Inhibition of CYP2B4 by 2-ethynylnaphthalene: evidence for the co-binding of substrate and inhibitor within the active site

2-Ethynylnaphthalene (2EN) is an effective mechanism-based inhibitor of CYP2B4. There are two inhibitory components: (1) irreversible inactivation of CYP2B4 (a typical time-dependent inactivation), and (2) a reversible component. The reversible component was unusual in that the degree of inhibition was not simply a characteristic of the enzyme-inhibitor interaction, but dependent on the size of the substrate molecule used to monitor residual activity. The effect of 2EN on the metabolism of seven CYP2B4 substrates showed that it was not an effective reversible inhibitor of substrates containing a single aromatic ring; substrates with two fused rings were competitively inhibited by 2EN; and larger substrates were non-competitively inhibited. Energy-based docking studies demonstrated that, with increasing substrate size, the energy of 2EN and substrate co-binding in the active site became unfavorable precisely at the point where 2EN became a competitive inhibitor. Hierarchical docking revealed potential allosteric inhibition sites separate from the substrate binding site.     

The emerging role for Cullin 4 family of E3 ligases in tumorigenesis

As a member of the Cullin-RING ligase family, Cullin-RING ligase 4 (CRL4) has drawn much attention due to its broad regulatory roles under physiological and pathological conditions, especially in neoplastic events. Based on evidence from knockout and transgenic mouse models, human clinical data, and biochemical interactions, we summarize the distinct roles of the CRL4 E3 ligase complexes in tumorigenesis, which appears to be tissue- and context-dependent. Notably, targeting CRL4 has recently emerged as a noval anti-cancer strategy, including thalidomide and its derivatives that bind to the substrate recognition receptor cereblon (CRBN), and anticancer sulfonamides that target DCAF15 to suppress the neoplastic proliferation of multiple myeloma and colorectal cancers, respectively. To this end, PROTACs have been developed as a group of engineered bi-functional chemical glues that induce the ubiquitination-mediated degradation of substrates via recruiting E3 ligases, such as CRL4 (CRBN) and CRL2 (pVHL). We summarize the recent major advances in the CRL4 research field towards understanding its involvement in tumorigenesis and further discuss its clinical implications. The anti-tumor effects using the PROTAC approach to target the degradation of undruggable targets are also highlighted.     

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.     

The critical role of lipopolysaccharide in the upregulation of aquaporin 4 in glial cells treated with Shiga toxin

Background

In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells.

Results

Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema.

Conclusions

We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood–brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0184-5) contains supplementary material, which is available to authorized users.     

Coordinated and selective recruitment of eIF4E and eIF4G factors for potyvirus infection in Arabidopsis thaliana

The translation initiation factors eIF4E and eIF(iso)4E play a key role during virus infection in plants. During mRNA translation, eIF4E provides the cap-binding function and is associated with the protein eIF4G to form the eIF4F complex. Susceptibility analyses of Arabidopsis mutants knocked-out for At-eIF4G genes showed that eIF4G factors are indispensable for potyvirus infection. The colonization pattern by a viral recombinant carrying GFP indicated that eIF4G is involved at a very early infection step. Like eIF4E, eIF4G isoforms are selectively recruited for infection. Moreover, the eIF4G selective involvement parallels eIF4E recruitment. This is the first report of a coordinated and selective recruitment of eIF4E and eIF4G factors, suggesting the whole eIF4F recruitment.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

Adipose tissue inflammation and cancer cachexia: possible role of nuclear transcription factors

Cancer cachexia is a multifaceted syndrome whose aetiology is extremely complex and is directly related to poor patient prognosis and survival. Changes in lipid metabolism in cancer cachexia result in marked reduction of total fat mass, increased lipolysis, total oxidation of fatty acids, hyperlipidaemia, hypertriglyceridaemia, and hypercholesterolaemia. These changes are believed to be induced by inflammatory mediators, such as tumour necrosis factor-α (TNF-α) and other factors.Attention has recently been drawn to the current theory that cachexia is a chronic inflammatory state, mainly caused by the host’s reaction to the tumour. Changes in expression of numerous inflammatory mediators, notably in white adipose tissue (WAT), may trigger several changes in WAT homeostasis. The inhibition of adipocyte differentiation by PPARγ is paralleled by the appearance of smaller adipocytes, which may partially account for the inhibitory effect of PPARγ on inflammatory gene expression. Furthermore, inflammatory modulation and/or inhibition seems to be dependent on the IKK/NF-κB pathway, suggesting that a possible interaction between NF-κB and PPARγ is required to modulate WAT inflammation induced by cancer cachexia.In this article, current literature on the possible mechanisms of NF-κB and PPARγ regulation of WAT cells during cancer cachexia are discussed. This review aims to assess the role of a possible interaction between NF-κB and PPARγ in the setting of cancer cachexia as well as its significant role as a potential modulator of chronic inflammation that could be explored therapeutically.     

