The oxidation of HSP70 is associated with functional impairment and lack of stimulatory capacity.

Expression of intracellular HSP70 is associated with cytoprotective effects against a wide range of stressful stimuli, such as inflammation, oxidative stress, hypoxia, endotoxins, infections, and fever. This cytoprotective effect is mainly attributed to their ability to stabilize protein structures through chaperone-like reversible interactions. HSP70 was recently detected in the extracellular medium, and its presence in serum is commonly associated with pathological situations, where it exerts modulatory effects on cells of the immune system. Previously, we have described the relationship between serum HSP70 levels, oxidant status, and clinical outcome of septic patients; the group of patients with higher prooxidant status and higher serum HSP70 had also higher mortality. To investigate the possible association between oxidized HSP70 and cytoprotection or cell death, we incubated RAW 264.7 macrophages with oxidized HSP70 and evaluated nitrite production, cell proliferation, cell viability, TNF-α release, and phagocytic activity. We also evaluated structural modifications caused by oxidation in purified HSP70. Oxidation of HSP70 altered its protein structure; besides, the modulatory effect of oxidized HSP70 on RAW264.7 cells was different from that of native HSP70. Macrophages treated with oxidized HSP70 presented lower proliferation and viability, lower phagocytic activity, and lower TNF-α release. These results indicate that oxidation of extracellular HSP70 modified its signaling properties, causing alterations on its modulatory effects on macrophage function and viability.

Alzheimer's and Parkinson's diseases: an environmental proteomic point of view.

Alzheimer's and Parkinson's diseases are severe neurodegenerative conditions triggered by complex biochemical routes. Many groups are currently pursuing the search for valuable biomarkers to either perform early diagnostic or to follow the disease's progress. Several studies have reported relevant findings regarding environmental issues and the progression of such diseases. Here the etiology and mechanisms of these diseases are briefly reviewed. Approaches that might reveal candidate biomarkers and environmental stressors associated to the diseases were analyzed under a proteomic perspective. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Quercetin promotes glioma growth in a rat model.

We have previously demonstrated that quercetin (Quer), a polyphenol widely found in vegetables, decreased glioma cell growth in vitro. Here, we asked whether this compound could affect glioma growth in an in vivo rat glioma model. We found that daily intraperitoneal Quer (50 mg/kg) injections lead to a concentration of 0.15 µg of Quer per gram of brain tissue, which increased the tumor volume in a time dependent manner. We observed a small reduction in lymphocytic infiltration, a marker of good prognosis in gliomas that was accompanied by a small reduction in cell viability of peripheral T-cells. Moreover, after Quer treatment neither body weight alteration nor liver pathology markers were detected. Although in vitro studies and massive literature reports point to the antitumoral properties of Quer, the present results indicate that great caution has to be taken in the design of clinical trials and the indiscriminate use of this polyphenol as dietary supplement.

Global quantitative proteomics using spectral counting: an inexpensive experimental and bioinformatics workflow for deep proteome coverage.

As the field of proteomics shifts from qualitative identification of protein "subfractions" to quantitative comparison of proteins from complex biological samples, it is apparent that the number of approaches for quantitation can be daunting for the result-oriented biologist. There have been many recent reviews on quantitative proteomic approaches, discussing the strengths and limitations of each. Unfortunately, there are few detailed methodological descriptions of any one of these quantitative approaches. Here we present a detailed bioinformatics workflow for one of the simplest, most pervasive quantitative approach-spectral counting. The informatics and statistical workflow detailed here includes newly available freeware, such as SePro and PatternLab which post-process data according to false discovery rate parameters, and statistically model the data to detect differences and trends.

An overview of proteomics approaches applied to biopharmaceuticals and cyclotides research.

The evolution in proteomics approaches is notable, including quantitative proteomics and strategies for elucidation of post-translational modifications. Faster and more accurate mass spectrometers as well as cleverer bioinformatics tolls are making the difference in such advancement. Among the wide range of research in plant proteomics, biopharmaceutical production using plants as "biofactories" and the screening of new activities of new molecules, in this case, peptides, are quite important regarding translational proteomics. The present review is focused on "recombinant proteins and bioactive peptides", with biopharmaceuticals and cyclotides chosen as examples. Their application and challenges are focused on a "translational proteomics" point of view, in order to exemplify some new areas of research based on proteomics strategies. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Characterization of JBURE-IIb isoform of Canavalia ensiformis (L.) DC urease.

