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

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The cellular modifier MOAG-4/SERF drives amyloid formation through charge complementation

Pras, Anita; Houben, Bert; Aprile, Francesco A.; Seinstra, Renée; Gallardo, Rodrigo; Janssen, Leen; Hogewerf, Wytse; Gallrein, Christian; De Vleeschouwer, Matthias; Mata-Cabana, Alejandro; Koopman, Mandy; Stroo, Esther; de Vries, Minke; Louise Edwards, Samantha; Kirstein, Janine; Vendruscolo, Michele; Falsone, Salvatore Fabio; Rousseau, Frederic; Schymkowitz, Joost; Nollen, Ellen A. A.
EMBO J.
Nov 2021
10.15252/embj.2020107568
While aggregation-prone proteins are known to accelerate aging and cause age-related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG-4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid-promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG-4 to neutralize charge. Our data indicate that MOAG-4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation-prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age-related protein toxicity.

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Jaishankar, Dinesh; Cosgrove, Cormac; Ramesh, Prathyaya; Mahon, James; Shivde, Rohan; Dellacecca, Emilia R.; Yang, Shiayin F.; Mosenson, Jeffrey; Guevara-Patiño, José A.; Le Poole, I. Caroline
Cell Stress and Chaperones.
Sep 2021
10.1007/s12192-021-01229-x
Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70iQ435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70iQ435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70iQ435A in mice affects anti-tumor responses. We found that HSP70iQ435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70iQ435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70iQ435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70iQ435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.

Serum Peptide Immunoglobulin G Autoantibody Response in Patients with Different Central Nervous System Inflammatory Demyelinating Disorders

Lee, Hye Lim; Park, Jin-Woo; Seok, Jin Myoung; Jeon, Mi Young; Kim, Hojin; Lim, Young-Min; Shin, Ha Young; Kang, Sa-Yoon; Kwon, Oh-Hyun; Lee, Sang-Soo; Seok, Hung Youl; Min, Ju-Hong; Lee, Sung-Hyun; Kim, Byung-Jo; Kim, Byoung Joon
Diagnostics.
Jul 2021
10.3390/diagnostics11081339
Previous efforts to discover new surrogate markers for the central nervous system (CNS) inflammatory demyelinating disorders have shown inconsistent results; moreover, supporting evidence is scarce. The present study investigated the IgG autoantibody responses to various viral and autoantibodies-related peptides proposed to be related to CNS inflammatory demyelinating disorders using the peptide microarray method. We customized a peptide microarray containing more than 2440 immobilized peptides representing human and viral autoantigens. Using this, we tested the sera of patients with neuromyelitis optica spectrum disorders (NMOSD seropositive, n = 6; NMOSD seronegative, n = 5), multiple sclerosis (MS, n = 5), and myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD, n = 6), as well as healthy controls (HC, n = 5) and compared various peptide immunoglobulin G (IgG) responses between the groups. Among the statistically significant peptides based on the pairwise comparisons of IgG responses in each disease group to HC, cytomegalovirus (CMV)-related peptides were most clearly distinguishable among the study groups. In particular, the most significant differences in IgG response were observed for HC vs. MS and HC vs. seronegative NMOSD (p = 0.064). Relatively higher IgG responses to CMV-related peptides were observed in patients with MS and NMOSD based on analysis of the customized peptide microarray.

Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target

Zhang, Zhiqiang; Cottignie, Ines; Van Zeebroeck, Griet; Thevelein, Johan M.
Biochem J.
Jan 2021
10.1042/BCJ20200722
Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor–Sch9–TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor–Sch9 interaction under conditions of nutrient starvation or other environmental challenges.

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein:Protein Interactions with Human Survivin

Garcia-Bonete, Maria-Jose; Jensen, Maja; Recktenwald, Christian V.; Rocha, Sandra; Stadler, Volker; Bokarewa, Maria; Katona, Gergely
Sci Rep.
Dec 2017
10.1038/s41598-017-17071-0
A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

A recombinant BBSome core complex and how it interacts with ciliary cargo

Klink, Björn Udo; Zent, Eldar; Juneja, Puneet; Kuhlee, Anne; Raunser, Stefan; Wittinghofer, Alfred
Nov 2017
10.7554/eLife.27434
Cilia are small, antenna-like structures on the surface of eukaryotic cells that harbor a unique set of sensory proteins, including GPCRs and other membrane proteins. The transport of these proteins involves the BBSome, an eight-membered protein complex that is recruited to ciliary membranes by the G-protein Arl6. BBSome malfunction leads to Bardet-Biedl syndrome, a ciliopathy with severe consequences. Short ciliary targeting sequences (CTS) have been identified that trigger the transport of ciliary proteins. However, mechanistic studies that relate ciliary targeting to BBSome binding are missing. Here we used heterologously expressed BBSome subcomplexes to analyze the complex architecture and to investigate the binding of GPCRs and other receptors to the BBSome. A stable heterohexameric complex was identified that binds to GPCRs with interactions that only partially overlap with previously described CTS, indicating a more complex recognition than anticipated. Arl6•GTP does not affect these interactions, suggesting no direct involvement in cargo loading/unloading.

