Specific targeting of a pore-forming toxin (listeriolysin O) to LHRH-positive cancer cells using LHRH targeting peptide.

Conventional drug delivery systems have many limitations including cytotoxicity and affecting non-specific cells. Cell-targeting peptides (CTPs) as a potential class of targeting moiety have some advantages over previous targeting moieties such as monoclonal antibodies, offer additional benefits to design systems using CTPs. Here we have engineered listeriolysin O (LLO) pore-forming toxin by adding a luteinizing hormone-releasing hormone (LHRH) targeting peptide to its N-terminus. Two versions of the toxin, with and without targeting peptide, were sub-cloned into a bacterial expression plasmid. BL21 DE3 cells were used for induction of expression and recombinant proteins were purified using nickel-immobilized metal affinity chromatography column. In order to treat MDA-MB-231 and SKOV3 cell lines as LHRH receptor positive and negative cells, two mentioned LLO toxins were used to evaluate their cytotoxicity and specificity. Our results reveal that the IC50 of LLO toxin on MDA-MB-231 and SKOV3 cells was 0.32 and 0.41 µg/ml respectively. Furthermore, IC50 of fusion LHRH-LLO toxin on the cells was 0.88 and 19.55 µg/ml. Cytotoxicity of engineered LHRH-LLO toxin on negative cells was significantly 48-fold lower than wild-type LLO toxin. But this difference has been lowered to only 2.7-fold less cytotoxicity in positive cells. To the best of our knowledge, the current work as the first study regarding engineered toxin revealed that CDC family members could be used to target the specific cell-type.

Mutants of listeriolysin O for enhanced liposomal delivery of macromolecules.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

Development of human hepatocellular carcinoma cell-targeted protein cages.

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.

Molecular design of protein-based nanocapsules for stimulus-responsive characteristics.

Hsp16.5, a small heat-shock protein (sHSP) from hyperthermophilic archaeon, forms a homogeneous complex comprised of 24 subunits with a molecular mass of 400 kDa. This complex self-organizes under physiological conditions, and the structure of the complex is a nanoscale spherical capsule with small pores. Furthermore, this natural nanocapsule exhibits very high thermal stability. In this paper, we functionalized the nanocapsule to control the structure in response to external stimuli such as a protease signal and temperature. For this purpose, several mutations (Mut1-10) to create a cleavage site for a specific protease, Factor Xa, were introduced on the outer surface of the nanocapsule using a genetic engineering strategy. The resulting mutants were expressed to high levels in Escherichia coli. One of these mutants, Mut6, which has the most accessible cleavage site located at the triangular pore on the surface of the capsule, formed a spherical assembly similar to that observed for the wild-type protein. Mut6 showed the highest sensitivity to Factor Xa, and the structure of the protease digested Mut6 disassembled irreversibly after heating. In contrast, the nanocapsule comprising the wild-type Hsp16.5 was not influenced by the dual stimuli. These results suggest that Mut6 acts as a stimulus-responsive nanocapsule. Such a characteristic of the protein-based nanocapsule has attractive potential as a versatile intelligent system.

Heavy metal accumulation, heat shock protein expression and cytogenetic changes in Tetrix tenuicornis (L.) (Tetrigidae, Orthoptera) from polluted areas.

The orthopteran insect Tetrix tenuicornis, collected from polluted and unpolluted areas, was used to study heavy metal accumulation and its impact on stress protein levels and on changes in the number and morphology of chromosomes in mitotic and meiotic cells. During two consecutive years, insects were collected from polluted areas of zinc-lead mine spoils near Boleslaw (Poland) and from unpolluted areas near Busko and Staszów (Poland). T. tenuicornis from the polluted area showed 1.5, 4.03, 4.32 and 41.73 times higher concentrations of copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd), respectively, than insects of the same species collected from unpolluted areas. Insects exposed to heavy metals showed only small changes, and rather a decrease in the concentration of constitutive and inducible heat shock proteins Hsp70, the level of which increases under stress conditions. A cytogenetic study of T. tenuicornis revealed intra-population anomalies in chromosome number and morphology in mitotic and meiotic cells and the presence of an additional B chromosome in germinal cells. In 50% of females collected from polluted areas, mosaic oogonial mitotic chromosome sets and diploid, hypo- or hypertetraploid, tetraploid, and octoploid chromosome numbers were detected. In turn, 14.6% of males showed a heterozygous deficiency of chromatin in L2 and M3 bivalents in addition to the presence of B chromosomes.

Cytosolic delivery of viral nucleoprotein by listeriolysin O-liposome induces enhanced specific cytotoxic T lymphocyte response and protective immunity.

