Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix-Loop-Helix Peptides with Cell Penetrating and Tumor Suppressor Properties.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, breast cancer, and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity, and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross, and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully exploit their potential to develop targeted therapies.

A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system.

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo. A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states i.e. (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out via FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur in lieu of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR).

Axonal accumulation of synaptic markers in APP transgenic Drosophila depends on the NPTY motif and is paralleled by defects in synaptic plasticity.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and extracellular plaques, which consist mainly of beta-amyloid derived from the beta-amyloid precursor protein (APP). An additional feature of AD is axonopathy, which might contribute to impairment of cognitive functions. Specifically, axonal transport defects have been reported in AD animal models, including mice and flies that overexpress APP and tau. Here we demonstrate that the APP-induced traffic jam of vesicles in peripheral nerves of Drosophila melanogaster larvae depends on the four residues NPTY motif in the APP intracellular domain. Furthermore, heterologous expression of Fe65 and JIP1b, scaffolding proteins interacting with the NPTY motif, also perturb axonal transport. Together, these data indicate that JIP1b or Fe65 may be involved in the APP-induced axonal transport defect. Moreover, we have characterized neurotransmission at the neuromuscular junction in transgenic larvae that express human APP. Consistent with the observation that these larvae do not show any obvious movement deficits, we found no changes in basal synaptic transmission. However, short-term synaptic plasticity was affected by overexpression of APP. Together, our results show that overexpression of APP induces partial stalling of axonal transport vesicles, paralleled by abnormalities in synaptic plasticity, which may provide a functional link to the deterioration of cognitive functions observed in AD.

Homo- and heterodimerization of APP family members promotes intercellular adhesion.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.