12-mer Peptide Research Articles

Molecular Mechanism of Selective Binding of Peptides to Silicon Surface

Despite extensive recent research efforts on material-specific peptides, the fundamental problem to be explored yet is the molecular interactions between peptides and inorganic surfaces. Here we used computer simulations (density functional theory and classical molecular dynamics) to investigate the adsorption mechanism of silicon-binding peptides and the role of individual amino acids in the affinity of peptides for an n-type silicon (n(+)-Si) semiconductor. Three silicon binding 12-mer peptides previously elaborated using phage display technology have been studied. The peptides' conformations close to the surface have been determined and the best-binding amino acids have been identified. Adsorption energy calculations explain the experimentally observed different degrees of affinity of the peptides for n(+)-Si. Our residual scanning analysis demonstrates that the binding affinity relies on both the identity of the amino acid and its location in the peptide sequence.

Rindopepimut: a promising immunotherapeutic for the treatment of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive glial cell-derived primary tumor. Current standard of care for patients with GBM includes maximal tumor resection plus adjuvant radiotherapy and temozolomide chemotherapy, increasing median overall survival to a mere 15 months from diagnosis. Because these therapies are inherently nonspecific, there is an increased likelihood of off-target and incomplete effects; therefore, targeted modalities are required for enhanced safety and efficacy. Rindopepimut is emerging as a safe and potentially effective drug for the treatment of GBM. Rindopepimut consists of a 14-mer peptide that spans the length of EGF receptor variant III, a mutant variant of EGF receptor found on approximately 30% of primary GBM, conjugated to the carrier protein keyhole limpet hemocyanin. Vaccination with rindopepimut has been shown to specifically eliminate cells expressing EGF receptor variant III. Phase II clinical trials have suggested that vaccination of newly diagnosed GBM patients with rindopepimut plus adjuvant granulocyte-macrophage colony-stimulating factor results in prolonged progression-free and overall survival with minimal toxicity. This review will outline the development of rindopepimut, as well as the current status of this vaccine.

Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3.

Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.

Unusual antigen recognition provides clue to pathogenicity of diabetogenic CD8 T cell (APP2P.101)

Abstract The defined length restriction as well as preference for amino acids at particular positions for each MHC allele is imposed by the physical as well as chemical nature of the peptide binding groove of the MHC molecule. Accordingly, the preferred peptide for the mouse MHC class I allele H-2Db is a 9mer with anchor residues N at P5 and M/I or L at position 9. However, in this study, we found that AI4, one of the most pathogenic CD8 T cell clones that participates in type 1 diabetes pathogenesis, recognizes an unusual 7mer peptide from the β cell specific protein insulin (InsA14-20) in the context of H-2Db. Further investigation showed that a majority of NOD mice had islet infiltrating T cells that recognized InsA14-20 or mimotopes derived from it, making the AI4-like T cells a very prevalent population. Crystallographic analysis revealed that InsA14-20 shares two solvent-exposed residues with all previously identified AI4 ligands which could explain the promiscuous nature of the AI4 T cell receptor. In addition, accessory anchoring residues were observed to compensate for the lack of the C-terminal anchor residue. In sum, the study demonstrates that less than ideal peptide-MHC interaction can still trigger T cell reactivity and contribute to disease pathogenesis, aided further by lack of thymic negative selection due to weak binding affinity.

A synthetic peptide from Trypanosoma cruzi mucin-like associated surface protein as candidate for a vaccine against Chagas disease

