Protection of tight junction between RPE cells with tissue factor targeting peptide

AIM：To investigate the effect of tissue factor targeting peptide（TF-TP）on retinal pigment epithelium（RPE）cells tight junctions.METHODS：Cell counting kit-8（CCK-8）was used to measure the proliferation of ARPE-19 cells.Expression of tight junction,ZO-1 in ARPE-19 cells was measured by Western blot and immunofluorescent staining.Western blot was also used to detect the expression of tissue factor（TF）.CEC Transmigration Assay was used to measure the migration of ARPE-19 cells.The transport of fluorescent markers [fluorescein isothiocyanate dextrans of 4,10,20（FD4,FD10,FD20） ]and the transepithelial electrical resistance（TEER）were used to measure in ARPE-19 cell RESULTS：CCK-8 assay showed that 5μmol/L TF-TP can inhibit ARPE-19 cells abnormally proliferation stimulated by lipopolysaccharide（LPS;P〈0.05）.LPS increased the transport of fluorescent markers（FD4,FD10,FD20）and decreased TEER levels in ARPE-19 cells,respectively,which were prevented by 5μmol/L TF-TP pretreatment（P〈0.05）. Furthermore,LPS significantly up-regulated the expression of TF and downregulated the expression of ZO-1（P〈0.05）in ARPE-19 cell which was inhibited by the TF-TP（P〈0.05）.In addition,TF-TP inhibited the abnormal migration induced by LPS in ARPE-19 cell（P〈0.05）.CONCLUSION：Our findings suggest that TF-TP suppressed proliferation and migration of ARPE-19 cells induced by LPS,and maintained the RPE tight junctions through inhibition of TF expression and increased expression of ZO-1.

Converting antimicrobial into targeting peptides reveals key features governing protein import into mitochondria and chloroplasts

We asked what peptide features govern targeting to the mitochondria versus the chloroplast,using antimicrobial peptides as a starting point.This approach was inspired by the endosymbiotic hypothesis that organelle-targeting peptides derive from antimicrobial amphipathic peptides delivered by the host cell,to which organelle progenitors became resistant.To explore the molecular changes required to convert antimicrobial into targeting peptides,we expressed a set of 13 antimicrobial peptides in Chlamydomonas reinhardtii.Peptides were systematically modified to test distinctive features of mitochondrion-and chloroplast-targeting peptides,and we assessed their targeting potential by following the intracellular localization and maturation of a Venus fluorescent reporter used as a cargo protein.Mitochondrial targeting can be achieved by some unmodified antimicrobial peptide sequences.Targeting to both organelles is improved by replacing lysines with arginines.Chloroplast targeting is enabled by the presence of flanking unstructured sequences,additional constraints consistent with chloroplast endosymbiosis having occurred in a cell that already contained mitochondria.If indeed targeting peptides evolved from antimicrobial peptides,then required modifications imply a temporal evolutionary scenario with an early exchange of cationic residues and a late acquisition of chloroplast-specific motifs.

Dual Targeting to Mitochondria and Chloroplasts： Characterization of Thr-tRNA Synthetase Targeting Peptide

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.

A novel PD-L1 targeting peptide self-assembled nanofibers for sensitive tumor imaging and photothermal immunotherapy in vivo

Programmed death 1(PD-1)and its ligand PD-L1 are two typical immune checkpoints.Antibody-based immune checkpoint blockade(ICB)strategy targeting PD-1/PD-L1 achieved a significant therapeutic effect on cancer.However,due to the impenetrability of antibody drugs and the occurrence of immune-related adverse events,only a minority of patients benefit from this treatment.Peptides multimerization has been widely proved to be an effective method to improve receptor binding affinity through a multivalent synergistic effect.In this study,we report a novel peptide-aggregation-induced emission(AIE)hybrid supramolecular TAP,which can self-assemble into nanofibers through non-covalent interactions such as hydrogen bonds,with a specific nanomolar affinity to PD-L1 in vivo and in vitro.Combined with near-infrared agents,it can be used for tumor imaging and photothermal therapy,which enables photothermal ablation of cancer cells for generating tumor-associated antigen(TAA)and triggering a series of immunological events.Collectively,our work suggests that synthetic self-assembled peptide nanofibers can be developed as attractive platforms for active photothermal immunotherapies against cancer.

