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Silk-based gene carriers with cell membrane destabilizing peptides.

ABSTRACT: Complexes of recombinant silk-polylysine molecules with ppTG1 peptide, a lysine-rich cell membrane destabilizing peptide to bind plasmid DNA (pDNA), are designed as less-cytotoxic and highly efficient gene carriers. The peptide destabilizes the cell membrane and promotes gene transfer. Our particular interest is in how ppTG1 enhances transfection efficiency of the silk-based delivery system into human cells. Genetically engineered silk proteins containing polylysine and the monomeric and dimeric ppTG1 sequences are synthesized in Escherichia coli , followed by transfection experiments. The pDNA complexes of silk-polylysine-ppTG1 dimer recombinant proteins prepared at an N/P 2 (the ratio of number of amines/phosphates from pDNA) shows the highest transfection efficiency into human embryonic kidney (HEK) cells, the level of which is comparable to the transfection reagent Lipofectamine 2000. The assemblies show a globular morphology with an average hydrodynamic diameter of 99 nm and almost no ?-sheet structure. Additionally, the silk-based pDNA complexes demonstrate excellent DNase resistance as well as efficient release of the pDNA by enzymes that degrade silk proteins. Also, comparison with ?-sheet induced silk-based pDNA complexes indicates that the ?-sheet structure content of the silk sequence of the pDNA complexes controls the enzymatic degradation rate of the complexes and, hence, can regulate the release profile of genes from the complexes. The bioengineered silk-based gene delivery vehicles containing cell membrane destabilizing peptides are therefore concluded to have potential for a less-toxic and controlled-release gene delivery system.

SUBMITTER: Numata K

PROVIDER: S-EPMC3037451 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Silk-based gene carriers with cell membrane destabilizing peptides.

Numata Keiji K Kaplan David L DL

Biomacromolecules 20101013 11

Complexes of recombinant silk-polylysine molecules with ppTG1 peptide, a lysine-rich cell membrane destabilizing peptide to bind plasmid DNA (pDNA), are designed as less-cytotoxic and highly efficient gene carriers. The peptide destabilizes the cell membrane and promotes gene transfer. Our particular interest is in how ppTG1 enhances transfection efficiency of the silk-based delivery system into human cells. Genetically engineered silk proteins containing polylysine and the monomeric and dimeric ...[more]

PMID: 20942485

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Project description:The novel neuroproteasome is localized to the neuronal plasma membrane to degrade intracellular proteins into peptides that are released to the extracellular space. Selective inhibition of this neuronal membrane proteasome (NMP) complex ceased the release of peptides and rapidly attenuated neuronal transmission. Based on these findings, we hypothesize that these neuron-specific peptides mediate a novel form of communication through unique peptide-receptor interactions to promote intracellular signaling cascades relevant to neuronal development and function. Our work indicates that NMP peptides can rapidly induce N-methyl-D-aspartate receptor (NMDAR)-dependent calcium influx from dendrites to the soma, leading to rapid and sustained phosphorylation of the well-defined activity-dependent transcription factor cAMP response element-binding protein (CREB). We also determined that the gene expression program drastically changes upon NMP peptide treatment of neurons with an increase in expression of immediate early genes (e.g., Fos, Npas4, Egr4) known to have critical neuroregulatory roles. These data support our current thinking that NMP peptides are endogenous and selective activators of synaptic NMDA receptors and are critical for promoting activity-dependent gene expression. This pathway is orthogonal to the classic neurotransmitters previously described to activate NMDARs and points to NMP and its resulting peptides as key contributors to the development and function of the nervous system. However, the unique peptide sequences leading to neuronal activation are still poorly understood. Here, we show that the NMP peptides have tremendous sequence diversity through an unbiased peptidomic approach, and the current ongoing effort is to identify unique active peptide sequences with distinct receptor specificity. Elucidating the mechanism of the NMP and its active peptide products is crucial to understanding the role of this novel signaling process in the nervous system.

Dataset Information

Silk-based gene carriers with cell membrane destabilizing peptides.

Publications

Silk-based gene carriers with cell membrane destabilizing peptides.

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