Short Polypeptide Research Articles

The Peptidoglycan Biosynthesis Gene murC in Frankia: Actinorhizal vs. Plant Type.

Nitrogen-fixing Actinobacteria of the genus Frankia can be subdivided into four phylogenetically distinct clades; members of clusters one to three engage in nitrogen-fixing root nodule symbioses with actinorhizal plants. Mur enzymes are responsible for the biosynthesis of the peptidoglycan layer of bacteria. The four Mur ligases, MurC, MurD, MurE, and MurF, catalyse the addition of a short polypeptide to UDP-N-acetylmuramic acid. Frankia strains of cluster-2 and cluster-3 contain two copies of murC, while the strains of cluster-1 and cluster-4 contain only one. Phylogenetically, the protein encoded by the murC gene shared only by cluster-2 and cluster-3, termed MurC1, groups with MurC proteins of other Actinobacteria. The protein encoded by the murC gene found in all Frankia strains, MurC2, shows a higher similarity to the MurC proteins of plants than of Actinobacteria. MurC2 could have been either acquired via horizontal gene transfer or via gene duplication and convergent evolution, while murC1 was subsequently lost in the cluster-1 and cluster-4 strains. In the nodules induced by the cluster-2 strains, the expression levels of murC2 were significantly higher than those of murC1. Thus, there is clear sequence divergence between both types of Frankia MurC, and Frankia murC1 is in the process of being replaced by murC2, indicating selection in favour of murC2. Nevertheless, protein modelling showed no major structural differences between the MurCs from any phylogenetic group examined.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Application of short hydrophobic elastin-like polypeptides for expression and purification of active proteins.

To investigate the application of short elastin-like polypeptides (ELPs) in the purification of bioactive proteins, short hydrophobic ELP[I] n (n = 30, 40, 50) tags were constructed. Both the ELP[I] n tags and the ELP[I] n -Trx fusion proteins could be stably expressed in Escherichia coli and purified by inverse transition cycling, respectively. Total protein concentrations determined by BCA protein assay showed that the yield of the fusion proteins decreased with increasing ELP length. Measurements of the inverse transition temperature (T t) of the ELP[I] n -Trx under different salts or PEG8000 concentrations showed decreased T t upon elevated concentrations; while, all the T ts were suitable for generating proteins from 4 to 37.5ºC. Furthermore, to identify a linker peptide for bioactive protein production without the need to remove the ELP[I] n tag, the activity of eGFP protein fused with ELP[I]30 tag by either a poly-N or a G4S linker was quantified using a fluorescence spectrophotometer. The results indicated that the ELP[I]30-eGFP fusion proteins with the poly-N linker showed higher fluorescence levels than those with the G4S linker. Our results demonstrated that short ELP[I] n tags with low T t were useful in protein expression and purification, and poly-N linker played the key role in producing bioactive proteins without the need to remove the ELPs.

Effective Strategies to Overcome the Insolubility of Recombinant ScFv Antibody against EpCAM Extracellular Domain in E. coli

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein overexpressed in epithelial-derived neoplasms and therefore is a highly interesting target for antibody therapy in a wide range of carcinomas. Single chain variable fragment (ScFv) antibodies, generated by the association of the variable heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker, retain the binding properties of classical antibodies. Due to its characteristics, Escherichia coli (E. coli) has inspired a great deal of interest for production of antibody fragments with high concentrations. Here, ScFv against EpCAM extracellular domain (EpEX) was expressed in E. coli BL21™(DE3) strain. The effect of different expression conditions on the total protein level was also investigated. Moreover, an attempt was made to overcome the problem of insolubility of the recombinant protein with alterations of expression condition like inducer concentration and temperature as well as addition of the solubility-enhancing agents. Our results showed that the maximum total protein expression was attained 7 h after induction at 37 °C with 0.5 mM IPTG (663.53 ± 7.33 mg/l). Moreover, the expressed antiEpEX-ScFv protein was 39.8% or 29.1% soluble in the presence of 50% glycerol, and Tween20 plus 50% glycerol respectively. Although the solubility of recombinant protein was significantly increased from 39.8% at 37 °C to 43.7% at 16 °C, the maximum level of soluble recombinant protein was attained at 37 °C. Consequently, we report a strategy combining different culture conditions and the solubility-enhancing additives such as glycerol for improving protein solubility.

