Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry

The nitration of protein tyrosine residues represents an important post-translational modification during development, oxidative stress, and biological aging. To rationalize any physiological changes with such modifications, the actual protein targets of nitration must be identified by proteomic methods. While several studies have used proteomics to screen for 3-nitrotyrosine-containing proteins in vivo, most of these studies have failed to prove nitration unambiguously through the actual localization of 3-nitrotyrosine to specific sequences by mass spectrometry. In this paper we have applied sequential solution isoelectric focusing and SDS-PAGE for the proteomic characterization of specific 3-nitrotyrosine-containing sequences of nitrated target proteins in vivo using nanoelectrospray ionization-tandem mass spectrometry. Specifically, we analyzed proteins from the skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well accepted animal model for biological aging. We identified the 3-nitrotyrosine-containing sequences of 11 proteins, including cytosolic creatine kinase, tropomyosin 1, glyceraldehyde-3-phosphate dehydrogenase, myosin light chain, aldolase A, pyruvate kinase, glycogen phosphorylase, actinin, gamma-actin, ryanodine receptor 3, and neurogenic locus notch homolog. For creatine kinase and neurogenic locus notch homolog, two 3-nitrotyrosine-containing sequences were identified, i.e. at positions 14 and 20 for creatine kinase and at positions 1175 and 1205 for the neurogenic locus notch homolog. The selectivity of the in vivo nitration of creatine kinase at Tyr14 and Tyr20 does not correspond to the product selectivity in vitro, where exclusively Tyr82 was nitrated when creatine kinase was exposed to peroxynitrite. The latter experiments demonstrate that the in vitro exposure of an isolated protein to peroxynitrite may not always be a good model to mimic protein nitration in vivo.     

Mapping the tropomyosin isoform 5 binding site on human erythrocyte tropomodulin: further insights into E-Tmod/TM5 interaction

Actin protofilaments in the erythrocyte membrane skeleton are uniformly approximately 37nm. This length may be in part attributed to a "molecular ruler" made of erythrocyte tropomodulin (E-Tmod) and tropomyosin (TM) isoforms 5 or 5b. We previously mapped the E-Tmod binding site to TM5 N-terminal heptad repeat residues "a" (I(7), I(14)), "d" (V(10)) and "f" (R(12)). We now map the TM5 binding site to E-Tmod residues at L(116), E(117) and/or E(118) by identifying among 35 deletion clones and a series of point mutations that no longer bind to human TM5 and rat TM5b. Upstream residues 71-104 contain an actin binding site. The N-terminal "KRK ring" may participate in balancing electrostatic force with hydrophobic interaction in dimerization of TM and its binding to E-Tmod.     

Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical-physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TMwt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TMKK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TMKK elutes much earlier than TMwt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.     

Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells

We investigated the mechanism of 8-Br-cAMP-mediated phospholipase D (PLD) activation using a primary cell culture system of human endometrial stromal cells (ES cells). PLD activity was increased by the treatment of ES cells with 8-Br-cAMP, maximally at 5 min. To determine whether the effects of 8-Br-cAMP on PLD occurred as a consequence of PKC activation, ES cells were preincubated for 15 min with RO320432 (1 microM) and GF109203X (1 microM), the PKC inhibitors, or they were pretreated for 24h with phorbol myristate acetate (100 nM) to downregulate PKC. However, these treatments had no effects on PLD activation induced by 8-Br-cAMP. Furthermore, 8-Br-cAMP had no effects on the subcellular distribution of PKC alpha and PKC betaI, confirming no involvement of PKC. 8-Br-cAMP activated ERK1/2, maximally at 5 min, and PD98059 (MEK inhibitor: 50 microM) and transfection of ES cells with dominant negative (DN)-MEK completely inhibited 8-Br-cAMP-induced PLD activation, suggesting that ERK1/2 mediates the PLD activation. To investigate the involvement of protein kinase A (PKA), Src, and Ras in 8-Br-cAMP-induced PLD activation, we used PKA inhibitor, H89 and Rp-cAMPs, and transfections of DN-Src and DN-Ras. H-89 and Rp-cAMPs completely blocked 8-Br-cAMP-mediated PLD and ERK activation, implying the involvement of PKA in this PLD activation. In addition, transfection of ES cells with DN-Src, or DN-Ras partially inhibited 8-Br-cAMP-induced ERK1/2 and consequently PLD activation, whereas cotransfection of DN-Src and DN-Ras completely inhibited ERK1/2 and PLD activation, suggesting that Src and Ras independently regulate ERK/PLD activation. Taken together, these results demonstrate a novel pathway in ES cells that 8-Br-cAMP activate PLD through PKA and ERK1/2 and this ERK/PLD activation by 8-Br-cAMP is mediated by Src and Ras, separately.     

Selective activation of adrenergic beta1 receptors induces heme oxygenase 1 production in RAW264.7 cells

We hypothesized that catecholamines through beta-adrenoceptor might modulate macrophage function. We showed that isoproterenol concentration-dependently induced HO-1 production through beta(1)-but not beta(2)-adrenoceptor. Production was increased by forskolin and inhibited by pretreatment with the PKA inhibitor, H-89. Furthermore, induction of HO-1 by isoproterenol effectively protected RAW264.7 cells from effects of glucose oxidase treatment, which was abrogated either by HO-1 inhibitor, ZnPP IX and beta-adenoceptor antagonist, propranolol. Thus, stimulation of HO-1 production through beta(1)-adenoceptors, and via the PKA pathways by isoproterenol, can enable RAW264.7 cells to resist oxidant stress, suggesting that catecholamine hormones may be necessary, at least, to maximize defending role of macrophages.     

Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.     

Annexin A3 is a potential angiogenic mediator

Angiogenesis is a complex process that is regulated by a variety of angiogenic activators and inhibitors. Disruption of the balanced angiogenesis leads to the progress of diseases such as tumor growth, rheumatoid arthritis, and various blood vessel-related disorders. Even though a number of proteins involved in angiogenesis have been identified so far, more protein factors remain to be identified due to complexity of the process. Here we report that annexin A3 (ANXA3) induces migration and tube formation of human umbilical vein endothelial cells. High level of vascular endothelial growth factor (VEGF), a prominent angiogenic factor, is also detected in conditioned medium obtained from cells transfected with ANXA3 expression plasmid. Reporter assays show that ANXA3 enhances hypoxia-inducible factor-1 (HIF-1) transactivation activity. Taken together, our results suggest that ANXA3 is a novel angiogenic factor that induces VEGF production through the HIF-1 pathway.     

Forster resonance energy transfer in liposomes: measurements of transmembrane helix dimerization in the native bilayer environment

The lipid bilayer vesicle is a model of the cellular membrane. Even in this simple system, however, measuring the thermodynamics of membrane protein association is a challenge. Here we discuss Forster resonance energy transfer (FRET) in liposomes as a method to probe the dimerization of transmembrane helices in a membrane environment. Although the measurements are labor intensive, FRET in liposomes can be measured accurately provided that attention is paid to sample homogeneity and sample equilibration. One must also take into account statistical expectations and the FRET that results from random colocalization of donors and acceptors in the bilayer. Without careful attention to these details, misleading results are easy to obtain in membrane FRET experiments. The results that we obtain in model systems are reproducible and depend solely on the concentration of the protein in the bilayer (i.e., on the protein-to-lipid ratio), thereby yielding thermodynamic parameters that are directly relevant to processes in biological membranes.     

Expression of p57 in mouse and human testes

The expression of cyclin-dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.