Chagas disease, caused by Trypanosoma cruzi, is responsible for producing significant morbidity and mortality throughout Latin America. The disease has recently become a public health concern to nonendemic regions like the U.S. and Europe. Currently there are no fully effective drugs or vaccine available to treat the disease. The mucin-associated surface proteins (MASPs) are glycosylphosphatidylinositol (GPI)-anchored glycoproteins encoded by a multigene family with hundreds of members. MASPs are among the most abundant antigens found on the surface of the infective trypomastigote stage of T. cruzi, thus representing an attractive target for vaccine development. Here we used immunoinformatics to select a 20-mer peptide with several predicted overlapping B-cell, MHC-I, and MHC-II epitopes, from a MASP family member expressed on mammal-dwelling stages of T. cruzi. The synthetic MASP peptide conjugated to keyhole limpet hemocyanin (MASPpep-KLH) was tested in presence or not of an adjuvant (alum, Al) as a vaccine candidate in the C3H/HeNsd murine model of T. cruzi infection. In considerable contrast to the control groups receiving placebo, Al, or KLH alone or the group immunized with MASPpep-KLH/Al, the group immunized with MASPpep-KLH showed 86% survival rate after challenge with a highly lethal dose of trypomastigotes. As evaluated by quantitative real-time polymerase chain reaction, MASPpep-KLH-immunized animals had much lower parasite load in the heart, liver, and spleen than control animals. Moreover, protected animals produced trypanolytic, protective antibodies, and a cytokine profile conducive to resistance against parasite infection. Finally, in vivo depletion of either CD4+ or CD8+ T cells indicated that the latter are critical for protection in mice immunized with MASPpep-KLH. In summary, this new peptide-based vaccine with overlapping B- and T-cell epitopes is able to control T. cruzi infection in mice by priming both humoral and cellular immunity.

Crotalphine: fluorescent analogues, structural analysis and its effect on co‐cultures of peritoneal macrophages and DRG neurons (LB615)

Crotalphine (CRP) is a 14‐mer peptide isolated from the venom of C. durissus terrificus “rattlesnake” that triggers long‐lasting antinociception (3‐5 days). This effect is mediated by activation of peripheral CB2 cannabinoid receptors, the subsequent release of dynorphin A and activation of opioid receptors. In inflamed tissues, granulocytes and macrophages are the main source of opioid peptides, which play a substantial role in the modulation of pain via interactions with opioid receptors located in peripheral sensory nerve fibers. In this work, we synthesized CRP and fluorescent analogues, analyzed its structure and investigated its effect on peritoneal macrophages and co‐cultures of dorsal root ganglia (DRG) neurons and peritoneal macrophages. All procedures were approved by ethics committee of the Butantan Institute (CEUAIB: 946/12) and performed in accordance to the International Association for the Study of Pain guidelines. CRP and the obtained analogues [Glu1]‐CRP, carboxyfluorescein (CF)‐[Glu1]‐CRP, CF‐[Gln1]‐CRP were able to induce antinociception. Circular dichroism spectra of CRP and [Glu1]‐CRP have shown bands indicative of the presence of random and helix structure and has allowed the construction of a 3D‐structure model of CRP. Fluorescence microscopy and mass spectra analysis of peritoneal resident or LPS‐activated macrophages treated with CF‐[Gln1]‐CRP (1 µM) showed that this peptide internalizes and is distributed in the cytoplasm of the cells, releasing dynorphin A (1‐13) and Met‐enkephalin to extracellular medium. Internalization of κ‐opioid receptors into small DRG neurons in co‐cultures of peritoneal macrophages and DRG neurons treated with bradykinin (10 μM) and CRP (1 μM), was also observed. On the other hand, we do not observed internalization of κ‐opioid receptors into macrophages. In conclusion, the data suggest a functional crosstalk between macrophages and DRG neurons in the presence of CRP. This phenomenon could contribute to the in vivo antinociceptive effect of this molecule.Grant Funding Source: Supported by FAPESP (2011/09333‐7), CeTICs/CEPID/FAPESP (2013/07467‐1) and INCTTox/CNPq (150848/2010‐2)

