Probing the redox-active residues in cytochrome c peroxidase

The reaction of cytochrome c peroxidase (CCP) with H 2 O 2 results in compound I formation, where the two oxidizing equivalents of H 2 O 2 are stored as an oxyferryl heme and a Trp191 radical. Ferrocytochrome c normally reduces compound I back to the resting enzyme, but in the absence of exogenous donors, CCP can reduce up to 20 equivalents of H 2 O 2 . Compound I of horseradish peroxidase (HRP) does not form a protein radical, and is unlikely to store oxidizing equivalents on its polypeptide. The conformational states in denaturants of recombinant CCP [CCP(MI)], HRP and their CN-ligated forms were investigated to probe the structural basis of peroxidase polypeptide vs heme reactivity. Despite similar structures, the kinetic stabilities and conformational states of CCP(MI) and HRP were found to be significantly different. The role of Trp residues as endogenous electron donors in yeast CCP, CCP(MI), and two active site mutants (W51F and W191F) was examined by protein steady-state fluorescence. Compound I and more highly oxidized forms were formed by adding 2, 6, and 20 equivalents of H 2 O 2 to the proteins in the absence of exogenous donors. Loss of protein fluorescence following protein denaturation in 8 M urea at pH 1.5 was correlated with Trp oxidation. The fluorescence data confirmed Trp191 radical formation in compound I, suggested that Trp5l becomes redox active when >2 equivalents of H 2 O 2 are reduced, and that V3, 4, 2.5 and 2 Trps were lost in CCP, CCP(MI), W51F and W191F, respectively, following addition of 20 equivalents of H 2 O 2 . Activity loss in the H 2 O 2 -oxidized proteins paralleled Trp loss, and correlated with their H 2 O 2 titers. SDS-PAGE revealed 40-75% crosslinking in H 2 O 2 -oxidized W51F, 0-35% in CCP and CCP(MI), and 30-35% in W191F. On-line HPLC-ESI-MS analysis of proteolytic, digests of crosslinked W191F revealed that peptides T 6 (residues 30-48) and T 26 (residues 227-243) formed T 6 --T 6 and T 6 --T 26 crosslinks, suggesting that oxidation of exposed Tyr residues in T 6 (Tyr36, 39, 42) is necessary for crosslinking. H 2 O 2 oxidation of CCP(MI) in the presence of the spin trap, MNP, revealed that spin adducts were formed on peptides T 6 , T 21 (residues 150-155; Tyr153) and T 26 (Tyr229, 236). Tyr236 was identified as the major site of spin adduct formation in CCP(MI) by MS sequencing

Analysis of networks of host proteins in the early time points following HIV transduction

Background: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. Results: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. Conclusion: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.Hungarian Scientific Research Fund [NKFI-6, 125238]; Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary [GINOP-2.3.3-15-2016-00020]; Janos Bolyai Research Scholarship of the Hungarian Academy of Sciences; NIEHS [ES06694]; NIH/NCI [CA023074]; BIO5 Institute of the University of Arizona; NIH/NCRR [1S10 RR028868-01]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Engineering of N. benthamiana L. plants for production of N-acetylgalactosamine-glycosylated proteins - towards development of a plant-based platform for production of protein therapeutics with mucin type O-glycosylation

<p>Abstract</p> <p>Background</p> <p>Mucin type O-glycosylation is one of the most common types of post-translational modifications that impacts stability and biological functions of many mammalian proteins. A large family of UDP-GalNAc polypeptide:N-acetyl-α-galactosaminyltransferases (GalNAc-Ts) catalyzes the first step of mucin type O-glycosylation by transferring GalNAc to serine and/or threonine residues of acceptor polypeptides. Plants do not have the enzyme machinery to perform this process, thus restricting their use as bioreactors for production of recombinant therapeutic proteins.</p> <p>Results</p> <p>The present study demonstrates that an isoform of the human GalNAc-Ts family, GalNAc-T2, retains its localization and functionality upon expression in <it>N. benthamiana </it>L. plants. The recombinant enzyme resides in the Golgi as evidenced by the fluorescence distribution pattern of the GalNAc-T2:GFP fusion and alteration of the fluorescence signature upon treatment with Brefeldin A. A GalNAc-T2-specific acceptor peptide, the 113-136 aa fragment of chorionic gonadotropin β-subunit, is glycosylated <it>in vitro </it>by the plant-produced enzyme at the "native" GalNAc attachment sites, Ser-121 and Ser-127. Ectopic expression of GalNAc-T2 is sufficient to "arm" tobacco cells with the ability to perform GalNAc-glycosylation, as evidenced by the attachment of GalNAc to Thr-119 of the endogenous enzyme endochitinase. However, glycosylation of highly expressed recombinant glycoproteins, like magnICON-expressed <it>E. coli </it>enterotoxin B subunit:<it>H. sapiens </it>mucin 1 tandem repeat-derived peptide fusion protein (LTBMUC1), is limited by the low endogenous UDP-GalNAc substrate pool and the insufficient translocation of UDP-GalNAc to the Golgi lumen. Further genetic engineering of the GalNAc-T2 plants by co-expressing <it>Y. enterocolitica </it>UDP-GlcNAc 4-epimerase gene and <it>C. elegans </it>UDP-GlcNAc/UDP-GalNAc transporter gene overcomes these limitations as indicated by the expression of the model LTBMUC1 protein exclusively as a glycoform.</p> <p>Conclusion</p> <p>Plant bioreactors can be engineered that are capable of producing Tn antigen-containing recombinant therapeutics.</p

