Peptide Mimics Research Articles

Abstract 1922: MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells

Abstract Adenoid Cystic Cancer (ACC) is the most aggressive salivary gland tumor with no effective systemic therapy. MYB/MYBL1 fusion event underlies the etiology of ACC with few recurrent variants focused on NOTCH and chromatin remodeling pathways and low overall mutational tumor burden. No chemotherapy or targeted drugs have been approved for ACC. Therefore, there is an urgent need to develop novel therapeutic strategies. To study MYB biology and test new therapeutic agents we have developed and characterized the phenotype of a new murine conditional MYB-NFIB transgene model and have also generated a mammary tumor cell line expressing the human MYB fusion transgene with ACC-like features. We have also generated new human MYB-NFIB positive ACC tumor cell lines and xenografts from patient samples and have tested these tumor cells with newly developed MYB mimic peptides. We analyzed the phenotype of mice carrying conditional MMTV-CRE MYB-NFIB transgene alone or after crossing to heterozygosity with p16/ARF null alleles. We observed shortened survival in two different mouse transgene strains due to development of B-cell leukemia manifested by elevated WBC, low platelets, and marked splenomegaly. We also noted sporadic solid tumors including the development of a murine mammary tumor which generated a MYB-NFIB positive tumor cell line for testing new therapies. In addition, we have generated patient derived xenografts (PDX) and the successful generation of a human ACC tumor cell line from fresh ACC patient biopsies. Using shRNA reagents we observed reduced tumor cell growth and viability assayed by MTS assay following Notch1 and MYB knockdown. We also tested four candidate MYB inhibitors, celastrol, all-trans retinoic acid, and the peptidomimetic inhibitors, MYBMIM and CRYBMIM, against murine and human ACC tumor samples with immortalized normal cells as controls and will discuss these results. Citation Format: Yue Jiang, Lauren Forbes, Jianping Li, Shelby Freeberg, Chunxia Cao, Maria Zajac-Kaye, Jonathan Licht, Alex Kentsis, Frederic Kaye. MYB mimic peptides targeting human and murine MYB-NFIB positive tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1922.

Peptoids as tools and sensors.

A review of molecular tools and sensors assembled on N-substituted glycine, or α-peptoid, oligomers between 2013 and November 2018 with the following sections: (a) Peptoids as crystal growth modifiers, (b) Peptoids as catalysts, (c) Ion and molecule sequestration and transport, (d) Peptoid sensors, (e) Macromolecule recognition, (f) Cellular transporters, (g) Medical imaging, (h) Future direction and (i) Summary and outlook. Peptoids are a promising class of peptide mimic making them an excellent platform for functional molecule preparation. Attributes of peptoid oligomers include: (a) the ease of precise sequence definition and mono-dispersity; (b) access to a vast chemical space within simple and repeating chemical preparative steps and (c) thermal, chemical and biological stability all lending support for their application in a number of areas, with some that have been realised to date. The peptoid tool and sensor examples selected have realised practical utility. They serve to illustrate the rapidity of new insight that can generate in many disparate areas of science and technology, enabling the quick assembly of design criteria for efficient peptoid molecular tools and sensors.

Influence of the Assembly State on the Functionality of a Supramolecular Jagged1-Mimicking Peptide Additive.

Expanding the bioactivation toolbox of supramolecular materials is of utmost relevance for their broad applicability in regenerative medicines. This study explores the functionality of a peptide mimic of the Notch ligand Jagged1 in a supramolecular system that is based on hydrogen bonding ureido-pyrimidinone (UPy) units. The functionality of the peptide is studied when formulated as an additive in a supramolecular solid material and as a self-assembled system in solution. UPy conjugation of the DSLJAG1 peptide sequence allows for the supramolecular functionalization of UPy-modified polycaprolactone, an elastomeric material, with UPy-DSLJAG1. Surface presentation of the UPy-DSLJAG1 peptide was confirmed by atomic force microscopy and X-ray photoelectron spectroscopy analyses, but no enhancement of Notch activity was detected in cells presenting Notch1 and Notch3 receptors. Nevertheless, a significant increase in Notch-signaling activity was observed when DSLJAG1 peptides were administered in the soluble form, indicating that the activity of DSLJAG1 is preserved after UPy functionalization but not after immobilization on a supramolecular solid material. Interestingly, an enhanced activity in solution of the UPy conjugate was detected compared with the unconjugated DSLJAG1 peptide, suggesting that the self-assembly of supramolecular aggregates in solution ameliorates the functionality of the molecules in a biological context.