Retargeting Adenovirus Serotype 48 Fiber Knob Domain by Peptide Incorporation

Adenovirus type 5 (Ad5) is a commonly used vector for gene therapy, but its efficacy is limited by high seroprevalence and off-target hepatic and splenic sequestration. In order to circumvent these limitations, the use of vectors derived from rare species adenoviruses is appealing. The opportunity to retarget rare species vectors to defined cell types through the incorporation of peptide ligands would be advantageous, particularly in targeting tumors and disseminated metastases. We used predictive structural modeling to assess the CD, DG, HI, and IJ loops of the Ad48 fiber knob and identify optimal incorporation locales for the 20-mer peptide, A20FMDV2 (A20). A20FMDV2 targets ανβ6 integrin, which is overexpressed in human carcinomas. Recombinant Ad48 fiber knob proteins Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20, and Knob48-IJ-A20 were engineered and purified after expression in Escherichia coli. We confirmed that Knob48, Knob48-CD-A20, and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 and Knob-HI-A20 failed to form a trimer. All A20-modified knob proteins blocked the transduction of Ad5-EGFPA20 via ανβ6, demonstrating that the inserted A20 peptide was functional. In conclusion, we show that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. Interestingly, in vitro gene transfer mediated by the non-factor-X-binding Ad48 vector was not sensitive to immunoglobulins and complement when incubated in the presence of mouse serum, unlike Ad5. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer favorable characteristics for targeted delivery in vivo.

Hepatitis B splice-generated protein antibodies in Syrian chronic hepatitis B patients: incidence and significance.

Background:Previous studies have suggested hepatitis B splice-generated protein (HBSP), when expressed, is involved in the pathogenesis of HBV infection.Objectives:We aimed to evaluate anti-HBSP incidence and association with several HBV infection parameters in a group of Syrian chronic hepatitis B patients.Patients and Methods:Eighty treatment-naïve HBsAg-positive adult chronic hepatitis B patients' sera were included in our prospective targeted study. Liver function, virological and histological tests results were obtained from patients’ medical files. Three variants of a 20-mer HBSP-derived peptide were designed based on HBV genome sequences obtained from Syrian patients' sera (GenBank Accession No. JN257148-JN257217). Microtiter plate wells were coated with the synthetic peptides and used to detect anti-HBSP antibodies by an optimized indirect enzyme-linked immunosorbent assay (ELISA). Samples were considered positive when showed optical density (OD) values higher than the cut-off value for at least one peptide variant.Results:Seven out of eighty (9%) CHB patients were positive for anti-HBSP antibodies. Mean OD values were not significantly different between HBeAg-positive and -negative patients (P > 0.05). OD values showed weak positive correlation with ALT and AST values (P < 0.05), and weak to moderate positive correlation with liver biopsy staging ranks (P < 0.05). No significant correlation was revealed with viral load values or liver biopsy grading ranks (P > 0.05).Conclusions:We introduced an anti-HBSP antibodies ELISA, designed for locally circulating HBV strains. Correlation observed of Anti-HBSP with liver fibrosis staging regardless of viral replication and liver inflammation suggests anti-HBSP antibodies as possible indicator for HBV-associated liver fibrosis.

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity.Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.

Identification and characterization of a salivary-pellicle-binding peptide by phage display

ObjectiveDental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. MethodsA library of 2×109 phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. ResultsTen unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. ConclusionsUse of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii.

Selection of peptides binding to metallic borides by screening M13 phage display libraries

BackgroundMetal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.ResultsIn this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.ConclusionsThis study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

Investigation of the mechanism of action of novel amphipathic peptides: Insights from solid-state NMR studies of oriented lipid bilayers

We have investigated in the present study the effect of both non-selective and selective cationic 14-mer peptides on the lipid orientation of DMPC bilayers by 31P solid-state nuclear magnetic resonance (NMR) spectroscopy. Depending on the position of substitution, these peptides adopt mainly either an α-helical structure able to permeabilize DMPC and DMPG vesicles (non-selective peptides) or an intermolecular β-sheet structure only able to permeabilize DMPG vesicles (selective peptides). Several systems have been investigated, namely bilayers mechanically oriented between glass plates as well as bicelles oriented with their normal perpendicular or parallel to the external magnetic field. The results have been compared with spectral simulations with the goal of elucidating the difference in the interaction of these two types of peptides with zwitterionic lipid bilayers. The results indicate that the perturbation induced by selective peptides is much greater than that induced by non-selective peptides in all the lipid systems investigated, and this perturbation has been associated to the aggregation of the selective β-sheet peptides in these systems. On the other hand, the oriented lipid spectra obtained in the presence of non-selective peptides suggest the presence of toroidal pores. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage-displayed 12-mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma.

