High yield purification of JNK1β1 and activation by in vitro reconstitution of the MEKK1 → MKK4 → JNK MAPK phosphorylation cascade

The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated protein kinase (MAPK) signaling pathways comprising a sequential three-tiered kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y residues within their activation loops, respectively. While improvements in the purification of large quantities of active JNKs have recently been made, inadequacies in their yield, purity, and the efficiency of their phosphorylation still exist. We describe a novel and robust method that further improves upon the purification of large yields of highly pure, phosphorylated JNK1β1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1β1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1β1 and its substrate ATF2 were both purified to >99% purity as GST fusion proteins using GSH-agarose affinity chromatography and each cleaved from GST using thrombin, constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. coli as thioredoxin-His₆-tagged proteins and purified using urea refolding and Ni²⁺-IMAC, respectively. Activation of JNK1β1 was then achieved by successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was used to activate MKK4, which in turn was used to efficiently phosphorylate and activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency.     

Assembly of microfibrils in vivo and in vitro from (1»3)-β-d-glucan synthesized by protoplasts of Saccharomyces cerevisiae

Polymer chains of (13)--d-glucan were dissolved with 1 M NaOH at 4° C from native microfibrillar protoplast nets. The chains associated into microfibrils during NaOH neutralization or dialysis. In contrast to the native microfibrils which are of uniform width individually (10 to 20 nm) and arranged in flat bundles, the microfibrils formed in vitro showed no band formation and consisted of fibrous spindle-shaped subunits of variable width or loose elementary fibrils about 1.7 nm wide. X-ray diagrams of native nets indicated a fairly high crystallinity and were different for wet and dry specimens. They corresponded to those of paramylon. Precipitated glucans produced diagrams different from the former and revealing a lower crystallinity especially with the dry samples.The X-ray pattern, combined with other data, allowed the precipitated microfibrils to be identified as aggregates of molecular strands composed each of three intertwined helical glucan chains. Since these triple helical chains are about 1.7 nm wide the elementary fibrils of this width can represent only single triple-helical strands. These helices have 7 glucose residues per turn and therefore a low symmetry which explains the poor crystallizing properties. The 7 membered helix represents a basic difference with the well crystallized native glucan which is built of highly symmetrical triple helices with 6 glucose residues per turn. Since 6₁ helical conformation is not formed in vitro at normal temperatures its generation in vivo must be due to the action of synthesizing enzymes at the protoplast membrane. The intertwining of these helices and crystallization of the strands are determined by their symmetry and physical properties of the chains. This characterizes the native microfibrils as products of self-assembly of enzymegenerated 6₁ helices.     

The influence of Congo red on the cell wall and (1 → 3)-β-d-glucan microfibril biogenesis in Saccharomyces cerevisiae

Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1 » 3)--d-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1 » 3)--d-glucan elementary fibrils depicted by electron microscopy.The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.In memory of Dr. D. R. Kreger of the University of Groningen, The Netherlands, who died on 7 January 1992     

The effect of dissolved oxygen concentrations on (1 → 3)- and (1 → 6)-β-glucanase production by Acremonium sp. IMI 383068 in batch culture

The influence of dissolved oxygen tension (DOT) on the production of extracellular (1 → 3)- and (1 → 6)-β-glucanases by the fungus Acremonium sp. IMI 383068 was investigated in batch culture. The controlled DOT levels at which cultures were grown affected the measured (1 → 3)-β-glucanase specific activities, but these effects were less marked on the measured (1 → 6)-β-glucanase specific activities. There appeared to be no direct link between the branching frequency of the fungus as reflected by its hyphal growth unit, and changing DOT levels. Whether pustulan or scleroglucan were used as the sole carbon source also affected production of these enzymes and their response to varying DOT. The measured (1 → 3)-β-glucanase specific activities were generally higher with scleroglucan, mainly because of the production of an extra active (1 → 3)-β-glucanase whereas pustulan grown cells produced a corresponding inactive protein with an identical electrophoretic mobility by SDS-PAGE and N-terminal amino acid sequence. With pustulan grown cells, DOT levels had comparatively little influence on the optimal measured specific activities of the (1 → 3)-β-glucanases, while with scleroglucan, they increased as DOT increased.     

Combination of induced autolysis and sodium hypochlorite oxidation for the production of Saccharomyces cerevisiae (1 → 3)-β-D-glucan

(1 3)--D-Glucans have received much attention with respect to their biological functions. A novel method to extract (1 3)--D-glucan from Saccharomyces cerevisiae cell wall is proposed in present work, which is based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL 1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal conditions were 52 °C, pH 5.5 and 1.5% (v/v) ethyl acetate. The kinetic behaviour of the yeast autolytic process under the optimized conditions was further studied. After 36 h of autolysis, 42.0% (w/w) cellular substances were released while the cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL 1 was treated by the novel method.     

