Structure-activity relationships of pyrrole based S-nitrosoglutathione reductase inhibitors: pyrrole regioisomers and propionic acid replacement

S-Nitrosoglutathione reductase (GSNOR) is a member of the alcohol dehydrogenase family (ADH) that regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). GSNO and SNOs are implicated in the pathogenesis of many diseases including those in respiratory, cardiovascular, and gastrointestinal systems. The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious GSNOR inhibitor which is currently undergoing clinical development. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on scaffold modification and propionic acid replacement. We identified equally potent and novel GSNOR inhibitors having pyrrole regioisomers as scaffolds using a structure based approach.     

Characterization of a double dockerin from the cellulosome of the anaerobic fungus Piromyces equi

The assembly into supramolecular complexes of proteins having complementary activities is central to cellular function. One such complex of considerable biological and industrial significance is the plant cell wall-degrading apparatus of anaerobic microorganisms, termed the cellulosome. A central feature of bacterial cellulosomes is a large non-catalytic protein, the scaffoldin, which contains multiple cohesin domains. An array of digestive enzymes is incorporated into the cellulosome through the interaction of the dockerin domains, present in the catalytic subunits, with the cohesin domains that are present in the scaffoldin. By contrast, in anaerobic fungi, such as Piromyces equi, the dockerins of cellulosomal enzymes are often present in tandem copies; however, the identity of the cognate cohesin domains in these organisms is unclear, hindering further biotechnological development of the fungal cellulosome. Here, we characterise the solution structure and function of a double-dockerin construct from the P. equi endoglucanase Cel45A. We show that the two domains are connected by a flexible linker that is short enough to keep the binding sites of the two domains on adjacent surfaces, and allows the double-dockerin construct to bind more tightly to cellulosomes than a single domain and with greater coverage. The double dockerin binds to the GH3 beta-glucosidase component of the fungal cellulosome, which is thereby identified as a potential scaffoldin.     

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN = 116 ± 10 s⁻¹ at 1 μM NO, T = 37 °C). The activity is [O₂]-dependent and characterized by an apparent K_M,O2 = 22 ± 7 μM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with ?5 mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.     

Specific transport of S-nitrosocysteine in human red blood cells: Implications for formation of S-nitrosothiols and transport of NO bioactivity within the vasculature

The transport of various S-nitrosothiols, NO and NO donors in human red blood cells (RBC) and the formation of erythrocytic S-nitrosoglutathione were investigated. Of the NO species tested only S-nitrosocysteine was found to form S-nitrosoglutathione in the RBC cytosol. L-Serine, L-cysteine and L-lysine inhibited formation of S-nitrosoglutathione. Incubation of RBC pre-incubated with S-[15N]nitroso-L-cysteine with native plasma or platelet-rich plasma led to formation of S-[15N]nitrosoalbumin and inhibited platelet aggregation, respectively. The specific transporter system of S-nitroso-L-cysteine in the RBC membrane may have implications for formation of S-nitrosoalbumin and S-nitrosohemoglobin and for transport of NO bioactivity within the vasculature.     

The relation between sphingomyelinase activity, lipid peroxide oxidation and NO-releasing in mice liver and brain

We used animal models to study connection between oxidating system and sphingomyelin signaling cascade, because this models are more close related to people disease. Activation of n-sphingomyelinase (n-SMase) in mice liver and brain is coincided in time with increased level of peroxide products (conjugated dienes) after injection of tumor necrosis factor alpha (TNF-alpha). We found that ceramide can induce peroxide oxidation and lead to accumulation of TNF-alpha in animal organs. Nitric oxide (NO) donors (S-nitrosoglutathione and dinitrosyl iron complex) reversibly inhibited activity of n-SMase and decreased level of lipid peroxidation products. This data proposed that both SMase and messengers of oxidative systems could be targets for NO-derived oxidants.     

