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Parkinson's disease (PD) is characterized mainly by the loss of dopaminergic neurons in the substantia nigra (SN) mediated via oxidative stress. Although glutaredoxin-1 (GLRX1) is known as one of the antioxidants involved in cell survival, the effects of GLRX1 on PD are still unclear. In this study, we investigated whether cell-permeable PEP-1-GLRX1 inhibits dopaminergic neuronal cell death induced by 1-methyl-4-phenylpyridinium (MPP + ) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We showed that PEP-1-GLRX1 protects cell death and DNA damage in MPP + -exposed SH-SY5Y cells via the inhibition of MAPK, Akt, and NF-κB activation and the regulation of apoptosis-related protein expression. Furthermore, we found that PEP-1-GLRX1 was delivered to the SN via the blood-brain barrier (BBB) and reduced the loss of dopaminergic neurons in the MPTP-induced PD model. These results indicate that PEP-1-GLRX1 markedly inhibited the loss of dopaminergic neurons in MPP + - and MPTP-induced cytotoxicity, suggesting that this fusion protein may represent a novel therapeutic agent against PD.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 μmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The mechanism of hair loss caused by aging is related to mitochondrial dysfunction. Pep-1-mediated mitochondrial transplantation is a potential therapeutic application for mitochondrial disorders, but its efficacy against hair aging remains unknown. This study compared platelet-rich plasma (PRP) therapy with mitochondrial transplantation for hair restoration and examined the related regulation in naturally aging mice. After dorsal hair removal, 100-week-old mice received weekly unilateral injections of 200 μg of allogeneic mitochondria-labeled 5-bromo-2'-deoxyuridine with (P-Mito) or without Pep-1 conjugation (Mito) or human PRP with a stamp-type electric injector for 1 month. The contralateral sides were used as corresponding sham controls. Compared with the control and corresponding sham groups, all treatments stimulated hair regrowth, and the effectiveness of P-Mito was equal to that of PRP. However, histology revealed that only P-Mito maintained hair length until day 28 and yielded more anagen follicles with abundant dermal collagen equivalent to that of the PRP group. Mitochondrial transplantation increased the thickness of subcutaneous fat compared with the control and PRP groups, and only P-Mito consistently increased mitochondria in the subcutaneous muscle and mitochondrial DNA copies in the skin layer. Therefore, P-Mito had a higher penetrating capacity than Mito did. Moreover, P-Mito treatment was as effective as PRP treatment in comprehensively reducing the expression of aging-associated gene markers, such as IGF1R and MRPS5, and increasing antiaging Klotho gene expression. This study validated the efficacy of mitochondrial therapy in the restoration of aging-related hair loss and demonstrated the distinct effects of PRP treatment., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Sirtuin proteins are a highly conserved class of nicotinamide adenine dinucleotide (NAD + )-dependent lysine deacylases. The pleiotropic human isoform 2 of Sirtuins (SIRT2) has been engaged in the pathogenesis of cancer in a plethora of reports around the globe. Thus, SIRT2 modulation is deemed as a promising approach for pharmaceutical intervention. Previously, we reported S-Trityl-l-Cysteine (STLC)-ornamented dimethylaminopyridine chemical entity named STC4 with a significant SIRT2 inhibitory capacity; this was separate from the conventional application of STLC scaffold as a kinesin-5 inhibitor. An interactive molecular docking study of SIRT2 and STC4 showed interaction between Asn168 of SIRT2 and the methyl ester of STC4, that appears to hinder STC4 to reach the selective pocket of the protein unlike strong SIRT2 inhibitor SirReal2. To improve its activity, herein, we utilized S-trityl cysteamine pharmacophore lacking the methyl ester. Nine compounds were synthesized and assayed affording three biopertinent SIRT2 inhibitors, and two of them, STCY1 and STCY6 showed higher inhibitory activity than STC4. These compounds have pronounced anti-proliferative activities against different cancer cell lines. A molecular docking study was executed to shed light on the supposed binding mode of the lead compound, STCY1, into the selective pocket of SIRT2 by interaction of the nitrogen of pyridine ring of the compound and Ala135 of the protein. The outcome of the study exposes that the active compounds are effective intermediates to construct more potent biological agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and β-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1β), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response., (Copyright © 2020. Published by Elsevier Ltd.)

Glutaredoxin 1 (GLRX1) has been recognized as an important regulator of redox signaling. Although GLRX1 plays an essential role in cell survival as an antioxidant protein, the function of GLRX1 protein in inflammatory response is still under investigation. Therefore, we wanted to know whether transduced PEP-1-GLRX1 protein inhibits lipopolysaccharide (LPS)- and 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced inflammation. In LPS-exposed Raw 264.7 cells, PEP-1-GLRX1 inhibited cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), activation of mitogen activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB) expression levels. In a TPA-induced mouse-ear edema model, topically applied PEP-1-GLRX1 transduced into ear tissues and significantly ameliorated ear edema. Our data reveal that PEP-1-GLRX1 attenuates inflammation in vitro and in vivo, suggesting that PEP-1-GLRX1 may be a potential therapeutic protein for inflammatory diseases. [BMB Reports 2020; 53(2): 106-111].

In a previous study, we utilized a proteomic approach and found a significant reduction in phosphatidylethanolamine-binding protein 1 (PEBP1) protein level in the spinal cord at 3 h after ischemia. In the present study, we investigated the role of PEBP1 against oxidative stress in NSC34 cells in vitro, and ischemic damage in the rabbit spinal cord in vivo. We generated a PEP-1-PEBP1 fusion protein to facilitate the penetration of blood-brain barrier and intracellular delivery of PEBP1 protein. Treatment with PEP-1-PEBP1 significantly decreased cell death and the induction of oxidative stress in NSC34 cells. Furthermore, administering PEP-1-PEBP1 did not show any significant side effects immediately before and after ischemia/reperfusion. Administration of PEP-PEBP1 improved the Tarlov's neurological score at 24 and 72 h after ischemia, and significantly improved neuronal survival at 72 h after ischemia based on neuronal nuclei (NeuN) immunohistochemistry, Flouro-Jade B staining, and western blot study for cleaved caspase 3. PEP-1-PEBP1 administration decreased oxidative stress based on malondialdehyde level, advanced oxidation protein products, and 8-iso-prostaglandin F2α in the spinal cord. In addition, inflammation based on myeloperoxidase level, tumor necrosis factor-α level, and high mobility group box 1 level was decreased by PEP-1-PEBP1 treatment at 72 h after ischemia. Thus, PEP-1-PEBP1 treatment, which decreases oxidative stress, inflammatory cytokines, and neuronal death, may be an effective therapeutic strategy for spinal cord ischemia., Competing Interests: All authors have approved the manuscript for submission, and we have no conflicts of interest to disclose.

