Peptide Aa Research Articles

Synthetic peptide optimization improves the inhibition of dengue NS2B-NS3 protease and dengue replication in vitro.

Dengue virus (DENV) infection is one of the most widely-spread flavivirus infections with no effective antiviral drugs available. Peptide inhibitors have been considered as one of the best drug candidates due to their high specificity, selectivity in their interactions and minimum side effects. In this study, we employed computational studies using YASARA, HADDOCK server and PyMOL software to generate short and linear peptides based on a reference peptide, CP5-46A, to block DENV NS2B-NS3 protease. The inhibition potencies of the peptides were evaluated using in-house DENV2 serine protease and fluorogenic peptide substrates. In vitro analyses were performed to determine the peptides cytotoxicity and the inhibitory effects against DENV2 replication in WRL-68 cells. Our computational analyses revealed that the docking energy of AYA3, a 16 amino acid (aa) (-81.2 ± 10.6 kcal/mol) and AYA9, a 15 aa peptide (-83.8 ± 6.8 kcal/mol) to DENV NS2B-NS3 protease were much lower than the reference peptide (46 aa; -70.9 ± 7.8 kcal/mol) and the standard protease inhibitor, aprotinin (58 aa; -48.2 ± 10.6 kcal/mol). Both peptides showed significant inhibition against DENV2 NS2B-NS3 protease activity with IC50 values of 24 µM and 23 µM, respectively. AYA3 and AYA9 peptides also demonstrated approximately 68% and 83% of viral plaque reduction without significantly affecting cell viability at 50 µM concentration. In short, we generated short linear peptides with lower cytotoxic effect and substantial antiviral activities against DENV2. Further studies are required to investigate the inhibitory effects of these peptides in vivo. Keywords: peptide inhibitors; dengue virus; NS2B-NS3 protease; plaque reduction.

Systematic Evolution of Ligands by Exosome Enrichment: A Proof-of-Concept Study for Exosome-Based Targeting Peptide Screening.

Selection of a peptide that binds preferentially to targeted cells or tissues is a prerequisite for targeted therapy. Although in vivo phage display is a high-throughput method, it is restricted in identifying target ligands specific for different vascular beds. In this study, the exosomes are repurposed for targeting peptide screening. Briefly, the signal peptide region of Lamp2b (a membrane protein on the exosomes) in the N-terminus is engineered to fuse with 10 aa long random peptides, while the C-terminus of Lamp2b is fused with the MS2 coating protein (MCP). Then, the whole Lamp2b-MCP open reading frame (ORF) is further engineered to harbor a 3'UTR sequence consisting of MS2. The resultant exosomes from engineered Lamp2b-MCP expressing cells display the 10 aa peptides on the outside while containing the genetic information inside. By proof-of-principle experiments, the exosomes with different peptides could preferentially distribute to different tissues besides the spleen and liver. Furthermore, detailed target sequences for different tissues are enriched by rounds of selection. In summary, the established novel targeted peptide screening strategy, namely, "exosome display," has broad applicability, especially for displaying and screening targeted peptides for the cells outside the capillary with condense barriers, like the neurons in the brain.

The structure of Legionella effector protein LpnE provides insights into its interaction with Oculocerebrorenal syndrome of Lowe (OCRL) protein