Nitric oxide and dihydrolipoic acid modulate the activity of caspase 3 in HepG2 cells

Herein, we report that dihydrolipoic acid and lipoic acid (LA) plus lipoamide dehydrogenase and NADH denitrosate S-nitrosocaspase 3 (CASP-SNO). In HepG2 cells, S-nitroso-l-cysteine ethyl ester (SNCEE) impeded the activity of caspase 3 (CASP-SH), while a subsequent incubation of the cells in SNCEE-free medium resulted in endogenous denitrosation and reactivation of CASP-SH. The latter process was inhibited in thioredoxin reductase-deficient HepG2 cells, in which, however, LA markedly reactivated CASP-SH. The data obtained are discussed with focus on low molecular mass dithiols that mimic the activity of thioredoxin in reactions of protein S-denitrosation.     

Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration

Background

Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported.

Methods

We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches.

Results

Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H₂O₂, NO, GSH and peroxynitrite showed that the ONOO⁻ molecule caused the highest inhibition of activity (51% at 5 mM SIN-1), with 5 mM H₂O₂ having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.

Conclusion

These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.

General significance

This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.     

Structural Analysis of Cysteine S-Nitrosylation: A Modified Acid-Based Motif and the Emerging Role of Trans-Nitrosylation

S-Nitrosylation, the selective and reversible addition of nitric oxide (NO) moiety to cysteine (Cys) sulfur in proteins, regulates numerous cellular processes. In recent years, proteomic approaches that are capable of identifying nitrosylated Cys residues have been developed. However, the features underlying the specificity of Cys modification with NO remain poorly defined. Previous studies suggested that S-nitrosylated Cys may be flanked by an acid-base motif or hydrophobic areas and show high reactivity, low pK_a, and high sulfur atom exposure. In the current study, we prepared an extensive, manually curated data set of proteins with S-nitrosothiols, accounting for a variety of biochemical functions, organisms of origin, and physiological responses to NO. Analysis of this generic NO-Cys data set revealed that proximal acid-base motif, Cys pK_a, sulfur atom exposure, and Cys conservation or hydrophobicity in the vicinity of the modified Cys do not define the specificity of S-nitrosylation. Instead, this analysis revealed a revised acid-base motif, which is located more distantly to the Cys and has its charged groups exposed. We hypothesize that, rather than being strictly used for direct activation of Cys, the modified acid-base motif is engaged in protein-protein interactions thereby contributing to trans-nitrosylation as an important and widespread mechanism for reversible modification of Cys with NO moiety. For proteins lacking the revised motif, we discuss alternative mechanisms including a potential role of nitrosoglutathione as a trans-acting agent.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation

Male accessory glands (MAGs) of insects are responsible for the production of many of the seminal fluid proteins and peptides that elicit physiological and behavioral responses in the post-mated female. In the yellow fever mosquito, Aedes aegypti, seminal fluid components are responsible for stimulating egg production, changing female behavior away from host-seeking toward egg-laying and mating refractoriness, but hitherto no behavior-modifying molecule from the MAGs has been structurally characterized. We now show using mass spectrometry and HPLC/ELISA that the MAG is a major site of synthesis of the biologically active decapeptide, Aea-HP-1 (pERPhPSLKTRFamide) that was first characterized by Matsumoto and colleagues in 1989 from mosquito head extracts and shown to have host-seeking inhibitory properties. The peptide is localized to the anterior portion of the MAG, occurs at high concentrations in the gland and is transferred to the female reproductive tract on copulation. Aea-HP-1 has a pyroglutamic acid at the N-terminus, an amidated carboxyl at the C-terminus and an unusual 4-hydroxyproline in position 4 of the peptide. The structure of the peptide with its blocked N- and C-termini confers resistance to metabolic inactivation by MAG peptidases; however the peptide persists for less than 2h in the female reproductive tract after copulation. Aea-HP-1 is not a ligand for the mosquito sex peptide/myoinhibitory peptide receptor. A. aegypti often mate close to the host and therefore it is possible that male-derived Aea-HP-1 induces short-term changes to female host-seeking behavior to reduce potentially lethal encounters with hosts soon after insemination.     

