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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.

Anti-allodynic effect of intrathecal antibodies against macrophage-inducible C-type lectin in spinal nerve ligation model in rat

Kang, Dong Ho; Kim, Woong Mo; Bae, Hong Beom; Yang, Jihoon; Choi, Jeong Il
Heliyon.
Nov 2024
10.1016/j.heliyon.2024.e40694
*Introduction* Macrophage-inducible C-type lectin (Mincle) has emerged as a potential contributor to neuropathic pain induction and neuroinflammatory responses within the spinal cord. Moreover, evidence suggests a close association between toll-like receptor (TLR) and Mincle expression in myeloid cells. This study evaluated the effectiveness of Mincle antibodies in neuropathic pain and identified the epitope of these antibodies. In addition, the mode of interaction between Mincle and TLR inhibition was explored using isobolographic analysis. *Methods* Three different Mincle antibodies and a specific TLR4 inhibitor (TAK-242) were intrathecally administered, and mechanical allodynia was evaluated using the von Frey test in a rat model of spinal nerve ligation (SNL). Isobolographic analysis was conducted on the effect of combination of TAK-242 and Mincle Ab. Microarray analysis examined the specific region of Mincle targeted by the antibodies. *Results* All Mincle antibodies and TAK-242 significantly alleviated mechanical allodynia in a dose-dependent manner. However, the maximal possible effects (MPE) produced by the antibodies ranged widely from 37.1% to 91.8%, comparable to that of TAK-242 (88.7%). The combination of TAK-242 and the antibody with the highest MPE resulted in an additive interaction for their anti-allodynic effects. Epitope mapping revealed that each antibody targeted the extracellular domain, with epitope lengths ranging from 5 to 15 amino acids. *Conclusions* The current study demonstrates the anti-allodynic effect of Mincle antibodies and additive interaction with TLR4 inhibition in spinal nerve ligation model, suggesting the potential of blocking of Mincle signaling with its antibodies as a novel treatment strategy for neuropathic pain.

Discovery of a novel highly specific, fully human PSCA antibody and its application as an antibody-drug conjugate in prostate cancer

Chu, Xiaojie; Shin, Seungmin; Baek, Du-San; Zhang, Liyong; Conard, Alex; Shi, Megan; Kim, Ye-Jin; Adams, Cynthia; Hines, Maggie; Liu, Xianglei; Chen, Chuan; Sun, Zehua; Jelev, Dontcho V.; Mellors, John W.; Dimitrov, Dimiter S.; Li, Wei
mAbs.
Aug 2024
10.1080/19420862.2024.2387240
Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63–69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.

Antigen-Heterologous Vaccination Regimen Triggers Alternate Antibody Targeting in SARS-CoV-2-DNA-Vaccinated Mice

Frische, Anders; Krogfelt, Karen Angeliki; Fomsgaard, Anders; Lassaunière, Ria
Vaccines.
Feb 2024
10.3390/vaccines12030218
An in-depth analysis of antibody epitopes following vaccination with different regimens provides important insight for developing future vaccine strategies. B-cell epitopes conserved across virus variants may be ideal targets for vaccine-induced antibodies and therapeutic drugs. However, challenges lie in identifying these key antigenic regions, and directing the immune system to target them. We previously evaluated the immunogenicity of two candidate DNA vaccines encoding the unmodified spike protein of either the SARS-CoV-2 Index strain or the Beta variant of concern (VOC). As a follow-on study, we characterized here the antibody binding profiles of three groups of mice immunized with either the DNA vaccine encoding the SARS-CoV-2 Index strain spike protein only, the Beta VOC spike protein only, or a combination of both as an antigen-heterologous prime-boost regimen. The latter induced an antibody response targeting overlapping regions that were observed for the individual vaccines but with additional high levels of antibody directed against epitopes in the SD2 region and the HR2 region. These heterologous-vaccinated animals displayed improved neutralization breadth. We believe that a broad-focused vaccine regimen increases neutralization breadth, and that the in-depth analysis of B-cell epitope targeting used in this study can be applied in future vaccine research.

A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by Escherichia coli in human and environmental samples

He, Xiaohua; Patfield, Stephanie; Hnasko, Robert; Rasooly, Reuven; Mandrell, Robert E.
PLoS ONE.
Oct 2013
10.1371/journal.pone.0076368
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) are frequent causes of severe human diseases ranging from diarrhea to hemolytic uremic syndrome. The existing strategy for detection of STEC relies on the unique sorbitol-negative fermentation property of the O157 strains, the most commonly identified serotype has been E. coli O157. It is becoming increasingly evident, however, that numerous non-O157 STEC serotypes also cause outbreaks and severe illnesses. It is necessary to have new methods that are capable of detecting all STEC strains. METHODS AND FINDINGS: Here we describe the development of a sandwich ELISA assay for detecting both O157 and non-O157 STECs by incorporating a novel polyclonal antibody (pAb) against Stx2. The newly established immunoassay was capable of detecting Stx2a spiked in environmental samples with a limit of detection between 10 and 100 pg/mL in soil and between 100 and 500 pg/mL in feces. When applied to 36 bacterial strains isolated from human and environmental samples, this assay detected Stx2 in all strains that were confirmed to be stx2-positive by real-time PCR, demonstrating a 100% sensitivity and specificity. CONCLUSIONS: The sandwich ELISA developed in this study will enable any competent laboratory to identify and characterize Stx2-producing O157 and non-O157 strains in human and environmental samples, resulting in rapid diagnosis and patient care. The results of epitope mapping from this study will be useful for further development of a peptide-based antibody and vaccine.

