Search

Background. Mesenchymal stem/stromal cells (MSCs) have been widely used for their therapeutic properties in many clinical applications including osteoarthritis. Despite promising preclinical results showing the ability of MSC to reduce the clinical severity of osteoarthritis (OA) in experimental animal models, the benefits of intra-articular injection of MSC in OA patients are limited to the short term. In this regard, it is anticipated that improving the properties of MSC may collectively enhance their long-term beneficial effects on OA. Methods and Results. Recently, we have shown that PPARβ/δ inhibition using a commercially available antagonist in murine MSC increases their immunoregulatory potential in vitro as well as their therapeutic potential in an experimental murine arthritis model. Here, we relied on an innovative strategy to inhibit PPARβ/δ:NF-κB TF65 subunit interaction in human MSC by designing and synthesizing an interfering peptide, referred to PP11. Through RT-qPCR experiments, we evidenced that the newly synthesized PP11 peptide reduced the expression level of PDK4, a PPARβ/δ target gene, but did not modify the expression levels of ACOX1 and CPT1A, PPARα target genes, and FABP4, a PPARγ target gene compared with untreated human MSC. Moreover, we showed that human MSCs pretreated with PP11 exhibit a significantly higher capacity to inhibit the proliferation of activated PBMC and to decrease the frequency of M1-like macrophages. Conclusions. We designed and synthesized an interfering peptide that potently and specifically blocks PPARβ/δ activity with concomitant enhancement of MSC immunoregulatory properties.

A mitochondrion is a cellular organelle able to produce cellular energy in the form of adenosine triphosphate (ATP). As in the nucleus, mitochondria contain their own genome: the mitochondrial DNA (mtDNA). This genome is particularly susceptible to mutations that are at the basis of a multitude of disorders, especially those affecting the heart, the central nervous system and muscles. Conventional clinical practice applied to mitochondrial diseases is very limited and ineffective; a clear need for innovative therapies is demonstrated. Gene therapy seems to be a promising approach. The use of mitochondrial DNA as a therapeutic, optimized by peptide-based complexes with mitochondrial targeting, can be seen as a powerful tool in the reestablishment of normal mitochondrial function. In line with this requirement, in this work and for the first time, a mitochondrial-targeting sequence (MTS) has been incorporated into previously researched peptides, to confer on them a targeting ability. These peptides were then considered to complex a plasmid DNA (pDNA) which contains the mitochondrial gene ND1 (mitochondrially encoded NADH dehydrogenase 1 protein), aiming at the formation of peptide-based nanoparticles. Currently, the ND1 plasmid is one of the most advanced bioengineered vectors for conducting research on mitochondrial gene expression. The formed complexes were characterized in terms of pDNA complexation capacity, morphology, size, surface charge and cytotoxic profile. These data revealed that the developed carriers possess suitable properties for pDNA delivery. Furthermore, in vitro studies illustrated the mitochondrial targeting ability of the novel peptide/pDNA complexes. A comparison between the different complexes revealed the most promising ones that complex pDNA and target mitochondria. This may contribute to the optimization of peptide-based non-viral systems to target mitochondria, instigating progress in mitochondrial gene therapy.

Abstract Background Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. Results Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. Conclusions This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.

We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors"), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers") that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

The article offers information on a study which examined the comprehensive and cost-efficient genetic diagnosis of cardiomyopathies through the use of microarray-based target enrichment approach followed by SOLiD next-generation sequencing (NGS). The researchers developed an array-based subgenomic enrichment diagnostic approach to determine mutations in patients dilated cardiomayopathy (DCM) and hypertrophic cardiomyopathy (HCM). It notes that the approach resulted to high sequence coverage of cardiomyopathy genes.

