Directed Evolution of Insoluble Arabidopsis thaliana Zeta Class Glutathione S-Transferase Mutants for Higher Solubility in Escherichia coli

Expression of recombinant protein in Escherichia coli (E.coli) is generally considered as one of the ideal systems to produce proteins for industrial production.However,the majority of proteins usually fail to fold into their native state and accumulate as insoluble inclusion bodies with no biological activity in E.coli (Yang et al.,2003).Although numerous strategies and approaches are proposed to solve the problem (Nygaard and Harlow,2001;Mogk et al.,2002;Austin,2003),they still fail to improve the solubility of protein and are not ideal for high-throughput applications (Waldo,2003).Furthermore,with the expression condition becoming stricter,the procedures become more complex and the costs grow higher,thus making them inappropriate for application in industrial production.     

Multiple Forms and Distribution of Calcium/Calmodulin-Stimulated Protein Kinase II in Brain

In recent years, the enzyme Ca²⁺/calmodulin-stimulated protein kinase II¹ (CaM-PK II) as attracted a great deal of interest. CaM-PK II is the most abundant calmodulin-stimulated protein kinase in brain, where it is particularly enriched in neurons (Ouimet et al., 1984; Erondu and Kennedy, 1985; Lin et al., 1987; Scholz et al., 1988). Neuronal CaM-PK II has been suggested to be involved in several phenomena associated with synaptic plasticity (Lisman and Goldring, 1988; Kelly, 1992), including long-term potentiation (Malinow et al., 1988; Malenka et al.,1989), neurotransmission (Nichols et al., 1990; Siekevitz, 1991), and learning (for review, see Rostas, 1991). This enzyme has also been postulated to be selectively vulnerable in several pathological condition, including epilepsy/kindling (Bronstein et al.,1990; Wu et al., 1990), cerebral ischemia (Taft et al., 1988), and organophosphorus toxicity (Abou-Donia and Lapadula, 1990).     

Identification and characterization of the Drosophila tau homolog

A pathological hallmark of neurodegenerative tauopathies, including Alzheimer's disease and a group of clinically heterogeneous frontotemporal dementias, is the presence of intracellular neurofibrillary protein lesions (reviewed in Spillantini and Goedert, TINS 10 (1998) 428). The principal component of these structures is the microtubule-associated protein tau. Although tau is normally a highly soluble protein enriched in axons, in these deposits, it is abnormally hyperphosphorylated, insoluble, and redistributed to the somatodendritic compartments of neurons. Through ultrastructual analyses, it has been determined that the tau protein in these lesions is filamentous and organized into paired-helical filaments, straight filaments, or ribbon-like filaments (Goedert et al., The Molecular and Genetic Basis of Neurological Disease (1997) 613). By the dynamic binding of microtubules, tau is thought to promote the structural stability of axons, but whether tau aggregates contribute to neurodegeneration through a direct toxicity on normal cellular functions such as organelle transport or an indirect effect on microtubule stability, is currently unknown. The identification of mutations in the tau locus in patients with familial frontotemporal dementia and Parkinsonism linked to chromosome 17 has demonstrated that mutations in tau are sufficient to cause neurodegenerative disease (Poorkaj et al., Ann. Neurol. 43 (1998) 815; Hutton et al., Nature 393 (1998) 702). To elucidate the mechanisms by which tau dysfunction contributes to neuronal loss, we have sought to model human tauopathies in a genetically tractable organism. Here we describe the isolation of a Drosophila tau cDNA (GenBank accession number AY032977), the production of antibodies that recognize the encoded protein, and their use in determining the expression and subcellular localization of the fly tau protein.     

GERp95, a membrane-associated protein that belongs to a family of proteins involved in stem cell differentiation.

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.     

Relationship between solubility of grifolan, a fungal 1,3-beta-D-glucan, and production of tumor necrosis factor by macrophages in vitro.