Specific Sequence of a Beta Turn in Human La Protein May Contribute to Species Specificity of Hepatitis C Virus

Human La protein is known to be an essential host factor for translation and replication of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that residues responsible for interaction of human La protein with the HCV internal ribosomal entry site (IRES) around the initiator AUG within stem-loop IV form a β-turn in the RNA recognition motif (RRM) structure. In this study, sequence alignment and mutagenesis suggest that the HCV RNA-interacting β-turn is conserved only in humans and chimpanzees, the species primarily known to be infected by HCV. A 7-mer peptide corresponding to the HCV RNA-interacting region of human La inhibits HCV translation, whereas another peptide corresponding to the mouse La sequence was unable to do so. Furthermore, IRES-mediated translation was found to be significantly high in the presence of recombinant human La protein in vitro in rabbit reticulocyte lysate. We observed enhanced replication with HCV subgenomic and full-length replicons upon overexpression of either human La protein or a chimeric mouse La protein harboring a human La β-turn sequence in mouse cells. Taken together, our results raise the possibility of creating an immunocompetent HCV mouse model using human-specific cell entry factors and a humanized form of La protein. Hepatitis C virus is known to infect only humans and chimpanzees under natural conditions. This has prevented the development of a small-animal model, which is important for development of new antiviral drugs. Although a number of human-specific proteins are responsible for this species selectivity and some of these proteins--mostly entry factors--have been identified, full multiplication of the virus in mouse cells is still not possible. In this study, we show that a turn in the human La protein that is responsible for the interaction with the viral RNA is highly specific for the human sequence. Replacement of the corresponding mouse sequence with the human sequence allows the mouse La to behave like its human counterpart and support viral growth in the mouse cell efficiently. This observation, in combination with previously identified cell entry factors, should open up the possibility of creating a mouse model of hepatitis C.

The influence of ZnO-binding 12-mer peptides on bio-inspired ZnO formation

ZnO precipitation experiments were carried out in a Trizma (tris(hydroxymethyl)aminomethane)-buffered aqueous solution at 37 °C and in the presence of three ZnO-binding 12-mer peptide pairs, which have nearly the same isoelectric point (pI). With this new approach, the influence of peptide sequences on ZnO mineralization under moderate conditions was investigated. Previous work was focused on electrostatic interactions between inorganic-binding peptides and inorganic surfaces. The precipitates were characterized by X-ray diffractometry (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Furthermore, the peptide binding to crystallographic planes of ZnO crystals was modeled by molecular dynamics (MD) simulation in explicit water. The binding free energies of all ZnO-binding peptides were calculated. The combined experimental and modeled results demonstrated a direct correlation between the binding strength of the peptide and the morphology of the ZnO particle, due to differences in their specific binding strength towards polar ZnO (001) and nonpolar (100) surfaces. As a consequence, the ZnO-binding peptides inhibited the growth of ZnO crystals by selective adsorption on the polar or nonpolar ZnO surfaces. This was demonstrated by comparison with a precipitation experiment under peptide-free conditions.

Spectroscopic Studies of Membrane Structure and Interactions of α-Synuclein 71-82

Parkinson's disease is a neurodegenerative disease affecting approximately 100,000 people in Canada, including 25,000 in Quebec. Symptoms such as tremor at rest, rigidity, balance problems, akinesia and bradykinesia, come from the drastic reduction of dopamine. Currently, prescription drugs only reduce these symptoms, but do not stop or slow down the progression of the disease. Several studies have demonstrated the presence of aggregates in the nervous tissues, specifically in the presynaptic terminals. These aggregates cause tissue degeneration. They are made of an amyloid protein called α-synuclein which, depending on the conditions, adopts a structure rich in β sheets and forms protofibrils and fibrils. It comprises 140 amino acids including a hydrophobic core. This section plays a crucial role in the aggregation of the protein. It has been shown that amino acids 71-82 are essential for fibril formation (71VTGVTAVAQKTV82). In the present study, we have investigated the membrane interactions of this 12-mer peptide using 31P solid-state NMR spectroscopy as well as infrared spectroscopy while the peptide structure was studied by infrared spectroscopy. The phospholipids used have different acyl chain lengths and different polar headgroups. The results show that the peptide tends to aggregate and specifically interacts with the negatively charged membranes.

