A metal-coded affinity tag approach to quantitative proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.     

Genome-wide CRISPR Screens in T Helper Cells Reveal Pervasive Crosstalk between Activation and Differentiation

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Nematodes found in Nile crocodiles in the Kruger National Park,South Africa,with redescriptions of Multicaecum agile (Wedl, 1861) (Heterocheilidae) and Camallanus kaapstaadi Southwell & Kirshner, 1937 (Camallanidae)

Sixteen Nile crocodiles were collected in the Kruger National Park, South Africa and vicinity during 2010 and 2011. A total of 11 nematode species representing six families were recovered. Heterocheilids were the dominant group, comprising five species, with Dujardinascaris madagascariensis (Chabaud & Caballero, 1966) being the most prevalent (75%), followed by Ingwenascaris sprenti Junker & Mutafchiev, 2017 (68.8%), which was also the second most numerous nematode. While less prevalent (31.3%), Typhlophoros kwenae Junker & Mutafchiev, 2017 was the most abundant species. Micropleura huchzermeyeri Junker & Mutafchiev, 2017 (Micropleuridae) was collected from five crocodiles and Crocodylocapillaria sp. (Capillariidae) occurred in a single host. Three nematodes, Camallanus kaapstaadi Southwell & Kirshner, 1937, Spirocamallanus sp. (both Camallanidae) and Ascarophis sp. (Cystidicolidae), are considered accidental infections, likely ingested with the hosts’ prey. Our findings of D. dujardini (Travassos, 1920), D. madagascariensis and Multicaecum agile (Wedl, 1861) in South Africa constitute new geographical records. Crocodylocapillaria sp. represents a new host and geographical record, while T. kwenae, I. sprenti and M. huchzermeyeri have been described as new species during the course of this survey. Multicaecum agile is here redescribed based on light and scanning electron microscopy. Previously undescribed morphological characters of C. kaapstaadi, typically a parasite of Xenopus spp. (Amphibia: Pipidae), but here found in two Nile crocodiles, are also presented.

     

Expression of the Gm1-Species, [NeuN]-GM1, in a Case of Human Glioma

Altered glycosylation is a common feature in tumors of various kind and particular interest has been focused on the expression of tumor-associated gangliosides. We have previously identified some human glioma-associated gangliosides and in this study yet another, not previously described, ganglioside has been isolated. The ganglioside was prepared from human glioma tissue taken at autopsy. The new ganglioside bound cholera-toxin B-subunit and its structure was confirmed by fast atom bombardment—mass spectrometry to be NeuN-GM1 (II³NeuNH₂-GgOse₄Cer). In the dissected tumor specimen, the concentration of NeuN-GM1 was 0.1 mol/g wet weight and accounted for approximately 20% of the monosialoganglioside fraction. Normal human brain tissue specimens (n = 10) did not contain detectable (>0.5 nmol/g wet weight of tissue) amounts of NeuN-GM1, indicating that this ganglioside might be associated with human glioma. However, none of the 17 other tumour specimens reveal any detectable amounts of this ganglioside. In conclusion, NeuN GM1 is a glioma-associated ganglioside but its exceptional expression limits its relevance as a molecule involved in general tumor biology.     

Thioredoxin can influence gene expression by affecting gyrase activity

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.     

Inhibition of basal and interleukin-1-induced VCAM-1 expression by phospholipid hydroperoxide glutathione peroxidase and 15-lipoxygenase in rabbit aortic smooth muscle cells

Cytokines or hydroperoxides upregulate cell adhesion molecules (CAM) in early stages of atherosclerosis. VCAM-1 expression was therefore investigated in rabbit aortic smooth muscle cells (SMC) stably transfected either with phospholipid hydroperoxide glutathione peroxidase (PHGPx; SMCPHGPx) as a hydroperoxide-reducing enzyme or with 15-lipoxygenase (15-LOX; SMCLOX) as a hydroperoxide-producing enzyme. Transfected cells showed up to 3-fold enhanced PHGPx and a marked LOX activity, respectively, that was absent in controls. Intracellular hydroperoxides were 6-fold higher in SMCLOX than in SMC or SMCPHGPx. Intracellular protein thiols were decreased by 50 and 90% in SMCPHGPx and SMCLOX, respectively. Glutathione mixed disulfides were tentatively increased from SMC via SMCPHGPx to SMCLOX, accordingly. Thiol reduction with tris(2-carboxyethyl)phosphine completely restored protein thiols in SMCPHGPx, whereas in SMCLOX only 60% of control values were recovered. Basal VCAM-1 mRNA levels were decreased by 50% in SMCPHGPx and 75% in SMCLOX. VCAM-1-inducibility was abrogated in SMCLOX but not in SMCPHGPx. Accordingly, NFkappaB-driven reporter gene activation by IL-1 was unaffected in SMCPHGPx but abolished in SMCLOX. The data confirm that PHGPx overexpression dampens CAM expression either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein modification. But hydroperoxides, when constitutively overproduced as in SMCLOX, inhibit CAM expression and render cells refractory to IL-1 stimulation likely due to oxidation of protein thiols of the signaling system.