The cryopreservation of cells underpins many areas of biotechnology, healthcare, and fundamental science by enabling the banking and distribution of cells. Cryoprotectants are essential to prevent cold-induced damage. Here, we demonstrate that extracellular localization of antifreeze proteins can significantly enhance post-thaw recovery of mammalian cell monolayers cryopreserved using dimethyl sulfoxide, whereas they show less benefit in suspension cryopreservation. A type III antifreeze protein (AFPIII) was used as the macromolecular ice recrystallization inhibitor and its intra/extracellular locations were controlled by using Pep-1, a cell-penetrating peptide. Flow cytometry and confocal microscopy confirmed successful delivery of AFPIII. The presence of extracellular AFPIII dramatically increased post-thaw recovery in a challenging 2-D cell monolayer system using just 0.8 mg·mL -1 , from 25% to over 60%, whereas intracellularly delivered AFPIII showed less benefit. Interestingly, the antifreeze protein was less effective when used in suspension cryopreservation of the same cells, suggesting that the cryopreservation format is also crucial. These observations show that, in the discovery of macromolecular cryoprotectants, intracellular delivery of ice recrystallization inhibitors may not be a significant requirement under "slow freezing" conditions, which will help guide the design of new biomaterials, in particular, for cell storage.

Phosphoprotein enriched in astrocytes 15 (PEA15) plays a multi-functional role in neuronal cell survival, however the effects of PEA15 against inflammation have not been investigated yet. To examine the effects of PEP-1-PEA15 protein against lipopolysaccharide (LPS)-induced inflammatory responses in Raw 264.7 cells and in a 12-O-tetradecanoylphobol 13-acetate (TPA)-induced mouse model, we constructed and purified PEP-1-PEA15 protein, which can transduce into cells or tissues. PEP-1-PEA15 inhibited LPS-induced damage in cells including that caused by reactive oxygen species (ROS) production and DNA fragmentation. PEP-1-PEA15 also significantly suppressed activation of mitogen activated protein kinases (MAPKs), pro-inflammatory mediator proteins and various cytokines. In a TPA-induced mouse ear edema model, PEP-1-PEA15 significantly reduced ear weight and thickness as well as MAPK activation as well as the expression levels of COX-2, iNOS, IL-6, IL-1β, and TNF-α. These results demonstrated that PEP-1-PEA15 showed anti-inflammatory effect in cells and animal model suggesting that this fusion protein protects cells or skin tissues from inflammatory response., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Pancreatic beta cell destruction and dysfunction induced by cytokines is a major cause of type 1 diabetes. Paraoxonase 1 (PON1), an arylesterase with antioxidant activity, has been shown to play an important role in preventing the development of diabetes in transgenic mice. However, no studies have examined the anti-diabetic effect of PON1 delivered to beta cells using protein transduction. In this study, we expressed the cell-permeable PON1 fused with PEP-1 protein transduction domain (PEP-1-PON1) to investigate whether transduced PEP-1-PON1 protects beta cells against cytokine-induced cytotoxicity. PEP-1-PON1 was effectively delivered to INS-1 cells and prevented cytokine-induced cell destruction in a dose-dependent manner. Transduced PEP-1-PON1 significantly reduced the levels of reactive oxygen species (ROS) and nitric oxide (NO), DNA fragmentation, and expression of inflammatory mediators, endoplasmic reticulum (ER) stress proteins, and apoptosis-related proteins in cytokine-treated cells. Moreover, transduced PEP-1-PON1 restored the decrease in basal and glucose-stimulated insulin secretion induced by cytokines. These data indicate that PEP-1-PON1 protects beta cells from cytokine-induced cytotoxicity by alleviating oxidative/nitrosative stress, ER stress, and inflammation. Thus, PEP-1-mediated PON1 transduction might be an effective method to reduce the extent of destruction and dysfunction of pancreatic beta cells in autoimmune diabetes. [BMB Reports 2018; 51(10): 539-544].

Cystamine is commonly used as a transglutaminase inhibitor. This disulphide undergoes reduction in vivo to the aminothiol compound, cysteamine. Thus, the mechanism by which cystamine inhibits transglutaminase activity in vivo could be due to either cystamine or cysteamine, which depends on the local redox environment. Cystamine inactivates transglutaminases by promoting the oxidation of two vicinal cysteine residues on the enzyme to an allosteric disulphide, whereas cysteamine acts as a competitive inhibitor for transamidation reactions catalyzed by this enzyme. The latter mechanism is likely to result in the formation of a unique biomarker, N -(γ-glutamyl)cysteamine that could serve to indicate how cyst(e)amine acts to inhibit transglutaminases inside cells and the body., (© 2018 The Author(s).)

Oxidative stress is known to be a primary risk factor for neuronal diseases. Glutaredoxin (GLRX)‑1, a redox‑regulator of the thioredoxin superfamily, is known to exhibit an important role in cell survival via various cellular functions. However, the precise roles of GLRX1 in brain ischemia are still not fully understood. The present study investigated whether transduced PEP‑1‑GLRX1 protein has protective effects against oxidative stress in cells and in an animal model. Transduced PEP‑1‑GLRX1 protein increased HT‑22 cell viability under oxidative stress and this fusion protein significantly reduced intracellular reactive oxygen species and levels of DNA damage. In addition, PEP‑1‑GLRX1 protein regulated RAC‑a serine/threonine‑protein kinase and mitogen‑activated protein kinase signaling, in addition to apoptotic signaling including B cell lymphoma (Bcl)‑2, Bcl‑2 associated X, apoptosis regulator, pro‑caspase‑9 and p53 expression levels. In an ischemic animal model, it was verified that PEP‑1‑GLRX1 transduced into the Cornu Ammonis 1 region of the animal brain, where it markedly protected against ischemic injury. These results indicate that PEP‑1‑GLRX1 attenuates neuronal cell death resulting from oxidative stress in vitro and in vivo. Therefore, PEP‑1‑GLRX1 may exhibit a beneficial role in the treatment of neuronal disorders, including ischemic injury.

Skin sensitization risk assessment of botanical ingredients is necessary for consumers' protection and occupational hazard identification. There are currently very few available alternative methods that can assist in the evaluation of complex mixtures. Chemical methods can provide essential information in a timely manner and thus help to reduce the need for in vivo testing, and they can complement and facilitate targeted in vitro assays. In the present work, the applicability of the high-throughput screening with dansyl cysteamine (DCYA) method for the systematic evaluation of skin sensitization of complex botanicals was explored. Botanical ingredients of four unrelated plant species were obtained and tested with the high-throughput fluorescence method at three concentrations. To illustrate the minimal matrix effects of the tested extracts on the developed method, the least DCYA-reactive extract (Rosa canina) was spiked with known sensitizers at different concentrations. The data obtained from the four plant extracts and the spiking experiments with known sensitizers, suggest that the high-throughput screening-DCYA method can be successfully applied for estimating the skin sensitization potential of complex botanical matrices. This is the first report of an attempt to develop a versatile in chemico method for the rapid detection of reactive skin sensitizers in complex botanical extracts, which could complement the battery of existing validated, non-animal methods., (Copyright © 2018 John Wiley & Sons, Ltd.)