Legionella pneumophila is a freshwater bacterium that replicates in predatory amoeba and alveolar macrophage. The ability of L.pneumophila to thrive in eukaryotic host cells is conferred by the Legionella containing vacuole (LCV). Formation and intracellular trafficking of the LCV are governed by an arsenal of effector proteins, many of which are secreted by the Icm/Dot Type 4 Secretion System. One such effector, known as LpnE (L.pneumophila Entry), has been implicated in facilitating bacterial entry into host cells, LCV trafficking, and substrate translocation. LpnE belongs to a subfamily of tetratricopeptide repeat proteins known as Sel1-like repeats (SLRs). All eight of the predicted SLRs in LpnE are required to promote host cell invasion. Herein, we report that LpnE(1-375) localizes to cis-Golgi in HEK293 cells via its signal peptide (aa 1-22). We further verify the interaction of LpnE(73-375) and LpnE(22-375) with Oculocerebrorenal syndrome of Lowe protein (OCRL) residues 10-208, restricting the known interacting residues for both proteins. To further characterize the SLR region of LpnE, we solved the crystal structure of LpnE(73-375) to 1.75Å resolution. This construct comprises all SLRs, which are arranged in a superhelical fold. The α-helices forming the inner concave surface of the LpnE superhelix suggest a potential protein-protein interaction interface. DATABASE: Coordinates and structure factors were deposited in the Protein Data Bank with the accession number 6DEH.

Identification of Small Peptides that Inhibit NADPH Oxidase (Nox2) Activation.

Nicotinamide adenine phosphate (NADPH) oxidase type 2 (Nox2), a major source of reactive oxygen species in lungs, plays an important role in tissue damage associated with acute inflammatory diseases. The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6), called aiPLA2, is required for Nox2 activation through its role in the cellular generation of Rac, a key cytosolic component of the activation cascade. Lung surfactant protein A (SP-A) binds to Prdx6, inhibits its aiPLA2 activity, and prevents activation of Nox2. Based on protein docking software, we previously identified a 16 amino acid (aa) peptide derived from rat SP-A as the Prdx6 binding motif. We now identify the minimal effective sequences of rat/mouse and human SP-A as 9-aa sequences that we have called PLA2-inhibitory peptide (PIP).These sequences are PIP-1, rat/mouse; PIP-2, human; and PIP-3, a hybrid of PIPs 1&2. aiPLA2 activity in vitro was inhibited by 50% with ~7–10 µg PIP/µg Prdx6. Inhibition of the aiPLA2 activity and Nox2 activation of lungs in vivo was similar for intratracheal (IT) and intravenous (IV) administration of PIP-2, but required its incorporation into liposomes as a delivery vehicle; tissue ½ time for decrease of the in vivo inhibition of aiPLA2 activity after PIP-2 administration was ~50 h. These properties suggest that PIP-2 could be an effective therapeutic agent to prevent tissue injury associated with lung inflammation.

Abstract 055: ND-13, a DJ-1-Derived Peptide, Protects Against the Renal Fibrosis and Inflammation Associated With Unilateral Ureter Obstruction

Oxidative stress and inflammation are important players in the pathogenesis of cardiovascular and renal diseases. DJ-1 is a redox-sensitive chaperone that regulates the expression of several antioxidant genes. Activation of the DJ-1/Nrf2 pathway in the kidney inhibits the development and progression of several renal diseases. The 20 aa peptide ND-13 consists of 13 highly conserved aa from the DJ-1 sequence and a TAT- derived 7 aa sequence to help in cell penetration. ND-13 prevents neuronal degeneration in mice; however, its effects on kidney damage remain unknown. We hypothesized that treatment with ND-13 would prevent the renal damage and inflammation associated with unilateral ureter obstruction (UUO). C57Bl/6 mice and DJ1 -/- mice underwent UUO and were divided in 3 groups: control (no UUO), UUO+scrambled peptide (SP) or UUO+ND-13 (3 mg/kg, s.c. daily). After 14 days of treatment, urine and kidneys were collected for analysis of renal damage. ND-13 treatment prevented the development of fibrosis in C57Bl/6 mice (UUO+SP: 702±189% of control, UUO+ND-13: 264±8% of control, n=2-4/group, p<0.05), suggesting that ND-13 is protective against connective tissue deposition in the kidney. Treatment with ND-13 decreased renal mRNA expression of TNF- α ( fold change from control: 101±46 in UUO+SP; 18±7 in UUO+ND-13, n=4-5/group, p<0.05), IL-6 (6.7±2 in UUO+SP; 1.54±0.3 in UUO+ND-13, p<0.05), TGF- β ( 3.3±1.12 UUO+SP; 1.4±0.03 UUO+ND-13, p<0.05) and Colagen1 α 1 (79±16 UUO+SP; 33±3.8 in UUO +ND-13, p<0.05) in C57Bl/6 mice. In DJ1 -/- mice, treatment with ND-13 similarly decreased expression of TNF-α, IL-6 and TGF-β, but, in contrast, failed to prevent renal fibrosis or kidney expression of col1 α 1 . UUO also led to elevated urinary NGAL, marker of proximal tubular injury, in DJ-1 -/- mice and ND-13 treatment prevented that increase (71±17% UUO vs control: -18±21% UUO+ND-13 vs control, n=5/group). Our results suggest that ND-13 has protective effects on renal injury, fibrosis and inflammation, crucial mechanisms in the pathogenesis of renal disease. Thus, ND-13 treatment may be a new therapeutic approach for the prevention of renal injury, fibrosis and inflammation in renal diseases. Funded by T32DK007545 to CDM and 5P01 HL074940-10, 7R01 DK039308-31 to PAJ.

