hours post-fire were analyzed using untargeted liquid-chromatography and high-resolution mass spectrometry. Changes in metabolite abundance after a fire were estimated with fixed effects linear regression, with false discovery rate (FDR) adjustment. Partial least squares discriminant analysis (PLS-DA) was also used, and variable important projection (VIP) scores were extracted. Systemic changes were evaluated using pathway enrichment for highly discriminating metabolites. Metabolome-wide-association-study (MWAS) identified 268 metabolites associated with firefighting activity at FDR q < 0.05. Of these, 20 were annotated with high confidence, including the amino acids taurine, proline, and betaine; the indoles kynurenic acid and indole-3-acetic acid; the known uremic toxins trimethylamine n-oxide and hippuric acid; and the hormone 7a-hydroxytestosterone. Partial least squares discriminant analysis (PLS-DA) additionally implicated choline, cortisol, and other hormones. Significant pathways included metabolism of urea cycle/amino group, alanine and aspartate, aspartate and asparagine, vitamin b3 (nicotinate and nicotinamide), and arginine and proline. Firefighters show a broad metabolic response to fires, including altered excretion of indole compounds and uremic toxins. Implicated pathways and features, particularly uremic toxins, may be important regulators of firefighter's increased risk for urinary tract cancers.

Identification of Mosquito Eggshell Proteins from Aedes aegypti by Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Proteomic Analysis.

The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner.

Tandem Mass Tagging Based Identification of Proteome Signatures for Reductive Stress Cardiomyopathy.

Nuclear factor erythroid 2-related factor 2 (NRF2), a redox sensor, is vital for cellular redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2-TG) exhibit reductive stress (RS). In this study, we identified novel protein signature for RS-induced cardiomyopathy using Tandem Mass Tag (TMT) proteomic analysis in heart tissues of TG (CaNrf2-TG) mice at 6-7 months of age. A total of 1,105 proteins were extracted from 22,544 spectra. About 560 proteins were differentially expressed in TG vs. NTg hearts, indicating a global impact of RS on the myocardial proteome. Over 32 proteins were significantly altered in response to RS -20 were upregulated and 12 were downregulated in the hearts of TG vs. NTg mice, suggesting that these proteins could be putative signatures of RS. Scaffold analysis revealed a clear distinction between TG vs. NTg hearts. The majority of the differentially expressed proteins (DEPs) that were significantly altered in RS mice were found to be involved in stress related pathways such as antioxidants, NADPH, protein quality control, etc. Interestingly, proteins that were involved in mitochondrial respiration, lipophagy and cardiac rhythm were dramatically decreased in TG hearts. Of note, we identified the glutathione family of proteins as the significantly changed subset of the proteome in TG heart. Surprisingly, our comparative analysis of NGS based transcriptome and TMT-proteome indicated that ~50% of the altered proteins in TG myocardium was found to be negatively correlated with their transcript levels. In association with the altered proteome the TG mice displayed pathological cardiac remodeling.

Neutrophil cathepsin G proteolysis of protease-activated receptor 4 generates a novel, functional tethered ligand.

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome.

Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects' DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European-American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.

Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo-electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein--not an mRNA--determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ("CAT tails").

The 3T3-L1 adipocyte glycogen proteome.

BACKGROUND: Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. RESULTS: Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). CONCLUSIONS: These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle.

The atypical histone macroH2A1.2 interacts with HER-2 protein in cancer cells.

Because HER-2 has been demonstrated in the nuclei of cancer cells, we hypothesized that it might interact with transcription factors that activate ERBB2 transcription. Macrohistone 2A1 (H2AFY; mH2A1) was found to interact with HER-2 in cancer cells that overexpress HER-2. Of the two human mH2A1 isoforms, mH2A1.2, but not mH2A1.1, interacted with HER-2 in human cancer cell lines. Overexpression of mH2A1.2, but not mH2A1.1, in cancer cells significantly increased HER-2 expression and tumorigenicity. Inhibition of HER-2 kinase activity diminished mH2A1 expression and mH2A1.2-induced ERBB2 transcription in cancer cells. Chromatin immunoprecipitation of mH2A1.2 in cancer cells stably transfected with mH2A1.2 showed enrichment of mH2A1.2 at the HER-2 promoter, suggesting a role for mH2A1.2 in driving HER-2 overexpression. The evolutionarily conserved macro domain of mH2A1.2 was sufficient for the interaction between HER-2 and mH2A1.2 and for mH2A1.2-induced ERBB2 transcription. Within the macro domain of mH2A1.2, a trinucleotide insertion (-EIS-) sequence not found in mH2A1.1 was essential for the interaction between HER-2 and mH2A1.2 as well as mH2A1.2-induced HER-2 expression and cell proliferation.