Identification of Streptococcus cristatus peptides that repress expression of virulence genes in Porphyromonas gingivalis

Ho, Meng-Hsuan; Lamont, Richard J.; Xie, Hua
Sci Rep.
May 2017
10.1038/s41598-017-01551-4
Dental plaque is a complex multispecies biofilm, and is a direct precursor of periodontal disease. The virulence of periodontal pathogens, such as Porphyromonas gingivalis, is expressed in the context of this polymicrobial community. Previously, we reported an antagonistic relationship between Streptococcus cristatus and P. gingivalis, and identified arginine deiminase (ArcA) of S. cristatus as the signaling molecule to which P. gingivalis responds by repressing the expression and production of FimA protein. Here we demonstrate that direct interaction between P. gingivalis and S. cristatus is necessary for the cell-cell communication. Two surface proteins of P. gingivalis, PGN_0294 and PGN_0806, were found to interact with S. cristatus ArcA. Using a peptide array analysis, we identified several P. gingivalis-binding sites of ArcA, which led to the discovery of an 11-mer peptide with the native sequence of ArcA that repressed expression of fimbriae and of gingipains. These data indicate that a functional motif of ArcA is sufficient to selectively alter virulence gene expression in P. gingivalis, and PGN_0294 and PGN_0806 may serve as receptors for ArcA. Our findings provide a molecular basis for future rational design of agents that interfere with the initiation and formation of a P. gingivalis-induced pathogenic community.

Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays

Weber, Laura K.; Palermo, Andrea; Kügler, Jonas; Armant, Olivier; Isse, Awale; Rentschler, Simone; Jaenisch, Thomas; Hubbuch, Jürgen; Dübel, Stefan; Nesterov-Mueller, Alexander; Breitling, Frank; Loeffler, Felix F.
Journal of Immunological Methods.
Apr 2017
10.1016/j.jim.2017.01.012
The antibody species that patrol in a patient’s blood are an invaluable part of the immune system. While most of them shield us from life-threatening infections, some of them do harm in autoimmune diseases. If we knew exactly all the antigens that elicited all the antibody species within a group of patients, we could learn which ones correlate with immune protection, are irrelevant, or do harm. Here, we demonstrate an approach to this question: First, we use a plethora of phage-displayed peptides to identify many different serum antibody binding peptides. Next, we synthesize identified peptides in the array format and rescreen the serum used for phage panning to validate antibody binding peptides. Finally, we systematically vary the sequence of validated antibody binding peptides to identify those amino acids within the peptides that are crucial for binding “their” antibody species. The resulting immune fingerprints can then be used to trace them back to potential antigens. We investigated the serum of an individual in this pipeline, which led to the identification of 73 antibody fingerprints. Some fingerprints could be traced back to their most likely antigen, for example the immunodominant capsid protein VP1 of enteroviruses, most likely elicited by the ubiquitous poliovirus vaccination. Thus, with our approach, it is possible, to pinpoint those antibody species that correlate with a certain antigen, without any pre-information. This can help to unravel hitherto enigmatic diseases.

Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis

Qendro, Veneta; Bugos, Grace A.; Lundgren, Debbie H.; Glynn, John; Han, May H.; Han, David K.
Proteomics.
Mar 2017
10.1002/pmic.201600322
In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases.

Acquired Factor XIII inhibitor associated with mantle cell lymphoma: ACQUIRED FXIII INHIBITOR

Nixon, Christian P.; Prsic, Elizabeth H.; Guertin, Christine A.; Stevenson, Ryan L.; Sweeney, Joseph D.
Transfusion.
Mar 2017
10.1111/trf.13947
BACKGROUND Acquired Factor (F)XIII deficiency is a very rare bleeding diathesis with a potentially fatal outcome, previously described in the context of autoimmune disorders and leukemias. There is minimal information on autoantibody characterization and the role of antifibrinolytic therapy in patient management. CASE REPORT A 79-year-old woman with a 3-month history of bruising and heavy menorrhagia presented with ongoing vaginal bleeding, symptomatic anemia, and a right thigh hematoma. Initial management included an axillary lymph node biopsy and coagulation evaluation. Pathologic examination of the biopsy specimen revealed mantle cell lymphoma. Clot solubility assay was consistent with a FXIII activity of less than 3%. An anti-FXIII inhibitor was suspected, the epitope specificity of which was mapped by micropeptide array analysis to regions in the β-sandwich and catalytic core domain of the FXIII-A subunit. Management with cryoprecipitate, steroids, rituximab, and antifibrinolytic therapy resolved the bleeding diathesis and suppressed the inhibitor. CONCLUSION This is the first reported case of an acquired FXIII inhibitor associated with mantle cell lymphoma in which the epitope specificity of the pathologic autoantibody was accurately defined. Antifibrinolytic therapy played a prominent role in the prevention of bleeding complications in the window period between initiation of immunosuppression and disappearance of the pathologic anti-FXIII autoantibody.

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