Cell-penetrating Peptide Pep-1 Research Articles

Effects of Substituting Disubstituted Amino Acids into the Amphipathic Cell Penetrating Peptide Pep-1.

Amphipathic cell-penetrating peptides based on the pep-1 sequence were synthesized by replacing the three hydrophilic glutamic acid residues with disubstituted, non-proteinogenic, hydrophobic amino acids. These substitutions facilitated maintenance of the peptides' secondary structure in a helical conformation, even in aqueous solution. Stability against enzymatic degradation was improved through the use of disubstituted amino acids. The resultant peptides exhibited high membrane permeability that remained relatively stable during prolonged incubation times. The results of this study indicate that the use of non-proteinogenic amino acids may be an effective approach to improve the cell membrane permeability for existing amphiphilic peptides.

Insight into the Mechanism of Internalization of the Cell-Penetrating Carrier Peptide Pep-1 by Conformational Analysis.

Different secondary structures of the pep-1 protein were blamed for transmembrane internalization process of drugs and drug deliveries. But which structure will be important for transmembrane delivery was still not clear. In this study, interactions between pep-1 and cell membranes were studied. Pep-1 in the buffer (Pep-1) and pep-1 on graphene (PDS/G) or they on graphene oxide (PDS/GO) were composed as the transmembrane delivery system to study the different secondary structure of pep-1 that influence for their transmembrane delivery. The curves of chirascan circular dichroism (CD) and all-atom discontinuous molecular dynamics (DMD) simulations illuminate that, in a buffer environment, most pep-1 formed 3-10 helix structures. Meanwhile, when Pep-1 composed graphene slice and formed PDS/G, 3-10 helix and alpha-helix structures can be found in small quantities. When they on graphene oxide and formed PDS/GO, coil or type II beta-turn structure can be found from most of the pep-1 and 3-10 helix structure disappeared. By using sum-frequency generation (SFG) vibrational spectroscopy, we found that pep-1 with 3-10 helix structures in buffer solutions damaged the lipid bilayer violently. PDS/G with less 3-10 helix structures will change the orientation of lipid bilayer effectively but slightly. Pep-1 with coil or type II Beta-turn in PDS/GO cannot influence the structure of lipid bilayers. Hemolysis experiments also proved that when pep-1 composed as PDS/G, they will change the orientation of the plasma membrane of red blood cells effectively but slightly. When they attach on the GO and formed PDS/GO, the plasma membrane of red blood cells cannot be influenced. In conclusion, 3-10 helix structures will be positively correlated with disturbance of membranes. These results will be effectively guided the clinic application of pep-1 as a transporter of the drug delivery system.

Mitochondrial transplantation regulates antitumour activity, chemoresistance and mitochondrial dynamics in breast cancer

