Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.     

Role of lipid droplet proteins in liver steatosis

Five proteins of the perilipin (Plin) family such as Plin1 (perilipin) Plin2 (adipose differentiation-related protein), Plin3 (tail-interacting protein of 47 kDa), Plin4 (S3-12), and Plin5 (myocardial lipid droplet protein) are characterized as lipid droplet (LD) proteins in adipocytes. Recent reports have demonstrated that fat-specific protein 27 (FSP27) and hypoxia-inducible protein 2 (HIG2) are also thought to be novel LD proteins in addition to proteins of the Plin family. Growing evidence have shown that LD proteins play a role in the pathophysiology in the fatty liver disease which is characterized by hepatocytes containing LD with excessive neutral lipid. Studies showed LD proteins such as Plin1, Plin2, Plin3, Plin5, FSP27, and HIG2 are expressed in the liver steatosis. Among them, a high fat diet increases expression of Plin2 and/or FSP27 through activation of peroxisome proliferator-activated receptor γ to develop fatty liver. In this article, recent advances on the role of LD proteins in pathophysiology of fatty liver diseases are summarized.     

Analysis of the role of the Shc and Grb2 proteins in signal transduction by the v-ErbB protein.

The epidermal growth factor receptor, EGFR, has been implicated in cell transformation in both mammalian and avian species. The v-ErbB oncoprotein is an oncogenic form of the chicken EGFR. The tyrosine kinase activity of this oncoprotein is required for transformation, but no transformation-specific cellular substrates have been described to date. Recently activation of the ras signal transduction pathway by the EGFR has been shown to involve the Shc and Grb2 proteins. In this communication, we demonstrate that the Shc proteins are phosphorylated on tyrosine residues and are complexed with Grb2 and the chicken EGFR following ligand activation of this receptor. In fibroblasts and erythroid cells transformed by the avian erythroblastosis virus (AEV) strains H and ES4, the Shc proteins are found to be constitutively phosphorylated on tyrosine residues. The tyrosine-phosphorylated forms of the AEV strain H v-ErbB protein are found in a complex with Shc and Grb2, but the Shc proteins do not bind to the AEV strain ES4 v-ErbB protein. Mutant forms of the v-ErbB protein (in which several of the tyrosines that become autophosphorylated have been deleted by truncation) are unable to transform erythroid cells but can still transform fibroblasts. Analysis of cells transformed by one of these mutants revealed that the truncated v-ErbB protein could no longer bind to either Shc or Grb2, but this oncoprotein still gave rise to tyrosine-phosphorylated Shc proteins that complexed with Grb2 and led to activation of mitogen-activated protein (MAP) kinase. The results suggest that stable binding of Grb2 and Shc to the v-ErbB protein is not necessary to activate this signal transduction pathway and assuming that the mutant activate MAP kinase in erythroid cells in a manner similar to that of fibroblasts, that activation of this pathway is not sufficient to transform erythroid cells.     

Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.     

Reactive oxygen species and protein kinases modulate the level of phospho-MEK-like proteins during human sperm capacitation

Capacitation is an essential process by which spermatozoa acquire fertilizing ability. Reactive oxygen species (ROS), protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases (PTKs), and the extracellular signal-regulated protein kinase (ERK or mitogen-activated protein kinase [MAPK]) pathway regulate sperm capacitation. Our aim was to evaluate the phosphorylation of MEK (MAPK kinase or MAP2K) or MEK-like proteins in human sperm capacitation and its modulation by ROS and kinases. Immunoblotting using an anti-phospho-MEK antibody indicated that the phosphorylation of three protein bands (55, 94, and 115 kDa) increased in spermatozoa treated with fetal cord serum ultrafiltrate (FCSu), BSA, or isobutylmethylxanthine plus dibutyryl cAMP as capacitating agents. These phospho-MEK-like proteins are localized along the sperm flagellum. The MEK-inhibitors PD98059 and U126 prevented this phosphorylation, suggesting that these proteins are MEK-like proteins. The ROS scavengers prevented, and the addition of H(2)O(2) or spermine-NONOate (nitric oxide donor) triggered, the increase of phospho-MEK-like proteins. The capacitation-related increases in phospho-MEK-like proteins induced by FCSu, H(2)O(2), and spermine-NONOate were similarly modulated by PKA, PKC, and PTK, suggesting ROS as mediators in this phenomenon. These results indicate that phospho-MEK-like proteins are modulated by ROS and kinases and probably represent an intermediary step between the early events and the late tyrosine phosphorylation associated with capacitation.     

