Proteomic Differences between Tellurite-Sensitive and Tellurite–Resistant E.coli

Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level.In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins.Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control.The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.     

In-gel total protein quantification using a ninhydrin-based method

Precise in-gel quantification of total protein amount of bands or spots in gels is the basis of subsequent biochemical, molecular biological and immunological analyses. Though several methods have been designed to evaluate relative amounts of proteins, these methods are of limited reliability because (semi-) quantifications depend on the amount of protein migrating into the gel and different proteins may lead to different absorptions/intensities of stained bands or spots. In the present study, we described a method to quantify both, hydrophilic and hydrophobic proteins using in-gel digestion with proteinase K, subsequent extraction and acid hydrolysis followed by the use of the ninhydrin reaction. The protocol is accurate and compatible with mass spectrometric characterization of proteins. Reproducible in-gel protein quantification was performed from SDS-PAGE and IEF/SDS-PAGE gels using bovine serum albumin as a standard protein. Bacteriorhodopsin separated on SDS-PAGE gel was quantified in addition in order to show that the method is also suitable for quantification of hydrophobic protein. This protocol for reliable in-gel protein quantification, which not only provides “arbitrary units of optical density”, can also be completed in a minimum of 4 days or maximum 1 week depending on the type of electrophoresis with the disadvantage of being time consuming.     

Manufacturing of peptides exhibiting biological activity

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87–95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171–182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17–24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551–1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.     

Increased kynurenic acid in the brain after neonatal asphyxia

In the brain, L-kynurenine is an intermediate for the formation of kynurenic acid, a metabolite with neuroprotective activities, and a substrate for the synthesis of 3-hydroxy-kynurenine, a metabolite with neurotoxic properties. In the present study, alterations of L-kynurenine, 3-hydroxy-kynurenine and kynurenic acid levels were examined in the brain of neonatal (10 minutes old) rats after 5, 10, 15 or 20 minutes of asphyxia, and in the brain of the corresponding caesarean-delivered controls, using sensitive high-performance liquid chromatographic methods. Among kynurenines we found a marked time-dependent increase of kynurenic acid levels, a moderately delayed increase of 3-hydroxy-kynurenine, and a trend for a decrease of L-kynurenine content. Thus, the brain reacted rapidly to the oxygen deficit by increasing kynurenic acid levels by 44% already after 5 minutes of asphyxia, and the most prominent elevation of kynurenic acid (302% of control) was found after 20 minutes of asphyxia--the critical time limit of survival.     

Derangement of Hypothetical Proteins in Fetal Down's Syndrome Brain

The success of the Human Genome Project (HGP) enables prediction of proteins by computer programs from nucleic acid sequences and for which there is no experimental evidence. Clues for function of hypothetical proteins are provided by sequence similarity with proteins of known function in model organisms. The availability of this bulk of new data is of immediate importance to Down's syndrome (DS) research. DS is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and is characterized by somatic anomalies and mental retardation. In addition, overexpression of chromosome 21 genes is directly or indirectly responsible for mental retardation and other phenotypic abnormalities of DS. To allow insight into how trisomy 21 represents the phenotype of DS, we constructed a two-dimensional protein map and investigated expression of 8 hypothetical proteins in fetal DS (n = 7) and control (n = 7) brains (cortex). Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption/ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Quantitative analysis of hypothetical protein FLJ10849, hypothetical protein FLJ20113, and activator of hsp90 ATPase homologue 1 (AHA1) revealed levels comparable between DS and controls. By contrast, expression levels of hypothetical protein KIAA1185, hypothetical protein 55.2 kDa, hypothetical protein 58.8 kDa, actin-related protein 3beta (ARP3beta), and putative GTP-binding protein PTD004 were significantly decreased (P < 0.05) in fetal DS brain, and domain analysis suggests involvement in cytoskeleton, signaling, and chaperone system abnormalities.     

Regulation of PCNA and CAF-1 expression by the two tuberous sclerosis gene products

Tuberous sclerosis is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 individuals. Two genes have been shown to be responsible for this disease: TSC1, encoding hamartin and TSC, encoding tuberin. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. In this study, we performed two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after overexpression of TSC1 or TSC2. We found expression of PCNA and the p48 subunit of CAF-1 to be regulated by two tuberous sclerosis gene products. CAF-1 and PCNA interact as major regulators of chromatin assembly during DNA repair. We suggest that deregulation of the control of chromatin assembly might contribute to development of tumors in tuberous sclerosis patients and provide important new insights into the molecular development, especially since deregulation of chromatin assembly and DNA repair results in genomic instability, a hallmark of tumor development.     

Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.     

Reduction of actin-related protein complex 2/3 in fetal Down syndrome brain

Down syndrome (DS) patients present with morphological abnormalities in brain development, leading to mental retardation. Given the importance of actin cytoskeleton to form the basis of various cell functions, the regulation of actin system is crucial during brain development. We therefore aimed to study the expression levels of actin binding proteins in fetal DS and control cortex. We evaluated the levels of eight actin binding proteins using the proteomic approach of two-dimensional gel electrophoresis with subsequent mass spectroscopical identification of protein spots. In fetal DS brain we found a significant reduction of the actin-related protein complex 2/3 (Arp2/3) 20 kDa subunit and the coronin-like protein p57, which are involved in actin filament cross-linking and nucleation and capping of actin filaments. We conclude that deficient levels of these proteins may, at least partially, be involved in the dysgenesis of the brain in DS.     

Protein levels of genes encoded on chromosome 21 in fetal Down syndrome brain: challenging the gene dosage effect hypothesis (Part I)

Summary.  Down syndrome (DS) is the most significant genetic disorder with mental retardation and is caused by trisomy 21. The phenotype of DS is thought to result from overexpression of a gene(s) located on the triplicated chromosome (region). An increasing body of evidence that challenge this “gene dosage effect” hypothesis, however, has been reported indicating that this hypothesis still remains to be elucidated. The availability of the complete sequence of genes on chromosome 21 could have an immediate impact on DS research, but no conclusions can be drawn from nucleic acid levels. This made us evaluate protein levels of six proteins, gene products, encoded on chromosome 21 (T-cell lymphoma invasion and metastasis inducing Tiam1 protein, holocarboxylase synthetase, human interferon-regulated resistance GTP-binding protein MxA, Pbx regulating protein 1, autoimmune regulator, and pericentrin) in fetal cortex from DS and controls at 18–19 weeks of gestational age using Western blot technique. None of the investigated proteins showed overexpression in DS compared to controls. Our present data showing unaltered expression of six proteins on chromosome 21 in fetal DS brain suggest that the existence of the trisomic state is not involved in abnormal development of fetal DS brain and that the gene dosage effect hypothesis is not sufficient to fully explain the DS phenotype. We are in the process of quantifying all gene products of chromosome 21 and our first results do not support the gene dosage hypothesis. Received June 27, 2002 Accepted July 19, 2002 Published online November 14, 2002 Authors' address: Prof. Dr. Gert Lubec, CChem, FRSC (UK), Department of Pediatrics, University of Vienna, Waehringer Guertel 18, A-1090 Vienna, Austria, Fax: +43-1-40400-3194, E-mail: gert.lubec@akh-wien.ac.at Abbreviations: AIRE, autoimmune regulator; DS, Down syndrome; HCS, holocarboxylase synthetase; Prep1, Pbx regulating protein 1; Tiam1, T-cell lymphoma invasion and metastasis 1