细胞穿透肽:一种新型非病毒载体

细胞膜的选择通透性对维持细胞内环境的稳定起着非常重要的作用,但细胞膜的这种特性限制了一些生物大分子和药物进入细胞内,不利于对一些细胞内疾病的诊断和药物靶向治疗的应用。如何将一些具有诊断和治疗潜力的生物大分子、药物通过细胞膜进入细胞内一直是医学界研究的热点和难点。细胞穿透肽是一类能够携带多肽、蛋白质、核酸、纳米颗粒、病毒颗粒及药物等穿过细胞膜进入细胞,导致完整载物内化的短肽,为生物大分子和药物进入细胞内部提供了有力的运载工具,其作为载体具有的高转导效率和低毒性特点,已经得到了广泛关注和大量研究。目前,细胞穿透肽作为生物分子和药物细胞内化的运载体已经在荧光成像,肿瘤治疗,抗炎治疗及药物靶向治疗中发挥了潜在的诊断和治疗作用,显示出其诱人的应用前景。     

Membrane permeability commonly shared among arginine-rich peptides

Delivery of proteins and other macromolecules using membrane-permeable carrier peptides is a recently developed novel technology, which enables us to modulate cellular functions for biological studies with therapeutic potential. One of the most often used carrier peptides is the arginine-rich basic peptide derived from HIV-1 Tat protein [HIV-1 Tat (48-60)]. Using this peptide, efficient intracellular delivery of molecules including proteins, oligonucleic acids and liposomes has been achieved. We have demonstrated that these features were commonly shared among many arginine-rich peptides such as HIV-1 Rev (34-50) and octaarginine. Not only the linear peptides but also branched-chain peptides showed efficient internalization with an optimum number of arginines (approximately eight residues). The structural and mechanistic features of the translocation of these membrane-permeable arginine-rich peptides are reviewed.     

Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.     

Computational protein design promises to revolutionize protein engineering

Natural evolution has produced an astounding array of proteins that perform the physical and chemical functions required for life on Earth. Although proteins can be reengineered to provide altered or novel functions, the utility of this approach is limited by the difficulty of identifying protein sequences that display the desired properties. Recently, advances in the field of computational protein design (CPD) have shown that molecular simulation can help to predict sequences with new and improved functions. In the past few years, CPD has been used to design protein variants with optimized specificity of binding to DNA, small molecules, peptides, and other proteins. Initial successes in enzyme design highlight CPD's unique ability to design function de novo. The use of CPD for the engineering of potential therapeutic agents has demonstrated its strength in real-life applications.     

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy.

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Intracellular delivery of a protein antigen with an endosomal-releasing polymer enhances CD8 T-cell production and prophylactic vaccine efficacy

Protein-based vaccines have significant potential as infectious disease and anticancer therapeutics, but clinical impact has been limited in some applications by their inability to generate a coordinated cellular immune response. Here, a pH-responsive carrier incorporating poly(propylacrylic acid) (PPAA) was evaluated to test whether improved cytosolic delivery of a protein antigen could enhance CD8+ cytotoxic lymphocyte generation and prophylactic tumor vaccine responses. PPAA was directly conjugated to the model ovalbumin antigen via reducible disulfide linkages and was also tested in a particulate formulation after condensation with cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA). Intracellular trafficking studies revealed that both PPAA-containing formulations were stably internalized and evaded exocytotic pathways, leading to increased intracellular accumulation and potential access to the cytosolic MHC-1 antigen presentation pathway. In an EG.7-OVA mouse tumor protection model, both PPAA-containing carriers robustly inhibited tumor growth and led to an approximately 3.5-fold increase in the longevity of tumor-free survival relative to controls. Mechanistically, this response was attributed to the 8-fold increase in production of ovalbumin-specific CD8+ T-lymphocytes and an 11-fold increase in production of antiovalbumin IgG. Significantly, this is one of the first demonstrated examples of in vivo immunotherapeutic efficacy using soluble protein-polymer conjugates. These results suggest that carriers enhancing cytosolic delivery of protein antigens could lead to more robust CD8+ T-cell response and demonstrate the potential of pH-responsive PPAA-based carriers for therapeutic vaccine applications.     

Staphylococcal surface display of metal-binding polyhistidyl peptides

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni(2+)- and Cd(2+)-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.     

Cell-penetrating peptides: Application in vaccine delivery

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.     

Computational design of peptide ligands

Peptides possess several attractive features when compared to small molecule and protein therapeutics, such as high structural compatibility with target proteins, the ability to disrupt protein-protein interfaces, and small size. Efficient design of high-affinity peptide ligands via rational methods has been a major obstacle to the development of this potential drug class. However, structural insights into the architecture of protein-peptide interfaces have recently culminated in several computational approaches for the rational design of peptides that target proteins. These methods provide a valuable alternative to experimental high-resolution structures of target protein-peptide complexes, bringing closer the dream of in silico designed peptides for therapeutic applications.     