Phosphorylation of smg p21, a ras p21-like GTP-binding protein, by cyclic AMP-dependent protein kinase in a cell-free system and in response to prostaglandin E1 in intact human platelets

We have separated multiple small Mr GTP-binding proteins (G proteins) from bovine brain membranes by several column chromatographies and purified to near homogeneity four of them, including a novel Mr 24,000 G protein (smg p25A), a novel Mr 22,000 G protein (smg p21), the rho protein (rho p20), and the c-Ki-ras protein (c-Ki-ras p21). Among these small Mr G proteins, only smg p21 is phosphorylated stoichiometrically by cAMP-dependent protein kinase (protein kinase A), and c-Ki-ras p21 is phosphorylated to a small extent by protein kinase A in a cell-free system. None of smg p25A, rho p20, and other partially purified small Mr G proteins is phosphorylated by protein kinase A. Neither smg p21 nor other small Mr G proteins are phosphorylated by protein kinase C. About 1 mol of phosphate is maximally incorporated into 1 mol of smg p21 by protein kinase A. Only serine residue(s) are phosphorylated. The guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-bound and GDP-bound forms of smg p21 are phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affects neither its GTP gamma S-binding nor GTPase activity. smg p21 is found in human platelets, and this human platelet smg p21 is also phosphorylated by protein kinase A at the same site(s) as bovine brain smg p21 in a cell-free system. When intact human platelets are stimulated by prostaglandin E1 known to elevate the cAMP level, four proteins with apparent Mr values of 240,000, 50,000, 24,000, and 22,000 are phosphorylated. These four proteins are also phosphorylated by the action of dibutyryl cAMP but not by the action of thrombin, Ca2+ ionophore A23187, or 12-O-tetradecanoylphorbol-13-acetate. Among the four proteins, the Mr 22,000 protein is identified as smg p21. The site(s) of phosphorylation of smg p21 by protein kinase A in a cell-free system are identical to that phosphorylated in response to prostaglandin E1 in intact platelets. These results indicate that among many small Mr G proteins, smg p21 is selectively phosphorylated by protein kinase A and that this G protein is also phosphorylated by this protein kinase in response to prostaglandin E1 in intact human platelets.     

Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry

Two-dimensional electrophoresis (2-DE) is a highly resolving technique for arraying proteins by isoelectric point and molecular mass. To date, the resolving ability of 2-DE for protein separation is unsurpassed, thus ensuring its use as the fundamental separation method for proteomics. When immobilized pH gradients (IPGs) are used for isoelectric focusing in the first dimension, excellent reproducibility and high protein load capacity can be achieved. While this has been beneficial for separations of soluble and mildly hydrophobic proteins, separations of membrane proteins and other hydrophobic proteins with IPGs have often been poor. Stimulated by the growing interest in proteomics, recent developments in 2-DE methodology have been aimed at rectifying this situation. Improvements have been made in the area of protein solubilization and sample fractionation, leading to a revamp of traditional approaches for 2-DE of membrane proteins. This review explores these developments.     

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.     

Identification of a family of endocytic proteins that define a new alpha-adaptin ear-binding motif

Endocytosis by clathrin-coated vesicles (CCVs) is an important mechanism mediating protein internalization. Here, we show that two proteins identified through a proteomics analysis of CCVs are new components of the endocytic machinery. The proteins, named NECAP (adaptin-ear-binding coat-associated protein) 1 and 2, are paralogues that display no sequence similarity or common domains with any known protein. Both are enriched in CCV coats, and further analysis of the brain-enriched isoform, NECAP 1, shows its partial localization to clathrin-coated pits and direct binding to the globular ear domain of the α-adaptin subunit (α-ear) of the adaptor protein 2 (AP-2) complex. Intriguingly, this interaction is mediated by a new motif, WVQF, that uses a distinct α-ear interface relative to known α-ear-binding partners. Disruption of this interaction blocks clathrin-mediated endocytosis. Together, our studies identify a new family of endocytic proteins that define a unique AP-2-binding motif.     