Humanin gene expression in fibroblast of Down syndrome subjects.

Down syndrome (DS) is characterized by trisomy of chromosome 21 and peculiar phenotype. Humanin (HN) is a mitochondrial short 24-residue polypeptide whit anti-apoptotic and neuroprotective effects. In this study we evaluated HN protein expression and HN mRNA levels in cultured fibroblasts from DS patients and normal controls. Our results obtained by immunocytochemistry, western-blot and qRT-PCR analysis show a significant HN up-regulation in DS patients. These results confirm previous studies and suggest a role for HN may in the DS phenotype.

PH-responsive hydrogels based on the self-assembly of short polypeptides for controlled release of peptide and protein drugs

In this study, a pH-responsive hydrogel consisting of a 4-arm poly(ethylene glycol)-block-poly(L-glutamic acid) (4a-PEG-PLG) copolymer was developed and used for the controlled release of peptide and protein drugs. It was found that the mechanical properties and degradation processes of the hydrogels could be tuned by changing the polymer concentrations. In vitro drug release results revealed that the release of insulin (or BSA) from hydrogel was highly dependent on the pH, i.e., less than 20% of insulin (or BSA) was released in the artificial gastric fluid (AGF) at 72 h, while close to 100% of insulin (or BSA) was released in the artificial intestinal fluid (AIF). It was because that the deprotonation of carboxyl groups in PLG block caused the disassembly, and even disintegration of the hydrogel in AGF, thereby resulting in accelerated drug release. Circular dichroism spectra showed that the bioactivities of insulin and BSA released from hydrogels were obviously unchanged compared to those of native insulin and BSA, respectively. Mouse fibroblast L929 cells were cultured on the surface of hydrogels and the viabilities of cultured cells were above 90% after incubation for 24 h, indicating that the hydrogels had good cytocompatibilities. Moreover, in vivo degradation evaluation disclosed that the formed hydrogels will completely degrade after 8 days, and the H&E staining study demonstrated the excellent biocompatibility of the as-prepared hydrogels. Therefore, the biocompatible and biodegradable 4a-PEG-PLG hydrogel may serve as a promising platform for pH-responsive drug delivery.

Tuning the Photophysical Features of Self-Assembling Photoactive Polypeptides for Light-Harvesting

The LH1 complex is the major light-harvesting antenna of purple photosynthetic bacteria. Its role is to capture photons, and then store them and transfer the excitation energy to the photosynthetic reaction center. The structure of LH1 is modular and it cooperatively self-assembles from the subunits composed of short transmembrane polypeptides that reversibly bind the photoactive cofactors: bacteriochlorophyll and carotenoid. LH1 assembly, the intra-complex interactions and the light-harvesting features of LH1 can be controlled in micellar media by varying the surfactant concentration and by adding carotenoid and/or a co-solvent. By exploiting this approach, we can manipulate the size of the assembly, the intensity of light absorption, and the energy and lifetime of its first excited singlet state. For instance, via the introduction of Ni-substituted bacteriochlorophyll into LH1, the lifetime of this electronic state of the antenna can be shortened by almost three orders of magnitude. On the other hand, via the exchange of carotenoid, light absorption in the visible range can be tuned. These results show how in a relatively simple self-assembling pigment-polypeptide system a sophisticated functional tuning can be achieved and thus they provide guidelines for the construction of bio-inspired photoactive nanodevices.

Conformational Preference of Macrocycles Investigated by Ion-Mobility Mass Spectrometry and Distance Geometry Modeling.