The cell-penetrating peptide (CPP) Pep-1 presents a great potential in drug delivery due to its intrinsic property to cross plasma membrane. However, its mechanism of entry into the cell remains unresolved. In this study, we compare the selectivity of Pep-1 towards vesicles mimicking normal and cancer cell membranes. The interaction was performed in a wide range of peptide-to-lipid molar ratios using infrared (IR), fluorescence, scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques. At low peptide concentration, fluorescence experiments show that lipid-phosphatidylserine (PS) seems to enable Pep-1 translocation into cancer cell membrane as evidenced by the blue shift of its maximal emission wavelength. DSC data show that Pep-1 induces segregation of lipids. At high peptide concentration, IR data indicate that the interaction of Pep-1 is relatively stronger with normal cell membrane than with cancer cell membrane through the phosphate groups, while the interaction is weaker with normal cell membrane than with cancer cell membrane through the carbonyl groups. TGA and DSC data reveal that vesicles of normal cell membrane are thermally more stable than vesicles of cancer cell membrane. This suggests that the additional lipid PS included in cancer cell membrane has a destabilizing effect on the membrane structure. SEM images reveal that Pep-1 form superstructures including spherical particles and fibrils in the presence of both model membranes. PS seems to enhance peptide transport across cellular membranes. The biophysical techniques in this study provide valuable insights into the properties of CPPs in drug delivery systems., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glutaredoxin-1 (GRX-1), belonging to the oxidoreductase family, is a component of the endogenous antioxidant defense system. In this study, we evaluated the effects of PEP-1-GRX-1 in rabbit articular chondrocytes. We found that PEP-1-GRX-1 causes a loss of the differentiated chondrocyte phenotype. PEP-1-GRX-1-treated cells exhibited decreases in type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. PEP-1-GRX-1 causes endoplasmic reticulum (ER)-stress, as evidenced by increases in ER stress marker proteins, i.e., glucose-regulated protein (GRP) 78, GRP 94, and phospho-eukaryotic initiation factor 2 (eIF2) α. These effects were inhibited by ER stress inhibitors. PEP-1-GRX-1 increased the phosphorylation of Akt, extracellular signal-regulated kinase (ERK)-1/2, and p38. Inhibition of ERK-1/2 by PD98059 prevented PEP-1-GRX-1-induced dedifferentiation and inhibited ER stress. The blockage of PI-3K/Akt or p38 kinase with SB203580 and LY294002 accelerated PEP-1-GRX-1-induced dedifferentiation, but did not have any effect on PEP-GRX-1-induced ER stress. Our results indicate that the ERK-1/2 pathway mediates chondrocyte dedifferentiation by PEP-GRX-1-induced ER stress. The PI-3K and p38 kinase pathways regulate PEP-1-GRX-1-induced chondrocyte dedifferentiation by an ER stress-independent pathway., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Chronic mercury intoxication is a severe health issue and occurs especially in gold mining communities. Common chelators used for improving mercury elimination are not everywhere available and challenged by poor cell wall penetration. This study is part of a feasibility trial and the aim was to gather first information about the efficacy of the newly developed chelator N,N'bis-(2-mercaptoethyl) isophthalamide (NBMI) on chronic mercury intoxication., Methods: In this three-armed, placebo-controlled randomized trial, 36 miners with mercury urine levels exceeding 15 μg/l were administered 100 mg NBMI, 300 mg NBMI or placebo for 14 days. Levels of mercury in urine [μg/l and μg/g creatinine] and plasma l were analyzed. Therapeutic effect was assessed using the medical intoxication score (MIS) and its single health outcomes (e.g. excessive salivation, sleeping problems), fatigue scores, a neuromotoric test battery (CATSYS) and a neurological outcome (Finger to nose test)., Results: Physical fatigue was significantly decreased in the 300 mg NBMI group compared to the control. Mercury concentration in urine following 300 mg NBMI treatment was significantly lowered compared to control, however, this effect was less distinct with adjustment for creatinine., Conclusion: NBMI showed an effect on physical fatigue and there were indications to positive effects on other symptoms as well. More comprehensive studies are mandatory to verify the effects of NBMI as a novel tool for treating mercury intoxications., Trial Registration: ClinicalTrials.gov Identifier: NCT02486289 . Date of registration: June 24, 2015.

Background: Current options to treat clinical relapse in inflammatory central nervous system (CNS) conditions such as cerebral ischemia-reperfusion injury are limited, and agents that are more effective are required. Disruption of the blood-brain barrier is an early feature of lesion formation that correlates with clinical exacerbation and facilitates the entry of inflammatory medium and inflammatory cells. Interleukin-1 receptor antagonist (IL-1RA) is a naturally occurring anti-inflammatory antagonist of the interleukin-1 (IL-1) family. The broad-spectrum anti-inflammatory effects of IL-1RA have been investigated against various forms of neuroinflammation. However, the effect of IL-1RA on blood-brain barrier disruption following ischemia-reperfusion has not been reported., Methods: In this study, we investigated the effects of IL-1RA and a novel protein (IL-1RA-PEP) that was fused to IL-1RA with a cell penetrating peptide, on blood-brain barrier integrity, in male rats subjected to transient middle cerebral artery occlusion., Results: After intravenous administration, IL-1RA-PEP (50 mg/kg) penetrated cerebral tissues more effectively than IL-1RA. Moreover, it preserved blood-brain barrier integrity, attenuated changes in expression and localization of tight junction proteins and matrix metalloproteinases, and enhanced angiogenesis in ischemic brain tissue. Further study suggested that the effects of IL-1RA-PEP on preserving blood-brain barrier integrity might be closely correlated with the p65/NF-κB pathway, as evidenced by the effects of the inhibitor JSH-23., Conclusions: Collectively, our results demonstrated that IL-1RA-PEP could effectively penetrate the brain of rats with middle cerebral artery occlusion and ameliorate blood-brain barrier disruption. This finding might represent its novel therapeutic potential in the treatment of the cerebral ischemia-reperfusion injury.

