Lysine Carbamylation Research Articles

Anti-carbamylated protein antibodies drive AEC II toward a profibrotic phenotype by interacting with carbamylated TLR5.

This study looked at the role of anti-carbamylated protein (anti-CarP) antibodies in contributing to lung fibrosis in CTD-associated interstitial lung disease (ILD) in an autoantigen-dependent manner. ELISA was used to test serum samples, including 89 from the CTD-ILD group and 170 from the non-CTD-ILD group, for anti-CarP levels. Male C57BL/6 mice were used for the pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5) and patient serum-derived or commercialized anti-CarP was used for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and co-immunoprecipitation followed by mass spectrometry (MS) analysis. Quantitative RT-PCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. A native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites. A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and microfibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5. Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progression.

The pattern of apolipoprotein A-I lysine carbamylation reflects its lipidation state and the chemical environment within human atherosclerotic aorta

Protein lysine carbamylation is an irreversible post-translational modification resulting in generation of homocitrulline (N-ε-carbamyllysine), which no longer possesses a charged ε-amino moiety. Two distinct pathways can promote protein carbamylation. One results from urea decomposition, forming an equilibrium mixture of cyanate (CNO−) and the reactive electrophile isocyanate. The second pathway involves myeloperoxidase (MPO)-catalyzed oxidation of thiocyanate (SCN−), yielding CNO− and isocyanate. Apolipoprotein A-I (apoA-I), the major protein constituent of high-density lipoprotein (HDL), is a known target for MPO-catalyzed modification in vivo, converting the cardioprotective lipoprotein into a proatherogenic and proapoptotic one. We hypothesized that monitoring site-specific carbamylation patterns of apoA-I recovered from human atherosclerotic aorta could provide insights into the chemical environment within the artery wall. To test this, we first mapped carbamyllysine obtained from in vitro carbamylation of apoA-I by both the urea-driven (nonenzymatic) and inflammatory-driven (enzymatic) pathways in lipid-poor and lipidated apoA-I (reconstituted HDL). Our results suggest that lysine residues within proximity of the known MPO-binding sites on HDL are preferentially targeted by the enzymatic (MPO) carbamylation pathway, whereas the nonenzymatic pathway leads to nearly uniform distribution of carbamylated lysine residues along the apoA-I polypeptide chain. Quantitative proteomic analyses of apoA-I from human aortic atheroma identified 16 of the 21 lysine residues as carbamylated and suggested that the majority of apoA-I carbamylation in vivo occurs on “lipid-poor” apoA-I forms via the nonenzymatic CNO− pathway. Monitoring patterns of apoA-I carbamylation recovered from arterial tissues can provide insights into both apoA-I structure and the chemical environment within human atheroma.

Global chemical modifications comparison of human plasma proteome from two different age groups

In this study, two groups of human plasma proteome at different age groups (old and young) were used to perform a comparison of global chemical modifications, as determined by tandem mass spectrometry (MS/MS) combined with non-limiting modification identification algorithms. The sulfhydryl in the cysteine A total of 4 molecular modifications were found to have significant differences passing random grouping tests: the succinylation and phosphorylation modification of cysteine (Cys, C) and the modification of lysine (Lys, K) with threonine (Thr, T) were significantly higher in the old group than in the young group, while the carbamylation of lysine was lower in the young group. We speculate that there is an increase in certain modified proteins in the blood of the old people which, in turn, changes the function of those proteins. This change may be one of the reasons why old people are more likely than young people to be at risk for age-related diseases, such as metabolic diseases, cerebral and cardiovascular diseases, and cancer.

Structure, catalytic mechanism, posttranslational lysine carbamylation, and inhibition of dihydropyrimidinases.

Dihydropyrimidinase catalyzes the reversible hydrolytic ring opening of dihydrouracil and dihydrothymine to N-carbamoyl-β-alanine and N-carbamyl-β-aminoisobutyrate, respectively. Dihydropyrimidinase from microorganisms is normally known as hydantoinase because of its role as a biocatalyst in the synthesis of d- and l-amino acids for the industrial production of antibiotic precursors and its broad substrate specificity. Dihydropyrimidinase belongs to the cyclic amidohydrolase family, which also includes imidase, allantoinase, and dihydroorotase. Although these metal-dependent enzymes share low levels of amino acid sequence homology, they possess similar active site architectures and may use a similar mechanism for catalysis. By contrast, the five human dihydropyrimidinase-related proteins possess high amino acid sequence identity and are structurally homologous to dihydropyrimidinase, but they are neuronal proteins with no dihydropyrimidinase activity. In this chapter, we summarize and discuss current knowledge and the recent advances on the structure, catalytic mechanism, and inhibition of dihydropyrimidinase.