Molecular Cloning and Docking of speB Gene Encoding Cysteine Protease With Antibiotic Interaction in Streptococcus pyogenes NBMKU12 From the Clinical Isolates.

Streptococcus pyogenes causes a variety of diseases ranging from mild diseases to severe invasive infections which result in significant morbidity and mortality. This study focuses on the antibiotic resistance of S. pyogenes and their interaction with cysteine protease. Around 36 beta-hemolytic isolates were collected from the clinical lab, of which seven isolates (19.4%) were identified as Streptococcus pyogenes. One of the seven isolates was collected from a urinary tract infection, which was identified by antibody agglutination and MALTI-TOF-MS, and it is designated as S. pyogenes NBMKU12. Around 8.3 to 66.6 % of the isolates were found to be resistant to one or more antimicrobial agents, especially, penicillin-G resistance was exhibited by 29.1% of the isolates. In the NBMKU12 isolate, the beta lactem (TEM) gene was detected among the 13 antibiotic genes for which it was tested. Furthermore, when analysis for presence of 13 virulence genes were carried out in NBMKU12 isolate, only speJ and speB were detected. The speB (streptococcal pyrogenic exotoxin B) encoding cysteine protease gene was cloned. This was followed by performing DNA sequencing to understand the putative cysteine protease interaction with antibiotics, inhibitors, and substrate. The speB gene consists of 1197 nucleotides and encodes a protein with multiple domains, including a signal peptide (aa 1–22), an inhibitor region (aa 27–156), and a catalytic cysteine domain (aa 160–367). The signal peptide cleavage site is predicted between Ala22 and Asn23. The putative 398 amino acid residues were found to have a theoretical pI of 8.76 and a molecular mass of 43,204.36 Da. The tested culture supernatants of NBMKU12 isolate exhibited the proteolytic activity against casein, papaya and pineapple used as substrates. The proteolytic activity suggests the expression of speB gene. Molecular docking analysis of cysteine protease showed that erythromycin (bond length 2.41 Å), followed by chloramphenicol (2.51 Å), exhibited a strong interaction; while penicillin-G (3.24 Å) exhibited a weak interaction, and this factor could be considered as a cause for penicillin-G resistance. The present study contributes to a better understanding of speB gene encoding cysteine protease, antibiotic resistance, and their interaction in the isolate, S. pyogenes NBMKU12. The antibiotics and cysteine protease interaction study confirms the resistance or sensitivity of S. pyogenes. Hence, it could be hypothesized that the isolate NBMKU12 is resistant to most of the tested antibiotics, and this resistance might be a cause for mutation.

