L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression

Abstract

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch^®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch^® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.     

Complex formation between the UL16 and UL21 tegument proteins of pseudorabies virus

The products of the UL16 and UL21 genes represent tegument proteins which are conserved throughout the mammalian herpesviruses. To identify and functionally characterize the respective proteins in the alphaherpesvirus pseudorabies virus, monospecific antisera against bacterially expressed fusion proteins were generated. In immunoblots the UL16 antiserum detected a ca. 40-kDa protein in infected cells and purified virion preparations, whereas the anti-UL21 serum recognized a protein of approximately 60 kDa. Interestingly, in immunoprecipitations using either antiserum, both proteins were coprecipitated, demonstrating the formation of a physical complex. To investigate protein function, viruses lacking either UL16, UL21, or both were constructed. Mutant viruses could be propagated on noncomplementing cells, indicating that these proteins, either alone or in combination, are not required for viral replication in cell culture. However, plaque sizes and viral titers were reduced. Electron microscopy showed only slight alterations in cytoplasmic virion morphogenesis, whereas intranuclear maturation stages were not affected. Similar results were obtained with a triple mutant simultaneously lacking the three conserved tegument proteins UL11, UL16, and UL21. In summary, our results uncover a novel interaction between conserved herpesvirus tegument proteins that increases the complexity of the intricate network of protein-protein interactions involved in herpesvirus morphogenesis.     

Mutagenesis of the active-site cysteine in the ubiquitin-specific protease contained in large tegument protein pUL36 of pseudorabies virus impairs viral replication in vitro and neuroinvasion in vivo

Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C(26)S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C(26)S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.     

High-performance liquid chromatographic analysis for the determination of a novel polymer-bound camptothecin derivative (MAG-camptothecin) and free camptothecin in human plasma

A selective HPLC assay is described for the determination of free and total (free plus polymer-bound) camptothecin (CPT) in human plasma after administration of the anti-tumor drug MAG-CPT (polymer bound camptothecin). Total CPT levels were determined after hydrolysis and free CPT was extracted from acidified plasma using Oasis solid-phase extraction material. Extracts were analyzed on a Zorbax SB-C₈ analytical column, using a mixture of acetonitrile–25 mM phosphate buffer (pH 4.0) as the eluent. Detection was performed fluorimetrically. Concentrations of polymer-bound CPT were calculated by subtraction of free from total CPT. The lower limits of quantitation of the methods were 100 ng/ml for total and 1.0 ng/ml for free CPT using 50 μl and 250 μl plasma, respectively. Special attention was paid to the stability of the analytes. The presented method was successfully applied in a clinical pharmacokinetic study in our institute.     

Implication of HMGR in homeostasis of sequestered and de novo produced precursors of the iridoid biosynthesis in leaf beetle larvae

Insects employ iridoids to deter predatory attacks. Larvae of some Chrysomelina species are capable to produce those cyclopentanoid monoterpenes de novo. The iridoid biosynthesis proceeds via the mevalonate pathway to geranyl diphospate (GDP) subsequently converted into 8-hydroxygeraniol-8-O-beta-D-glucoside followed by the transformation into the defensive compounds. We tested whether the glucoside, its aglycon or geraniol has an impact on the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key regulatory enzyme of the mevalonate pathway and also the iridoid biosynthesis. To address the inhibition site of the enzyme, initially a complete cDNA encoding full length HMGR was cloned from Phaedon cochleariae. Its catalytic portion was then heterologously expressed in Escherichia coli. Purification and characterization of the recombinant protein revealed attenuated activity in enzyme assays by 8-hydroxygeraniol whereas no effect has been observed by addition of the glucoside or geraniol. Thus, the catalytic domain is the target for the inhibitor. Homology modeling of the catalytic domain and docking experiments demonstrated binding of 8-hydroxygeraniol to the active site and indicated a competitive inhibition mechanism. Iridoid producing larvae are potentially able to sequester glucosidically bound 8-hydroxygeraniol whose cleavage of the sugar moiety results in 8-hydroxygeraniol. Therefore, HMGR may represent a regulator in maintenance of homeostasis between de novo produced and sequestered intermediates of iridoid metabolism. Furthermore, we demonstrated that HMGR activity is not only diminished in iridoid producers but most likely prevalent within the Chrysomelina subtribe and also within the insecta.     

Phenotypic differences in behavior, physiology and neurochemistry between rats selected for tameness and for defensive aggression towards humans

To better understand the biology of tameness, i.e. tolerance of human presence and handling, we analyzed two lines of wild-derived rats (Rattus norvegicus) artificially selected for tameness and defensive aggression towards humans. In response to a gloved human hand, tame rats tolerated handling, whereas aggressive rats attacked. Cross-fostering showed that these behavioral differences are not caused by postnatal maternal effects. Tame rats were more active and explorative and exhibited fewer anxiety-related behaviors. They also had smaller adrenal glands, larger spleens and lower levels of serum corticosterone. Blood glucose levels were lower in tame rats, whereas the concentrations of nine amino acids were higher. In the brain, tame rats had lower serotonin and higher taurine levels than aggressive rats. Our findings reinforce the notion that tameness is correlated with differences in stress response and will facilitate future efforts to uncover the genetic basis for animal tameness.