The Proteasome Metabolizes Peptide-Mediated Nonviral Gene Delivery Systems
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Gene Therapy (2005) 12, 1581–1590 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt RESEARCH ARTICLE The proteasome metabolizes peptide-mediated nonviral gene delivery systems J Kim, C-P Chen and KG Rice Division of Medicinal and Natural Product Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA, USA The proteasome is a multisubunit cytosolic protein complex directly with proteasome inhibition, and was not the result of responsible for degrading cytosolic proteins. Several studies lysosomal enzyme inhibition. The enhanced transfection was have implicated its involvement in the processing of viral specific for peptide DNA condensates, whereas Lipofecta- particles used for gene delivery, thereby limiting the mine- and polyethylenimine-mediated gene transfer were efficiency of gene transfer. Peptide-based nonviral gene significantly blocked. A series of novel gene transfer peptides delivery systems are sufficiently similar to viral particles in containing intrinsic GA proteasome inhibitors (CWK18(GA)n, their size and surface properties and thereby could also be where n ¼ 4, 6, 8 and 10) were synthesized and found to recognized and metabolized by the proteasome. The present inhibit the proteasome. The gene transfer efficiency mediated study utilized proteasome inhibitors (MG 115 and MG 132) by these peptides in four different cell lines established that a to establish that peptide DNA condensates are metabolized GA repeat of four is sufficient to block the proteasome and by the proteasome, thereby limiting their gene transfer significantly enhance the gene transfer. Together, these efficiency. Transfection of HepG2 or cystic fibrosis/T1 (CF/T1) results implicate the proteasome as a previously undiscov- cells with CWK18 DNA condensates in the presence of ered route of metabolism of peptide-based nonviral gene MG 115 or MG 132 resulted in significantly enhanced gene delivery systems and provide a rationale for the use of expression. MG 115 and MG 132 increased luciferase proteasome inhibition to increase gene transfer efficiency. expression 30-fold in a dose-dependent manner in HepG2 Gene Therapy (2005) 12, 1581–1590. doi:10.1038/ and CF/T1. The enhanced gene expression correlated sj.gt.3302575; published online 30 June 2005 Keywords: peptide-mediated gene delivery; metabolism; proteasome; gene transfer Introduction subunits having multiple proteolytic activities.11,12 The chymotrypsin-like activity, trypsin-like activity and The premature metabolism of plasmid DNA limits both peptidyl-glutamyl peptide hydrolyzing activity residing the level and duration of transient gene expression by within the 20S proteasome are responsible for degrading nonviral gene delivery systems.1 However, no prior studies polypeptides.13–15 have determined which enzymes are involved in dissociat- Recently, proteasomal processing has been implicated ing and degrading nonviral gene delivery polymers prior in the intracellular trafficking and metabolism of to the metabolism of plasmid DNA. The accepted dogma is viral vectors used for gene delivery. In the presence that peptide-based nonviral gene delivery systems are of proteasome inhibitors, adeno-associated virus (AAV) degraded by lysosomal proteases.2 Since nonviral gene mediated 50-fold higher gene expression in cell culture delivery systems represent unique combinations of poly- and enhanced the level of AAV-mediated transduction mer, lipid, peptide and DNA, there are likely multiple in the liver and lung.16 pathways that account for the intracellular metabolism of Based on the evidence that viruses are metabolized individual nonviral gene delivery systems. by the proteasome, we speculated that peptide DNA Proteasomes are multisubunit protease complexes condensates could also undergo a similar route of that play a major role in the selective degradation metabolism due to their comparable size and suscept- of intracellular proteins3–5 such as proteins that are miss ibility to serine proteases. In support of this hypothesis, folded or oxidatively damaged, proteins involved in the following investigation demonstrates that low mole- signal transduction, transcription factors and the proces- cular weight proteasome inhibitors (MG 115 and MG sing of foreign proteins for antigen presentation.6–10 The 132) block peptide DNA condensate metabolism and proteolytic core, 20S proteasome, has a cylindrical simultaneously enhance gene transfer efficiency. Further structure composed of a stack of four heptameric support for this hypothesis comes from the finding that proteasome inhibition enhances peptide-mediated gene transfer but not gene transfer mediated by other carriers Correspondence: Dr KG Rice, Division