CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis.

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.

An allosteric HTRA1-calpain 2 complex with restricted activation profile.

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.

The RNA Binding Protein Tristetraprolin Contributes to CFTR mRNA Stability in Cystic Fibrosis.

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.

LIX1 Controls MAPK Signaling Reactivation and Contributes to GIST-T1 Cell Resistance to Imatinib.

Gastrointestinal stromal tumor (GIST), the most common sarcoma, is mainly caused by an oncogenic mutation in the KIT receptor tyrosine kinase. Targeting KIT using tyrosine kinase inhibitors, such as imatinib and sunitinib, provides substantial benefit; however, in most patients, the disease will eventually progress due to KIT secondary mutations leading to treatment failure. Understanding how GIST cells initially adapt to KIT inhibition should guide the selection of appropriate therapies to overcome the emergence of resistance. Several mechanisms have been broadly implicated in the resistance to imatinib anti-tumoral effects, including the reactivation of MAPK signaling upon KIT/PDGFRA targeted inhibition. This study provides evidence that LImb eXpression 1 (LIX1), a protein we identified as a regulator of the Hippo transducers YAP1 and TAZ, is upregulated upon imatinib or sunitinib treatment. LIX1 silencing in GIST-T1 cells impaired imatinib-induced MAPK signaling reactivation and enhanced imatinib anti-tumor effect. Our findings identified LIX1 as a key regulator of the early adaptative response of GIST cells to targeted therapies.

WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents.

Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure-activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.

PIP30/FAM192A is a novel regulator of the nuclear proteasome activator PA28γ.

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.

Peptide-Based Nanoparticles to Rapidly and Efficiently "Wrap 'n Roll" siRNA into Cells.

Delivery of small interfering RNA (siRNA) as a therapeutic tool is limited due to critical obstacles such as the cellular barrier, the negative charges of the siRNA molecule, and its instability in serum. Several siRNA delivery systems have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells, especially by forming nanoparticles. In this study, we have developed a new family of short (15mer or 16mer) tryptophan-(W) and arginine-(R) rich Amphipathic Peptides (WRAP) able to form stable nanoparticles and to enroll siRNA molecules into cells. The lead peptides, WRAP1 and WRAP5, form defined nanoparticles smaller than 100 nm as characterized by biophysical methods. Furthermore, they have several benefits as oligonucleotide delivery tools such as the rapid encapsulation of the siRNA, the efficient siRNA delivery in several cell types, and the high gene silencing activity, even in the presence of serum. In conclusion, we have designed a new family of CPPs specifically dedicated for siRNA delivery through nanoparticle formation. Our results indicate that the WRAP family has significant potential for the safe, efficient, and rapid delivery of siRNA for diverse applications.

Optimization of the process of inverted peptides (PIPEPLUS) to screen PDZ domain ligands.

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 PIPEPLUS 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.

A retro-inverso cell-penetrating peptide for siRNA delivery.

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.

Cellular trafficking determines the exon skipping activity of Pip6a-PMO in mdx skeletal and cardiac muscle cells.

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.

Upgrading Mitochondria-Targeting Peptide-Based Nanocomplexes for Zebrafish In Vivo Compatibility Assays.

