Enrichment of phosphoproteins for proteomic analysis using immobilized Fe(III)-affinity adsorption chromatography

We described an efficient protocol to strongly enrich phosphoproteins from mixtures of total cellular proteins using homemade, recyclable Fe(III)-affinity columns. An integral feature of the method is the use of a detergent cocktail that allows use of different pHs for total protein extraction (pH 6.8) and for subsequent affinity capture of phosphoproteins (pH 3.4). Affinity captured proteins from rat fibroblasts were fractionated on 2D gels and random selection was identified by mass spectrometry. More than 85% of identified proteins were previously known to be phosphorylated. The specificity of the method was further validated by isolating proteins from (32)P labeled cells. Our comparison of the clusters of acidic residues in the captured proteins with acidic clusters in proteins of the rat genome indicates that affinity for phosphate groups dominates over adsorption of proteins with acidic clusters.     

Ultra-high sensitivity multi-photon detection imaging in proteomics analyses

We report on the use of 125I and 131I labeling and of new, multicolor, multi-photon detection (MPD) methods to routinely and quantitatively detect protein spots on two-dimensional gel electrophoresis plates in the zeptomole to attomole range. We demonstrate that the MPD methodology can be used to detect radioactive labels on two-dimensional gels and has several characteristics that are advantageous for functional proteomics. First, by using single particle detectors, the sensitivity for detection of radiolabels can be improved dramatically. Second, because single particle detectors can differentiate the particle energies produced by different decay processes, it is possible to choose combinations of radioisotopes that can be detected and quantified individually on the same 2-D gel. Third, the MPD technology is essentially linear over six to seven orders of magnitude, i.e., it is possible to accurately quantify radiolabeled proteins over a range from at least 60 zeptomoles to 60 femtomoles. Finally for radionuclides that decay by electron capture, e.g., with emission of both beta and gamma rays, co-incident detection of two particles/photons can be used to detect such radionuclides well below background radiation levels. These methods are used to monitor acidic/phosphorylated proteins in as little as 60 ng of HeLa cells proteins.     

Perspectives in spicing up proteomics with splicing

In the post-genomics era there has been an acceleration of understanding of cellular and organismal biology and this acceleration has moved the goalposts for proteomics. Higher eukaryotes use alternative promoters, alternative splicing, RNA editing and post-translational modification to produce multiple isoforms of proteins from single genes. Switching amongst these isoforms is a major mechanism for control of cellular function. At present fundamental limitations in sensitivity, in absolute quantitation of proteins and in the characterization of protein structure at functionally important levels strongly limit the applicability of proteomics to higher eukaryotes. Recent developments suggest that quantitative, top-down proteomics analyses of complete proteins at sub-attomole levels are necessary for physiologically relevant studies of higher eukaryotes. New proteomics technologies which will ensure the future of proteomics as an important technology in medicine and cellular biology of higher eukaryotes are becoming available.     

Tissue zinc dynamics during the immune reaction in mice

Owing to the importance of zinc for the functioning of the immune system, the role of endogenous Zn, located both in lymphoid and nonlymphoid organs, was investigated during the standard humoral and cellular types of immune response. For this purpose, the dynamics of hepatic, thymic, splenic, and renal Zn content was determined in mice sensitized with (a) sheep red blood cells and (b) semiallogeneic lymphocytes during the local host vs graft reaction (HVGR). The data obtained by ion-coupled plasma spectrometry revealed that the humoral type of immunity is characterized by a significant increase of Zn concentration in the liver and in the thymus. Simultaneously, linear regression analysis showed that the generation of plaque-forming cells in the individual mouse was highly positively correlated with Zn concentration in the liver (r = 0.897), and spleen (r = 0.833), and negatively with Zn concentration in the thymus (r = -0.624). Similar relationships between the intensity of local immune reaction and tissue Zn levels were found in local HVGR at the fifth day in the liver and spleen (r = 0.861 andr = 0.695, respectively), at the seventh day in the thymus (r = -0.797), and at the tenth day in the liver (r = -0.859). The data emphasize the necessity of Zn for the development of normal immune response and point to the existence of a Zndependent hepato-thymic axis during the humoral and cellular types of immune reactivity.     

Probing of the porcine serum lipoprotein surfaces by Mn(II) binding: an e.s.r. study

Mn(II) ions were used for probing the surfaces of porcine LDL₁, LDL₂ and HDL. From the intensity of the e.p.r. lines corresponding to the unbound Mn(II) the percentage of the ions bound to the lipoprotein surface is determined. From the titration curves the binding parameters, dissociation constant. K_d, and the number of binding sites, n, in all the three lipoproteins studied have been derived. There are at least two types of binding sites in each lipoprotein class. The ”weak’ binding sites are charaterized by approximately the same value of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(≈ 6.2 × 10}{}^{\mathrm{?3}}\text{mol l}{}^{\mathrm{?1}}$ and different values for n (n = 114 for LDL₁, n = 135 for LDL₂ and n = 28 for HDL). Similarly, for the ”strong’ binding sites K_d ≈ 1.6 × 10^?4 mol l^?1 and the number of binding sites is 15, 20 and 5 for LDL₁, LDL₂ and HDL respectively. It is concluded that the binding sites are probably located in the protein part of the lipoproteins and that they are mainly associated with the negatively charged amino acids.     