BackgroundThe transfer of whole mitochondria that occurs during cell contact has been found to support cancer progression. However, the regulatory role of mitochondria alone is difficult to elucidate due to the complex microenvironment. Currently, mitochondrial transplantation is an available approach for restoring mitochondrial function in mitochondrial diseases but remains unclear in breast cancer. Herein, effects of mitochondrial transplantation via different approaches in breast cancer were investigated.MethodsWhole mitochondria (approximately 10.5 μg/ml) were transported into MCF-7 breast cancer cells via passive uptake or Pep-1-mediated delivery. Fresh mitochondria isolated from homeoplasmic 143B osteosarcoma cybrids containing mitochondrial DNA (mtDNA) derived from health individuals (Mito) or mtDNA with the A8344G mutation (Mito8344) were conjugated with cell-penetrating peptide Pep-1 (P-Mito) or not conjugated prior to cell co-culture. Before isolation, mitochondria were stained with MitoTracker dye as the tracking label. After 3 days of treatment, cell viability, proliferation, oxidative stress, drug sensitivity to Doxorubicin/Paclitaxel and mitochondrial function were assessed.ResultsCompared with P-Mito, a small portion of Mito adhered to the cell membrane, and this was accompanied by a slightly lower fluorescent signal by foreign mitochondria in MCF-7 cells. Both transplantations induced cell apoptosis by increasing the nuclear translocation of apoptosis-inducing factor; inhibited cell growth and decreased oxidative stress in MCF-7 cells; and increased the cellular susceptibility of both the MCF-7 and MDA-MB-231 cell lines to Doxorubicin and Paclitaxel. Mitochondrial transplantation also consistently decreased Drp-1, which resulted in an enhancement of the tubular mitochondrial network, but a distinct machinery through the increase of parkin and mitochondrial fusion proteins was observed in the Mito and P-Mito groups, respectively. Furthermore, although there were no differences in energy metabolism after transplantation of normal mitochondria, metabolism was switched to the energetic and glycolytic phenotypes when the mitochondria were replaced with dysfunctional mitochondria, namely, Mito8344 and P-Mito8344, due to dramatically induced glycolysis and reduced mitochondrial respiration, respectively. Consequently, transplant-induced growth inhibition was abolished, and cell growth in the Mito8344 group was even higher than that in the control group.ConclusionThis study reveals the antitumour potential of mitochondrial transplantation in breast cancer via distinct regulation of mitochondrial function.

Vesicles mimicking normal and cancer cell membranes exhibit differential responses to the cell-penetrating peptide Pep-1

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.

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Attenuates Lung Injury in Septic Shock Rats.

Oxidative stress and inflammation have been identified to play a vital role in the pathogenesis of lung injury induced by septic shock. Heme oxygenase-1 (HO-1), an effective antioxidant and anti-inflammatory and antiapoptotic substance, has been used for the treatment of heart, lung, and liver diseases. Thus, we postulated that administration of exogenous HO-1 protein transduced by cell-penetrating peptide PEP-1 has a protective role against septic shock-induced lung injury. Septic shock produced by cecal ligation and puncture caused severe lung damage, manifested in the increase in the lung wet/dry ratio, oxidative stress, inflammation, and apoptosis. However, these changes were reversed by treatment with the PEP-1-HO-1 fusion protein, whereas lung injury in septic shock rats was alleviated. Furthermore, the septic shock upregulated the expression of Toll-like receptor 4 (TLR4) and transcription factor NF-κB, accompanied by the increase of lung injury. Administration of PEP-1-HO-1 fusion protein reversed septic shock-induced lung injury by downregulating the expression of TLR4 and NF-κB. Our study indicates that treatment with HO-1 protein transduced by PEP-1 confers protection against septic shock-induced lung injury by its antioxidant, anti-inflammatory, and antiapoptotic effects.

Calorimetric and Spectroscopic Studies of the Effects of the Cell Penetrating Peptide Pep-1 and the Antimicrobial Peptide Combi-2 on Vesicles Mimicking Escherichia coli Membrane.

The objective of this study is to measure and compare the effects of the cell penetrating peptide (CPP) Pep-1 and the antimicrobial peptide (AMP) combi-2 on vesicles of membranes mimicking Escherichia coli (E. coli). To characterize the effects of Pep-1 and combi-2 on E. coli membrane vesicles, a combination of five biophysical techniques was employed: fluorescence, infrared, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Upon addition of E. coli membranes, tryptophan fluorescence intensity of Pep-1 showed a sudden blue-shift and decreased in a nonconcentration-dependent manner while the intensity of combi-2 decreased in a concentration-dependent manner, most significantly for a very low peptide-to-lipid ratio of 1:40. Complexes of Pep-1 and combi-2 with E. coli membrane mimicking vesicles having shown a significant blue-shift in fluorescence intensity were then prepared and studied in freeze-dried states. IR results indicate that Pep-1 and combi-2 adopt a major 310-helix structure in the presence of E. coli membrane mimicking vesicles at low peptide concentration. Pep-1 and combi-2 have a similar effect on E. coli membrane mimicking vesicles at low concentration even though combi-2 is in the interfacial region of the bilayer while Pep-1 is located between the interfacial region and the hydrophobic region. Combi-2 at low concentration acts as a CPP. TGA and DSC results reveal that combi-2 has a stabilizing effect on E. coli at any concentration while Pep-1 stabilizes the E. coli membrane only at high concentration. Both peptides show a preferential interaction with one of the anionic lipids leading to clustering in E. coli membrane. SEM images reveal that Pep-1 and combi-2 form superstructures including fibrils in the presence of E. coli membrane mimicking vesicles. Calorimetric and spectroscopic techniques may be used in a complementary way with imaging techniques to gain more insights into peptide-lipid interactions.