Pep-1 is a cell-penetrating peptide that represents a powerful strategy for delivering large, hydrophilic therapeutic molecules into cells. Model membranes, such as lipid vesicles and planar bilayers, have been useful for investigating the direct translocation of cell-penetrating peptides. Here, we present a droplet interface bilayer-based approach to quantify pep-1-mediated β-galactosidase translocation. We found that β-galactosidase translocation is driven only by the negative transmembrane potential resulting from the asymmetric bilayers. The asymmetric droplet interface bilayer method may be generally applicable for high-throughput screening of the efficacy of cell-penetrating peptides.

Bacteria have the natural ability to install protective postsynthetic modifications onto its bacterial peptidoglycan (PG), the coat woven into bacterial cell wall. Peptidoglycan O-acetyltransferase B (PatB) catalyzes the O-acetylation of PG in Gram (-) bacteria, which aids in bacterial survival, as it prevents autolysins such as lysozyme from cleaving the PG. We explored the mechanistic details of PatB's acetylation function and determined that PatB has substrate specificity for bioorthgonal short N-acetyl cysteamine (SNAc) donors. A variety of functionality including azides and alkynes were installed on tri-N-acetylglucosamine (NAG) 3 , a PG mimic, as well as PG isolated from various Gram (+) and Gram (-) bacterial species. The bioorthogonal modifications protect the isolated PG against lysozyme degradation in vitro. We further demonstrate that this postsynthetic modification of PG can be extended to use click chemistry to fluorescently label the mature PG in whole bacterial cells of Bacillus subtilis. Modifying PG postsynthetically can aid in the development of antibiotics and immune modulators by expanding the understanding of how PG is processed by lytic enzymes.

In this study, we have established an effective and novel nanocarrier system for the effective treatment of glioma. We have established the glioma tissue penetrating nanocarrier system by conjugating Pep-1 as a targeting ligand on the liposome surface to enhance the anticancer efficacy of cilengitide (CGL). The particles were nanosized and exhibited a controlled release of drug in both the pH conditions. The cellular uptake assay showed that conjugation of Pep-1 on the liposome surface remarkably increased the cellular uptake. The uptake of CGT-loaded Pep-1 peptide-conjugated liposome (PeCNL) increased to 89.8% compared to 47.5% for CNL indicating the efficient internalization of the nanocarriers. Consistently, PeCNL exhibited a significantly higher cytotoxic effect in cancer cells compared to that of non-targeted CGT-loaded liposome (CNL). PeCNL exhibited a higher apoptosis of cancer cells (∼35%) compared to that of CNL. Most importantly, PeCNL exhibited a significantly superior anticancer effect with tumor volume as low as ∼350mm 3 indicating the superior anticancer potential of targeted formulations. Similarly, PeCNL showed the lowest staining for Ki67 indicating that the targeted NP has the maximum effect in controlling the proliferation of cancer cells. Taken together, Pep-1 conjugated liposome could exhibit better antitumor efficacy when applied to IL-13R2 receptor overexpressed specific brain glioma., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer that invariably results in a dismal prognosis. Chemotherapy and radiotherapy have not been completely effective as standard treatment options for patients due to recurrent disease. We and others have therefore developed molecular strategies to specifically target interleukin 13 receptor alpha 2 (IL13RA2), a GBM restricted receptor expressed abundantly on over 75% of GBM patients. In this work, we evaluated the potential of Pep-1L, a novel IL13RA2 targeted peptide, as a platform to deliver targeted lethal therapies to GBM. To demonstrate GBM-specificity, we radiolabeled Pep-1L with Copper-64 and performed in vitro cell binding studies, which demonstrated specific binding that was blocked by unlabeled Pep-1L. Furthermore, we demonstrated real-time GBM localization of [64Cu]Pep-1L to orthotopic GBMs using small animal PET imaging. Based on these targeting data, we performed an initial in vivo safety and therapeutic study using Pep-1L conjugated to Actinium-225, an alpha particle emitter that has been shown to potently and irreversibly kill targeted cells. We infused [225Ac]Pep-1L into orthotopic GBMs using convection-enhanced delivery and found no significant adverse events at injected doses. Furthermore, our initial data also demonstrated significantly greater overall, median and mean survival in treated mice when compared to those in control groups (p < 0.05). GBM tissue extracted from mice treated with [225Ac]Pep-1L showed double stranded DNA breaks, lower Ki67 expression and greater propidium iodide internalization, indicating anti-GBM therapeutic effects of [225Ac]Pep-1L. Based on our results, Pep-1L warrants further investigation as a potential targeted platform to deliver anti-cancer agents.

Background: The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model., Methods: Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model., Results: PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema., Conclusion: These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

In the present study, we investigated the ability of Cu, Zn-superoxide dismutase (SOD1) to improve the therapeutic potential of adipose tissue-derived mesenchymal stem cells (Ad-MSCs) against ischemic damage in the spinal cord. Animals were divided into four groups: the control group, vehicle (PEP-1 peptide and artificial cerebrospinal fluid)-treated group, Ad-MSC alone group, and Ad-MSC-treated group with PEP-1-SOD1. The abdominal aorta of the rabbit was occluded for 30 min in the subrenal region to induce ischemic damage, and immediately after reperfusion, artificial cerebrospinal fluid or Ad-MSCs (2 × 10 5 ) were administered intrathecally. In addition, PEP-1 or 0.5 mg/kg PEP-1-SOD1 was administered intraperitoneally to the Ad-MSC-treated rabbits. Motor behaviors and NeuN-immunoreactive neurons were significantly decreased in the vehicle-treated group after ischemia/reperfusion. Administration of Ad-MSCs significantly ameliorated the changes in motor behavior and NeuN-immunoreactive neuronal survival. In addition, the combination of PEP-1-SOD1 and Ad-MSCs further increased the ameliorative effects of Ad-MSCs in the spinal cord after ischemia. Furthermore, the administration of Ad-MSCs with PEP-1-SOD1 decreased lipid peroxidation and maintained levels of antioxidants such as SOD1 and glutathione peroxidase compared to the Ad-MSC alone group. These results suggest that combination therapy using Ad-MSCs and PEP-1-SOD1 strongly protects neurons from ischemic damage by modulating the balance of lipid peroxidation and antioxidants.

The role of cell penetrating peptides (CPPs) has been challenged in recent years for drug delivery to ocular tissues for the targeting of both anterior and posterior segments. The enhancement of trans-corneal transport for anterior segment targeting is a very important issue possibly leading to important outcomes on efficacy and to the opportunity of topical administration of molecules with unfavorable penetration properties. The aim of the present work was the design and synthesis of new CPPs, deriving from the structure of PEP-1 peptide. Synthesized peptides were labeled with 5-carboxyfluorescein (5-FAM), and their diffusion behavior and distribution inside the cornea were evaluated by a validated ex vivo model and a confocal microscopy approach. Newly synthesized peptides showed similar corneal permeation profiles as PEP-1 (P app = 0.75 ± 0.56 × 10 -6 cm/s), about 2.6-fold higher than 5-FAM (P app = 0.29 ± 0.08 × 10 -6 cm/s) despite the higher molecular weight. Confocal microscopy experiments highlighted the tendency of PEP-1 and its derived peptides to localize in the intercellular space and/or in the plasma membrane. Noteworthy, using penetratin as positive control, a higher trans-corneal permeation (P app = 6.18 ± 1.46 × 10 -6 cm/s) was evidenced together with a diffusion by intracellular route and a different accumulation between wings and basal epithelial cells, probably depending on the stage of cell development. Finally, PEP-1 and pep-7 proved to be safe and well tolerated when tested on human conjuctival cell line.