The setup of synthetic biological systems involving millions of bases is still limited by the required high quality of synthetic DNA. Important drivers to further open up the field are the accuracy and scale of chemical DNA synthesis and the downstream processing of longer DNA assembled from short fragments. We developed a new, highly parallel and miniaturized method for the preparation of high quality DNA termed “Megacloning” by using Next Generation Sequencing (NGS) technology in a preparative way. We demonstrate our method by processing both conventional and microarray-derived DNA oligonucleotides in combination with a bead-based high throughput pyrosequencing platform, gaining a 500-fold error reduction for microarray oligonucleotides in a first embodiment. We also show the assembly of synthetic genes as part of the Megacloning process. In principle, up to millions of DNA fragments can be sequenced, characterized and sorted in a single Megacloner run, enabling many new applications.

Cystic Fibrosis (CF) is the most common inherited disorder and is characterized by an inflammatory phenotype. Here, we found that in bronchial epithelium reconstituted form lung tissue biopsies from patients with CF, the RNA-binding protein tristetraprolin (TTP), a key regulator of inflammation, is dysregulated in cells that strongly express cytokines and interleukins. TTP activity is regulated by extensive post-translational modifications, particularly phosphorylation. We found that in addition to mRNA downregulation, phosphorylated TTP (which cannot bind to mRNA) accumulated in CF cultures, suggesting that the imbalance in TTP phosphorylation status could contribute to the inflammatory phenotype in CF. We confirmed TTP destabilizing role on IL8 mRNA through its 3'UTR sequence in CF cells. We next demonstrated that TTP phosphorylation is mainly regulated by MK2 through activation of ERK, which also was hyperphosphorylated. TTP is considered a mRNA decay factor with some exception, and we present a new positive role of TTP in CF cultures. We determined that TTP binds to specific ARE motifs on the 3'UTR of mRNA sequences and also, for the first time, to the 3'UTR of Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) where TTP binding stabilizes the mRNA level. This study identified new partners that can be targeted in CF and proposes a new way to control CFTR gene expression.

Background: Mesenchymal stem/stromal cells (MSCs) have been widely used for their therapeutic properties in many clinical applications including osteoarthritis. Despite promising preclinical results showing the ability of MSC to reduce the clinical severity of osteoarthritis (OA) in experimental animal models, the benefits of intra-articular injection of MSC in OA patients are limited to the short term. In this regard, it is anticipated that improving the properties of MSC may collectively enhance their long-term beneficial effects on OA., Methods and Results: Recently, we have shown that PPAR β / δ inhibition using a commercially available antagonist in murine MSC increases their immunoregulatory potential in vitro as well as their therapeutic potential in an experimental murine arthritis model. Here, we relied on an innovative strategy to inhibit PPAR β / δ :NF- κ B TF65 subunit interaction in human MSC by designing and synthesizing an interfering peptide, referred to PP11. Through RT-qPCR experiments, we evidenced that the newly synthesized PP11 peptide reduced the expression level of PDK4 , a PPAR β / δ target gene, but did not modify the expression levels of ACOX1 and CPT1A , PPAR α target genes, and FABP4 , a PPAR γ target gene compared with untreated human MSC. Moreover, we showed that human MSCs pretreated with PP11 exhibit a significantly higher capacity to inhibit the proliferation of activated PBMC and to decrease the frequency of M1-like macrophages., Conclusions: We designed and synthesized an interfering peptide that potently and specifically blocks PPAR β / δ activity with concomitant enhancement of MSC immunoregulatory properties., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Gautier Tejedor et al.)

SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.