Macrocycles provide intricate shape manifolds that leverage the depth of the modern organic chemistry toolbox. Novel chemistry can be introduced via new bond types and unique torsional angles inaccessible by traditional small molecules and biomolecules. In this work, we investigate the conformational space of a class of biscationic macrocycles in protic and aprotic solvents using a combination of ion-mobility spectrometry mass spectrometry, distance geometry modeling, and quantum mechanical calculations. We identify at least three major conformations of the macrocycles. Two of the conformations are rotational isomers in which the amide (carbonyl amide) N-C bond of the acyl hydrazine can adopt either E- or Z-configuration. The E- and Z-rotational isomers were separately observed in previous X-ray crystallography studies on the same set of macrocycles, but both isomers were never proved to exist for the same molecule. We show that low-dielectric solvents and counterions, such as Cl- or PF6-, appear to stabilize the Z-conformation. Lastly, desolvation of the macrocycles in the absence of bound counterions yields a gas-phase "flat" Z-conformation. Our results suggest that the macrocycles are flexible and behave much like short polypeptides. The combination of ion-mobility spectrometry mass spectrometry and distance geometry modeling provides a versatile and robust approach to unravel fundamental information on the flexible chemical space of macrocycles.

PerioVax3, a key antigenic determinant with immunoprotective potential against periodontal pathogen

Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.

A hybrid, bottom-up, structurally accurate, Go¯-like coarse-grained protein model.

Coarse-grained (CG) protein models in the structural biology literature have improved over the years from being simple tools to understand general folding and aggregation driving forces to capturing detailed structures achieved by actual folding sequences. Here, we ask whether such models can be developed systematically from recent advances in bottom-up coarse-graining methods without relying on bioinformatic data (e.g., protein data bank statistics). We use relative entropy coarse-graining to develop a hybrid CG but Go¯-like CG peptide model, hypothesizing that the landscape of proteinlike folds is encoded by the backbone interactions, while the sidechain interactions define which of these structures globally minimizes the free energy in a unique native fold. To construct a model capable of capturing varied secondary structures, we use a new extended ensemble relative entropy method to coarse-grain based on multiple reference atomistic simulations of short polypeptides with varied α and β character. Subsequently, we assess the CG model as a putative protein backbone forcefield by combining it with sidechain interactions based on native contacts but not incorporating native distances explicitly, unlike standard Go¯ models. We test the model's ability to fold a range of proteins and find that it achieves high accuracy (∼2 Å root mean square deviation resolution for both short sequences and large globular proteins), suggesting the strong role that backbone conformational preferences play in defining the fold landscape. This model can be systematically extended to non-natural amino acids and nonprotein polymers and sets the stage for extensions to non-Go¯ models with sequence-specific sidechain interactions.

Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions.

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.

Identifying peptides that specifically bind to MDA-MB-468 breast cancer cells*

Abstract Objective To use phage display technique to screen for small polypeptides that specifically bind to MDAMB-468 cells. Methods A random heptapeptide phage display library was used for in vitro screening against target MDA-MB-468 cells. SC1180 cells were used for subtractive selection. High-affinity phage DNA was extracted, and peptides were sequenced. Results (1) The original library capacity of the polypeptide library was 2 × 1013 pfu/mL, and phage titer was determined over 4 rounds. The average library capacity was 1.8 × 1013 pfu/mL. (2) Subtractive screening showed that the phage library volume of each round was 1.8 × 1012 pfu/mL, and that there was an enrichment effect in each subsequent round. Screening was stopped after the fourth round. (3) PCR results showed that the size of 39 products (78.0%) and 11 products (22%), were 300 bp and 258 bp, respectively. Thirty positive phages were selected for DNA extraction and sequencing, and the corresponding amino acid sequence was LMTRXSK. The sequence had no homology with known genes or proteins. Conclusion Using the phage display technique, we identified that the short polypeptide, LMTRXSK, specifically binds MDA-MB-468 human breast cancer cells.