Regulatory effects of simvastatin and apoJ on APP processing and amyloid-β clearance in blood-brain barrier endothelial cells

Amyloid-β peptides (Aβ) accumulate in cerebral capillaries indicating a central role of the blood-brain barrier (BBB) in the pathogenesis of Alzheimer's disease (AD). Although a relationship between apolipoprotein-, cholesterol- and Aβ metabolism is evident, the interconnecting mechanisms operating in brain capillary endothelial cells (BCEC) are poorly understood. ApoJ (clusterin) is present in HDL that regulates cholesterol metabolism which is disturbed in AD. ApoJ levels are increased in AD brains and in plasma of cerebral amyloid angiopathy (CAA) patients. ApoJ may bind, prevent fibrillization, and enhance clearance of Aβ. We here define a connection of apoJ and cellular cholesterol homeostasis in amyloid precursor protein (APP) processing/Aβ metabolism at the BBB. Silencing of apoJ in primary porcine (p)BCEC decreased intracellular APP and Aβ oligomer levels while the addition of purified apoJ to pBCEC increased intracellular APP and enhanced Aβ clearance across the pBCEC monolayer. Treatment of pBCEC with Aβ_(1–40) increased expression of apoJ and receptors involved in amyloid transport including lipoprotein receptor-related protein 1 [LRP1]. In accordance, cerebromicrovascular endothelial cells isolated from 3 × Tg AD mice showed elevated expression levels of apoJ and LRP1 as compared to Non-Tg animals. Treatment of pBCEC with HMGCoA-reductase inhibitor simvastatin markedly increased intracellular and secreted apoJ levels, in parallel increased secreted Aβ oligomers and reduced Aβ uptake and cell-associated Aβ oligomers. Simvastatin effects on apoJ, APP processing, and LRP1 expression in BCEC were confirmed in the mouse model. We suggest a close and complex interaction of apoJ, cholesterol homeostasis, and APP/Aβ processing and clearance at the BBB.     

The Unusual Amino Acid Triplet Asn–Ile–Cys Is a Glycosylation Consensus Site in Human α-Lactalbumin

Human -lactalbumin has not been described as a glycoprotein, despite the fact that several -lactalbumins of both ruminant and nonruminant species are known to be glycosylated. In all these species the glycosylation site is the ⁴⁵Asn in the usual triplet ⁴⁵Asn–Gly/Gln–⁴⁷Ser. We have found that human -lactalbumin is glycosylated and the glycosylation site has been determined by protein sequencing and mass spectrometry. We report an unusual glycosylation site at ⁷¹Asn in the triplet ⁷¹Asn–Ile–⁷³Cys, which is conserved in all known -lactalbumins except red-necked wallaby. That a relatively small proportion of the protien is glycosylated (about 1%) may reflect the importance of this region of the protein sequence to the molten globule state of -lactalbumin.     

Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines.

Integrin-mediated cell adhesion cooperates with growth factor receptors in the control of cell proliferation, cell survival, and cell migration. One mechanism to explain these synergistic effects is the ability of integrins to induce phosphorylation of growth factor receptors, for instance the epidermal growth factor (EGF) receptor. Here we define some aspects of the molecular mechanisms regulating integrin-dependent EGF receptor phosphorylation. We show that in the early phases of cell adhesion integrins associate with EGF receptors on the cell membrane in a macromolecular complex including the adaptor protein p130Cas and the c-Src kinase, the latter being required for adhesion-dependent assembly of the macromolecular complex. We also show that the integrin cytoplasmic tail, c-Src kinase, and the p130Cas adaptor protein are required for phosphorylation of EGF receptor in response to integrin-mediated adhesion. We show that integrins induce phosphorylation of EGF receptor on tyrosine residues 845, 1068, 1086, and 1173, but not on residue 1148, a major site of phosphorylation in response to EGF. In addition we find that integrin-mediated adhesion increases the amount of EGF receptor expressed on the cell surface. Therefore these data indicate that integrin-mediated adhesion induces assembly of a macromolecular complex containing c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosine residues.     

Synthesis,anti-bacterial and anti-protozoal activities of amidinobenzimidazole derivatives and their interactions with DNA and RNA

Amidinobenzimidazole derivatives connected to 1-aryl-substituted 1,2,3-triazole through phenoxymethylene linkers 7a–7e, 8a–8e, and 9a–9e were designed and synthesised with the aim of evaluating their anti-bacterial and anti-trypanosomal activities and DNA/RNA binding affinity. Results from anti-bacterial evaluations of antibiotic-resistant pathogenic bacteria revealed that both o-chlorophenyl-1,2,3-triazole and N-isopropylamidine moieties in 8c led to strong inhibitory activity against resistant Gram-positive bacteria, particularly the MRSA strain. Furthermore, the non-substituted amidine and phenyl ring in 7a induced a marked anti-bacterial effect, with potency against ESBL-producing Gram-negative E. coli better than those of the antibiotics ceftazidime and ciprofloxacin. UV–Vis and CD spectroscopy, as well as thermal denaturation assays, indicated that compounds 7a and 8c showed also binding affinities towards ctDNA. Anti-trypanosomal evaluations showed that the p-methoxyphenyl-1,2,3-triazole moiety in 7b and 9b enhanced inhibitory activity against T. brucei, with 8b being more potent than nifurtimox, and having minimal toxicity towards mammalian cells.