Disulfide reduction abolishes tissue factor cofactor function

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Biological control of phytopathogenic bacteria Pantoea agglomerans and Erwinia chrysanthemi using 100 essential oils

Plants interact with a wide variety of pathogenic organisms by virtue of their sessile lifestyle. The Pantoea agglomerans and Erwinia chrysanthemi are major plant pathogen amongst them. They are known to cause significant losses in many crop plants. In the present study, bacteria isolated from infected Cajanus cajan and Arachis hypogaea seed are identified by 16S rDNA sequencing as P. agglomerans and E. chrysanthemi, respectively. In vitro antimicrobial properties of 100 essential oils (EOs) were evaluated against P. agglomerans and E. chrysanthemi. The chemical composition of most active EOs was investigated by gas chromatography–mass spectral analysis. The potential properties of these EOs as ecofriendly and economical biocontrol in agriculture is discussed.     

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.     

Identification of a novel IVD mutation in a consanguineous family with isovaleric acidemia

Isovaleric acidemia (IVA) is a rare autosomal recessive disorder caused by a deficiency of isovaleryl-CoA dehydrogenase encoded by IVD gene. In this case study we report the first Saudi IVA patients from a consanguineous family with a novel transversion (p.G362V) and briefly discuss likely phenotype–genotype correlation of the disease in the Saudi population. We explored the functional consequences of the mutation by using various bioinformatics prediction algorithms and discussed the likely mechanism of the disease caused by the mutation.     

Molecular cloning and expression of Hedychium coronarium farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral and wounding/herbivory induced leaf volatile sesquiterpenoids

Farnesyl pyrophosphate synthase (FPPS EC 2.5.1.10) catalyzes the production of farnesyl pyrophosphate (FPP), which is a key precursor for many sesquiterpenoids such as floral scent and defense volatiles against herbivore attack. Here we report a new full-length cDNA encoding farnesyl diphosphate synthase from Hedychium coronarium. The open reading frame for full-length HcFPPS encodes a protein of 356 amino acids, which is 1068 nucleotides long with calculated molecular mass of 40.7 kDa. Phylogenetic tree analysis indicates that HcFPPS belongs to the plant FPPS super-family and has strong relationship with FPPS from Musa acuminata. Expression of the HcFPPS gene in Escherichia coli yielded FPPS activity. Tissue-specific and developmental analyses of the HcFPPS mRNA and corresponding volatile sesquiterpenoid levels in H. coronarium flowers revealed that the HcFPPS might play a regulatory role in floral volatile sesquiterpenoid biosynthesis. The emission of the FPP-derived volatile terpenoid correlates with strong expression of HcFPPS induced by mechanical wounding and Udaspes folus-damage in leaves, which suggests that HcFPPS may have an important ecological function in H. coronarium vegetative organ.     

A Novel Integrated Method Tor Large-scale Detection,Identification, and Quantification of Widely Targeted Metabolites： Application in the Study of Rice Metabolomics

Liquid chromatography-mass spectrometry （LC-MS）-based metabolomics has been facilitated by the con- struction of MSz spectral tag （MS2T） library from the total scan ESI MS/MS data, and the development of widely targeted metabolomics method using MS/MS data gathered from authentic standards. In this report, a novel strategy called step- wise multiple ion monitoring-enhanced product ions （stepwise MIM-EPI） was developed to construct the MS2T library, in which stepwise MIM was used as survey scans to trigger the acquisition of EPI. A total number of 698 （almost） non- redundant metabolites with MS2 spectra were obtained, of which 135 metabolites were identified/annotated. Integrating the data gathered from our MS2T library and other available multiple reaction monitoring （MRM） information, a widely targeted metabolomics method was developed to quantify 277 metabolites, including some phytohormones. Evaluation of the dehydration responses and natural variations of these metabolites in rice leaf not only suggested the coordinated regulation of abscisic acid （ABA） with metabolites such as serotonin derivative（s）, polyamine conjugates under drought stress, but also revealed some C-glycosylated flavones as the potential markers for the discrimination of indica and japonica rice subspecies. The new MS2T library construction and widely targeted metabolomics strategy could be used as a tool for rice functional genomics.     

The byssus of the zebra mussel (Dreissena polymorpha): spatial variations in protein composition

The notorious biofouling organism Dreissena polymorpha (the zebra mussel) attaches to a variety of surfaces using a byssus, a series of protein threads that connect the animal to adhesive plaques secreted onto hard substrata. Here, the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize the composition of different regions of the byssus is reported. All parts of the byssus show mass peaks corresponding to small proteins in the range of 3.7–7 kDa, with distinctive differences between different regions. Indeed, spectra from thread and plaques are almost completely non-overlapping. In addition, several peaks were identified that are unique to the interfacial region of the plaque, and therefore likely represent specialized adhesive proteins. These results indicate a high level of control over the distribution of proteins, presumably with different functions, in the byssus of this freshwater species.     

Proteomics: A Paradigm Shift

ABSTRACT

Proteome—the protein complement of a genome—has become the protein renaissance and a key research tool in the post-genomic era. The basic technology involves the routine usage of gel electrophoresis and spectrometry procedures for deciphering the primary protein sequence/structure as well as knowing certain unique post-translational modifications that a particular protein has undergone to perform a specific function in the cell. However, the recent advancements in protein analysis have ushered this science to provide deeper, bigger and more valuable perspectives regarding performance of subtle protein-protein interactions. Applications of this branch of molecular biology are as vast as the subject is and include clinical diagnostics, pharmaceutical and biotechnological industries. The 21st century hails the use of products, procedures and advancements of this science as finer touches required for the grooming of fast-paced technology.     

Serum proteomic profiling for the early diagnosis of colorectal cancer

No ideal serum biomarker currently exists for the early diagnosis of colorectal cancer (CRC). Magnetic bead‐based fractionation coupled with MALDI‐TOF MS was used to screen serum samples from CRC patients, healthy controls, and other cancer patients. A diagnostic model with five proteomic features (m/z 1778.97, 1866.16, 1934.65, 2022.46, and 4588.53) was generated using Fisher algorithm with best performance. The Fisher‐based model could discriminate CRC patients from the controls with 100% (46/46) sensitivity and 100% (35/35) specificity in the training set, 95.6% (43/45) sensitivity and 83.3% (35/42) specificity in the test set. We further validated the model with 94.4% (254/269) sensitivity and 75.5% (83/110) specificity in the external independent group. In other cancers group, the Fisher‐based model classified 25 of 46 samples (54.3%) as positive and the other 21 as negative. With FT‐ICR‐MS, the proteomic features of m/z 1778.97, 1866.16, 1934.65, and 2022.46, of which intensities decreased significantly in CRC, were identified as fragments of complement C3f. Therefore, the Fisher‐based model containing five proteomic features was able to effectively differentiate CRC patients from healthy controls and other cancers with a high sensitivity and specificity, and may be CRC‐specific. Serum complement C3f, which was significantly decreased in CRC group, may be relevant to the incidence of CRC. J. Cell. Biochem. 114: 448–455, 2013. © 2012 Wiley Periodicals, Inc.