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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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Systematic analysis of the RGS2 degron reveals characteristics of substrate recognition by the F-box protein FBXO44

McNabb, Harrison J.; Cho, Eugene; Pitman, Mary; Rushton, Phillip S.; Mobley, David; Sjögren, Benita
Journal of Biological Chemistry.
Nov 2025
10.1016/j.jbc.2025.110757
Regulator of G protein signaling 2 (RGS2) negatively modulates signaling downstream of G protein–coupled receptors by accelerating GTP hydrolysis at Gα subunits of heterotrimeric G proteins. Decreased RGS2 levels are implicated in numerous diseases, including cardiovascular disease and asthma. Thus, identifying selective means of enhancing RGS2 protein levels would be a viable therapeutic strategy. RGS2 is rapidly degraded through the ubiquitin–proteasomal pathway, and we previously identified F-box only protein 44 (FBXO44) as the substrate recognition component of the E3 ligase responsible for facilitating RGS2 degradation. As such, the RGS2–FBXO44 interaction is a potential target for pharmacological intervention. Detailed information on the FBXO44 recognition site (degron) in RGS2 will aid in structure-based small-molecule inhibitor design, as well as in identifying additional FBXO44 targets, which would help predict possible side effects of targeting this interaction. Thus, the goal of this study was to dissect the molecular properties for FBXO44 binding of the RGS2 degron. We used a peptide array utilizing systematic residue substitution, combined with AlphaFold modeling and molecular dynamics simulations, to identify several amino acid changes that altered binding both positively and negatively. Finally, we experimentally confirmed our results in cells through coimmunoprecipitation and proteasomal inhibition, using full-length RGS2. Altogether, these results provide structural insights into RGS2–FBXO44 binding, which will aid in structure-guided drug discovery efforts. It also provides a framework for building a consensus recognition motif for FBXO44, which could aid in identifying more substrates for this understudied F-box protein.

Preclinical characterization of an active immunotherapy targeting calcitonin gene-related peptide

Boyd, Justin D.; Wang, Shixia; Lin, Hsiao-Wen; Hsieh, Yueh-Ting; Sun, Yu Shuang; Thibodeaux, Brett A.; Lu, Hanxin; Sahni, Jaya; Wiggins, Jonathan; Longo, Matthew S.; Brooks, Jeanne K.; Vroom, Madeline M.; Chang, Yi-Pin; Liu, Zhi; Ding, Shuang; Dodart, Jean-Cosme
Commun Med.
Apr 2025
10.1038/s43856-025-00870-2
Abstract **Background** The success of passive immunotherapies targeting Calcitonin gene-related peptide (CGRP) for managing migraine has prompted our efforts towards developing an active immunotherapy that induces the production of endogenous antibodies against CGRP. Achieving efficacious antibody titers via immunization could provide a more convenient and cost-effective treatment alternative to anti-CGRP monoclonal antibody (mAb) therapies. However, immunization against endogenous CGRP faces multiple challenges such as breaking immune tolerance, inducing sufficient antibody titers, and avoiding immune response-associated toxicity. **Methods** Synthetic peptide immunogens formulated in adjuvants were delivered intramuscularly. Serum samples were collected post immunization and used to measure antibody titers as well as for the isolation of antibodies specific to CGRP. Antibodies were characterized for their binding affinities and specificities. The capsaicin-induced increase in dermal blood flow model was used in rats for the assessment of the pharmacodynamic effect of immunization. **Results** Here we demonstrate that a peptide-based active immunotherapy designed to induce antibodies against CGRP promotes robust antibody titers across preclinical species. Characterization of the immune response strongly suggests that this peptide immunogen primarily stimulates a humoral response and only induced CGRP-specific antibodies. Antibodies produced by immunization are primarily IgG1 and demonstrate binding and activity potencies similar to marketed monoclonal antibodies against CGRP. Finally, immunization demonstrates in vivo efficacy in a rat pharmacodynamic model. **Conclusion** Our results strongly suggest that a peptide-based active immunotherapy against CGRP could provide an affordable and convenient therapeutic for the prevention of migraine.

Identification of Tripeptide Modulators of ACE2 Activity Using a High Throughput Screen (Abstract ID: 165381)

