Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking.

Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.

Widespread displacement of DNA- and RNA-binding factors underlies toxicity of arginine-rich cell-penetrating peptides.

Due to their capability to transport chemicals or proteins into target cells, cell-penetrating peptides (CPPs) are being developed as therapy delivery tools. However, and despite their interesting properties, arginine-rich CPPs often show toxicity for reasons that remain poorly understood. Using a (PR)n dipeptide repeat that has been linked to amyotrophic lateral sclerosis (ALS) as a model of an arginine-rich CPP, we here show that the presence of (PR)n leads to a generalized displacement of RNA- and DNA-binding proteins from chromatin and mRNA. Accordingly, any reaction involving nucleic acids, such as RNA transcription, translation, splicing and degradation, or DNA replication and repair, is impaired by the presence of the CPPs. Interestingly, the effects of (PR)n are fully mimicked by protamine, a small arginine-rich protein that displaces histones from chromatin during spermatogenesis. We propose that widespread coating of nucleic acids and consequent displacement of RNA- and DNA-binding factors from chromatin and mRNA accounts for the toxicity of arginine-rich CPPs, including those that have been recently associated with the onset of ALS.

Enhancement of adeno-associated virus serotype 6 transduction into T cells with cell-penetrating peptides.

BACKGROUND: Adeno-associated viruses (AAVs) are gaining interest in the development of cellular immunotherapy. Compared to other viral vectors, AAVs can reduce the risk of insertional oncogenesis. AAV serotype 6 (AAV6) shows the highest efficiency for transducing T cells. Nevertheless, a multiplicity of infection (MOI) of up to one million viral genomes per cell is required to transduce the target cells effectively. Cell-penetrating peptides (CPPs) are short, positively charged peptides that easily translocate the plasma membranes and can facilitate the cellular uptake of a wide variety of cargoes, including small molecules, nucleic acids, drugs, proteins and viral vectors. METHODS: The present study evaluated five CPPs (Antp, TAT-HA2, LAH4, TAT1 and TAT2) on their effects on enhancing transduction of AAV6 packaging a green fluorescent protein transgene into Jurkat T cell line. RESULTS: Vector incubation with peptides TAT-HA2 and LAH4 at a final concentration of 0.2 mm resulted in an approximately two-fold increase in transduced cells. At the lowest MOI tested (1.25 × 104 ), using LAH4 resulted in a 10-fold increase in transduction efficiency. The peptide LAH4 increased the uptake of AAV6 viral particles in both Jurkat cells and mouse primary T cells. Regardless of the large size of the AAV6-LAH4 complexes, their internalization does not appear to depend on macropinocytosis. CONCLUSIONS: Overall, the present study reports an approach to significantly improve the delivery of transgenes into T cells using AAV6 vectors. Notably, the peptides TAT-HA2 and LAH4 contribute to improving the use of AAV6 as a gene delivery vector for the engineering of T cells.

Cell-Penetrating Peptide TAT-HuR-HNS3 Suppresses Proinflammatory Gene Expression via Competitively Blocking Interaction of HuR with Its Partners.

Proinflammatory cytokines/chemokines are commonly regulated by RNA-binding proteins at posttranscriptional levels. Human Ag R (HuR)/embryonic lethal abnormal vision-like 1 (ELAVL1) is one of the well-characterized RNA-binding proteins that increases the stability of short-lived mRNAs, which encode proinflammatory mediators. HuR employs its nucleocytoplasmic shuttling sequence (HNS) domain, interacting with poly(ADP-ribose) polymerase 1 (PARP1), which accounts for the enhanced poly-ADP-ribosylation and cytoplasmic shuttling of HuR. Also by using its HNS domain, HuR undergoes dimerization/oligomerization, underlying the increased binding of HuR with proinflammatory cytokine/chemokine mRNAs and the disassociation of the miRNA-induced silencing complex from the targets. Therefore, competitively blocking the interactions of HuR with its partners may suppress proinflammatory mediator production. In this study, peptides derived from the sequence of the HuR-HNS domain were synthesized, and their effects on interfering HuR interacting with PARP1 and HuR itself were analyzed. Moreover, cell-penetrating TAT-HuR-HNS3 was delivered into human and mouse cells or administered into mouse lungs with or without exposure of TNF-α or LPS. mRNA levels of proinflammatory mediators as well as neutrophil infiltration were evaluated. We showed that TAT-HuR-HNS3 interrupts HuR-PARP1 interaction and therefore results in a lowered poly-ADP-ribosylation level and decreased cytoplasmic distribution of HuR. TAT-HuR-HNS3 also blocks HuR dimerization and promotes Argonaute 2-based miRNA-induced silencing complex binding to the targets. Moreover, TAT-HuR-HNS3 lowers mRNA stability of proinflammatory mediators in TNF-α-treated epithelial cells and macrophages, and it decreases TNF-α-induced inflammatory responses in lungs of experimental animals. Thus, TAT-HuR-HNS3 is a promising lead peptide for the development of inhibitors to treat inflammation-related diseases.

