The effects of helix breaking mutations in the diphtheria toxin transmembrane domain helix layers of the fusion toxin DAB389IL-2

The fusion protein toxin DAB389IL-2 is composed of the catalytic and transmembrane domains of diphtheria toxin genetically linked to human interleukin 2 (IL-2). This fusion toxin is selectively toxic for eukaryotic cells which express the high-affinity form of the IL-2 receptor and the mechanism of intoxication parallels that of native diphtheria toxin. We used site-directed mutagenesis to introduce Pro residues into each of the three helical layers of the transmembrane domain. Although each of the mutations results in the complete loss of cytotoxic activity, individual mutants were found to vary with respect to channel formation in planar lipid bilayers, binding affinity and melting temperature. We propose that each of the three helix layers plays a critical role in the productive delivery of the catalytic domain to the cell cytosol.      

The effects of helix breaking mutations in the diphtheria toxin transmembrane domain helix layers of the fusion toxin DAB389IL-2

Irritable bowel syndrome is frequently encountered in clinical practice, and it has been repeatedly suggested that abnormal colonic motor activity is one of the major pathophysiological mechanisms responsible for the origin of symptoms in such disorder. If this statement is true, then high-amplitude propagated colonic contractions (HAPCs), i.e. the mass movements, may play an important role. To test this hypothesis, we conducted an investigation by recording colonic motility for a prolonged (24 h) period in 25 patients with irritable bowel syndrome and in 18 healthy volunteers, to compare the number of mass movements over 24 h in patients (constipation-predominant, alternating bowel habits) and controls. The overall amount of motility was also assessed in twelve patients and 13 controls. We also looked for the possible changes in mass movements and motility which may occur with defecation and after a meal. The results showed that 1) with respect to HAPCs and motility index, neither group was significantly different from controls; 2) HAPCs and the motility index were significantly reduced during sleep in all groups tested; 3) HAPCs were significantly more common before as compared to after defecation and after as compared to before meals; 4) HAPCs are not independent from the segmental contractile activity; 5) the motility index/24 h was lower in the constipation-predominant group of patients with respect to controls. We conclude that in patients with irritable bowel syndrome colonic motility per se may play a pathophysiological role in the genesis of the symptoms, although other mechanisms are likely to concur, or to be responsible for the complaints of these patients. However, colonic prolonged recordings are very useful for studying physiological and pathophysiological correlates of sleep, eating, and defecation.     

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T- (Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine- dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.      

Structure of the metal-ion-activated diphtheria toxin repressor/tox operator complex

The virulent phenotype of the pathogenic bacterium Corynebacterium diphtheriae is conferred by diphtheria toxin, whose expression is an adaptive response to low concentrations of iron. The expression of the toxin gene (tox) is regulated by the repressor DtxR, which is activated by transition metal ions. X-ray crystal structures of DtxR with and without (apo-form) its coordinated transition metal ion have established the general architecture of the repressor, identified the location of the metal-binding sites, and revealed a metal-ion-triggered subunit-subunit 'caliper-like' conformational change. Here we report the three-dimensional crystal structure of the complex between a biologically active Ni(II)-bound DtxR(C102D) mutant, in which a cysteine is replaced by an aspartate at residue 102, and a 33-base-pair DNA segment containing the toxin operator toxO. This structure shows that DNA interacts with two dimeric repressor proteins bound to opposite sides of the tox operator. We propose that a metal-ion-induced helix-to-coil structural transition in the amino-terminal region of the protein is partly responsible for the unique mode of repressor activation by transition metal ions.     

Genetic construction and characterization of the diphtheria toxin-related interleukin 15 fusion protein DAB389 sIL-15

A gene fusion encoding DAB³⁸⁹ sIL-15 was constructed in whichthe catalytic and transmembrane domains of native diphtheriatoxin (DAB³⁸⁹) are genetically linked to the N-terminus of simianinterleukin 15 (sIL-15). It was demonstrated that the cytotoxicaction of DAB³⁸⁹ sIL-15 is mediated through the IL-15 receptor.Since toxicity may be blocked with chloroquine, it was concludedthat following binding to the IL-15 receptor, the fusion toxinis internalized by receptor-mediated endocytosis and must passthrough an acidic compartment in order to facilitate the deliveryof the catalytic domain to the cytosol of target cells. As anon-toxic control, the ADP-ribosyltransferase defective mutantDA(E149S)B³⁸⁹ sIL-15 was constructed. It was demonstrated thatboth sIL-15 and DA(E149S)B³⁸⁹ sIL-15 stimulate protein and DNAsynthesis in IL-15 receptor-positive CTLL-2 cells in vitro Received July 8, 1995; revised September 25, 1995; accepted October 10, 1995.     