Nitrosative stress in plants

Nitrosative stress has become a usual term in the physiology of nitric oxide in mammalian systems. However, in plants there is much less information on this type of stress. Using olive leaves as experimental model, the effect of salinity on the potential induction of nitrosative stress was studied. The enzymatic l-arginine-dependent production of nitric oxide (NOS activity) was measured by ozone chemiluminiscence. The specific activity of NOS in olive leaves was 0.280nmol NOmg(-1) proteinmin(-1), and was dependent on l-arginine, NADPH and calcium. Salt stress (200mM NaCl) caused an increase of the l-arginine-dependent production of nitric oxide (NO), total S-nitrosothiols (RSNO) and number of proteins that underwent tyrosine nitration. Confocal laser scanning microscopy analysis using either specific fluorescent probes for NO and RSNO or antibodies to S-nitrosoglutathione and 3-nitrotyrosine, showed also a general increase of these reactive nitrogen species (RNS) mainly in the vascular tissue. Taken together, these findings show that in olive leaves salinity induces nitrosative stress, and vascular tissues could play an important role in the redistribution of NO-derived molecules during nitrosative stress.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation

Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE-C3 rat hepatoma cells. NO donors such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH-ethylester (GSH-EE) completely blocked the GSNO-mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO-mediated S-nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO-treated cells and subjected to immunoblot analysis. The anti-nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO-treated samples. All these results indicate that S-nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity.     

YccW is the m5C methyltransferase specific for 23S rRNA nucleotide 1962

Methylation at the 5-position of cytosine [m⁵C (5-methylcytidine)] occurs at three RNA nucleotides in Escherichia coli. All these modifications are at highly conserved nucleotides in the rRNAs, and each is catalyzed by its own m⁵C methyltransferase enzyme. Two of the enzymes, RsmB and RsmF, are already known and methylate 16S rRNA at nucleotides C967 and C1407, respectively. Here, we report the identity of the third E. coli m⁵C methyltransferase. Analysis of rRNAs by matrix-assisted laser desorption/ionization mass spectrometry showed that inactivation of the yccW gene leads to loss of m⁵C methylation at nucleotide 1962 in E. coli 23S rRNA. This methylation is restored by complementing the knockout strain with a plasmid-encoded copy of the yccW gene. Purified recombinant YccW protein retains its specificity for C1962 in vitro and methylates naked 23S rRNA isolated from the yccW knockout strain. However, YccW does not methylate assembled 50S subunits, and this is somewhat surprising as the published crystal structures show nucleotide C1962 to be fully accessible at the subunit interface. YccW-directed methylation at nucleotide C1962 is conserved in bacteria, and loss of this methylation in E. coli marginally reduces its growth rate. YccW had previously eluded identification because it displays only limited sequence similarity to the m⁵C methyltransferases RsmB and RsmF and is in fact more similar to known m⁵U (5-methyluridine) RNA methyltransferases. In keeping with the previously proposed nomenclature system for bacterial rRNA methyltransferases, yccW is now designated as the rRNA large subunit methyltransferase gene rlmI.     

A Non-Active-Site SET Domain Surface Crucial for the Interaction of MLL1 and the RbBP5/Ash2L Heterodimer within MLL Family Core Complexes

