Immunization with dendritic cells (DCs) transfected with genes encoding tumor-associated antigens (TAAs) is a highly promising approach to cancer immunotherapy. We have developed a system, using complexes of plasmid DNA expression constructs with the cationic peptide CL22, that transfects human monocyte-derived DCs much more efficiently than alternative nonviral agents. After CL22 transfection, DCs expressing antigens stimulated autologous T cells in vitro and elicited primary immune responses in syngeneic mice, in an antigen-specific manner. Injection of CL22-transfected DCs expressing a TAA, but not DCs pulsed with a TAA-derived peptide, protected mice from lethal challenge with tumor cells in an aggressive model of melanoma. The CL22 system is a fast and efficient alternative to viral vectors for engineering DCs for use in immunotherapy and research.
Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure vari-
To investigate the phosphorylation of human endothelin-converting enzyme-1 (hECE-1) and identify potential residues involved, both in vivo and in vitro phosphorylation labeling assays of hECE-1 isoforms were performed in combination with site-directed mutagenesis and mass spectrometric analyses. Initial studies found that endogenous hECE-1 was constitutively phosphorylated in a primary endothelial cell line. The four known isoforms of hECE-1 expressed in this cell line (1a, 1b, 1c, and 1d) were then cloned by reverse transcription-PCR to determine which isoform(s) may be phosphorylated. The isoforms differ only in the first portion of their short aminoterminal cytoplasmic domains whereas their transmembrane domains and ectodomains of the proteins are identical. Isoforms 1b, 1c, and 1d but not 1a, were constitutively phosphorylated in vivo when expressed in Chinese hamster ovary cells and casein kinase I readily phosphorylated the immunopurified isoforms in vitro. Site-directed mutagenesis established that two conserved serine residues, Ser 18 and Ser 20, (numbering based on isoform 1c) form at least one phosphorylation site in these three isoforms. Mutant forms of 1b, 1c, and 1d were constructed in which a single alanine was introduced at either serine residue and a double mutant for each isoform was constructed as well in which both serines were replaced with alanine. Phosphorylation of the single mutants was greatly reduced and was nearly abolished in the double mutants in both in vivo and in vitro labeling assays. Analysis by MALDI-MS of 32 P-labeled proteolytic peptides derived from wild type 1c and the 1c mutants supported both Ser 18 and Ser 20 as phosphorylated residues. These data demonstrate the first finding that hECE-1 is constitutively phosphorylated within its cytoplasmic domain in an isoform-specific manner.
Coenzyme Q 0 (Q 0 ), a strong electrophile, is toxic to insulin-producing cells. Q 0 was incubated with rat and human pancreatic islets and INS-1 insulinoma cells, and its attachment to cellular proteins was studied with Western analysis using antiserum raised against the benzoquinone ring structure of ubiquinone (anti-Q). Q 0 covalently bonded to two proteins, one of 50 kDa and another of 70 kDa. Both proteins were found to be mitochondrial in human and rat islet cells and in many rat organs. Mitochondria were incubated with Q 0 , and affinity-purified anti-Q was used to immunoprecipitate the 50-kDa protein. Amino acid sequencing identified it as dihydrolipoamide succinyltransferase, the E2 component of the ␣-ketoglutarate dehydrogenase complex (KDC). Western analysis also showed that Q 0 bonds to the E2 components of the purified KDC and the pyruvate dehydrogenase complex (PDC). Dihydrolipoamide acetyltransferase, the E2 of the PDC, has a molecular mass of 70 kDa, and the 70-kDa protein was inferred to be this enzyme. Q 0 was found to bond only to proteins containing dihydrolipoate, and in preparations of mitochondria, thiol reducing agents facilitated the attachment of Q 0 , but oxidizing agents prevented it, suggesting that Q 0 bonds to thiols of dihydrolipoamide. Incubation of human or pig PDC with Q 0 followed by matrix-assisted laser desorption ionization time-of-flight and liquid chromatography/electrospray ionization mass spectrometry analyses of chymotrypsin-digested peptides of PDC E2 confirmed that Q 0 bonds to the dihydrolipoamide in these proteins. In mitochondria, coenzymes Q 1 and Q 2 did not bond to the 50-kDa protein but competed with the bonding of Q 0 to this protein. The prevention by Q 1 of the bonding of Q 0 to KDC E2, as well as other characteristics of the Q 0 effect, are reminiscent of the action of Q 0 on the mitochondrial permeability transition pore described previously (Fontaine, E., Ichas, F., and Bernardi, P. (1998) J. Biol. Chem. 273, 25734 -25740).Ubiquinone analogs (1, 2) and other quinones (3-6) stimulate insulin release from pancreatic islets, but they also kill the insulin-producing cell. The insulin release is not simply due to insulin leaked from dying cells because numerous inhibitors of cellular respiration that kill the cell, such as rotenone, antimycin A, cyanide, and dinitrophenol, inhibit insulin release rather than stimulate it (7, 8). The injurious effects of these compounds may be due, in part, to redox cycling that collapses the proton gradient that maintains the electrical potential of the inner mitochondrial membrane. However, many quinones, because of their electrophilicity, can induce various patterns of oxidative damage to cells. Also, some ubiquinone analogs influence the mitochondrial permeability transition pore (PTP) 1 (9 -11). To learn more about the roles of quinones in these processes, we raised antisera to the ring structure of ubiquinone (anti-Q antibodies) and used them in Western analysis and for immunoprecipitation. This enabled us to identify se...
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