Structural and functional consequences of age-related isomerization in α-crystallins

Long-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as side-chain isomerization. Recently, tandem MS has enabled identification and characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here, we examined the impact of isomerization of aspartic acid or epimerization of serine at four sites mapping to crucial oligomeric interfaces in human αA- and αB-crystallin, the most abundant chaperone proteins in the eye lens. To characterize the effect of isomerization on quaternary assembly, we utilized synthetic peptide mimics, enzyme assays, molecular dynamics calculations, and native MS experiments. The oligomerization of recombinant forms of αA- and αB-crystallin that mimic isomerized residues deviated from native behavior in all cases. Isomerization also perturbs recognition of peptide substrates, either enhancing or inhibiting kinase activity. Specifically, epimerization of serine (αASer-162) dramatically weakened inter-subunit binding. Furthermore, phosphorylation of αBSer-59, known to play an important regulatory role in oligomerization, was severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp-62. Similarly, isomerization of αBAsp-109 disrupted a vital salt bridge with αBArg-120, a contact that when broken has previously been shown to yield aberrant oligomerization and aggregation in several disease-associated variants. Our results illustrate how isomerization of amino acid residues, which may seem to be only a minor structural perturbation, can disrupt native structural interactions with profound consequences for protein assembly and activity.

Consideration of Binding Kinetics in the Design of Stapled Peptide Mimics of the Disordered Proteins Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 and Eukaryotic Translation Initiation Factor 4G.

Protein disorder plays a crucial role in signal transduction and is key for many cellular processes including transcription, translation, and cell cycle. Within the intrinsically disordered protein interactome, the α-helix is commonly used for binding, which is induced via a disorder-to-order transition. Because the targeting of protein-protein interactions (PPIs) remains an important challenge in medicinal chemistry, efforts have been made to mimic this secondary structure for rational inhibitor design through the use of stapled peptides. Cap-dependent mRNA translation is regulated by two disordered proteins, 4E-BP1 and eIF4G, that inhibit or stimulate the activity of the m7G cap-binding translation initiation factor, eIF4E, respectively. Both use an α-helical motif for eIF4E binding, warranting the investigation of stapled peptide mimics for manipulating eIF4E PPIs. Herein, we describe our efforts toward this goal, resulting in the synthesis of a cell-active stapled peptide for further development in manipulating aberrant cap-dependent translation in human diseases.

Bicyclic Pyrrolidine-Isoxazoline γ Amino Acid: A Constrained Scaffold for Stabilizing α-Turn Conformation in Isolated Peptides.

Unnatural amino acids have tremendously expanded the folding possibilities of peptides and peptide mimics. While α,α-disubstituted and β-amino acids are widely studied, γ-derivatives have been less exploited. Here we report the conformational study on the bicyclic unnatural γ amino acid, 4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-3-carboxylic acid 1. In model peptides, the (+)-(3aR6aS)-enantiomer is able to stabilize α-turn conformation when associated to glycine, as showed by 1H-NMR, FT-IR, and circular dichroism experiments, and molecular modeling studies. α-turn is a structural motif occurring in many biologically active protein sites, although its stabilization on isolated peptides is quite uncommon. Our results make the unnatural γ-amino acid 1 of particular interest for the development of bioactive peptidomimetics.

Structure-Activity Study of Antibacterial Poly(ester urethane)s with Uniform Distribution of Hydrophobic and Cationic Groups.

