Characterization of PGua4, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis.

To investigate the structure-cellular penetration relationship of guanidinium-rich transporters (GRTs), we previously designed PGua4, a five-amino acid peptoid containing a conformationally restricted pattern of eight guanidines, which showed high cell-penetrating abilities and low cell toxicity. Herein, we characterized the cellular uptake selectivity, internalization pathway, and intracellular distribution of PGua4, as well as its capacity to deliver cargo. PGua4 exhibits higher penetration efficiency in HeLa cells than in six other cell lines (A549, Caco-2, fibroblast, HEK293, Mia-PaCa2, and MCF7) and is mainly internalized by clathrin-mediated endocytosis and macropinocytosis. Confocal microscopy showed that it remained trapped in endosomes at low concentrations but induced pH-dependent endosomal membrane destabilization at concentrations ≥10 µM, allowing its diffusion into the cytoplasm. Importantly, PGua4 significantly enhanced macropinocytosis and the cellular uptake and cytosolic delivery of large IgGs following noncovalent complexation. Therefore, in addition to its peptoid nature conferring high resistance to proteolysis, PGua4 presents characteristics of a promising tool for IgG delivery and therapeutic applications.

Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein-Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System.

Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints-lactam, hydrocarbon, triazole, double triazole and thioether-on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2).

Label-free cell signaling pathway deconvolution of angiotensin type 1 receptor reveals time-resolved G-protein activity and distinct AngII and AngIIIIV responses.

Angiotensin II (AngII) type 1 receptor (AT1R) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. AT1R has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human AT1R. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of AT1R. First, we assessed the contributions of Gq, G12/13, Gi, Gßγ, ERK1/2 and ß-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with ß-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the AT1R-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous AT1R ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested AT1R biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties.

iRAGE as a novel carboxymethylated peptide that prevents advanced glycation end product-induced apoptosis and endoplasmic reticulum stress in vascular smooth muscle cells.

Advanced glycation end-products (AGE) and the receptor for AGE (RAGE) have been linked to numerous diabetic vascular complications. RAGE activation promotes a self-sustaining state of chronic inflammation and has been shown to induce apoptosis in various cell types. Although previous studies in vascular smooth muscle cells (VSMC) showed that RAGE activation increases vascular calcification and interferes with their contractile phenotype, little is known on the potential of RAGE to induce apoptosis in VSMC. Using a combination of apoptotic assays, we showed that RAGE stimulation with its ligand CML-HSA promotes apoptosis of VSMC. The formation of stress granules and the increase in the level of the associated protein HuR point toward RAGE-dependent endoplasmic reticulum (ER) stress, which is proposed as a key contributor of RAGE-induced apoptosis in VSMC as it has been shown to promote cell death via numerous mechanisms, including up-regulation of caspase-9. Chronic NF-κB activation and modulation of Bcl-2 homologs are also suspected to contribute to RAGE-dependent apoptosis in VSMC. With the goal of reducing RAGE signaling and its detrimental impact on VSMC, we designed a RAGE antagonist (iRAGE) derived from the primary amino acid sequence of HSA. The resulting CML peptide was selected for the high glycation frequency of the primary sequence in the native protein in vivo. Pretreatment with iRAGE blocked 69.6% of the increase in NF-κB signaling caused by RAGE activation with CML-HSA after 48h. Preincubation with iRAGE was successful in reducing RAGE-induced apoptosis, as seen through enhanced cell survival by SPR and reduced PARP cleavage. Activation of executioner caspases was 63.5% lower in cells treated with iRAGE before stimulation with CML-HSA. To our knowledge, iRAGE is the first antagonist shown to block AGE-RAGE interaction and we propose the molecule as an initial candidate for drug discovery.

Using carboxyfluorescein diacetate succinimidyl ester to monitor intracellular protein glycation.

Protein glycation is a ubiquitous process involved in vascular complications observed in diabetes. Glyoxal (GO), an intracellular reactive oxoaldehyde that is one of the most potent glycation agents, readily reacts with amines present on proteins to produce the lysine-derived adduct carboxymethyllysine, which is a prevalent advanced glycation end-product (AGE). Our group previously showed that cell exposure to GO leads to an alteration in the cell contractile activity that could occur as a result of the glycation of various proteins regulating the cell contractile machinery. Here, we measured the extent of glycation on three functionally distinct proteins known to participate in cell contraction and cytoskeletal organization-Rho-kinase (ROCK), actin, and gelsolin (GSN)-using an assay based on the reaction of the cell membrane-permeable fluorescent probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), which reacts with primary amine groups of proteins. By combining CFDA-SE fluorescence and Western blot detection, we observed (following GO incubation) increased glycation of actin and ROCK as well as an increased interaction between actin and GSN as observed by co-immunoprecipitation. Thus, we conclude that the use of the fluorescent probe CFDA-SE offers an interesting alternative to perform a comparative analysis of the extent of intracellular protein glycation in live cells.