Minimum amino acid residues of an α-helical peptide leading to lipid nanodisc formation.

Nanodiscs are a relatively new class of nanoparticles composed of amphiphilic α-helical scaffold peptides and a phospholipid bilayer, and find potential applications in various fields. In order to identify the minimum number of amino acid residues of an amphiphilic α-helical peptide that leads to nanodisc formation, seven peptides differing in lengths (22-, 18-, 14-, 12-, 10-, 8-, and 6-mers) that mimic and modify the C-terminal domain of apoA-I (residues 220-241) were synthesized. At a concentration of 0.3 mM, the 6- and 8-mer peptides did not present any surface activity. In case of the 10-mer peptide, the aqueous surface tension initially decreased and reached a constant value of 51.9 mN/m with the 14-, 18-, and 22-mer peptides. Moreover, upon mixing the surface-active peptides (14-, 18-, and 22-mers) with dipalmitoylphosphatidylcholine (DMPC) liposomes (2.5:1, peptide : DMPC), the turbid DMPC liposome solution rapidly became transparent. Further analysis of this solution by negative-stain transmission electron microscopy (NS-TEM) indicated the presence of disk-like nanostructures. The average diameter of the nanodiscs formed was 9.5 ± 2.7 nm for the 22-mer, 8.1 ± 2.7 nm for the 18-mer, and 25.5 ± 8.5 nm for the 14-mer peptides. These results clearly demonstrate that the surface properties of peptides play a critical role in nanodisc formation. Furthermore, the minimum length of an amphiphilic peptide from the C-terminal of apoA-I protein that can lead to nanodisc formation is 14 amino acid residues.

In vitro histone lysine methylation by NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L

Histone lysine methylation has a pivotal role in regulating the chromatin. Histone modifiers, including histone methyl transferases (HMTases), have clear roles in human carcinogenesis but the extent of their functions and regulation are not well understood. The NSD family of HMTases comprised of three members (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L) are oncogenes aberrantly expressed in several cancers, suggesting their potential to serve as novel therapeutic targets. However, the substrate specificity of the NSDs and the molecular mechanism of histones H3 and H4 recognition and methylation have not yet been established. Herein, we investigated the in vitro mechanisms of histones H3 and H4 recognition and modifications by the catalytic domain of NSD family members. In this study, we quantified in vitro mono-, di- and tri- methylations on H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20 by the carboxyl terminal domain (CTD) of NSD1, NSD2 and NSD3, using histone as substrate. Next, we used a molecular modelling approach and docked 6-mer peptides H3K4 a.a. 1-7; H3K9 a.a. 5-11; H3K27 a.a. 23-29; H3K36 a.a. 32-38; H3K79 a.a. 75-81; H4K20 a.a. 16-22 with the catalytic domain of the NSDs to provide insight into lysine-marks recognition and methylation on histones H3 and H4. Our data highlight the versatility of NSD1, NSD2, and NSD3 for recognizing and methylating several histone lysine marks on histones H3 and H4. Our work provides a basis to design selective and specific NSDs inhibitors. We discuss the relevance of our findings for the development of NSD inhibitors amenable for novel chemotherapies.

Screening of Peptide Ligands for Pyrroloquinoline Quinone Glucose Dehydrogenase Using Antagonistic Template-Based Biopanning

We have developed a novel method, antagonistic template-based biopanning, for screening peptide ligands specifically recognizing local tertiary protein structures. We chose water-soluble pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH-B) as a model enzyme for this screening. Two GDH-B mutants were constructed as antagonistic templates; these have some point mutations to induce disruption of local tertiary structures within the loop regions that are located at near glucose-binding pocket. Using phage display, we selected 12-mer peptides that specifically bound to wild-type GDH-B but not to the antagonistic templates. Consequently, a peptide ligand showing inhibitory activity against GDH-B was obtained. These results demonstrate that the antagonistic template-based biopanning is useful for screening peptide ligands recognizing the specific local tertiary structure of proteins.