Infections associated with antibiotic-resistant bacteria have become a threat to the global public health. Antimicrobial polymers, which are synthetic mimics of antimicrobial peptides, have gained increasing attention, as they may have a lower chance of inducing resistance. The cationic-hydrophobic balance and distribution of cationic and hydrophobic moieties of these polymers is known to have a major effect on antimicrobial activity. We studied the properties of a series of facially amphiphilic antimicrobial surfactant-like poly(ester urethane)s with different hydrophobic pendant groups (P1, P2, and P3) and cationic groups distributed uniformly along the polymer chain. These polymers exhibited bactericidal activity against Gram-negative Escherichia coli and Pseudomonas aeruginosa, as well as Gram-positive Staphylococcus aureus and Staphylococcus epidermidis. Microscopy and dye release assays demonstrated that these polymers cause membrane disruption, which is dependent on the cationic-hydrophobic ratio in the polymer. Membrane permeability assays revealed that these polymers can permeabilize the outer membrane of E. coli and damage the cytoplasmic membrane of both E. coli and S. aureus. In addition, our results indicate that the three polymers exhibit a different extent of membrane disruption against E. coli. P1 caused minor damage to the cytoplasmic membrane integrity, but it was able to dissipate the cytoplasmic membrane potential, leading to cell death. P2 and P3 depolarized the cytoplasmic membrane and also caused significant damage to the cytoplasmic membrane. Overall, we showed a new class of broad-spectrum bactericidal polymers whose membrane disrupting ability against E. coli correlates with the structural differences of the hydrophobic pendant groups.

New orthogonally trifunctionalized morpholine nucleosides

Three novel uracil-based morpholine nucleosides each containing three different orthogonally reactive functional groups, namely, amino, carboxy and acetylene ones, were synthesized. The obtained monomers are intended for the synthesis of labeled nucleotide, nucleic acid or peptide mimics.

Ovarian Tumor Cell Expression of Claudin-4 Reduces Apoptotic Response to Paclitaxel.

A significant factor contributing to poor survival rates for patients with ovarian cancer is the insensitivity of tumors to standard-of-care chemotherapy. In this study, we investigated the effect of claudin-4 expression on ovarian tumor cell apoptotic response to cisplatin and paclitaxel. We manipulated claudin-4 gene expression by silencing expression [short hairpin RNA (shRNA)] in cells with endogenously expressed claudin-4 or overexpressing claudin-4 in cells that natively do not express claudin-4. In addition, we inhibited claudin-4 activity with a claudin mimic peptide (CMP). We monitored apoptotic response by caspase-3 and Annexin V binding. We examined proliferation rate by counting the cell number over time as well as measuring the number of mitotic cells. Proximity ligation assays, immunoprecipitation (IP), and immunofluorescence were performed to examine interactions of claudin-4. Western blot analysis of tubulin in cell fractions was used to determine the changes in tubulin polymerization with changes in claudin-4 expression. Results show that claudin-4 expression reduced epithelial ovarian cancer (EOC) cell apoptotic response to paclitaxel. EOCs without claudin-4 proliferated more slowly with enhanced mitotic arrest compared with the cells expressing claudin-4. Furthermore, our results indicate that claudin-4 interacts with tubulin, having a profound effect on the structure and polymerization of the microtubule network. In conclusion, we demonstrate that claudin-4 reduces the ovarian tumor cell response to microtubule-targeting paclitaxel and disrupting claudin-4 with CMP can restore apoptotic response. IMPLICATIONS: These results suggest that claudin-4 expression may provide a biomarker for paclitaxel response and can be a target for new therapeutic strategies to improve response.

Targeting Lipooligosaccharide (LOS) for a Gonococcal Vaccine.

The increasing incidence of gonorrhea worldwide and the global spread of multidrug-resistant strains of Neisseria gonorrhoeae, constitute a public health emergency. With dwindling antibiotic treatment options, there is an urgent need to develop safe and effective vaccines. Gonococcal lipooligosaccharides (LOSs) are potential vaccine candidates because they are densely represented on the bacterial surface and are readily accessible as targets of adaptive immunity. Less well-understood is whether LOSs evoke protective immune responses. Although gonococcal LOS-derived oligosaccharides (OSs) are major immune targets, often they undergo phase variation, a feature that seemingly makes LOS less desirable as a vaccine candidate. However, the identification of a gonococcal LOS-derived OS epitope, called 2C7, that is: (i) a broadly expressed gonococcal antigenic target in human infection; (ii) a virulence determinant, that is maintained by the gonococcus and (iii) a critical requirement for gonococcal colonization in the experimental setting, circumvents its limitation as a potential vaccine candidate imposed by phase variation. Difficulties in purifying structurally intact OSs from LOSs led to “conversion” of the 2C7 epitope into a peptide mimic that elicited cross-reactive IgG anti-OS antibodies that also possess complement-dependent bactericidal activity against gonococci. Mice immunized with the 2C7 peptide mimic clear vaginal colonization more rapidly and reduce gonococcal burdens. 2C7 vaccine satisfies criteria that are desirable in a gonococcal vaccine candidate: broad representation of the antigenic target, service as a virulence determinant that is also critical for organism survival in vivo and elicitation of broadly cross-reactive IgG bactericidal antibodies when used as an immunogen.