Abstract 3446: Deciphering key cancer and inflammation signaling pathways with homogeneous bioluminescent cell-based kinase activity assays

Abstract Signaling pathways activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences, causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential to better understand normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, nonhomogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two-subunit system based on NanoLuc luciferase that was successfully demonstrated for protein-protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF-κB, BTK and JAK/STAT). In very few steps we detected the expected biologic response of these pathways either by monitoring BTK autophosphorylation, assessing phosphorylation and degradation of IκB or phosphorylation of STAT3 upon activation of the cells with TNFα, or IL6, respectively. We also tested different small- or large-molecule inhibitors of these pathways or their corresponding node kinase (BTK, IKK, or JAKs) and obtained the expected pharmacology. The bioluminescent system presented here has many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis, allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors. Citation Format: Hicham Zegzouti, Brian Hwang, Nidhi Nath, Said Goueli. Deciphering key cancer and inflammation signaling pathways with homogeneous bioluminescent cell-based kinase activity assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3446.

P-310 - A peptide derived from naturally occurring surfactant protein A (SP-A) inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) and NOX2 activation and prevents mouse lung injury following intratracheal LPS

We have developed a peptide based on the naturally occurring 26 kDa SP-A protein that inhibits the PLA2 but not the GPx activity of Prdx6. A 16 aa sequence was predicted by the ZDOCK program as the molecular site for binding of SP-A to Prdx6 and was confirmed using ITC and CD measurements.The peptide (called PLA2-inhibitory peptide or PIP) inhibited the PLA2 activity of recombinant human Prdx6 by ~70% and similarly inhibited Prdx6-associated PLA2 activity in homogenates of mouse lungs while presence of the peptide prevented the activation of lung NOX2.We have found since that a 9 aa peptide, named PIP-2,retains the Prdx6-inhibitory properties of both the protein and PIP. PIP was tested in a mouse model of acute lung injury (ALI) generated by the intratracheal instillation of bacterial lipopolysaccharide (LPS).PIP was encapsulated in liposomes for intracellular delivery and administered IV to mice either co-incidentally with or at 12–14 h after LPS. Mouse lungs evaluated at 24 h after LPS showed a marked decrease of lung leukocytic infiltration, of altered permeability, and of the oxidation of tissue macromolecules in the animals receiving PIP. These studies suggest that PIP might be a useful therapeutic for the prevention and treatment of ALI.

Deciphering Key Cancer and Inflammation Signaling Pathways with a Novel Homogeneous Bioluminescent Cell Based Kinase Activity Assays

Signaling pathway activation leads to a multitude of cellular responses including modulation of enzyme activity, altered gene expression, and protein translocation or degradation. Specific phosphorylation events by specific kinases in the cell constitute important nodes in signaling pathways. Altered normal kinase activity leads to several undesirable consequences causing diseases such as cancer and inflammation. Thus, monitoring these signaling events is essential for better understanding of normal cell behavior and disease states. Current assays to analyze cellular kinase activities can be tedious, non‐homogeneous, and not easily adaptable to HTS or suffer from generation of false hits due to fluorescence interference, signal quenching, or require special instruments for detection. To overcome these shortcomings, we developed a simple homogeneous assay platform to detect phosphorylation of specific endogenous proteins in cells. We took advantage of the NanoLuc Binary Technology (NanoBiT), a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated a detection system containing secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments (11 aa peptide and 18 kDa). Cell lysate containing the phosphorylated protein is incubated with two primary antibodies that recognize separate epitopes on a single protein. These will bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of target protein phosphorylation. We tested this system by monitoring the activation of signaling pathways involved in cancer, immune and inflammatory responses (NF‐kB, JAK/STAT, and BTK). With very few steps, we detected the expected biological response of these pathways by monitoring phosphorylation/degradation of IkB, phosphorylation of STAT3, and BTK autophosphorylation. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, JAKs, and BTK) and obtained the expected pharmacology. The bioluminescent system presented here have many advantages including “Add and Read” format, no cell engineering required as the phosphorylation of endogenous substrate is detected in any cell type including primary cells, and only a simple luminometer is required for detection. Our results demonstrate that this technology can be adapted to any signaling pathway analysis allowing scientists to analyze signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of duplicated Midkine genes and their functional regulation in blunt snout bream (Megalobrama amblycephala).