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups,because of an enhanced reactivity of its thiol ester

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α₂-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s'' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1''s thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1''s protease inhibition at pH 5, indicating that A2ML1''s hydroxyl reactivity is not an adaption to its acidic epidermal environment.     

Translation initiation factors eIF4E and eIFiso4E are required for polysome formation and regulate plant growth in tobacco

Eukaryotic initiation factor eIF4E plays a pivotal role in translation initiation. As a component of the ternary eIF4F complex, eIF4E interacts with the mRNA cap structure to facilitate recruitment of the 40S ribosomal subunit onto mRNA. Plants contain two distinct cap-binding proteins, eIF4E and eIFiso4E, that assemble into different eIF4F complexes. To study the functional roles of eIF4E and eIFiso4E in tobacco, we isolated two corresponding cDNAs, NteIF4E1 and NteIFiso4E1, and used these to deplete cap-binding protein levels in planta by antisense downregulation. Antibodies raised against recombinant NteIF4E1 detected three distinct cap-binding proteins in tobacco leaf extracts; NteIF4E and two isoforms of NteIFiso4E. The three cap-binding proteins were immuno-detected in all tissues analysed and were coordinately regulated, with peak expression in anthers and pollen. Transgenic tobacco plants showing significant depletion of either NteIF4E or the two NteIFiso4E isoforms displayed normal vegetative development and were fully fertile. Interestingly, NteIFiso4E depletion resulted in a compensatory increase in NteIF4E levels, whereas the down-regulation of NteIF4E did not trigger a reciprocal increase in NteIFiso4E levels. The antisense depletion of both NteIF4E and NteIFiso4E resulted in plants with a semi-dwarf phenotype and an overall reduction in polyribosome loading, demonstrating that both eIF4E and eIFiso4E support translation initiation in planta, which suggests their potential role in the regulation of plant growth.     

Structure and functions of the translation initiation factor eIF4E and its role in cancer development and treatment

In eukaryotic cells, protein synthesis is a complex and multi-step process that has several mechanisms to start the translation including cap-dependent and cap-independent initiation. The translation control of eukaryotic gene expression occurs principally at the initiation step. In this context, it is critical that the eukaryotic translation initiation factor eIF4E bind to the 7-methylguanosine (m7G) cap present at the 5′-UTRs of most eukaryotic mRNAs. Combined with other initiation factors, eIF4E mediates the mRNA recruitment on ribosomes to start the translation. Moreover, the eIF4E nuclear bodies are involved in the export of specific mRNAs from the nucleus to the cytoplasm. In this review, we focus on the eIF4E structure and its physiological functions, and describe the role of eIF4E in cancer development and progression and the current therapeutic strategies to target eIF4E.     

Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of FcepsilonRI and their association with detergent-resistant membranes.

Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (FcepsilonRI) by the Src family kinase Lyn is the first known biochemical step that occurs during activation of mast cells and basophils after cross-linking of FcepsilonRI by antigen. The hypothesis that specialized regions in the plasma membrane, enriched in sphingolipids and cholesterol, facilitate the coupling of Lyn and FcepsilonRI was tested by investigating functional and structural effects of cholesterol depletion on Lyn/FcepsilonRI interactions. We find that cholesterol depletion with methyl-beta-cyclodextrin substantially reduces stimulated tyrosine phosphorylation of FcepsilonRI and other proteins while enhancing more downstream events that lead to stimulated exocytosis. In parallel to its inhibition of tyrosine phosphorylation, cholesterol depletion disrupts the interactions of aggregated FcepsilonRI and Lyn on intact cells and also disrupts those interactions with detergent-resistant membranes that are isolated by sucrose gradient ultracentrifugation of lysed cells. Importantly, cholesterol repletion restores receptor phosphorylation together with the structural interactions. These results provide strong evidence that membrane structure, maintained by cholesterol, plays a critical role in the initiation of FcepsilonRI signaling.     