In-depth quantitative profiling of post-translational modifications of Timothy grass pollen allergome in relation to environmental oxidative stress

An association between pollution (e.g., from traffic emissions) and the increased prevalence of respiratory allergies has been observed. Field-realistic exposure studies provide the most relevant assessment of the effects of the intensity and diversity of urban and industrial contamination on pollen structure and allergenicity. The significance of in-depth post-translational modification (PTM) studies of pollen proteomes, when compared with studies on other aspects of pollution and altered pollen allergenicity, has not yet been determined; hence, little progress has been made within this field. We undertook a comprehensive comparative analysis of multiple polluted and environmentally preserved Phleum pratense (Timothy grass) pollen samples using scanning electron microscopy, in-depth PTM profiling, determination of organic and inorganic pollutants, analysis of the release of sub-pollen particles and phenols/proteins, and analysis of proteome expression using high resolution tandem mass spectrometry. In addition, we used quantitative enzyme-linked immunosorbent assays (ELISA) and immunoglobulin E (IgE) immunoblotting. An increased phenolic content and release of sub-pollen particles was found in pollen samples from the polluted area, including a significantly higher content of mercury, cadmium, and manganese, with irregular long spines on pollen grain surface structures. Antioxidative defense-related enzymes were significantly upregulated and seven oxidative PTMs were significantly increased (methionine, histidine, lysine, and proline oxidation; tyrosine glycosylation, lysine 4-hydroxy-2-nonenal adduct, and lysine carbamylation) in pollen exposed to the chemical plant and road traffic pollution sources. Oxidative modifications affected several Timothy pollen allergens; Phl p 6, in particular, exhibited several different oxidative modifications. The expression of Phl p 6, 12, and 13 allergens were downregulated in polluted pollen, and IgE binding to pollen extract was substantially lower in the 18 patients studied, as measured by quantitative ELISA. Quantitative, unrestricted, and detailed PTM searches using an enrichment-free approach pointed to modification of Timothy pollen allergens and suggested that heavy metals are primarily responsible for oxidative stress effects observed in pollen proteins.

PON-1 carbamylation is enhanced in HDL of uremia patients

High-density lipoprotein (HDL) carbamylation has been known in uremia patients. Paraoxonase-1 (PON-1) is an important HDL protein responsible for HDL anti-oxidant, arylesterase and lactonase activities. PON-1 carbamylation in uremic HDL has never been explored. We isolated HDL from uremia patients and control healthy subjects for study. Sandwich ELISA was used to estimate carbamylated PON-1 protein expression in HDL, and nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) was applied to identify the amino acid in PON-1 carbamylated. PON-1 enzyme activities were estimated by substrates conversion method. HDL anti-oxidant activity was gauged by fluorescence changes of indicator dye in the presence of H2O2. Our study results proved that the degree of PON-1 carbamylation was higher in uremic HDL than in control HDL. Sandwich ELISA study showed that carbamylated PON-1 concentration in uremic HDL was 1.49 ± 0.08 fold higher than that in HDL from controls (p < 0.05). The nanoLC-MS/MS showed that the carbamylation of lysine 290 (K290) of PON-1, a residue adjacent to PON-1 activity determining site, was detected in uremic HDL but not detected in control HDL. K290 carbamylation leads to local conformation changes that reduce accessible solvent accessibility. The HDL paraoxonase, arylesterase, and lactonase activities were all significantly lower in uremia patients than in control subjects. Additionally, HDL anti-antioxidant ability was also lower in uremia patients. Carbamylation of PON-1 in uremia patients could be one of the factors in impairing PON-1 enzyme activities and HDL anti-oxidation function.

Crystal structures of monometallic dihydropyrimidinase and the human dihydroorotase domain K1556A mutant reveal no lysine carbamylation within the active site

