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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.

Antigenic characteristics of glycosylated protein 3 of highly pathogenic porcine reproductive and respiratory syndrome virus

Wang, Xinglong; Dang, Ruyi; Liu, Wenkai; Yang, Zengqi; Du, Enqi; Zhang, Shuxia
Virus Research.
Aug 2014
10.1016/j.virusres.2014.04.016
Highly pathogenic (HP)-porcine reproductive and respiratory syndrome virus (PRRSV) emerged in 2006 and has now become a global threat to pig farms. Despite extensive characterization of HP-PRRSV proteins by direct analysis and comparison with typical PRRSV, immune recognition remain poorly understood. Glycosylated protein 3 (GP3) has an important function in inducing protective immune response. To analyze the antigenic character of HP-PRRSV GP3, a total of 217 peptides were printed on a chip and used to react with HP-PRRSV specific serum. The reactions of these peptides to HP-PRRSV specific pig serum were scanned and quantified using the software PepSlide® Analyzer by fluorescence intensity. The intensity plots showed various reactions in different parts of GP3. The highest reaction intensity value reached 29,184.5 with the peptide sequence of CSENDHDELGFMVPP. Conversely, 88 peptides showed no reaction with 0 florescence intensity. A further analysis based on the result of the peptide microarray revealed an antigen reaction active region (AR) from Y51 to S106 in GP3. The AR had four parts of variation that may be a significant mutation of the typical PRRSV to HP-PRRSV. Acquired data may be useful for understanding HP-PRRSV variation and its GP3 immune recognition.

Anti-ADAMTS13 IgG autoantibodies present in healthy individuals share linear epitopes with those in patients with thrombotic thrombocytopenic purpura

Grillberger, R.; Casina, V. C.; Turecek, P. L.; Zheng, X. L.; Rottensteiner, H.; Scheiflinger, F.
Haematologica.
Apr 2014
10.3324/haematol.2013.100685

Histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and alters HagB-induced chemokine responses

Borgwardt, Derek S.; Martin, Aaron D.; Van Hemert, Jonathan R.; Yang, Jianyi; Fischer, Carol L.; Recker, Erica N.; Nair, Prashant R.; Vidva, Robinson; Chandrashekaraiah, Shwetha; Progulske-Fox, Ann; Drake, David; Cavanaugh, Joseph E.; Vali, Shireen; Zhang, Yang; Brogden, Kim A.
Sci Rep.
Jan 2014
10.1038/srep03904
Histatins are human salivary gland peptides with anti-microbial and anti-inflammatory activities. In this study, we hypothesized that histatin 5 binds to Porphyromonas gingivalis hemagglutinin B (HagB) and attenuates HagB-induced chemokine responses in human myeloid dendritic cells. Histatin 5 bound to immobilized HagB in a surface plasmon resonance (SPR) spectroscopy-based biosensor system. SPR spectroscopy kinetic and equilibrium analyses, protein microarray studies and I-TASSER structural modeling studies all demonstrated two histatin 5 binding sites on HagB. One site had a stronger affinity with a KD1 of 1.9 μM and one site had a weaker affinity with a KD2 of 60.0 μM. Binding has biological implications and predictive modeling studies and exposure of dendritic cells both demonstrated that 20.0 μM histatin 5 attenuated (p < 0.05) 0.02 μM HagB-induced CCL3/MIP-1α, CCL4/MIP-1β and TNFα responses. Thus histatin 5 is capable of attenuating chemokine responses, which may help control oral inflammation.

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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