Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner

Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products.     

Identification of a replication-independent replacement histone H3 in the basidiomycete Ustilago maydis

Ustilago maydis is a haploid basidiomycete with single genes for two distinct histone H3 variants. The solitary U1 gene codes for H3.1, predicted to be a replication-independent replacement histone. The U2 gene is paired with histone H4 and produces a putative replication-coupled H3.2 variant. These predictions were evaluated experimentally. U2 was confirmed to be highly expressed in the S phase and had reduced expression in hydroxyurea, and H3.2 protein was not incorporated into transcribed chromatin of stationary phase cells. Constitutive expression of U1 during growth produced ~25% of H3 as H3.1 protein, more highly acetylated than H3.2. The level of H3.1 increased when cell proliferation slowed, a hallmark of replacement histones. Half of new H3.1 incorporated into highly acetylated chromatin was lost with a half-life of 2.5 h, the fastest rate of replacement H3 turnover reported to date. This response reflects the characteristic incorporation of replacement H3 into transcribed chromatin, subject to continued nucleosome displacement and a loss of H3 as in animals and plants. Although the two H3 variants are functionally distinct, neither appears to be essential for vegetative growth. KO gene disruption transformants of the U1 and U2 loci produced viable cell lines. The structural and functional similarities of the Ustilago replication-coupled and replication-independent H3 variants with those in animals, in plants, and in ciliates are remarkable because these distinct histone H3 pairs of variants arose independently in each of these clades and in basidiomycetes.     

Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.     

Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

Phylogenetic reconstruction at the species and intraspecies levels in the genus Pisum (L.) (peas) using a histone H1 gene

A phylogenetic analysis of the genus Pisum (peas), embracing diverse wild and cultivated forms, which evoke problems with species delimitation, was carried out based on a gene coding for histone H1, a protein that has a long and variable functional C-terminal domain. Phylogenetic trees were reconstructed on the basis of the coding sequence of the gene His5 of H1 subtype 5 in 65 pea accessions. Early separation of a clear-cut wild species Pisum fulvum is well supported, while cultivated species Pisum abyssinicum appears as a small branch within Pisum sativum. Another robust branch within P. sativum includes some wild and almost all cultivated representatives of P. sativum. Other wild representatives form diverse but rather subtle branches. In a subset of accessions, PsbA-trnH chloroplast intergenic spacer was also analysed and found less informative than His5. A number of accessions of cultivated peas from remote regions have a His5 allele of identical sequence, encoding an electrophoretically slow protein product, which earlier attracted attention as likely positively selected in harsh climate conditions. In PsbA-trnH, a 8bp deletion was found, which marks cultivated representatives of P. sativum.     

On the species-specific biotransformation of dibenzo[a,l]pyrene

We were aimed at investigating the activation of the carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) in Chinese hamster V79 cells that express single human, rat or fish cytochrome P450 (CYP) enzymes. DB[a,l]P is detectable in environmental samples and has been characterized as the most potent carcinogenic species among all PAHs as yet tested in rodent bioassays. Metabolite profiles and metabolite-dependent cytotoxic and clastogenic activities were monitored. The total turnover of CYP-mediated transformation of DB[a,l]P was as follows: human CYP1B1>fish CYP1A1 approximately human CYP1A1>rat CYP1A2>rat CYP1A1. By contrast, enzyme forms that are not classified as being members of family CYP1, such as CYP2A6, 2E1, 2B1, and 3A4, failed to catalyze any detectable conversion of this substrate. All CYP1A1 enzymes tested formed both the K-region trans-8,9- and the trans-11,12-dihydrodiol, whereas human CYP1B1 failed to catalyze K-region activation. In cells expressing human or fish CYP1A1, human CYP1B1, and rat CYP1A2, the (-)-trans-11,12-dihydrodiol was formed enantiospecifically. DB[a,l]P-dependent cytotoxicities (EC(50)) were found in the following order: human CYP1A1 (12 nM)>fish CYP1A1 (30 nM)>human CYP1B1 (45 nM)>other forms. In addition, an appreciable micronuclei formation was detected in human CYP1A1- and 1B1-expressing cells during exposure to DB[a,l]P. Our study demonstrates that human CYP1A1, 1B1 and fish CYP1A1 are able to transform DB[a,l]P into genotoxic derivatives in appreciable amounts. In contrast, CYP enzymes from rat predominantly target the K-region of DB[a,l]P and thus are serving more a rather protective route of biotransformation. Together our data suggest that humans might be more susceptible to DB[a,l]P-induced carcinogenicity than rats.     

