Analysis Of Cells Penetrating Peptides(CPPs) Prokaryotic Expression Library And Studies Of Mechanism And Function Of CPP With High Frequency

The plasma membrane of eukaryotic cells,which is a semi-permeable membrane,lays the foundation for material exchanges between extracellular milieu and the cellular constituents,and therefore is essential to cell survival and function.Yet,the natural barrier also presents a major challenge for intracellular delivery of cargos which have valuable therapeutic and diagnosis potential such as the non-lipid soluble protein,antibody,peptides,nucleic acids,polar molecule,probe and the imaging agent.Over the past decades,cell penetrating peptides(CPPs)has been discovered and further studied,opens a new era of intracellular transport carrier,which have also become one of the most popular and efficient techniques for achieving intracellular access.A large number of studies confirm that CPPs can successfully delivered hydrophilic protein,peptides,nucleic acid,polarity chemical molecules and even quantum dots in variety of cell lines with biological activities and minimal toxicity.Especially,some CPPs can carry cargo molecules cross the major human body hurdle,such as blood-brain barrier,blood testosterone and placental barrier in an active form.Therefore,CPPs acts as powerful tools for bioactive molecules intracellular transport have extensive and important application prospect in cell biology,immunology,drug development and gene biological treatment and tumor targeting therapy field.Since the discovery of CPPs provides excellent research tool for understanding of plasma membrane structure and function,the mechanism of CPPs uptake has been the subject of numerous studies.Now it is generally accepted that the process of CPPs internalization basically can be divided into two stages:first,bind to cell surface through electrostatic interaction,then penetrate the cell membrane to the inside,released into the cytoplasm and nucleus.As a result,containing large positive charge is characterized in amino acid sequence for majority of CPPs.In the second stage,the CPPs uptake mechanism mainly involves two pathways:one is endocytosis independent,means a direct translocation of the plasma membrane or occurs via pore formation,carpet-like perturbations,or inverted micelles formed in the bilayer;another is endocytosis mediated model,which may involve clathrin,caveolin and pinocytosis mediated pathway.In the above two,to date endocytic uptake is the main pathway for most known CPPs,is also the most intensively studied.But for a certain CPP pathway,a unifying pathway for translocation remains elusive,due to different experiment methods and conditions utilized by groups and organizations.It has been suggested that various properties of CPPs,properties of the associated cargo,such as size,charge and concentration,as well as cell type,can have a significant impact on the mechanism of uptake.Due to the above factors,combined with the difference in research method and conditions,the exact mechanism of CPPs uptake is rather complicated.At present,the number of known CPPs sequence has grown rapidly up to more than one hundred,its source mainly includes the transcription factors,viral proteins,signal peptides,antimicrobial peptides and designed CPPs.Considering part of nature derived CPPs has faults,such as efficiency difference and lack of cell specificity,prediction and reconstruction new one based on natural frame using molecular imitation and design by the aids of modern bioinformatics methods has become an important direction.According to the structure of a certain CPP,new candidate with much stronger permeability has been synthesized in laboratory.But as a whole,known CPPs are characterized by widely distribution,length difference,sequence diversity,and origin variety.So far,the understanding of common structure only stay in:most CPPs contain arginine and lysine(quite a number of positive charge in physiological condition),classified as amphipathic(alpha-helix or beta-sheet)and hydrophobic,etc.Considering the limitations in understanding of CPPs structure characteristics,molecular modification and prediction are lack of useful guides and clear directions.In addition to the insufficient knowledge on the mechanism and structure,the CPPs application also faces some obstacles and challenges.A major obstacle to CPP mediated drug delivery is thought to consist in the often rapid metabolic passing the enzymatic barriers of epithelia and endothelia.Until today,however,despite its general relevance to bioavailability,information on the momentous subject of enzymatic stability and degradation of CPPs is rare.What's more,massive differences in CPP translocation between "leaky" and "non-leaky" cell culture models were demonstrated."Leaky" cell culture models,such as Hela cells,lack the ability to form TJs and,therefore,grow to quite leaky and highly permeable monolayers.On the other hand,when grown to "non-leaky" epithelial type cell cultures,as represented by confluent MDCK monolayers,cells are connected by junctional complexes encircling the apex of each cell.High-resistance epithelial/endothelial cell barrier restrict the passage of CPPs cargoed from the intestine,respiratory tract and the circulating blood into the human body.To overcome the