Effect of heme oxygenase-1 transduced by cell penetrating peptide PEP-1 on renal injury in rats with acute paraquat poisoning

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.

Light-Controlled Delivery of Monoclonal Antibodies for Targeted Photoinactivation of Ki-67.

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.

Transduced PEP-1-Heme Oxygenase-1 Fusion Protein Reduces Remote Organ Injury Induced by Intestinal Ischemia/Reperfusion

BackgroundA fusion protein composed of heme oxygenase-1 (HO-1) and cell-penetrating peptide PEP-1 has been shown to reduce local intestinal injury after intestinal ischemia/reperfusion (I/R). In this study, we investigated the effects of PEP-1-HO-1 fusion protein on remote organ injury induced by intestinal I/R in rats.Material/methodsWe randomly assigned 24 male Sprague-Dawley rats to 3 groups: Sham, I/R, and I/R plus PEP-1-HO-1 treatment (HO). The model of intestinal I/R was established by occluding the superior mesenteric artery for 45 min followed by 120-min reperfusion. In HO group, PEP-1-HO-1 was administered intravenously 30 min before ischemia, while animals in the Sham and I/R groups received the equal volume of physiological saline. At the end of the experiment, lung, liver, and blood samples were collected and analyzed.ResultsMalondialdehyde levels and histological injury scores were increased, and superoxide dismutase activities were decreased in the lung and liver tissues in the I/R group compared with the Sham group (P<0.05). Serum levels of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, interleukin-6, and lung tissue wet weight to dry weight ratio were increased in the I/R group compared with the Sham group (P<0.05). NF-κB expression in intestinal tissues was significantly higher in the I/R group than in the Sham group. These changes were significantly reversed by treatment with PEP-1-HO-1.ConclusionsThis study demonstrates that administration of PEP-1-HO-1 has a protective role against lung and liver injury after intestinal I/R, attributable to the reduction of released proinflammatory cytokines regulated by NF-κB.

Treatment of human cells derived from MERRF syndrome by peptide-mediated mitochondrial delivery

Background aimsThe feasibility of delivering mitochondria using the cell-penetrating peptide Pep-1 for the treatment of MERRF (myoclonic epilepsy with ragged red fibers) syndrome, which is caused by point mutations in the transfer RNA genes of mitochondrial DNA, is examined further using cellular models derived from patients with MERRF syndrome. MethodsHomogenesis of mitochondria (wild-type mitochondria) isolated from normal donor cells with about 83.5% preserved activity were delivered into MERRF fibroblasts by Pep-1 conjugation (Pep-1-Mito). ResultsDelivered doses of 52.5 μg and 105 μg Pep-1-Mito had better delivered efficiency and mitochondrial biogenesis after 15 days of treatment. The recovery of mitochondrial function in deficient cells receiving 3 days of treatment with peptide-mediated mitochondrial delivery was comprehensively demonstrated by restoration of oxidative phosphorylation subunits (complex I, III and IV), mitochondrial membrane potential, adenosine triphosphate synthesis and reduction of reactive oxygen species production. The benefits of enhanced mitochondrial regulation depended on the function of foreign mitochondria and not the existence of mitochondrial DNA and can be maintained for at least 21 days with dramatically elongated mitochondrial morphology. In contrast to delivery of wild-type mitochondria, the specific regulation of Pep-1-Mito during MERRF syndrome progression in cells treated with mutant mitochondria was reflected by the opposite performance, with increase in reactive oxygen species production and matrix metalloproteinase activity. ConclusionsThe present study further illustrates the feasibility of mitochondrial intervention therapy using the novel approach of peptide-mediated mitochondrial delivery and the benefit resulting from mitochondria-organelle manipulation.