Midkine (Mdk) is a heparin-binding growth factor that is involved in regulating cell growth, differentiation and migration. Here, we report the isolation and characterization of duplicated mdk genes in blunt snout bream (Megalobrama amblycephala). The mdka and -b genes encode 146 aa and 147 aa peptides, respectively, sharing a sequence identity of 64%. During embryogenesis, mdka mRNA is detectable after 12 h post-fertilization (hpf) and mdkb mRNA can be detected after 8 hpf, about 4 h prior to mdka mRNA. Whole-mount in situ hybridization demonstrated that two paralogs of mdk mRNA were detected in the brain and dorsal neural tube at 16 hpf. At 22 hpf, mdka mRNA was abundant in the brain and dorsal neural tube, whereas mdkb mRNA were transcribed in the brain and tailbud. Later, at 55 hpf, both paralogs were mainly expressed in the brain. Furthermore, both the mdk genes were highly expressed in multiple adult tissues except in the skin and a low expression of mdka in the muscle. In addition, they were differentially inhibited in the liver and intestine with exogenous recombinant human growth hormone, while their mRNA levels were up-regulated in the brain. During starvation, both the mdk genes were significantly up-regulated in the intestine, brain and liver and returned to the control levels following 6 days of refeeding. Our results suggest that duplicated mdk genes may play conserved and divergent roles in embryonic development and tissue growth regulation in blunt snout bream.

Bifidobacterium bifidum Extracellular Sialidase Enhances Adhesion to the Mucosal Surface and Supports Carbohydrate Assimilation.

ABSTRACTBifidobacterium is a natural inhabitant of the human gastrointestinal (GI) tract. We studied the role of the extracellular sialidase (SiaBb2, 835 amino acids [aa]) from Bifidobacterium bifidum ATCC 15696 in mucosal surface adhesion and carbohydrate catabolism. Human milk oligosaccharides (HMOs) or porcine mucin oligosaccharides as the sole carbon source enhanced B. bifidum growth. This was impaired in a B. bifidum ATCC 15696 strain harboring a mutation in the siabb2 gene. Mutant cells in early to late exponential growth phase also showed decreased adhesion to human epithelial cells and porcine mucin relative to the wild-type strain. These results indicate that SiaBb2 removes sialic acid from HMOs and mucin for metabolic purposes and may promote bifidobacterial adhesion to the mucosal surface. To further characterize SiaBb2-mediated bacterial adhesion, we examined the binding of His-tagged recombinant SiaBb2 peptide to colonic mucins and found that His-SiaBb2 as well as a conserved sialidase domain peptide (aa 187 to 553, His-Sia) bound to porcine mucin and murine colonic sections. A glycoarray assay revealed that His-Sia bound to the α2,6-linked but not to the α2,3-linked sialic acid on sialyloligosaccharide and blood type A antigen [GalNAcα1-3(Fucα1-2)Galβ] at the nonreducing termini of sugar chains. These results suggest that the sialidase domain of SiaBb2 is responsible for this interaction and that the protein recognizes two distinct carbohydrate structures. Thus, SiaBb2 may be involved in Bifidobacterium-mucosal surface interactions as well as in the assimilation of a variety of sialylated carbohydrates.