In vivo evaluation of the antimutagenic and antigenotoxic effects of β-glucan extracted from Saccharomyces cerevisiae in acute treatment with multiple doses

Ample evidence suggests that cancer is triggered by mutagenic damage and diets or supplements capable of reducing such incidences can be related to the prevention of neoplasy development or to an improvement in life quality of patients who undergo chemotherapy. This research aimed to evaluate the antimutagenic and antigenotoxic activity of β-glucan. We set up 8 experimental groups: control (Group 1), cyclophosphamide (Group 2), Groups 3–5 to assess the effect of β-glucan administration, and Groups 6–8 to evaluate the association between cyclophosphamide and β-glucan. The intraperitonial concentrations of β-glucan used were 100, 150 and 200 mg/kg. Micronucleus and comet assays showed that within the first week of treatment β-glucan presented a damage reduction rate between 100–62.04% and 94.34–59.52% for mutagenic and genotoxic damages, respectively. This activity decreased as the treatment was extended. During the sixth week of treatment antimutagenicity rates were reduced to 59.51–39.83% and antigenotoxicity was not effective. This leads to the conclusion that the efficacy of β-glucan in preventing DNA damage is limited when treatment is extended, and that its use as a chemotherapeutic adjuvant need to be better clarified.     

Sulfation of (1→3)-β-d-glucan from the fruiting bodies of Russula virescens and antitumor activities of the modifiers

A water-insoluble (1→3)-β-d-glucan isolated from the fresh fruiting bodies of Russula virescens was sulfated using sulfur trioxide-pyridine complex as reagent in dimethyl sulfoxide. Depending on the reaction conditions, the products showed different degrees of sulfation (DS) ranging from 0.17 to 1.17 and different weight average molecular weights (M_ws) ranging from 2.5 × 10⁴ to 1.2 × 10⁵ Da. Moreover, the antitumor activities of the five sulfated derivatives against Sarcoma 180 tumor cell were tested both in vitro and in vivo. The results indicated that the native (1→3)-β-d-glucan did not show antitumor activity, while the sulfated derivatives exhibited enhanced antitumor activities. This study demonstrated that DS and M_w could influence the antitumor activities of the sulfated derivatives.     

Response of the ArcCHECK® device at 6 MV and 15 MV for VMAT and IMRT quality control

PurposeTo study the response of the ArcCHECK® device as VMAT and IMRT verification system.MethodsVarious tests analyzing the linearity, the repeatability and the angular dependence of the device response, its dependence with the pulse repetition rate and the leakage losses were performed. The long-term response in dose measurements and the uniformity of the detectors conforming the system were controlled using a statistical process control program. The Elekta Infinity™ 6 and 15 MV photon beams were used.ResultsThe device showed excellent repeatability and linearity. The differences between the responses obtained for any pair of angular incidences were less than 2%. The absorbed dose increased by 3% when the pulse repetition rate varied from 50 to 600 MU/min. Results are in overall agreement with those found in previous works for the ArcCHECK®, in which a reduced number of the device diodes were analyzed, and for the MapCheck®, an older 2D device that used the same diodes. Charge losses were found to be negligible except for some of the diodes of the device. The statistical process control program is a very useful tool to control the correct functioning of the device in the long term.ConclusionsThe results of the analysis carried out indicate that the working and stability conditions of the ArcCHECK® device are adequate for its purpose. The dependence with the pulse repetition rate should be considered when VMAT or similar treatments are evaluated. A control program for the statistical monitoring of the device would be desirable and useful.     

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp,from rye arabinoxylan

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-l-arabinofuranosidase Axh43A indicated that U1 was α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp and U2 was α-l-Araf-(1 → 2)-d-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the −1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite −1. This is the first report of a xylanase capable of producing α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp from highly arabinosylated xylan.     

Methyl-β-cyclodextrin is a useful compound for extraction and purification of prenylated enzymes from the retinal disc membrane

cGMP phosphodiesterase 6 (PDE6) and rhodopsin kinase (GRK1) are quantitatively minor prenylated proteins involved in vertebrate phototransduction. Here, we report that methyl-β-cyclodextrin (MCD), a torus-shaped oligosaccharide with a hydrophobic pore, can be used as a selective extractant for such prenylated proteins from frog retinal disc membranes, and that MCD makes it possible to purify frog PDE6 holoenzyme with very simple procedure. The EC50s of MCD for the extraction of GRK1 and PDE6 from the cytoplasmic surface of the disc membrane were 0.17 and 5.1 mM, respectively. By successive extraction of the membrane by 1 mM and then 20 mM MCD, we obtained crude GRK1 and PDE6, respectively. From the 20mM extract, we were able to purify the PDE6 holoenzyme using one-step anion-exchange column chromatography. From 1mM MCD extract, GRK1 was further purified by an affinity column. Following the removal of MCD by ultrafiltration, we were able to confirm integrity of these enzymes by reconstituting phototransduction system in vitro. We have therefore demonstrated that MCD is a useful compound for selective extraction and purification of prenylated peripheral membrane proteins from the cytoplasmic surface of biological membranes.     

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

In Trichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, the dpm2 and dpm3 genes coding for the DPMII and DPMIII subunits of T. reesei DPM synthase were cloned and functionally analyzed after expression in the Saccharomyces cerevisiae dpm1Δ [genotype (BY4743; his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterized S. cerevisiae strains expressing the dpm1, 2, 3 or dpm1, 3 genes and analyzed the consequences of dpm expression on protein O-glycosylation in vivo and on the cell wall composition.     

