The fission yeast COP9/signalosome is involved in cullin modification by ubiquitin-related Ned8p

Background

The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex.

Results

We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to ～ 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN.

Conclusions

Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.     

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

Background

Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies.

Results

Canine TK1 phosphorylated the thymidine (dThd) analog 3'-azido-thymidine (AZT) as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26?kDa (dog) and 25?kDa (human) for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active.

Conclusions

These results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.     

Identification of cellular pathways affected by Sortin2, a synthetic compound that affects protein targeting to the vacuole in Saccharomyces cerevisiae

Background

Sortin2 is a low mass compound that interferes with vacuolar delivery of proteins in plants and yeast. The Sortin2 phenotype was tested in Arabidopsis thaliana and found to be reversible upon drug removal, demonstrating the ability of chemical genomics to induce reversible phenotypes that would be difficult to achieve using conventional genetics [1]. However, standard genetic methods can be used to identify drug target pathways in a high-throughput manner.

Results

In this study, we analyzed structure-function relationships of Sortin2 using structural analogues. The results show the key roles of sulphite substitution and a benzoic acid group. A Sortin 2 hypersensitivity screen for the induced secretion of a vacuolar cargo protein was done utilizing a yeast haploid deletion library. Using bioinformatics approaches, we highlighted functional information about the cellular pathways affected by drug treatment which included protein sorting and other endomembrane system-related processes.

Conclusion

Chemical, genomic and genetics approaches were used to understand the mode of action of Sortin2, a bioactive chemical that affects the delivery of a vacuolar protein. Critical features of Sortin2 structure necessary for bioactivity suggest a binding pocket that may recognize two ends of Sortin2. The genome-wide screen shows that Sortin2 treatment in yeast affects primarily components within the endomembrane system. This approach allowed us to assign putative functions in protein sorting for fifteen genes of previously unknown function.     

DNA as a Target for Anticancer Phen-Imidazole Pd(II) Complexes

Imidazole ring is a known structure in many natural or synthetic drug molecules and its metal complexes can interact with DNA and do the cleavage. Hence, to study the influence of the structure and size of the ligand on biological behavior of metal complexes, two water-soluble Pd(II) complexes of phen and FIP ligands (where phen is 1,10-phenanthroline and FIP is 2-(Furan-2-yl)-1H–Imidazo[4,5-f][1, 10]phenanthroline) with the formula of [Pd(phen)(FIP)](NO₃)₂ and [Pd(FIP)₂]Cl₂, that were activated against chronic myelogenous leukemia cell line, K562, were selected. Also, the interaction of these anticancer Pd(II) complexes with highly polymerized calf thymus DNA was extensively studied by means of electronic absorption, fluorescence, and circular dichroism in Tris-buffer. The results showed that the binding was positive cooperation and [Pd(phen)(FIP)](NO₃)₂ (K _f = 127 M^-1 G = 1.2) exhibited higher binding constant and number of binding sites than [Pd(FIP)₂]Cl₂ (K _f = 13 M^-1 G = 1.03) upon binding to DNA. The fluorescence data indicates that quenching effect for [Pd(phen)(FIP)](NO₃)₂ (K _SV = 58 mM^?1) was higher than [Pd(FIP)₂]Cl₂ (K _SV = 12 mM^?1). Also, [Pd(FIP)₂]Cl₂ interacts with ethidium bromide-DNA, as non-competitive inhibition, and can bind to DNA via groove binding and [Pd(phen)(FIP)](NO₃)₂ can intercalate in DNA. These results were confirmed by circular dichroism spectra. Docking data revealed that longer complexes have higher interaction energy and bind to DNA via groove binding.

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Graphical Abstract Two anticancer Pd(II) complexes of imidazole derivative have been synthesized and interacted with calf thymus DNA. Modes of binding have been studied by electronic absorption, fluorescence, and CD measurements. [Pd(FIP)₂]Cl₂ can bind to DNA via groove binding while intercalation mode of binding is observed for [Pd(phen)(FIP)](NO₃)₂.

     

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

Background

The inorganic (P_i) phosphate transporter (PiT) family comprises known and putative Na⁺- or H⁺-dependent P_i-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with P_i to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na⁺-dependent P_i (NaP_i) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins.

Results

We show that the human PiT2 histidine, H₅₀₂, and the human PiT1 glutamate, E₇₀, - both conserved in eukaryotic PiT family members - are critical for P_i transport function. Noticeably, human PiT2 H₅₀₂ is located in the C-terminal PiT family signature sequence, and human PiT1 E₇₀ is located in ProDom domains characteristic for all PiT family members. A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR₂₅₄-V₄₈₃), was found to be a fully functional P_i transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL₁₈₃-V₄₈₃) did also support P_i transport albeit at very low levels.

Conclusions

The results suggest that the overall structure of the P_i-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship.     

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes

Background

The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.

Results

To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo.

Conclusions

Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.     

Presence of thiamine pyrophosphate in mammalian peroxisomes

Background

Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the α-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.