defects,it has become particularly necessary to looking for new CPPs with better properties besides structure modification.Phage display is defined as a proteinomics technology for screening functional polypeptide,in which exogenous peptides or proteins were fusion expressed with phage capsid protein,unifies the pheno-type(protein)and gene-type(DNA).Phage random peptide library constructed from mixture of certain length random peptides covers information of all possible amino acids arrangement mode fitting for given length.If the peptides displayed on the outside of phage particles can recognize and bind to target molecules(target cells),the phages will attach to the surface of receptor and are separated from no bindings when the random peptide library added.After several rounds of biopanning,phage possessing specific binding peptides will be obtained efficiently,quickly and easily.By phage lysis,DNA extract and PCR and sequencing reaction,amino acid sequence information of binding peptides will be available.In this thesis,we employed this technology in whole cell mode,combined the CPPs permeability and phage amplification,and realized the enrichment of translocation peptides and construct CPPs prokaryotic expression library.At last we completed the collection and analysis of large amounts of data output of high-throughput screening methods by using powerful bioinformatics.Our work and result in detail is as follows:(1)Using phage random peptide library in combination with whole-cell mode,we successfully obtained phages which can internalize Hela cells.Results showed that the recovery rate increased round by round,enrichment effect is ideal;(2)Thinking about the balance of "enrichment degree" and "sequence diversity",we adopt the products of 3rd round as template,design the PCR primers paired with common sequence of vectors,insert a bulk of a large number of CPPs DNA in the prokaryotic expression vector.Therefore,CPPs prokaryotic expression library fused with enhanced green fluorescent protein(EGFP)had been established;(3)After picking up monoclone from library,CPPs and EGFP fusion protein was yielded by the ways of prokaryotic expression and purification.We verified its internalization capacity through fluorescence observation.Results showed that the clone positive rate in the library(that is the proportion of CPPs confirmed)is as high as 70%,a total of 152 independence CPPs was achieved;(4)With the help of bioinformatics tools,we carried out analysis on physical and chemical properties,homology and characteristic motif of 152 CPPs.It was found that in the library pI value distributed between 3.00?12.99.In particular peptides had more tendency to be mildly acid(pH5.00?6.99)and weak alkali(pH8.00?9.99),a small part of them showed partial strong acid.Net charge ranged from-1,0 and +1,rare sequence was rich in positive and negative charge.Grand average of hydropathicity(GRAVY)value distributed between-3.999-3.999,most of them standed from-1.999-0.000 and showed weak hydrophilic.There were many difference between the properties as described above and well-known knowledge of CPPs structure.In the view of clustal analysis,152 CPPs can be summarized into three categories,which mean that individuals within one group shared higher sequence similarity.70 characteristic tripeptide sequences were identified by dissection analysis.Characteristic tetrapeptide,pentapeptide and hexapeptide motif containg tripeptide sequence were also obtained,which were likely to be the smallest functional unit(motor sequence)of CPPs;(5)Considering its electronegativity and weak hydrophilcity,we determined the highest abundance pDHY in CPPs library as the research object,chose the classic Tat as comparison to seek similarities and differences on internalization characteristics(distribution,cell toxicity,metabolism and cell specificity),mechanism and function between them under the same conditions.At first,the data demonstrated that pDHY and Tat can internalize cell in time-and concentration-dependent manner,the dynamics characteristic of both is consistent;as for distribution inside,pDHY cannot enter into the Hela nucleus,other than Tat.This difference was also obvious when PC-3(human prostate cancer cell line),SH-SY-5Y(human neuroblastoma cell line),A549(human lung adeno-carcinoma cell line),3T3(mouse embryo fibroblasts),HUVEC(human umbilical vein endothelial cell line)and neonatal rat cardiomyocytes tested.But in murine macrophage cell line Raw264.7,both of the two peptides behaviored with some similarities.Secondly,trypan blue dye experiment results showed that pDHY does no harm to cell in quite a wide range of concentration with high safety in usage,which is similar to Tat.All data were analyzed with SPSS 13.0 software,using One-way ANOVA method,for pDHY analysis of variance results was F=0.600,P=0.700,there was no statistically significant difference.Analysis of variance results of Tat treatment group was F=0.371,P=0.863,there was no statistically significant difference neither.Then,when delivering large cargo,weight similar with EGFP(27 kDa),Tat was mainly trapped in endosome and degraded earlier,while pDHY tended to distribute in diffusion state with better stability.The half-life of Tat-EGFP within cells was about 2-3 hours;however pDHY-EGFP can reach nearly six