A mitochondrion is a cellular organelle able to produce cellular energy in the form of adenosine triphosphate (ATP). As in the nucleus, mitochondria contain their own genome: the mitochondrial DNA (mtDNA). This genome is particularly susceptible to mutations that are at the basis of a multitude of disorders, especially those affecting the heart, the central nervous system and muscles. Conventional clinical practice applied to mitochondrial diseases is very limited and ineffective; a clear need for innovative therapies is demonstrated. Gene therapy seems to be a promising approach. The use of mitochondrial DNA as a therapeutic, optimized by peptide-based complexes with mitochondrial targeting, can be seen as a powerful tool in the reestablishment of normal mitochondrial function. In line with this requirement, in this work and for the first time, a mitochondrial-targeting sequence (MTS) has been incorporated into previously researched peptides, to confer on them a targeting ability. These peptides were then considered to complex a plasmid DNA (pDNA) which contains the mitochondrial gene ND1 (mitochondrially encoded NADH dehydrogenase 1 protein), aiming at the formation of peptide-based nanoparticles. Currently, the ND1 plasmid is one of the most advanced bioengineered vectors for conducting research on mitochondrial gene expression. The formed complexes were characterized in terms of pDNA complexation capacity, morphology, size, surface charge and cytotoxic profile. These data revealed that the developed carriers possess suitable properties for pDNA delivery. Furthermore, in vitro studies illustrated the mitochondrial targeting ability of the novel peptide/pDNA complexes. A comparison between the different complexes revealed the most promising ones that complex pDNA and target mitochondria. This may contribute to the optimization of peptide-based non-viral systems to target mitochondria, instigating progress in mitochondrial gene therapy.

The field of gene therapy still attracts great interest due to its potential therapeutic effect towards the most deadly diseases, such as cancer. For cancer gene therapy to be feasible and viable in a clinical setting, the design and development of a suitable gene delivery system is imperative. Peptide based vectors, in particular, reveal to be promising for therapeutic gene release. Following this, two different peptides, RALA and WRAP5, have been investigated mainly regarding their ability to form complexes with a p53 encoding plasmid (pDNA) with suitable properties for gene delivery. To address this issue, and after an initial screening study focused on the dependence of pDNA complexation capacity with the nitrogen to phosphate groups (N/P) ratio, a design of experiments (DoE) tool has been employed. For each peptide/pDNA system, parameters such as, the buffer pH and the N/P ratio were considered the DoE inputs and the vector size, zeta potential and pDNA complexation capacity (CC) were monitored as DoE outputs. The main goal was to find the optimal experimental conditions to minimize particle sizes, as well as, to maximize the positive surface charges of the formulated nanosystems and maximize the pDNA CC. Through the DoE method applied, the optimal RALA/pDNA and WRAP5/pDNA formulations were revealed and show interesting features related to peptide structure and pDNA complexation ability. This work illustrates the great utility of experimental design tools in optimizing the formulation of peptide/pDNA vectors in a minimum number of experiments providing relevant knowledge for the development of more suitable and efficient gene delivery systems. The new insights achieved on these carriers clearly instigate deeper research on gene therapy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cell-penetrating peptides (CPP) are broadly recognized as efficient non-viral vectors for the internalization of compounds such as peptides, oligonucleotides or proteins. Characterizing these carriers requires reliable methods to quantify their intracellular uptake. Flow cytometry on living cells is a method of choice but is not always applicable (e.g. big or polarized cells), so we decided to compare it to fluorescence spectroscopy on cell lysates. Surprisingly, for the internalization of a series of TAMRA-labeled conjugates formed of either cationic or amphipathic CPPs covalently coupled to a decamer peptide, we observed important differences in internalization levels between both methods. We partly explained these discrepancies by analyzing the effect of buffer conditions (pH, detergents) and peptide sequence/structure on TAMRA dye accessibility. Based on this analysis, we calculated a correction coefficient allowing a better coherence between both methods. However, an overestimated signal was still observable for both amphipathic peptides using the spectroscopic detection, which could be due to their localization at the cell membrane. Based on several in vitro experiments modeling events at the plasma membrane, we hypothesized that fluorescence of peptides entrapped in the membrane bilayer could be quenched by the tryptophan residues of close transmembrane proteins. During cell lysis, cell membranes are disintegrated liberating the entrapped peptides and restoring the fluorescence, explaining the divergences observed between flow cytometry and spectroscopy on lysates. Overall, our results highlighted major biases in the fluorescently-based quantification of internalized fluorescently-labeled CPP conjugates, which should be considered for accurate uptake quantification., (Copyright © 2019 Elsevier B.V. All rights reserved.)