Dihydropyrimidinase (DHPase) is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase (DHOase), hydantoinase, and imidase. Almost all of these zinc metalloenzymes possess a binuclear metal center in which two metal ions are bridged by a post-translational carbamylated Lys. Crystal structure of Tetraodon nigroviridis DHPase reveals that one zinc ion is sufficient to stabilize Lys carbamylation. In this study, we found that one metal coordination was not sufficient to fix CO2 to the Lys in bacterial DHPase. We prepared and characterized mono-Zn DHPase from Pseudomonas aeruginosa (PaDHPase), and the catalytic activity of mono-Zn PaDHPase was not detected. The crystal structure of mono-Zn PaDHPase determined at 2.23 Å resolution (PDB entry 6AJD) revealed that Lys150 was no longer carbamylated. This finding indicated the decarbamylation of the Lys during the metal chelating process. To confirm the state of Lys carbamylation in mono-Zn PaDHPase in solution, mass spectrometric (MS) analysis was carried out. The MS result was in agreement with the theoretical value for uncarbamylated PaDHPase. Crystal structure of the human DHOase domain (huDHOase) K1556A mutant was also determined (PDB entry 5YNZ), and the structure revealed that the active site of huDHOase K1556A mutant contained one metal ion. Like mono-Zn PaDHPase, oxygen ligands of the carbamylated Lys were not required for Znα binding. Considering the collective data from X-ray crystal structure and MS analysis, mono-Zn PaDHPase in both crystalline state and solution was not carbamylated. In addition, structural evidences indicated that post-translational carbamylated Lys was not required for Znα binding in PaDHPase and in huDHOase.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Brain ureido degenerative protein modifications are associated with neuroinflammation and proteinopathy in Alzheimer\u2019s disease with cerebrovascular disease

BackgroundBrain degenerative protein modifications (DPMs) are associated with the apparition and progression of dementia, and at the same time, Alzheimer’s disease with cerebrovascular disease (AD + CVD) is the most prevalent form of dementia in the elder population. Thus, understanding the role(s) of brain DPMs in this dementia subtype may provide novel insight on the disease pathogenesis and may aid on the development of novel diagnostic and therapeutic tools. Two essential DPMs known to promote inflammation in several human diseases are the ureido DPMs (uDPMs) arginine citrullination and lysine carbamylation, although they have distinct enzymatic and non-enzymatic origins, respectively. Nevertheless, the implication of uDPMs in the neuropathology of dementia remains poorly understood.MethodsIn this study, we use the state-of-the-art, ultracentrifugation-electrostatic repulsion hydrophilic interaction chromatography (UC-ERLIC)-coupled mass spectrometry technology to undertake a comparative characterization of uDPMs in the soluble and particulate postmortem brain fractions of subjects diagnosed with AD + CVD and age-matched controls.ResultsAn increase in the formation of uDPMs was observed in all the profiled AD + CVD brains. Citrulline-containing proteins were found more abundant in the soluble fraction of AD + CVD whereas homocitrulline-containing proteins were preferentially abundant in the particulate fraction of AD + CVD brains. Several dementia-specific citrulline residues were also identified in soluble proteins previously categorized as pro-immunogenic, which include the receptor P2X7, alpha-internexin, GFAP, CNP, MBP, and histones. Similarly, diverse dementia-specific homocitrulline residues were also observed in the particulate fractions of AD + CVD in proteins that have been vastly implicated in neuropathology. Intriguingly, we also found that the amino acids immediately flanking arginine residues may specifically influence the increase in protein citrullination.ConclusionsTaken together, these results indicate that uDPMs widely contribute to the pathophysiology of AD + CVD by promoting neuroinflammation and proteinopathy. Furthermore, the obtained results could help to identify disease-associated proteins that can act as potential targets for therapeutic intervention or as novel biomarkers of specific neuropathology.

Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation.

Comprehensive analysis of post-translational modifications (PTMs) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. We report that the immuno-purification of lysine-acetylated peptides is hampered by the copurification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay.

Proteins and their modifications in a medieval mummy.

Proteins and their modifications of the natural mummy of Cangrande della Scala (Prince of Verona, Northern Italy, 1291-1329) were studied. The nano-LC-Q-TOF analysis of samples of rib bone and muscle from the mummy showed the presence of different proteins including Types I, III, IV, V, and XI collagen, hemoglobin (subunits alpha and beta), ferritin, biglycan, vitronectin, prothrombin, and osteocalcin. The structure of Type I and Type III collagen was deeply studied to evaluate the occurrence of modifications in comparison with Type I and Type III collagen coming from tissues of recently died people. This analysis showed high percentage of asparaginyl and glutaminyl deamidation, carbamylation and carboxymethylation of lysine, as well as oxidation and dioxidation of methionine. The most common reaction during the natural mummification process was oxidation-the majority of lysine and proline of collagen Type I was hydroxylated whereas methionine was oxidated (oxidated or dioxidated). To the best of our knowledge, this is the first study which reports the protein profile of a natural mummified human tissue and the first one which describes the carbamylation and carboxymethylation of lysine in mummified tissues.

