Altered tubulin assembly dynamics with N-homocysteinylated human 4R/1N tau in vitro

Tau isoforms promote neuronal integrity through binding and stabilization of microtubule proteins (MTP). It has been shown that hyperphosphorylation of tau contributes to Alzheimer’s disease (AD) pathology and related tauopathies. However, other pathogenic modifications of tau have not been well characterized. It is well accepted that elevated level of homocysteine (Hcy) is associated with neurodegenerative diseases such as AD. As a result of N-homocysteinylation of lysine residues, Hcy becomes a component of proteins, as a protein–homocystamide adduct, which affects protein structure and function. Here we demonstrate that N-homocysteinylation of human tau (4R/1N isoform) inhibits its function via impaired tau–tubulin specific binding and MTP assembly dynamics in vitro.     

Identification and origin of Nε-homocysteinyl-lysine isopeptide in humans and mice

Homocysteine (Hcy) metabolites, Hcy-thiolactone and N-Hcy-proteins, have been linked to the pathology of human cardiovascular and neurodegenerative diseases. Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. N-Hcy-protein, in which Hcy is linked via isopeptide bond to ε-amino group of a protein lysine residue, forms in a post-translational reaction of Hcy-thiolactone with proteins. Here, we identify a novel metabolite, Nε-Hcy-Lys, in human and mouse plasma, and show that this metabolite is elevated in genetic (cystathionine β-synthase deficiency in humans and mice, methylenetetrahydrofolate reductase deficiency in mice) or dietary (high Met diet in mice) deficiencies in Hcy metabolism. We also show that Nε-Hcy-Lys is generated by proteolytic degradation of N-Hcy-protein in mouse liver extracts. Our data indicate that free Nε-Hcy-Lys is an important pathology-related component of Hcy metabolism in humans and mice.     

Site-specific modification of positively-charged surfaces on human serum albumin by malondialdehyde

Malondialdehyde (MDA), a lipid peroxidation product, reacts with lysine residues in proteins. Human serum albumin (HSA) is a major target of MDA-modification of serum proteins. To identify, the modification sites of HSA by MDA in vitro, MDA-treated HSA was digested with a protease and the resulting peptides were subjected to liquid chromatography-tandem mass spectrometry. We identified six peptides, which contained a N-propenal adduct at Lys136, Lys174, Lys240, Lys281, Lys525, and Lys541, and revealed that Lys525 is the most reactive residue for MDA modification. Analysis of electrostatic surface potential of a 3-D model structure of HSA indicates that Lys525 is located at the center of positively charged grooves. The results of this study indicate that the modification of proteins by lipid-derived aldehydes may be influenced by the electrostatic potential of the protein surface.     

Any use in proteomics for low-tech approaches? Detecting fibrinogen chains of different animal species in two-dimensional electrophoresis patterns

We characterized the two-dimensional electrophoretic patterns of fibrinogen chains α, β, and γ from the plasma of six animal species – Bos taurus, Canis familiaris, Equus caballus, Felis catus, Gallus domesticus and Sus scrofa. Comparing the spots resolved from serum and plasma samples, or exploiting the cross-reactivity of animal fibrinogen with an antiserum raised against the human protein could detect only some of the fibrinogen chains. Conversely, the analysis of the precipitate obtained by heating plasma for some minutes at 56 °C was adequate for the recognition of all fibrinogen chains in all samples. Physicochemical properties of the homologous proteins were found to extensively vary across species, with complete separation among the mapping areas for α, β and γ chains and maximal heterogeneity among β chains.     

Simultaneous characterization of sequence polymorphisms,glycosylation and phosphorylation of fibrinogen in a direct analysis by LC–MS