Grifolan, GRN, is a fungal antitumor beta-glucan isolated from Grifola frondosa. Various studies suggested that the underlying mechanism of the antitumor activity of GRN is strongly related to immune modulation. In the previous publication (Adachi et al., 1994; Okazaki et al., 1995), we have shown that GRN activates macrophages to produce tumor necrosis factor (TNF) in vitro. In this study, the structural unit essential to produce TNF was examined by chemical modifications of GRN. GRN suspended in distilled water was treated at 150 degrees C for up to 3 h. Addition of the resulting turbid solution to the RAW 264.7 macrophage-like cell line produced TNF, and the relative activity was diminished in relation to the heat treatment period. The fractions with a heating period longer than 15 min did not show any activity. After centrifugation of the resulting solution, significant activity was shown by precipitate fractions, suggesting that the insoluble form of GRN is important for TNF production. Interestingly, the precipitate fraction obtained from 9 min of treatment also had significant activity. In addition, admixing the soluble fraction with the particles significantly inhibited the TNF production. In contrast to these observations, the high-molecular-mass subfraction of the soluble fraction prepared by ultrafiltration produced significant amounts of TNF. Similar phenomena were shown with sodium hydroxide treatment and dimethylsulfoxide treatment. These facts strongly suggested that insoluble as well as a high molecular mass soluble form of GRN are required for TNF production by macrophages.     

Myoglobin as an ultrastructural probe for studying permeability of the nephron.

Hemeproteins, like cytochrome c (12,500 M.W.; Karnovsky and Rice, 1969) and myoglobin (17,816 M.W.; Anderson, 1972; Simionescu et al., 1973) are advantageous over the true peroxidases with larger molecular weights (e.g. horseradish peroxidase, ca. 40,000 M.W) as ultrastructural probes in that they do not elicit vascular leakage in the inflammatory response (Cotran and Karnovsky, 1967) and are relatively nontoxic immunologically inert substances. The main disadvantage in using cytochrome c and myoglobin is that they have weak peroxidatic activity compared to the true peroxidases (Nakamura et al., 1960; Keilin, 1961; Kurozimi et al., 1961). These hemeproteins, however, offer the following advantages: 1) they retain sufficient peroxidatic activity after aldehyde-fixation to oxidize 3,3'-diaminobenzidine (DAB), 2) they may be localized by virtue of an insoluble reaction product (osmium black) deposited at the site of hemeprotein immobilization by fixation, and 3) they represent low molecular weight probes. This brief report emphasizes the advantages of myoglobin in the study of glomerular permeability, transport by endocytosis in proximal tubules and translocation of protein in the lower segments of the nephron.     

Separation of synaptic junctional complexes from thin layers of rabbit cerebral cortex

Synaptic junctional fractions were separated from rabbit brain by procedures based on combining the methods of Cotman and Taylor [4], Orosz et al. [16, 17] and Lisman et al. [13]. Thin layers of cerebral cortices were homogenized to obtain a crude mitochondrial-synaptosomal fraction. The sedimentation rates of mitochondria and mitochondria containing synaptosomes were increased by raising the density of mitochondria with an insoluble dense formazan deposit inside mitochondria after iodo-nitrotetrazolium treatment. The synaptic plasma membrane fraction isolated by this method contained no mitochondrial contamination. After Triton X-100 treatment the insoluble residues of the detergent were centrifuged through discontinuous sucrose gradients. A great enrichment of morphologically identifiable intact synaptic junctions was observed in some of the obtained interface layers.     