Insights on Structure-Passive Permeability Relationship in Pyrrole and Furan-Containing Macrocycles.

Macrocycles have recognized therapeutic potential, but their limited cellular permeability can hinder their development as oral drugs. To better understand the structure-permeability relationship of heterocycle-containing, semipeptidic macrocycles, a library was synthesized. These compounds were created by developing two novel reactions described herein: the reduction of activated oximes by LiBH4 and the aqueous reductive mono-N-alkylation of aldehydes using catalytic SmI2 and stoichiometric Zn. The permeability of the macrocycles was evaluated through a parallel artificial membrane permeability assay (PAMPA), and the results indicated that macrocycles with a furan incorporated into the structure have better passive permeability than those with a pyrrole moiety. Compounds bearing a 2,5-disubstituted pyrrole (endo orientation) were shown to be implicated in intramolecular H-bonds, enhancing their permeability. This study highlighted the impact of heterocycles moieties in semipeptides, creating highly permeable macrocycles, thus showing promising avenues for passive diffusion of drugs beyond the rule-of-five chemical space.

Surface micropatterning for the formation of an in vitro functional endothelial model for cell-based biosensors.

Label-free biosensing, such as with surface plasmon resonance (SPR), is a highly efficient method for monitoring the responses of living cells exposed to pharmacological agents and biochemical stimuli in vitro. Conventional cell culture protocols used in cell-based biosensing generally provide little direct control over cell morphologies and phenotypes. Surface micropatterning techniques have been exploited for the controlled immobilization and establishment of well-defined cell morphologies and phenotypes. In this article, surface adhesion micropatterns are used to control the adhesion of endothelial cells within adjacent hexagonal microstructures to promote the emergence of a well-controlled and standardized cell layer phenotype onto SPR sensor surfaces. We show that the formation of cell-cell junctions can be controlled by tuning the inter-cellular spacing in groups of 3 neighbouring cells. Fluorescence microscopy was used to confirm the formation of vascular endothelium cadherin junctions, a structural marker of a functional endothelium. In order to confirm the functionality of the proposed model, the response to thrombin, a modulator of endothelium integrity, was monitored by surface plasmon resonance imaging (SPRI). Experiments demonstrate the potential of the proposed model as a primary biological signal transducer for SPRI-based analysis, with potential applications in cell biology, pharmacology and diagnostic.

Anticancer drugs affect the alternative splicing of Bcl-x and other human apoptotic genes.

Inducing an apoptotic response is the goal of most current chemotherapeutic interventions against cancer. However, little is known about the effect of chemotherapeutic agents on the alternative splicing of apoptotic genes. Here, we have tested 20 of the mainstream anticancer drugs for their ability to influence the production of Bcl-x splice isoforms. We find that many drugs shift splicing toward the proapoptotic Bcl-x(S) splice variant in 293 cells. The drugs modulate splicing decisions most likely through signaling events because the splicing switch is not compromised by inhibiting de novo protein synthesis or the activity of caspases. Several drugs also shift Bcl-x splicing in cancer cell lines (MCF-7, HeLa, PC-3, PA-1, and SKOV-3), but the set of active drugs varies between cell lines. We also examined the effect of anticancer agents on the alternative splicing of 95 other human apoptotic genes in different cell lines. Almost every drug can alter a subset of alternative splicing events in each cell line. Although drugs of the same class often influence the alternative splicing of the same units in individual cell lines, these units differ considerably between cell lines, indicating cell line-specific differences in the pathways that control splicing.

The signaling signature of the neurotensin type 1 receptor with endogenous ligands.