PA28γ is a nuclear activator of the 20S proteasome involved in the regulation of several essential cellular processes, such as cell proliferation, apoptosis, nuclear dynamics, and cellular stress response. Unlike the 19S regulator of the proteasome, which specifically recognizes ubiquitylated proteins, PA28γ promotes the degradation of several substrates by the proteasome in an ATP- and ubiquitin-independent manner. However, its exact mechanisms of action are unclear and likely involve additional partners that remain to be identified. Here we report the identification of a cofactor of PA28γ, PIP30/FAM192A. PIP30 binds directly and specifically via its C-terminal end and in an interaction stabilized by casein kinase 2 phosphorylation to both free and 20S proteasome-associated PA28γ. Its recruitment to proteasome-containing complexes depends on PA28γ and its expression increases the association of PA28γ with the 20S proteasome in cells. Further dissection of its possible roles shows that PIP30 alters PA28γ-dependent activation of peptide degradation by the 20S proteasome in vitro and negatively controls in cells the presence of PA28γ in Cajal bodies by inhibition of its association with the key Cajal body component coilin. Taken together, our data show that PIP30 deeply affects PA28γ interactions with cellular proteins, including the 20S proteasome, demonstrating that it is an important regulator of PA28γ in cells and thus a new player in the control of the multiple functions of the proteasome within the nucleus., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

PDZ domains play crucial roles in cell signaling processes and are therefore attractive targets for the development of therapeutic inhibitors. In many cases, C-terminal peptides are the physiological binding partners of PDZ domains. To identify both native ligands and potential inhibitors we have screened arrays synthesized by the process of inverted peptides (PIPE), a variant of SPOT synthesis that generates peptides with free C-termini. Here, we present the development of a new functionalized cellulose membrane as solid support along with the optimized PIPE PLUS technology. Improved resolution and accuracy of the synthesis were shown with peptide arrays containing both natural and non-natural amino acids. These new screening possibilities will advance the development of active, selective and metabolically stable PDZ interactors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases., Results: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression., Conclusions: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.

RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation and intracellular delivery. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. In this context, the secondary amphipathic peptide CADY has shown to form stable siRNA complexes and to improve their cellular uptake independent of the endosomal pathway. In the present work, we have described the parameters influencing CADY nanoparticle formation (buffers, excipients, presence of serum, etc.), and have followed in details the CPP:siRNA self-assembly. Once optimal conditions were determined, we have compared the ability of seven different CADY analogues to form siRNA-loaded nanoparticles compared to CADY:siRNA. First of all, we were able to show by biophysical methods that structural polymorphism (α-helix) is an important prerequisite for stable nanoparticle formation independently of occurring sequence mutations. Luciferase assays revealed that siRNA complexed to CADY-K (shorter version) shows better knock-down efficiency on Neuro2a-Luc(+) and B16-F10-Luc(+) cells compared to CADY:siRNA. Altogether, CADY-K is an ideal candidate for further application especially with regards to ex vivo or in vivo applications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cell-penetrating peptide (CPP)-mediated delivery of phosphorodiamidate morpholino oligomers (PMO) results in efficient exon skipping and has shown great promise as a potential therapy for Duchenne muscular dystrophy (DMD). However, large differences in efficiency have been observed between CPPs and in delivery to different tissues. Cellular trafficking has appeared to be an important determinant of activity. This chapter provides details of experimental procedures to monitor exon skipping efficiency and cellular trafficking of Pip6a-PMO, a recently developed and particularly efficient conjugate, in skeletal H2k cells and in primary cardiomyocytes from mdx mice. Similar procedures may be used in principle to evaluate any free or vector-associated oligonucleotide for exon skipping.

