Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

BACKGROUND: Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells. RESULTS: In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC) or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH(6)). The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF)(3)K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes), and did not require localization of the enzyme to the nucleus. CONCLUSIONS: The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells

Cell-Permeable Parkin Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals

<div><p>Parkinson’s disease (PD) is a neurodegenerative disorder of complex etiology characterized by the selective loss of dopaminergic neurons, particularly in the substantia nigra. Parkin, a tightly regulated E3 ubiquitin ligase, promotes the survival of dopaminergic neurons in both PD and Parkinsonian syndromes induced by acute exposures to neurotoxic agents. The present study assessed the potential of cell-permeable parkin (CP-Parkin) as a neuroprotective agent. Cellular uptake and tissue penetration of recombinant, enzymatically active parkin was markedly enhanced by the addition of a hydrophobic macromolecule transduction domain (MTD). The resulting CP-Parkin proteins (HPM₁₃ and PM₁₀) suppressed dopaminergic neuronal toxicity in cells and mice exposed to 6-hydroxydopamine (6-OHDH) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These included enhanced survival and dopamine expression in cultured CATH.a and SH-SY5Y neuronal cells; and protection against MPTP-induced damage in mice, notably preservation of tyrosine hydroxylase-positive cells with enhanced dopamine expression in the striatum and midbrain, and preservation of gross motor function. These results demonstrate that CP-Parkin proteins can compensate for intrinsic limitations in the parkin response and provide a therapeutic strategy to augment parkin activity in vivo.</p></div

CP-Parkin protects neuronal cells from 6-OHDA-induced apoptosis.

<p>(<b>A</b>) Suppression of apoptosis in dopaminergic CATH.a cells. CATH.a cells at 5% (Low) or 70% (High) confluence were incubated with 50 µM 6-hydroxydopamine (6-OHDA, Agonist) for 1 hr, treated for 2.5 hrs with 2.5 µM HP or HPM₁₃ and assessed for apoptosis by TUNEL staining. The micrographs (left panels) are representative of three independent experiments, plotted (right panels) as means ± S.D. Experimental differences between groups were assessed by a Student’s two-paired <i>t</i>-test (*<i>p</i><0.001). (<b>B</b>) Suppression of apoptosis in SH-SY5Y cells. Apoptosis in SH-SY5Y treated with 6-OHDA with and without PM₁₀ was assessed as described in (<b>A</b>). (<b>C</b>) HPM₁₃ enhances dopamine release from CATH.a cells. The cells were incubated with 80 µM tyrosine for 24 hrs, treated for 5 hrs with 2.5 µM HP or HPM₁₃, and levels of secreted dopamine were measured by ELISA. The data are presented as means ± S.D. of 4 independent experiments. Experimental differences between groups were assessed by a Student’s two-paired <i>t</i>-test (*<i>p</i><0.01 and **<i>p</i> 0.05).</p

Structure and expression of MTD-parkin fusion proteins.

<p>(<b>A</b>) Structures of parkin fusion proteins. 6xHis-tagged parkin proteins (left) contained His-tag (HP) only or together with MTD01 (HPM₀₁) or MTD13 (HPM₁₃) sequences. Parkin proteins without the 6xHis tag (right) included native parkin (P) and proteins containing C-terminal MTD10 (PM₁₀), MTD13 (PM₁₃) MTD151 (PM₁₅₁) and MTD174 (PM₁₇₄) sequences. (<b>B–C</b>) Protein expression in <i>E. coli</i>. SDS PAGE analysis of cell lysates before (−) and after (+) IPTG induction; aliquots of Ni²⁺ affinity purified proteins (P); and molecular weight standards (M). The size (number of amino acids), yield (mg/L) and solubility of each recombinant protein are indicated. Solubility was scored on a 4-point scale from highly soluble, with little tendency to precipitate (++++), to largely insoluble proteins (+).</p

MTD-mediated parkin delivery to the brain.

<p>(<b>A–B</b>) Immunoblotting of parkin proteins in the cerebellum. Sagittal sections through the cerebellum were immunostatined with anti-parkin (<b>A</b>) or anti-MTD10 (<b>B</b>) antibody 2 hrs after IP injection of 200 µg of diluent alone or His-tagged parkin proteins without (HP) or with the MTD13 or MTD10 sequences (HPM₁₃ or PM₁₀) (<b>C–D</b>) Western blot analysis of brain parkin. Lysates were prepared from brain samples 2 hrs (<b>C</b>) and 30 hrs (<b>D</b>) after IV administration of diluent alone or 200 µg His-tagged parkin proteins without (HP) or with the MTD01 (HPM₀₁) or MTD13 (HPM₁₃) sequences (<b>C</b>) or with untagged parkin protein containing MTD10 (PM₁₀) (<b>D</b>) and analyzed by western blotting using anti-parkin and anti-β-actin antibodies.</p

Auto-ubiquitination activity of recombinant parkin proteins.

<p>E3 ligase activity of purified recombinant parkin proteins was assessed by an auto-ubiquitination assay. 1 µg of each of the indicated proteins was incubated for 1 hr at 37°C with 1 µM E1, 50 µM E2, 1 mM histidine-tagged Ubiquitin and 10 mM Mg-ATP, and the reaction products were fractionated by SDS PAGE and immunoblotted against an anti-Ubiquitin antibody. Parkin proteins (P, HP, PM₁₀, PM₁₃, PM₁₅₁ and PM₁₇₄) are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102517#pone-0102517-g001" target="_blank">Figure 1</a>. Samples without individual components or containing an unrelated protein, 6xHis-NM23 (HN), were used as negative controls.</p