Peptidoglycan Recognition by Wheat Germ Agglutinin. A View by NMR

Wheat germ agglutinin (WGA) is a lectin composed of 4 homologous hevein domains. It has been shown that WGA binds N-acetyl glucosamine (GlcNAc)-related oligosaccharides and has applications as commercial reagent to detect glycans containing such modified residues. Peptidoglycan (PGN), the main component of the bacterial cell wall, is a polymeric material made of repeating disaccharide units of GlcNAc- N-acetylmuramic acid cross-linked with short polypeptide fragments. Wheat germ agglutinin is able to bind bacterial cells, a phenomenon that could correlate with its plant-defense capacities, but there is no information at the molecular level about how WGA binds to the PGN. Herein, we present structural data on the binding of a short PGN fragment to WGA by means of saturation transfer difference nuclear magnetic resonance studies. The results show that the GlcNAc residue establishes the major contacts with WGA, followed by the N-acetylmuramic acid residue. In contrast, the peptide moiety displays minor contacts at the binding site.

Non-reciprocal light-harvesting nanoantennae made by nature

Most of our current understanding of mechanisms of photosynthesis comes from spectroscopy. However, the classical definition of a radio antenna can be extended to the optical regime to discuss the function of light-harvesting antennae. Further to our previously proposed model of a loop antenna, we provide several more physical explanations in considering the nonreciprocal properties of light harvesters of bacteria. We explain the function of the nonheme iron at the reaction center and present reasons for each module of the light harvester being composed of one carotenoid, two short α-helical polypeptides, and three bacteriochlorophylls; we also explained the toroidal shape of the light harvester, the upper bound of the characteristic length of the light harvester, the functional role played by the observed long-lasting spectrometric signal, and the observed photon antibunching. Based on these analyses, two mechanisms that might be used by radiation-durable bacteria, Deinococcus radiodurans; and the nonreciprocity of an archaeon, Haloquadratum walsbyi, are analyzed. The physical lessons involved are useful for designing artificial light harvesters, optical sensors, wireless power chargers, passive superPlanckian heat radiators, photocatalytic hydrogen generators, and radiation protective cloaks. In particular, it can predict what kind of particles should be used to separate sunlight into a photovoltaically and thermally useful range to enhance the efficiency of solar cells.

Ionic polypeptide tags for protein phase separation.

Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. The protein design factors that govern complex coacervation under varying conditions are still relatively unexplored. Recent work indicates that proteins with an intrinsically disordered region, a higher net charge, or a patch of charged residues are more likely to undergo a phase transition. Based on these design parameters, polyionic coacervation tags were designed and assessed for their ability to promote protein complex coacervation with oppositely charged polyelectrolytes. The phase behavior of a panel of engineered proteins was evaluated with the strong polycation poly(4-vinyl N-methyl pyridinium iodide). Proteins containing the ionic tags formed liquid coacervate droplets, while isotropically charged protein variants formed solid precipitates. The ionic tags also promoted phase separation at higher salt concentrations than an isotropic distribution of charge on the protein surface. The salt dependence of the protein complex coacervation could be predicted independently for tagged or isotropic variants by the ratio of negative-to-positive residues on the proteins and universally by calculating the distance between like charges. The addition of just a six residue polyionic tag generated a globular protein capable of liquid-liquid phase separation at physiological pH and ionic strength. This model system has provided the initial demonstration that short, ionic polypeptide sequences (6-18 amino acids) can drive the liquid-liquid phase separation of globular proteins.

Generation of a Vaccine against Group B Streptococcal Infection on the Basis of a Cold-Adapted Influenza A Virus

The aim of this study was to develop a cold-adapted influenza A (H7N9) strain-based vector vaccine expressing epitopes of conserved lipoprotein of Streptococcus agalactiae. Recombinant hemagglutinin (HA) genes of live attenuated influenza vaccine (LAIV) strain A/Anhui/1/2013(H7N9) with insertions of group B Streptococcus (GBS) surface antigen ScaAB gene parts of different length in-frame between signal peptide and HA1 were constructed by means of reverse genetics. Viral vector vaccine H7-ScaAB-85 expressing the shortest (85 amino acids) polypeptide from lipoprotein ScaAB linked to HA via a flexible linker was genetically stable, replicated in embryonated chicken eggs and MDCK cells at the same level as cold-adapted influenza A (H7N9) strain. An increase of ScaAB polypeptide insertion length up to 141 amino acids resulted in a decrease of the viral replication level in both susceptible systems and intense mutagenesis of hybrid HA-gene of recombinant influenza A virus in the gene region encoding foreign bacterial lipoprotein. These findings suggest that H7-ScaAB-85 is a promising vaccine candidate, which should be further assessed using a mouse model.