Transduction of PEP-1–Heme Oxygenase-1 Fusion Protein Reduces Myocardial Ischemia/Reperfusion Injury in Rats

Recent studies have uncovered that overexpression of heme oxygenase-1 (HO-1) by induction or gene transfer provides myocardial protection. In the present study, we investigated whether HO-1 protein mediated by cell-penetrating peptide PEP-1 could confer cardioprotection in a rat model of myocardial ischemia/reperfusion (I/R) injury. Male Sprague-Dawley rats were subjected to 30 minutes of ischemia by occluding the left anterior descending coronary artery and to 120 minutes of reperfusion to prepare the model of I/R. Animals were randomized to receive PEP-1-HO-1 fusion protein or saline 30 minutes before a 30-minute occlusion. I/R increased myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and reduced myocardial superoxide dismutase activity. Administration of PEP-1-HO-1 reduced myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and increased myocardial superoxide dismutase and HO-1 activities. His-probe protein was only detected in PEP-1-HO-1-transduced hearts. In addition, transduction of PEP-1-HO-1 markedly reduced elevated myocardial tissue nuclear factor-κB induced by I/R. The results suggested that transduction of PEP-1-HO-1 fusion protein decreased myocardial reperfusion injury, probably by attenuating the production of oxidants and proinflammatory cytokines regulated by nuclear factor-κB.

Effects of heme oxygenase-1 transduced by cell penetrating peptide PEP-1 on renal ischemia/reperfusion injury in rats

Objective To investigate the effects of heme oxygenase-1 (HO-1) transduced by cell penetrating peptide PEP-1 on renal ischemia/reperfusion (I/R) injury in rats.Methods Eighteen male Sprague-Dawley rats,aged 7-9 weeks,weighing 210-260 g,were randomly divided into 3 groups (n =6 each):sham operation group (group S),renal I/R injury group (group I/R) and fusion protein PEP-1/HO-I + I/R group (group HO).I/R injury was produced by occluding bilateral renal arteries for 45 min followed by reperfusion for 6 h.The fusion protein PEP-1/HO-1 was injected via the left iliac vein 30 min prior to ischemia in group HO.Bilateral renal arteries were only exposed but not occluded in group C.Blood samples were collected from the right common carotid artery at 6 h of reperfusion for determination of serum blood urea nitrogen (BUN) and creatinine (Cr) concentrations.The malondialdehyde (MDA) content,superoxide dismutase (SOD) activity and HO-1 expression in renal tissues were measured.Results Compared with group S,the levels of MDA,serum BUN and Cr were significantly increased,the SOD activity was decreased,and HO-1 expression was up-regulated in groups I/R and HO (P ＜0.05).Compared with group I/R,the levels of MDA,serum BUN and Cr were significantly decreased,the SOD activity was increased,and HO-1 expression was up-regulated in group HO (P ＜ 0.05).Conclusion HO-1 protein can be successfully transduced into renal tissues by PEP-1 and transduced HO-1 protein reduces renal I/R injury by inhibiting lipid peroxidation response. Key words: Heme oxygenase (decyclizing) ; Reperfusion injury; Kidney; Cell penetrating peptide

Functional Recovery of Human Cells Harbouring the Mitochondrial DNA Mutation MERRF A8344G via Peptide-Mediated Mitochondrial Delivery