整合素α6靶向自组装促凋亡纳米多肽对中枢神经系统急性淋巴细胞白血病的靶向治疗

There is currently no effective targeted therapeutic strategy for the treatment of central nervous system acute lymphoblastic leukemia(CNS-ALL).Integrinα6 is considered a potential target for CNS-ALL diagnosis and therapy because of its role in promoting CNS-ALL disease progression.The targeted peptide D(RWYD)(abbreviated RD),with nanomolar affinity to integrinα6 was identified by peptide scanning techniques such as alanine scanning,truncation,and D-substitution.Herein,we developed a therapeutic nanoparticle based on the integrinα6-targeted peptide for treating CNS-ALL.The self-assembled proapoptotic nanopeptide_(D)(RWYD)-_(D)(KLAKLAK)_(2)-G_(D)(FFY)(abbreviated RD-KLA-Gffy)contains the integrinα6-targeted peptide RD,the well-known proapoptotic peptide_(D)(KLAKLAK)_(2)(abbreviated KLA),and the self-assembling tetrapeptide GD(FFY)(abbreviated Gffy).The functional mechanism of RD-KLA-Gffy is clarified using different experiments.Our results demonstrate that RD-KLA-Gffy is highly enriched in CNS-ALL lesions and induces tumor cell apoptosis,thus reducing CNS-ALL disease burden and prolonging the survival of CNS-ALL mice without obvious toxicity.Moreover,the combined use of RD-KLA-Gffy and methotrexate(MTX)shows a potent antitumor effect in treating CNS-ALL,indicating that RD-KLA-Gffy plays an important role in suppressing CNS-ALL progression either as a single agent or in combination with MTX,which shows promise for application in CNS-ALL therapy.

Phage display screening of therapeutic peptide for cancer targeting and therapy

Recently,phage display technology has been announced as the recipient of Nobel Prize in Chemistry 2018.Phage display technique allows high affinity target-binding peptides to be selected from a complex mixture pool of billions of displayed peptides on phage in a combinatorial library and could be further enriched through the biopanning process;proving to be a powerful technique in the screening of peptide with high affinity and selectivity.In this review,we will first discuss the modifications in phage display techniques used to isolate various cancer-specific ligands by in situ,in vitro,in vivo,and ex vivo screening methods.We will then discuss prominent examples of solid tumor targeting-peptides;namely peptide targeting tumor vasculature,tumor microenvironment(TME)and overexpressed receptors on cancer cells identified through phage display screening.We will also discuss the current challenges and future outlook for targeting peptidebased therapeutics in the clinics.

A composite peptide-supramolecular microneedle system for melanoma immunotherapy

Despite recent advances in melanoma treatment through the use of antibody immunotherapy,the clinical benefit remains restricted by its inefficient infiltration and immunosuppression within the tumor microenvironment(TME).In addition,immunerelated adverse events(irAEs)have often occurred due to the off-target binding of therapeutic drugs to normal tissues after systematic administration.Herein,we constructed an integrated and cascaded drug delivery system for the treatment of melanoma.In addition to blocking the programmed cell death protein 1 or its ligand(PD-1/PD-L1)axis,the PD-L1 targeting peptide(FE)with spherical micelle self-assembly characteristics could also effectively encapsulate the immune adjuvant resiquimod(R848),and form a complete nano drug.FE^(R)was further integrated into tumor-responsive microneedles(MNs)to establish FE^(R)@MN and could reach the cascaded functions.FE^(R)could be sustainedly released from the MN system and disassemble into monomers,achieving PD-1/PD-L1 axis blockade whilst reprogramming the immunosuppressive TME.Notably,FE^(R)@MN permits the controllable release and retention enhancement of the targeting peptide in the TME,thus causing prolonged PD-L1 blockade effect.It is demonstrated that this synergistic treatment could efficiently inhibit melanoma growth,providing a new strategy for the combination treatment of melanoma.