A Small Cyclic β-Hairpin Peptide Mimics the Rbfox2 RNA Recognition Motif and Binds to the Precursor miRNA 20b.

The RNA recognition motif (RRM), which is the most abundant RNA-binding motif in eukaryotes, is a well-structured domain of about 90 amino acids, yet the β2β3 hairpin, corresponding to strands 2 and 3 of the β-sheet, and the intervening loop make essential interactions with RNA in many RRM complexes. A series of small cyclic peptide mimics of the β2β3 hairpin of Rbfox2 protein that recognize the terminal loop of precursor miR-20b have been designed to investigate whether the full RNA-binding protein can be mimicked with a minimal structurally preorganized peptide. Within a small library of seven cyclic peptides, a peptide with low-micromolar affinity for the miR-20b precursor was found. NMR spectroscopy titration data suggest that this peptide specifically targets the apical loop of pre-miR-20b. This work shows that it is possible to mimic RNA-binding proteins with designed stable peptides, which provide a starting point for designing or evolving small peptide mimetics of RRM proteins.

Probing Anti-inflammatory Properties Independent of NF-κB Through Conformational Constraint of Peptide-Based Interleukin-1 Receptor Biased Ligands.

Interleukin-1β (IL-1β) binds to the IL-1 receptor (IL-1R) and is a key cytokine mediator of inflammasome activation. IL-1β signaling leads to parturition in preterm birth (PTB) and contributes to the retinal vaso-obliteration characteristic of oxygen-induced retinopathy (OIR) of premature infants. Therapeutics targeting IL-1β and IL-1R are approved to treat rheumatoid arthritis; however, all are large proteins with clinical limitations including immunosuppression, due in part to inhibition of NF-κB signaling, which is required for immuno-vigilance and cytoprotection. The all-D-amino acid peptide 1 (101.10, H-d-Arg-d-Tyr-d-Thr-d-Val-d-Glu-d-Leu-d-Ala-NH2) is an allosteric IL-1R modulator, which exhibits functional selectivity and conserves NF-κB signaling while inhibiting other IL-1-activated pathways. Peptide 1 has proven effective in experimental models of PTB and OIR. Seeking understanding of the structural requirements for the activity and biased signaling of 1, a panel of twelve derivatives was synthesized employing the various stereochemical isomers of α-amino-γ-lactam (Agl) and α-amino-β-hydroxy-γ-lactam (Hgl) residues to constrain the D-Thr-D-Val dipeptide residue. Using circular dichroism spectroscopy, the peptide conformation in solution was observed to be contingent on Agl, Hgl, and Val stereochemistry. Moreover, the lactam mimic structure and configuration influenced biased IL-1 signaling in an in vitro panel of cellular assays as well as in vivo activity in murine models of PTB and OIR. Remarkably, all Agl and Hgl analogs of peptide 1 did not inhibit NF-κB signaling but blocked other pathways, such as JNK and ROCK2 phosphorylation contingent on structure and configuration. Efficacy in preventing preterm labor correlated with a capacity to block IL-1β-induced IL-1β synthesis. Furthermore, the importance of inhibition of JNK and ROCK2 phosphorylation for enhanced activity was highlighted for prevention of vaso-obliteration in the OIR model. Taken together, lactam mimic structure and stereochemistry strongly influenced conformation and biased signaling. Selective modulation of IL-1 signaling was proven to be particularly beneficial for curbing inflammation in models of preterm labor and retinopathy of prematurity (ROP). A class of biased ligands has been created with potential to serve as selective probes for studying IL-1 signaling in disease. Moreover, the small peptide mimic prototypes are promising leads for developing immunomodulatory therapies with easier administration and maintenance of beneficial effects of NF-κB signaling.

