Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study

Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG(neg)) and sialic acid-deficient (SA(neg)) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA(neg)>WT>GAG(neg). The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA(neg) and GAG(neg) cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG(neg) cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering

Angiomotin Regulates YAP Localization during Neural Differentiation of Human Pluripotent Stem Cells

Summary: Leveraging the extraordinary potential of human pluripotent stem cells (hPSCs) requires an understanding of the mechanisms underlying cell-fate decisions. Substrate elasticity can induce differentiation by signaling through the transcriptional coactivator Yes-associated protein (YAP). Cells cultured on surfaces mimicking brain elasticity exclude YAP from their nuclei and differentiate to neurons. How YAP localization is controlled during neural differentiation has been unclear. We employed CRISPR/Cas9 to tag endogenous YAP in hPSCs and used this fusion protein to identify YAP's interaction partners. This engineered cell line revealed that neural differentiation promotes a change in YAP interactors, including a dramatic increase in angiomotin (AMOT) interaction with YAP. AMOT regulates YAP localization during differentiation. AMOT expression increases during neural differentiation and leads to YAP nuclear exclusion. Our findings that AMOT-dependent regulation of YAP helps direct hPSC fate provide insight into the molecular mechanisms by which the microenvironment can induce neural differentiation. : Kiessling and colleagues employed CRISPR/Cas9 to generate an hPSC line in which YAP is tagged, thus facilitating affinity purification of endogenous-level YAP complexes for analysis. This strategy uncovered proteins that interact with YAP during self-renewal and differentiation and identified angiomotin as a key regulator of YAP localization during neural differentiation. Keywords: YAP, mechanosensing, neuronal differentiation, angiomotin, human pluripotent stem cells, embryonic stem cells, neural progenitor cells, ubiquitination, CRISPR genome engineering, proteomic

Formation of a lipid bilayer containing WT membrane fragments and penetratin interaction with this membrane monitored by PWR.

<p>Panels A) and B) correspond to the PWR spectra obtained for the buffer (1) and the lipid bilayer (2) and Panels C) and D) for spectra obtained after addition of about 0.1 µM of penetratin (+) to the bilayer (solid line) obtained for p- and s-polarized light, respectively. The resonance position shifts obtained for p- (•) and s- (▪) polarizations for the incremental addition of penetratin are represented in Panel E, together with the hyperbolic binding (affinity constants are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024096#pone-0024096-t002" target="_blank">table 2</a>). Panel F corresponds to the kinetic measurements obtained for the p-pol light upon addition of penetratin (0.05 µM) to the lipid bilayer (rate constants are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024096#pone-0024096-t003" target="_blank">Table 3</a>).</p

(A) Isothermal calorimetric titration of 50 nmol penetratin to a suspension of 1.5 million CHO cells at 37°C.

<p>(B) Released heat measured after the addition of 50 nmol (white, final concentration 32 µM) and 7.75 nmol (black, final concentration 5 µM) penetratin to WT, GAG^neg and SA^neg cells.</p

Direction, magnitude and graphical analysis of the spectral changes observed by PWR upon penetratin interaction with the synthetic lipid model membrane.

<p>Direction, magnitude and graphical analysis of the spectral changes observed by PWR upon penetratin interaction with the synthetic lipid model membrane.</p

Amount (pmoles) of A) internalized and B) high-affinity membrane bound penetratin in WT, GAG^neg and SA^neg cells.

<p>Corresponding intracellular penetratin concentrations (µM) are also indicated (A) taken the volume of one cell as being 1 pL.</p

Kinetic analysis of the interaction of penetratin with lipid membranes; data obtained by PWR spectroscopy.

<p>Note: experiment was repeated 3 times.</p

Parallel in&nbsp;vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience

Gene Ontology analyses of autism spectrum disorders (ASD) risk genes have repeatedly highlighted synaptic function and transcriptional regulation as key points of convergence. However, these analyses rely on incomplete knowledge of gene function across brain development. Here we leverage Xenopus tropicalis to study in&nbsp;vivo ten genes with the strongest statistical evidence for association with ASD. All genes are expressed in developing telencephalon at time points mapping to human mid-prenatal development, and mutations lead to an increase in the ratio of neural progenitor cells to maturing neurons, supporting previous in silico systems biological findings implicating cortical neurons in ASD vulnerability, but expanding the range of convergent functions to include neurogenesis. Systematic chemical screening identifies that estrogen, via Sonic hedgehog signaling, rescues this convergent phenotype in Xenopus and human models of brain development, suggesting a resilience factor that may mitigate a range of ASD genetic risks