Thyclotides, tetrahydrofuran-modified peptide nucleic acids that efficiently penetrate cells and inhibit microRNA-21.

Peptide nucleic acids (PNAs) are promising therapeutic molecules for gene modulation; however, they suffer from poor cell uptake. Delivery of PNAs into cells requires conjugation of the PNA to another large molecule, typically a cell-penetrating peptide or nanoparticle. In this study, we describe a new PNA-based molecule with cyclic tetrahydrofuran (THF) backbone modifications that in some cases considerably improve cell uptake. We refer to these THF-PNA oligomers as thyclotides. With THF groups at every position of the oligomer, the cell uptake of thyclotides targeted to miR-21 is enhanced compared with the corresponding unmodified PNA based on an aminoethylglycine backbone. An optimized thyclotide can efficiently enter cells without the use of cell-penetrating peptides, bind miR-21, its designated microRNA target, decrease expression of miR-21 and increase expression of three downstream targets (PTEN, Cdc25a and KRIT1). Using a plasmid with the PTEN-3'UTR coupled with luciferase, we further confirmed that a miR-21-targeted thyclotide prevents miR-21 from binding to its target RNA. Additionally, the thyclotide shows no cytotoxicity when administered at 200 times its active concentration. We propose that thyclotides be further explored as therapeutic candidates to modulate miRNA levels.

Cell-penetrating peptide-conjugated Morpholino rescues SMA in a symptomatic preclinical model.

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)2XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy.

Cell-Penetrating Peptides as Vehicles for Delivery of Therapeutic Nucleic Acids. Mechanisms and Application in Medicine.

Currently, nucleic acid therapeutics are actively developed for the treatment and prophylactic of metabolic disorders and oncological, inflammatory, and infectious diseases. A growing number of approved nucleic acid-based drugs evidences a high potential of gene therapy in medicine. Therapeutic nucleic acids act in the cytoplasm, which makes the plasma membrane the main barrier for the penetration of nucleic acid-based drugs into the cell and requires development of special vehicles for their intracellular delivery. The optimal carrier should not only facilitate internalization of nucleic acids, but also exhibit no toxic effects, ensure stabilization of the cargo molecules, and be suitable for a large-scale and low-cost production. Cell-penetrating peptides (CPPs), which match all these requirements, were found to be efficient and low-toxic carriers of nucleic acids. CPPs are typically basic peptides with a positive charge at physiological pH that can form nanostructures with negatively charged nucleic acids. The prospects of CPPs as vehicles for the delivery of therapeutic nucleic acids have been demonstrated in numerous preclinical studies. Some CPP-based drugs had successfully passed clinical trials and were implemented into medical practice. In this review, we described different types of therapeutic nucleic acids and summarized the data on the use of CPPs for their intracellular delivery, as well as discussed, the mechanisms of CPP uptake by the cells, as understanding of these mechanisms can significantly accelerate the development of new gene therapy approaches.

Design of a new cell penetrating peptide for DNA, siRNA and mRNA delivery.

BACKGROUND: Delivery systems, including peptide-based ones, that destabilize endosomes in a pH-dependent manner are increasingly used to deliver cargoes of therapeutic interest, such as nucleic acids and proteins into mammalian cells. METHODS: The negatively charged amphipathic alpha-helicoidal forming peptide named HELP (Helical Erythrocyte Lysing Peptide) is a derivative from the bee venom melittin and was shown to have a pH-dependent activity with the highest lytic activity at pH 5.0 at the same time as becoming inactive when the pH is increased. The present study aimed to determine whether replacement in the HELP peptide of the glutamic acid residues by histidines, for which the protonation state is sensitive to the pH changes that occur during endosomal acidification, can transform this fusogenic peptide into a carrier able to deliver different nucleic acids into mammalian cells. RESULTS: The resulting HELP-4H peptide displays high plasmid DNA, small interfering RNA and mRNA delivery capabilities. Importantly, in contrast to other cationic peptides, its transfection activity was only marginally affected by the presence of serum. Using circular dichroism, we found that acidic pH did not induce significant conformational changes for HELP-4H. CONCLUSIONS: In summary, we were able to develop a new cationic histidine rich peptide able to efficiently deliver various nucleic acids into cells.

