Identification of dynamic proteome changes upon ligand activation of Trk-receptors using two-dimensional fluorescence difference gel electrophoresis and mass spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.     

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn

Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent‐exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.     

Comparison of different approaches for evaluation of the detection and quantitation limits of a purity method: a case study using a capillary isoelectrofocusing method for a monoclonal antibody

Several different techniques suggested by the International Conference on Harmonization (ICH) Q2R1 guideline were used to assess the signal and concentration at the limit of detection (LOD) and limit of quantitation (LOQ) for a purity method. These approaches were exemplified with a capillary isoelectrofocusing (cIEF) method, which has been developed to quantify the distribution of the charge isoforms of a monoclonal antibody. The charge isoforms are the result of incomplete posttranslational processing of C-terminal lysine residues of the heavy chain by carboxypeptidase. Results showed no significant discrepancy between LOD/LOQ obtained by the different techniques. Validation experiments corroborated the calculated LOQ. The results indicate that any single technique can provide meaningful values for the LOD and LOQ. Finally, important points to consider when applying these techniques to purity methods are discussed.     

Photoinactivation of the photosynthetic electron transport chain by accumulation of over-saturating light pulses given to dark adapted pea leaves

The effect of cumulative over-saturating pulses (OSP) of white light (1 s, >10 000 μmol photons m⁻² s⁻¹), applied every 20 min on pea leaves, was investigated during a complete diurnal cycle of 24 h. In dark-adapted leaves, this treatment leads to a progressive decline of the optimum Photosystem II (PS II) quantum yield. Continuous low background light (except far-red light) had a protective effect against this OSP-induced photoinactivation. The lack of far-red effect could be due to its absorption mainly in PS I and not in PS II, but could be also due to the general low absorption in this wavelength region. The photoinactivation was enhanced in leaves that had been previously infiltrated with chloramphenicol. The quantum yield of CO₂ assimilation, but not its maximal capacity, was inhibited by the OSP treatment. The most spectacular effects observed, in addition to an irreversible quenching of Fm, was a strong inhibition of Q_A ⁻ reoxidation revealed by a large increase in the Fs level and consequently by a decrease of ΔF/Fm′. Under such conditions, we observed that the electron flow deduced from ΔF/Fm′ underestimated the real electron flow to CO₂. Time-resolved Chlorophyll a fluorescence measurements showed that the reduced capacity of Q_A ⁻ reoxidation in OSP treated leaves was accompanied by the appearance of a 4.7 ns component attributed to PS II charge recombination. We suggest that a modification at the Q_B site may influence the redox potential of Q_A/Q_A ⁻, facilitating the reversion of the primary charge separation. In addition, a 1.2 ns fluorescence component accumulated, which appeared to be responsible for the underestimation of PS II electron flow. The observed photoinactivation seemed to be different from the photoinhibition often described in the literature, which occurs under continuous light. This revised version was published online in June 2006 with corrections to the Cover Date.     

Application of native-state electrospray mass spectrometry to identify zinc-binding sites on engineered hemoglobin

We report the utility of native-state mass spectrometry to detect zinc ion binding to the engineered hemoglobin rHb52. Various preparations of this recombinant hemoglobin had significantly different oxygen affinities. Detailed characterization of denatured globins did not show any difference between analyzed hemoglobin molecules. However, when solutions of intact hemoglobin pseudotetramers were analyzed by native-state electrospray mass spectrometry, a significant shift in the mass spectrum was observed, indicating labile modification of hemoglobin. Using collision-induced dissociation (CID), we found a mass gain of 63 Da located on the beta-globin. EDTA treatment of modified hemoglobin prior to the infusion removed the modification and restored the predicted oxygen affinity. Ion-trap fragmentation of the +8 charged ion of modified beta-globin showed a neutral loss of 96+/-1 Da, consistent with neutral loss of zinc sulfide. These findings indicated zinc binding to the beta-globin through a cysteine residue. Involvement of Cys93 was confirmed by kinetics of cysteine residue reactivity with dithiodipyridine and peptide mapping. Presence of zinc was confirmed by ICP-MS metal analysis.     

Hypoxia affects root sodium and chloride concentrations and alters water conductance in salt-treated jack pine (<Emphasis Type="Italic">Pinus banksiana</Emphasis>) seedlings

The effects of NaCl were studied in 6-month-old jack pine (Pinus banksiana Lamb.) seedlings growing in solution culture under hypoxic (approximately 2 mg l^у O₂) and well-aerated (approximately 8 mg l^у O₂) conditions. The results showed that hypoxia led to further reduction of stomatal conductance (g_s) in plants treated with 45 mM NaCl. This effect was likely due to a reduction in root hydraulic conductance by both stresses. When applied individually or together, neither 45 mM NaCl nor hypoxia affected cell membrane integrity of needles as measured by tissue electrolyte leakage. Hypoxia did not alter shoot Na⁺ and Cl^m concentrations in NaCl-treated plants. However, root Na⁺ concentrations were lower in NaCl-treated hypoxic plants, suggesting that hypoxia affected the ability of roots to store Na⁺. Hypoxia also induced root electrolyte leakage from NaCl-treated and control plants. The higher root Cl^m concentrations compared with Na⁺ and the positive correlation between root Cl^m concentrations and electrolyte leakage suggest that Cl^m played a major role in salt injury observed in jack pine seedlings. Roots of well-aerated plants treated for 1 week with NaCl contained almost two-fold higher concentration of total non-structural carbohydrates compared with plants from other experimental treatments and these concentrations decreased in subsequent weeks. We suggest that under prolonged hypoxic conditions, roots lose the ability to prevent Cl^m uptake resulting in the increase in root Cl^m concentration, which has damaging effects on root cell membranes.     

Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA

The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis.     

The Molecular Biology Toolkit (MBT): a modular platform for developing molecular visualization applications

Background  

The large amount of data that are currently produced in the biological sciences can no longer be explored and visualized efficiently with traditional, specialized software. Instead, new capabilities are needed that offer flexibility, rapid application development and deployment as standalone applications or available through the Web.     

Thermodynamics of the alpha-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium

Amphipathic alpha-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an alpha-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the alpha-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the alpha-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of DeltaH(helix) approximately -0.7 kcal/mol per residue, an entropy change of DeltaS(helix) approximately -1.9 cal/molK residue and a free energy change of DeltaG(helix)=-0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.     

Rapid Stimulation of an Oxidative Burst during Elicitation of Cultured Plant Cells : Role in Defense and Signal Transduction

Stimulation of cultured plant cells with elicitors of the defense response leads to the rapid destruction of a variety of water-soluble compounds including indoleacetic acid and certain fluorescent dyes. This destructive activity, which is often vigorously manifested within 5 minutes of elicitor addition, is shown to derive from the rapid production of H₂O₂ and its use by extracellular peroxidases. Because of its speed of appearance, this oxidative burst may qualify as the first induced line of defense against invading pathogens. Since H₂O₂ has been implicated as a second messenger of hormone-stimulated metabolic changes in some animal cells, its possible role in transduction of the defense signal in plants was also examined. Not only did exogenous H₂O₂ alone stimulate phytoalexin production in the plant cell suspension, but inhibition of elicitor-stimulated phytoalexin production was observed upon addition of catalase and other inhibitors of the oxidative burst. Furthermore, for inhibition to occur, the presence of catalase was required during elicitor addition, since if introduction of the enzyme was delayed until 1 hour after addition of the elicitor, no inhibition resulted. These results suggest that H₂O₂ also plays an important role in inducing subsequent defense responses such as phytoalexin production.