Glioblastoma multiforme (GBM) is the most common and aggressive primary central nervous system (CNS) tumor with a short survival time. The failure of chemotherapy is ascribed to the low transport of chemotherapeutics across the Blood Brain Tumor Barrier (BBTB) and poor penetration into tumor tissue. In order to overcome the two barriers, small nanoparticles with active targeted capability are urgently needed for GBM drug delivery. In this study, we proposed PEGylated Polyamidoamine (PAMAM) dendrimer nanoparticles conjugated with glioma homing peptides (Pep-1) as potential glioma targeting delivery system (Pep-PEG-PAMAM), where PEGylated PAMAM dendrimer nanoparticle was utilized as carrier due to its small size and perfect penetration into tumor and Pep-1 was used to overcome BBTB via interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis. The preliminary availability and safety of Pep-PEG-PAMAM as a nanocarrier for glioma was evaluated. In vitro results indicated that a significantly higher amount of Pep-PEG-PAMAM was endocytosed by U87 MG cells. In vivo fluorescence imaging of U87MG tumor-bearing mice confirmed that the fluorescence intensity at glioma site of targeted group was 2.02 folds higher than that of untargeted group (**p<0.01), and glioma distribution experiment further revealed that Pep-PEG-PAMAM exhibited a significantly enhanced accumulation and improved penetration at tumor site. In conclusion, Pep-1 modified PAMAM was a promising nanocarrier for targeted delivery of brain glioma., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1-SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1-SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1-SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1-SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Thioesters play essential roles in many biosynthetic pathways to fatty acids, esters, polyketides, and non-ribosomal peptides. Coenzyme A (CoA) and related phosphopantetheine thioesters are typically employed as activated acyl units for diverse C-C, C-O, and C-N coupling reactions. To study and control these enzymatic assembly lines in vitro and in vivo structurally simplified analogs such as N-acetylcysteamine (NAC) thioesters have been developed. This review gives an overview on experimental strategies enabled by synthetic NAC thioesters, such as the elucidation of complex biosynthetic pathways and enzyme mechanisms as well as precursor-directed biosynthesis and mutasynthesis. The review also summarizes synthetic protocols and protection group strategies to access these versatile synthetic tools, which are reactive and often unstable compounds. In addition, alternative phosphopantetheine thioester mimics are presented that can be used as protein tags or suicide inhibitors for protein crosslinking and off-loading probes to elucidate polyketide intermediates., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To study the effects of heme oxygenase-1 transduced by cell penetrating peptide PEP-1 on renal injury in acute paraquat-induced rats. Methods: The fusion protein PEP-1/HO-1 was manufactured by genetic engineering methods. One hundred and twenty-six healthy adult Sprague-Dawley (SD) rats (63 male, 63 female) were randomly divided into three groups: (1) Control group: 42 rats, (2) Poisoned group: 42 rats, (3) PEP-1/HO-1 intervention group: 42 rats. The rats in Poisoned group and intervention group were treated intraperitoneally with paraquat (25 mg/kg) deliquated with normal saline. Control group rats were treated with the same way of normal saline as the others. Intervention group rats were injected 1 mg of the fusion protein PEP-1/HO-1 into the left iliac vein at 30 minutes before the administration of the paraquat. After abdominal dissection with ether anaesthesia, six rats respectively from each of three groups were taken tissue samples from kidney at 1 st、6 th、12 th、24 th、36 th、48 th、and 72 nd hour respectively. Tissue Superoxide Dismutase (SOD) activity and Malondialdehyde (MDA) content were measured, and renal tissue was quickly remained to observe the expression of Heme Oxygenase-1 (HO-1) by the method of immunohistochemistry. Blood samples were collected from the abdominal aorta for determination of Blood Urea Nitrogen (BUN) and Creatinine (Cr) concentrations in plasma. Results: To prepare high purity fusion protein PEP-1/HO-1 Successfully. The levels of the plasma Bun and Cr in Poisoned and intervention group were significantly higher than that in Control group in the six to seventy-two hours ( P <0.01) ; Whereas the increases of Bun and Cr were markedly intibited in PEP-1/HO-1 intervention group, in which, the levels of Bun and Cr in the six to seventy-two hours were significantly lower than Poisoned group ( P <0.05). Compared with control group, the levels of MDA in poisoned groups were increased in the one to forty-eight hours and the levels of it in groups PEP-1/HO-1 in the one to thirty-six ( P <0.01 or P <0.05). The T-SOD activity of the poisoned group was decreased in the one to thirty-six hours than that of the control group ( P <0.05). Compared with poisoned group, the T-SOD activity at various points in time was increased in group PEP-1/HO-1 ( P <0.01). In control group, there was only very weak expressions of HO-1 in the normal renal tissue. In poisoned and intervention group, the expressions of HO-1 were significantly higher than that in control group in all time ( P <0.01). The HO-1 expressions of PEP-1/HO-1 group in all time was significantly higher than that in poisoned group ( P <0.01 or P <0.05). Conclusion: The HO-1 protein Can be successfully transduced into renal tissue by cell penetrating peptide PEP-1 and the transduced HO-1 protein reduces renal injury of the acute paraquat-induced rats by inhibiting lipid peroxidation response.

Reactive oxygen species generated under oxidative stress are involved in neuronal diseases, including ischemia. Glutathione S-transferase pi (GSTpi) is a member of the GST family and is known to play important roles in cell survival. We investigated the effect of GSTpi against oxidative stress-induced hippocampal HT-22 cell death, and its effects in an animal model of ischemic injury, using a cell-permeable PEP-1-GSTpi protein. PEP-1-GSTpi was transduced into HT-22 cells and significantly protected against H2O2-treated cell death by reducing the intracellular toxicity and regulating the signal pathways, including MAPK, Akt, Bax, and Bcl-2. PEP-1-GSTpi transduced into the hippocampus in animal brains, and markedly protected against neuronal cell death in an ischemic injury animal model. These results indicate that PEP-1-GSTpi acts as a regulator or an antioxidant to protect against oxidative stressinduced cell death. Our study suggests that PEP-1-GSTpi may have potential as a therapeutic agent for the treatment of ischemia and a variety of oxidative stress-related neuronal diseases. [BMB Reports 2016; 49(7): 382-387].