Abstract 4495: Elucidating BCL11A’s function in triple negative breast cancer

Abstract It has been recently shown that the B-cell CLL/lymphoma 11A (BCL11A) transcription factor is highly expressed in triple-negative breast cancer (TNBC) and has critical functions in promoting tumor growth and mammary stem cell maintenance. Studies in our group have found that the first twelve amino acids (aa 1-12) of BCL11A are a conserved sequence that allows binding to retinoblastoma binding protein 4 (RBBP4). RBBP4, a histone binding protein, is involved in many epigenetic complexes, including the Polycomb repressive complex 2 (PRC2), the nucleosome remodeling and deacetylase (NuRD) complex, and SIN3A complex. Through these interactions BCL11A may exert transcriptional and epigenetic regulation. Herein we investigate the importance of BCL11A’s interaction with RBBP4 and its function in TNBC. BCL11A-complex interactions were observed through pull-down experiments of TNBC cell lysate utilizing aa 2-16 of BCL11A and a scramble control sequence. Our results demonstrate that this 16-amino acid sequence is sufficient to bind to RBBP4 and pull down the PRC2, NuRD, and SIN3A complexes. Furthermore, the NuRD and Sin3a complexes pulled down by BCL11A aa 2-16 retained their functionality as observed through post pull-down histone deacetylase (HDAC) activity assays. These results indicate significantly higher HDAC activity in the wildtype sequence as compared to the scrambled sequence. To further investigate this interaction, protein crystallization was conducted, the results of which validate the binding between BCL11A and RBBP4. Additionally, a BCL11A peptide (aa 2-12) and a scramble control were transfected into the TNBC cell line SUM149. Analysis via flow cytometry shows a reduction of the ALDH+ cell population in the BCL11A peptide transfected cells versus scramble control. We are currently investigating the necessity of this 12-amino acid sequence in interactions with RBBP4, PRC2, NuRD and SIN3A via co-Immunoprecipitation experiments using an N-terminal truncated BCL11A protein (del 2-12). The effects of full-length BCL11A and del 2-12 on the ALDH+ cell population are also being examined. These experiments will further delineate this sequence’s importance in BCL11A-RBBP4 interactions. Citation Format: Nicholas O. Stevers, Samantha L. Tinsley, Rebecca Moody, Chang-Ching Lin, Jennifer Meagher, Jeanne A. Stuckey, Miao-Chia Lo, Duxin Sun. Elucidating BCL11A’s function in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2017-4495

Synthetic peptide TEKKRRETVEREKE derived from ezrin induces differentiation of NIH/3T3 fibroblasts

Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-β1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-β1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds.

Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species.

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3'UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC50. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

Ameloblastin Peptides Modulates the Osteogenic Capacity of Human Mesenchymal Stem Cells.

During amelogenesis the extracellular enamel matrix protein AMBN is quickly processed into 17 kDa (N-terminus) and 23 kDa (C-terminus) fragments. In particular, alternatively spliced regions derived by exon 5/6 within the N-terminus region are known to be critical in biomineralization. Human mesenchymal stem cells (hMSC) also express and secrete AMBN, but it is unclear if this expression has effects on the hMSC themselves. If, as suggested from previous findings, AMBN act as a signaling molecule, such effects could influence hMSC growth and differentiation, as well as promoting the secretion of other signaling proteins like cytokines and chemokines. If AMBN is found to modulate stem cell behavior and fate, it will impact our understanding on how extracellular matrix molecules can have multiple roles during development ontogenesis, mineralization and healing of mesenchymal tissues. Here we show that synthetic peptides representing exon 5 promote hMSC proliferation. Interestingly, this effect is inhibited by the application of a 15 aa peptide representing the alternatively spliced start of exon 6. Both peptides also influence gene expression of RUNX2 and osteocalcin, and promote calcium deposition in cultures, indicating a positive influence on the osteogenic capacity of hMSC. We also show that the full-length AMBN-WT and N-terminus region enhance the secretion of RANTES, IP-10, and IL-8. In contrast, the AMBN C-terminus fragment and the exon 5 deleted AMBN (DelEx5) have no detectable effects on any of the parameters investigated. These findings suggest the signaling effect of AMBN is conveyed by processed products, whereas the effect on proliferation is differentially modulated through alternative splicing during gene expression.