Kinetic isotope effects reveal the presence of significant secondary structure in the transition state for the folding of the N-terminal domain of L9

Our present understanding of the nature of the transition state for protein folding depends predominantly on studies where individual side-chain contributions are mapped out by mutational analysis (phi value analysis). This approach, although extremely powerful, does not in general provide direct information about the formation of backbone hydrogen bonds. Here, we report the results of amide H/D isotope effect studies that probe the development of hydrogen bonded interactions in the transition state for the folding of a small alpha-beta protein, the N-terminal domain of L9. Replacement of amide protons by deuterons in a solvent of constant isotopic composition destabilized the domain, decreasing both its T(m) and Delta G(0) of unfolding. The folding rate also decreased. The parameter Phi(H/D), defined as the ratio of the effect of isotopic substitution upon the activation free energy to the equilibrium free energy was determined to be 0.6 in a D(2)O background and 0.75 in a H(2)O background, indicating that significant intraprotein hydrogen bond interactions are developed in the transition state for the folding of NTL9. The value is in remarkably good agreement with more traditional measures of the position of the transition state, which report on the relative burial of surface area. The results provide a picture of a compact folding transition state containing significant secondary structure. Indirect analysis argues that the bulk of the kinetic isotope effect arises from the beta-sheet-rich region of the protein, and suggests that the development of intraprotein hydrogen bonds in this region plays a critical role in the folding of NTL9.     

Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors

The pH-dependent insertion of pHLIP across membranes is proving to be a useful property for targeting acidic tissues or tumors and delivering drugs attached to its C-terminus. It also serves as a model peptide for studies of protein insertion into membranes, so further elucidation of the insertion mechanism of pHLIP and its features is desirable. We examine how the peptide perturbs a model phosphatidylcholine membrane and how it associates with the lipid bilayer using an array of fluorescence techniques, including fluorescence anisotropy measurements of TMA-DPH anchored in bilayers, quenching of pHLIP fluorescence by brominated lipids and acrylamide, and measurements of energy transfer between aromatic residues of pHLIP and TMA-DPH. When pHLIP is bound to the surface of bilayers near neutral pH, the membrane integrity is preserved whereas the elastic properties of bilayers are changed as reported by an increase of membrane viscosity. When it is inserted, there is little perturbation of the lipids. The results also suggest that pHLIP can bind to the membrane surface in a shallow or a deep mode depending on the phase state of the lipids. Using parallax analysis, the change of the penetration depth of pHLIP was estimated to be 0.4 Å from the bilayer center and 2.8 Å from the membrane surface after the liquid-to-gel phase transition.     

Molecular dissection of the inhibitor binding pocket of mitotic kinesin Eg5 reveals mutants that confer resistance to antimitotic agents

The mitotic kinesin Eg5 plays an essential role in establishing the bipolar spindle. Recently, several antimitotic inhibitors have been shown to share a common binding region on Eg5. Considering the importance of Eg5 as a potential drug target for cancer chemotherapy it is essential to understand the molecular mechanism, by which these agents block Eg5 activity, and to determine the "key residues" crucial for inhibition. Eleven residues in the inhibitor binding pocket were mutated and the effects were monitored by kinetic analysis and mass spectrometry. Mutants R119A, D130A, P131A, I136A, V210A, Y211A and L214A abolish the inhibitory effect of monastrol. Results for W127A and R221A are less striking, but inhibitor constants are still considerably modified compared to wild-type Eg5. Only one residue, Leu214, was found to be essential for inhibition by STLC. W127A, D130A, V210A lead to increased K(i)(app) values, but binding of STLC is still tight. R119A, P131A, Y211A and R221A convert STLC into a classical rather than a tight-binding inhibitor with increased inhibitor constants. These results demonstrate that monastrol and STLC interact with different amino acids within the same binding region, suggesting that this site is highly flexible to accommodate different types of inhibitors. The drug specificity is due to multiple interactions not only with loop L5, but also with residues located in helices alpha2 and alpha3. These results suggest that tumour cells might develop resistance to Eg5 inhibitors, by expressing Eg5 point mutants that retain the enzyme activity, but prevent inhibition, a feature that is observed for certain tubulin inhibitors.     