Two human papillomavirus (HPV) viral oncoproteins, E6 and E7 represent ideal targets for development of a therapeutic HPV vaccine. It is important to reduce the rate of HPV-associated malignancies through improvement of vaccine modalities. In this study, we used a short amphipathic peptide carrier, Pep-1, for delivery of the full-length HPV16 E7 protein into mammalian cells and evaluated immune responses and protective effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7/Pep-1 protein form stable nanoparticles through noncovalent binding with an average size of 120 to 250 nm. The efficient delivery of E7 protein by Pep-1 at molar ratio of 1:20 was detected in HEK-293T cell line for 1 h and 3 h post-transfection. Immunization with E7/Pep-1 nanoparticles at a ratio of 1:20 induced a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein in a murine tumor model. These data suggest that Pep-1 peptide would indicate promising applications for improvement of HPV therapeutic vaccines. © 2016 IUBMB Life, 68(6):459-467, 2016., (© 2016 International Union of Biochemistry and Molecular Biology.)

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides  mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated allogeneic mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress and apoptotic death in the rat PC12 cells. The remaining cells exhibited a greater capability of neurite outgrowth. Furthermore, allogeneic and xenogeneic transplantation of peptide-labeled mitochondria after 3 months improved the locomotive activity in the PD rats. This increase was accompanied by a marked decrease in dopaminergic neuron loss in the substantia nigra pars compacta (SNc) and consistent enhancement of tyrosine hydroxylase-positive immunoreaction of dopaminergic neurons in the SNc and striatum. We also observed that in the SN dopaminergic neuron in the treated PD rats, mitochondrial complex I protein and mitochondrial dynamics were restored, thus ameliorating the oxidative DNA damage. Moreover, we determined signal translocation of graft allogeneic mitochondria from the MFB to the calbindin-positive SN neuron, which demonstrated the regulatory role of mitochondrial transport in alleviating 6-OHDA-induced degeneration of dopaminergic neurons., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Glial cell line-derived neurotrophic factor (GDNF) is a potential therapeutic protein on a variety of central nervous system diseases including ischemic stroke. However, GDNF is a large molecule that cannot cross the blood-brain barrier (BBB), which is still intact in the early hours after stroke when neural rescue is possible. PEP-1 protein transduction domain can deliver protein cargo across the cell membrane and the BBB. In the present study, we generated a novel fusion protein PEP-1-GDNF and examined whether PEP-1-GDNF is protective in focal cerebral ischemia. PEP-1-GDNF (200 μg/kg) or PBS was intravenously applied over 5 min immediately after reperfusion of 90 min transient middle cerebral artery occlusion (MCAO). After 28 days, rats were deeply anesthetized and decapitated. Behavioral tests were performed during this period. The results showed that PEP-1-GDNF significantly reduced the infarct volume and improved behavioral function. Further, PEP-1-GDNF promoted the cell proliferation and differentiation in the dentate gyrus of the hippocampus and attenuated ischemia-induced learning and memory damage., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

To overcome the major disadvantages of cysteamine, the only registered treatment for the rare genetic disease cystinosis, nine prodrugs of γ-glutamyl-cysteamine (4) were synthesized for evaluation. Esterification of the thiol conferred oxidative stability, while sufficient lipophilicity for oral bioavailability was achieved by acylation of the α-carboxyl group of γ-glutamyl-cysteamine (4). Low cytotoxicity was observed in cultured HaCaT keratinocytes using the MTT assay, with EC50 values higher than or similar to that of cysteamine. Successful uptake of the esterified prodrugs and the subsequent release of cysteamine into cultured human proximal tubule epithelial cells were demonstrated using CMQT derivatisation and HPLC with UV detection. These prodrugs show potential as novel delivery vehicles of cysteamine to improve the treatment of the genetic disorder nephropathic cystinosis., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50=5.7±0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Administration of macromolecule compositions in medicine and cosmetics always exhibited low bioavailability due to the limitation of transmembrane transport. Here, human epidermal growth factor (hEGF) was fused with glutathione S-transferase (GST) and Pep-1, the first commercial cell-penetrating peptide, in Escherichia coli. The fusion protein was firstly purified with the affinity chromatography, and then the GST tag was released by TEV protease. Final purification was achieved by the ion exchange chromatography. The biological activities and the transmembrane ability of the obtained products were determined using scratch wound-healing assay, MTT analysis, and immunofluorescence assay. The results showed that both rhEGF and Pep-1-fused hEGF were soluble expressed in E. coli. The fusion of Pep-1 could markedly increase the transmembrane ability of EGF, whereas it did not interfere with the growth-stimulating and migration-promoting functions of hEGF on fibroblasts. This research provided a novel strategy for the transmembrane transport of protein-derived cosmetics or drugs.

SIRT2 is a member of the mammalian sirtuin protein family, primarily found in the cytoplasm. It regulates numerous cellular processes including aging, DNA repair, cell cycle, and survival under stress conditions. However, the biological function and mechanism of the SIRT2 protein was not well understood in normal cells such as primary chondrocytes. In this study, we examined the effects of SIRT2 on differentiation and inflammation in rabbit articular chondrocytes by using a cell-permeative PEP-1-SIRT2 protein. Our results indicate that PEP-1-SIRT2-induced a loss of type II collagen and decreased sulfate proteoglycan levels in a dose- and time-dependent manner, as examined by Western blotting, alcian blue staining, and immunohistochemistry. Furthermore, PEP-1-SIRT2 caused an inflammatory response by inducing the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). In addition, after treatment with PEP-1-SIRT2, phosphorylation of both p38 and ERK was observed. Inhibition of ERK with PD98059 (PD) suppressed PEP-1-SIRT2-induced dedifferentiation and COX-2 expression. Reduction in PEP-1-SIRT2-induced inflammatory response was observed upon inhibition of p38 by SB203580 (SB). The same pattern was demonstrated in PEP-1-SIRT2-induced dedifferentiation and inflammatory response during culture with serial passages. During expansion to four passages, levels of type II collagen decreased, whereas levels of COX-2 and SIRT2 increased and activated ERK and p38. Furthermore, PEP-1-SIRT2 enhances dedifferentiation through the ERK pathway and inflammatory response through the ERK and p38 pathways in rabbit chondrocytes in vitro. These findings suggest that PEP-1-SIRT2 induces dedifferentiation via the ERK pathway and inflammation through the p38 and ERK pathways in rabbit articular chondrocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