Lysine carbamylation for enzymatic function: metal and structural requirements

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. Carbamylation on lysine extends the residue length by ~2 Å and changes the side chain from a positive to negative charge at neutral pH. The proteins involved with lysine carbamylation are found to be related to the human diseases, such as type 2 diabetes, developmental delay, metabolic acidosis, mental retardation, hypotonia and seizures. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apo enzyme as well as two forms of the holo enzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified with possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala69–Arg74 and Met158–Met165, located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the mono-metal TnDhp, and in the "closed form" in the di-metal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala69–Arg74 dynamic loop. In addition, the structures of the di-metal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine and N-carbamyl-β-amino isobutyrate, as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.

Crystal Structures of Vertebrate Dihydropyrimidinase and Complexes from Tetraodon nigroviridis with Lysine Carbamylation: METAL AND STRUCTURAL REQUIREMENTS FOR POST-TRANSLATIONAL MODIFICATION AND FUNCTION

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala(69)-Arg(74) and Met(158)-Met(165), located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the monometal TnDhp, and in the "closed form" in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala(69)-Arg(74) dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine, and N-carbamyl-β-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodiacetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions.

Crystal Structure of the Narrow-Spectrum OXA-46 Class D β-Lactamase: Relationship between Active-Site Lysine Carbamylation and Inhibition by Polycarboxylates

Class D beta-lactamases represent a heterogeneous group of active-site serine beta-lactamases that show an extraordinary panel of functional features and substrate profiles, thus representing relevant models for biochemical and structural studies. OXA-46 is a narrow-spectrum enzyme belonging to the OXA-2 subgroup which was found in a Pseudomonas aeruginosa clinical isolate from northern Italy. In this work, we obtained the three-dimensional structure of OXA-46, which shows the overall fold of active serine beta-lactamases and a dimeric quaternary structure. Significant differences with currently available structures of class D beta-lactamases were found in the loops located close to the active site, which differ in length and conformation. Interestingly, the three subunits present in the asymmetric unit showed some structural heterogeneity, only one of which presented a carbamylated lysine recognized as an important functional feature of class D enzymes. The carbamylation state of residue Lys75 appeared to be associated with different shapes and dimensions of the active site. Moreover, a tartrate molecule from the crystallization buffer was found in the active site of the noncarbamylated subunits, which interacts with catalytically relevant residues. The OXA-46 crystal asymmetric units thus interestingly present the structures of the free carbamylated active site and of the ligand-bound uncarbamylated active site, offering the structural basis for investigating the potential of new scaffolds of beta-lactamase inhibitors.

Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Quantitative carbamylation as a stable isotopic labeling method for comparative proteomics

A method was developed that uses urea to both solublize and isotopically label biological samples for comparative proteomics. This approach uses either light or heavy urea ((12)CH(4)(14)N(2)O or (13)CH(4)(15)N(2)O, respectively) at a concentration of 8 M and a pH of 7 to dissolve the samples prior to digestion. After the sample is digested using standard proteomic protocols and dried, isotopic labeling is completed by resuspending the sample in a solution of 8 M urea at a pH of 8.5, using the same isotopic species of urea as used for digestion and incubating for 4 h at 80 degrees C. Under these conditions, carbamylation occurs only on the primary amines of the peptides. The effects of complete carbamylation on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) (collision-induced dissociation (CID)) were examined. Peptides that had a C-terminal carbamylated lysine residue were found to have a reduced intensity when viewed by MALDI-TOFMS. CID of a tryptic peptide that was carbamylated on both the N-terminus and the C-terminus was found to have a more uniform distribution of b- and y-ions, as well as prominent ions from loss of water. Reversed-phase chromatography coupled to ESI-MS/MS was used to identify and quantify the isotopically labeled standard proteins, bovine serum albumin (BSA), bovine transferrin, and bovine alpha-casein. Quantitative error between theoretical and observed data ranged from 1.7-10.0%. Relative standard deviations for protein quantitation ranged from 5.2-27.8% over a dynamic range from 0.1-10 (L/H). The development of a method utilizing urea-assisted carbamylation of lysines and N-termini to globally labeled samples for comparative proteomics may prove useful for samples that require a strong chaotrope prior to proteolysis.

A Chlorophyll a/b-binding Protein Homolog That Is Induced by Iron Deficiency Is Associated with Enlarged Photosystem I Units in the Eucaryotic Alga Dunaliella salina

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.

Do chemical modifications dissociate between the enzymatic and pharmacological activities of β bungarotoxin and notexin?