Fibrinogen is an abundant plasma glycoprotein involved in pathologically important processes like blood clotting, hemostasis and angiogenesis. Sequence polymorphisms and posttranslational modification (PTM) status of fibrinogen are important factors of cardiovascular disease. We aim for the simultaneous analysis of fibrinogen subunits for sequence polymorphisms (SNPs), phosphorylation and glycosylation by top-down mass spectrometry. Fibrinogen was isolated from human plasma of twelve individuals and subunits of fibrinogen were separated by RP-HPLC and subsequently analyzed by high resolution ESI mass spectrometry. Two coding single nucleotide polymorphisms on the Aα- and Bβ-subunit could be identified on the basis of their mass shifts: Three individuals are heterozygous and two are homozygous for Thr312Ala on the Aα-subunit, three individuals are heterozygous for Arg448Lys on the Bβ-subunit. For the Aα-subunit we find mono- and diphosphorylation amounting to about 55% to 71% and O-glycosylation (likely sialyl-T-antigen) from 10% to 17%. N-glycosylation is present with one or two sialic acids in a ratio of about 3:2 and 3:1 for the Bβ and the γ-subunit, respectively. Both SNPs and the PTMs are associated with fibrinogen levels, clotting behavior and thus the risk for cardiovascular diseases. The homozygosity of the SNP at position 312 in the alpha chain for example nearly doubles the risk for ischemic stroke. Isolation and analysis of fibrinogen can be achieved in a few hours from only one drop of blood plasma, and thus the method presented here should assist in a quick assessment and prevention of stroke and infarction.     

A study on human serum albumin influence on glycation of fibrinogen

Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [¹³C₆] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen.     

Characterization of N-homocysteinylated Albumin Adducts

Increased plasma homocysteine levels are considered an important risk factor for vascular disease. Homocysteine, an intermediate compound in methionine metabolism, is an amino acid that includes a thiol group, and circulates as different species. One of them, Homocysteine thiolactone (HTL) forms adducts through irreversible reactions with epsilon-NH₂ groups of lysine residues. These processes can alter the structure and biological function of diverse proteins that may be involved in the detrimental effects of homocysteine. Particularly, in this work we evaluated HTL-mediated molecular changes in human serum albumin (HSA) through electrophoretic techniques. Albumin and HTL were incubated (37 °C, 6 h) at HSA:HTL molar ratios of 1:25, 1:50 and 1:100. Polyacrylamid gel electrophoresis showed that electrophoretic mobility was increased in the treated HSA respect to control, in an HTL-concentration-dependent manner. That anodic shift of the treated samples was also observed in crossed immunoelectrophoresis profiles. As expected, a decrease in the isoelectric point of the homocysteinylated albumin (pI 4.7) in comparison to that of control (pI 4.8) was shown by the isoelectric focusing technique. Moreover, the electropherogram acquired by capillary zone electrophoresis indicated that migration times and full width at half height were enhanced with the rise of HTL concentration. We propose that the in vitro structural changes of albumin described in the present work would be involved in the harmful effects of the N-homocysteinylation process.     

A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation

Background

Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.

Methods

AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.

Results

Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (> 200 kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.

Conclusions

A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.

General significance

These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.     

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.     

Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.     

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein.     

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.     

Distinct Roles of N-Glycosylation at Different Sites of Corin in Cell Membrane Targeting and Ectodomain Shedding

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.     

A centrifugal ultrafiltration strategy for isolating the low-molecular weight (≤ 25 K) component of human plasma proteome

The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. In this study, a separation and enrichment strategy based on centrifugal ultrafiltration was developed for the LMF (i.e., ≤ 25 K) of plasma routinely prepared from normal, healthy volunteers. Four commercially-available filter membranes of similar nominal molecular weight cut-off (NMWC), but differing membrane chemistries and filter orientations (Microcon®, Millipore; Centrisart®, Sartorius; Amicon Ultra®, Millipore; Vivaspin®, Sartorius), were evaluated. Of these filtration devices, only the Sartorius Vivaspin® tangential membrane, NMWC 20 K was effective in the non-retention of M_r > 50 K, and recovery and enrichment of low-M_r components from human plasma. This filter membrane device was further optimized with respect to plasma buffer composition, centrifugal force, duration and temperature. Optimal ultrafiltration conditions were obtained using 100 µL of normal plasma in 10% acetonitrile, and a centrifugation force of 4000 × g for 35 min at 20 °C. In this LMF, 44 proteins (from 266 unique peptides) were identified using a combination of 1D-SDS-PAGE / nano-LC-MS/MS and a stringent level of identification (FDR < 1%). We report the identification of several proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization (HUPO) Plasma Proteome Project datasets. When compared with the low-M_r human plasma/serum proteome datasets of Zhou et al. (Electrophoresis, 2004. 25, 1289–98), Gundry et al. (Proteomics Clin. Appl., 2007. 1, 73–88) and Villanueva et al. (Anal Chem, 2004. 76, 1560–70), 64% of our identifications (28 proteins) were novel; these include cofilin-1, PPIase A, and the SH3 domain-binding glutamic acid-rich-like protein 3. In addition to intact proteins, many peptide fragments from high-abundance proteins (e.g., fibrinogen, clusterin, Factor XIIIa, transferrin, kinogen-1, and inter-alpha-trypsin inhibitor), presumably derived by ex vivo proteolysis, were observed.     