Photo-Control of RAS GDP-GTP Exchange using the SOSαH Mimicking Peptides Modified with Spiropyran Derivative

Ras is a kind of GTPase protein, and it regulates cellular signal transduction. For example, some information such as cell growth and cell differentiation from in vitro to in vivo of downstream by cycling between GTP active form and GDP inactive form. GDP-GTP exchange of Ras is induced by binding of GEF(Guanine Exchange Factor) to Ras. Recently some kinds of peptide which interfere binding of GEF resulting in inhibition of GDP-GTP exchange, were reported. Synthetic peptide mimicking αH helix of SOS inhibited the Ras GDP-GTP exchange in the presence of SOS (Son Of Sevenless). Photochromic molecule, spiropyran isomerize between ring-closed spiro (SP) and ring-opened merocyanine (MC) forms photo-reversibly upon visible and ultraviolet lights. It is known that the zwitterion of MC forms exhibit dimerization. Previously, it has been demonstrated that the peptides modified with spiropyran derivatives change their secondary structure accompanied by formation of spiropyran dimerization photoreversibly. In this study, we designed and synthesized the peptides which have two cysteines and analogous to the SOS αH-helix. The two cysteine residues of the peptides were modified with SP-maleimide(SP-MA) stoichiometrically. The spiropyran incorporated into the peptide exhibited SP-MC isomerization upon visible light and ultraviolet light irradiations. The secondary structural change of the SOS mimic peptide induce by photo-isomerization of SP-MA were examined. Furthermore, photoreversible inhibition of Ras GDP-GTP exchange in the presence of SOS with the photochromic peptide was analyzed using fluorescent GDP analogues.

The Role of Connexin-43 in the Inflammatory Process: A New Potential Therapy to Influence Keratitis.

The studies outlined in this review highlight the relationship between inflammatory signaling molecules and connexin-43 (Cx43). Gap junction (GJ) channels and hemichannels (HCs) participate in the metabolic activity between intra- and extracellular space. Some ions and small molecules are exchanged from cell to cell or cell to extracellular space to affect the process of inflammation via GJ. We analyzed the effects of signaling molecules, such as innate immunity messengers, transcription factors, LPS, cytokine, inflammatory chemokines, and MMPs, on Cx43 expression during the inflammatory process. At the same time, we found that these signaling molecules play a critical role in the pathogenesis of keratitis. Thus, we assessed the function of Cx43 during inflammatory corneal disease. Corneal healing plays an essential role in the late stage of keratitis. We found that Cx43 is involved in wound healing. Studies have shown that the decrease of Cx43 can decrease the time of healing. We also report several Cx43 mimic peptides which can inhibit the activity of Cx43 Hc to mediate the releasing of adenosine triphosphate (ATP), which may in turn influence the inflammatory process.

Folding and Assembly of Vanilloid Receptor Secondary-Structure Peptide with Hexahistidine Linker at Nickel-Nitrilotriacetic Acid Monolayer for Capsaicin Recognition.