Single-Molecule Discrimination of Labeled DNAs and Polypeptides Using Photoluminescent-Free TiO2 Nanopores.

Multicolor fluorescence substantially expands the sensing capabilities of nanopores by complementing or substituting the resistive pulsing signals. However, to date single-fluorophore detection in multiple color channels has proven to be challenging primarily due to high photoluminescence (PL) emanating from the silicon nitride (SiN x) membrane. We hypothesize that the high bandgap of titanium oxide (TiO2) would eliminate the PL background when used as a substrate for a nanopore, and hence enable individual fluorophore sensing during the fast passage of biomolecules through the pore. Herein, we introduce a method for fabricating locally supported, free-standing, TiO2 membranes, in which solid-state nanopores can be readily drilled. These devices produce essentially no PL in the blue-to-red visible spectral range, even when excited by multiple lasers simultaneously. At the same time, the TiO2 nanopores exhibit low electrical noise comparable with standard SiN x devices. Importantly, the optical signal-to-background ratio (SBR) in single-molecule sensing is improved by an order of magnitude, enabling the differentiation among labeled DNA molecules of similar length based solely on their labeling scheme. Finally, the increased SBR of the TiO2 devices allows detection of single fluorophores conjugated to the lysine or cysteine residues of short polypeptides, thus introducing the possibility for optical based peptide/protein discrimination in nanopores.

Molecular structure, expression and antibacterial characterization of a novel antimicrobial peptide NK-lysin from the large yellow croaker Larimichthys crocea

A commercially important marine fish of the Chinese aquaculture industry, the large yellow croaker (Larimichthys crocea), has been seriously threatened by white spot disease and bacterial infection caused by a ciliate protozoan parasite Cryptocaryon irritans. Chemical drugs have a low efficiency in preventing this disease and have side effects on the environment; therefore, new methods for disease treatment are urgently needed. NK-lysin is the component of natural killer cells (NK) and cytotoxic T lymphocytes (CTLs), and is considered a potential antimicrobial peptide. In the current study, we describe LCNKL2 in the large yellow croaker, a novel NK-lysin gene obtained from a liver transcriptome database. The open reading frame of its cDNA sequence was 417 bp, which coded for 138 amino acids. Six completely conserved cysteine residues and a Saposin B domain were found in its deduced amino acid sequence. Compared with NK-lysins reported in other teleosts and higher vertebrates, the polypeptide structure of LCNKL2 is more variable, but still contains the characteristics seen in other NK-lysin members, providing clear evidence it belongs to the NK-lysin family. In healthy large yellow croaker, the LCNKL2 gene was mainly detected in head kidney, spleen and gill, and not detected in brain. During pathogen stimulation, the expression of LCNKL2 gene significantly changed, suggesting a role in the fishes immune response. A short polypeptide from the core region of the LCNKL2 mature peptide sequence was synthesized and its antibacterial activity was tested. The result showed that this short peptide could inhibit Staphylococcus aureus and Vibrio harveyi growth effectively, but was not effective against Aeromonas hydrophila and Photobacteria damselae. Our results indicate LCNKL2 is a novel member of the teleosts NK-lysin family, and as an immune-related factor is involved in the immune response induced by pathogens.

The Putative Peptide Gene FEP1 Regulates Iron Deficiency Response in Arabidopsis.

