Consensus Peptide Sequence Research Articles

Functional mimetic peptide discovery isolated by phage display interacts selectively to fibronectin domain and inhibits gelatinase.

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cellinvasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies.

Proteomic studies of bone and skeletal health outcomes.

Proteins are an essential part of essentially all biological processes, and there is enormous variation in protein forms and concentrations that is not reflected in DNA or RNA. Recently there have been rapid advances in the ability to measure protein sequence, modification and concentration, particularly with methods based in mass spectrometry. Global measures of proteins in tissues or in the circulation provide a broad assessment of the proteome that can be extremely useful for discovery, and targeted proteomic measures can yield specific and sensitive assessments of specific peptides and proteins. While most proteomic measures are directed at the detection of consensus peptide sequences, mass spectrometry based proteomic methods also allow a detailed examination of the peptide sequence differences that result from genetic variants and that may have important effects on protein function. In evaluating proteomic data, a number of analytical considerations are important, including an understanding of missing data, the challenge of multiple testing and replication, and the use of rapidly evolving methods in systems biology. While proteomics has not yet had a major impact in skeletal research, interesting recent research has used these approaches in the study of bone cell biology and the discovery of biomarkers of skeletal disorders. Proteomics can be expected to have an increasing influence in the study of bone biology and pathophysiology.

Abstract B166: Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients

Abstract EBV transformed lymphoblastoid cell lines (LCL) derived from patients with Epstein Barr virus (EBV)-related cancer have been used as "feeder cells" after terminally irradiation in multiple cell therapy protocols targeting EBV-related cancers. These cells are subsequently co-cultured with patient peripheral blood mononuclear cells (PBMC) and are sufficient expansion of autologous cell product, given therapeutically back to patients. LCL are known to present both host genome derived cancer-associated antigens relevant for cancer, alongside EBV antigens. PBMC that are co-cultured with LCL have been used in multiple small studies across multiple EBV related cancers. This study was performed in the context of LCL used as feeder cells in the context of EBV-related nasopharyngeal cancer that was treated with autologous cell product after co-culture with LCL as part of a phase II clinical trial in Singapore. The aims of this study were, firstly, to perform unbiased identification of MHC peptides using mass spectrometry; secondly, to map the peptides to respective HLA alleles using publicly available HLA peptide binding prediction algorithms; and thirdly, to look specifically at HLA A*11 allele specific EBV-derived peptides. 6 patients with HLA A*11 were identified and LCL from these patients were grown in cell culture for 1-3 months each, until 5E8 cells were available. Cells were treated in suspension with acidification in order to enable acid elution of MHC peptides. Mass spectrometric analysis was performed for unbiased evaluation of MHC peptides. Based on the consensus peptide sequences derived from 8-mer and 9-mer peptides, HLA A*11:01 appeared to be a dominant allele with at least 20% of peptides having basic end residues that are typical for binding to HLA allele. Between 3,000 to 5,000 distinct peptide sequences across lengths characteristic for both class I and II MHC alleles were identified for each sample, including multiple peptides derived host genome DNA, and some from EBV proteins including LMP1, BARF1, EBNA6, and TTK. There was some overlap of peptides seen across the samples. For example, 2 patient samples, with 3,636 and 4,033 distinct peptides identified respectively, had 1,025 overlapping peptides. 2 patient samples with HLA A*11:01 and HLA A*11:02 respectively, had 1 overlapping EBV protein derived set of peptides that demonstrated differences in trimming of peptides derived from the same epitope across these 2 HLA classes. Only 1 EBV protein was found to be consistently represented in the MHC peptidome across the patient samples, with the same epitope represented across all the samples within the scope of this study. This small study demonstrates that mass spectrometric analysis of large numbers of cells is able to directly deconvolute the MHC peptide, and this is relevant in the context of adoptive cell therapies that utilize "feeder cells" that are required to reliably present clinically relevant antigens to generate autologous cell product. Furthermore, in the absence of reliable peptide prediction algorithms for HLA alleles that are more prevalent in Asia, such as HLA A*11:02, this approach is useful for direct characterization of peptides that bind. Citation Format: Amit Jain. Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B166.