Walker, David F.; Karamyan, Vardan T.
The Journal of Pharmacology and Experimental Therapeutics.
Mar 2025
10.1016/j.jpet.2024.100697
Angiotensin converting enzyme 2 (ACE2) works in the renin angiotensin aldosterone system to decrease circulating levels of angiotensin II by removing the C-terminal phenylalanine and converting it to angiotensin (1-7). In addition, ACE2 has received increased interest in research due to its role in COVID-19 pathogenesis, as the binding site and cell entry gate for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While ACE2 inhibitors have been primarily used as pharmacological tools to study the renin-angiotensin system, small molecule ACE2 enhancers (aka activators) are highly desired because of their hypothesized therapeutic potential. This study was designed to identify peptide-based enhancers of ACE2. First, binding of human recombinant ACE2 to all possible tripeptides composed of the 20 proteinogenic amino acids, was evaluated using a proprietary immunofluorescence-based peptide microarray. Binding of 6xHis-tagged ACE2 to the 8000 tripeptides immobilized on a microchip was evaluated at 10 µg/ml and 100 µg/ml concentrations of the peptidase using a DyLight680-conjugated anti-6xHis-tag antibody. Hemagglutinin (HA) immobilized on the microchip served as a positive control peptide in the microarray and it was tracked using a DyLight800-conjugated anti-HA antibody. The read-out was performed with an Innopsys InnoScan 710-IR Microarray Scanner at scanning gains of 50/10 (red/green). In the result of the microarray a number of tripeptides were identified as potential ACE2 binders. Among them, 22 tripeptides were selected to represent several the most pronounced binders as well as a number of structurally similar tripeptides that did not show appreciable binding to ACE2 to serve as negative control. The effect of the selected peptides (at 1, 10 and 100 µM) on activity of human recombinant ACE2 was tested in a continuous enzymatic assay using a fluorogenic substrate. Contrary to our expectation, none of the peptides affected the activity of ACE2 in a significant manner. These results suggest that the selected peptides do not alter activity of ACE2, but they do not exclude the possibility that some of the peptides may still bind to the peptidase. Our subsequent experiments will apply differential scanning fluorometry (DSF) to determine whether these peptides physically interact with recombinant ACE2.

HCV immunodominant peptide mapping reveals unique HLA-A*02-restricted signatures: insights for CD8+ T-cell-based vaccines and immunotherapies

Cardoso Corrêa-Dias, Laura; Lopes-Ribeiro, Ágata; Marques-Ferreira, Geovane; Gomes-de-Pontes, Letícia; Pereira-Santos, Thaiza Aline; De Sousa Reis, Erik Vinicius; Silva Moraes, Thaís De Fátima; Assis Martins-Filho, Olindo; Figueiredo Barbosa-Stancioli, Edel; Guimarães Da Fonseca, Flávio; Coelho-dos-Reis, Jordana Grazziela
Immunogenetics.
Jan 2025
10.1007/s00251-025-01370-2
Several barriers for the development of an HCV vaccine still exist, including the genetic diversity of the virus, and the shortage of assessable models for in vitro and in vivo assays. Therefore, in this study, HCV epitope mapping was performed for 59 polyprotein sequences from 7 HCV genotypes. Around 2,880 peptides were considered epitopes for CD8+ T cells. The peptide induction of cytokines from Th1 and/or Th2 axes of the cellular immune response was assessed, indicating a tendency for Th2 axis. In vitro evaluation was performed using peptide microarray and a recombinant HLA-A*02:01 molecule. A total of 615 peptides of high reactivity to HLA-A*02:01 were identified, with predominance of leucine and tryptophan residues, highlighting their importance for TCR-epitope binding and CD8+ T activation. Finally, HCV-derived peptide patterns restricted to HLA-A2*02:01 observed in this study provide important information for the development of a multi-epitope-based pan-genotypic vaccine against the virus.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein

Gardner, Thomas J.; Stein, Kathryn R.; Duty, J. Andrew; Schwarz, Toni M.; Noriega, Vanessa M.; Kraus, Thomas; Moran, Thomas M.; Tortorella, Domenico
Nat Commun.
Dec 2016
10.1038/ncomms13627
The prototypic β-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies’ epitope as an ‘antigenic hot spot’ critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

A single amino acid substitution alter antigenicity of Glycosylated protein 4 of HP-PRRSV