The human neurotensin 1 receptor (hNTS1) is a G protein-coupled receptor involved in many physiological functions, including analgesia, hypothermia, and hypotension. To gain a better understanding of which signaling pathways or combination of pathways are linked to NTS1 activation and function, we investigated the ability of activated hNTS1, which was stably expressed by CHO-K1 cells, to directly engage G proteins, activate second messenger cascades and recruit ß-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the Gαq-, Gαi1-, GαoA-, and Gα13-protein signaling pathways as well as the recruitment of ß-arrestins 1 and 2. Using pharmacological inhibitors, we further demonstrated that all three ligands stimulated the production of inositol phosphate and modulation of cAMP accumulation along with ERK1/2 activation. Interestingly, despite the functional coupling to Gαi1 and GαoA, NT was found to produce higher levels of cAMP in the presence of pertussis toxin, supporting that hNTS1 activation leads to cAMP accumulation in a Gαs-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive Gi/o-c-Src signaling pathway and PLCß-DAG-PKC-Raf-1-dependent pathway downstream of Gq. Finally, the whole-cell integrated signatures monitored by the cell-based surface plasmon resonance and changes in the electrical impedance of a confluent cell monolayer led to identical phenotypic responses between the three ligands. The characterization of the hNTS1-mediated cellular signaling network will be helpful to accelerate the validation of potential NTS1 biased ligands with an improved therapeutic/adverse effect profile.

Amplification of AngII-dependent cell contraction by glyoxal: implication of cell mechanical properties and actomyosin activity.

Glyoxal (GO), a highly reactive metabolite of glucose, is associated with diabetic vascular complications via the formation of advanced glycation end-products. Considering its ability to react with proteins' amino acids and its crosslinking potential, we suggest that GO affects cellular mechanical functions such as contractility. Therefore, we tested the effects of GO on cellular contractile response following AngII stimulation of human embryonic kidney cells over-expressing the AT1 receptor (HEK 293 AT1aR). Prior to cell stimulation with AngII, cells exposed to GO exhibited carboxymethyllysine-adduct formation and an increase in cellular stiffness, which could be prevented by pre-treatment with aminoguanidine. The time-dependent cellular contractile response to AngII was measured by monitoring cell membrane displacement by atomic force atomic force microscopy (AFM) and by quantifying myosin light chain phosphorylation (p-MLC) via immunoblotting. Interestingly, short-term GO exposure increased by 2.6 times the amplitude of cell contraction induced by AngII and this was also associated with a sustained rise in p-MLC. This increased response to AngII induced by GO appears to be linked to its glycation potential, as aminoguanidine pre-treatment prevented this increased cellular mechanical response. Our results also suggest that GO could have an impact on ROCK activity, as ROCK inhibition with Y-27632 blocked the enhanced contractile response (p = 0.011) measured under GO conditions. Together, these results indicate that GO enhances the cellular response to AngII and modifies cellular mechanical properties via a mechanism that relies on its glycation potential and on the activation of the ROCK-dependent pathway.

Cancer-associated regulation of alternative splicing.

Alternative splicing of pre-mRNA increases the diversity of protein functions. Here we show that about half of all active alternative splicing events in ovarian and breast tissues are changed in tumors, and many seem to be regulated by a single factor; sequence analysis revealed binding sites for the RNA binding protein FOX2 downstream of one-third of the exons skipped in cancer. High-resolution analysis of FOX2 binding sites defined the precise positions relative to alternative exons at which the protein may function as either a silencer or an enhancer. Most of the identified targets were shifted in the same direction by FOX2 depletion in cell lines as they were in breast and ovarian cancer tissues. Notably, we found expression of FOX2 itself is downregulated in ovarian cancer and its splicing is altered in breast cancer samples. These results suggest that the decreased expression of FOX2 in cancer tissues modulates splicing and controls proliferation.

Multiple and specific mRNA processing targets for the major human hnRNP proteins.

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.

Identification of alternative splicing markers for breast cancer.

Breast cancer is the most common cause of cancer death among women under age 50 years, so it is imperative to identify molecular markers to improve diagnosis and prognosis of this disease. Here, we present a new approach for the identification of breast cancer markers that does not measure gene expression but instead uses the ratio of alternatively spliced mRNAs as its indicator. Using a high-throughput reverse transcription-PCR-based system for splicing annotation, we monitored the alternative splicing profiles of 600 cancer-associated genes in a panel of 21 normal and 26 cancerous breast tissues. We validated 41 alternative splicing events that significantly differed in breast tumors relative to normal breast tissues. Most cancer-specific changes in splicing that disrupt known protein domains support an increase in cell proliferation or survival consistent with a functional role for alternative splicing in cancer. In a blind screen, a classifier based on the 12 best cancer-associated splicing events correctly identified cancer tissues with 96% accuracy. Moreover, a subset of these alternative splicing events could order tissues according to histopathologic grade, and 5 markers were validated in a further blind set of 19 grade 1 and 19 grade 3 tumor samples. These results provide a simple alternative for the classification of normal and cancerous breast tumor tissues and underscore the putative role of alternative splicing in the biology of cancer.