Adamts-18 Is a Novel Candidate Gene Of Vascular Development That Is Related To Aggravated Thrombosis: Evidence From a Adamts-18 Knock Out Mice

HIV-ITP patients have a unique antibody (Ab) against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). The search for a physiologic ligand that could induce this reaction was undertaken by panning the GPIIIa49-66 peptide with a phage surface display 7-mer peptide library. From 20 positive clones, 1 had 70% identity with a C-terminal region of ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin (TSR)-like motif 18), which is secreted by endothelial cell (EC). The recombinant C-terminal fragment of ADAMTS-18 can completely dissolve platelet aggregates formed in vitro. Moreover, this fragment lyses thrombi formed in the cerebral artery of mice and reduces infarction and neurologic impairment in murine ischemic stroke model (Blood 113: 6051, 2009). However, whether ADAMTS-18 represents the dominant physiologic mechanism controlling thrombus dissolution in vivo remains to be clarified.Here, we used ADAMTS-18-deficient (ADAMTS-18-/-) mice to study the contributions of ADAMTS-18 to thrombus formation in vivo. To investigate possible functional differences between WT and ADAMTS-18-/- platelets, we tested WT and ADAMTS-18-/- platelets in a model of pulmonary thromboembolism induced by infusion of a mixture of platelet agonist collagen (25 µg per mouse) and epinephrine (1 µg per mouse). In lung tissue Hematoxylin and eosin-stained (HE) slides, the mean number of thrombi per lung was same in the ADAMTS-18-/- group compared with WT group (163.7 ±14.38 vs 174.9 ±11.73, n=30/group, P=0.5480). In vitro, there is no difference between WT and ADAMTS-18-/- platelet aggregation trace and activation initiated by various platelet agonists ADP (10 µM) or collagen (2 µg/mL). No difference was noted on WT and ADAMTS-18-/- platelet adhesion on immobilized ligand (fibrinogen). These results indicated ADAMTS-18 had no effect on platelet function.We next evaluate the effect of ADAMTS-18 on thrombus formation in a second well-established carotid artery thrombosis model, which is induced by 10% FeCl3 patch. In the process of surgical operation, we unexpectedly observed that all ADAMTS-18-/- mice have premature common carotid artery bifurcation compared with WT mice. A Doppler flow monitor showed ADAMTS-18-/- mice exhibited significantly reduced carotid artery blood flow than WT mice (ADAMTS-18-/- vs WT, 0.5 ± 0.11 vs 0.75 ± 0.21 mL/min, n=7/group, P=0.0298), which results in shortened time of thrombus formation (ADAMTS-18-/- vs WT, 452.17 ± 68.88 vs 611.43 ± 92.02 sec, n=7/group, P=0.0005 ). Immunohistochemistry staining showed that the common carotid artery of ADAMTS-18-/- mice had increased adventitial collagen deposition compared with WT mice. In vivo matrigel plug assay demonstrated that ADAMTS-18-/- mice had significantly lower density of blood vessels compared to the WT mice. Since the middle cerebral artery arises from the internal carotid artery, we conjecture that ADAMTS-18-/- mice would have aggravated brain infarction for the less cerebral blood flow supplying. This proved to be true. In transient middle cerebral artery occlusion (tMCAO) model, the infarction size in ADAMTS-18-/- mice was significantly larger than in WT mice (mean infarction %, 25.68 ± 4.13 vs 17.41 ± 3.24, n=8, P=0.0012). Taken together, these observations suggest vasculature is the potential site of action of ADAMTS-18. To our knowledge, this is the first validation study of linkage and association of ADAMTS-18 as a pro-vasculature gene that is related to aggravated thrombosis. Disclosures:No relevant conflicts of interest to declare.