Utilizing clathrin triskelions as carriers for spatially controlled multi-protein display.

The simultaneous and spatially controlled display of different proteins on nanocarriers is a desirable property not often achieved in practice. Here, we report the use of clathrin triskelions as a versatile platform for functional protein display. We hypothesized that site-specific molecular epitope recognition would allow for effective and ordered protein attachment to clathrin triskelions. Clathrin binding peptides (CBPs) were genetically fused to mCherry and green fluorescent protein (GFP), expressed, and loaded onto clathrin triskelions by site-specific binding. Attachment was confirmed by surface plasmon resonance. mCherry fusion proteins modified with various CBPs displayed binding affinities between 470 nM and 287 µM for the clathrin triskelions. Simultaneous attachment of GFP-Wbox and mCherry-Cbox fusion constructs to the clathrin terminal domain was verified by Förster resonance energy transfer. The circulating half-lives, area under the curve, and the terminal half-lives of GFP and mCherry were significantly increased when attached to clathrin triskelions. Clathrin triskelion technology is useful for the development of versatile and multifunctional carriers for spatially controlled protein or peptide display with tremendous potential in nanotechnology, drug delivery, vaccine development, and targeted therapeutic applications.

Targeted Delivery of Deoxycytidine Kinase to Her2-Positive Cells Enhances the Efficacy of the Nucleoside Analog Fludarabine.

Cytotoxic drugs, such as nucleoside analogs and toxins, commonly suffer from off-target effects. One approach to mitigate this problem is to deliver the cytotoxic drug selectively to the intended site. While for toxins this can be achieved by conjugating the cell-killing moiety to a targeting moiety, it is not an option for nucleoside analogs, which rely on intracellular enzymes to convert them to their active triphosphorylated form. To overcome this limitation, and achieve site-targeted activation of nucleoside analogs, we fused the coding region of a prodrug-activating enzyme, deoxycytidine kinase (dCK), to affinity reagents that bind to the Her2 cell surface protein. We evaluated dCK fusions to an anti-Her2 affibody and Designed Ankyrin Repeat Protein (DARPin) for their ability to kill cancer cells by promoting the activation of the nucleoside analog fludarabine. Cell staining and flow cytometry experiments with three Her2 positive cancer cell lines (BT-474-JB, JIMT-1 and SK-OV-3) indicate dCK fusions binding and cellular internalization. In contrast, these reagents bind only weakly to the Her2 negative cell line, MCF-7. Cell proliferation assays indicate that SK-OV-3 and BT-474-JB cell lines exhibit significantly reduced proliferation rates when treated with targeting-module fused dCK and fludarabine, compared to fludarabine alone. These findings demonstrate that we have succeeded in delivering active dCK into the Her2-positive cells, thereby increasing the activation of fludarabine, which ultimately reduces the dose of nucleoside analog needed for cell killing. This strategy may help establish the therapeutic index required to differentiate between healthy tissues and cancer cells.

Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and ß2 GABAA subunits. We isolated three anti-ß2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

Single chain variable fragment antibodies block aggregation and toxicity induced by familial ALS-linked mutant forms of SOD1.

Approximately 10% of amyotrophic lateral sclerosis (ALS) cases are familial (known as FALS) with an autosomal dominant inheritance pattern, and ~25% of FALS cases are caused by mutations in Cu/Zn superoxide dismutase (SOD1). There is convincing evidence that mutant SOD1 (mtSOD1) kills motor neurons (MNs) because of a gain-of-function toxicity, most likely related to aggregation of mtSOD1. A number of recent reports have suggested that antibodies can be used to treat mtSOD1-induced FALS. To follow up on the use of antibodies as potential therapeutics, we generated single chain fragments of variable region antibodies (scFvs) against SOD1, and then expressed them as 'intrabodies' within a motor neuron cell line. In the present study, we describe isolation of human scFvs that interfere with mtSOD1 in vitro aggregation and toxicity. These scFvs may have therapeutic potential in sporadic ALS, as well as FALS, given that sporadic ALS may also involve abnormalities in the SOD1 protein or activity.

Thy-1 mRNA destabilization by norepinephrine a 3' UTR cAMP responsive decay element and involves RNA binding proteins.

Thy-1 is a cell surface protein important in immunologic and neurologic processes, including T cell activation and proliferation, and neuronal outgrowth. In murine thymocytes, Thy-1 is downregulated in response to norepinephrine (NE) through posttranscriptional destabilization of its mRNA mediated by ßAR/AC/cAMP/PKA signaling. In this study we investigated factors involved in NE/cAMP-mediated Thy-1 mRNA destabilization in S49 thymoma cells, and identified a region containing two copies of the AUUUA regulatory element (ARE), a motif commonly associated with mRNA decay, in the Thy-1 mRNA 3' UTR. Insertion of the Thy-1 ARE region into a reporter gene, resulted in cAMP induced destabilization of the reporter gene mRNA. RNA-protein binding studies revealed multiple Thy-1 ARE binding proteins, including AUF1, HuR, and TIAR. RNA silencing of HuR enhanced cAMP-mediated downregulation of Thy-1 mRNA, in contrast, silencing AUF1 had no effect. Immunoblotting revealed multiple proteins phosphorylated by PKA as a result of NE or cAMP signaling. These results reveal that the machinery of NE/cAMP modulation of Thy-1 mRNA decay involves a cAMP responsive ARE in its 3' UTR and multiple site specific ARE binding proteins. These findings add to our knowledge of Thy-1 mRNA regulation and provide insight into the regulation of ARE containing mRNAs, which impacts stress-related immunosuppression.

Intracellular folding of the Tetrahymena group I intron depends on exon sequence and promoter choice.

The Tetrahymena group I intron splices 20 to 50 times faster in Tetrahymena than in vitro, implying that the intron rapidly adopts its active conformation in the cell. The importance of cotranscriptional folding and the contribution of the rRNA exons to the stability of the active pre-RNA structure were investigated by comparing the activity of minimal pre-RNAs expressed in Escherichia coli. Pre-RNAs containing exons derived from E. coli 23 S rRNA were three to four times more active than the wild-type Tetrahymena pre-RNA. E. coli transcripts of the chimeric E. coli pre-RNA were two to eight times more active than were T7 transcripts. However, the effect of cotranscriptional folding depends on exon sequences. Unexpectedly, the unspliced pre-RNA decays more slowly than predicted from the rate of splicing. This observation is best explained by partitioning of transcripts into active and inactive pools. We propose that the active pool splices within a few seconds, whereas the inactive pool is degraded without appreciable splicing.