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

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Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity

Waizenegger, Anja; Urulangodi, Madhusoodanan; Lehmann, Carl P.; Reyes, Teresa Anne Clarisse; Saugar, Irene; Tercero, José Antonio; Szakal, Barnabas; Branzei, Dana
Nov 2020
10.1038/s41467-020-19503-4
The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.

Compounds and Methods Targeting Interleukin-19

Higgs Jr., Richard Earl; Konrad, Robert John; Nickoloff, Brian Jeffrey; Siegel II, Robert William; Mertz, Prema Maria
Nov 2020
The present invention provides compounds and methods targeting human interleukin-19, including therapeutic antibodies, pharmaceutical compositions and diagnostic applications useful in the field of immune-mediated diseases including psoriasis, atopic dermatitis, psoriatic arthritis, bronchial asthma and diabetic nephropathy.

Novel Anti-Cd40 Antibodies and Use Thereof

Park, Chung Gyu; Kim, Jung Sik; Lucas, Zachariah
Sep 2020
The present invention relates to novel anti-CD40 antibodies and a use thereof and, more specifically, provided are a pharmaceutical composition for treating or preventing autoimmune diseases and a composition for inhibiting immune rejection during organ transplantation, both compositions containing, as an active ingredient, novel anti-CD40 antibodies that specifically bind to a novel epitope of CD40. Novel anti-CD40 antibodies of the present invention directly target CD40, but not CD40 ligands, and block the signaling of CD40-CD154 without stimulating platelets so as to exhibit excellent antagonistic effects, thereby being expected to be usable as a preparation effective in the treatment of autoimmune diseases and the inhibition of organ transplantation rejection.

Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays

Heiss, Kirsten; Heidepriem, Jasmin; Fischer, Nico; Weber, Laura K; Dahlke, Christine; Jaenisch, Thomas; Loeffler, Felix F
J. Proteome Res..
Sep 2020
10.1021/acs.jproteome.0c00484
Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 500 000 deaths globally. Outbreaks caused by viruses such as SARS-CoV-2, HIV, Ebola, influenza, and Zika have increased over the last decade, underlining the urgent need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides. Using these highly diverse libraries, covering tens of thousands of peptides, allow for the in-depth analysis of antibody signatures in a multiplexed, high-throughput fashion. In this review, we highlight synthesis platforms that facilitate fast and highly flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines, to quickly encounter pandemic threats.

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk

King, Rhodri J.; Schuett, Katharina; Tiede, Christian; Jankowski, Vera; John, Vicky; Trehan, Abhi; Simmons, Katie; Ponnambalam, Sreenivasan; Storey, Robert F.; Fishwick, Colin W.G.; McPherson, Michael J.; Tomlinson, Darren C.; Ajjan, Ramzi A.
Haematologica.
Apr 2020
10.3324/haematol.2019.239558
Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen β chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen β-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.

On‐Chip Neo‐Glycopeptide Synthesis for Multivalent Glycan Presentation

Mende, Marco; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Mattes, Daniela S.; Eickelmann, Stephan; Bordoni, Vittorio; Wawrzinek, Robert; Fuchsberger, Felix F.; Seeberger, Peter H.; Rademacher, Christoph; Delbianco, Martina; Mallagaray, Alvaro; Loeffler, Felix F
Chem. Eur. J..
Apr 2020
10.1002/chem.202001291
Single glycan–protein interactions are often weak, such that glycan binding partners commonly utilize multiple, spatially defined binding sites to enhance binding avidity and specificity. Current array technologies usually neglect defined multivalent display. Laser-based array synthesis technology allows for flexible and rapid on-surface synthesis of different peptides. By combining this technique with click chemistry, neo-glycopeptides were produced directly on a functionalized glass slide in the microarray format. Density and spatial distribution of carbohydrates can be tuned, resulting in well-defined glycan structures for multivalent display. The two lectins concanavalin A and langerin were probed with different glycans on multivalent scaffolds, revealing strong spacing-, density-, and ligand-dependent binding. In addition, we could also measure the surface dissociation constant. This approach allows for a rapid generation, screening, and optimization of a multitude of multivalent scaffolds for glycan binding.

