Repair of methyl lesions in DNA and RNA by oxidative demethylation.

It was established several decades ago that it is crucial for all organisms to repair their DNA to maintain genome integrity and numerous proteins are dedicated to this purpose. However, it is becoming increasingly clear that it is also important to prevent and repair lesions in the macromolecules encoded by the DNA, i.e. RNA and protein. Many neurological disorders such as Alzheimer's disease and Parkinson's disease are associated with the aggregation of defective, misfolded proteins, and several mechanisms exist to prevent such aggregation, both through direct protein repair and through the elimination and repair of faulty or damaged RNAs. A few years ago, it was discovered that the E. coli AlkB protein represented an iron and 2-oxoglutarate dependent oxygenase capable of repairing methyl lesions in DNA by a novel mechanism, termed oxidative demethylation. Furthermore, it was found that both human and bacterial AlkB proteins were able to demethylate lesions also in RNA, thus representing the first example of RNA repair. In the present review, recent findings on the AlkB mechanism, as well as on RNA damage in general, will be discussed.

Design of toxins that can be activated by cell-specific proteases and their potential use in targeted cell killing.

Protein toxins designed to eliminate specific cell types, e.g. disease-associated cells, have mainly made by linking the active domain of the toxin to a protein that only binds to certain cells. A different approach for the construction of toxins capable of killing disease-associated cells is suggested here, based on the knowledge that many of these cells express specific proteases that are not expressed in normal tissue. The construction of toxins that become activated through cleavage by the protease (HIV-1 PR) expressed by the HIV-1 virus is described. These toxins contain a signal for degradation by the N-end rule pathway, which is cleaved off by HIV-1 PR, resulting in increased toxicity. Alternative strategies for the construction of toxins that can be activated by proteases are discussed.

Externally added aFGF mutants do not require extensive unfolding for transport to the cytosol and the nucleus in NIH/3T3 cells.

Acidic fibroblast growth factor (aFGF) is transported to the cytosol and the nucleus when added to cells expressing FGF receptors, implying that aFGF must cross cellular membranes. Since protein translocation across membranes commonly requires extensive unfolding of the protein, we were interested in testing whether this is also necessary for membrane translocation of aFGF. We therefore constructed mutant growth factors with intramolecular disulfide bonds to prevent complete unfolding. Control experiments demonstrated that translocation of aFGF by the diphtheria toxin pathway, which requires extensive unfolding of the protein, was prevented by disulfide bond formation, indicating that the introduced disulfide bonds interfered with the unfolding of the growth factor. On the other hand, when the growth factor as such was added to cells expressing FGF receptors, the disulfide-bonded mutants were translocated to the cytosol and the nucleus equally well as wild-type aFGF. The possibility that the translocation of the mutants was due to reduction of the disulfide bonds prior to translocation was tested in experiments using an irreversibly cross-linked mutant. Also this mutant was transported to the cytosol and to the nucleus. The results suggest that extensive unfolding is not required for membrane translocation of aFGF.

Penetration of protein toxins into cells.

AB toxins deliver their enzymatically active A domain to the cytosol. Some AB-toxins are able to penetrate cellular membranes from endosomes where the low pH triggers their translocation. One such toxin is diphtheria toxin and important features of its translocation mechanism have been unraveled during the last year. Other toxins depend on retrograde transport through the secretory pathway to the ER before translocation, and recent findings suggest that these toxins take advantage of the ER translocation machinery normally used for transport of cellular proteins. In addition, the intracellular targets of many of these toxins have been identified recently.

Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin.

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.

Toxins that are activated by HIV type-1 protease through removal of a signal for degradation by the N-end-rule pathway.

Diphtheria toxin enters the cytosol of mammalian cells where it inhibits cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work we describe the construction of toxins that contain a signal for N-end-rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed.

Characterization of membrane translocation by anthrax protective antigen.