The role of particles on the biogeochemical cycling of domoic acid and its isomers in natural water matrices

The adsorption of dissolved domoic acid (DA) and its geometrical isomers was assessed in aqueous solutions containing various types of particles. In one series of experiments carried out in coastal seawater, detectable net adsorption of 100 nM DA occurred only onto natural seawater particles (unfiltered seawater) and 0.5 g L⁻¹ chromatographic silica (18%) in 0.2 μm-filtered seawater. Some net adsorption (<5%) also occurred in the 0.5 g L⁻¹ suspension of estuarine sediment and 0.5 g L⁻¹ solution of humic acid in filtered seawater. No losses were seen in 0.5 g L⁻¹ suspensions of illite, kaolinite, montmorillonite, and silica sand. Biological degradation accounted for small losses (8–10%) in filtered seawater without particles. A second series of experiments using organic-free, <5 μm fractions of kaolinite and montmorillonite in deionized water (DIW) demonstrated that 70% of DA adsorbed onto kaolinite, but only 5% onto montmorillonite. Geometrical isomers of DA (iso-DA D, E, and F) showed negligible adsorption (0–8%) onto a variety of particles in filtered seawater, suggesting that major ions in seawater neutralize electrostatic attractions between particles and DA isomers. These results suggest that DA and its isomers are relatively hydrophilic and not particle reactive. Our data suggest that photochemical and biological degradation of dissolved DA and its isomers appears to occur in bulk surface seawater and its transport to bottom sediments must be mainly biologically driven.     

Investigation of the role of cytochrome P450 2B4 active site residues in substrate metabolism based on crystal structures of the ligand-bound enzyme

Based on the X-ray crystal structures of 4-(4-chlorophenyl)imidazole (4-CPI)- and bifonazole (BIF)-bound P450 2B4, eight active site mutants at six positions were created in an N-terminal modified construct termed 2B4dH and characterized for enzyme inhibition and catalysis. I363A showed a >4-fold decrease in differential inhibition by BIF and 4-CPI (IC(50,BIF)/IC(50,4-CPI)). F296A, T302A, I363A, V367A, and V477A showed a 2-fold decreased k(cat) for 7-ethoxy-4-trifluoromethylcoumarin O-deethylation, whereas V367A and V477F showed an altered K(m). T302A, V367L, and V477A showed >4-fold decrease in total testosterone hydroxylation, whereas I363A, V367A, and V477F showed altered stereo- and regioselectivity. Interestingly, I363A showed a 150-fold enhanced k(cat)/K(m) with testosterone, and yielded a new metabolite. Furthermore, testosterone docking into three-dimensional models of selected mutants based on the 4-CPI-bound structure suggested a re-positioning of residues 363 and 477 to yield products. In conclusion, our results suggest that the 4-CPI-bound 2B4dH/H226Y crystal structure is an appropriate model for predicting enzyme catalysis.     

Association between genes encoding components of the IL-4/IL-4 receptor pathway and dermatitis in children

Objective

To determine whether IL-4, IL-4Rα and STAT6 polymorphisms are associated with susceptibility to dermatitis in Egyptian children.

Methods

We genotyped three groups of children, consisting of 106 atopic dermatitis (AD) children, 95 non-AD children, and 100 of healthy controls, for IL-4 (− 590 C/T), (− 33 C/T), IL-4Rα (I50V), (Q576R) and STAT6 (2964 G/A), (2892 C/T) gene polymorphisms using PCR-RFLP assay. Total serum IgE and serum IL-4 levels were detected by ELISA.

