CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core.

pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions. CcdB remains dimeric at pH 4 as shown by multiple techniques, such as size-exclusion chromatography coupled to multiangle light scattering, analytical ultracentrifugation, and electron paramagnetic resonance. Comparative analysis using two-dimensional 15N-1H heteronuclear single-quantum coherence NMR spectra of CcdB at pH 4 and 7 suggests that the pH 4 and native state have similar but nonidentical structures. Hydrogen-exchange-mass-spectrometry studies demonstrate that the pH 4 state has substantial but anisotropic changes in local stability with core regions close to the dimer interface showing lower protection but some other regions showing higher protection relative to pH 7.

Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy.

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.

Methotrexate-gelonin conjugate - an inhibitor of MCF-7 cells expressing the dihydrofolate receptor.

An immunotoxin composed of gelonin, a basic ribosome-inactivating protein, type I of 30 kDa, isolated from the seeds of the Indian plant Gelonium multiflorum and methotrexate (MTX) has been studied as a potential tool of gelonin delivery into the cytoplasm of MTX-responsive cells. On the average, about five molecules of methotrexate were chemically coupled to gelonin via an N-hydroxysuccinimide ester of the drug. The MTX-gelonin conjugate was able to reduce the viability of MCF-7 cells in a dose-dependent manner with ID50 of 10 nm, whereas gelonin or MTX alone showed none or very little effects. Besides its ribosome-inactivating activity, which is about ten-fold lower in an in vitro translation assay (IC50 of 50.5 ng/ml as compared to 4.6 ng/ml), the conjugate also significantly induced direct and oxidative DNA damage as shown by the alkaline comet assay. Hence, MTX-gelonin conjugates are promising candidates for the treatment of MTX-responsive cancers.

Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity.

The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargoes into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2 µg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents.

Conformational changes of the chaperone SecB upon binding to a model substrate--bovine pancreatic trypsin inhibitor (BPTI).

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

Novel immunotoxin: a fusion protein consisting of gelonin and an acetylcholine receptor fragment as a potential immunotherapeutic agent for the treatment of Myasthenia gravis.

In continuation of our attempts for antigen-specific suppression of the immune system [I.L. Urbatsch, R.K.M. Sterz, K. Peper, W.E. Trommer, Eur. J. Immunol. 23(1993) 776-779] a novel fusion protein composed of amino acids 4-181 of the extracellular domain of the alpha-subunit of the human muscle acetylcholine receptor and the plant toxin gelonin was expressed in Escherichia coli. The fusion protein formed inclusion bodies but could be solubilized in the presence of guanidinium hydrochloride. After a simple two step purification and refolding procedure, it exhibited a native structure at least in the main immunogenic region as shown by antibodies recognizing a conformational epitope. Half maximal inhibition of translation was achieved at 46 ng/ml as compared to 4.6 ng/ml for native and 2.4 for recombinant gelonin. Its use as therapeutic agent for the treatment of Myasthenia gravis was investigated in an animal model. Female Lewis rats were immunized with complete acetylcholine receptor from the electric ray Torpedo californica and developed thereafter experimental autoimmune M. gravis. Quantitative assessment of the disease was achieved by repetitive stimulation of the Nervus tibialis. Rats showed no symptoms of M. gravis, neither visually nor electrophysiologically after treatment with the fusion protein as determined one and seven weeks after the second application. This approach may also be useful for the therapy of further autoimmune diseases by substituting other autoantigens for the AchR fragment in the fusion protein.

On the contentious sequence and glycosylation motif of the ribosome inactivating plant protein gelonin.

The amino acid sequence and the glycosylation motif of the ribosome inactivating protein (RIP) gelonin are identified by Fourier transform ion cyclotron resonance mass spectrometry. Intact gelonin as isolated from the seeds of Gelonium multiflorum consists of at least three different post-translational modified forms: analysis of gelonin peptides as obtained by proteolytic digestion is consistent with the amino acid sequence published by Nolan et al. High resolution mass determination established a glycosylation pattern of GlcNAc2Man(3-5)Xyl. N189 was identified as glycosylation site. The proposed glycan structure is consistent with a standard plant N-glycosylation pattern as found in other RIP. Based on these results we suggest that gelonin is located in the vacuole of Gelonium multiflorum seeds.

Soluble expression and affinity purification of functional domain of human acetylcholine receptor alpha-subunit by the modulation of maltose binding protein.

