-
[show abstract]
[hide abstract]
ABSTRACT: The rise in antibiotic resistance has led to an increased research focus on discovery of new antibacterial candidates. While broad-spectrum antibiotics are widely pursued, there is evidence that resistance arises in part from the wide spread use of these antibiotics. Our group has developed a system to produce protein affinity agents, called synbodies, which have high affinity and specificity for their target. In this report, we describe the adaptation of this system to produce new antibacterial candidates towards a target bacterium. The system functions by screening target bacteria against an array of 10,000 random sequence peptides and, using a combination of membrane labeling and intracellular dyes, we identified peptides with target specific binding or killing functions. Binding and lytic peptides were identified in this manner and in vitro tests confirmed the activity of the lead peptides. A peptide with antibacterial activity was linked to a peptide specifically binding Staphylococcus aureus to create a synbody with increased antibacterial activity. Subsequent tests showed that this peptide could block S. aureus induced killing of HEK293 cells in a co-culture experiment. These results demonstrate the feasibility of using the synbody system to discover new antibacterial candidate agents.
PLoS ONE 01/2013; 8(1):e54162. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A practical diagnostic test is needed for early Alzheimer's disease (AD) detection. Immunosignaturing, a technology that employs antibody binding to a random-sequence peptide microarray, generates profiles that distinguish transgenic mice engineered with familial AD mutations (APPswe/PSEN1-dE9) from non-transgenic littermates. It can also detect an AD-like signature in humans. Here, we assess the changes in the immunosignature at different time points of the disease in mice and humans. We also evaluate the accuracy of the late-stage signature as a test to discriminate between young mice with familial AD mutations from non-transgenic littermates. Plasma samples from AD patients were assayed 3-12months apart, while APPswe/PSEN1-dE9 and non-transgenic controls supplied plasma at monthly intervals until they reached 15months of age. Microarrays with 10,000 random-sequence peptides were used to compare antibody binding patterns. These patterns gradually changed over the life-span of mice. Strong, characteristic signatures were observed in transgenic mice at early, mid and late stages, but these profiles had minimal overlap. The signature of young transgenic mice had an error rate of 18% at classifying plasma samples from late-stage transgenic mice. Conversely, the late-stage transgenic mice signature discriminated between young transgenic mice and littermates with an error rate of 21%. Less distinctive profiles were recognizable throughout the transgenic mice lifespan, being detectable as early as 2months. The human signature had minimal change on short-term follow-up. Our results call for a reappraisal of the way incipient AD is studied, as biomarkers seen in late-stages of the disease may not be relevant in earlier stages.
Journal of neuroimmunology 10/2012; · 2.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The development of arrays of human proteins has been a huge boon to the search for autoantibody diagnostics. Typically, slides with thousands of recombinant human proteins arrayed in an addressable fashion are incubated with sera from diseased or normal people. If an antibody binds a protein more in the diseased than in the normal cohort it is considered an autoantibody response. It is usually presumed that the autoantibody was elicited by the protein bound on the array. However, our studies using human protein and random peptide arrays indicate that antibody specificity may not be as high as commonly thought. Therefore we have tested the assumption of the source of autoantibodies. One test was to generate antibodies to two totally random peptides and bind these antibodies to a human protein array. One of the antibodies generated bound two human proteins. A second test was to generate an antibody to a frameshift peptide occurring in cancers. This antibody also bound several proteins on the array. We conclude that one should be cautious about assuming a particular autoantibody target on an array which elicited the original immune response.
Experimental and Molecular Pathology 03/2012; 92(3):304-11. · 2.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To address the need for a universal system to assess health status, we previously described a method termed "immunosignaturing" which splays the entire humoral antibody repertoire across a peptide microarray. Two important issues relative to the potential broad use of immunosignatures are sample preparation and stability. In the present study, we compared the immunosignatures developed from serum, plasma, saliva, and antibodies eluted from blood dried onto filter paper. We found that serum and plasma provide identical immunosignatures. Immunosignatures derived from dried blood also correlated well with those from nondried serum from the same individual. Immunosignatures derived from dried blood were capable of distinguishing naïve mice from those infected with influenza virus. Saliva was applied to the arrays, and the IgA immunosignature correlated strongly with that from dried blood. Finally, we demonstrate that dried blood retains immunosignature information even when exposed to high temperature. This work expands the potential diagnostic uses for immunosignatures. These features suggest that different forms of archival samples can be used for diagnosis development and that in prospective studies samples can be easily procured.
Clinical and vaccine immunology: CVI 03/2012; 19(3):352-8. · 2.37 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible "immunosignature" on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.
Molecular & Cellular Proteomics 01/2012; 11(4):M111.011593. · 7.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task.
GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance.
GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.
BMC Bioinformatics 01/2012; 13:1. · 2.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: One approach to prepare protein binding ligands is to join two low-affinity ligands that bind different sites on the target protein to create a high-affinity bivalent ligand. This typically requires some knowledge of the ligand binding site and requires exquisite orientation of the ligands in order to achieve maximum binding affinity. Here, we explored the limit of affinity improvement possible with no a priori knowledge of peptide binding site and with minimal effort spent in linking the lead peptides. We compared the affinity enhancement from linking two peptides with low affinity for tumor necrosis factor-α (TNFA) to the affinity enhancement from linking affinity improved versions of these peptides using several different scaffolds. We found that we achieved the highest affinity gain not by the precise positioning of the peptides, but rather by using affinity improved versions of the lead peptides to produce synbodies with apparent K(D)'s of 9 to 48 nM. Kinetic analysis showed that the binding kinetics of the synbody are strongly influenced by the kinetics of the starting peptide. This suggests that careful selection of peptides based on their kinetic profile prior to linking will influence the kinetics of the final binding agent.
Bioconjugate Chemistry 08/2011; 22(8):1473-8. · 4.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Accurate assessment of Alzheimer's disease (AD), both presymptomatically and at different disease stages, will become increasingly important with the expanding elderly population. There are a number of indications that the immune system is engaged in AD. Here we explore the ability of an antibody-profiling technology to characterize AD and screen for peptides that may be used for a simple diagnostic test.
We developed an array-based system to profile the antibody repertoire of transgenic mice with cerebral amyloidosis (TG) and elderly individuals with or without AD. The array consists of 10,000 random sequence peptides (20-mers) capable of detecting antibody binding patterns, allowing the identification of peptides that mimic epitopes targeted by a donor's serum.
TG mice exhibited a distinct immunoprofile compared to nontransgenic littermates. Further, we show that dementia patients, including autopsy-confirmed AD subjects, have distinguishable profiles compared to age-matched nondemented people. Using antibodies to different forms of Aβ peptide and blocking protocols, we demonstrate that most of this signature is not due to the subject's antibodies raised against Aβ.
We propose that "immunosignaturing" technology may have potential as a diagnostic tool in AD.
Annals of Neurology 08/2011; 70(2):286-95. · 11.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A universal platform for efficiently mapping antibody epitopes would be of great use for many applications, ranging from antibody therapeutic development to vaccine design. Here we tested the feasibility of using a random peptide microarray to map antibody epitopes. Although peptide microarrays are physically constrained to ∼10(4) peptides per array, compared with 10(8) permitted in library panning approaches such as phage display, they enable a much more high though put and direct measure of binding. Long (20 mer) random sequence peptides were chosen for this study to look at an unbiased sampling of sequence space. This sampling of sequence space is sparse, as an exact epitope sequence is unlikely to appear. Commercial monoclonal antibodies with known linear epitopes or polyclonal antibodies raised against engineered 20-mer peptides were used to evaluate this array as an epitope mapping platform. Remarkably, peptides with the most sequence similarity to known epitopes were only slightly more likely to be recognized by the antibody than other random peptides. We explored the ability of two methods singly and in combination to predict the actual epitope from the random sequence peptides bound. Though the epitopes were not directly evident, subtle motifs were found among the top binding peptides for each antibody. These motifs did have some predictive ability in searching for the known epitopes among a set of decoy sequences. The second approach using a windowing alignment strategy, was able to score known epitopes of monoclonal antibodies well within the test dataset, but did not perform as well on polyclonals. Random peptide microarrays of even limited diversity may serve as a useful tool to prioritize candidates for epitope mapping or antigen identification.
Molecular & Cellular Proteomics 11/2010; 10(3):M110.000786. · 7.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme activity. A polyvinyl alcohol solution was applied to array surfaces to limit the diffusion of product molecules released from enzymatic reactions, allowing the simultaneous measurement of enzyme activity and binding at each peptide spot. For proof of concept, it was possible to identify peptides that bound to horseradish peroxidase, alkaline phosphatase, and beta-galactosidase and substantially altered enzyme activity by comparing the binding level of peptide to enzyme and bound enzyme activity. This basic technique may be generally applicable to find peptides or other small molecules that modify enzyme activity.