Results

Because of the crucial role of the cofactor TPP, we reanalyzed its subcellular localization in rat liver. In addition to the known mitochondrial and cytosolic pools, we demonstrated, for the first time, that peroxisomes contain TPP (177 ± 2 pmol/mg protein). Subsequently, we verified whether TPP could be synthesized from its precursor thiamine, in situ, by a peroxisomal thiamine pyrophosphokinase (TPK). However, TPK activity was exclusively recovered in the cytosol.

Conclusion

Our results clearly indicate that mammalian peroxisomes do contain TPP but that no pyrophosphorylation of thiamine occurs in these organelles, implying that thiamine must enter the peroxisome already pyrophosphorylated. Consequently, TPP entry may depend on a specific transport system or, in a bound form, on HACL1 translocation.     

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

Background

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model.

Results

Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β₁ subunit protein levels. Both sGC activity and β₁ subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β₁ subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β₁ subunits.

Conclusions

These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β₁ subunit stability.     

Identification of glycosylated marker proteins of epithelial polarity in MDCK cells by homology driven proteomics

Background

MDCK cells derived from canine kidney are an important experimental model system for investigating epithelial polarity in mammalian cells. Monoclonal antibodies against apical gp114 and basolateral p58 have served as important tools in these studies. However, the molecular identity of these membrane glycoproteins has not been known.

Results

We have identified the sialoglycoprotein gp114 as a dog homologue of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Gp114 was enriched from tissue culture cells by subcellular fractionation and immunoaffinity chromatography. The identification was based on tandem mass spectrometry and homology based proteomics. In addition, the p58 basolateral marker glycoprotein was found to be the β subunit of Na⁺K⁺-ATPase.

Conclusion

Gp114 has been characterized previously regarding glycosylation dependent trafficking and lipid raft association. The identification as a member of the canine CEACAM family will enable synergy between the fields of epithelial cell biology and other research areas. Our approach exemplifies how membrane proteins can be identified from species with unsequenced genomes by homology based proteomics. This approach is applicable to any model system.     

Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

Background

The repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity. Though not absolutely required, non-core regions of RAG1 and RAG2 have been shown to influence the efficiency and fidelity of the recombination reaction.

Results

Using a partial proteolysis approach in combination with bioinformatics analyses, we identified the domain boundaries of a structural domain that is present in the 380-residue N-terminal non-core region of RAG1. We term this domain the Central Non-core Domain (CND; residues 87-217).

Conclusions

We show how the CND alone, and in combination with other regions of non-core RAG1, functions in nuclear localization, zinc coordination, and interactions with nucleic acid. Together, these results demonstrate the multiple roles that the non-core region can play in the function of the full length protein.     

Template-Free Hydrothermal Synthesis of Hollow α-FeOOH Urchin-Like Spheres and Their Conversion to α-Fe2O3 Under Low-Temperature Thermal Treatment in Air

Hollow α-FeOOH urchin-like spheres were synthesized by a simple hydrothermal method at 160 °C for 12 h and their thermal conversion to hollow α-Fe₂O₃ urchin-like spheres was performed at 300 °C for 2 h in air. The results from X-ray diffraction and electron microscopy analyses reveal that hollow α-FeOOH urchin-like spheres were completely transformed to hollow α-Fe₂O₃ urchin-like spheres without a significant morphological change. Also, the effect of hydrothermal treatment temperature (170–200 °C for 12 h) on the phase structure and morphology of the final product was investigated. Pure α-FeOOH, the mixture of α-FeOOH and α-Fe₂O₃, and pure α-Fe₂O₃ with different morphologies were obtained at <180, 180–190 and 200 °C, respectively. The obtained materials can be used in the photodegradation of organic pollutants under visible light irradiation.     

Apoptosis in BEL-7402 cells induced by ruthenium(II) complexes through a ROS-mediated mitochondrial pathway

Three Ru(II) polypyridyl complexes [Ru(dmb)₂(HMSPIP)](ClO₄)₂ (1), [Ru(phen)₂(HMSPIP)](ClO₄)₂ (2) and [Ru(dmp)₂(HMSPIP)](ClO₄)₂ (3) were synthesized and characterized. The cytotoxicity in vitro, apoptosis, cell cycle arrest, reactive oxygen species and mitochondrial membrane potential were assayed. The IC₅₀ values of complexes 1, 2 and 3 toward BEL-7402, A549, MG-63 and SK-BR-3 cell lines ranged from 10.9 ± 1.6 to 42.0 ± 3.4 μM. Complexes 1, 2 and 3 can effectively induce apoptosis and inhibit the growth of BEL-7402 cells at the G2/M phase. These complexes can enhance the level of reactive oxygen species and induce decrease in the mitochondrial membrane potential. Additionally, complex 2 can down-regulate the expression of antiapoptotic protein of Bcl-2 protein and up-regulate the levels of proapoptotic protein Bim in BEL-7402 cells.     