An open reading frame of the alpha-subunit 1-205 residues (alpha205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR alpha205 as the template and inserted into vector pMAL-c2X. The constructed pMAR alpha205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR alpha205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR alpha205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR alpha205 and AChR alpha205 were similar to that of AChR alpha-subunit from Torpedo.

Vitamin E supplementation does not increase the vitamin C radical concentration at rest and after exhaustive exercise in healthy male subjects.

BACKGROUND: Extensive exercise may promote the formation of reactive oxygen species and subsequently contribute to tissue damage. A compound which can protect cells and tissues is vitamin E. The vitamin E radical, formed during the radical scavenging process, can be reduced by vitamin C resulting in a higher level of the vitamin C radical (semidehydroascorbate free radical). An increase of the vitamin C radical, however, is assumed to exert prooxidative effects. AIM OF THE STUDY: To elucidate whether supplementation of vitamin E and exhaustive exercise lead to an increase of the vitamin C radical in human plasma. METHODS: A placebo controlled, cross over study with 13 male volunteers was carried out. After an 8 day supplementation period with 500 I.U. D-alpha-tocopherol, the subjects performed two exhaustive treadmill runs. Blood samples were collected at rest, 0, 0.25, 1, 3, 24 and 48 h after exercise. Serum was separated and concentrations of D-alpha-tocopherol and ascorbic acid were determined by HPLC. Vitamin C radical levels in plasma were assessed by electron paramagnetic resonance (EPR). RESULTS: Vitamin E and C both showed a tendency to decrease between 3 h and 24 h after exercise. Vitamin C radical level remained stable during the whole period. Neither vitamin E supplementation nor exercise had any influence on the plasma concentration of the vitamin C radical. CONCLUSIONS: Vitamin E supplementation under conditions of mild oxidative stress does not result in an increased vitamin C radical concentration.

Association of alpha-subunits with nucleotide-modified beta-subunits induces asymmetry in the catalytic sites of the F1-ATPase alpha3beta3-hexamer.

The photoaffinity spin-labeled ATP analog, 2-N3-SL-adenosine triphosphate (ATP), was used to covalently modify isolated beta-subunits from F1-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated alpha-subunits to the modified beta-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2:1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated beta-subunits, binding of alpha-subunits induces asymmetry in the catalytic sites even in the absence of the gamma-subunit.

Lipid peroxidation-independent mechanisms of vitamin e-mediated protection against cyclosporine a-induced hepatocellular toxicity and apoptosis.

Cyclosporine A (CsA) produces oxidative stress and apoptosis in rat hepatocytes, but it is not known whether membrane lipid peroxidation plays a role in CsA toxicity. The objective of the present study was to determine whether iron-catalyzed hydroxyl or membrane alkoxyl radical formation is causally involved in the prooxidative cell injury and apoptosis. As previously reported, cultured rat hepatocytes exposed to CsA exhibited concentration-dependent signs of apoptotic cell injury, including chromatin condensation and fragmentation, increased caspase-3 activity, and release of cytosolic lactate dehydrogenase. Addition of the ferric iron chelator desferrioxamine or the novel potent lipophilic iron chelator CGP72670 did not protect against CsA-induced cell death, indicating that iron-catalyzed lipid peroxidation is unlikely to be involved in CsA-mediated apoptosis. In contrast, the classical chain-breaking lipid peroxidation inhibitor alpha-tocopherol was able to rescue cells from CsA-induced cytotoxicity and apoptosis. alpha-Tocopherol not only effectively inhibited the production of thiobarbituric acid-reactive substances and the formation of reactive oxygen species, but it also prevented proteins from being oxidized and forming mixed disulfides. Furthermore, alpha-tocopherol inhibited the cellular uptake of extracellular calcium (45 Ca 2+) into cells, similar to the reducing agent dithiothreitol. By decreasing the extracellular calcium concentrations or by adding calcium channel blockers (diltiazem, nifedipine) or a cell-permeable calcium chelator Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tertraacetic acid (BAPTA), both CsA-induced caspase-3 activation and apoptosis were inhibited, indicating a pivotal role of Ca 2+ in mediating CsA-induced cell injury. These results suggest that alpha-tocopherol protects from CsA-mediated apoptosis and cytotoxictiy by preventing the oxidation of redox-sensitive Ca 2+ -ATPase. Thus, it is the attenuation of increased calcium influx and, hence, the inhibition of caspase-3 activation, rather than the downstream inhibition of lipid peroxidation, that is a key mechanism of the protection provided by alpha-tocopherol against CsA-induced cell injury.

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes.

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.