Antipain microinjection prevents progesterone to inhibit adenyl cyclase in Xenopus oocytes

Microinjection of antipain, an inhibitor of thiol and Ca2+-dependent proteases, in immature Xenopus oocytes inhibited meiotic maturation induced by progesterone, but not by transfer of cytoplasm taken from maturing oocytes. Oocytes could be released from antipain inhibition by increasing progesterone concentration. alpha-32P-ATP was microinjected to study adenylcyclase in ovo. As already reported, neosynthesis of cAMP was decreased following progesterone application. This decrease was not observed, or it was considerably reduced, in oocytes previously injected with antipain. In amphibian, full-grown ovarian oocytes are arrested at first meiotic prophase, and have a large nucleus known as the germinal vesicle. Progesterone induces the production of a cytoplasmic maturation-promoting factor (MPF), which itself triggers germinal vesicle breakdown (GVBD), and subsequent events of meiotic maturation (Masui and Markert, 1971; Gerhart et al., 1984). A considerable body of evidences support the view that release from prophase block is due to inactivation of a cAMP-dependent protein kinase (reviewed by Maller, 1983). On the other hand, progesterone has been shown to induce a transient decrease in cAMP level (Speaker and Butcher, 1977; Schorderet-Slatkine et al., 1982; Cicirelli et al., 1985), and this initial drop of cAMP, along with a number of studies indicating a decrease in adenylate cyclase activity (Mulner et al., 1979; Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et al., 1981; Jordana et al., 1981), provided key support to the theory that an early drop in cAMP led to the dephosphorylation of a hypothetical protein which initiates maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeting of proteins into the peroxisomal matrix

Summary During the last few years much has been learned regarding signals that target proteins into peroxisomes. The emphasis in the near future will undoubtedly shift towards the elucidation of the mechanism of import. The use of mammalian and yeast cells deficient in peroxisome assembly and/or import (Zoeller & Raetz, 1986; Erdmann et al., 1989; Cregg et al., 1990; Morand et al., 1990; Tsukamoto, Yokota & Fujiki, 1990) should provide a handle on the genes (Erdmann et al., 1991; Tsukamoto et al., 1991) involved in these processes. This will have to be coupled with further development of in vitro systems which will permit the dissection of the steps in the translocation of proteins into peroxisomes. Though some progress has been made in the development of such assays (Imanaka et al., 1987; Small et al., 1987, 1988; Miyazawa et al., 1989), the fragility of peroxisomes and the absence of biochemical hallmarks of import (such as protein modifications or proteolytic processing) have hindered progress. Since peroxisomes exist in the form of a reticulum in mammalian cells (Gorgas, 1984), all peroxisome purification schemes (from mammalian cells at least) must undoubtedly rupture the peroxisomes, which then reseal to form vesicular structures. Additionally, the reliance on the latency of catalase alone as a major criterion for the integrity of peroxisomes ignores the fact that many other matrix proteins leak out of peroxisomes at vastly different rates during purification of the organelles (Thompson & Krisans, 1990). In view of these problems, the development of peroxisomal transport assays with semi-intact cells would also constitute an important advance. It is very likely that in the next few years we will witness some major advances in our understanding of the mechanism by which proteins enter this organelle.I would like to thank all the members of my lab and my collaborators, past and present, whose hard work provided the material for this review. This work has been supported by grants from the March of Dimes Foundation (#1081) and the NIH (DK41737).     

The impact of molecular systematics on hypotheses for the evolution of root nodule symbioses and implications for expanding symbioses to new host plant genera

Current taxonomic schemes place plants that can participate in root nodule symbioses among disparate groups of angiosperms. According to the classification scheme of Cronquist (1981) which is based primarily on the analysis of morphological characters, host plants of rhizobial symbionts are placed in subclasses Rosidae and Hamamelidae, and those of Frankia are distributed among subclasses Rosidae, Hamamelidae, Magnoliidae and Dilleniidae. This broad phylogenetic distribution of nodulated plants has engendered the notion that nitrogen fixing endosymbionts, particularly those of actinorhizal plants, can interact with a very broad range of unrelated host plant genotypes. New angiosperm phylogenies based on DNA sequence comparisons reveal a markedly different relationship among nodulated plants and indicate that they form a more coherent group than has previously been thought (Chase et al., 1993; Swensen et al., 1994; Soltis et al., 1995). Molecular data support a single origin of the predisposition for root nodule symbiosis (Soltis et al., 1995) and at the same time support the occurrence of multiple origins of symbiosis within this group (Doyle, 1994; Swensen, 1996; Swensen and Mullin, In Press).     