Cell-penetrating peptide-mediated delivery of phosphorodiamidate morpholino oligomers (PMOs) has shown great promise for exon-skipping therapy of Duchenne Muscular Dystrophy (DMD). Pip6a-PMO, a recently developed conjugate, is particularly efficient in a murine DMD model, although mechanisms responsible for its increased biological activity have not been studied. Here, we evaluate the cellular trafficking and the biological activity of Pip6a-PMO in skeletal muscle cells and primary cardiomyocytes. Our results indicate that Pip6a-PMO is taken up in the skeletal muscle cells by an energy- and caveolae-mediated endocytosis. Interestingly, its cellular distribution is different in undifferentiated and differentiated skeletal muscle cells (vesicular versus nuclear). Likewise, Pip6a-PMO mainly accumulates in cytoplasmic vesicles in primary cardiomyocytes, in which clathrin-mediated endocytosis seems to be the pre-dominant uptake pathway. These differences in cellular trafficking correspond well with the exon-skipping data, with higher activity in myotubes than in myoblasts or cardiomyocytes. These differences in cellular trafficking thus provide a possible mechanistic explanation for the variations in exon-skipping activity and restoration of dystrophin protein in heart muscle compared with skeletal muscle tissues in DMD models. Overall, Pip6a-PMO appears as the most efficient conjugate to date (low nanomolar EC50), even if limitations remain from endosomal escape.

PDZ domain interactions are involved in signaling and trafficking pathways that coordinate crucial cellular processes. Alignment-based PDZ binding motifs identify the few most favorable residues at certain positions along the peptide backbone. However, sequences that bind the CAL (CFTR-associated ligand) PDZ domain reveal only a degenerate motif that overpredicts the true number of high-affinity interactors. Here, we combine extended peptide-array motif analysis with biochemical techniques to show that non-motif "modulator" residues influence CAL binding. The crystallographic structures of 13 CAL:peptide complexes reveal defined, but accommodating stereochemical environments at non-motif positions, which are reflected in modulator preferences uncovered by multisequence substitutional arrays. These preferences facilitate the identification of high-affinity CAL binding sequences and differentially affect CAL and NHERF PDZ binding. As a result, they also help determine the specificity of a PDZ domain network that regulates the trafficking of CFTR at the apical membrane., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Spore formation in Bacillus subtilis relies on a regulated intramembrane proteolysis (RIP) pathway that synchronizes mother-cell and forespore development. To address the molecular basis of this SpoIV transmembrane signaling, we carried out a structure-function analysis of the activating protease CtpB. Crystal structures reflecting distinct functional states show that CtpB constitutes a ring-like protein scaffold penetrated by two narrow tunnels. Access to the proteolytic sites sequestered within these tunnels is controlled by PDZ domains that rearrange upon substrate binding. Accordingly, CtpB resembles a minimal version of a self-compartmentalizing protease regulated by a unique allosteric mechanism. Moreover, biochemical analysis of the PDZ-gated channel combined with sporulation assays reveal that activation of the SpoIV RIP pathway is induced by the concerted activity of CtpB and a second signaling protease, SpoIVB. This proteolytic mechanism is of broad relevance for cell-cell communication, illustrating how distinct signaling pathways can be integrated into a single RIP module., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have recently reported that cell-penetrating peptides (CPPs) and novel chimeric peptides containing CPP (referred as B peptide) and muscle-targeting peptide (referred as MSP) motifs significantly improve the systemic exon-skipping activity of morpholino phosphorodiamidate oligomers (PMOs) in dystrophin-deficient mdx mice. In the present study, the general mechanistic significance of the chimeric peptide configuration on the activity and tissue uptake of peptide conjugated PMOs in vivo was investigated. Four additional chimeric peptide-PMO conjugates including newly identified peptide 9 (B-9-PMO and 9-B-PMO) and control peptide 3 (B-3-PMO and 3-B-PMO) were tested in mdx mice. Immunohistochemical staining, RT-PCR and western blot results indicated that B-9-PMO induced significantly higher level of exon skipping and dystrophin restoration than its counterpart (9-B-PMO), further corroborating the notion that the activity of chimeric peptide-PMO conjugates is dependent on relative position of the tissue-targeting peptide motif within the chimeric peptide with respect to PMOs. Subsequent mechanistic studies showed that enhanced cellular uptake of B-MSP-PMO into muscle cells leads to increased exon-skipping activity in comparison with MSP-B-PMO. Surprisingly, further evidence showed that the uptake of chimeric peptide-PMO conjugates of both orientations (B-MSP-PMO and MSP-B-PMO) was ATP- and temperature-dependent and also partially mediated by heparan sulfate proteoglycans (HSPG), indicating that endocytosis is likely the main uptake pathway for both chimeric peptide-PMO conjugates. Collectively, our data demonstrate that peptide orientation in chimeric peptides is an important parameter that determines cellular uptake and activity when conjugated directly to oligonucleotides. These observations provide insight into the design of improved cell targeting compounds for future therapeutics studies.Molecular Therapy-Nucleic Acids (2013) 2, e124; doi:10.1038/mtna.2013.51; published online 24 September 2013.