Journal of the American Chemical Society 05/2010; 132(18):6419-24. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A universal system to diagnose disease, characterize infection or evaluate the response to a vaccine would be useful. Towards this end we introduce a machine-readable platform that we term "Immunosignaturing". Rather than attempt to identify antibodies one by one, we splay the entire immune response across an array of 10,000 random sequence peptides. This segregates serum antibodies sufficiently to group and characterize responses caused by disease or vaccination. In the present study, we explore in detail the murine immunosignature to influenza A/PR/8/34 immunization and subsequent challenge. Even though the peptides are random sequence, the response to immunization and challenge is quite apparent. We find that the immunosignatures contained information not evident in whole virus ELISA. Antibody recognition of 283 influenza-specific peptides increased upon immunization and remained elevated for 211 days post-challenge. A set of 65 peptides, which overlapped 39 of the peptides that were consistent across time, was capable of distinguishing mice based on infectious dose, while whole virus ELISA could not. These peptide populations are consistently recognized in independent biological replicates of infection and are largely, but not solely, composed of virus reactive antibodies. The immunosignaturing analysis was expanded to analysis of human recipients of the 2006/2007 seasonal influenza vaccine. We find that 30 peptides are significantly recognized by all donors 21 days post-immunization and have the power to distinguish immune from pre-immune samples. Taken together the data suggest that immunosignaturing on a random peptide array can serve as a universal platform to assess antibody status in ways that cannot be replicated by conventional immunological assays.
Vaccine 05/2010; 28(28):4529-37. · 3.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: There is a pressing need for high-affinity protein binding ligands for all proteins in the human and other proteomes. Numerous groups are working to develop protein binding ligands but most approaches develop ligands using the same strategy in which a large library of structured ligands is screened against a protein target to identify a high-affinity ligand for the target. While this methodology generates high-affinity ligands for the target, it is generally an iterative process that can be difficult to adapt for the generation of ligands for large numbers of proteins.
We have developed a class of peptide-based protein ligands, called synbodies, which allow this process to be run backwards--i.e. make a synbody and then screen it against a library of proteins to discover the target. By screening a synbody against an array of 8,000 human proteins, we can identify which protein in the library binds the synbody with high affinity. We used this method to develop a high-affinity synbody that specifically binds AKT1 with a K(d)<5 nM. It was found that the peptides that compose the synbody bind AKT1 with low micromolar affinity, implying that the affinity and specificity is a product of the bivalent interaction of the synbody with AKT1. We developed a synbody for another protein, ABL1 using the same method.
This method delivered a high-affinity ligand for a target protein in a single discovery step. This is in contrast to other techniques that require subsequent rounds of mutational improvement to yield nanomolar ligands. As this technique is easily scalable, we believe that it could be possible to develop ligands to all the proteins in any proteome using this approach.
PLoS ONE 01/2010; 5(5):e10728. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: There is a significant need for affinity reagents with high target affinity/specificity that can be developed rapidly and inexpensively. Existing affinity reagent development approaches, including protein mutagenesis, directed evolution, and fragment-based design utilize large libraries and/or require structural information thereby adding time and expense. Until now, no systematic approach to affinity reagent development existed that could produce nanomolar affinity from small chemically synthesized peptide libraries without the aid of structural information.
Based on the principle of additivity, we have developed an algorithm for generating high affinity peptide ligands. In this algorithm, point-variations in a lead sequence are screened and combined in a systematic manner to achieve additive binding energies. To demonstrate this approach, low-affinity lead peptides for multiple protein targets were identified from sparse random sequence space and optimized to high affinity in just two chemical steps. In one example, a TNF-α binding peptide with K(d) = 90 nM and high target specificity was generated. The changes in binding energy associated with each variation were generally additive upon combining variations, validating the basis of the algorithm. Interestingly, cooperativity between point-variations was not observed, and in a few specific cases, combinations were less than energetically additive.
By using this additivity algorithm, peptide ligands with high affinity for protein targets were generated. With this algorithm, one of the highest affinity TNF-α binding peptides reported to date was produced. Most importantly, high affinity was achieved from small, chemically-synthesized libraries without the need for structural information at any time during the process. This is significantly different than protein mutagenesis, directed evolution, or fragment-based design approaches, which rely on large libraries and/or structural guidance. With this algorithm, high affinity/specificity peptide ligands can be developed rapidly, inexpensively, and in an entirely chemical manner.
PLoS ONE 01/2010; 5(11):e15432. · 4.09 Impact Factor
-
Nature 10/2009; 461(7265):E8-E8. · 36.28 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An application of high density random sequence peptide microarrays for rapid and reliable identification of artificial carbohydrate receptors is reported.
The Analyst 05/2009; 134(4):650-2. · 4.23 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Current analytical methods have been slow in addressing the growing need for glyco-analysis. A new generation of more empirical high-throughput (HTP) tools is needed to aid the advance of this important field. To this end, we have developed a new HTP screening platform for identification of surface-immobilized peptides that specifically bind O-antigenic glycans of bacterial lipopolysaccharides (LPS). This method involves screening of random sequence peptide libraries in addressable high-density microarray format with the newly developed luminescent LPS-quantum dot micelles. Screening of LPS fractions from O111:B4 and O55:B5 serotypes of E. coli on a microarray consisting of 10,000 20-mer peptide features revealed minor differences, while comparison of LPS from E. coli O111:B4 and P. aeruginosa produced sets of highly specific peptides. Peptides strongly binding to the E. coli LPS were highly enriched in aromatic and cationic amino acids, and most of these inhibited growth of E. coli. Flow cytometry and isothermal titration calorimetry (ITC) experiments showed that some of these peptides bind LPS in-solution with a K(d) of 1.75 microM. Peptide selections against P. aeruginosa were largely composed of hydrogen-bond forming amino acids in accordance with dramatic compositional differences in O-antigenic glycans in E. coli and P. aeruginosa. While the main value of this approach lies in the ability to rapidly differentiate bacterial and possibly other complex glycans, the peptides discovered here can potentially be used off-array as antiendotoxic and antimicrobial lead compounds, and on-array/on-bead as diagnostic and affinity reagents.
ChemBioChem 03/2009; 10(5):877-88. · 3.94 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bacterial endotoxins or lipopolysaccharides (LPS) are among the most potent activators of the innate immune system, yet mechanisms of their action and in particular the role of glycans remain elusive. Efficient non-invasive labeling strategies are necessary for studying interactions of LPS glycans with biological systems. Here we report a new method for labeling LPS and other lipoglycans with luminescent quantum dots. The labeling is achieved by partitioning of hydrophobic quantum dots into the core of various LPS aggregates without disturbing the native LPS structure. The biofunctionality of the LPS-Qdot conjugates is demonstrated by the labeling of mouse monocytes. This simple method should find broad applicability in studies concerned with visualization of LPS biodistribution and identification of LPS binding agents.
Biochemical and Biophysical Research Communications 02/2009; 380(1):1-4. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent analysis of a Gal4 mutant (Gap71) carrying three point mutations (S22D, K23Q and K25F) in its DNA-binding domain (DBD), has demonstrated that it cannot occupy GAL promoters efficiently in cells and that it is not mono-ubiquitylated, suggesting a functional link between this modification and stable DNA binding in cells. The mechanistic underpinning of this phenotype is that this protein is hypersensitive to a newly discovered activity of the proteasomal ATPases--their ability to actively dissociate transcription factor-DNA complexes after direct interaction with the activation domain. In this paper, we examine the roles of each of the three point mutations contained in Gap71 individually. These experiments have revealed that serine 22 is a site of phosphorylation in the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo. These data represent the first report of an essential phosphorylation event that is critical for the activity of this paradigmatic transcription factor.
Molecular BioSystems 12/2008; 4(11):1116-25. · 3.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The Saccharomyces cerevisiae Gal4 protein is a paradigmatic transcriptional activator containing a C-terminal acidic activation domain (AD) of 34 amino acids. A mutation that results in the truncation of about two-thirds of the Gal4AD (gal4D) results in a crippled protein with only 3% the activity of the wild-type activator. We show here that although the Gal4D protein is not intrinsically deficient in DNA binding, it is nonetheless unable to stably occupy GAL promoters in vivo. This is because of the activity of the proteasomal ATPases, including Sug1/Rpt6, which bind to Gal4D via the remainder of the AD and strip it off of DNA. A mutation that suppressed the Gal4D "no growth on galactose" phenotype repressed the stripping activity of the ATPase complex but not other activities. We further demonstrate that Gal4D is hypersensitive to this stripping activity because of its failure to be monoubiquitylated efficiently in vivo and in vitro. Evidence is presented that the piece of the AD that is deleted in Gal4D protein is likely a recognition element for the E3 ubiquitin-protein ligase that modifies Gal4. These data argue that acidic ADs comprise at least two small peptide subdomains, one of which is responsible for activator monoubiquitylation and another that interacts with the proteasomal ATPases, coactivators and other transcription factors. This study validates the physiological importance of Gal4 monoubiquitylation and clarifies its major role as that of protecting the activator from being destabilized by the proteasomal ATPases.
Journal of Biological Chemistry 06/2008; 283(18):12614-23. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Destabilization of activator-DNA complexes by the proteasomal ATPases can inhibit transcription by limiting activator interaction with DNA. Modification of the activator by monoubiquitylation protects the activator from this destabilization activity. In this study, we probe the mechanism of this protective effect of monoubiquitylation. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and Rpt1. This interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. A model is proposed in which activator monoubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.
Journal of Biological Chemistry 06/2008; 283(31):21789-98. · 4.77 Impact Factor