Presence of Phosphatidylserine Synthesizing Enzymes in Triton Insoluble Floating Fractions from Cerebrocortical Plasma Membranes

Mammals synthesize phosphatidylserine (PS), a binding PKC molecule, by exchanging the nitrogen base of phosphatidylethanolamine or phosphatidylcholine with free serine. Serine base exchange enzyme (SBEE) was found in Triton insoluble floating fractions (TIFFs) from rat cerebellum which contained PKC. Consequently, SBEE might modulate PS levels in the PKC binding area (Buratta et al., J Neurochem 103:942–951, 2007). In the present study, we determined whether SBEE and PKC were localised in rat cerebral cortex TIFFs (cx-TIFFs) and in rat cerebrocortical plasma membrane-TIFFs (PM-TIFFs) which are more directly involved in signal transduction than intracellular membranes. Cx-and PM-TIFFs expressed SBEE activity and contained PKC. SBEE used ethanolamine as free exchanging base which may modulate PS level in the PKC binding area, transforming PS into PE and vice versa. The slight decrease in [¹⁴C]serine incorporation in the presence of choline indicated the existence of a SBEE isoform which may play a peculiar role in this brain area.     

Haemolysin-derived synthetic peptides with pore-forming and haemolytic activity

Escherichia coli haemolysin (Hlya) is a pore-forming protein which belongs to the family of 'Repeat-toxins' (RTX) (Lo et al., 1987; Lally et al., 1989; Kraig et al., 1990). A model for the pore-forming structure of HlyA has been proposed (Ludwig et al., 1991) which consists of eight transmembrane segments all present in this hydrophobic region of HlyA. We report here that two synthetic peptides of 10 and 8 amino acids in length (Pep1 and Pep2, respectively), which are derived from transmembrane segment V, are able to form pores in an artificial lipid bilayer. In addition, Pep1 exhibits strong haemolytic activity when tested on human red blood cells (HRBCs). The haemolytic activity of Pep1 and of E. coli haemolysin is completely inhibited by antibodies raised against Pep1.     

PFDB: a generic protein family database integrating the CATH domain structure database with sequence based protein family resources

MOTIVATION: The PFDB (Protein Family Database) is a new database designed to integrate protein family-related data with relevant functional and genomic data. It currently manages biological data for three projects-the CATH protein domain database (Orengo et al., 1997; Pearl et al., 2001), the VIDA virus domains database (Albà et al., 2001) and the Gene3D database (Buchan et al., 2001). The PFDB has been designed to accommodate protein families identified by a variety of sequence based or structure based protocols and provides a generic resource for biological research by enabling mapping between different protein families and diverse biochemical and genetic data, including complete genomes. RESULTS: A characteristic feature of the PFDB is that it has a number of meta-level entities (for example aggregation, collection and inclusion) represented as base tables in the final design. The explicit representation of relationships at the meta-level has a number of advantages, including flexibility-both in terms of the range of queries that can be formulated and the ability to integrate new biological entities within the existing design. A potential drawback with this approach-poor performance caused by the number of joins across meta-level tables-is avoided by implementing the PFDB with materialized views using the mature relational database technology of Oracle 8i. The resultant database is both fast and flexible. This paper presents the principles on which the database has been designed and implemented, and describes the current status of the database and query facilities supported.     

Neutrophils promote the release of alpha-6-fucosyltransferase from blood platelets through the action of cathepsin G and elastase

Human blood platelets release alpha-6-fucosyltransferase during coagulation of blood or after stimulation with thrombin or other agonists that cause platelet activation (Antoniewicz et al., FEBS Lett. 244 (1989) 388-390). However, in the absence of neutrophils the thrombin-stimulated platelets release only a small fraction of alpha-6-fucosyltransferase activity (Ko?cielak et al., Acta Biochim. Polon. 42 (1995) 35-40). We show that the effect of neutrophils is reproduced by cathepsin G or (less efficiently) by elastase, the two enzymes that are released by neutrophils during coagulation of blood. We have also localized alpha-6-fucosyltransferase to membrane and alpha-granule fractions of platelets that had been disrupted by nitrogen cavitation. It is concluded that thrombin-activated neutrophils release cathepsin G and elastase that promote degranulation of platelets and hence the secretion of alpha-6-fucosyltransferase.     