Peptide selected by phage display increases survival of SH-SY5Y neurons comparable to brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a well-known neuroprotectant and a potent therapeutic candidate for neurodegenerative diseases. However, there are several clinical concerns about its therapeutic applications. In the current study, we designed and developed BDNF-mimicking small peptides as an alternative to circumvent these problems. A phage-displayed peptide library was screened using BDNF receptor (neurotrophic tyrosine kinase receptor type2 [NTRK2]) and evaluated by ELISA. The peptide sequences showed similarity to loop2 of BDNF, they were recognized as discontinuous epitopes though. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and the surface residues of the NTRK2 protein at the IgC2 domain. A consensus peptide sequence was then synthesized to generate a mimetic construct (named as RNYK). The affinity binding and function of this construct was confirmed by testing against the native structure of NTRK2 in SH-SY5Y cells in vitro using flow-cytometry and MTT assays, respectively. RNYK at 5 ng/mL prevented neuronal degeneration of all- trans-retinoic acid-treated SH-SY5Y with equal efficacy to or even better than BDNF at 50 ng/mL.

Phage Display Selection, In Vitro Characterization, and Correlative PET Imaging of a Novel HER3 Peptide.

HER3 (ERBB3) is a receptor tyrosine kinase that is implicated in treatment resistance across multiple cancers, including those of the breast, lung, and prostate. Overexpression of HER3 following targeted therapy can occur rapidly and heterogeneously both within a single lesion and across sites of metastasis, making protein quantification by biopsy highly challenging. A global, non-invasive methodology such as positron emission tomography (PET) imaging can permit serial quantification of HER3, providing a useful approach to monitor HER3 expression across the entire tumor burden both prior to and following treatment. PET imaging of HER3 expression may permit a more personalized approach to targeted therapy by allowing for detection of HER3-mediated resistance, in addition to informing clinical trial patient selection for novel therapies targeting HER3. Phage display selection targeting the HER3 extracellular domain was performed in order to develop a peptide with optimal blood clearance and highly accurate HER3 quantification. The selection converged to a consensus peptide sequence that was subsequently found to bind HER3 with an affinity of 270±151nM. The peptide, termed HER3P1, was bound with high selectivity to HER3 over other similar receptor tyrosine kinases such as EGFR and HER2. Furthermore, HER3P1 was able to distinguish between high and low HER3-expressing cells in vitro. The peptide was radiolabeled with Ga-68 and demonstrated to specifically bind HER3 by in vivo PET imaging. Uptake of [68Ga]HER3P1 was highly specific for HER3-positive tumors, with tumor-to-background ratios ranging from 1.59-3.32, compared to those of HER3-negative tumors, ranging from 0.84-0.93. The uptake of [68Ga]HER3P1 also demonstrated high (P<0.001) correlation with protein expression as quantified by Western blot and confirmed by biodistribution. HER3P1 accurately quantifies expression of HER3 by PET imaging and has potential utility as a clinical imaging agent.

Proteomic composition of the acrostyle: Novel approaches to identify cuticular proteins involved in virus–insect interactions

The acrostyle is a distinct anatomical region present on the cuticle at the inner face of the common food/salivary canal at the tip of aphid maxillary stylets. This conserved structure is of particular interest as it harbors the protein receptors of at least 1 plant virus, Cauliflower mosaic virus, and presumably has other roles in plant–insect interactions. Previously we reported immunolabeling of a highly conserved motif of cuticular proteins from the CPR family (named for the presence of a Rebers and Riddiford consensus) within the acrostyle. Here we report the development of novel tools to further study the proteomic composition of this region and to identify proteins involved in insect‐virus interactions. Using a series of antibodies against cuticular proteins from the RR‐2 subfamily, we identified additional peptides present within the acrostyle. Our results demonstrated that the acrostyle is a complex structure containing multiple domains of cuticular proteins accessible for interaction. In addition, an array of overlapping peptides, which covers the diversity of the majority of the RR‐2 subfamily, was developed as a generic tool to characterize cuticular protein/pathogen interactions. Upon probing this array with Cucumber mosaic virus particles, consensus peptide sequences from hybridizing peptides were identified. Use of these novel tools has extended our knowledge of the proteomic composition of insect maxillary stylets and identified sequences that could be involved in virus binding, thus contributing to further elucidation of the various properties and functions of the acrostyle.