The mixed lineage leukemia-1 (MLL1) enzyme is a histone H3 lysine 4 (H3K4) monomethyltransferase and has served as a paradigm for understanding the mechanism of action of the human SET1 family of enzymes that include MLL1–MLL4 and SETd1a,b. Dimethylation of H3K4 requires a sub-complex including WRAD (WDR5, RbBP5, Ash2L, and DPY-30), which binds to each SET1 family member forming a minimal core complex that is required for multiple lysine methylation. We recently demonstrated that WRAD is a novel histone methyltransferase that preferentially catalyzes H3K4 dimethylation in a manner that is dependent on an unknown non-active-site surface from the MLL1 SET domain. Recent genome sequencing studies have identified a number of human disease-associated missense mutations that localize to the SET domains of several MLL family members. In this investigation, we mapped many of these mutations onto the three-dimensional structure of the SET domain and noticed that a subset of MLL2 (KMT2D, ALR, MLL4)-associated Kabuki syndrome missense mutations map to a common solvent-exposed surface that is not expected to alter enzymatic activity. We introduced these mutations into the MLL1 SET domain and observed that all are defective for H3K4 dimethylation by the MLL1 core complex, which is associated with a loss of the ability of MLL1 to interact with WRAD or with the RbBP5/Ash2L heterodimer. Our results suggest that amino acids from this surface, which we term the Kabuki interaction surface or KIS, are required for formation of a second active site within SET1 family core complexes.     

Genetically Derepressed Nucleoplasmic Stellate Protein in Spermatocytes of D. melanogaster Interacts with the Catalytic Subunit of Protein Kinase 2 and Carries Histone-Like Lysine-Methylated Mark

Summary

The X-chromosome-linked clusters of the tandemly repeated testis-specific Stellate genes of Drosophila melanogaster, encoding proteins homologous to the regulatory β-subunit of the protein kinase casein kinase 2 (CK2), are repressed in wild-type males. Derepression of Stellate genes in the absence of the Y chromosome or Y-linked crystal locus (crystal line) causes accumulation of abundant protein crystals in testes and different meiotic abnormalities, which lead to partial or complete male sterility. To understand the cause of abnormalities in chromosome behavior owing to Stellate overexpression, we studied subcellular localization of Stellate proteins by biochemical fractionation and immunostaining of whole testes. We showed that, apart from the known accumulation of Stellate in crystalline form, soluble Stellate was located exclusively in the nucleoplasm, whereas Stellate crystals were located mainly in the cytoplasm. Coimmunoprecipitation experiments revealed that the α-subunit of the protein kinase CK2 (CK2α) was associated with soluble Stellate. Interaction between soluble Stellate and CK2α in the nucleus could lead to modulations in the phosphorylation of nuclear targets of CK2 and abnormalities in the meiotic segregation of chromosomes. We also observed that Stellate underwent lysine methylation and mimicked trimethyl-H3K9 epigenetic modification of histone H3 tail.     

Structural evidence for co-evolution of the regulation of contraction and energy production in skeletal muscle

Skeletal muscle phosphorylase kinase (PhK) is a Ca²⁺-dependent enzyme complex, (αβγδ)₄, with the δ subunit being tightly bound endogenous calmodulin (CaM). The Ca²⁺-dependent activation of glycogen phosphorylase by PhK couples muscle contraction with glycogen breakdown in the “excitation-contraction-energy production triad.” Although the Ca²⁺-dependent protein-protein interactions among the relevant contractile components of muscle are well characterized, such interactions have not been previously examined in the intact PhK complex. Here we show that zero-length cross-linking of the PhK complex produces a covalent dimer of its catalytic γ and CaM subunits. Utilizing mass spectrometry, we determined the residues cross-linked to be in an EF hand of CaM and in a region of the γ subunit sharing high sequence similarity with the Ca²⁺-sensitive molecular switch of troponin I that is known to bind actin and troponin C, a homolog of CaM. Our findings represent an unusual binding of CaM to a target protein and supply an explanation for the low Ca²⁺ stoichiometry of PhK that has been reported. They also provide direct structural evidence supporting co-evolution of the coordinate regulation by Ca²⁺ of contraction and energy production in muscle through the sharing of a common structural motif in troponin I and the catalytic subunit of PhK for their respective interactions with the homologous Ca²⁺-binding proteins troponin C and CaM.