Mechanisms of chaperone-mediated autophagy

Chaperone-mediated autophagy is one of several lysosomal pathways of proteolysis. This pathway is activated by physiological stresses such as prolonged starvation. Cytosolic proteins with particular peptide sequence motifs are recognized by a complex of molecular chaperones and delivered to lysosomes. No vesicular traffic is required for this protein degradation pathway, so it differs from microautophagy and macroautophagy. Protein substrates bind to a receptor in the lysosomal membrane, the lysosome-associated membrane protein (lamp) type 2a. Levels of lamp2a in the lysosomal membrane are controlled by alterations in the lamp2a half-life as well as by the dynamic distribution of the protein between the lysosomal membrane and the lumen. Substrate proteins are unfolded before transport into the lysosome lumen, and the transport of substrate proteins requires a molecular chaperone within the lysosomal lumen. The exact roles of this lysosomal chaperone remain to be defined. The mechanisms of chaperone-mediated autophagy are similar to mechanisms of protein import into mitochondria, chloroplasts, and the endoplasmic reticulum.     

High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.     

Biochemical characterization of a truncated penta-EF-hand Ca2+ binding protein from maize

Plants possess multiple genes encoding calcium sensor proteins that are members of the penta-EF-hand (PEF) family. Characterized PEF proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit function in diverse cellular processes in a calcium-dependent manner by interacting with their target proteins at either their N-terminal extension or Ca2+ binding domains. We have identified a previously unreported class of PEF proteins in plants that are notable because they do not possess the hydrophobic amino acid rich N-terminal extension that is typical of these PEF proteins. We demonstrate that the maize PEF protein without the N-terminal extension has the characteristics of known PEF proteins; the protein binds calcium in the 100 nM range and, as a result of calcium binding, displays an increase in hydrophobicity. Characterization of the truncated maize PEF protein provides insights into the role of the N-terminal extension in PEF protein signaling. In the context of the current model of how PEF proteins are activated by calcium binding, these results demonstrate that this distinctive class of PEF proteins could function as calcium sensor proteins in plants even in the absence of the N-terminal extension.     

Exchange and stability of HeLa ribosomal proteins in vivo.

The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.     

The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid.

The preS/S coding region of hepatitis B virus encodes two polypeptides (preS1 and preS2) that are larger in size but less abundant than the major viral surface antigen (S) protein. Unlike the preS2 and S proteins, the preS1 protein is preferentially localized on circulating virus particles but is not efficiently secreted from mammalian cells in culture. To search for differences in protein processing that might relate to these properties, we determined whether any of the hepatitis B virus surface proteins are acylated with long-chain fatty acids. Transfected COS cells expressing all three proteins were incubated with 3H-palmitate or 3H-myristate, and the cell extracts were examined by immunoprecipitation. While none of these proteins was labeled with 3H-palmitate, the preS1 protein but not the preS2 or S protein incorporated 3H-myristate via a hydroxylamine-resistant amide linkage. Comparison of the N-terminal amino acid sequences of hepadnaviral preS1 proteins with those of known myristylated proteins suggests that this unusual modification may be a common feature of all hepadnaviral preS1 proteins.     

Lipocortins are major substrates for protein kinase C in extracts of human neutrophils.

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.     

Specificity of binding of NH2-terminal residue of proteins to ubiquitin-protein ligase. Use of amino acid derivatives to characterize specific binding sites