Wang, Xinglong; Wang, Zhenbin; Xu, Hongyu; Biao, Xiang; Yang, Zengqi
Virol J.
Jul 2016
10.1186/s12985-016-0586-3
Background Porcine reproductive and respiratory syndrome (PRRS) is an important pig endemic disease in pork-producing countries worldwide. The etiology, porcine reproductive and respiratory syndrome virus (PRRSV), is characterized by fast antigen variability. Glycosylated protein 4 (GP4) is a minor protein in PRRSV virion, but contributes to induce protective immune responses. However, the antigenic characterization of PRRSV GP4 and the role of the mutations in this protein in PRRSV evolution are not clear. Methods Peptides chip scanning and peptide based ELISA was used to analyze the antigenic characterization of HP-PRRSV GP4. A total of 142 peptides printed on a chip were used to reveal the antigen reaction characteristics of the HP-PRRSV. The reactions of these peptides with HP-PRRSV-specific pig serum were scanned and quantified using the software PepSlide® Analyzer by fluorescence intensity. The active reaction regions (AR) were identified based on the scanning results and then the amino acids (aa) sequences of AR(s) is aligned among PRRSV strains for further identify the key aa site(s) impact the antigenicity of the protein. Peptide based ELISA is then reacted with PRRSV positive sera derived from pig inoculated with different PRRSV strains for further analysis the role of specific amino acid in AR. Results The intensity plot was used to show the reactions of the peptides with PRRSV serum and it showed that enormously different response happened to various parts of GP4. The highest reaction intensity value reached 6401.5 against one peptide with the sequence DIKTNTTAASDFVVL. An AR from S29 to G56 was identified. Sequence alignment revealed various mutations in site 43 and possibly played an important role in this AR. Peptides ELISA reaction with sera from pigs inoculated with different PRRSV strain revealed that the change of aa in site 43 reduced the reaction of the peptide with PRRSV positive sera derived from pigs inoculated with the peptide related PRRSV strains. Conclusion In this study, one AR covering S29 to G56 was identified in GP4. The aa in site 43 play an important role in determining the antigenic character of GP4. The continual mutations (S → G → D → N) occurred in this site alter the antigenicity of PRRSV GP4.

High-Density Peptide Arrays for Malaria Vaccine Development

Loeffler, Felix F.; Pfeil, Johannes; Heiss, Kirsten
Apr 2016
10.1007/978-1-4939-3387-7_32
The development of an efficacious and practicable vaccine conferring sterile immunity towards a Plasmodium infection represents a not yet achieved goal. A crucial factor for the impact of a given anti-plasmodial subunit vaccine is the identification of the most potent parasitic components required to induce protection from both infection and disease. Here, we present a method based on a novel high-density peptide array technology that allows for a flexible readout of malaria antibodies. Peptide arrays applied as a screening method can be used to identify novel immunogenic antibody epitopes under a large number of potential antigens/peptides. Ultimately, discovered antigen candidates and/or epitope sequences can be translated into vaccine prototype design. The technology can be further utilized to unravel antibody-mediated immune responses (e.g., involved in the establishment of semi-immunity) and moreover to confirm vaccine potency during the process of clinical development by verifying the induced antibody responses following vaccination.

HIV-1 gp140 epitope recognition is influenced by immunoglobulin DH gene segment sequence

Wang, Yuge; Kapoor, Pratibha; Parks, Robert; Silva-Sanchez, Aaron; Alam, S. Munir; Verkoczy, Laurent; Liao, Hua-Xin; Zhuang, Yingxin; Burrows, Peter; Levinson, Michael; Elgavish, Ada; Cui, Xiangqin; Haynes, Barton F.; Schroeder, Harry
Immunogenetics.
Feb 2016
10.1007/s00251-015-0890-x
Complementarity Determining Region 3 of the immunoglobulin (Ig) H chain (CDR-H3) lies at the center of the antigen-binding site where it often plays a decisive role in antigen recognition and binding. Amino acids encoded by the diversity (DH) gene segment are the main component of CDR-H3. Each DH has the potential to rearrange into one of six DH reading frames (RFs), each of which exhibits a characteristic amino acid hydrophobicity signature that has been conserved among jawed vertebrates by natural selection. A preference for use of RF1 promotes the incorporation of tyrosine into CDR-H3 while suppressing the inclusion of hydrophobic or charged amino acids. To test the hypothesis that these evolutionary constraints on DH sequence influence epitope recognition, we used mice with a single DH that has been altered to preferentially use RF2 or inverted RF1. B cells in these mice produce a CDR-H3 repertoire that is enriched for valine or arginine in place of tyrosine. We serially immunized this panel of mice with gp140 from HIV-1 JR-FL isolate and then used enzyme-linked immunosorbent assay (ELISA) or peptide microarray to assess antibody binding to key or overlapping HIV-1 envelope epitopes. By ELISA, serum reactivity to key epitopes varied by DH sequence. By microarray, sera with Ig CDR-H3s enriched for arginine bound to linear peptides with a greater range of hydrophobicity but had a lower intensity of binding than sera containing Ig CDR-H3s enriched for tyrosine or valine. We conclude that patterns of epitope recognition and binding can be heavily influenced by DH germ line sequence. This may help explain why antibodies in HIV-infected patients must undergo extensive somatic mutation in order to bind to specific viral epitopes and achieve neutralization.

Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen

Schmidt, Ronny; Jacak, Jaroslaw; Schirwitz, Christopher; Stadler, Volker; Michel, Gerd; Marmé, Nicole; Schütz, Gerhard J.; Hoheisel, Jörg D.; Knemeyer, Jens-Peter
J. Proteome Res..
Jan 2011
10.1021/pr101070j

Combinatorial Synthesis of Peptide Arrays onto a Microchip

Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
Science.
Dec 2007
10.1126/science.1149751
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.

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