Analysis of hepatic glycogen-associated proteins.

Glycogen particles are associated with a population of proteins that mediate its biological functions, including: management of glucose flux into and out of the glycogen particle, maintenance of glycogen structure and regulation of particle size, number, and cellular location. A survey of the glycogen-associated proteome would be predicted to identify the relative representation of known members of this population, and associations with unexpected proteins that have the potential to mediate other functions of the glycogen particle. We therefore purified glycogen particles from both mouse and rat liver, using different techniques, and analyzed the resulting tryptic peptides by MS. We also specifically eluted glycogen-binding proteins from the pellet using malto-oligosaccharides. Comparison of the rat and mouse populations, and analysis of specifically eluted proteins allow some conclusions to be made about the hepatic glycogen sub-proteome. With the exception of glycogen branching enzyme all glycogen metabolic proteins were detected. Novel associations were identified, including ferritin and starch-binding domain protein 1, a protein that contains both a transmembrane endoplasmic reticulum signal peptide and a carbohydrate-binding module. This study therefore provides insight into the organization of the glycogen proteome, identifies other associated proteins and provides a starting point to explore the dynamic nature and cellular distribution of this metabolically important protein population.

The nuclear matrix shell proteome of human epidermis.

BACKGROUND: Proteomic approaches have identified cancer specific biomarker proteins in the nuclear matrix fraction of cancer cells. We wanted to determine whether a similar approach could be used to investigate melanoma biomarkers. OBJECTIVE: Since it was not clear that a nuclear matrix fraction could be isolated from the intact human epidermis, we first wanted to determine whether a nuclear matrix fraction could be isolated from the intact epidermis of human skin. If this was possible, we secondarily wanted to compare the proteome of cultured melanoma and carcinoma cells to that of the intact epidermis. METHODS: We applied two-dimensional electrophoresis (2DGE) and LC/MS/MS to identify proteins isolated in the nuclear matrix shell protein fraction isolated from the human epidermis and from cultured primary skin and cancer cells. RESULTS: A subcellular fractionation of intact epidermis succeeded in yielding a nuclear matrix shell which made up approximately 40% of total tissue protein. Only 5-10% of total cell protein was fractionated in the nuclear matrix shell of cultured skin cells. The nuclear matrix shell of the intact epidermis was distinguishable from cultured keratinocytes or HaCaT cells by expression of keratin 1. The nuclear matrix of the epidermis was distinguishable from melanocytes and melanoma cells by expression of vimentin in melanocyte-derived cells and by expression of desmoplakin in the intact epidermis. CONCLUSION: The nuclear matrix-intermediate filament system can be isolated from the intact human epidermis. A careful examination of the protein composition of this subcellular fraction from the epidermis and skin cancers may identify useful cancer specific biomarkers.

A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go).

When a eukaryotic mRNA sequence specifying an amino acid motif known as 2A is directly followed by a proline codon, two nonoverlapping proteins are synthesized. From earlier work, the second protein is known to start with this proline codon and is not created by proteolysis. Here we identify the C-terminal amino acid of an upstream 2A-encoded product from Perina nuda picorna-like virus that is glycine specified by the last codon of the 2A-encoding sequence. This is an example of recoding where 2A promotes unconventional termination after decoding of the glycine codon and continued translation beginning with the 3' adjacent proline codon.

Human eosinophil major basic protein 2: location of disulfide bonds and free sulfhydryl groups.

Eosinophil granule major basic protein 2 (MBP2 or major basic protein homolog) is a paralog of major basic protein (MBP1) and, similar to MBP1, is cytotoxic and cytostimulatory in vitro. MBP2, a small protein of 13,433 Da molecular weight, contains 10 cysteine residues. Mass spectrometry shows two cystine disulfide linkages (Cys20-Cys115 and Cys92-Cys107) and 6 cysteine residues with free sulfhydryl groups (Cys2, Cys23, Cys42, Cys43, Cys68, and Cys96). MBP2, similar to MBP1, has conserved motifs in common with C-type lectins. The disulfide bond locations are conserved among human MBP1, MBP2 and C-type lectins.

Thioredoxin reductase is required for the inactivation of tumor suppressor p53 and for apoptosis induced by endogenous electrophiles.

Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation and apoptosis were compared and contrasted with the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone-quinone attenuates p53 function in human MCF7 cells.