Herein, we report the self-assembly of a synthetic vanilloid receptor (VR) peptide that selectively binds capsaicin. We synthesized a 26-mer peptide-YSEILFFVQS-HHHHHH-LAMGWTNMLY (S3HS4)-comprising two chemoreceptor domains of transient receptor potential channel (TRPV1) linked by a hexahistidine sequence. High-speed atomic force microscopy (AFM) imaging in water revealed that the peptide structures alternated rapidly between wedge shape and linear forms. Circular dichroism spectroscopy showed that 65% of the amide units in the peptide chain adopted an α-helix structure, which was ascribed to the chemoreceptor domains. S3HS4 developed well-packed monolayers at the Ni-treated thiolated nitrilotriacetic acid self-assembled monolayers by chelation of the hexahistidine segment, as characterized by infrared spectroscopy and AFM, which exhibited statistically constant specific height. Therefore, S3HS4 was expected to fold spontaneously upon chelation, and the resulting helix-turn-helix conformers developed films while uniformly oriented: the tilt angle was 69° from the surface normal to the substrate. According to microgravimetric analysis using a quartz crystal microbalance (QCM), the adsorption was 84 ± 47 pmol cm-2 ( n = 3), which was almost consistent with the saturation adsorption of an α-helix unit. We also used a QCM to investigate the host-guest reactions of S3HS4 and found that the S3HS4-attached QCM-chip-bound capsaicin with an apparent binding constant of (4.2 ± 3.6) × 104 M-1 ( n = 4), whereas there was no evidence of binding to vanillin or acetophenone. Two controls-a blank chip without S3HS4 and a chip modified with a single helical peptide (LAMGWTNMLY-HHHHHH)-produced no capsaicin response. To the best of our knowledge, S3HS4 is the first example of a synthetic VR mimic peptide. We believe that the present surface-directed structure-based design can be used to exploit the α-helix bundle in hexahistidine-linked bishelical peptides.

Structural and functional characterization of HIV-1 cell fusion inhibitor T20.

The peptide drug T20 (enfuvirtide), derived from the C-terminal heptad repeat region of HIV-1 gp41, is the only membrane fusion inhibitor available for treatment of viral infection; however, its mechanism of action remains elusive and its structural basis is lacking. We focused on determining the crystal structure of T20 in complex with N39, a target mimic peptide derived from the N-terminal heptad repeat region of gp41. On the basis of the structural information, the mechanisms of action of T20 and its resistance were further characterized. A panel of peptides was synthesized. The T20/N39 complex was assembled for crystallization studies. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), native polyacrylamide gel electrophoresis (N-PAGE), and mutational analysis were applied to analyze the structural and functional properties. A crystal structure of six-helical bundle (6-HB) structure formed by T20 and N39 was determined with a resolution limit of 2.3 Å, which revealed the critical intrahelical and interhelical interactions underlying the mechanism of action of T20 and its resistance mutations. Although the structural properties in the C-terminal tryptophan-rich motif (TRM) of T20 and the fusion peptide proximal region (FPPR) of N39 could not be finely defined by the structure, the data from biophysical and mutational analyses verified the essential roles of the TRM and FPPR motifs for the binding and inhibitory activities of T20. For the first time, our studies provide a structural basis of T20, which help our understanding on the mechanisms of HIV-1 fusion and its inhibition.

Involvement of sphingosine-1-phosphate receptors 2/3 in IR-induced sudden cardiac death.

It has been demonstrated that S1P receptors affect heart ischaemia-reperfusion (IR) induced injury. However, whether S1P receptors affect IR-induced cardiac death has not been investigated. The aim of this paper is to demonstrate the role of S1P receptors in IR-induced cardiac death. Healthy adult male Sprague-Dawley rats were assigned to the following groups: non-operation control group, sham operation group, IR group, IR group pretreated with DMSO, IR group pretreated with S1P3 agonist, IR group pretreated with an antagonist of S1P3, IR group pretreated with S1P2 and S1P3 antagonists, IR group pretreated with heptanol and antagonists of S1P2/3, and IR group pretreated with Gap26 and antagonists of S1P2/3 (heptanol acts as a Cx43 uncoupler and the mimic peptide Gap26 as Cx43 blocker). The groups with S1P2 or S1P3 agonist application before reperfusion were used to assess whether these can be used for therapy of IR. The haemodynamics, electrocardiograms (ECG), infarction area, and mortality rates were recorded. Immunohistological connexin 43 (Cx43) expression in the heart was detected in each group. Blocking S1P2/3 receptors with specific antagonists resulted in an increment of IR-induced mortality, increased infarction size, redistribution of Cx43 expression, as well as affecting the heart function. The infarction size, heart function, and mortality were totally or partially restored in the S1P2, S1P3 agonist-pretreated IR group, and the heptanol/Gap26-treated S1P2/3-blocked IR group. The S1P receptor S1P2/3 and Cx43 are involved in the IR-induced cardiac death.