Previous studies have indicated that at least part of the selection of proteins for degradation takes place at a binding site on ubiquitin-protein ligase, to which the protein substrate is bound prior to ligation to ubiquitin. It was also shown that proteins with free NH2-terminal alpha-NH2 groups bind better to this site than proteins with blocked NH2 termini (Hershko, A., Heller, H., Eytan, E., and Reiss, Y. (1986) J. Biol. Chem. 261, 11992-11999). In the present study, we used simple derivatives of amino acids, such as methyl esters, hydroxamates, or dipeptides, to examine the question of whether the protein binding site of the ligase is able to distinguish between different NH2-terminal residues of proteins. Based on specific patterns of inhibition of the binding to ligase by these derivatives, three types of protein substrates could be distinguished. Type I substrates are proteins that have a basic NH2-terminal residue (such as ribonuclease and lysozyme); these are specifically inhibited by derivatives of the 3 basic amino acids (His, Arg, and Lys) with respect to degradation, ligation to ubiquitin, and binding to ligase. Type II substrates (such as beta-lactoglobulin or pepsinogen, that have a Leu residue at the NH2 terminus) are not affected by the above compounds, but are specifically inhibited by derivatives of bulky hydrophobic amino acids (Leu, Trp, Phe, and Tyr). In these cases, the amino acid derivatives apparently act as specific inhibitors of the binding of the NH2-terminal residue of proteins, as indicated by the following observations: (a) derivatives in which the alpha-NH2 group is blocked were inactive and (b) in dipeptides, the inhibitory amino acid residue had to be at the NH2-terminal position. An additional class (Type III) of substrates comprises proteins that have neither basic nor bulky hydrophobic NH2-terminal amino acid residues; the binding of these proteins is not inhibited by homologous amino acid derivatives that have NH2-terminal residues similar to that of the protein. It is concluded that Type I and Type II proteins bind to distinct and separate subsites of the ligase, specific for basic or bulky hydrophobic NH2-terminal residues, respectively. On the other hand, Type III proteins apparently predominantly interact with the ligase at regions of the protein molecule other than the NH2-terminal residue.     

Interacting domains of the HN and F proteins of newcastle disease virus

The activation of most paramyxovirus fusion proteins (F proteins) requires not only cleavage of F(0) to F(1) and F(2) but also coexpression of the homologous attachment protein, hemagglutinin-neuraminidase (HN) or hemagglutinin (H). The type specificity requirement for HN or H protein coexpression strongly suggests that an interaction between HN and F proteins is required for fusion, and studies of chimeric HN proteins have implicated the membrane-proximal ectodomain in this interaction. Using biotin-labeled peptides with sequences of the Newcastle disease virus (NDV) F protein heptad repeat 2 (HR2) domain, we detected a specific interaction with amino acids 124 to 152 from the NDV HN protein. Biotin-labeled HR2 peptides bound to glutathione S-transferase (GST) fusion proteins containing these HN protein sequences but not to GST or to GST containing HN protein sequences corresponding to amino acids 49 to 118. To verify the functional significance of the interaction, two point mutations in the HN protein gene, I133L and L140A, were made individually by site-specific mutagenesis to produce two mutant proteins. These mutations inhibited the fusion promotion activities of the proteins without significantly affecting their surface expression, attachment activities, or neuraminidase activities. Furthermore, these changes in the sequence of amino acids 124 to 152 in the GST-HN fusion protein that bound HR2 peptides affected the binding of the peptides. These results are consistent with the hypothesis that HN protein binds to the F protein HR2 domain, an interaction important for the fusion promotion activity of the HN protein.     

Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary fibrosis

Oxygen-derived free radicals produced by phagocytes have been postulated to contribute to lung tissue damage occurring during diffuse lung diseases (DLD). The two-dimensional electrophoretic (2-DE) analysis of bronchoalveolar lavage (BAL) protein composition revealed different protein profiles in sarcoidosis (S), idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) with a significant increase of low molecular weight proteins in IPF. Some of these proteins are involved in antioxidant processes. The aims of this report were to analyse the oxidative stress occurring in patients with DLD through determination of BAL protein carbonyl content and to identify target proteins of oxidation by a proteomic approach (2-DE combined with immunoblotting with specific antibodies for carbonyl groups). Carbonylated proteins detected by enzyme-linked immunosorbent assay (ELISA) were increased in BAL of patients with S, IPF and SSc compared to healthy controls with a significant difference for S and IPF. The proteomic approach to the analysis of BAL revealed that protein carbonylation was a process involving specific carbonylation-sensitive proteins and that in IPF a greater number of proteins target of oxidation were present. In conclusion, to our knowledge, this is the first report providing a database of proteins target of oxidation in BAL of patients with sarcoidosis, idiopathic pulmonary fibrosis and systemic sclerosis.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.