Optimised ‘on demand’ protein arraying from DNA by cell free expression with the ‘DNA to Protein Array’ (DAPA) technology

Schmidt, Ronny; Cook, Elizabeth A.; Kastelic, Damjana; Taussig, Michael J.; Stoevesandt, Oda
Journal of Proteomics.
Aug 2013
10.1016/j.jprot.2013.02.002
We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. Biological significance: DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.

Provocation of an Autoimmune Response to Cardiac Voltage-Gated Sodium Channel NaV1.5 Induces Cardiac Conduction Defects in Rats

Korkmaz, Sevil; Zitron, Edgar; Bangert, Anna; Seyler, Claudia; Li, Shiliang; Hegedüs, Peter; Scherer, Daniel; Li, Jin; Fink, Thomas; Schweizer, Patrick A.; Giannitsis, Evangelos; Karck, Matthias; Szabó, Gábor; Katus, Hugo A.; Kaya, Ziya
Journal of the American College of Cardiology.
Jul 2013
10.1016/j.jacc.2013.04.041
Objectives: This study sought to test the hypothesis that inducing an autoimmune response against the cardiac sodium channel (NaV1.5) induces arrhythmias. Background: Sporadic evidence supports the concept that autoantibodies may cause cardiac arrhythmias but substantial experimental investigations using in vivo models have been lacking to date. The NaV1.5 is essential for cardiac impulse propagation and its dysfunction has been linked to conduction disease. Methods: Rats were immunized with a peptide sequence derived from the third extracellular loop of the first domain of NaV1.5. After 28 days, we evaluated in vivo both the electrical and mechanical parameters of cardiac function. Histopathology, myocardial gene and protein expression were assessed. Whole-cell patch-clamp was used to measure sodium current (INa) density in isolated cardiomyocytes. Results: NaV1.5-immunized rats had high titers of autoantibodies against NaV1.5. On ECG recording, NaV1.5-immunized animals showed significantly prolonged PR-intervals. During Holter ECG-monitoring we observed repeated prolonged episodes of third-degree atrioventricular and sinoatrial block in every NaV1.5-immunized animal, but not in controls. Immunization had no effect on cardiac function. In comparison to controls, myocardial NaV1.5 mRNA and protein levels were decreased in immunized rats. INa density was reduced in cardiomyocytes incubated with sera from NaV1.5-immunized rats and from patients with idiopathic atrioventricular block (AVB) in comparison to sera from respective controls. In patients with idiopathic AVB, we observed autoantibodies against NaV1.5 that were absent in sera from healthy controls. Conclusions: Provocation of an autoimmune response against NaV1.5 induces conductance defects probably caused by a reduced expression level and an inhibition of NaV1.5 by autoantibodies, resulting in decreased INa.

Complex Formation between NheB and NheC Is Necessary to Induce Cytotoxic Activity by the Three-Component Bacillus cereus Nhe Enterotoxin

Heilkenbrinker, Uta; Dietrich, Richard; Didier, Andrea; Zhu, Kui; Lindbäck, Toril; Granum, Per Einar; Märtlbauer, Erwin
PLoS ONE.
Apr 2013
10.1371/journal.pone.0063104
The nonhemolytic enterotoxin (Nhe) is known as a major pathogenicity factor for the diarrheal type of food poisoning caused by Bacillus cereus. The Nhe complex consists of NheA, NheB and NheC, all of them required to reach maximum cytotoxicity following a specific binding order on cell membranes. Here we show that complexes, formed between NheB and NheC under natural conditions before targeting the host cells, are essential for toxicity in Vero cells. To enable detection of NheC and its interaction with NheB, monoclonal antibodies against NheC were established and characterized. The antibodies allowed detection of recombinant NheC in a sandwich immunoassay at levels below 10 ng ml−1, but no or only minor amounts of NheC were detectable in natural culture supernatants of B. cereus strains. When NheB- and NheC-specific monoclonal antibodies were combined in a sandwich immunoassay, complexes between NheB and NheC could be demonstrated. The level of these complexes was directly correlated with the relative concentrations of NheB and NheC. Toxicity, however, showed a bell-shaped dose-response curve with a plateau at ratios of NheB and NheC between 50:1 and 5:1. Both lower and higher ratios between NheB and NheC strongly reduced cytotoxicity. When the ratio approached an equimolar ratio, complex formation reached its maximum resulting in decreased binding of NheB to Vero cells. These data indicate that a defined level of NheB-NheC complexes as well as a sufficient amount of free NheB is necessary for efficient cell binding and toxicity. Altogether, the results of this study provide evidence that the interaction of NheB and NheC is a balanced process, necessary to induce, but also able to limit the toxic action of Nhe.

Purification of High-Complexity Peptide Microarrays by Spatially Resolved Array Transfer to Gold-Coated Membranes

Schirwitz, Christopher; Loeffler, Felix F.; Felgenhauer, Thomas; Stadler, Volker; Nesterov-Mueller, Alexander; Dahint, Reiner; Breitling, Frank; Bischoff, F. Ralf
Adv. Mater..
Mar 2013
10.1002/adma.201203853
A method for the one-step purification of high-complexity peptide microarrays is presented. The entire peptide library is transferred from the synthesis support to a gold coated polyvinylidenfluoride (PVDF) membrane, whereby only full-length peptides covalently couple to the receptor membrane via an N-terminally added cysteine. Highly resolved peptide transfer and purification of up to 10 000 features per cm2 is demonstrated.

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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