Iron is an essential element for all organisms, and plants have developed sophisticated systems to acquire iron and maintain iron homeostasis. We found that an Arabidopsis thaliana ABA-hypersensitive mutant, aba hypersensitive germination2-1 (ahg2-1), that is known to be defective in mitochondrial mRNA regulation, had increased expression of iron deficiency response genes. The ahg2-1 mutant had lower heme levels than the wild type. Transcriptome data further revealed that novel genes encoding short polypeptides were highly expressed in this mutant. The expression of one of these genes, which we named FE-UPTAKE-INDUCING PEPTIDE 1 (FEP1), was induced under iron-deficient conditions and was observed in the vascular tissues of the leaves and roots, as well as in leaf mesophyll cells. Notably, deletion or insertion mutations of FEP1 exhibited impaired iron accumulation in shoots but normal iron levels in roots. Artificially induced expression of FEP1 was sufficient to induce iron deficiency response genes, such as basic HELIX-LOOP-HELIX 38 (bHLH38), bHLH39, IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2), and led to iron accumulation in planta. Further analysis confirmed that the encoded peptide, but not the FEP1 RNA, was responsible for this activity. Remarkably, the activation of bHLH39 by FEP1 was independent of FER-LIKE IRON DEFICIENCY INDUCED (FIT), a key transcription factor in the iron deficiency response. Taken together, our results indicate that FEP1 functions in iron homeostasis through a previously undescribed regulatory mechanism for iron acquisition in Arabidopsis.

Abstract 5718: Ex vivo ATLAS-identification of neoantigens for personalized cancer immunotherapy in mouse melanoma

Abstract Neoantigens are emerging as attractive vaccine targets for personalized cancer immunotherapy. As opposed to tumor-associated antigens, neoantigens contain non-synonymous mutations that enable their identification as foreign targets not subject to central tolerance in the thymus. Personalized cancer vaccines leverage neoantigens to specifically direct the immune system to recognize cancer cells for the coordinated attack and destruction of tumors. While in silico methods are commonly used to predict immunogenic neoantigens primarily via putative binding to major histocompatibility complexes (MHC), the positive predictive value of these approaches is low as they cannot account for the complexity of antigen processing, the diversity of MHC class I and class II alleles, and the additional steps of T cell activation. Ex vivo technologies have the potential to overcome the limitations of neoantigen identification by utilizing biologically-relevant testing. ATLAS™ is an unbiased immune response profiling platform that enables comprehensive screening of a tumor mutanome by using a patient's own autologous immune cells, specifically monocyte-derived dendritic cells (MDDC) as antigen presenting cells (APCs) and sorted CD8+ and CD4+ T cells. By utilizing autologous APCs and T cells, ATLAS is agnostic to MHC diversity and assesses preexisting T cell responses to any given mutation. Patient MDDC are pulsed with an ordered array of Escherichia coli expressing patient-specific mutations as short polypeptides. CD8+ and CD4+ T cell response screening is performed using APCs and E. coli with and without pore-forming lysteriolysin O (cLLO) facilitating MHC class I or class II presentation, respectively. Thus, preexisting patient T cell responses to cancer antigens can be characterized by inflammatory cytokine secretion. We utilized a mouse melanoma model to demonstrate the capability of the ATLAS platform for identification of vaccine neoantigens. Whole exome sequencing was performed on B16F10 melanomas resected from C57BL/6 mice, identifying >1600 non-somatic, non-silent mutations. E. coli libraries individually expressing all mutations were constructed and used to screen APCs and T cells from the spleens of B16F10 tumor-bearing mice. Biologically relevant neoantigens were identified by their ability to modulate the secretion of inflammatory cytokines by CD4+ and CD8+ T cells. The significance of the identified neoantigens in comparison to predicted and previously reported B16F10 antigens is described. Top neoantigen candidates were selected and manufactured as synthetic long peptides. Therapeutic vaccination with ATLAS-identified neoantigens in tumor challenge studies is planned and progress will be reported. These studies demonstrate a biologically-relevant approach to improve neoantigen selection for personalized cancer vaccine design enabling improved therapeutic efficacy. Citation Format: Hanna Starobinets, Catarina Nogueira, Kyle Ferber, Huilei Xu, Abha Dhaneshwar, Jason R. Dobson, James Loizeaux, James Foti, Michael O'Keefe, Erick Donis, Wendy Broom, Pamela Carroll, Paul Kirschmeier, Jessica B. Flechtner, Hubert Lam. Ex vivo ATLAS-identification of neoantigens for personalized cancer immunotherapy in mouse melanoma [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 5718.