We explored the feasibility of mitochondrial therapy using the cell-penetrating peptide Pep-1 to transfer mitochondrial DNA (mtDNA) between cells and rescue a cybrid cell model of the mitochondrial disease myoclonic epilepsy with ragged-red fibres (MERRF) syndrome. Pep-1-conjugated wild-type mitochondria isolated from parent cybrid cells incorporating a mitochondria-specific tag were used as donors for mitochondrial delivery into MERRF cybrid cells (MitoB2) and mtDNA-depleted Rho-zero cells (Mitoρ°). Forty-eight hours later, translocation of Pep-1-labelled mitochondria into the mitochondrial regions of MitoB2 and Mitoρ° host cells was observed (delivery efficiencies of 77.48 and 82.96%, respectively). These internalized mitochondria were maintained for at least 15 days in both cell types and were accompanied by mitochondrial function recovery and cell survival by preventing mitochondria-dependent cell death. Mitochondrial homeostasis analyses showed that peptide-mediated mitochondrial delivery (PMD) also increased mitochondrial biogenesis in both cell types, but through distinct regulatory pathways involving mitochondrial dynamics. Dramatic decreases in mitofusin-2 (MFN2) and dynamin-related protein 1/fission 1 were observed in MitoB2 cells, while Mitoρ° cells showed a significant increase in optic atrophy 1 and MFN2. These findings suggest that PMD can be used as a potential therapeutic intervention for mitochondrial disorders.

Molecular interactions between cell penetrating peptide Pep-1 and model cell membranes.

We investigated the molecular interactions of a cell penetrating peptide (CPP) Pep-1 with model cell membranes using sum frequency generation (SFG) vibrational spectroscopy, supplemented by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Hydrogenated and deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG and dDPPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) were used in the experiments to represent gel-phase and fluid-phase lipid bilayers, respectively. Our SFG results indicated that Pep-1 molecules adopted a β-sheet conformation when adsorbed to the surface of gel-phase DPPG lipid bilayers. When interacting with fluid-phase POPG lipid bilayers, Pep-1 adopted a mix of α-helical and β-sheet structures over a broad range of peptide concentrations. The orientation distribution of the α-helical Pep-1 segment associated with the fluid-phase bilayers was found to depend on the peptide concentration. SFG orientation analysis showed that Pep-1 molecules adopted an orientation nearly perpendicular to the plane of the bilayer for peptide concentrations of 0.28 and 1.4 μM. When the Pep-1 concentration was increased to 7.0 μM, combined SFG and ATR-FTIR measurements showed that Pep-1 molecules were associated with the bilayer with a broad orientation distribution. Our results demonstrated that lipid bilayer phase and peptide concentration affect the conformation and orientation of Pep-1 molecules associated with model cell membranes, which is crucial to the translocation process of CPPs. A combination of SFG and ATR-FTIR studies can be used to determine the conformation and orientation of CPPs interacting with model cell membranes in situ.

Effects of heme oxygenase-1 mediated by cell penetrating peptide PEP-1 on intestinal Ischemia/reperfusion injury in rats

Objective To investigate the effects of heme oxygenase-1 (HO-1) mediated by cell penetrating peptide PEP-1 on intestinal ischemia/reperfusion (I/R) injuiy in tats. Methods Eighteen male SD rats aged 7-9 weeks weighing 210-260 g were randomly divided into 3 groups (re = 6 each): sham operation group (group S) , I/R group and PEP-1/HO-1 + I/R group (group HO) . To establish a model of intestinal I/R injury, intestines were exteriorized and the superior mesenteric artery was exposed and occluded for 45 min ischemia, and then the clamp was removed for 120 min reperfusion. The PEP-1/HO-1 fusion protein 0.5 mg was injected via the left iliac vein 30 min prior to ischemia in group HO. The superior mesenteric artery was exposed but not occluded in group S. At the end of reperfusion, the rats were sacrificed and intestinal tissues obtained to determine the intestinal wet/ dry ratio, malondialdehyde (MDA) level, activities of superoxide dismutase (SOD) and HO-1, and HO-1 protein expression. The histological changes in the intestinal mucosa were examined and the injuiy was scored. Results Compared with group S, the intestinal wet/dry ratio, MDA level, HO-1 activity, HO-1 protein expression and injury score were significantly increased, while the SOD activity was significantly decreased in groups I/R and HO ( P ＜ 0.05) . Compared with group I/R, the intestinal wet/dry ratio, MDA level and injury score were significantly decreased, while the SOD activity, HO-1 activity and HO-1 protein expression increased in group HO ( P ＜ 0.05) . The pathologic changes were significantly attenuated in group HO compared with group I/R.Conclusion HO-1 protein can be successfully delivered into intestinal tissues by PEP-1 and has protective effects against intestinal I/R injury. Key words: Heme Oxygenase-1; Intestine, small; Reperfusion injury; Cell penetrating peptide