P. Rosenberg, A. Ghassemi, E. Condrea, D. Dhillon and C.-C. Yang. Do chemical modifications dissociate between the enzymatic and pharmacological activities of β bungarotoxin and notexin? Toxicon 27, 137–159, 1989.—We have measured enzymatic, hemolytic and anticoagulant activities, lethal potencies and effects on contractions of the phrenic nerve-diaphragm preparation, by chemically modified derivatives of β bungarotoxin (βBuTX) and notexin, two presynaptically acting toxins which have PLA 2 activity. The following chemical modifications of βBuTX were tested: alkylation and methylation of histidine 48, alkylation of tryptophan 19, sulfonylation of tyrosine 68, oxidation of methionines 6 and 8, semicarbazide addition under varied conditions to carboxyl groups, varied extents of carbamylation or trinitrophenylation of lysines and guanidination of all lysines with or without trinitrophenylation of the N-terminal asparagine. Only the histidine, tryptophan and tyrosine residues were modified in notexin. The results obtained were compared with those previously obtained using chemically modified derivatives of Naja nigricollis and Naja naja atra PLA 2 enzymes which do not have a specific presynaptic site of action. The results with oxidized methionine and lysine-modified derivatives of βBuTX are supportive of the suggestions of others that the N-terminal region and basic residues away from the enzymatic active region contribute towards the β type presynaptic neurotoxicity of the PLA 2 toxins. Using modified derivatives of βBuTX and notexin, the dissociations between enzymatic activities and pharmacological properties were not as marked as previously observed with N. nigricollis and N. n. atra PLA 2; nevertheless, several dissociations were noted. We conclude that, just as with non-presynaptically acting PLA 2 enzymes, some pharmacological actions of presynaptically acting PLA 2 toxins may occur independently of phospholipid hydrolysis.

Relation between binding and the action of phospholipases A 2 on Escherichia coli exposed to the bactericidal/permeability-increasing protein of neutrophils

Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A 2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A 2 to E. coli in the presence and absence of BPI. Phospholipases A 2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A 2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A 2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH 2-terminal amino sequences of the active and inactive phospholipase A 2 suggests that this portion of the phospholipase A 2 molecule plays a role in BPI-dependent binding and hydrolysis.

The relationship between high-affinity noncatalytic binding of snake venom phospholipases A 2 to brain synaptic plasma membranes and their central lethal potencies

The basic phospholipase A 2 from Naja nigricollis (African spitting cobra) snake venom is enzymatically less active but more toxic than the acidic phospholipase A 2 from Naja naja atra (Taiwan cobra) snake venom, following injection into the right lateral ventricle of the brain of rats. When radiolabeled with 125I, these phospholipases A 2 retained enzymatic activities and lethal potencies. Both enzymes bound with high affinity and specificity to brain synaptic plasma membrane preparations in vitro even in the absence of calcium, suggesting a non-catalytic binding. The acidic enzyme, in a calcium-free medium, had two binding components with K d values of 1.10·10 −10 and 2.75·10 −8 M and B max values of 6·10 −13 and 3.4·10 −11 mol/mg, respectively. Multiple specific and nonspecific binding components were observed for each phospholipase A 2; saturability for all of the binding sites was conclusively demonstrated only for the N. naja atra phospholipase A 2 in a calcium-free medium ( B max = 3.4·10 −11 mol/mg ). The levels of specific and total binding were 150 pmol/mg and 450 pmol/mg, respectively, for the comparatively toxic enzyme and 15 pmol/mg and 35 pmol/mg, respectively, for the comparatively nontoxic enzyme at a concentration of 2.5·10 −8 M. These levels of binding (both total and specific) were directly correlated with the intraventricular lethal potencies of the phospholipases A 2 (0.5 and 5.0 μg/rat for the N. nigricollis and N. naja atra phospholipases A 2, respectively), suggesting a possible relationship between binding and lethal potency. Carbamylation of lysines reduced the levels of binding and the lethal potencies of both enzymes to a greater extent than their enzymatic activities. Pretreatment with high temperature, proteinases, phospholipases A 2 or C suggested that radiolabeled phospholipase A 2 binds to phospholipids rather than proteins. However, only the N. naja atra phospholipase A 2 manifested a strict dependence on a divalent cation (Ca 2+ or Sr 2+) for most of its binding. The N. nigricollis enzyme demonstrated a much lower rate of dissociation from synaptic plasma membranes than did N. naja atra phospholipase A 2, suggesting that hydrophobic interactions are more important in the binding of the more toxic enzyme as compared to the less toxic enzyme. It is proposed that differences in the extent of high-affinity noncatalytic binding to membrane phospholipids may be at least partly responsible for the marked difference in central toxicities of these two phospholipases A 2.