The lack of effective delivery systems has slowed the development of mitochondrial gene therapy. Delivery systems based on cell-penetrating peptides (CPPs) like the WRAP (tryptophan and arginine-rich peptide) family conjugated with a mitochondrial targeting sequence (MTS) have emerged as adequate carriers to mediate gene expression into the mitochondria. In this work, we performed the PEGylation of WRAP/pDNA nanocomplexes and compared them with previously analyzed nanocomplexes such as (KH)9/pDNA and CpMTP/pDNA. All nanocomplexes exhibited nearly homogeneous sizes between 100 and 350 nm in different environments. The developed complexes were biocompatible and hemocompatible to both human astrocytes and lung smooth muscle cells, ensuring in vivo safety. The nanocomplexes displayed mitochondria targeting ability, as through transfection they preferentially accumulate into the mitochondria of astrocytes and muscle cells to the detriment of cytosol and lysosomes. Moreover, the transfection of these cells with MTS-CPP/pDNA complexes produced significant levels of mitochondrial protein ND1, highlighting their efficient role as gene delivery carriers toward mitochondria. The positive obtained data pave the way for in vivo research. Using confocal microscopy, the cellular internalization capacity of these nanocomplexes in the zebrafish embryo model was assessed. The peptide-based nanocomplexes were easily internalized into zebrafish embryos, do not cause harmful or toxic effects, and do not affect zebrafish's normal development and growth. These promising results indicate that MTS-CPP complexes are stable nanosystems capable of internalizing in vivo models and do not present associated toxicity. This work, even at an early stage, offers good prospects for continued in vivo zebrafish research to evaluate the performance of nanocomplexes for mitochondrial gene therapy.

Evidence That a Peptide-Drug/p53 Gene Complex Promotes Cognate Gene Expression and Inhibits the Viability of Glioblastoma Cells.

Glioblastoma multiform (GBM) is considered the deadliest brain cancer. Conventional therapies are followed by poor patient survival outcomes, so novel and more efficacious therapeutic strategies are imperative to tackle this scourge. Gene therapy has emerged as an exciting and innovative tool in cancer therapy. Its combination with chemotherapy has significantly improved therapeutic outcomes. In line with this, our team has developed temozolomide-transferrin (Tf) peptide (WRAP5)/p53 gene nanometric complexes that were revealed to be biocompatible with non-cancerous cells and in a zebrafish model and were able to efficiently target and internalize into SNB19 and U373 glioma cell lines. The transfection of these cells, mediated by the formulated peptide-drug/gene complexes, resulted in p53 expression. The combined action of the anticancer drug with p53 supplementation in cancer cells enhances cytotoxicity, which was correlated to apoptosis activation through quantification of caspase-3 activity. In addition, increased caspase-9 levels revealed that the intrinsic or mitochondrial pathway of apoptosis was implicated. This assumption was further evidenced by the presence, in glioma cells, of Bax protein overexpression-a core regulator of this apoptotic pathway. Our findings demonstrated the great potential of peptide TMZ/p53 co-delivery complexes for cellular transfection, p53 expression, and apoptosis induction, holding promising therapeutic value toward glioblastoma.

Computational design of a PDZ domain peptide inhibitor that rescues CFTR activity.

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.

Delivery Systems for Mitochondrial Gene Therapy: A Review.

Mitochondria are membrane-bound cellular organelles of high relevance responsible for the chemical energy production used in most of the biochemical reactions of cells. Mitochondria have their own genome, the mitochondrial DNA (mtDNA). Inherited solely from the mother, this genome is quite susceptible to mutations, mainly due to the absence of an effective repair system. Mutations in mtDNA are associated with endocrine, metabolic, neurodegenerative diseases, and even cancer. Currently, therapeutic approaches are based on the administration of a set of drugs to alleviate the symptoms of patients suffering from mitochondrial pathologies. Mitochondrial gene therapy emerges as a promising strategy as it deeply focuses on the cause of mitochondrial disorder. The development of suitable mtDNA-based delivery systems to target and transfect mammalian mitochondria represents an exciting field of research, leading to progress in the challenging task of restoring mitochondria's normal function. This review gathers relevant knowledge on the composition, targeting performance, or release profile of such nanosystems, offering researchers valuable conceptual approaches to follow in their quest for the most suitable vectors to turn mitochondrial gene therapy clinically feasible. Future studies should consider the optimization of mitochondrial genes' encapsulation, targeting ability, and transfection to mitochondria. Expectedly, this effort will bring bright results, contributing to important hallmarks in mitochondrial gene therapy.

Mycobacterium abscessus alkyl hydroperoxide reductase C promotes cell invasion by binding to tetraspanin CD81.