As a novel carrier for folate receptor (FR)-targeted intracellular delivery, we designed two types of targetable liposomal systems using Pep-1 peptide (Pep1) and folic acid as a cell-penetrating peptide (CPP) and target molecule, respectively. Folate-linked Pep1 (Fol-Pep1) was synthesized by solid phase peptide synthesis (SPPS) and verified using (1)H NMR and far-ultraviolet (UV) circular dichroism (CD). The chimeric ligand (Fol-Pep1)-modified liposome (cF-P-L) was prepared by coupling Fol-Pep1 to maleimide-derivatized liposomes at various ratios. The dual ligand (folate and Pep1)-modified liposome (dF/P-L) was prepared by separately attaching both ligands to the liposomal surface via a short (PEG2000) or long (PEG3400) linker. The physical and conformational characteristics including vesicle size, zeta potential, and the number of conjugated ligands were determined. Intracellular uptake specificities of various fluorescent probe-containing cF-P-L and dF/P-L systems were assessed using FR-positive HeLa and FR-negative HaCaT cells. Cellular uptake behavior was visualized by confocal laser scanning microscopy (CLSM). Internalization was time-dependent. Fol-Pep1 and Pep-1 cytotoxicities were negligible up to 25 μM in FR-positive and FR-negative cells. Empty cF-P-L and dF/P-L were nontoxic at the concentration used. The optimized dF3/P2(450/90) system carrying 450 PEG3400-linked folate and 90 PEG2000-linked Pep1 molecules could be a good candidate for FR-specific intracellular drug delivery.

Skin sensitization is an important toxicological end-point in the risk assessment of chemical allergens. Because of the complexity of the biological mechanisms associated with skin sensitization, integrated approaches combining different chemical, biological and in silico methods are recommended to replace conventional animal tests. Chemical methods are intended to characterize the potential of a sensitizer to induce earlier molecular initiating events. The presence of an electrophilic mechanistic domain is considered one of the essential chemical features to covalently bind to the biological target and induce further haptenation processes. Current in chemico assays rely on the quantification of unreacted model nucleophiles after incubation with the candidate sensitizer. In the current study, a new fluorescence-based method, 'HTS-DCYA assay', is proposed. The assay aims at the identification of reactive electrophiles based on their chemical reactivity toward a model fluorescent thiol. The reaction workflow enabled the development of a High Throughput Screening (HTS) method to directly quantify the reaction adducts. The reaction conditions have been optimized to minimize solubility issues, oxidative side reactions and increase the throughput of the assay while minimizing the reaction time, which are common issues with existing methods. Thirty-six chemicals previously classified with LLNA, DPRA or KeratinoSens™ were tested as a proof of concept. Preliminary results gave an estimated 82% accuracy, 78% sensitivity, 90% specificity, comparable to other in chemico methods such as Cys-DPRA. In addition to validated chemicals, six natural products were analyzed and a prediction of their sensitization potential is presented for the first time., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Oxidative stress is closely associated with cardiac fibrosis. However, the effect of copper, zinc-superoxide dismutase (SOD1) as a therapeutic agent is limited due to the insufficient transduction. This study was aimed to investigate the effect of PEP-1-SOD1 fusion protein on angiotensin II (ANG II)-induced collagen metabolism in rat cardiac myofibroblasts (MCFs)., Methods: MCFs were pretreated with SOD1 or PEP-1-SOD1 fusion protein for 2 h followed by incubation with ANG II for 24 h. Cell proliferation was measured by Cell Counting Kit-8. Superoxide anion productions were detected by both fluorescent microscopy and Flow Cytometry. MMP-1 and TIMP-1 were determined by ELISA. Intracellular MDA content and SOD activity were examined by commercial assay kits. Protein expression was analyzed by western blotting., Results: PEP-1-SOD1 fusion protein efficiently transduced into MCF, scavenged intracellular O2 (-), decreased intracellular MDA content, increased SOD activity, suppressed ANG II-induced proliferation, reduced expression of TGF-β1, α-SMA, collagen type I and III, restored MMP-1 secretion, and attenuated TIMP-1 secretion., Conclusion: PEP-1-SOD1 suppressed MCF proliferation and differentiation and reduced production of collagen type I and III. Therefore, PEP-1-SOD1 fusion protein may be a potential novel therapeutic agent for cardiac fibrosis.

Background: Methionine sulfoxide reductase A (MsrA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. However, therapeutic use of exogenous MsrA in oxidative stress-induced diseases is limited, because it cannot enter the cells. The aim of this study is to investigate whether MsrA with PEP-1, a cell penetrating peptide, fused to its N-terminus can protect against oxidative stress in macrophages and can attenuate atherosclerosis in apolipoprotein E deficient (apoE(-/-)) mice., Methods: MsrA and the fusion protein PEP-1-MsrA were expressed and purified using a pET28a expression system. Transduction of the fusion protein into macrophages was confirmed by Western blot and immunofluorescence staining. Intracellular reactive oxygen species (ROS) and apoptosis levels were measured by flow cytometry. In in vivo study, MsrA or PEP-1-MsrA proteins were intraperitoneally injected into apoE(-/-) mice fed a Western diet for 12 weeks. Plasma lipids levels, inflammatory gene expression, and paraoxonase-1 (PON1) and superoxide dismutase (SOD) activities were assessed. Atherosclerotic lesions were analyzed by Oil Red O staining and immunohistochemistry., Results: PEP-1-MsrA could penetrate the cells and significantly reduced intracellular ROS levels and apoptosis in H2O2-treated macrophages. It also decreased TNFα and IL-1β mRNA levels and increased the IL-10 mRNA level in lipopolysaccharide-treated macrophages. In in vivo study, PEP-1-MsrA injection significantly increased plasma PON1 and SOD activities and decreased plasma monocyte chemoattractant protein 1 (MCP-1) level compared to the injection of vehicle control or MsrA. In PEP-1-MsrA injected mice, hepatic PON1 levels were increased, while the expression of TNFα and IL-6 mRNA in the liver was suppressed. Although plasma total cholesterol and triglyceride levels did not change, the aortic atherosclerosis in PEP-1-MsrA treated mice was significantly reduced. This was accompanied by a reduction of total and apoptotic macrophages in the lesions., Conclusion: Our study provides evidence that PEP-1-MsrA may be a potential therapeutic agent for atherosclerosis-related cardiovascular diseases.