Cell-Penetrating Peptide-Peptide Nucleic Acid Conjugates as a Tool for Protein Functional Elucidation in the Native Bacterium.

Approximately 30% or more of the total proteins annotated from sequenced bacteria genomes are annotated as hypothetical or uncharacterized proteins. However, elucidation on the function of these proteins is hindered by the lack of simple and rapid screening methods, particularly with novel or hard-to-transform bacteria. In this report, we employed cell-penetrating peptide (CPP) -peptide nucleotide acid (PNA) conjugates to elucidate the function of such uncharacterized proteins in vivo within the native bacterium. Paenibacillus, a hard-to-transform bacterial genus, was used as a model. Two hypothetical genes showing amino acid sequence similarity to ι-carrageenases, termed cgiA and cgiB, were identified from the draft genome of Paenibacillus sp. strain YYML68, and CPP-PNA probes targeting the mRNA of the acyl carrier protein gene, acpP, and the two ι-carrageenase candidate genes were synthesized. Upon direct incubation of CPP-PNA targeting the mRNA of the acpP gene, we successfully observed growth inhibition of strain YYML68 in a concentration-dependent manner. Similarly, both the function of the candidate ι-carrageenases were also inhibited using our CPP-PNA probes allowing for the confirmation and characterization of these hypothetical proteins. In summary, we believe that CPP-PNA conjugates can serve as a simple and efficient alternative approach to characterize proteins in the native bacterium.

In vivo delivery of a multiepitope peptide and Nef protein using novel cell-penetrating peptides for development of HIV-1 vaccine candidate.

OBJECTIVES: A potent HIV vaccine should overcome some limitations such as polymorphism of human HLA, the diversity of HIV-1 virus, and the lack of an effective delivery system. In this study, a DNA construct encoding Nef60-84, Nef126-144, Vpr34-47, Vpr60-75, Gp16030-53, Gp160308-323, and P248-151 epitopes was designed using bioinformatics tools. The pcDNA3.1-nef-vpr-gp160-p24 and pcDNA3.1-nef constructs were prepared in large scale as endotoxin-free form. Moreover, the recombinant Nef-Vpr-Gp160-p24 polypeptide and Nef protein were generated inE. coli. These constructs were delivered using cell penetrating peptides (CPPs) in vivo, and immune responses were assessed for different modalities in BALB/c mice. RESULTS: The recombinant DNA constructs were confirmed as the ~ 867 bp and ~ 648 bp bands related tonef-vpr-gp160-p24 andnef genes on agarose gel. Moreover, the purified Nef-Vpr-Gp160-p24 polypeptide and Nef protein showed the ~ 32 kDa and ~ 30 kDa bands on SDS-PAGE, respectively. The results of immune responses indicated that the heterologous prime/boost regimens using both Nef-Vpr-Gp160-P24 and Nef antigens induced significantly the secretion of IgG2a, IgG2b, IFN-γ and Granzyme B compared to other groups. The levels of Granzyme B in mice immunized with Nef antigen were higher than those immunized with Nef-Vpr-Gp160-P24 antigen. The CPPs showed the same potency with Montanide adjuvant for eliciting immune responses. CONCLUSIONS: The heterologous prime/boost regimens for both antigens could significantly direct immune responses toward Th1 and CTL activity compared to other regimens. Comparing the efficiency of Nef-Vpr-Gp160-P24 and Nef constructs, the Nef-Vpr-Gp160-P24 constructs delivered by CPPs showed promising results as an HIV vaccine candidate.

PSD-95: An Effective Target for Stroke Therapy Using Neuroprotective Peptides.

Therapies for stroke have remained elusive in the past despite the great relevance of this pathology. However, recent results have provided strong evidence that postsynaptic density protein-95 (PSD-95) can be exploited as an efficient target for stroke neuroprotection by strategies able to counteract excitotoxicity, a major mechanism of neuronal death after ischemic stroke. This scaffold protein is key to the maintenance of a complex framework of protein interactions established at the postsynaptic density (PSD) of excitatory neurons, relevant to neuronal function and survival. Using cell penetrating peptides (CPPs) as therapeutic tools, two different approaches have been devised and advanced to different levels of clinical development. First, nerinetide (Phase 3) and AVLX-144 (Phase 1) were designed to interfere with the coupling of the ternary complex formed by PSD-95 with GluN2B subunits of the N-methyl-D-aspartate type of glutamate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS). These peptides reduced neurotoxicity derived from NMDAR overactivation, decreased infarct volume and improved neurobehavioral results in different models of ischemic stroke. However, an important caveat to this approach was PSD-95 processing by calpain, a pathological mechanism specifically induced by excitotoxicity that results in a profound alteration of survival signaling. Thus, a third peptide (TP95414) has been recently developed to interfere with PSD-95 cleavage and reduce neuronal death, which also improves neurological outcome in a preclinical mouse model of permanent ischemia. Here, we review recent advancements in the development and characterization of PSD-95-targeted CPPs and propose the combination of these two approaches to improve treatment of stroke and other excitotoxicity-associated disorders.

Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice.

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.

Enhanced osteogenic differentiation of human mesenchymal stem cells by direct delivery of Cbfß protein.

Core binding factor ß (Cbfß) is a non-DNA binding cofactor of Runx2 that potentiates DNA binding. Previously, it has been reported that Cbfß plays an essential role in osteogenic differentiation and skeletal development by inhibition adipogenesis. Here, we delivered the recombinant Cbfß protein into human mesenchymal stem cells (MSCs) and triggered osteogenic lineage commitment. The efficient delivery of Cbfß was achieved by fusing 30Kc19 protein, which is a cell-penetrating protein derived from the silkworm. After the production of the recombinant Cbfß-30Kc19 protein in the Escherichia coli expression system, and confirmation of its intracellular delivery, MSCs were treated with the Cbfß-30Kc19 once or twice up to 300 µg/ml. By investigating the upregulation of osteoblast-specific genes and phenotypical changes, such as calcium mineralization, we demonstrated that Cbfß-30Kc19 efficiently induced osteogenic differentiation in MSCs. At the same time, Cbfß-30Kc19 suppressed adipocyte formation and downregulated the expression of adipocyte-specific genes. Our results demonstrate that the intracellularly delivered Cbfß-30Kc19 enhances osteogenesis in MSCs, whereas it suppresses adipogenesis by altering the transcriptional regulatory network involved in osteoblast-adipocyte lineage commitment. Cbfß-30Kc19 holds great potential for the treatment of bone-related diseases, such as osteoporosis, by allowing transcriptional regulation in MSCs, and overcoming the limitations of current therapies.

Nerve Growth Factor Gene Delivery across the Blood-Brain Barrier to Reduce Beta Amyloid Accumulation in AD Mice.

The therapeutic potential of the nerve growth factor (NGF) gene using brain-targeted liposomal nanoparticles was investigated for the treatment of Alzheimer's disease (AD). We designed brain-targeted gene delivery systems with prolonged systemic circulation and enhanced cellular penetration by conjugating the transferrin (Tf) ligand and the penetratin (Pen) peptide to liposomes via a 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) phospholipid. In vitro characterization studies showed that the nanoparticles had homogeneous particle size and positive zeta potential and were able to protect the plasmid DNA against enzymatic degradation. In vivo brain targeting efficiency of designed liposomes was mimicked using an in vitro triple coculture blood-brain barrier (BBB) model. Liposomal nanoparticles dual-modified with Tf and Pen encasing plasmid NGF efficiently crossed the in vitro BBB model and, subsequently, transfected the primary neuronal cells. Increasing NGF expression in primary neuronal cells following treatment with liposomes increased the levels of the presynaptic marker synaptophysin in vitro. A dose-response study in vivo was performed in order to select the appropriate dose of plasmid NGF to induce significant NGF expression and, consequently, a therapeutic effect. Administration of PenTf-liposomes containing pNGF to amyloid precursor protein (APP)/PS1 mice (aged 3 months) for 4 weeks (one injection per week) significantly decreased (p < 0.05) the levels of toxic soluble and insoluble Aß peptides as compared to those levels in untreated APP/PS1 mice. Additionally, the treatment stimulated cell proliferation and increased the levels of synaptic markers, synaptophysin and PSD-95. These data suggest the therapeutic potential of PenTf-liposome-mediated NGF gene therapy, and this system can be considered a candidate for the treatment of AD.

Dual Peptide-Based Gene Delivery System for the Efficient Transfection of Plant Callus Cells.

Owing to their diverse functions and tunable physicochemical properties, peptides are promising alternatives to the conventional gene delivery tools that are available for plant systems. However, peptide-mediated gene delivery is limited by low transfection efficiency in plants because of the insufficient cytosolic translocation of DNA cargo. Here, we report a dual peptide-based gene delivery system for the efficient transfection of plant callus cells. This system is based on the combination of an artificial peptide composed of cationic cell-penetrating and hydrophobic endosomal escape domains with a gene carrier peptide composed of amphiphilic cell-penetrating and cationic DNA-binding domains. Cellular internalization and transfection studies revealed that this dual peptide-based system enables more efficient transfection of callus cells than does a carrier peptide alone by enhancing the endocytic uptake and subsequent cytosolic translocation of a carrier peptide/DNA complex. The present strategy will expand the utility of peptide-mediated plant gene delivery for a wide range of applications and basic research.