Cytoplasmic dynein associated peptide-linked nanoparticles for non-viral vector applications

Event Abstract Back to Event Cytoplasmic dynein associated peptide-linked nanoparticles for non-viral vector applications Ercia Carbone1, Komal Rajpura1, Ho Man Kan1, Tao Jiang1 and Wai Hong Lo1 1 University of Connecticut Health Center, Institute for Regenerative Engineering, United States Statement of Purpose: Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NP) hold great promise for gene delivery applications. Non-viral polymeric nanoscale vectors offer several advantages over viral vectors[1]. However, most non-viral vectors suffer from significantly lower transfection efficiencies than viral vectors. This is because during their way to the nuclei of cells, non-viral vectors must overcome a series of diffusional and enzymatic barriers [1]. Interestingly, it has been shown that a number of viruses exploit microtubules by interacting with cytoplasmic dynein motor protein (a motor protein responsible for retrograde movement along microtubules) in order to enter the nucleus of the host cell. Cytoplasmic dynein is a large, multisubunit molecular motor that translocates various cargoes toward the nucleus. The consensus peptide sequence “K/R-X-T-Q-T” has been shown to be important for interacting with one of the dynein subunits [2]. Here, we develop dynein associated peptide-linked polymeric nanoparticles for non-viral vector applications. It is hypothesized that the novel dynein peptide-linked nanoparticles will hijack the endogenous dynein motor system intracellularly, and consequently move along microtubules toward the nucleus (-ve end of microtubule) (Fig.1). Methods: Dynein-associated peptide “LysGluThrGlnThrCys (KETQTC)” and the control peptide “LysGluThrAlaThrCys (KETATC)” were synthesized and labeled with FITC commercially at the N-terminus of the peptides (LifeTein, Inc). Note that the additional Cys residue is to facilitate the conjugation of the Asp to PLGA NPs; Maleimide-PEG-PLGA and Methoxy-PEG-PLGA were purchased from Polyscitech. This NP was fabricated using a water-in-oil-in-water (W/O/W) double emulsion technique using a mixture of 1:9 (Maleimide-PEG-PLGA : Methoxy-PEG-PLGA) [3]. The particle size distribution and zeta potential measurements of the NPs were determined by dynamic laser scattering. Conjugation of the peptides to the NP was performed as previously described [3]. Equal amounts of peptide-linked NPs or control peptide-linked NPs were mixed with purified dynein LC8 protein (1 mg) (ProSpec) for 1 hour at 4°C. The complex, i.e. peptide-linked NPs bound to LC8, was precipitated by centrifugation. The precipitated NPs were washed 3x with PBS and subsequently boiled with 2x sample buffer (Bio-rad). The proteins were resolved by SDS-PAGE (Bio-rad) and subsequently analyzed by immunoblot analysis with anti-LC8 antibody (Millipore). Human MG63 osteoblasts (ATCC) were cultured on coverslips with dynein peptide-linked NPs or control peptide-linked NPs for various time points. After removing the NPs and washing the coverslips 4x with PBS, the cells were fixed with 4% PFA (Santa Cruz). Nuclei were stained with popdium iodide (PI) nucleic acid stain. Cells were then mounted by a mounting solution, and fluorescent signals on the cells were observed via confocal microscopy (Zeiss LSM ConfoCor 3). Results: The average diameter of the NPs is 143.8 nm and the zeta potential is -26.43 mV. Biochemical binding analysis showed that purified recombinant dynein LC8 protein efficiently binds to KETQT-NPs. In contrast, LC8 protein does not bind well to the control peptide (KETAT)-NPs (Fig.2A). We further demonstrated that the KETQT-NPs can interact with endogenous dynein LC8 protein in MG63 cell lysates (Fig. 2B). To investigate the localization of the peptide-NPs in cells, we incubated the NPs with cells for various time points. Figure 2C shows that the FITC signals from KETQT-NPs (green) are colocalized with cells’ nuclei (red), whereas the FITC signals from the control NPs do not. Conclusions: Taken together, our data demonstrated that the KETQT-NPs can bind to dynein subunit (LC8) in vitro and in vivo, and these NPs can be enriched in nuclei region when they were incubated with cell culture. Future research including live-cell imaging will be used to trace the movement of the NPs in cells. Our dynein associated NPs may have a significant impact in non-viral vector development for gene therapy applications. This work was supported by funding from the State of Connecticut Stem Cell Research Foundation.