A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo

Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well‐characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA‐based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost‐effective method that can be used to prevent unnecessary animal experiments.     

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

Introduction

Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.

Methods

An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.

Results

Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.

Conclusions

We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.     

Familial hypofibrinogenaemia associated with heterozygous substitution of a conserved arginine residue; Bβ255 Arg→His (Fibrinogen Merivale)

Sequencing of all three fibrinogen genes from an individual with hypofibrinogenaemia led to the identification of two new point mutations in the Bβ gene. Family studies showed the mutations Bβ255 Arg→His (Fibrinogen Merivale) and Bβ148 Lys→Asn (Fibrinogen Merivale II) were on different alleles and that only the Bβ255 Arg→His mutation segregated with hypofibrinogenaemia. Three simple heterozygotes for this mutation had mean fibrinogen concentrations of 1.4 mg/ml, while heterozygotes for the Bβ148 Lys→Asn mutation had normal fibrinogen concentrations. ESI MS analysis of endoproteinase Asp-N digests of Bβ chains showed that the Bβ255 Arg→His substitution was not expressed in plasma, confirming it as the cause of the hypofibrinogenaemia. The Bβ148 Lys→Asn chains, on the other hand, were equally expressed with wild-type Bβ chains in simple heterozygotes. Genotype analysis failed to detect either substitution in 182 healthy controls. Arg²⁵⁵ is located in the first strand of the five-stranded sheet that forms the main feature of the βD domain and appears to form an essential H bond with Gly⁴¹⁴. Both the Arg and Gly are absolutely conserved, not only in all known Bβ chains, but also in all homologous αE and γ chains and in all fibrinogen-related proteins. Protein instability from loss of this contact could easily explain the association of this mutation with hypofibrinogenaemia.     

Recombinant human fibrinogen expressed in the yeast Pichia pastoris was assembled and biologically active

Fibrinogen is a large plasma glycoprotein with a molecular mass of 340 kDa that plays a critical role in the final stage of blood coagulation. Human plasma fibrinogen is a dimeric molecule comprising two sets of three different polypeptides (Aα, 66 kDa; Bβ, 55 kDa; γ, 48 kDa). To express recombinant human fibrinogen in the methylotrophic yeast Pichia pastoris, we constructed an expression vector containing three individual fibrinogen chain cDNAs under the control of the mutated AOX2 (mAOX2) promoter. First, P. pastoris GTS115 was transformed with the vector, but the expressed recombinant fibrinogen suffered severe degradation by yeast-derived proteases under conventional nutrient culture conditions. Fibrinogen degradation was prevented by using the protease A-deficient strain SMD1168 as a host strain and regulating the pH of the culture to between 5.5 and 7.0. Western blot analysis revealed that the Aα, Bβ and γ chains of recombinant fibrinogen were assembled and secreted as a complete molecule. The Bβ chain of the recombinant fibrinogen was N-glycosylated but the Aα chain, as in plasma fibrinogen, was not. The γ chains however were heterologous, one being N-glycosylated and the other not. The recombinant fibrinogen was capable of forming a thrombin-induced clot in the presence of factor XIIIa and both the glycosylated and the non-glycosylated γ chains were involved in the formation of cross-linking fibrin. The present study indicates that the recombinant fibrinogen expressed in P. pastoris, although different from plasma fibrinogen in post-translational modification, is correctly assembled and biologically active.     

N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system

Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.