of Medicinal and Natural Product such as polyethylenimine (PEI) and Lipofectamine. This Chemistry, College of Pharmacy, 115 S. Grand Ave., University of Iowa, Iowa City, IA 52242, USA could explain differences in gene transfer efficiency Received 22 December 2004; accepted 16 May 2005; published observed between nonviral delivery systems and also online 30 June 2005 provide a working hypothesis on how to improve the Proteasome metabolism of nonviral gene delivery systems J Kim et al 1582 gene transfer efficiency of peptide-mediated gene deliv- There are also mechanisms by which nonubiquinated ery by blocking metabolism. peptides are degraded by the proteasome. Peptide DNA condensates composed primarily of polylysine se- Results quences may also be recognized as foreign and processed by this pathway resulting in their metabolism. To Proteasomes degrade a variety of proteins by recogniz- investigate this possibility, proteasome inhibitors were ing polyubiquitin attached to their lysine side chains.17 applied to mammalian cells during in vitro gene transfer 18 with CWK18 DNA condensates. Proteasome inhibition was achieved using two tripeptidyl aldehyde analogues, MG 115 and MG 132, that block both the chymotryptic and tryptic-like activity19 and by epoxomicin, which selectively blocks the chymotrypsin-like activity of 20S proteasomes (Figure 1).20 Since proteasome inhibitors are known to induce apoptosis,21–23 the relationship between proteasome inhibitor concentration and cell viability was deter- mined. Treatment of HepG2 cells for 24 h with MG 115, MG 132 or epoxomicin resulted in a dose-dependent decrease in cell viability measured by the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Figure 2a–c). The LD50 was approximately 2 mM for MG 115 and MG 132 and was 0.5 mM for epoxomicin. Figure 1 Schematic of the proteasome and the site of inhibition by MG The percent inhibition of the proteasomal chymo- 115, MG 132 and epoxomicin. tryptic-like activity measured in HepG2 cell lysate was Figure 2 Influence of proteasome inhibitors on cell viability. HepG2 cells were treated in triplicate with varying concentrations of MG 115 (a), MG 132 (b) or epoxomicin (c) for 24 h and assessed for cytotoxicity by the MTT assay. The cytotoxicity of a cathepsin B inhibitor, CA-074 Me, on HepG2 cells was evaluated (d). Likewise, CF/T1 cells were treated with varying concentrations of MG 115 (e), MG 132 (f) for 24 h and assessed for cytotoxicity by the MTT 7 assay. The LD50 was estimated from the concentration of proteasome inhibitor that caused 50% apoptosis. Values are the mean s.d. (n ¼ 3). Gene Therapy Proteasome metabolism of nonviral gene delivery systems J Kim et al 1583 Figure 3 Inhibition of proteasomal activity. HepG2 cells were treated in triplicate with varying concentrations of MG 115 (a), MG 132 (b) or epoxomicin (c) for 24 h and assessed for proteasome activity. The cathepsin B activity and proteasome activity were determined following treatment of HepG2 cells with CA-074 Me (d). Likewise, CF/T1 cells were treated with varying concentrations of MG 115 (e) and MG 132 (f) for 24 h and assessed for proteasome activity. The proteasomal activity is expressed as the released AMC per milligram of total cell protein. Values are the mean7s.d. (n ¼ 3). directly related to the inhibitor dose applied to cells peptide specific, CWK18 was substituted with CWK17C, a (Figure 3a–c). Normalization of the proteasome activity more potent gene transfer peptide that undergoes DNA on a per mg of protein basis accounted for losses due template sulfhydryl crosslinking to form DNA conden- 18 to cell apoptosis. MG 115 and MG 132 produced an IC50 sates. Transfection of HepG2 cells with CWK17C DNA of 2 and 5 mM, respectively, whereas epoxomicin was in the presence of 10 mM MG 132 resulted in a 50-fold significantly more potent with an IC50 of 0.5 mM. enhancement in gene transfer efficiency (not shown). The The IC50 of each proteasome inhibitor was further relative magnitude change in gene expression was analyzed by adding inhibitors directly to HepG2 cell independently reproduced twice with triplicate analysis lysates. MG 115, MG 132 and epoxomicin each possessed for each experiment; however, the absolute magnitude in an IC50 of approximately 0.1 mM, indicating the cell gene expression was somewhat variable between experi- membrane was an uptake barrier to proteasome inhibi- ments which is a function of cell seeding number and cell tors. confluency at the time of transfection. The reporter gene expression mediated by CWK18 The influence of proteasome inhibitors on lysosomal DNA condensates was amplified by treating cells with enzyme activity was analyzed to determine if inadver- MG 115, 132 and epoxomicin as a function of increasing tent cathepsin B inhibition could be responsible