Acute myocardial infarction (AMI) is a frequent and disabling disease, which is the first cause of cardiovascular mortality worldwide. Infarct size is a major determinant of myocardial functional recovery and mortality after AMI. Limitation of infarct size thus appears as an appropriate strategy to prevent post-ischemic heart failure and improve survival. Reperfusion is the only treatment recommended to reduce infarct size but despite obvious benefits, it may also have deleterious effects called ischemia-reperfusion (IR) injury including myocyte cell death. Proteins involved in the apoptosis cascade generally interact over large surfaces lacking well-defined pockets. Therefore, inhibitory peptides are optimal biomolecules to target these large protein surfaces, they are often more selective to their target than conventional small organic molecules, and they can be tailored for optimal affinity or desired metabolic property. Since peptides do not cross freely biological membranes, they are generally administered in association with cell penetrating peptides (CPPs) and with homing peptides (HPs) for selective organs or tissues targeting. As a first approach in vivo, we made use of the already known BH4 peptidic inhibitor of the mitochondrial apoptotic pathway, which showed cardioprotective properties in a murine model of AMI after a single bolus of intravenous administration. More importantly, similar peptidic strategies and tools are likely to be adaptable to many other situations in which cells have to be protected from apoptosis such as stroke or organ transplantation.

Successful and effective cellular delivery remains a main obstacles in the medical field. The use of cell-penetrating peptides (CPPs) has become one of the most important tools for the internalisation of a wide range of molecules including pharmaceuticals. It is still difficult to choose one CPP for one biological application because there is no ubiquitous CPP meeting the diverse requirements. In our case, we are looking for a suitable CPP to deliver the pro-apoptotic KLA peptide (KLAKLAKKLAKLAK) by a simple co-incubation strategy. For that reason, we selected three different cell lines (fibroblastic, cancerous and macrophagic cells) and studied the uptake and subcellular localisation of six different CPPs alone as well as mixed with the KLA peptide. Furthermore, we used the CPPs with a carboxyamidated or a carboxylated C-terminus and analysed the impact of the C-termini on internalisation and cargo delivery. We could clearly showed that the cellular CPP uptake is not only dependent on the used CPP and cell line but also highly affected by its chemical nature of the C-terminus (uptake: carboxyamidated CPPs > carboxylated CPPs) and can influence its cellular localisation. We successfully delivered the KLA peptide in the three cell lines and learned that here as well, the C-terminus is crucial for an effective peptide delivery. Finally, we induced apoptosis in mouse leukaemic monocyte macrophage (RAW 264.7) and in human breast adenocarcinoma (MCF-7) cells using the mixture of amidated MPG peptide : KLA and in african green monkey kidney fibroblast (Cos-7) cells using carboxylated integrin peptide : KLA., (Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.)