Effects of heme oxygenase-1 mediated by cell penetrating peptide PEP-1 on myocardial ischemia/reperfusion injury in isolated rat hearts

Objective To investigate the protective effects of heme oxygenase-1 （HO-1） mediated by cell penetrating peptide PEP-1 on myocardium against ischemia/reperfusion （IR） injury in isolated rat hearts. Methods Healthy male SD rats weighing 220-280 g were anesthetized with intraperitoneal pentobarbital. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95%O2-5% CO2 at 37 ℃. Eighteen isolated rat hearts were randomly divided into 3 groups （ n = 6 each）： Ⅰ group sham operation （group S）;Ⅱ group IR and Ⅲ group PEP-1/HO-1 ＋ IR （group HO-1）. The isolated rat hearts were perfused with an oxygena-ted （95% O2-5% CO2 ） K-H solution at 37 ℃ in a Langendorff apparatus and were subjected to 40 min of global ischemia followed by 50 min of reperfusion after 30 min of stabilization. In group Ⅲ （group HO- 1 ） the isolated hearts were perfused with 50 μmol/L PEP-1/HO-1 for 15 min before ischemia. After 50 min of reperfusion, HO-1expression, MDA content and SOD activity in myocardial tissues were determined. The activities of creatine kinase （CK） and lactic dehydrogenase （LDH） in coronary effluent fluid were measured. Results The HO- 1 expression was significanfly higher in HO-1 group than in group IR. IR induced significant increase in MDA content and decrease in SOD activity in myocardium and CK and LDH activities in coronary effluent in group Ⅱ compared with group S. PEP-1/HO-1 significantly attenuated IR-induced changes. Conciusion HO-1 mediated by PEP-1 has protective effects on myocardium ngainst IR injury in rats. Key words: Heme oxygenase（decyclizing）; Cell penetrating peptides; Myocardial reperfusion injury

Cell selectivity and mechanism of action of short antimicrobial peptides designed from the cell‐penetrating peptide Pep‐1

Pep-1-K (PK) is a good cell-selective antimicrobial peptide designed from cell-penetrating peptide Pep-1. To develop novel short antimicrobial peptides with higher cell selectivity and shorter length compared with PK, several PK analogs were designed by the deletion, addition and/or substitution of amino acids. Among these analogs, PK-12-KKP (KKPWWKPWWPKWKK) showing the sequence and structure homology with a Trp/Pro-rich natural antimicrobial peptide, indolicidin (IN), displayed a 20-fold higher cell selectivity as compared to IN. Circular dichroism analysis revealed that PK-12-KKP adopts a folded structure combined with some portions of unordered structure. PK-12-KKP selectively binds to negatively charged bacterial membrane-mimetic vesicles, and its high phospholipid selectivity corresponds well with its high cell selectivity. Moreover, it showed very weak potential in depolarization of the cytoplasmic membrane of Staphylococcus aureus at 8 microM (4x minimal inhibitory concentration) and dye leakage from negatively charged liposomes. These results suggest that the ultimate target of our designed PK-12-KKP maybe the intracellular components (e.g. protein, DNA or RNA) rather than the cytoplasmic membranes. Collectively, our designed short Trp/Pro-rich peptide, PK-12-KKP, appears to be an excellent candidate for future development as a novel antimicrobial agent.