Pulmonary Biomarkers Based on Alterations in Protein Expression after Exposure to Arsenic

OBJECTIVE: Environmental exposure to arsenic results in multiple adverse effects in the lung. Our objective was to identify potential pulmonary protein biomarkers in the lung-lining fluid of mice chronically exposed to low-dose As and to validate these protein changes in human populations exposed to As. METHODS: Mice were administered 10 or 50 ppb As (sodium arsenite) in their drinking water for 4 weeks. Proteins in the lung-lining fluid were identified using two-dimensional gel electrophoresis (n = 3) or multidimensional protein identification technology (MUDPIT) (n = 2) coupled with mass spectrometry. Lung-induced sputum samples were collected from 57 individuals (tap water As ranged from ~ 5 to 20 ppb). Protein levels in sputum were determined by ELISA, and As species were analyzed in first morning void urine. RESULTS: Proteins in mouse lung-lining fluid whose expression was consistently altered by As included glutathione-S-transferase (GST)-omega-1, contraspin, apolipoprotein A-I and A-IV, enolase-1, peroxiredoxin-6, and receptor for advanced glycation end products (RAGE). Validation of the putative biomarkers was carried out by evaluating As-induced alterations in RAGE in humans. Regression analysis demonstrated a significant negative correlation (p = 0.016) between sputum levels of RAGE and total urinary inorganic As, similar to results seen in our animal model. CONCLUSION: Combinations of proteomic analyses of animal models followed by specific analysis of human samples provide an unbiased determination of important, previously unidentified putative biomarkers that may be related to human disease

The Competition of Charge Remote and Charge Directed Fragmentation Mechanisms in Quaternary Ammonium Salt Derivatized Peptides—An Isotopic Exchange Study

Derivatization of peptides as quaternary ammonium salts (QAS) is a promising method for sensitive detection by electrospray ionization tandem mass spectrometry (Cydzik et al. J. Pept. Sci.2011, 17, 445–453). The peptides derivatized by QAS at their N-termini undergo fragmentation according to the two competing mechanisms – charge remote (ChR) and charge directed (ChD). The absence of mobile proton in the quaternary salt ion results in ChR dissociation of a peptide bond. However, Hofmann elimination of quaternary salt creates an ion with one mobile proton leading to the ChD fragmentation. The experiments on the quaternary ammonium salts with deuterated N-alkyl groups or amide NH bonds revealed that QAS derivatized peptides dissociate according to the mixed ChR-ChD mechanism. The isotopic labeling allows differentiation of fragments formed according to ChR and ChD mechanisms

RasGRP1 is a causal factor in the development of l-DOPA-induced dyskinesia in Parkinson's disease.

The therapeutic effects of l-3,4-dihydroxyphenylalanine (l-DOPA) in patients with Parkinson's disease (PD) severely diminishes with the onset of abnormal involuntary movement, l-DOPA-induced dyskinesia (LID). However, the molecular mechanisms that promote LID remain unclear. Here, we demonstrated that RasGRP1 [(guanine nucleotide exchange factor (GEF)] controls the development of LID. l-DOPA treatment rapidly up-regulated RasGRP1 in the striatum of mouse and macaque model of PD. The lack of RasGRP1 in mice (RasGRP1-/- ) dramatically diminished LID without interfering with the therapeutic effects of l-DOPA. Besides acting as a GEF for Ras homolog enriched in the brain (Rheb), the activator of the mammalian target of rapamycin kinase (mTOR), RasGRP1 promotes l-DOPA-induced extracellular signal-regulated kinase (ERK) and the mTOR signaling in the striatum. High-resolution tandem mass spectrometry analysis revealed multiple RasGRP1 downstream targets linked to LID vulnerability. Collectively, the study demonstrated that RasGRP1 is a critical striatal regulator of LID

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. © 2013 Wysocka-Kapcinska et al