Solving the crystallographic structure of the ring-shaped heptamer formed by protective antigen (PA), the B moiety of anthrax toxin, has focused attention on understanding how this oligomer mediates membrane translocation of the toxin's A moieties. We have developed an assay for translocation in which radiolabeled ligands are bound to proteolytically activated PA (PA63) at the surface of CHO or L6 cells, and translocation across the plasma membrane is induced by lowering the pH. The cells are then treated with Pronase E to degrade residual surface-bound material, and protected ligands are quantified after fractionation by SDS-PAGE. Translocation was most efficient (35%-50%) with LFN, the N-terminal PA binding domain of the anthrax lethal factor (LF). Intact LF, edema factor (EF), or fusion proteins containing LFN fused to certain heterologous proteins [the diphtheria toxin A chain (DTA) or dihydrofolate reductase (DHFR)] were less efficiently translocated (15%-20%); and LFN fusions to several other proteins were not translocated at all. LFN with different N-terminal residues was found to be degraded according to the N-end rule by the proteasome, and translocation of LFN fused to a mutant form of DHFR with a low affinity for methotrexate (MTX) protected cells from the effects of MTX. Both results are consistent with a cytosolic location of protected proteins. Evidence that a protein must unfold to be translocated was obtained in experiments showing that (i) translocation of LFNDTA was blocked by introduction of an artificial disulfide into the DTA moiety, and (ii) translocation of LFNDHFR and LFNDTA was blocked by their ligands (MTX and adenine, respectively). These results demonstrate that the acid-induced translocation by anthrax toxin closely resembles that of diphtheria toxin, despite the fact that these two toxins are unrelated and form pores by different mechanisms.

Cloning of an intracellular protein that binds selectively to mitogenic acidic fibroblast growth factor.

In addition to its extracellular action, there is evidence that acidic fibroblast growth factor (aFGF) acts inside cells. To identify intracellular proteins interacting with aFGF, we screened a HeLa cell library in the yeast two-hybrid system using pLex-aFGF as a bait. A clone binding to aFGF, but not to the non-mitogenic mutant aFGF-K132E, was isolated and characterized. The insert contained an open reading frame corresponding to a novel protein of 42 kDa. The protein, termed aFGF intracellular binding protein (FIBP), is mainly hydrophilic and does not contain an N-terminal signal sequence. In vitro-translated FIBP bound specifically to a fusion protein of maltose-binding protein and aFGF. FIBP became post-translationally associated with microsomes added to the cell-free protein synthesizing system, and the membrane-associated protein bound aFGF with high efficiency. Immunoblots and fluorescence microscopy demonstrated that the protein is present in nuclei and, to a lesser extent, associated with mitochondria and other cytoplasmic membranes. The possibility is discussed that FIBP may be involved in the mitogenic action of aFGF.

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway.

The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process.

Effect of mutation of cytoplasmic receptor domain and of genistein on transport of acidic fibroblast growth factor into cells.

Acidic fibroblast growth factor (aFGF) binds to specific transmembrane receptors and is partly transported to a nuclear location. To study this transport we made a kinase-negative mutant of FGF receptor 4 as well as one where the major part of the cytoplasmic receptor domain was deleted, and expressed them in U2OSDr1 cells that lack functional FGF receptors. All receptors mediated endocytic uptake of aFGF. Translocation of the growth factor across cellular membranes was assayed using aFGF with a C-terminal CAAX-motif, which signals addition of a farnesyl group onto the protein once in the cytosol. CAAX-tagged aFGF was farnesylated when incubated with cells containing wild-type or kinase-negative receptors. It was not farnesylated in cells expressing the deleted receptor, or when the incubation was in the presence of genistein. aFGF incubated with cells transfected with wild-type or kinase-negative receptors, but not with the deleted receptor, was partly recovered from the nuclear fraction in the absence, but not in the presence of genistein. The data indicate that the cytoplasmic receptor domain, but not the active kinase, is required for transport of the growth factor into cells, and that genistein inhibits the process.

Retrograde transport of mutant ricin to the endoplasmic reticulum with subsequent translocation to cytosol.

Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of Na(2)(35)SO(4), the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.

Structure-function relationship of the ion channel formed by diphtheria toxin in Vero cell membranes.

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Deltagm, was initially determined. DT-AB induced a approximately 10-fold lower Deltagm than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Deltagm, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Deltagm. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Deltagm and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.

Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell.