Fluorescent sterols monitor cell penetrating peptide Pep-1 mediated uptake and intracellular targeting of cargo protein in living cells

Although cell-penetrating peptides (CPP) facilitate endocytic uptake of proteins, little is known regarding the extent to which CPPs facilitate protein cargo exit from endocytic vesicles for targeting to other intracellular sites. Since the plasma membrane and less so intracellular membranes contain cholesterol, the fluorescent sterol analogues dansyl-cholestanol (DChol) and dehydroergosterol (DHE) were used to monitor the uptake and intracellular distribution of fluorescent-tagged acyl coenzyme A binding protein (ACBP) into COS-7 cells and rat hepatoma cells. Confocal microscopy colocalized DChol and Texas Red-ACBP (TR-ACBP) with markers for the major endocytosis pathways, especially fluorescent-labeled cholera toxin (marker of ganglioside GM1 in plasma membrane lipid rafts) and dextran (macropinocytosis marker), but less so with transferrin (clathrin-mediated endocytosis marker). These findings were confirmed by multiphoton laser scanning microscopy colocalization of TR-ACBP with DHE (naturally-fluorescent sterol) and by double immunofluorescence labeling of native endogenous ACBP. Serum greatly and Pep-1 further 2.4-fold facilitated uptake of TR-ACBP, but neither altered the relative proportion of TR-ACBP colocalized with membranes/organelles (nearly 80%) vs cytoplasm and/or nucleoplasm (20%). Interestingly, Pep-1 selectively increased TR-ACBP associated with mitochondria while concomitantly decreasing that in endoplasmic reticulum. In summary, fluorescent sterols (DChol, DHE) were useful markers for comparing the distributions of both transported and endogenous proteins. Pep-1 modestly enhanced the translocation and altered the intracellular targeting of exogenous-delivered (TR-ACBP) in living cells.

Gramicidin A-based peptide vector for intracellular protein delivery

The development of the peptide-based vectors for the intracellular delivery of biologically active macromolecules has opened new prospects of their application in research and therapy. Earlier the amphipathic cell-penetrating peptide (CPP) Pep-1 was reported to mediate cellular uptake of proteins without covalent binding to them. In this work we studied the ability of a series of membrane-active amphipathic peptides, based on the gramicidin A sequence, to transport a model protein across the eukaryotic cell membrane. Among them the positively charged Cys-containing peptide P10C demonstrated the most effective β-galactosidase intracellular delivery. Besides, this peptide was shown to form noncovalent associates with β-galactosidase as judged from electrophoresis and enzymatic activity assays. In addition, a series of new gramicidin analogues were prepared and the effect of N-terminus modification of gramicidin on the protein transduction efficiency was studied.

Single-Molecule Imaging of the Association of the Cell-Penetrating Peptide Pep-1 to Model Membranes

Pep-1 is an amphiphatic peptide that can form noncovalent complexes with a cargo protein with subsequent delivery into a live cell. In this study, the behavior of Pep-1 was directly visualized by fluorescent imaging techniques at the single-molecule level of sensitivity. The interactions of Pep-1 and two of its labeled fluorescent analogues with large and cell-sized giant unilamellar vesicles and supported bilayers are reported. The role of the bilayer charge and ionic strength of the medium were examined. Pep-1 caused fusion and association of vesicles, and it perturbed the vesicle's membrane. The association of the peptide with neutral bilayers was promoted by anchoring of the cysteamine moiety. The association of the peptide with the structural defects of the neutral membrane was very efficient. The electrostatic forces were shown to be important for the association of the peptide only in low ionic strength solutions and were completely diminished at physiological ionic strength. Pep-1 did not induce the association to the model membrane of a number of proteins chosen to exhibit a range of properties. The results suggest that Pep-1 assisted delivery of cargo in living cells may result from cooperative effects.