AcGly-Phe-Asn(OH) and AcGly-Phe-Asn(NH2) tripeptides selectively affect the proliferation rate of MDA-MB 231 and HuDe cells.

There is increasing interest in the bioactivity of peptides carrying out antiproliferative, antihypertensive, antimicrobial, antioxidant, anticholesterolemic, opioid, and antidiabetic activities. The bioavailability of peptides depends on how readily they are digested by endopeptidases and their ability to pass through cell membranes, features that are determined by the peptide's chemical and physical structure. On the basis of structures present in peptides that have biological activity, particularly antiproliferative activity, the tripeptides AcGly-Phe-Asn(OH) and AcGly-Phe-Asn(NH2) have been designed and synthesized, then tested for their antiproliferative activity on human breast adenocarcinoma cells (MDA-MB 231) and human dermal fibroblasts (HuDe). The results show that the peptides significantly affect the proliferation of MDA-MB 231 and HuDe cells, with differentiated response between tumor and normal cells, and thus indicate that C-terminal amidation plays a role. Interestingly, the activity of both peptides in dermal fibroblasts follows the characteristic biphasic pattern of hormesis, a dose-response relationship.

Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.

A Mitochondrial VDAC1-Based Peptide Greatly Suppresses Steatosis and NASH-Associated Pathologies in a Mouse Model.

Non-alcoholic steatosis and non-alcoholic steatohepatitis (NASH) are liver pathologies characterized by severe metabolic alterations due to fat accumulation that lead to liver damage, inflammation, and fibrosis. We demonstrate that the voltage-dependent anion channel 1 (VDAC1)-based peptide R-Tf-D-LP4 arrested steatosis and NASH progression, as produced by a high-fat diet (HFD-32) in a mouse model, and reversed liver pathology to a normal-like state. VDAC1, a multi-functional mitochondrial protein, regulates cellular metabolic and energetic functions and apoptosis and interacts with many proteins. R-Tf-D-LP4 treatment eliminated hepatocyte ballooning degeneration, inflammation, and liver fibrosis associated with steatosis, NASH, and hepatocarcinoma, and it restored liver pathology-associated enzyme and glucose levels. Peptide treatment affected carbohydrate and lipid metabolism, increasing the expression of enzymes and factors associated with fatty acid transport to mitochondria, enhancing ß-oxidation and thermogenic processes, yet decreasing the expression of enzymes and regulators of fatty acid synthesis. The VDAC1-based peptide thus offers a promising therapeutic approach for steatosis and NASH.

HIF-1α-derived cell-penetrating peptides inhibit ERK-dependent activation of HIF-1 and trigger apoptosis of cancer cells under hypoxia.

Hypoxia is frequently encountered in the microenvironment of solid tumors. Hypoxia-inducible factors (HIFs), the main effectors of cell response to hypoxia, promote cancer cell survival and progression. HIF-1α, the oxygen-regulated subunit of HIF-1, is often correlated with oncogenesis and represents an attractive therapeutic target. We have previously reported that activation HIF-1α by ERK involves modification of two serine residues and masking of a nuclear export signal (NES), all inside a 43-amino acid domain termed ERK Targeted Domain (ETD). Overexpression of ETD variants including wild-type, phospho-mimetic (SE) or NES-less (IA) mutant forms caused HIF-1 inactivation in two hepatocarcinoma cell lines, while a phospho-deficient (SA) form was ineffective and acted as a sequence-specific negative control. To deliver these ETD forms directly into cancer cells, they were fused to the HIV TAT-sequence and produced as cell-permeable peptides. When the TAT-ETD peptides were added to the culture medium of Huh7 cells, they entered the cells and, with the exception of ETD-SA, accumulated inside the nucleus, caused mislocalization of endogenous HIF-1α to the cytoplasm, significant reduction of HIF-1 activity and inhibition of expression of specific HIF-1, but not HIF-2, gene targets under hypoxia. More importantly, transduced nuclear TAT-ETD peptides restricted migration, impaired colony formation and triggered apoptotic cell death of cancer cells grown under hypoxia, while they produced no effects in normoxic cells. These data demonstrate the importance of ERK-mediated activation of HIF-1 for low oxygen adaptation and the applicability of ETD peptide derivatives as sequence-specific HIF-1 and cancer cell growth inhibitors under hypoxia.