CBIO-20GLIOBLASTOMA SPECIFIC PROTEINS IDENTIFIED THROUGH COMBINED IN VITRO AND IN VIVO PHAGE DISPLAY BIOPANNING

Glioblastomas are the most common and lethal primary brain tumor with conventional therapy offering only palliation. Their resistance to therapy has been hypothesized to be due in part to the cancer stem cell, the target of which the development of safe and effective future therapeutics will be focused on. As phage display technology has been used in cancer to identify, target, and image tumor cells, we employed phage display technology to identify novel proteins specific to the functioning of glioblastoma stem cells (GSCs). We have previously shown that in vivo phage display biopanning against primary glioblastoma cell lines xenografted in immunocompromised mice is an effective strategy for isolating glioma stem cell (GSC) specific peptides sequences. Using this strategy, we identified Vav3, a guanine exchange factor that plays a role in the maintenance of GSCs. In order to further identify novel proteins that may play a role in GSC maintenance, we have employed an in vitro biopanning strategy against GSCs isolated from patient derived glioblastoma cell lines grown in culture. Several peptides isolated using this strategy matched peptides that were separately isolated using the in vivo biopanning technique, validating the ability of these selection strategies to achieve consensus peptide sequences that are specific for GSC binding. In silico analysis of these peptides using a BLAST search as previously described revealed CD48. Analysis of CD48 showed that it is preferentially expressed in glioblastoma cells with a potential role in tumor maintenance. Together, these studies validate the effectiveness of utilizing different phage display biopanning strategies to isolate novel proteins that may play a vital role in maintenance of glioblastomas.

Expression and Selection of Human Foxm1c Binding Peptides and Their Inhibitions on MCF7 Cancer Cells

Elevated expression of forkhead/winged helix transcription factor FOXM1 are closely involved in cancer initiation, invasion and metastasis. It is a potentially suitable target for anticancer drug discovery and therapy. In this study, we investigated the expression and selection of human Foxm1c binding peptides and their inhibitions on MCF7 cancer cells. The protein of DNA binding domain of human FoxM1c(DBDFoxM1c) was expressed in prokaryotic system and then purified and later used as the target for binding peptide selection based on the phage random dodecapeptide library. The phages were enriched after four rounds of selection and 18 different peptide sequences were obtained. seven possible consensus peptide sequences WHL/Q/D/N, DL/HL/NY, SSLWN/E; HLD/YY/E; YH/LE/D/E/D; YPH/L/S and F/NY/FN/YL were identified and they were found to dock well to the 3D structure model of DBDFoxM1c by molecular docking. One representative peptide was designed and in vitro study revealed that the viability of MCF7 cancer cells could be reduced to 34.30 % at the concentration of 100 µg/ml of the peptide. In conclusion, the consensus peptide sequences identified in our study were newly reported and it is the first report of the screening of binding peptides targeting DBDFoxM1c. We expect that the results of these studies will result in future development of FoxM1c based lead peptide drug as well as the further understanding of DBDFoxM1c.

Bioinformatics Analysis of the FREM1 Gene-Evolutionary Development of the IL-1R1 Co-Receptor, TILRR.

The TLRs and IL-1 receptors have evolved to coordinate the innate immune response following pathogen invasion. Receptors and signalling intermediates of these systems are generally characterised by a high level of evolutionary conservation. The recently described IL-1R1 co-receptor TILRR is a transcriptional variant of the FREM1 gene. Here we investigate whether innate co-receptor differences between teleosts and mammals extend to the expression of the TILRR isoform of FREM1. Bioinformatic and phylogenetic approaches were used to analyse the genome sequences of FREM1 from eukaryotic organisms including 37 tetrapods and five teleost fish. The TILRR consensus peptide sequence was present in the FREM1 gene of the tetrapods, but not in fish orthologs of FREM1, and neither FREM1 nor TILRR were present in invertebrates. The TILRR gene appears to have arisen via incorporation of adjacent non-coding DNA with a contiguous exonic sequence after the teleost divergence. Comparing co-receptors in other systems, points to their origin during the same stages of evolution. Our results show that modern teleost fish do not possess the IL-1RI co-receptor TILRR, but that this is maintained in tetrapods as early as amphibians. Further, they are consistent with data showing that co-receptors are recent additions to these regulatory systems and suggest this may underlie differences in innate immune responses between mammals and fish.