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3' and 5' ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.

Proteome databases and other online resources for chloroplast research in Arabidopsis.

Proteomics aimed at addressing sub cellular fractions, such as chloroplasts, are a complex challenge. In the past few years, several studies in different laboratories have identified and, more recently, quantified, thousands of proteins within whole chloroplasts or chloroplast fractions. A considerable number of these studies are available for querying, using online resources, such as databases containing the proteins identified, encoding genes, acquired spectra, and phosphopeptides. The main purpose of this review is to identity and highlight useful features of these online resourses, mainly focused in proteomics databases related to chloroplast research in Arabidopsis thaliana. Several web sites were consulted. Among them, 11 were selected and discussed herein. The databases were classified into Plastid Databases, General Organelle Proteome Databases, and General Arabidopsis Proteome Databases. Special care was taken to present information regarding protein identification, protein quantification, and data integration. A selected list of online resources is presented in two tables. The databases analyzed are a useful source of information for researchers in the plastid organelle and plant proteomics fields.

Proteomic comparison of plastids from developing embryos and leaves of Brassica napus.

Plastids are highly specialized organelles, responsible for photosynthesis and biosynthesis of various phytochemicals. To better understand plastid diversity and metabolism, a quantitative proteomic study of two plastid forms from Brassica napus (oilseed rape) was performed. Plastids were isolated from leaves (chloroplasts) of two-week-old plants and developing embryos (embryoplasts) three-weeks after flowering, using an approach avoiding protein storage vacuole contamination. Proteins from five different plastid preparations were prefractionated by SDS-PAGE and sectioned into multiple bands, and in-gel proteins were subjected to trypsin digestion. Tryptic peptides from each band were eluted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectra were searched against a comprehensive plant database. Proteins were quantified based on MS/MS spectral counting of unique, nonhomologous peptides. Functional classification and quantitative comparison of over 2000 redundant proteins (compiled to 675 nonredundant proteins) determined that light reaction proteins are more prominent in chloroplasts, while many Calvin cycle enzymes are more prominent in embryoplasts. Embryoplasts also contain a diversity of other metabolic enzymes undetected in chloroplasts. Many enzymes involved in de novo fatty acid and amino acid biosynthesis were detected in embryoplasts but not chloroplasts. Additionally, protein synthesis-related proteins were prominent in embryoplasts. Collectively, these results indicate that these two plastid types are distinct.

Global and targeted proteomics in developing jack bean (Canavalia ensiformis) seedlings: an investigation of urease isoforms mobilization in early stages of development.

Jack bean (Canavalia ensiformis) seeds are toxic for insects and the toxicity is due in part to an entomotoxic peptide enzymatically released from ureases in the midgut of susceptible insects. To characterize expression of urease isoforms in jack bean seed, particularly the more abundant urease isoform (JBU), quantitative proteomics was performed. Quiescent through 5-day germinating seeds were analyzed at 1-day intervals using a total proteomics approach (TPA) and also after co-immunoprecipitation (co-IP) with anti-JBU monoclonal antibodies. Jack bean proteins for TPA and co-IP were pre-fractionated by SDS-PAGE, segmented for in-gel trypsin digestion, and analyzed by liquid chromatography coupled to nanospray ionization tandem mass spectrometry (LC-MS/MS). Acquired MS(2) data were searched against a comprehensive plant database and the MEROPS peptidase database, in the absence of a jack bean EST database. Proteins detected in TPA were quantified by label-free spectral counting. A total of 234 and 106 non-redundant proteins were detected in TPA and co-IP, respectively. Mobilization of JBU was observed beginning 3-days after imbibition indicating that the entomotoxic peptide was not formed before this stage. A predicted urease isoform, JBURE-IIb, was detected in the co-IP study. Additionally, 46 plastid proteins, including RuBisCO and plastid ATPase were pulled down with JBU antibodies. These data shed new light on the behavior of urease isoforms during the early stages of plant development.

A comparative study of the expression of serine proteinases in quiescent seeds and in developing Canavalia ensiformis plants.

An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.