Results

There was a non-significant association of IL-4 − 590 C/T, − 33 C/T polymorphisms in the children with non-AD or those with AD when compared with the controls. We identified a significant association between IL-4Rα I50V, Q576R polymorphisms and dermatitis susceptibility in AD (p = 0.002, < 0.001 respectively), whereas no such association was observed in non-AD group (p = 0.52, 0.99 respectively). A significant association between STAT6 polymorphisms and both types of dermatitis was found. Patients who were carriers of IL4 − 590C, IL-4Rα I50V G, STAT6 2964 A and STAT6 2892 T had an increased risk of AD [OR and 95% CI: 3.2 (2.5–4.2), p = 0.005]. Furthermore, there was no relation between each polymorphism and serum IL-4 level (p > 0.05 for each) while homozygosity for the risk alleles of IL-4, IL-4Rα and STAT6 SNPs were significantly associated with increased total IgE levels in all subjects.

Conclusion

In Egyptian children, the IL-4Rα and the STAT6 polymorphism may play a role in susceptibility to AD. In addition, gene–gene interaction between the IL-4, the IL-4Rα and the STAT6 significantly increases an individual's susceptibility to AD.     

IL-4-induced upregulation of adenine nucleotide translocase 3 and its role in Th cell survival from apoptosis

We have identified mitochondrial adenine nucleotide translocase (ANT)3 as a novel target up-regulated by IL-4 in human T cells. The IL-4-induced ANT3 expression is dependent on tyrosine kinase, NF-kappaB, PI3K/Akt, and Erk pathways. In fact, IL-4 induced specific activation of NF-kappaB, Akt, and Erk in Jurkat T cells and partially rescued these cells from dexamethasone-induced apoptosis. The IL-4-mediated T cell survival was blocked by inhibitors of tyrosine kinase, NF-kappaB, PI3K/Akt, and Erk. During the IL-4-induced T cell rescue, there was a concomitant increase in ANT3, nuclear NF-kappaB, and Bcl-2 and a decrease in ANT1, I-kappaB, and mitochondrial Bax-alpha levels. Importantly, overexpression of ANT3 effectively protected T cells from dexamethasone-induced apoptosis, while forced expression of ANT1 caused apoptosis. In contrast, siRNA knock-out of ANT3 expression induced T cell apoptosis and blocked the IL-4-mediated cell survival. Together these results suggest that ANT3 has a potential role in Th cell survival and immune cell homeostasis.     

Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

The major role of glutathione in the metabolism and excretion of N,N-dimethyl-4-aminoazobenzene in the rat

In the normal rat given a single dose of one mg N,N-dimethyl-4-aminoazobenzene (DAB) via the hepatic portal vein the following biliary metabolites reached their maximal rates of excretion in the sequence: 4'-sulphonyloxy-DAB, N-(glutathione-S-methylene)-4-aminoazobenzene (GSCH2AB), 4'-sulphonyloxy-N-methyl-4-aminoazobenzene (4'-sulphonyloxy-MAB) 4'-sulphonyloxy-GSCH2AB and MAB-4'-beta-glucuronide. The unusual and relatively unstable N-methylene glutathione conjugates were major metabolites accounting for up to 70% of the whole. It was shown that all the 4-aminoazobenzene (AB) and perhaps all of the 4'-sulphonyloxy-AB, which may be observed in bile, are artefacts due to decomposition of GSCH2AB and 4'-sulphonyloxy-GSCH2AB respectively and that biliary excretion of N-methyl oxidised products of MAB and 4'-hydroxy-MAB is dependent on their conversion to the GSH conjugates, GSCH2AB and 4'-hydroxy-GSCH2AB respectively. Sulphotransferase inhibition by pentachlorophenol caused a reduction in the excretion of all sulphate conjugates, but biliary excretion as a whole was not reduced significantly due to a compensatory increase in the excretion of MAB-4'-beta-glucuronide and the appearance of 4'-OH-GSCH2AB. Glutathione (GSH) depletion by diethylmaleate caused a reduction in biliary metabolites of DAB by lowering the levels of GSH conjugates. This was because the amount of N-methyl oxidation of MAB and 4'-hydroxy-MAB were proportional to the amount of GSH present. The fall in N-methyl oxidation was not compensated for by an increase in 4'-hydroxylation and was accompanied by a delay in the appearance of 4'-hydroxylated metabolites. The administration of potential precursors of 4'-sulphonyloxy-GSCH2AB establishes the sequence of reactions resulting in its formation to be 4'-hydroxylation, N-methyl oxidation, GSH conjugation and O-sulphation.