Yeast Two‐hybrid Reveals a Potato Polypyrimidine Tract Binding (PTB) Protein Interacts with a Nova‐like Protein

PTB proteins are RNA‐binding proteins that contain four RNA recognition motifs. In potato, four PTB‐like genes, designated as PTB1, PTB6, TC201749 and PTB7, have been identified. In plants, these proteins play an important role in alternative splicing. In order to understand the multifunctional role of PTB proteins of plants we performed yeast two‐hybrid screening using PTB1 protein as bait. PTB1 protein was found to interact with a Nova‐like protein, termed StNova‐like protein. Nova (neuron‐oncological ventral antigen) protein is a RNA‐binding protein that contains three K‐homology domains. In humans, these proteins play an important role in regulation of neuronal RNA metabolism but the role of Nova‐like proteins in plants has not been explored. In this study, the StNova‐like gene has been cloned into the pMAL‐c2E vector, expressed in T7 express cells and purified over Ni‐NTA resin. Further, putative interactions between StPTB proteins and StNova were confirmed by pull‐down experiments and by blue‐native PAGE. Using a random 21‐mer phage‐peptide library, we also identified a consensus peptide sequence motif [S/G][I/L][L/I][S] that binds to PTB1, suggesting that the PTB1 protein might be interacting with the [S][I][I][S] sequence present in StNova‐like protein. Our studies suggest that in plants, Nova‐like proteins may be involved in processes of RNA metabolism mediated by PTB proteins.

Intradermal vaccination of HIV-infected patients with short HIV Gag p24-like peptides induces CD4 + and CD8 + T cell responses lasting more than seven years

Vacc-4x contains 4 HIV p24-like short peptides. In a previous phase II trial this immunized 90% of 38 patients on antiretroviral treatment (ART) after intradermal delivery in conjunction with local granulocyte-macrophage colony-stimulating factor (GM-CSF) as adjuvant. In this study, 22 responders were retested for cellular memory at a median 7.3 y after their last immunization. All had resumed effective ART after an interspersed ART-free median interval of 2.2 y. Vacc-4x as 15-mer overlapping by 2 amino acid panels and Vacc-4x consensus peptide sequences (4xCP) were used as antigens. Proliferation was determined as percentages of CFSE(dim)HLA-DR(+) 7AAD(-) CD3+ T cells of the CD4+ and CD8+ subsets after 6 days of culture. Frequencies of specific T cells in 6-h cultures were determined by interferon-γ (IFN)(+) CD4+ and IFN(+) CD8+, as well as degranulating bifunctional CD107a + IFN(+) CD8 + subsets. Proliferative CD4+ and CD8+ responses to Vacc-4x as well as 4xCP were still present in 95% and 68%, respectively. Proliferation correlated with the Vacc-4x delayed-type hypersensitivity test (DTH) obtained after completed immunizations (CD4 + r = 0.63 (p = 0.002) and CD8 + r = 0.47 (p = 0.03)), suggesting that they represent T cell memory recall responses. The proliferative CD8+ and possibly CD4 + subset responses to 4xCP peptides correlated with Vacc-4x (r = 0.46 (p = 0.03) and r = 0.38 (p = 0.08), respectively). Forty-one percent still had Vacc-4x-specific IFN + CD4 + T cells, which correlated to corresponding frequencies of 4xCP peptides (r = 0.50, p = 0.02). CD107a(+) IFN(+) CD8 + T cell responses against Vacc-4x were found in 55%. Evidence of long-lasting T cell memory recall responses to a peptide-based immunotherapeutic candidate for HIV-infected patients should enhance the focus on peptide-based intradermal vaccine delivery.

Conserved peptide sequences bind to actin and enolase on the surface ofPlasmodium bergheiookinetes

The description of Plasmodium ookinete surface proteins and their participation in the complex process of mosquito midgut invasion is still incomplete. In this study, using phage display, a consensus peptide sequence (PWWP) was identified in phages that bound to the Plasmodium berghei ookinete surface and, in selected phages, bound to actin and enolase in overlay assays with ookinete protein extracts. Actin was localized on the surface of fresh live ookinetes by immunofluorescence and electron microscopy using specific antibodies. The overall results indicated that enolase and actin can be located on the surface of ookinetes, and suggest that they could participate in Plasmodium invasion of the mosquito midgut.

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10 2 colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