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3

The ultimate goal in the management of inherited as well as acquired diseases is a rational therapy with the aim to eliminate the underlying biochemical defects, rather than a symptomatic treatment. Among other approaches somatic gene therapy is a promising candidate to meet these objectives and appears to have the potential to revolutionize modern medicine. Gene therapy is characterized by the transfer of genetic information to a patient through the use of recombinant DNA technology. Several strategies for the treatment of monogenetic disorders as well as chronical diseases like cancer and AIDS have been used in various somatic gene therapy projects. So far, 329 clinical studies (phases I, I/II and II) with over 2500 patients have been initiated worldwide since 1989. No significant toxicity and adverse side effects have been observed. To allow efficient transfer of the therapeutic genes, a variety of gene delivery techniques have been developed based on viral and non-viral vector systems. For the success of this technology it is vital to achieve regulated and sustained expression of foreign genes in specific target tissues. This will be crucial for the widespread application of somatic gene therapy. So far none for the gene delivery systems is able to meet the requirements of safety, efficiency and specificity demonstrating that vector research will be an important focus in the development of optimized transfer methods. From a regulatory point of view pharmaceutical DNA-products can be regarded as drugs and are therefore subject to the same regulations. Human gene therapy must, however, be limited to manipulations affecting somatic, differentiated cells to prevent the transferred gene from being transmitted to the individual's descendants. Applications for the purpose of 'enhancement' and not for the treatment of diseases are also not acceptable. Under these prerequisites, somatic gene therapy does not raise any new ethical concerns and can be interpreted as a special form of an organ transplantation. A comparison of the different regulatory situations of gene therapy in Europe and the United States demonstrates that for the European countries a uniform regulation is desired. Today somatic gene therapy is still in its infancy. It will continue to be scientifically and technically challenging until simple and effective procedures will have been developed. Demonstration of its clinical efficacy especially in the long term will have to be the next step. Looking at the history of biotechnology and the success of the biotechnology industry that is now providing safe and efficient products from recombinant DNA-technology there is little doubt that gene therapy will become a successful treatment for various indications in the next decade. The purpose of this article is to review the current status of the development in somatic gene therapy.     

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery

Neuropeptide Y (NPY) is the most abundant peptide present in the mammalian central and peripheral nervous system. NPY exhibits a variety of potent central and peripheral effects including those on feeding, memory, blood pressure, cardiac contractility and intestinal secretions. Classical pharmacological studies have shown that NPY effects are mediated by four different receptor subtypes, Y-1, Y-1-like, Y-2, and Y-3. However, the existence of numerous atypical activities provide strong evidence for the occurrence of additional NPY receptor subtypes. Pharmacological studies have further been facilitated by the recent cloning and expression of Y-1, Y-2, Y-4 (PP-1) and Y-5 receptors. Moreover, the cloned Y-5 receptor has been suggested to be the long awaited Y-1-like receptor involved in feeding. Structure-activity studies have laid a good foundation towards the development of receptor selective compounds, and to date potent Y-1 selective peptide and nonpeptide antagonists have been developed. The need to clone numerous receptor subtypes and to develop receptor selective compounds for physiological and perhaps clinical use is expected to keep NPY research active for many years to come.     

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Abstract The transmembrane (T) domain of diphtheria toxin (DT) comprisesnine -helices and has been shown to play an essential role inthe efficent delivery of the catalytic (C) domain ofDT acrossthe eukaryotic cell membrane and into the cytosol. We have demonstratedrecently thatthe first three amphipathic helixes of the T domain,although not necessary for either channel formation or receptorbinding, are required for the efficient transmembrane deliveryof the Cdomain.In the present study,we have performed a detailedstructure-function analysis of T domainhelix 1 (TH1) of theDT-related fusion protein DAB₃₈₉lL-2. We performed exchangeandsite-directed mutagenesis of TH1 and the resulting mutantfusion toxins were analyzed by gel electrophoresis and testedfor their efficiencies in the delivery of the C domain to thecell cytosol. We demonstrate that the overall charge distributionand hydrophobicity of amino acids in the amphipathic helix TH1,rather than a specific amino acid sequence, are critical forthe function of this helix. The insertion of a charged residuein the hydrophobic face of TH1 abolishes cytotoxic activity,whereas replacement of a hydrophobic residue by a charged aminoacid in the hydrophilic face of the helix has little, if any,effect on cytotoxic activity. In addition,we have identifiedSer220 by site-directed mutagenesis as a residue that appearsto be criticalfor correct folding of the fusion toxin. Mutationsin this position result in fusion proteins that are extremelysensitive to proteolytic attack.