MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function

Background  

Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function.     

Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases

Background  

The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised.     

Identification of archaeal proteins that affect the exosome function in vitro

Background  

The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors.     

Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2

Background  

Two main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA) have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues.     

Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners

Background  

Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity.     

Characterization of rubber particles and rubber chain elongation in <Emphasis Type="Italic">Taraxacum koksaghyz</Emphasis>

Background  

Natural rubber is a biopolymer with exceptional qualities that cannot be completely replaced using synthetic alternatives. Although several key enzymes in the rubber biosynthetic pathway have been isolated, mainly from plants such as Hevea brasiliensis, Ficus spec. and the desert shrub Parthenium argentatum, there have been no in planta functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion Taraxacum koksaghyz, which has long been considered as a potential alternative source of low-cost natural rubber, has a rapid life cycle and can be genetically transformed using a simple and reliable procedure. However, there is very little molecular data available for either the rubber polymer itself or its biosynthesis in T. koksaghyz.     

The effect of amino acid deletions and substitutions in the longest loop of GFP

Background  

The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.     

Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

Background  

Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it.     

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.     

Exploring laccase-like multicopper oxidase genes from the ascomycete <Emphasis Type="Italic">Trichoderma reesei</Emphasis>: a functional,phylogenetic and evolutionary study

Background  

The diversity and function of ligninolytic genes in soil-inhabiting ascomycetes has not yet been elucidated, despite their possible role in plant litter decay processes. Among ascomycetes, Trichoderma reesei is a model organism of cellulose and hemicellulose degradation, used for its unique secretion ability especially for cellulase production. T. reesei has only been reported as a cellulolytic and hemicellulolytic organism although genome annotation revealed 6 laccase-like multicopper oxidase (LMCO) genes. The purpose of this work was i) to validate the function of a candidate LMCO gene from T. reesei, and ii) to reconstruct LMCO phylogeny and perform evolutionary analysis testing for positive selection.     

Nanocrystalline magnesium oxide: a novel and efficient catalyst for facile synthesis of 2,4,5-trisubstituted imidazole derivatives

Abstract  

Nanocrystalline magnesium oxide with high specific surface area has been used as a novel and efficient catalyst for an improved and rapid synthesis of biologically active 2,4,5-trisubstituted imidazoles, by three-component, one-pot condensation of 1,2-diketones and aryl aldehydes, in excellent yields under solvent-free and conventional heating conditions. The method has several advantages, for example excellent yields, shorter reaction time, and use of a non-toxic and recyclable catalyst.     

The PLAC1-homology region of the ZP domain is sufficient for protein polymerisation

Background  

Hundreds of extracellular proteins polymerise into filaments and matrices by using zona pellucida (ZP) domains. ZP domain proteins perform highly diverse functions, ranging from structural to receptorial, and mutations in their genes are responsible for a number of severe human diseases. Recently, PLAC1, Oosp1-3, Papillote and CG16798 proteins were identified that share sequence homology with the N-terminal half of the ZP domain (ZP-N), but not with its C-terminal half (ZP-C). The functional significance of this partial conservation is unknown.     

Brain iron metabolism and its perturbation in neurological diseases

Abstract  

Enormous advances have been made in the last decade in understanding iron metabolism and iron homeostasis at both the cellular and the systemic level. This includes the identification of genes and proteins involved in iron transport, such as the ferric reductase DcytB, the proton-coupled ferrous (divalent) iron transporter DMT1, the iron exporter ferroportin and the membrane-bound ferroxidase hephaestin. The modulation of their translation by the iron regulatory protein (IRP) system has also been identified together with the impressive signalling cascades involved in regulating the chef d’orchestre of systemic iron homeostasis, hepcidin. However, exactly how the brain regulates fluxes and storage of iron between neurons, oligodendrocytes, astrocytes and microglial cells remains an enigma. In this review we discuss the possible mechanisms which may be involved in the transfer of iron across the blood–brain barrier, together with the possible role played by astrocytes. The consequences of iron deficiency and iron excess on brain function are described. Finally, various neurodegenerative diseases, where accumulation of iron may be important in the pathogenesis, are presented as well as the possible use of iron chelators to diminish disease progression.     

Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

Background  

Vaccinia virus gene B1R encodes a serine/threonine protein kinase. In vitro this protein kinase phosphorylates ribosomal proteins Sa and S2 and vaccinia virus protein H5R, proteins that become phosphorylated during infection. Nothing is known about the sites phosphorylated on these proteins or the general substrate specificity of the kinase. The work described is the first to address these questions.     

QSAR of α-campholenic derivatives with sandalwood odor, and molecular design

Abstract  

Quantitative structure–activity relationship (QSAR) studies have been carried out in a series of α-campholenic derivatives with sandalwood odor based on quantum-chemical data derived by use of the Hartree–Fock (HF) method. To build QSAR models, a multiple linear regression method was used. The models obtained have good predictive ability and are of high statistical significance, with good correlation coefficients, and p values less than 0.05. The models contribute also to identification of important quantum-chemical aspects of the sandalwood odor. On the basis of the QSAR models built, several new sandalwood compounds were designed and the best candidate for experimental synthesis was suggested.     

Comparison of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isocitrate dehydrogenases (ICD-1 and ICD-2) reveals differences in coenzyme affinity,oligomeric state,pH tolerance and phylogenetic affiliation

Background  

M.tb icd-1 and M.tb icd-2, have been identified in the Mycobacterium tuberculosis genome as probable isocitrate dehydrogenase (ICD) genes. Earlier we demonstrated that the two isoforms can elicit B cell response in TB patients and significantly differentiate TB infected population from healthy, BCG-vaccinated controls. Even though immunoassays suggest that these proteins are closely related in terms of antigenic determinants, we now show that M.tb icd-1 and M.tb icd-2 code for functional energy cycle enzymes and document the differences in their biochemical properties, oligomeric assembly and phylogenetic affiliation.     

Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri

Background

Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host.

Results

In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3) pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP), SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA) and Glutathione S-transferase (GST) improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed.

Conclusions

Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.     

Rapid determination of tricarboxylic acid cycle enzyme activities in biological samples

Background  

In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays.     

Determination of thermodynamic parameters of Xerocomus chrysenteron lectin interactions with N-acetylgalactosamine and Thomsen-Friedenreich antigen by isothermal titration calorimetry

Background  

Lectins are carbohydrate-binding proteins which potentially bind to cell surface glycoconjugates. They are found in various organisms including fungi. A lectin from the mushroom Xerocomus chrysenteron (XCL) has been isolated recently. It shows insecticidal activity and has antiproliferative properties.