Comparative investigation of the copper(II) complexes of (R)-, (S)- and (R,S)-1-phenyl-N,N-bis(pyridine-3-ylmethyl)ethanamine along with the related complex of (R,S)-1-cyclohexyl-N,N-bis(pyridine-3-ylmethyl)ethanamine. Synthetic, magnetic, and structural studies

The interaction of the enantiopure (R)- and (S)-1-phenyl-N,N-bis(pyridine-3- ylmethyl)ethanamine ligands, R-L ¹ and S-L ¹ , with copper(II) chloride followed by addition of hexafluorophosphate resulted in the isolation of the corresponding enantiomeric complexes [Cu(R-L ¹ )Cl](PF₆) (1), [Cu(S-L ¹ )Cl](PF₆) (2) and [Cu(S-L ¹ )Cl](PF₆)??0.5Et₂O (3), in which dimerization occurs through two long Cu??????Cl interactions, the ??-chloro bridges being thus strongly asymmetric. The organic ligand is bound to the metal centre via its N₃-donor dipyridylmethylamine fragment in a planar fashion, such that each copper centre is in a square planar environment (or distorted square pyramidal with a long axial bond length if the additional interaction is considered). When R,S-L ¹ was employed in a parallel synthesis, the similar racemic complex [Cu(R,S-L ¹ )Cl](PF₆)??0.5MeOH (4) was obtained, in which the L ¹ ligands in each dimeric unit have opposite hands. In contrast to the complexes of L ¹ , the reaction of Cu(II) chloride with the related ligand, (R)-1-cyclohexyl-N,N-bis(pyridine-3-ylmethyl)ethanamine (R-L ² ), yielded the mononuclear complex [Cu(R,S-L ² )Cl₂] (5), displaying a distorted square pyramidal coordination geometry. The structure of this product along with its corresponding circular dichroism spectrum revealed that racemisation of the starting R-L ² ligand has occurred under the relatively mild (basic) conditions employed for the synthesis. A temperature-dependent magnetic studies of the complexes 1, 2 and 5 indicate that a week ferromagnetic interaction is operative in each dicopper core in 1 and 2 with 2J?=?1.2?cm^?1. On the other hand, a week antiferromagnetic intermolecular interaction is operative for 5.     

Synthesis, Cation-Binding and Optical Spectral Studies of Photoemmitive Benzothiazole Crown Ethers

Crown ethers 1–4, encompassing a photoemittive benzothiazole chromophore have been synthesised using standard protocols. Alkali metal picrate extraction profiles reveal that compared to the known crown ethers, benzothiazole benzo-15-crown-5 1 and benzothiazole dibenzo-18-crown-6 3 exhibit relatively higher % extraction for K⁺ than Na⁺ ions. The UV-visible and fluorescence spectra of 1–4, in comparison to their neutral forms were found to be red shifted on protonation due to enhanced intramolecular charge transfer transition. The ortho-substituted benzothiazole crowns 2–4 showed higher Stokes shifts compared to the para analog 1 in the presence of CF₃CO₂H, presumably due to H-bond assisted conformational restriction. No changes were noticeable in the absorption spectra in the presence of alkali metal ions. Even, fluorescence properties of 1–4 were not found to be drastically perturbed by these ions. While 1 exhibited slight quenching at alkali metal ion concentration over 10-folds with respect to that of 1, interestingly, 2–4 showed a slight enhancement of fluorescent intensity at least up to 10-fold concentration of metal ions over those of 2–4. Further increase of metal ion concentrations produces quenching effects. This behavior has been tentatively explained by invoking electrostatic interaction between these cations and the benzothiazole nitrogen ligand.     

Crown-containing styryl dyes

Complexation of styryl dyes containing theN-phenylaza-15-crown-5 fragment (trans-2a,b) with Ca²⁺ ions in MeCN was studied. Unlike cationic dyetrans-2a, betainetrans-2b forms both complexes of 1∶1 stoichiometry and aggregates consisting of four dye molecules and one Ca²⁺ ion. Cation-induced aggregation was observed also for the analog of dyetrans-2b, which contains a dimethylamino group instead of an azacrown ether fragment (trans-3). Apparently, the interaction between a metal cation and sulfo groups of moleculestrans-2b ortrans-3 makes the main contribution to the stability of aggregates. The dependence of the stability of aggregates on the nature of the metal cation was studied.     

Evaluation of intermolecular interaction energy using one-range addition theorems for {\psi ^\alpha} – ETO and Coulomb-Yukawa like correlated interaction potentials with integer and noninteger indices

Using one-range addition theorems for complete orthonormal sets of ${\psi^\alpha }$ – exponential type orbitals (α = 1, 0, ?1, ?2, . . .) intruduced by the author, the series expansion relations are established for the energy of interaction between molecules which have any number of closed and open shells. The final results are expressed through the linear combination coefficients of molecular orbitals, the parameters of Coulomb-Yukawa like correlated interaction potentials with integer and noninteger indices and the multicenter overlap integrals. The formulas obtained are useful for the study of interaction between atomic-molecular systems when the ${\psi^\alpha}$ – exponential type orbital basis functions in Hartree-Fock-Roothaan and explicitly correlated methods are employed.