Time to give up traditional methods for the management of gastrointestinal neuroendocrine tumours

Neuroendocrine tumors(NETs)are a rare and heterogeneous disease group and constitute 0.5%of all malignancies.The annual incidence of NETs is increasing worldwide.The reason for the increase in the incidence of NETs is the detection of benign lesions,incidental detection due to the highest use of endoscopic and imaging procedures,and higher recognition rates of pathologists.There have been exciting developments regarding NET biology in recent years.Among these,first of all,somatostatin receptors and downstream pathways in neuroendocrine cells have been found to be important regulatory mechanisms for protein synthesis,hormone secretion,and proliferation.Subsequently,activation of the mammalian target of rapamycin pathway was found to be an important mechanism in angiogenesis and tumor survival and cell metabolism.Finally,the importance of proangiogenic factors(platelet-derived growth factor,vascular endothelial growth factor,fibroblastic growth factor,angiopoietin,and semaphorins)in the progression of NET has been determined.Using the combination of biomarkers and imaging methods allows early evaluation of the appropriateness of treatment and response to treatment.

Integrin-targeting with peptide-bioconjugated semiconductor-magnetic nanocrystalline heterostructures

Binary asymmetric nanocrystals （BNCs）, composed of a photoactive TiO2 nanorod joined with a superparamagnetic γ-Fe203 spherical domain, were embedded in polyethylene glycol modified phospholipid micelle and successfully bioconjugated to a suitably designed peptide containing an RGD motif. BNCs represent a relevant multifunctional nanomaterial, owing to the coexistence of two distinct domains in one particle, characterized by high photoactivity and magnetic properties, that is particularly suited for use as a phototherapy and hyperthermia agent as well as a magnetic probe in biological imaging. We selected the RGD motif in order to target integrin expressed on activated endothelial cells and several types of cancer cells. The prepared RGD-peptide/BNC conjugates, comprehensively monitored by using complementary optical and structural techniques, demon- strated a high stability and uniform dispersibility in biological media. The cytotoxicity of the RGD-peptide/BNC conjugates was studied in vitro. The cellular uptake of RGD-peptide conjugates in the cells, assessed by means of two distinct approaches, namely confocal microscopy analysis and emission spectroscopy determination in cell lysates, displayed selectivity of the RGD-peptide-BNC conjugate for the cw]33 integrin. These RGD-peptide-BNC conjugates have a high potential for theranostic treatment of cancer.

Serological Targeted Analysis of an ITIH4 Peptide Isoform: A Preterm Birth Biomarker and Its Associated SNP Implications

Over 11% of all pregnancies in the US result in preterm birth, greatly contributing to perinatal morbidity and mortality （Goldenberg and Rouse, 1998）. Preterm birth etiologies remain largely unknown, and effective prevention methods have yet to be developed. The use of biofluid （e.g., serum or urine） for the analysis of the naturally occurring peptidome （MW 〈 4000） as a source of biomarkers has been reported for different diseases （Villanueva et al., 2006; Ling et al., 2010a, 2010b, 2010c, 2011）. Mass spectrometry-based profiling of naturally occurring peptides can provide an extensive in- ventory of serum peptides derived from either high-abundant endogenous circulating proteins or cell and tissue proteins （Liotta and Petricoin, 2006）.

A bone-targeted engineered exosome platform delivering siRNA to treat osteoporosis

The complex pathogenesis of osteoporosis includes excessive bone resorption,insufficient bone formation and inadequate vascularization,a combination which is difficult to completely address with conventional therapies.Engineered exosomes carrying curative molecules show promise as alternative osteoporosis therapies,but depend on specifically-functionalized vesicles and appropriate engineering strategies.Here,we developed an exosome delivery system based on exosomes secreted by mesenchymal stem cells(MSCs)derived from human induced pluripotent stem cells(iPSCs).The engineered exosomes BT-Exo-siShn3,took advantage of the intrinsic anti-osteoporosis function of these special MSC-derived exosomes and collaborated with the loaded siRNA of the Shn3 gene to enhance the therapeutic effects.Modification of a bone-targeting peptide endowed the BT-Exo-siShn3 an ability to deliver siRNA to osteoblasts specifically.Silencing of the osteoblastic Shn3 gene enhanced osteogenic differentiation,decreased autologous RANKL expression and thereby inhibited osteoclast formation.Furthermore,Shn3 gene silencing increased production of SLIT3 and consequently facilitated vascularization,especially formation of type H vessels.Our study demonstrated that BT-Exo-siShn3 could serve as a promising therapy to kill three birds with one stone and implement comprehensive anti-osteoporosis effects.

PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein＇s function. We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups： chloroplast, thylakoid, mitochondrion, secretory pathway, and ＂other＂. When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL： http：//bioinformatics.biol.uoa.gr/PredSL/.