Blocking the Interaction between EphB2 and ADDLs by a Small Peptide Rescues Impaired Synaptic Plasticity and Memory Deficits in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an age-dependent neurodegenerative disorder and amyloid β-derived diffusible ligands (ADDLs) play a key role in triggering the early cognitive deficits that constitute AD. ADDLs may bind EphB2 and alter NMDA receptor trafficking and synaptic plasticity. Here, we identified the interaction sites of the EphB2 FN domain with ADDLs for the first time to develop a small (10 aa) peptide (Pep63) capable of blocking the EphB2-ADDL interaction. We found that Pep63 not only rescued the ADDL-induced depletion of EphB2 and GluN2B-containing NMDA receptors from the neuronal surface in cultured hippocampal neurons, but also improved impaired memory deficits in APPswe/PS1dE9 (APP/PS1) transgenic mice. Our results suggest that blocking the EphB2-ADDL interaction with Pep63 may be a promising strategy for AD treatment.

Characterization of Individual Histone Posttranslational Modifications and Their Combinatorial Patterns by Mass Spectrometry-Based Proteomics Strategies.

Histone posttranslational modifications (PTMs) play an essential role in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. Such PTMs are mostly localized on histone N-terminal tails where, as single units or in a combinatorial manner, they influence chromatin reader protein binding and fine-tune the abovementioned activities. Mass spectrometry (MS) is currently the most adopted strategy to characterize proteins and protein PTMs. We hereby describe the protocols to identify and quantify histone PTMs and their patterns using either bottom-up or middle-down proteomics. In the bottom-up strategy we obtain 5-20 aa peptides by derivatization with propionylation followed by trypsin digestion. The newly generated N-termini of histone peptides can be further derivatized with light or isotopically heavy propionyl groups to increase chromatographic retention and allow multiplexed analyses. Moreover, we describe how to perform derivatization and trypsin digestion of histones loaded into a gel, which is usually the final step of immunoprecipitation experiments. In the middle-down strategy we obtain intact histone tails of 50-60 aa by digestion with the enzyme GluC. This allows characterization of combinatorial histone PTMs on N-terminal tails.

Genomic organization, expression and antimicrobial activity of a hepcidin from taimen (Hucho taimen, Pallas).

Hepcidin, an antimicrobial peptide, plays a crucial role in innate immune system of teleost fish. As a cysteine-rich peptide, hepcidin possesses a dual function including iron regulation and innate immunity. In the present study, a full-length hepcidin cDNA (HtHep) was cloned and characterized by RT-PCR and RACE techniques from taimen (Hucho taimen, Pallas), which is a type of rare, precious and cold-water fish species in China. The cDNA contains an open reading frame (ORF) of 267 bp encoding 88 amino acid (aa), with 170 bp located in the 5(') untranslated region (UTR) and 151 bp in the 3' UTR. The genomic sequences analysis showed that the HtHep gene consisted of three exons and two introns (with the length 94 and 251 bp, respectively). With a predicted molecular mass of 2881.4Da and a theoretical pI of 8.53, the deduced amino acid encodes a signal peptide of 24 aa, prodomain of 39 aa and mature peptide of 25 aa. The signal peptidase (SA-VP) and the motif RX (K/R)R of propeptide convertase suggested the cleavage site of signal and mature peptide. Eight conserved cysteine residues were also identified and formed four disulfide bonds. Pair-wise alignments showed that HtHep clustered together with two fish species of Salmonidae family (Salmo salar and Oncorhynchus mykiss) in HAMP1 branch. Quantitative RT-PCR analysis indicated that the mRNA levels of HtHep were detected in a wide range of tissues and the highest level was detected in the liver. Its expression was also detected early during embryonic stage and could be up-regulated in the liver when challenged with pathogenic bacteria (Yersinia ruckeri). The recombinant HtHep (rHtHep) had antimicrobial activity against both gram-positive (Micrococcus lysodeikticus and Staphylococcus aureus) and gram-negative bacteria (Escherichia coli). Our results suggested that HtHep might be involved in the innate immune defense against bacterial pathogens in taimen.

Abstract 3091: Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction

Abstract Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.

The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus.

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely.