Binding Capacity Of Peptides Research Articles

Isolation and differential structure characteristics of calcium-binding peptides derived from Pacific cod bones by hydroxyapatite affinity

Calcium-chelating peptides were found in Pacific cod bone, but their binding structure and properties have not been elucidated. Novel calcium-binding peptides were isolated by hydroxyapatite affinity chromatography (HAC), and their binding structure and properties were investigated by isothermal titration calorimetry (ITC), multispectral techniques, and mass spectrometry. Based on multiple purifications, the calcium binding capacity (CBC) of Pacific cod bone peptides (PBPs) was increased from 1.71 ± 0.15 μg/mg to 7.94 ± 1.56 μg/mg. Peptides with a molecular weight of 1–2 kDa are closely correlated with CBC. After binding to calcium, the secondary structure of peptides transitioned from random coil to β-sheet, resulting in a loose and porous microstructure. Hydrogen bonds, electrostatic interaction, and hydrophobic interaction contribute to the formation of peptide‑calcium complexes. The F21 contained 42 peptides, with repeated “GE” motif. Differential structure analysis provides a theoretical basis for the targeted preparation of high CBC peptides.

Absence of specific autoantibodies in patients with narcolepsy type 1 as indicated by an unbiased random peptide-displayed phage screening.

Narcolepsy type 1 (NT1) is an enigmatic sleep disorder characterized by the selective loss of neurons producing orexin (also named hypocretin) in the lateral hypothalamus. Although NT1 is believed to be an autoimmune disease, the orexinergic neuron-specific antigens targeted by the pathogenic immune response remain elusive. In this study, we evaluated the differential binding capacity of various peptides to serum immunoglobin G from patients with NT1 and other hypersomnolence complaints (OHCs). These peptides were selected using an unbiased phage display technology or based on their significant presence in the serum of NT1 patients as identified from previous studies. Although the subtractive biopanning strategy successfully enriched phage clones with high reactivity against NT1 serum IgG, the 101 randomly selected individual phage clones could not differentiate the sera from NT1 and OHC. Compared to the OHC control group, serum from several NT1 patients exhibited increased reactivity to the 12-mer peptides derived from TRBV7, BCL-6, NRXN1, RXRG, HCRT, and RTN4 proteins, although not statistically significant. Collectively, employing both unbiased and targeted methodologies, we were unable to detect the presence of specific autoantibodies in our NT1 patient cohort. This further supports the hypothesis that the autoimmune response in NT1 patients likely stems primarily from T cell-mediated immunity rather than humoral immunity.

Pulsed electric field (PEF) activates immune activity in RAW 264.7 macrophages by altering pine nut peptide-TLR4 binding sites

The purpose of this study was to investigate the mechanism of PEF treatment on the regulation of pine nut peptide (VNAVLH) induced immune activity of RAW 264.7 based on C-terminal modification by molecular dynamics (MD). The results showed that PEF treatment can reduce the overall stability of pine nut peptides, enhance the flexibility of hydrophobic and positively charged residues in pine nut peptides, and make their residue structures more flexible. Active sites verification experiments showed that PEF treatment could improve the immune activity of histidine only when it was at C-terminal. Western blot and immunofluorescence analysis showed that PEF-treated VNAVLH could activate NF-κB pathway by promoting the nuclear translocation of NF-κB/p65 and the expression of IKKβ, and activate MAPK pathway by promoting the phosphorylation of JNK, ERK, and p38 in RAW 264.7. Furthermore, toll-like receptor 4 (TLR4), as the main receptor for VNAVLH to exert immune activity, exhibited a more prominent peptide-binding capacity and had more hydrogen-bonding sites to PEF-treated peptide compared to toll-like receptor 2 (TLR2). Therefore, enhanced immunoreactivity of VNAVLH may be due to the formation of a more stable complex with TLR4 after PEF treatment, which activates the MAPK/NF-κB signaling pathway causing an intracellular cascade response.

Immunostimulatory effects of Hsp70 fragments and Hsp27 in design of novel HIV-1 vaccine formulations.

Heat shock proteins (HSPs) as an adjuvant induce antigen-specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N-Hsp70(aa 1-387) with ATPase property and C-Hsp70 (aa 508-641) with peptide-binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV-1 Nef antigen-specific immunity in both in vitro and in vivo experiments. At first, the nanoparticles harbouring DNA fusion constructs (i.e. N-Hsp70-Nef, C-Hsp70-Nef and Hsp27-Nef) complexed with HIV Rev (34-50) cell-penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27-Nef, N-Hsp70-Nef and C-Hsp70-Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single-cycle replicable (SCR) HIV-1 virion-exposed splenocytes was investigated. Our data showed that the stable and non-toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp-Nef constructs than in mice receiving Nef antigen. The C-Hsp70 was also superior for inducing Nef-specific Th1 and CTL immunity compared with N-Hsp70 and Hsp27. The T-cell activity was maintained in the SCR-exposed splenocytes, especially the splenocytes of mice receiving the C-Hsp70-Nef regimen. Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen-specific immunity. Notably, the C-Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV-1 therapeutic vaccines.

Statistical methods and its applications in Whey protein binding (Bos Taurus)

Milk is made up of two proteins, such as casein (80%) and whey (20%). These proteins, which include membrane globular proteins, glycoproteins, and lipoproteins, are mostly found in the outer layer of milk fat globule membranes. WP can be separated from the casein in milk or formed as a byproduct of cheese making and contains all nine essential amino acids with a low lactose content. Whey protein has many health benefits, including improving strength and body composition, promoting weight loss, boosting metabolism, stifling hunger, maintaining good health, avoiding nourishing the bones, reducing inflammation, promoting quick wound healing, lowering blood pressure, repairing and preserving muscle tissues etc. Many dairy and food-based industries eagerly explore the accurate retention time for the formation of health related new byproducts. As of date, there are very few studies available in the world literature citation database and too many micro and macro studies on WP are currently being undertaken globally, they provide insight into the binding of particular amino acids (Seq. peptides) and their accurate lag time of binding to the formation of "helixes and sheaths. In this paradigm, statistical tool are very important to determine average time and WP algorithms for analysis of seq amino acids and the metabolic pathway's action. Overall, the present research attempts to extrapolate the Seq peptide's binding capacity and its retention time for amino acids in order to fill the aforementioned research gap. For the model demonstration we used the commonly cited whey protein peptide (Seq) NCBS, which was considered to prove the hypothesis. The Markova two state random and Monte Carlo simulation (superimposed artificial neural network) models were formulated to extract transient probabilities and retention times of Seq peptides. As per the results, the binding capacity was found to be excellent with a good retention time (5 to 11.50 minutes) in cross-linked and unlinked cases (10 to 13.50 minutes). Bioinformatics, the dairy industry, and life scientists will find these more recent discoveries to be very helpful for measuring likelihoods and transient probabilities of binding.

Genetically Engineered Hepatitis C Virus-like Particles (HCV-LPs) Tagged with SP94 Peptide to Acquire Selectivity to Liver Cancer Cells via Grp78.

Targeted cancer therapy is a challenging area that includes multiple chemical and biological vehicles. Virus-like particles (VLPs) combine safety and efficacy in their roles as potential vaccines and drug delivery vehicles. In this study, we propose a novel drug delivery system based on HCV-LPs engineered with SP94 and RGD peptides mediated by a specific molecular chaperone (Grp78) associated with cancer drug resistance. The PCR primers were designed for engineering two constructs, SP94-EGFP-CORE-HIS and RGD-EGFP-CORE-HIS, by sequential PCR reactions. The two fragments were cloned into pFastBac Dual under the polyhedrin promoter and then used to produce two recombinant baculoviruses (AcSP94 and AcRGD). The VLP’s expression was optimized by recombinant virus infection with different MOIs, ranging from 1 to 20 MOI. Recombinant VLP2 were purified by Ni-NTA and their sizes and shapes were confirmed with TEM. They were incubated with different types of cells prior to examination using the fluorescence microscope to test the binding specificity. The effect of the overexpression of the Grp78 on the binding affinity of the engineered VLPs was tested in HepG2 and HeLa cells. The protocol optimization revealed that MOI 10 produced the highest fluorescence intensities after 72 h for the two recombinant proteins (SP94-core and RGD-core). Moreover, the binding assay tested on different types of mammalian cells (HeLa, HEK-293T, and HepG2 cells) showed green fluorescence on the periphery of all tested cell lines when using the RGD-core protein; while, the SP94-core protein showed green fluorescence only with the liver cancer cells, HepG2 and HuH7. Overexpression of Grp78 in HepG2 and HeLa cells enhanced the binding efficiency of the engineered VLPs. We confirmed that the SP94 peptide can be specifically used to target liver cancer cells, while the RGD peptide is sufficiently functional for most types of cancer cells. The overexpression of the Grp78 improved the binding capacity of both SP94 and RGD peptides. It is worth noting that the SP94 peptide can function properly as a recombinant peptide, and not only as a chemically conjugated peptide, as heretofore commonly used.

Total predicted MHC-I epitope load is inversely associated with population mortality from SARS-CoV-2

SummaryPolymorphisms in MHC-I protein sequences across human populations significantly affect viral peptide binding capacity, and thus alter T cell immunity to infection. In the present study, we assess the relationship between observed SARS-CoV-2 population mortality and the predicted viral binding capacities of 52 common MHC-I alleles. Potential SARS-CoV-2 MHC-I peptides are identified using a consensus MHC-I binding and presentation prediction algorithm called EnsembleMHC. Starting with nearly 3.5 million candidates, we resolve a few hundred highly probable MHC-I peptides. By weighing individual MHC allele-specific SARS-CoV-2 binding capacity with population frequency in 23 countries, we discover a strong inverse correlation between predicted population SARS-CoV-2 peptide binding capacity and mortality rate. Our computations reveal that peptides derived from the structural proteins of the virus produce a stronger association with observed mortality rate, highlighting the importance of S, N, M, and E proteins in driving productive immune responses.

Neoantigen Landscape of Relapsed Acute Leukemia Following Allogeneic Stem Cell Transplantation

INTRODUCTION: The potent graft versus leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) is considered as a blueprint for cellular immunotherapy. However, failure of GVL leads to relapse of underlying leukemia, the major cause of death after allo-SCT. In solid tumors, higher tumor mutation burdens are associated with better response to check point inhibitors which implies the importance of neoantigen specific T-cell functions in cancer immunity. In contrast, the frequencies of somatic mutations in acute leukemia are generally low, therefore the role of neoantigens in GVL remains undetermined. Here, we developed a platform to screen for potential neoantigens by performing whole exome sequencing (WES) and RNA sequencing (RNAseq) in matched samples: leukemic blasts at relapse after allo-SCT, recipient T cell controls, and donor cells.METHODS: Leukemic blasts from patients in relapse were enriched by flow sorting from bone marrow aspirate or peripheral blood samples. Recipient T cells were isolated from pre-transplant peripheral blood as germline controls, and donor monocytes or CD34-positive cells were used as hematopoietic-lineage cell controls. WES was performed to 100X coverage, paired with RNAseq 40M reads per sample. Somatic mutations were detected with mutect and mpileup, followed by annotation with SnpEff. High confidence somatic mutations were subjected to pVAC-seq for neoantigen predictions.RESULTS: Six patients with relapsed acute leukemia (AML 5, ALL 1) after allo-SCT and their transplant donors (matched sibling 3, haplo-identical 3) had suitable samples available for analysis. On average, somatic mutations were identified in 297 genes (range 108- 609) by comparing leukemic blasts and germline control T cells. Among those mutations, potential candidates of neoantigen were identified in five out of six subjects. Allele frequencies of mutant genes varied. Most of neoantigens were predicted to bind HLA of both class I (median 5, range 0-15) and class II (median 6, range 0-12). One subject had only HLA class II restricted peptides as predicted neoantigens. Of interest majority of antigens were derived from molecules known to play important roles in leukemia or tumor biology which include ETV6, CCNY, IDH2, PTPN11, SF3B1, and TP53. Evolution analysis of neoantigen showed an emergence of new antigens in relapsed leukemia while a few driver gene mutations persisted after allo-SCT (Figure).CONCLUSION: Our in-silico analysis demonstrated the possibility that somatic mutation in acute leukemia could serve as putative neoantigens applicable for novel immunotherapy after allo-SCT. The binding capacity of mutant peptides to class I and II HLA implies the importance of both CD4 and CD8 contributions to anti-neoantigen immunity. Next, we will search for neoantigen specific T cells exerting an anti-leukemia effect to validate the GVL potential of these mutations in allo-SCT. DisclosuresNo relevant conflicts of interest to declare.

The critical amino acids of a nephritogenic epitope on human Goodpasture autoantigen for binding to HLA-DRB1*1501

BackgroundAnti-GBM disease is caused by autoimmunity to Goodpasture antigen on α3(IV)NC1 and had strong associations with HLA-DRB1*1501. Previous studies identified α3127-148 (P14: TDIPPCPHGWISLWKGFSFIMF) as a T cell epitope. The present study was aimed to investigate the binding capacity of P14 to HLA-DRB1*1501 and the critical amino acids for this binding. MethodsA line of EBV-transformed human B cells homozygous for HLA-DRB1*1501 was used to detect the binding capacity of peptides to HLA-DRB1*1501 using flow cytometry analysis. P14 was sequentially truncated into 8 peptides with 15 amino acids to identify the core binding motif. A set of alanine substituted peptides of P14-2 was then synthesized to identify its critical residues for binding to HLA-DRB1*1501. The structure of HLA-DR2b-Peptide-TCR complex was constructed by modeling to analyze the interaction of each amino acids of P14-2 with the HLA-DR2b molecule. ResultsP14 could bind to HLA-DRB1*1501 expressed on B cell surface. The N-terminus of P14 was the core binding motif and the truncated peptide P14-2 (DIPPCPHGWISLWKG) 128-142 had the strongest binding capacity. After sequential amino acid substitution, we found the binding capacity of P14-2 was completely lost by the substitution of cysteine (C) 132 and significantly decreased by the substitution of tryptophan (W) 136, lysine (K) 141, or glycine (G) 142, but still at a high level. The modeling showed that (C) 132 had a strong interaction with pocket 4 on the β chain of DR2b. Thus, C132, W 136, K141, and G142 were defined as the critical amino acid residues for the binding capacity of P14 to HLA-DRB1*1501. ConclusionWe identified α3128-142 (DIPPCPHGWISLWKG) as the core binding motif of P14 to HLA-DRB1*1501 molecule. And the critical amino acid residues for this binding were further defined as C132, W 136, K 141, and G 142.

Effect of secondary anchor amino acid substitutions on the immunogenic properties of an HLA-A*0201-restricted T cell epitope derived from the Trypanosoma cruzi KMP-11 protein

The TcTLE peptide (TLEEFSAKL) is a CD8+ T cell HLA-A*0201-restricted epitope derived from the Trypanosoma cruzi KMP-11 protein that is efficiently processed, presented and recognized by CD8+ T cells from chagasic patients. Since the immunogenic properties of wild-type epitopes may be enhanced by suitable substitutions in secondary anchor residues, we have studied the effect of introducing specific mutations at position 3, 6 and 7 of the TcTLE peptide. Mutations (E3L, S6V and A7F) were chosen on the basis of in silico predictions and in vitro assays were performed to determine the TcTLE-modified peptide binding capacity to the HLA-A*0201 molecule. In addition, the functional activity of peptide-specific CD8+ T cells in HLA-A2+ chagasic patients was also interrogated. In contrast to bioinformatics predictions, the TcTLE-modified peptide was found to have lower binding affinity and stability than the original peptide. Nevertheless, CD8+ T cells from chronic chagasic patients recognized the TcTLE-modified peptide producing TNF-α and INF-γ and expressing CD107a/b, though in less extension than the response triggered by the original peptide. Overall, although the amino acids at positions 3, 6 and 7 of TcTLE are critical for the peptide affinity, they have a limited effect on the immunogenic properties of the TcTLE epitope.

Epitope Mapping Using Peptide Microarray in Autoantibody Profiling.

The use of peptide microarrays for epitope mapping of autoantibodies greatly facilitates the early diagnosis of allergic, cytotoxin-associated diseases and especially inflammatory diseases. A common approach to create the microarrays utilizes nitrocellulose-coated glass slides for peptide probe binding, which is based on surface adsorption. Advantages of this method include excellent peptide binding capacity and long-term stability. To ensure equal accessibility to all antibodies on the peptide microarray during epitope mapping, all probes are immobilized in a random manner, thus avoiding concentration-dependent effects on signal intensity.In this chapter, we provide a step-by-step protocol on how to construct the peptide microarrays and perform epitope mapping of autoantibodies using them. Finally we present a comparative approach for the evaluation of the data.

Indirectly Recognized HLA-C Mismatches and Their Potential Role in Transplant Outcome

HLA-C mismatches are clearly associated to alloreactivity after hematopoietic stem-cell transplantation; in a number of large cohorts, HLA-C mismatches are correlated to an increased risk of acute graft-versus-host disease (GVHD) or even impaired survival. While for HLA-A and -B, both antigenic as well as allelic mismatches are associated with an increased risk of acute GVHD, such an increased risk is only observed for antigenic HLA-C mismatches and not for allelic mismatches. These observations raise the question what sets HLA-C apart from HLA-A and -B. The difference may well be related to the reduced levels of cell-surface expression of HLA-C as compared to HLA-A and -B, possibly due to, among other factors, a limited peptide-binding capacity. This limited peptide-binding capacity may retain HLA-C in the ER and enhance degradation of the HLA-C protein. Once degraded, HLA-C-derived peptides can be presented to the immune system via other HLA alleles and are thus available for indirect recognition. Indeed, such HLA-C-derived peptides have previously been eluted from other HLA alleles. We have recently developed an approach to predict indirect recognition of HLA molecules, by establishing the numbers of predicted indirectly recognizable HLA epitopes (PIRCHES). The number of PIRCHES presented on HLA class I and II (PIRCHE-I and -II, respectively), are highly correlated to clinical measures of alloreactivity, such as acute GVHD. In the present “Hypothesis & Theory,” we reviewed the current knowledge on HLA-C mismatches and alloreactivity. Moreover, we speculate about the role of direct and indirect recognition of HLA-C and the consequences for donor selection in HLA-C mismatched stem-cell transplantation.

Abstract A54: Isolation and characterization of cancer stem cells with peptides.

Abstract Background: Cancer as the most malignant disease still demands the more effective therapeutics methods although continuous efforts have been input to cancer therapies. Recent studies indicate that only a small subset of cancer cells termed cancer stem cells (CSCs) contributes to tumor progression, metastasis and drug-resistance. Drugs targeting to CSCs might become more effective approaches to cancer therapy. To date, isolation and characterization of CSCs have largely depended on surface markers which are not specific or well-recognized for individual tumor types. Our goal is to develop methods for isolating and characterizing a set of lung CSCs based on the specific binding peptides for CSCs without any recognized definitive specific surface markers. Materials and Methods: Stem-like cells were enriched from non-small cell lung cancer (NSCLC) cell line H460 by sphere-forming assay. Characterized these stem-like cells by colony-forming assay, drug-resistance assay and soft agar assay. Screening and identification of specific binding peptides for CSCs were performed with bacteria surface display methods. The affinity between the specific binding peptides and CSCs was determined with fluorescence microscope and flow cytometric analysis. The specificity of peptides binding with CSCs was examined through comparing the binding capacity of peptides with either H460 cells or other cell lines including stem cells. The isolation of CSCs was performed using specific fluorescent peptides with FACS machine. The further characterization of selected CSCs including exploring the source and tumorigenesis mechanism of CSCs was performed through miRNA analysis. Results: Stem-like cells divided and formed tumor spheres in a couple of weeks, while most of cancer cells died in the serum-free medium. These stem-like cells were able to grow indefinitely as tumor spheres in long-term culture (more than 20 passages). Meanwhile, sphere cells could form colonies in the soft agar plates and exhibit the drug resistance capacity, which indicated that these sphere cells behaved as CSCs. Three peptides binding specifically to those CSCs were identified with bacteria surface display method. They could seldom bind with other cell types including H460 cells, H1299 sphere cells, HLF cells, MSCs and ESCs. One selected peptides named HCBP-1 exhibited highly specific binding capacity to H460 CSCs. The apparent dissociation constant (KD) was 6.4 × 10-7 M with a flow cytometry-based assay. In the peptides binding subgroup of H460 cells, at least 10 miRNAs presented the different expression rate from the H460 cells. Conclusions: In our study, a panel of specific binding peptides was identified with bacteria surface display method which could be used for isolation and characterization of CSCs without definitive CSCs markers. Furthermore, this new method could be potentially expanded to characterize and isolate CSCs in any types of cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A54. Citation Format: Yimin Zhu, Anxin Wang. Isolation and characterization of cancer stem cells with peptides. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A54.

Regulation of the Expression of Chaperone gp96 in Macrophages and Dendritic Cells

The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD).

Abstract B32: Cell penetrating Myosin-Va fragment covering the binding motif of DLC2: Evaluation of pro-apoptotic properties

Abstract Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Recently, our research group has demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Besides, MVaf expression attenuated B16-F10 solid tumor growth in mice suggesting that this peptide may be useful as an apoptosis-inducing tool for basic cancer research. We suggest that MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. In view of such findings, we have proposed to develop a recombinant Tat-fusion MVaf to be explored in peptide-based therapeutic approaches. The native MVaf sequence was altered through the N-terminal addition of a transducible Tat-HA tag. EGFP was alternatively introduced into recombinant products to assess their subcellular localization. Bacterially expressed His-tagged fusion proteins were purified by immobilized metal chelate affinity chromatography on Ni-NTA beads, and transduction assays were initially performed in HeLa cells. The kinetics of Tat-HA-MVaf (THM, 13.7 kDa) and Tat-HA-EGFP-MVaf (THEM, 41.9 kDa) uptake, at concentrations ranging from 1 to 2 μM, was evaluated by confocal laser scanning microscopy (CLSM). Tat-HA-EGFP (THE, 33.9 kDa) was used as a control. Tat-HA-MVaf displayed a diffuse distribution throughout the cells in the first 5 min incubation, with a clear perinuclear accumulation after 1h incubation. THEM and THE fluorescence were detected in many developing vesicles adjacent to cell membrane, and distributed throughout the cytoplasm and nucleus after 5 min incubation, but became markedly weaker after 1h, suggesting proteolytic loss. Despite intense diffusion and intracellular accumulation of recombinant proteins, no significant changes in morphology and cell viability were detected by means of flow cytometry and MTT assay. Pull-down assay revealed that THEM but not THE binds to immobilized GST-DLC2 and to GST-DLC1 in a lesser extent, which confirms peptide-binding capacity to DLCs. On the other hand, pretreatment of cells with chloroquine, an inhibitor of endosomal acidification, sensitized cells to peptide treatment. We believe that Tat-mediated subcellular localization may have predominated, resulting in mislocalization of the peptide cargo. Also, one major drawback in using pTAT-conjugated peptides is their entrapment in cytoplasmic compartments and nucleus, resulting in limited availability and biological activity. One possible strategy is to produce recombinant peptides conjugated reversibly to Tat by a disulfide bond, which are disrupted upon intracellular delivery. Funding support. FAPESP / CNPq. Citation Format: Denise P. S. Leitão Mazzi, Cleidson P. Alves, Enilza Maria Espreafico. Cell penetrating Myosin-Va fragment covering the binding motif of DLC2: Evaluation of pro-apoptotic properties. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B32.

A Broad Profile of Co-Dominant Epitopes Shapes the Peripheral Mycobacterium tuberculosis Specific CD8+ T-Cell Immune Response in South African Patients with Active Tuberculosis

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.

Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli

The binding capacity of peptides with broad antimicrobial activity, or antimicrobial peptides (AMPs), to microbes has recently been applied to the specific detection of bacteria and viruses. We established a novel lateral flow assay (LFA) that combines AMPs labeled with colloidal gold and a target-specific antibody immobilized on a nitrocellulose membrane. α-Helical AMPs, especially cecropin P1 (CP1), magainin 2 (MG2), and ceratotoxin A (CtxA), were shown to have optimal properties as probes in LFA. We also established a multiplex LFA for the simultaneous detection and identification of three serogroups of Shiga toxin-producing Escherichia coli (STEC) using the CP1 probe with polyclonal antibodies anti-O157, anti-O26, and anti-O111. Each serogroup of E. coli could easily and rapidly be detected by multiplex LFA using CP1 and each was clearly visualized in a different position on the LFA strip. The multiplex LFA could detect all tested E. coli strains from serogroups O157 (22/22), O26 (17/17), and O111 (7/7), and the detection limit was 104CFU/mL. No other serogroups of E. coli, including STEC O45, O91, O103, O121, and O145, or non-E. coli strains, reacted. The multiplex LFA could detect E. coli O157, O26, and O111 in food samples at very low levels (6.3, 2.9, and 5.6CFU per 25g of ground beef, respectively) after 18-h enrichment, and these results were in accordance with the results of the culture method, immunochromatography (IC) strip, and PCR. Given the broad binding capacity, AMP probes in combination with specific antibodies in the novel multiplex LFA may have the potential to detect various microbes simultaneously with identification on a single strip.

Attaching the phage display-selected GLA peptide to liposomes: Factors influencing target binding

In our previous study, phage display selections were performed by in situ perfusion of a random peptide library through a mouse brain. This yielded two peptides (GLA and GYR) that showed significant binding to human brain endothelial cells (hCMEC/D3) when displayed on phage particles, but not to human umbilical vein endothelial cells (HUVECs). In the present study, these peptides were produced synthetically and coupled to liposomes to investigate the capacity of the peptides to act as ligands for targeting to hCMEC/D3 cells. Flow cytometry studies showed that these peptides when coupled to liposomes showed weak binding to the target brain endothelial cells. We hypothesized that the weak endothelial cell binding of the selected peptides when coupled to liposomes as compared to the binding of the peptides displayed on phage particles may be ascribed to: change of vehicle shape, change of peptide density, or change of peptide conformation. Peptide density on the liposomes influenced binding of the liposomes to the cells, however, this effect was minor. To study the influence of the peptide conformation, the GLA peptide was recombinantly produced fused to the N1–N2 domains of the phage p3 minor coat protein (p3-GLA) to mimic its conformation when displayed on phage particles. Binding of liposomes modified with either the GLA peptide or the p3-GLA protein to hCMEC/D3 cells was studied, and the p3-GLA-liposomes showed a higher binding to the cells compared to the GLA-liposomes. The experiments demonstrate that bringing the GLA peptide into the original phage protein environment restores and improves the peptide binding capacity and suggest that the GLA peptide, with some modifications, may be used as a brain-targeting ligand in the future.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Teoría de péptidos de alta unión de malaria al glóbulo rojo: Predicciones teóricas de nuevos péptidos de unión y mutaciones teóricas predictivas de aminoácidos críticos

The trial-error method has been used to study the binding capacity of peptides from merozoite proteins to red blood cell receptors.Physical-mathematical characterizations with probability and entropy have differentiated in an objective way high-affinity binding peptides from those that do not bind.Five theoretical peptides and 40 peptides from 4 merozoite surface proteins were selected. An induction was made by calculating the probability of grouped amino acids to happen, and consecutively that of two amino acids. By building a sample probability space with high-affinity binding peptides and another one with non-binding peptides, the predictive values for high-affinity and non-binding peptides was calculated. One hundred and sixteen non-binding peptides from the same proteins were quantified, confirming predictive values. The predictive values for overlapped peptides from MSP-2 were calculated, and some performance measures for predictions were quantified. Theoretical mutations for one high-affinity binding peptide were constructed, quantifying for each mutation the predictive values.A predictive methodology for the binding of merozoite surface proteins to red blood cell receptors was developed, with 95% sensibility, and 90% specificity, with respect to experimental results.The development of an a-causal theory founded in a physical-mathematical order, which replaced the molecular biology language and the assay-error method for a mental experiment, physical-mathematical laws and one theoretical induction lead to the creation of effective predictions of the experiments.