Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumour TRAIL sensitivity.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.

Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL.

BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2. METHODS: We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer. RESULTS: Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers. CONCLUSION: Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

Processing and functional display of the 86 kDa heterodimeric penicillin G acylase on the surface of phage fd.

The large heterodimeric penicillin G acylase from Alcaligenes faecalis was displayed on the surface of phage fd. We fused the coding sequence (alpha subunit-internal peptide-beta subunit) to the gene of a phage coat protein. A modified g3p signal sequence was used to direct the polypeptide to the periplasm. Here we show that a heterodimeric enzyme can be expressed as a fusion protein that matures to an active biocatalyst connected to the coat protein of phage fd, resulting in a phage to which the beta-subunit is covalently linked and the alpha-subunit is non-covalently attached. The enzyme can be displayed either fused to the minor coat protein g3p or fused to the major coat protein g8p. In both cases the penicillin G acylase on the phage has the same Michaelis constant as its freely soluble counterpart, indicating a proper folding and catalytic activity of the displayed enzyme. The display of the heterodimer on phage not only allows its further use in protein engineering but also offers the possibility of applying this technology for the excretion of the enzyme into the extracellular medium, facilitating purification of the protein. With the example of penicillin acylase the upper limit for a protein to become functionally displayed by phage fd has been further explored. Polyvalent display was not observed despite the use of genetic constructs designed for this aim. These results are discussed in relation to the pore size being formed by the g4p multimer.

SecDF of Bacillus subtilis, a molecular Siamese twin required for the efficient secretion of proteins.

In the present studies, we show that the SecD and SecF equivalents of the Gram-positive bacterium Bacillus subtilis are jointly present in one polypeptide, denoted SecDF, that is required to maintain a high capacity for protein secretion. Unlike the SecD subunit of the pre-protein translocase of Escherichia coli, SecDF of B. subtilis was not required for the release of a mature secretory protein from the membrane, indicating that SecDF is involved in earlier translocation steps. Strains lacking intact SecDF showed a cold-sensitive phenotype, which was exacerbated by high level production of secretory proteins, indicating that protein translocation in B. subtilis is intrinsically cold-sensitive. Comparison with SecD and SecF proteins from other organisms revealed the presence of 10 conserved regions in SecDF, some of which appear to be important for SecDF function. Interestingly, the SecDF protein of B. subtilis has 12 putative transmembrane domains. Thus, SecDF does not only show sequence similarity but also structural similarity to secondary solute transporters. Our data suggest that SecDF of B. subtilis represents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.

Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.

Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.

Evolution of the single copy alpha A-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3' non-coding regions.

alpha A-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat alpha A2-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The alpha A-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3'-untranslated regions of alpha A2-crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The alpha A2-mRNA 3'-non-coding regions of reptiles and birds are 300-550 bases longer than those of mammals. Some rodents produce next to the alpha A2-mRNA another messenger that encodes the alpha AIns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the alpha A2-polypeptide chain. alpha A2 and alpha AIns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides found in vivo and in vitro. The size heterogeneity of the alpha A2-mRNA from most mammals examined is due to the variable size of the poly(A) tail.

Primary and secondary structure of hamster vimentin predicted from the nucleotide sequence.

The nucleotide sequence of two recombinant plasmids containing hamster vimentin cDNA was determined. The sequence comprises 1,640 base pairs and reveals virtually the total primary structure of vimentin and a large part of the 3' noncoding region. Secondary structure prediction methods allow the characterization of two distinct regions of the polypeptide chain, 135 and 145 residues long, which are able to form alpha helices organized in "coiled coils." Three nonhelical domains can be distinguished: a very basic NH2-terminal domain of at least 67 residues, a nonhelical region of 45 amino acids separating the two helix domains, and a COOH-terminal region of 55 residues, which contains an excess of acidic amino acids. The meaning of each of these domains of the vimentin polypeptide for the subunit and filament formation is discussed.