Cytotoxic T lymphocytes (CTLs) are capable of conferring protection against intracellular pathogens and tumor. Protective antiviral immunity, mediated by the activation of antigenic epitope-specific CTL, can be achieved by delivering exogenous antigen into the cytosol of antigen-presenting cells. Cytosolic introduction of vaccine antigen, however, requires a specialized delivery strategy due to the membrane barrier limiting the access of macromolecules to the cytosol. In this study, we have investigated the potential ability of listeriolysin O-containing liposomes (LLO-liposomes) to deliver lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP), harnessing the intracellular invasion mechanism of Listeria monocytogenes, to stimulate a NP-specific CTL response. We have analyzed the ability of LLO-liposomes to induce an enhanced CTL response and determined the extent of CTL-mediated protection using an in vivo infection model. Mice immunized with LLO-liposomes containing NP generated a higher frequency of NP-specific CD8+ T cells with greater effector activity than the control groups immunized with either non-LLO-liposomal NP or LLO-liposomes containing control protein. Moreover, LLO-liposomal NP-immunized mice were completely protected against a lethal intracerebral challenge with a virulent strain of LCMV and were capable of clearing a chronic LCMV infection. Our study demonstrates that LLO-liposomes can be used as an efficient vaccine delivery system carrying a viral antigenic protein to generate protective antiviral immunity.

Effect of HSP47 on prolyl 4-hydroxylation of collagen model peptides.

Prolyl 4-hydroxylation, the most important post-translational modification in collagen biosynthesis, is catalyzed by prolyl 4-hydroxylase, an endoplasmic reticulum-resident enzyme. HSP47 is a collagen-binding stress protein which also resides in the endoplasmic reticulum (Nagata, K. and Yamada, K.M. (1986) J. Biol. Chem., 261, 7531-7536). Both prolyl 4-hydroxylase and HSP47 interact with procollagen alpha-chains during their folding and/or modification in the endoplasmic reticulum. Recent study has revealed that a simple collagen model peptide, (Pro-Pro-Gly)n, is recognized by HSP47 as well as by prolyl 4-hydroxylase in vitro (Koide et al., manuscript submitted). In the present study, we investigated the effect of HSP47 on the prolyl 4-hydroxylation of such collagen model peptides. To monitor the enzymatic hydroxylation of the peptides, we developed a non-RI assay system based on reversed-phase HPLC. When HSP47 was added to the reaction mixture, substrate and less-hydroxylated materials accumulated. This effect depended on the peptide-binding activity of HSP47, because a mutant HSP47 without collagen-binding activity did not show any inhibitory effect on prolyl 4-hydroxylation. Kinetic analysis and other biochemical analyses suggest that HSP47 retards the enzymatic reaction competing for the substrate peptide.

Heat shock proteins HSP25, HSP60, HSP72, HSP73 in isoosmotic cortex and hyperosmotic medulla of rat kidney.

The distribution of heat shock proteins (HSP) HSP60, HSP73, HSP72 and HSP25 in the isoosmotic cortex and the hyperosmotic medulla of the rat kidney was investigated using Western blot analysis and immunohistochemistry. HSP73 was homogeneously distributed throughout the whole kidney. The level of HSP60 was high in the renal cortex and low in the medulla. HSP25 and HSP72 were present in large amounts in the medulla. Only low levels of HSP25 and almost undetectable amounts of HSP72 were found in the cortex. HSP25 exists in one nonphosphorylated and several phosphorylated isoforms. Western blot analysis preceded by isoelectric focussing showed that HSP25 predominates in its nonphosphorylated form in the outer medulla but in its phosphorylated form in cortex and inner medulla. Although this intrarenal distribution pattern was not changed during prolonged anaesthesia (thiobutabarbital sodium), a shift from the nonphosphorylated to the phosphorylated isoforms of HSP25 occurred in the medulla. The characteristic intrarenal distribution of the constitutively expressed HSPs (HSP73, HSP60, HSP25) may reflect different states of metabolic activity in the isoosmotic (cortex) and hyperosmotic (medulla) zones of the kidney. The high content of inducible HSP72 in the medulla most likely is a consequence of the osmotic stress imposed upon the cells by the high urea and salt concentrations in the hyperosmotic medullary environment.

Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids.

The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.

Distribution in tissue sections of the human groEL stress-protein homologue.

A monoclonal antibody (ML30) raised against the 65 kDa heat-shock protein of mycobacteria showed widespread staining of sections from standard paraffin-embedded human tissues. The staining had a granular pattern and was particularly marked in cells with abundant mitochondria. Increased staining was observed in the synovial lining, histocytes and in the endothelium of reactive and rheumatoid synovium; it was also increased in the reactive lung alveolar lining. It is suggested that the antibody identifies an epitope in mitochondria of a protein homologous with the groEL heat-shock protein of bacteria.