Mycobacterium abscessus (Mab) is an increasingly recognized pulmonary pathogen. How Mab is internalized by macrophages and establishes infection remains unknown. Here, we show that Mab uptake is significantly reduced in macrophages pre-incubated with neutralizing anti-CD81 antibodies or in cells in which the large extracellular loop (LEL) of CD81 has been deleted. Saturation of Mab with either soluble GST-CD81-LEL or CD81-LEL-derived peptides also diminished internalization of the bacilli. The mycobacterial alkyl hydroperoxide reductase C (AhpC) was unveiled as a major interactant of CD81-LEL. Pre-exposure of macrophages with soluble AhpC inhibited mycobacterial uptake whereas overexpression of AhpC in Mab enhanced its internalization. Importantly, pre-incubation of macrophages with anti-CD81-LEL antibodies inhibited phagocytosis of AhpC-coated beads, indicating that AhpC is a direct interactant of CD81-LEL. Conditional depletion of AhpC in Mab correlated with decreased internalization of Mab. These compelling data unravel a previously unexplored role for CD81/AhpC to promote uptake of pathogenic mycobacteria by host cells.

Chemical biology approaches reveal conserved features of a C-terminal processing PDZ protease.

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.

The agony of choice: how to find a suitable CPP for cargo delivery.

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.

Tips and Tools to Understand Direct Membrane Translocation of siRNA-Loaded WRAP-Based Nanoparticles.

Cell-penetrating peptide (CPP)-based approaches are excellent method for delivering cell-impermeable compounds/therapeutics such as proteins, antibodies, antisense oligonucleotides, siRNAs, plasmids, and drugs, as covalently or noncovalently conjugated cargo into cells. Nowadays, it is generally accepted that cellular internalization of these CPP-cargoes or CPP-nanoparticles occur via endocytosis-dependent mechanisms or by direct cell translocation.Here, we describe a subset of biophysical and biological methods which can be used to dissect the internalization mechanism of CPPs. Presented protocols and results were shown for the recently developed siRNA-loaded WRAP-based nanoparticles. The rapid and efficient cell delivery of WRAP encapsulated siRNA could be attributed to the main direct cellular translocation of the nanoparticles even if, to some extent, endocytosis-dependent internalization occurred.Deciphering the internalization mechanism is still an important requirement to understand and to optimize the action mode of CPPs or CPP-based nanoparticles as transfection reagents.

Therapeutic Peptides to Treat Myocardial Ischemia-Reperfusion Injury.

Cardiovascular diseases (CVD) including acute myocardial infarction (AMI) rank first in worldwide mortality and according to the World Health Organization (WHO), they will stay at this rank until 2030. Prompt revascularization of the occluded artery to reperfuse the myocardium is the only recommended treatment (by angioplasty or thrombolysis) to decrease infarct size (IS). However, despite beneficial effects on ischemic lesions, reperfusion leads to ischemia-reperfusion (IR) injury related mainly to apoptosis. Improvement of revascularization techniques and patient care has decreased myocardial infarction (MI) mortality however heart failure (HF) morbidity is increasing, contributing to the cost-intense worldwide HF epidemic. Currently, there is no treatment for reperfusion injury despite promising results in animal models. There is now an obvious need to develop new cardioprotective strategies to decrease morbidity/mortality of CVD, which is increasing due to the aging of the population and the rising prevalence rates of diabetes and obesity. In this review, we will summarize the different therapeutic peptides developed or used focused on the treatment of myocardial IR injury (MIRI). Therapeutic peptides will be presented depending on their interacting mechanisms (apoptosis, necroptosis, and inflammation) reported as playing an important role in reperfusion injury following myocardial ischemia. The search and development of therapeutic peptides have become very active, with increasing numbers of candidates entering clinical trials. Their optimization and their potential application in the treatment of patients with AMI will be discussed.

PPARß/δ-Interfering Peptide Enhanced Mesenchymal Stromal Cell Immunoregulatory Properties.

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 PPARß/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 PPARß/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 PPARß/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 PPARß/δ activity with concomitant enhancement of MSC immunoregulatory properties.