Translocation of cytosol of exogenous, CAAX-tagged acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) added externally to cells has been proposed to enter the nucleus and stimulate DNA synthesis, but it has remained controversial whether or not exogenous aFGF has the capability of crossing cellular membranes. To test this, a novel principle to study translocation of proteins to the cytosol was developed by fusing a C-terminal farnesylation signal, a CAAX tag (C = Cys, A = an aliphatic amino acid, and X = any amino acid), onto aFGF. Farnesylation is only known to occur in the cytosol and possibly in the nucleus. When incubated with NIH3T3 cells overnight, about one-third of the cell-associated, CAAX-tagged growth factor was farnesylated, indicating that efficient translocation had taken place. Binding to specific FGF receptors was required for translocation to occur. Part of the farnesylated growth factor was found in the nuclear fraction. The data indicate that CAAX-tagged aFGF added externally to cells is able to cross cellular membranes and enter the cytosol and the nucleus.

Farnesylation of CaaX-tagged diphtheria toxin A-fragment as a measure of transfer to the cytosol.

Diphtheria toxin binds to receptor-positive cells through its B-fragment, the toxin is then endocytosed, and the low pH in endosomes triggers the translocation of the enzymatically active A-fragment to the cytosol. A synchronous release of A-fragments into the cytosol can be induced by exposing cells with surface-bound toxin to low pH. We have used this protein translocation system to develop a novel method to study whether or not a protein is exposed to the cytosol. Protein farnesylation is a cytosolic modification signaled by a C-terminal CaaX motif, and to visualize the translocation process, we added a farnesylation signal to the toxin A-fragment. The A-fragment with an added CaaX motif was farnesylated within 1 h after exposure of cells with surface-bound toxin to low pH, and also A-fragment translocated from endosomes was quantitatively farnesylated. The results indicate that all cell-mediated reduction of the toxin implicates translocation of the A-fragment to the cytosol. The farnesylation was inhibited by lovastatin, the alkylating agent NEM, and the peptidomimetic farnesylation inhibitor B581. Farnesylated A-fragment partitioned preferentially into the detergent phase upon extraction with Triton X-114. Our data suggest that farnesylation of a CaaX tag is generally applicable as a cytosolic marker, and this strategy for monitoring protein transfer to the cytosol may have considerable potential for studying the transport to the cytosol of proteins added externally to cells.

Cell-mediated reduction and incomplete membrane translocation of diphtheria toxin mutants with internal disulfides in the A fragment.

Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.

Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges.

Fragment A of diphtheria toxin is translocated to the cytosol when the toxin is presented to receptor-positive cells. The toxin binds to cell surface receptors through its B-fragment, and after endocytotic uptake, the low endosomal pH triggers translocation of the A-fragment across the membrane. Translocation can also be induced at the level of the plasma membrane by exposure to low pH medium. Based on the diphtheria toxin crystal structure, we made five double cysteine mutants of the A-fragment, each expected to form an intramolecular disulfide bond. Four of the double cysteine mutants efficiently formed an intramolecular disulfide bridge, and these same mutants showed a strong reduction in their translocating ability. The inhibition of translocation was observed both when the toxin was endocytosed and when translocation was induced by exposing surface-bound toxin to low pH. The data indicate that extensive unfolding of the A-fragment is required for translocation.

Dual mode of signal transduction by externally added acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF), fused to diphtheria toxin and translocated into cells, stimulated DNA synthesis in toxin-resistant cells lacking functional aFGF receptors while having a high number of diphtheria toxin receptors. In NIH 3T3 cells that lack diphtheria toxin receptors, but have receptors for aFGF, both aFGF and the fusion protein induced tyrosine phosphorylation, but only aFGF as such entered the nuclei and stimulated DNA synthesis. The results indicate that signaling occurs partly through cell surface receptors and partly by transport of the growth factor into the cell.

Subcloning and characterization of the binding domain of fragment B of diphtheria toxin.

The binding domain (R domain) of diphtheria toxin as defined from the recently published crystal structure [Choe, Bennett, Fujii, Curmi, Kantardjieff, Collier and Eisenberg (1992) Nature (London) 357, 216-222] was subcloned. The 17 kDa peptide containing amino acids 378-535 from fragment B of diphtheria toxin preceded by the tripeptide Met-His-Gly bound specifically and with high affinity to diphtheria-toxin receptors. It efficiently inhibited the toxicity of full-length toxin. The binding domain entered the detergent phase of Triton X-114 at pH values below 6, indicating that it exposed hydrophobic regions at acidic pH.