The selective inhibition of intracellular and nuclear molecules such as Ki-67 holds great promise for the treatment of cancer and other diseases. However, the choice of the target protein and the intracellular delivery of the functional agent remain crucial challenges. Main hurdles are (a) an effective delivery into cells, (b) endosomal escape of the delivered agents, and (c) an effective, externally triggered destruction of cells. Here we show a light-controlled two-step approach for selective cellular delivery and cell elimination of proliferating cells. Three different cell-penetrating nano constructs, including liposomes, conjugates with the nuclear localization sequence (NLS), and conjugates with the cell penetrating peptide Pep-1, delivered the light activatable antibody conjugate TuBB-9-FITC, which targets the proliferation associated protein Ki-67. HeLa cells were treated with the photosensitizer benzoporphyrin monoacid derivative (BPD) and the antibody constructs. In the first optically controlled step, activation of BPD at 690 nm triggered a controlled endosomal escape of the TuBB-9-FITC constructs. In more than 75% of Ki-67 positive, irradiated cells TuBB-9-FITC antibodies relocated within 24 h from cytoplasmic organelles to the cell nucleus and bound to Ki-67. After a second light irradiation at 490 nm, which activated FITC, cell viability decreased to approximately 13%. Our study shows an effective targeting strategy, which uses light-controlled endosomal escape and the light inactivation of Ki-67 for cell elimination. The fact that liposomal or peptide-assisted delivery give similar results leads to the additional conclusion that an effective mechanism for endosomal escape leaves greater variability for the choice of the delivery agent.

The heterologous epitope-peptide from different viruses may represent an attractive candidate vaccine. In order to evaluate the role of cell-permeable peptide (PEP-1) and Ii-Key moiety from the invariant chain (Ii) of MHC on the heterologous peptide chimeras, we linked the two vehicles to hybrid epitopes on the VP2 protein (aa197-209) of the infectious bursal disease virus and HN protein (aa345-353) of the Newcastle disease virus. The chimeric vaccines were prepared and injected into mice. The immune effects were measured by indirect ELISA. The results showed that the vehicle(s) could significantly boost immune effects against the heterologous epitope peptide. The Ii-Key-only carrier induced more effective immunological responses, compared with the PEP-1 and Ii-Key hybrid vehicle. The carrier-peptide hybrids all showed strong colocalization with major histocompatibility complex (MHC) class II molecules compared with the epitope-peptide (weakly-binding) after co-transfection into 293T cells. Together, our results lay the groundwork for designing new hybrid vaccines based on Ii-Key and/or PEP-1 peptides., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression.

Cationic cell-penetrating peptides have been widely used to enhance the intracellular delivery of various types of cargoes, such as drugs and proteins. These reagents are chemically similar to the multi-basic peptides that are known to be potent proprotein convertase inhibitors. Here, we report that both HIV-1 TAT47-57 peptide and the Chariot reagent are micromolar inhibitors of furin activity in vitro. In agreement, HIV-1 TAT47-57 reduced HT1080 cell migration, thought to be mediated by proprotein convertases, by 25%. In addition, cyclic polyarginine peptides containing hydrophobic moieties which have been previously used as transfection reagents also exhibited potent furin inhibition in vitro and also inhibited intracellular convertases. Our finding that cationic cell-penetrating peptides exert potent effects on cellular convertase activity should be taken into account when biological effects are assessed.

Ketoreductases (KRs) from modular polyketide synthases (PKSs) can perform stereospecific catalysis, selecting a polyketide with a D- or L-α-methyl substituent for NADPH-mediated reduction. In this report, molecular dynamics (MD) simulations were performed to investigate the interactions that control stereospecificity. We studied the A1-type KR from the second module of the amphotericin PKS (A1), which is known to be stereospecific for a D-α-methyl-substituted diketide substrate (dkD). MD simulations of two ternary complexes comprised of the enzyme, NADPH, and either the correct substrate, dkD, or its enantiomer (dkL) were performed. The coordinates for the A1/NADPH binary complex were obtained from a crystal structure (PDB entry 3MJS), and substrates were modeled in the binding pocket in conformations appropriate for reduction. Simulations were intended to reproduce the initial weak binding of the polyketide substrate to the enzyme. Long (tens of nanoseconds) MD simulations show that the correct substrate is retained in a conformation closer to the reactive configuration. Many short (up to a nanosecond) MD runs starting from the initial structures display evidence that Q364, three residues N-terminal to the catalytic tyrosine, forms a hydrogen bond to the incorrect dkL substrate to yield an unreactive conformation that is more favorable than the reactive configuration. This interaction is not as strong for dkD, as the D-α-methyl substituent is positioned between the glutamine and the reactive site. This result correlates with experimental findings [Zheng, J., et al. (2010) Structure 18, 913-922] in which a Q364H mutant was observed to lose stereospecificity.

Metals are essential components of all living cells, and in many cases cells trigger and utilize dynamic metal movements for signaling purposes. So, it is very critical to develop the biosensors for determination of metal ions in living systems with high selectivity and accuracy. In this work, taking Cu(2+) as a model, an accurate and selective ratiometric electrochemical biosensor was developed. First, the specific molecule, 2,2',2″-(2,2',2″-nitrilotris(ethane-2,1-diyl)-tris((pyridin-2-ylmethyl)azanediyl)triethanethiol (TPAASH), was designed and synthesized for specific recognition of Cu(2+). Meanwhile, electroactive molecule, 6-(ferrocenyl)hexanethiol (FcHT) was coimmobilized with TPAASH at one electrode as inner reference molecule to provide a built-in correction for avoiding the environmental effects. Thus, the developed biosensor demonstrated high accuracy and remarkable selectivity toward Cu(2+) against other metal ions, amino acids, and so on. In addition, the biosensor also showed high sensitivity due to the electrocatalytic activity of the nanostructured gold flowers. As a result, the present ratiometric electrochemical biosensor was successfully applied in detection of Cu(2+) in brain microdialysates of normal rat brain and that followed by global cerebral ischemia.

Introduction: A lipiodol solution of (188)Re-4-hexadecyl-2,2,9,9-tetramethyl-4,7-diaza-1,10-decanedithiol (HTDD) has been successfully developed for liver cancer therapy; however, its preparation requires a multi-step synthesis and it is characterized by a low labeling yield., Methods: We synthesized a new compound, 4-hexadecyl-4,7-diaza-1,10-decanedithioacetate (AHDD), without gem dimethyl groups to address these issues. AHDD was formulated into a kit and was labeled with (188)Re. Biodistribution study was performed using normal BALB/c mice., Results: The kit was labeled with (188)Re with a high efficiency (98.8±0.2%). After extraction with lipiodol, the overall yield of (188)Re-HDD/lipiodol was as high as 90.2±2.6%. A comparative biodistribution study of (188)Re-HTDD and (188)Re-HDD was performed in normal mice after intravenous injection. The lungs were identified as the main uptake site due to capillary-blockage. (188)Re-HDD/lipiodol showed a significantly higher lung uptake than that of (188)Re-HTDD/lipiodol (p<0.05)., Conclusion: The newly synthesized (188)Re-HDD/lipiodol showed improved radiolabeling yield and biodistribution results compared to (188)Re-HTDD/lipiodol, and may therefore be more suitable for liver cancer therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)