The Myc tag monoclonal antibody 9E10 displays highly variable epitope recognition dependent on neighboring sequence context

Schüchner, Stefan; Behm, Christian; Mudrak, Ingrid; Ogris, Egon
Sci. Signal..
Jan 2020
10.1126/scisignal.aax9730
Epitope tags are short, linear antibody recognition sequences that enable detection of tagged fusion proteins by antibodies. Epitope tag position and neighboring sequences potentially affect its recognition by antibodies, and such context-dependent differences in tag binding may have a wide-ranging effect on data interpretation. We tested by Western blotting six antibodies that recognize the c-Myc epitope tag, including monoclonal antibodies 9E10, 4A6, 9B11, and 71D10 and polyclonal antibodies 9106 and A-14. All displayed context-dependent differences in their ability to detect N- or C-terminal Myc-tagged proteins. In particular, clone 9E10, the most cited Myc-tag antibody, displayed high context-dependent detection variability, whereas others, notably 4A6 and 9B11, showed much less context sensitivity in their detection of Myc-tagged proteins. The very high context sensitivity of 9E10 was further substantiated by peptide microarray analyses. We conclude that recently developed, purpose-made monoclonal antibodies specific for Myc have much more uniform reactivity in diverse assays and are much less context sensitive than is the legacy antibody 9E10.

Molecular mimicry, genetic homology, and gene sharing proteomic “molecular fingerprints” using an EBV (Epstein-Barr virus)-derived microarray as a potential diagnostic method in autoimmune disease

Dreyfus, David H.; Farina, Antonella; Farina, Giuseppina Alessandra
Immunol Res.
Dec 2018
10.1007/s12026-018-9045-0
EBV (Epstein-Barr Virus) and other human DNA viruses are associated with autoimmune syndromes in epidemiologic studies. In this work, immunoglobulin G response to EBV-encoded proteins which share regions with human immune response proteins from the human host including ZEBRA (BZLF-1 encoded protein), BALF-2 recombinase expressed primarily during the viral lytic replication cycle, and EBNA-1 (Epstein-Barr Virus Nuclear Antigen) expressed during the viral latency cycle respectively were characterized using a laser-printed micro-array (PEPperprint.com). IgG response to conserved “A/T hooks” in EBV-encoded proteins such as EBNA-1 and the BALF-2 recombinase related to host DNA-binding proteins including RAG-1 recombinase and histones, and EBV-encoded virokines such as the IL-10 homologue BCRF-1 suggest further directions for clinical research. The author suggests that proteomic “molecular fingerprints” of the immune response to viral proteins shared with human immune response genes are potentially useful in early diagnosis and monitoring of autoantibody production and response to therapy in EBV-related autoimmune syndromes.

Regulatory T-cell deficiency leads to pathogenic bullous pemphigoid antigen 230 autoantibody and autoimmune bullous disease

Haeberle, Stefanie; Wei, Xiaoying; Bieber, Katja; Goletz, Stephanie; Ludwig, Ralf J.; Schmidt, Enno; Enk, Alexander H.; Hadaschik, Eva N.
Journal of Allergy and Clinical Immunology.
Dec 2018
10.1016/j.jaci.2018.04.006
Background Autoimmune bullous diseases/dermatoses (AIBDs) are severe autoantibody-mediated skin diseases. The pathogenic relevance of autoreactive CD4+ T cells for the induction of autoantibody production remains to be fully evaluated. Scurfy mice lack functional regulatory T (Treg) cells, experience spontaneous activation of autoreactive CD4+ T cells, and display severe erosive skin lesions suggestive of AIBDs. Objective We sought to determine whether AIBDs develop in Treg cell–deficient scurfy mice. Methods Histology, indirect immunofluorescence (IF) microscopy, direct IF, and ELISA were used to prove the presence of AIBDs in scurfy mice. Monoclonal autoantibodies from sera of scurfy mice were screened by using indirect IF on murine skin, and immunoprecipitation and mass spectrometry were used for target antigen identification, followed by confirmation in modified human embryonic kidney cells and murine keratinocytes. Pathogenicity was determined by injecting the autoantibody into neonatal mice and transferring scurfy CD4+ T cells into nu/nu mice. Results Autoantibodies against different known autoantigens of AIBDs spontaneously develop in scurfy mice. Histology reveals subepidermal blisters, and direct IF of skin of scurfy mice shows a predominant linear staining pattern. The mAb 20B12 shows a linear staining pattern in indirect IF, recognizes the murine hemidesmosomal protein bullous pemphigoid antigen 230 (BP230) as the target antigen, and cross-reacts with human BP230. Purified mAb 20B12 induces subepidermal blisters in neonatal mice. Transfer of scurfy CD4+ T cells is sufficient to induce antibodies with reactivity to AIBD autoantigens and subepidermal blisters in the skin of recipient T cell–deficient nu/nu mice. Conclusion We show that the absence of Treg cells leads to AIBDs by pathogenic autoantibodies targeting BP230.

Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

Lee, Yeon-Ah; Hahm, Dae-Hyun; Kim, Jung Yeon; Sur, Bonjun; Lee, Hyun Min; Ryu, Chun Jeih; Yang, Hyung-In; Kim, Kyoung Soo
Arthritis Res Ther.
Oct 2018
10.1186/s13075-018-1736-3
Background Different adiponectin isoforms appear to be differentially involved in the pathogenesis of various diseases. The purpose of this study was to generate monoclonal antibodies (mAbs) specific to different adiponectin isoforms and investigate whether these mAbs have potential as therapeutic agents for such diseases. Methods Hybridoma cells producing monoclonal antibodies were generated and screened using enzyme-linked immunosorbent assay and Western blotting for the production of mAbs recognizing human adiponectin isoforms. Results The mAb from hybridoma clone KH7–41 recognized both the middle molecular weight (MMW) (hexamer) and low molecular weight (LMW) (trimer) isoforms of adiponectin in human serum, whereas the KH7–33 mAb detected only MMW (hexamer) adiponectin. The KH4–8 clone recognized both the high molecular weight (HMW) (multimer) and MMW adiponectin isoforms. However, in mouse and rat sera, the abovementioned antibodies recognized only the MMW isomer. These mAbs also recognized adiponectin in various human tissues, such as lung, kidney, and adipose tissues, although the three mAbs had different staining intensities. The mAb from clone KH4–8 effectively inhibited increases in interleukin-6 (IL-6) and IL-8 expression in recombinant adiponectin-stimulated human osteoblasts and human umbilical vein endothelial cells. Also, the mAbs KH7–33 and KH4–8 significantly ameliorated rheumatic symptoms in a collagen-induced arthritis mouse model. This result suggests that these mAb treatments may ameliorate adiponectin-mediated inflammatory response. Conclusions mAbs against human adiponectin isomers can potentially be developed as therapeutic antibodies to target specific detrimental isoforms of adiponectin while maintaining the functions of beneficial isoforms.

Interaction of the Warsaw breakage syndrome DNA helicase DDX11 with the replication fork-protection factor Timeless promotes sister chromatid cohesion

Cortone, Giuseppe; Zheng, Ge; Pensieri, Pasquale; Chiappetta, Viviana; Tatè, Rosarita; Malacaria, Eva; Pichierri, Pietro; Yu, Hongtao; Pisani, Francesca M.
PLoS Genet.
Oct 2018
10.1371/journal.pgen.1007622
Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS.

Combinatorial Synthesis of Macromolecular Arrays by Microchannel Cantilever Spotting (µCS)

Atwater, Jordyn; Mattes, Daniela S.; Streit, Bettina; von Bojničić-Kninski, Clemens; Loeffler, Felix F.; Breitling, Frank; Fuchs, Harald; Hirtz, Michael
Adv. Mater..
Aug 2018
10.1002/adma.201801632
Surface-bound microarrays of multiple oligo- and macromolecules (e.g., peptides, DNA) offer versatile options in biomedical applications like drug screening, DNA analysis, or medical diagnostics. Combinatorial syntheses of these molecules in situ can save significant resources in regard to processing time and material use. Furthermore, high feature densities are needed to enable high-throughput and low sample volumes as generally regarded in combinatorial chemistry. Here, a scanning-probe-lithography-based approach for the combinatorial in situ synthesis of macromolecules is presented in microarray format. Feature sizes below 40 µm allow for the creation of high-density arrays with feature densities of 62 500 features per cm2. To demonstrate feasibility of this approach for biomedical applications, a multiplexed array of functional protein tags (HA- and FLAG-tag) is synthesized, and selective binding of respective epitope recognizing antibodies is shown. This approach uses only small amounts of base chemicals for synthesis and can be further parallelized, therefore, opening up a route to flexible, highly dense, and cost-effective microarrays.

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