Peptide vaccines and peptide libraries

Synthetic immunogens, containing built-in adjuvanticity, B cell, T helper cell and CTL epitopes or mimotopes, are ideal and invaluable tools to study the immune response with respect to antigen processing and presentation. This serves as a basis for the development of complete and minimal vaccines which do not need large carrier proteins, further adjuvants, liposome formulations or other delivery systems. Combinatorial peptide libraries, either completely random or characterized by one or several defined positions, are useful tools for the identification of the critical features of B cell epitopes and of MHC class I and class II binding natural and synthetic epitopes. The complete activity pattern of an O/Xn library with hundreds of peptide collections, each made up from billions of different peptides, represents the ranking of amino acid residues mediating contact to the target proteins of the immune system. Combinatorial libraries support the design of peptides applicable in vaccination against infectious agents as well as therapeutic tumour vaccines. Using the principle of lipopeptide vaccines, strong humoral and cellular immune responses could be elicited. The lipopeptide vaccines are heat-stable, non-toxic, fully biodegradable and can be prepared on the basis of minimized epitopes by modern methods of multiple peptide synthesis. The lipopeptides activate the antigen-presenting macrophages and B cells and have been recently shown to stimulate innate immunity by specific interaction with receptors of the Toll family.     

Manufacturing of recombinant therapeutic proteins in microbial systems

Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.     

Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

Antimicrobial peptides (AMPs) are regarded as attractive alternatives to conventional antibiotics, but their production in microbes remains challenging due to their inherent bactericidal nature. To address these limitations, we have developed a novel AMP fusion protein system based on an encapsulin nanocompartment protein and have demonstrated its utility in enhancing expression of HBCM2, an AMP with activity against Gram-negative bacteria. Here, HBCM2 was fused to the N-terminus of several Encapsulin monomer (Enc) variants engineered with multiple TEV protease recognition site insertions to facilitate proteolytic release of the fused HBCM2. Fusion of HBCM2 to the Enc variants, but not other common carrier proteins, enabled robust overexpression in Escherichia coli C43(DE3) cells. Interestingly, variants with a TEV site insertion following residue K71 in Enc exhibited the highest overexpression and HBCM2 release efficiencies compared to other variants but were deficient in cage formation. HBCM2 was purified from the highest expressing variant following TEV protease digestion and was found to be highly active in inhibiting E. coli growth (MIC = 5 μg/ml). Our study demonstrates the potential use of the Enc system to enhance expression of AMPs for biomanufacturing and therapeutic applications.     

Investigation of archaeosomes as carriers for oral delivery of peptides

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.     

Bacteriocins: perspective for the development of novel anticancer drugs

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.

     

The selected reaction monitoring/multiple reaction monitoring-based mass spectrometry approach for the accurate quantitation of proteins: clinical applications in the cardiovascular diseases

Selected reaction monitoring, also known as multiple reaction monitoring, is a powerful targeted mass spectrometry approach for a confident quantitation of proteins/peptides in complex biological samples. In recent years, its optimization and application have become pivotal and of great interest in clinical research to derive useful outcomes for patient care. Thus, selected reaction monitoring/multiple reaction monitoring is now used as a highly sensitive and selective method for the evaluation of protein abundances and biomarker verification with potential applications in medical screening. This review describes technical aspects for the development of a robust multiplex assay and discussing its recent applications in cardiovascular proteomics: verification of promising disease candidates to select only the highest quality peptides/proteins for a preclinical validation, as well as quantitation of protein isoforms and post-translational modifications.     

Cell-penetrating peptides: from molecular mechanisms to therapeutics

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure-function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.     

Liposomal dry powders as aerosols for pulmonary delivery of proteins

The purpose of this research was to develop liposomal dry powder aerosols for protein delivery. The delivery of stable protein formulations is essential for protein subunit vaccine delivery, which requires local delivery to macrophages in the lungs. β-Glucuronidase (GUS) was used as a model protein to evaluate dry powder liposomes as inhaled delivery vehicles. Dimyristoyl phosphatylcholine:cholesterol (7∶3) was selected as the liposome composition. The lyophilization of liposomes, micronization of the powders, aerosolization using a dry powder inhaler (DPI), and in vitro aerodynamic fine particle fraction upon collection in a twinstage liquid impinger were evaluated. After lyophilization and jet-milling, the total amount of GUS and its activity, representing encapsulation efficiency and stability, were evaluated. The GUS amount and activity were measured and compared with freshly-prepared liposomes in the presence of mannitol, 43% of initial GUS amount, 29% of GUS activity after lyophilization and 36% of GUS amount, 22% of activity after micronization were obtained. Emitted doses from dry powder inhaler were 53%, 58%, 66%, and 73% for liposome powder:mannitol carrier ratios of 1∶0, 1∶4, 1∶9, and 1∶19. Fifteen percent of the liposome particles were less than 6.4 μm in aerodynamic diameter. The results demonstrate that milled liposome powders containing protein molecules can be aerosolized effectively at a fixed flow rate. Influences of different cryoprotectants on lyophilization of protein liposome formulations are reported. The feasibility of using liposomal dry powder aerosols for protein delivery has been demonstrated but further optimization is required in the context of specific therapeutic proteins. Published: December 21, 2005     

Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications

Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.