Dug1p Is a Cys-Gly Peptidase of the ��-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

GSH metabolism in yeast is carried out by the γ-glutamyl cycle as well as by the DUG complex. One of the last steps in the γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the constitutent amino acids. Saccharomyces cerevisiae extracts carry Cys-Gly dipeptidase activity, but the corresponding gene has not yet been identified. We describe the isolation and characterization of a novel Cys-Gly dipeptidase, encoded by the DUG1 gene. Dug1p had previously been identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an alternate GSH degradation pathway and has also been suggested to function as a possible di- or tripeptidase based on genetic studies. We show here that Dug1p is a homodimer that can also function in a Dug2-Dug3-independent manner as a dipeptidase with high specificity for Cys-Gly and no activity toward tri- or tetrapeptides in vitro. This activity requires zinc or manganese ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly. Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We also show that the Dug1p Schizosaccharomyces pombe orthologue functions as the exclusive Cys-Gly peptidase in this organism. The human orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by complementation of the dug1Δ mutant and by biochemical characterization, which revealed a high substrate specificity and affinity for Cys-Gly. The results indicate that the Dug1p family represents a novel class of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide (l-γ-glutamyl-l-cysteinyl-glycine) present in almost all eukaryotes (barring a few protozoa) and in a few prokaryotes (1). In the cell, glutathione exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the millimolar range), a relatively low redox potential (-240 mV), and a high stability conferred by the unusual peptidase-resistant γ-glutamyl bond are three of the properties endowing GSH with the attribute of an important cellular redox buffer. GSH also contributes to the scavenging of free radicals and peroxides, the chelation of heavy metals, such as cadmium, the detoxification of xenobiotics, the transport of amino acids, and the regulation of enzyme activities through glutathionylation and serves as a source of sulfur and nitrogen under starvation conditions (2, 3). GSH metabolism is carried out by the γ-glutamyl cycle, which coordinates its biosynthesis, transport, and degradation. The six-step cycle is schematically depicted in Fig. 1 (2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism. γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two steps in glutathione biosynthesis. γ-glutamyltranspeptidase, γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are involved in glutathione catabolism. Activities responsible for γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and 5-oxoprolinase activities have not been detected, which has led to the suggestion of the presence of an incomplete, truncated form of the γ-glutamyl cycle (4) made of γ-glutamyl transpeptidase (γGT)⁴ and Cys-Gly dipeptidase and only serving a GSH catabolic function. Although γGT and Cys-Gly dipeptidase activities were detected in S. cerevisiae cell extracts, only the γGT gene (ECM38) has been identified so far. Cys-Gly dipeptidase activity has been identified in humans (5, 6), rats (7–10), pigs (11, 12), Escherichia coli (13, 14), and other organisms (15, 16), and most of them belong to the M17 or the M1 and M19 metallopeptidases gene families (17).S. cerevisiae has an alternative γGT-independent GSH degradation pathway (18) made of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex. Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its Schizosaccharomyces pombe homologue. We also show that the mammalian orthologue of DUG1, CNDP2, can complement the defective utilization of Cys-Gly as sulfur source of an S. cerevisiae strain lacking DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase activity in vitro, with a strong preference for Cys-Gly over all other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the metallopeptidases M20A family and have been known to carry carnosine (β-alanyl-histidine) and carnosine-like (homocarnosine and anserine) peptidase activity (20, 21). This study thus reveals that the metallopeptidase M20A family represents a novel Cys-Gly peptidase family, since only members of the M19, M1, and M17 family were known to carry this function.     

Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT).

Methods

Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed.

Results

Among the 4 probes examined in this study, ¹²⁵I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, ¹²³I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft.

Conclusion

¹²³I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.     

Performance of the auxotrophic Saccharomyces cerevisiae BY4741 as host for the production of IL-1β in aerated fed-batch reactor: role of ACA supplementation, strain viability, and maintenance energy

Background

Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.

Results

To investigate specific features of growth and protein secretion, we have sequenced the 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H⁺/glycerol transporters.

Conclusion

This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.     

Mode of action of endo-(1→3)-β-d-glucanases from marine molluscs on the laminarin from Laminaria cichorioides: The structure and the inhibitory effect of the resulting (1→3;1→6)-β-d-gluco-oligosaccharides

The oligosaccharides released by the action of endo-(1→3)-β-d-glucanases from the marine molluscs Chlamys albidus (laminarinase Lo) and Spisula sachalinensis (laminarinase LIV) on Laminaria laminarin have been studied. For laminarinase Lo, the branched products were shown to be 6²-β-d-glucopyranosyl-laminaribiose and 6³- and 6²-β-d-glucopyranosyl-laminaritrioses by methylation analysis and ¹³C-n.m.r. spectroscopy. It is suggested that one or two (1→3) linkages adjacent to (1→6) branch-points result in resistance to enzymic attack. 6³-β-d-Glucopyranosyl-laminaritriose inhibited laminarinases Lo and LIV (I₅₀ 1.2 × 10⁻³m and 1.5 × 10⁻³m, respectively).