Near-infrared fluorescent labeled CGRRAGGSC peptides for optical imaging of IL-11Rα in athymic mice bearing tumor xenografts

The interleukin-11 (IL-11) and the IL-11 receptor α-subunit (IL-11Rα) have been demonstrated to regulate the invasion and proliferation of tumor cells. Our study intends to evaluate a noninvasive imaging of IL-11Rα expression in breast tumors using near-infrared (NIR) fluorescent dye Cy7-labeled IL-11 mimic peptide CGRRAGGSC. This work evaluated the IL-11Rα expression of breast tumor cells and the binding status of this peptide to IL-11Rα in vitro and in vivo by using Western blotting, immunofluorescence staining and near-infrared fluorescence imaging. Our biochemical study showed that IL-11Rα was overexpressed in breast tumor cells (MCF-7). The cell-binding assay demonstrated specific binding of peptide CGRRAGGSC to MCF-7 cells in vitro. In vivo imaging results showed that NIR fluorescent signals of Cy7-CGRRAGGSC were selectively accumulated in tumor and metabolic organs. While in the blocking experiment, free CGRRAGGSC obviously blocked the concentration of the Cy7-CGRRAGGSC in the tumors. These results suggested that IL-11Rα may be used as a potential target for noninvasive imaging in IL-11Rα overexpressed tumors. Furthermore, the imaging agent of near-infrared fluorescent dye Cy7-labeled CGRRAGGSC is suitable for IL-11Rα expression imaging study in vivo.

Look Closely, the Beautiful May Be Small: Precursor-Derived Peptides in Plants.

During the past decade, a flurry of research focusing on the role of peptides as short- and long-distance signaling molecules in plant cell communication has been undertaken. Here, we focus on peptides derived from nonfunctional precursors, and we address several key questions regarding peptide signaling. We provide an overview of the regulatory steps involved in producing a biologically active peptide ligand that can bind its corresponding receptor(s) and discuss how this binding and subsequent activation lead to specific cellular outputs. We discuss different experimental approaches that can be used to match peptide ligands with their receptors. Lastly, we explore how peptides evolved from basic signaling units regulating essential processes in plants to more complex signaling systems as new adaptive traits developed and how nonplant organisms exploit this signaling machinery by producing peptide mimics.

Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress-induced endothelial dysfunction in renal microvessels.

Protein tyrosine (Tyr) nitration, the covalent addition of a nitro group (•NO2) to Tyr residues, is emerging as a candidate mechanism of endothelial dysfunction. Previous studies have shown that Tyr nitration is primarily induced by nitrosative stress, a process characterized by the production of reactive nitrogen species, especially peroxynitrite anion (ONOO-), which is considered a secondary product of NO in the presence of superoxide radicals (O2•-). However, the impact of nitrosative stress-induced Tyr nitration on endothelial dysfunction has not been thoroughly elucidated to date. We developed an endothelial dysfunction model, a process called "endothelial-to-mesenchymal transition (EndMT)," and evaluated the production of NO, O2•-, and protein nitration during EndMT. The results showed that TGF-β1 stimulation induced EndMT and elevated endothelial NO and O2•- production as well as nitration of the catalytic subunit of protein phosphatase (PP)2A. Mass spectrometry analysis showed that Tyr265 was the nitration site in the catalytic subunit of protein phosphatase (PP)2A, and this Tyr nitration increased PP2A activity and disrupted endothelial integrity. To devise an endothelial-targeted anti-PP2Ac nitration strategy, a mimic peptide, tyrosine 265 wild type (Y265WT), conjugated with the cell-penetrating peptide HIV-1 TAT protein (TAT) was synthesized. PP2Ac nitration and PP2A activity were significantly inhibited by pretreatment with TAT-265WT, and the integrity of endothelial cells was maintained. Furthermore, injection of TAT-265WT attenuated renal nitration formation and caused anticapillary rarefaction in a unilateral urethral obstructive nephropathy model. Taken together, these results offer preclinical proof of concept for TAT-265WT as a tractable agent to protect against nitrosative stress-induced endothelial dysfunction in renal microvessels.-Deng,Y., Cai, Y., Liu, L., Lin, X., Lu, P., Guo, Y., Han, M., Xu, G. Blocking Tyr265 nitration of protein phosphatase 2A attenuates nitrosative stress-induced endothelial dysfunction in renal microvessels.