hours.All data were analyzed with SPSS 13.0 software,the influence of different treatment and time point on fluorescence intensity was analyzed using factorial design analysis of variance,comparison between the two treatment groups at the same time point was analyzed using t-test of random design.Statistic results showed that the fluorescence intensity differences between treatment groups was statistically significant(F=233.331,P<0.001),between different time points was also statistically significant(F=177.582,P<0.001),the two factors of treatments and time points had interaction effect(Fgroup*time=10.128,P<0.001),the signal intensity in cells treated with pDHY-EGFP was higher than Tat-EGFP treatment,the difference was statistically significant(P<0.001).The results above suggested that the pDHY loading macromolecular is more stable intracellular than Tat under the same conditions.At last,mechanism study revealed that both of them didn't need energy and bivalent cations(like Ca2+)for uptake,furthermore pDHY translocation had nothing to do with electrostatic adsorption on the surface of the cell membrane.In endocytosis inhibitors and membrane/plasma protein tandem affinity purification experiment,it had been shown that pDHY internalization strongly depends on the cytoskeleton structure,such as cell microtubule,microfilament,as well as requires the participation of members of the Rho GTPases.(6)Given the high delivery of pDHY,we investigated the the efficiency and biological activity of apoptosis related proteins,plasmid DNA and RNA oligo carried by pDHY into PC-3 cell.Flow cytometry assay results showed that after treated for 48h,PC-3 cell apoptosis rate rised,the difference has statistical significance compared with normal group(P=0.009),while compared with the His-Vp3 treatment group differences are statistically significant(P=0.014)too.The results above suggested that fusion protein pDHY-Vp3 can effectively induce the PC-3 cells apoptosis,its effect was obviously better than that of Vp3 alone.For the statistical analysis of the data using SPSS 13.0 software,the One-way ANOVA analysis of variance was applied.Q-PCR,Western-blot and trypan blue test results showed that when pDHY was mixed with plasmid DNA and oligo RNA in appropriate proportion(pDHY:MiR-200b mimics is 10:1,pDHY:pcDNA3-TP73 is 50:1)and formed non-covalent bond compounds,the transduction efficiency was simliar to transfection reagents Lipofect 2000,there was no statistically significant difference(P=1.000 and P=0.378),but the cell toxicity was significantly lower than the transfection reagent Lipofect 2000,with difference has statistical significance(P<0.001).At the same time,when Lipofect 2000 transfection system was added pDHY according to the proportion,transfection efficiency was lifted,difference has statistical significance(P=0.005 and P=0.041),whereas cytotoxic effect was similar with Lipofect 2000 alone,with no statistically significant difference(P=1.000).For the statistical analysis of the data using SPSS 13.0 software,the One-way ANOVA analysis of variance was applied.CCK 8 experimental results showed that when the MiR-200b and TP73 were overexpressed in PC-3,cell proliferation rate decreased obviously,the difference had statistical significance(P<0.001).With the join of pDHY and improvement of transfection efficiency,MiR-200b and TP73 expression levels were higher than Lipofect 2000 alone,while the PC-3 cell proliferation rate decline was even more significant,with statistical significance(P<0.001)differences vs Lipofect 2000 used alone.The results above also verified the conclusion that expression level of MiR-200b and TP73 had correlation with AIPC cell proliferation.For the statistical analysis of the data using SPSS 13.0 software,the One-way ANOVA analysis of variance was applied.Taken together,based on the deficiencies and difficulties in the current sdudy of CPPs,in the thesis we took the advantage of functional proteomics tools and strategies,first used phage display random peptide library to build up a high-throughput,systematically method looking for CPPs and a high efficiency,simple process verifying its penetration ability.With the help of bioinformatics tools,we completed the arrangement of CPPs sequences in the library,summarized the physical and chemical properties,extraced the characteristic motif,namely the smallest functional unit(motor sequence).The significance of the above work is that study aimed to seek regularity of CPPs structure from the view of omics,provided power guidence to modification and prediction of CPPs.On the other hand,we carried out systemic research on the most abundant peptide-pDHY,including dynamics,cell toxicity,cell specificity,metabolism stability,internalization mechanism and biological function,and explored the application related to Human prostate carcinoma study and therapy.Due to the limited experiment means and knowledge level,new group CPPs represente by pDHY should undergo further study.However,this work will broaden our vision and supplyement knowledge about CPPs,extend and improve the understanding of plasm membrane transport mechanism.At the same time,this work has important practical significance for CPPs application in cell biology,drug development,genetic researches,biological therapy and tumor targeting therapy.