Monomer-dimer transition of the conserved N-terminal domain of the mammalian peroxisomal matrix protein import receptor, Pex14p

Pex14p is a central component of the peroxisomal matrix protein import machinery. In the recently determined crystal structure, a characteristic face consisting of conserved residues was found on a side of the conserved N-terminal domain of the protein. The face is highly hydrophobic, and is also the binding site for the WXXXF/Y motif of Pex5p. We report herein the dimerization of the domain in the isolated state. The homo-dimers are in equilibrium with the monomers. The homo-dimers are completely dissociated into monomers by complex formation with the WXXXF/Y motif peptide of Pex5p. A putative dimer model shows the interaction between the conserved face and the PXXP motif of another protomer. The model allows us to discuss the mechanism of the oligomeric transition of the full-length Pex14p modulated by the binding of other peroxins.     

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm “Ming” lethal egg mutant

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-e^m) lose water and become concaved around 1 h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e^m mutant to 360 kb on chromosome 10 using 2287 F₂ individuals. Using expression analysis and RNA interference, the best l-e^m candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~ 1.9 kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a > 100 kb DNA fragment in the l-e^m mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e^m mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e^m mutant. These results indicate that BmEP80 is responsible for the l-e^m mutation.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.     

Computational modeling of cell adhesion and movement using a continuum-kinetics approach

Adhesion of leukocytes to substrate involves the coupling of disparate length and timescales between molecular mechanics and macroscopic transport, and existing models of cell adhesion do not use full cellular information. To address these challenges, a multiscale computational approach for studying the adhesion of a cell on a substrate is developed and assessed. The cellular level model consists of a continuum representation of the field equations and a moving boundary tracking capability to allow the cell to change its shape continuously. At the receptor-ligand level, a bond molecule is mechanically represented by a spring. Communication between the macro/micro- and nanoscale models is facilitated interactively during the computation. The computational model is assessed using an adherent cell, rolling and deforming along the vessel wall under imposed shear flows. Using this approach, we first confirm existing numerical and experimental results. In this study, the intracellular viscosity and interfacial tension are found to directly affect the rolling of a cell. Our results also show that the presence of a nucleus increases the bond lifetime, and decreases the cell rolling velocity. Furthermore, it is found that a cell with a larger diameter rolls faster, and decreases the bond lifetime. This study shows that cell rheological properties have significant effects on the adhesion process contrary to what has been hypothesized in most literature.     

Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure

Due to their dynamic ensemble nature and a deficiency of experimental restraints, disordered states of proteins are difficult to characterize structurally. Here, we have expanded upon our previous work on the unfolded state of the Drosophila drk N-terminal (drkN) SH3 domain with our program ENSEMBLE, which assigns population weights to pregenerated conformers in order to calculate ensembles of structures whose properties are collectively consistent with experimental measurements. The experimental restraint set has been enlarged with newly measured paramagnetic relaxation enhancements from Cu(2+) bound to an amino terminal Cu(2+)-Ni(2+) binding (ATCUN) motif as well as nuclear Overhauser effect (NOE) and hydrogen exchange data from recent studies. In addition, two new pseudo-energy minimization algorithms have been implemented that have dramatically improved the speed of ENSEMBLE population weight assignment. Finally, we have greatly improved our conformational sampling by utilizing a variety of techniques to generate both random structures and structures that are biased to contain elements of native-like or non-native structure. Although it is not possible to uniquely define a representative structural ensemble, we have been able to assess various properties of the drkN SH3 domain unfolded state by performing ENSEMBLE minimizations of different conformer pools. Specifically, we have found that the experimental restraint set enforces a compact structural distribution that is not consistent with an overall native-like topology but shows preference for local non-native structure in the regions corresponding to the diverging turn and the beta5 strand of the folded state and for local native-like structure in the region corresponding to the beta6 and beta7 strands. We suggest that this approach could be generally useful for the structural characterization of disordered states.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Thermodynamic determination of the binding constants of angiotensin-converting enzyme inhibitors by a displacement method

Somatic angiotensin I-converting enzyme (s-ACE) plays a central role in blood pressure regulation and has been the target of most antihypertensive drugs. A displacement isothermal titration calorimetry method has been used to accurately determine the binding constant of three strong s-ACE inhibitors. Under the experimental conditions studied in this work, the relative potency of the inhibitors was determined to be enalaprilat>lisinopril>captopril. We analyze the thermodynamic behaviour of the binding process using the new structural information provided by the ACE structures, as well as the conformational changes that occur upon binding.