Inefficient nuclear delivery of plasmid DNA is thought to activity of apoptotic and lysosomal nucleases; (2) disposal be one of the daunting hurdles to gene transfer, utilizing a of microinjected plasmid DNA was inhibited in cytosolnonviral delivery system such as polycation-DNA complex.depleted cells or following the encapsulation of DNA in Following its internalization by endocytosis, plasmid DNA phospholipid vesicles; (3) generation and subsequent elimhas to be released into the cytosol before its nuclear entry ination of free 3Ј-OH ends could be detected by the tercan occur. However, the stability of plasmid DNA in the minal deoxynucleotidyl transferase-mediated dUTP nick cytoplasm, that may play a determinant role in the transfecend-labeling assay (TUNEL), reflecting the fragmentation tion efficiency, is not known. The turnover of plasmid DNA, of the injected DNA; and finally (4) isolated cytosol, delivered by microinjection into the cytosol, was deterobtained by selective permeabilization of the plasma memmined by fluorescence in situ hybridization (FISH) and brane, exhibits divalent cation-dependent, thermolabile quantitative single-cell fluorescence video-image analysis. nuclease activity, determined by Southern blotting and 32 PBoth single-and double-stranded circular plasmid DNA disrelease from end-labeled DNA. Collectively, these findings appeared with an apparent half-life of 50-90 min from the suggest that the metabolic instability of plasmid DNA, cytoplasm of HeLa and COS cells, while the amount of cocaused by cytosolic nuclease, may constitute a previously injected dextran (MW 70 000) remained unaltered. We prounrecognized impediment for DNA translocation into the pose that cytosolic nuclease(s) are responsible for the nucleus and a possible target to enhance the efficiency of rapid degradation of plasmid DNA, since (1) elimination of gene delivery. plasmid DNA cannot be attributed to cell division or to the Keywords: gene transfer; plasmid DNA; turnover; degradation; DNase; microinjection Introduction Liposome-mediated cellular transfer of plasmid DNA is a promising approach for gene therapy. However, despite the significant amount of lipid/DNA complexes internalized by the target cells, transgene expression remains undesirably low.1 Obstacles to nuclear accumulation of plasmid DNA include: the slow internalization process of the lipid/DNA complex in certain cells; 2 the entrapment of DNA in the endolysosomal compartment; 1,3,4 and the diffusional barrier of the nuclear envelope. 5The underlying mechanism of escape of internalized plasmid DNA from the endo-lysosomes is not fully understood. This process involves the destabilization of the limiting membrane of the endolysosomal compartment, the dissociation of the lipid/DNA complex and the release of plasmid DNA into the cytosol.6-8 Penetration of naked plasmid DNA into the cytosol was verified by using the T7 polymerase transfection system, which Correspondence: GL Lukacs, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canad...
Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5-to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.
The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a KD of 5.2 microM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (KD of 1.4 microM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.
Impaired biosynthetic processing of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, constitutes the most common cause of CF. Recently, we have identified a distinct category of mutation, caused by premature stop codons and frameshift mutations, which manifests in diminished expression of COOH-terminally truncated CFTR at the cell surface. Although the biosynthetic processing and plasma membrane targeting of truncated CFTRs are preserved, the turnover of the complex-glycosylated mutant is sixfold faster than its wild-type (wt) counterpart. Destabilization of the truncated CFTR coincides with its enhanced susceptibility to proteasome-dependent degradation from post-Golgi compartments globally, and the plasma membrane specifically, determined by pulse–chase analysis in conjunction with cell surface biotinylation. Proteolytic cleavage of the full-length complex-glycosylated wt and degradation intermediates derived from both T70 and wt CFTR requires endolysosomal proteases. The enhanced protease sensitivity in vitro and the decreased thermostability of the complex-glycosylated T70 CFTR in vivo suggest that structural destabilization may account for the increased proteasome susceptibility and the short residence time at the cell surface. These in turn are responsible, at least in part, for the phenotypic manifestation of CF. We propose that the proteasome-ubiquitin pathway may be involved in the peripheral quality control of other, partially unfolded membrane proteins as well.
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