Several proteases like the high temperature requirement A (HtrA) protein family containing internal or C-terminal PDZ domains play key roles in protein quality control in the cell envelope of Gram-negative bacteria. While several HtrA proteases have been extensively characterized, many features of C-terminal processing proteases such as tail-specific protease (Tsp) are still unknown. To fully understand these cellular control systems, individual domains need to be targeted by specific peptides acting as activators or inhibitors. Here, we describe the identification and design of potent inhibitors and activators of Tsp. Suitable synthetic substrates of Tsp were identified and served as a basis for the generation of boronic acid-based peptide inhibitors. In addition, a proteomic screen of E. coli cell envelope proteins using a synthetic peptide library was performed to identify peptides capable of amplifying Tsp's proteolytic activity. The implications of these findings for the regulation of PDZ proteases and for future mechanistic studies are discussed., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel mutated in patients with cystic fibrosis (CF). The most prevalent CFTR mutation, ΔF508, blocks folding in the endoplasmic reticulum. Recent work has shown that some ΔF508-CFTR channel activity can be recovered by pharmaceutical modulators ("potentiators" and "correctors"), but ΔF508-CFTR can still be rapidly degraded via a lysosomal pathway involving the CFTR-associated ligand (CAL), which binds CFTR via a PDZ interaction domain. We present a study that goes from theory, to new structure-based computational design algorithms, to computational predictions, to biochemical testing and ultimately to epithelial-cell validation of novel, effective CAL PDZ inhibitors (called "stabilizers") that rescue ΔF508-CFTR activity. To design the "stabilizers", we extended our structural ensemble-based computational protein redesign algorithm K* to encompass protein-protein and protein-peptide interactions. The computational predictions achieved high accuracy: all of the top-predicted peptide inhibitors bound well to CAL. Furthermore, when compared to state-of-the-art CAL inhibitors, our design methodology achieved higher affinity and increased binding efficiency. The designed inhibitor with the highest affinity for CAL (kCAL01) binds six-fold more tightly than the previous best hexamer (iCAL35), and 170-fold more tightly than the CFTR C-terminus. We show that kCAL01 has physiological activity and can rescue chloride efflux in CF patient-derived airway epithelial cells. Since stabilizers address a different cellular CF defect from potentiators and correctors, our inhibitors provide an additional therapeutic pathway that can be used in conjunction with current methods.

There is an obvious need to develop pharmacological strategies to protect the heart in patients suffering from acute myocardial infarction. Apoptosis was evidenced as a main contributor of myocardial ischemia-reperfusion (IR) injury. Our cardioprotective strategy was based on the use of four cell penetrating peptides (CPP: Tat, (RXR)4, Bpep and Pip2b) which were conjugated to the BH4-peptide, derived from the BH4 domain of the Bcl-xL anti-apoptotic protein. These CPP-BH4 conjugates were able to reduce staurosporine-induced apoptosis in primary cardiomyocytes in vitro. Although Pip2b-BH4 was more efficient in terms of cellular uptake, it was as efficient as Tat-BH4 for its anti-apoptotic activity. As required for potential therapeutic application their cardioprotective effects were evaluated in an in vivo mouse model of myocardial IR injury. Our results clearly show that a single low dose (1 mg/kg) injection of Tat-BH4 and Pip2b-BH4 administered intravenously 5 min before reperfusion was able to drastically reduce infarct size (~47%) and to inhibit apoptosis (~60%) in the left ventricle of treated mice. Importantly, these effects are not observed following the injection of CPP alone or scrambled version of BH4. This study evidences that the Pip2b CPP, designed for oligonucleotides translocation, as well as the widely used natural Tat CPP exhibit similar efficacy in vivo to deliver BH4 anti-apoptotic peptide to the reperfused myocardium and may thus become useful therapeutic tools to treat acute myocardial infarction in the clinical setting., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Progress in our understanding of the molecular pathogenesis of human malignancies has provided therapeutic targets amenable to oligonucleotide (ON)-based strategies. Antisense ON-mediated splicing regulation in particular offers promising prospects since the majority of human genes undergo alternative splicing and since splicing defects have been found in many diseases. However, their implementation has been hampered so far by the poor bioavailability of nucleic acids-based drugs. Cell-penetrating peptides (CPPs) now appear as promising non-viral delivery vector for non-permeant biomolecules. We describe here new CPPs allowing the delivery of splice redirecting steric-block ON using either chemical conjugation or non-covalent complexation. We also describe a convenient and robust splice redirecting assay which allows the quantitative assessment of ON nuclear delivery.