A sequence of seventy-three nucleotides from the coliphage R17 genome

1. A sequence of 73 nucleotides of the RNA genome from coliphage R17 was determined. It can be read through in only one translational frame. The fragment is not part of the coatprotein cistron (Min Jou et al., 1972), nor does it come from the untranslated sequences described previously (Steitz, 1969; Nichols, 1970; Cory et al., 1970; de Wachter et al., 1971; Contreras et al., 1971; Cory et al., 1972). It contains two sequences of 23 and 24 nucleotides, 22 of which are identical. This kind of reiteration is the first one found in bacteriophage nucleic acid. 2. Improved conditions were found and tested for blocking oligonucleotides with carbodi-imide and cleaving by ribonuclease A at cytidylate residues. 3. A synthetic medium is described which allows labelling in vivo with (32)P to give specific radioactivities higher than those obtained in the procedures used previously.     

Strategic Distribution of Protective Proteins within Bran Layers of Wheat Protects the Nutrient-Rich Endosperm

Bran from bread wheat (Triticum aestivum ‘Babbler’) grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.Wheat grain (Triticum aestivum) is a major cereal crop and staple food in many parts of the world. The endosperm is the main nutritional component and is extracted in milling to produce base ingredients such as flour and semolina. Crop yield and quality may be compromised by both environmental and biological stresses. Wheat varieties are known to vary in their resistance to such stresses, probably due to individual differences in defense protein levels (Demeke and Morris, 2002; Bonnin et al., 2005; Yarullina et al., 2005). Cereal grain contains many defense proteins that have been categorized according to their mode of action and structural similarities. A major class of these is the pathogenesis-related (PR) proteins, which include PR-1, PR-2 (β -1,3-glucanases), PR-3 (chitinases), PR-4 (wheatwin1), and PR-5 (thaumatin-like proteins; Selitrennikoff, 2001; Desmond et al., 2006). Other known defense proteins are xylanase inhibitor proteins (XIPs) and α -amylase inhibitor proteins (Mundy et al., 1984; Payan et al., 2003). All of these defense proteins have both general and specific roles that contribute to plant survival, although little is known of their location within the various grain tissues, particularly the multiple layers that constitute bran.Proteomic analysis of wheat grain has previously been applied to identify proteins in the germ and endosperm (Skylas et al., 2000; Wong et al., 2004; Mak et al., 2006), but analysis of bran and bran tissue fractions has not been reported. Collection of sufficiently pure bran tissue fractions has limited progress, mainly due to the strong bonds between the various bran tissue layers and endosperm in dry grain. Thus, a method to obtain bran layers free from contaminants, such as adjacent tissue and endosperm, is required to provide a sample suitable for proteomic analysis. Soaking whole grain in water causes the endosperm to soften, allowing it to be easily removed and washed from the bran; the bran becomes malleable enough to dissect. While this approach might not identify the proteome of dry grain fractions, it is the best available representation of the three distinct tissue fractions in grains, namely the outer layer (epidermis and hypodermis), intermediate layer (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells; Antoine et al., 2003, 2004). Using this method, water-soluble proteins that diffuse from the grain can be collected and identified.In this study we aimed (1) to dissect bran into the three separate tissue fractions described above and to identify the protein complement of each fraction using proteomics, (2) to confirm the location of three major defense proteins identified (one from each microfraction) using immunolocalization, and (3) to identify water-soluble proteins and assay any defense-related proteins for enzymatic activity.