Calcium induces electron transfer in NADPH oxidase 5

Superoxide generated by non‐phagocyte NADPH oxidases (NOXs), such as NOX5 and DUOX, is of growing importance for vascular physiology and pathology. NOX5 and DUOX enzymes consist of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. They appear to be regulated by self‐contained Ca2+ binding domains (EFD). The basis for their catalytic profiles and regulation is unclear. Here, we demonstrated that Ca2+ gates heme reduction NOX5 in anaerobic conditions. To better understand this interaction, we monitored its Ca2+ and terbium (Tb3+) bindings by fluorescent spectroscopy using wild type and mutants of the recombinant EFD. The titration of EFD yielded a [Ca2+]0.5 of 4.5 μM, which is similar to published results, but the Ca2+ bindings appears to be tighter and occur in a cooperative manner. About 10 μM Ca2+ is required to fully saturate EFD and completely activate NOX5 superoxide production. EFD binds terbium with a [Tb3+]0.5 of 20 μM. The site‐directed labeling on Cys107 of EFD with IAEDNS had the same response to the Ca2+ level as the non‐labeled EFD. It reveals an anisotropy value of 0.11 and will be used to gather the structural information of the EFD‐flavoprotein complex using FRET. NOX5 contains a consensus peptide sequence that interacts with Ca2+‐bound calmodulin (CaM), and it has been demonstrated that this CaM/Ca2+ binding increases its sensitivity for free Ca2+. We used Troponin C (TnC)‐CaM chimeras to pinpoint the structural‐functional relationship to understand how individual EF‐hands motifs in CaM affect the electron transfer in NOX5. We are currently performing similar studies in DUOX. This work was supported by Beginner‐Grant‐in‐Aid from the American Heart Association

Reduction of IgG in nonhuman primates by a peptide antagonist of the neonatal Fc receptor FcRn

The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG-hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.

An FcγRIIa-binding peptide that mimics the interaction between FcγRIIa and IgG

A disulphide-constrained peptide that binds to the low affinity Fc receptor, FcγRIIa (CD32) has been identified and its structure solved by NMR. Linear (7-mer and 12-mer) and disulphide-constrained (7-mer) phage display peptide libraries were panned on recombinant soluble FcγRIIa genetically fused to HSA (HSA–FcγRIIa). Peptides were isolated only from the constrained peptide library and these contained the consensus sequence, CWPGWxxC. Phage clones displaying variants of the peptide consensus sequence bound to FcγRIIa and the strongest binding clone C7C1 (CWPGWDLNC) competed with IgG for binding to FcγRIIa and was inhibited from binding to FcγRIIa by the FcγRIIa-blocking antibody, IV.3, suggesting that C7C1 and IgG share related binding sites on FcγRIIa. A synthetic disulphide-constrained peptide, pep-C7C1 bound to FcγRIIa by biosensor analysis, albeit with low affinity (KD∼100μM). It was significant that the FcγRIIa consensus peptide sequence contained a Proline (Pro3), which when substituted with alanine abrogated FcγRIIa binding, consistent with Pro3 contributing to receptor binding. Upon binding of IgG and IgE to their respective Fc receptors (FcγRs and FcɛRI) Pro329 in the Fc makes a critical interaction with two highly conserved Trp residues (Trp90 and Trp113) of the FcRs. The NMR structure of pep-C7C1 revealed a stabilizing type II β-turn between Trp2 and Trp5, with Pro3 solvent exposed. Modelling of the pep-C7C1 structure in complex with FcγRIIa suggests that Pro3 of C7C1 binds to FcγRIIa by inserting between Trp90 and Trp113 of FcγRIIa thereby mimicking the molecular interaction made between FcγRIIa and IgG.

The Burkholderia pseudomallei serine protease MprA is autoproteolytically activated to produce a highly stable enzyme

Burkholderia pseudomallei, a tropical pathogen and the causative agent of melioidosis, is known to secrete a serine metalloprotease (MprA) into the internal milieu of the infectious host. This protein has been shown to cause extensive damage to mammalian physiological proteins and its role in the pathogenesis of melioidosis is still under investigation. Previously, we have reported on the epitope mapping of B. pseudomallei protease that revealed a consensus peptide sequence of serine–methionine–alanine (SMA). The serine within this motif is involved in the serine protease catalytic triad and the SMA domain of the B. pseudomallei serine protease could be a major immunodominant domain. We undertook to further characterize the mprA gene and protein to gain deeper insight into the role and mechanism of this protein. Preliminary analysis showed that the crude lysate of expressed recombinant protease was able to hydrolyze gelatin, azocasein and skimmed milk. Further biochemical characterization demonstrated that the expressed protein maintained good proteolytic activity over a wide pH range of 5–11, is stable from 4 °C up to 68 °C and partially digested immunoglobulins A and G, transferrin and myosin. The proteolytic activity was strongly inhibited by phenylmethylsulfonyl fluoride. From our in vitro experimental evidence, we propose a proenzyme processing mechanism similar to that of subtilisin to produce the mature active protease.

Peptide ligands targeting integrin α3β1 in non-small cell lung cancer

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.