Identification of a 13 Amino Acid Peptide Mimetic of Erythropoietin and Description of Amino Acids Critical for the Mimetic Activity of EMP1

Johnson, Dana L.; Farrell, Francis X.; Barbone, Francis P.; McMahon, Frank J.; Tullai, Jennifer; Hoey, Kenway; Livnah, Oded; Wrighton, Nicholas C.; Middleton, Steven A.; Loughney, Deborah A.; Stura, E.A.; Dower, William J.; Mulcahy, Linda S.; Wilson, Ian A.; Jolliffe, Linda K.

doi:10.1021/bi971956y

Cited by 91 publications

(81 citation statements)

References 24 publications

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“…More information on the involvement of the specific amino acid residues in the binding of the peptides to the GD2-specific antibody may be gained from replacement and truncation experiments. Additionally, such analyses would allow further optimization of the obtained sequences (35). Other peptide mimicking GD2 gangliosides have already been published.…”

Section: Discussionmentioning

confidence: 99%

“…Modification of the peptide disulphide bonds was introduced in two steps (both performed for 30 min at 4˚C). In the first step, the appropriate wells were subjected to reducing conditions through the addition of 35 , and a washing step (6 times with TBS containing 0.5% Tween-20), the HRP-conjugated anti-mouse Ig antibodies were used to detect the bound 14G2a mAb (1 h, 37˚C). The secondary antibodies were diluted 1:4,000 in TBS containing 2% BSA and 0.1% Tween-20.…”

Section: Methodsmentioning

confidence: 99%

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Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library

Horwacik

Czaplicki²,

Talarek³

et al. 2007

Int J Mol Med

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Abstract. Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library

Horwacik

Czaplicki²,

Talarek³

et al. 2007

Int J Mol Med

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“…Our NGF dimeric peptidomimetic strategy has been further encouraged by the synthesis of small peptides mimicking the 34-kDa dimeric protein erythropoietin and the finding that erythropoietin receptor activation by peptidomi-metics also requires the dimeric form (30). These erythropoietin peptidomimetics have served as starting points for the design of erythropoietin small molecule compounds (31). Using strategies derived from our development of a NGF loop 1 dimeric peptidomimetic with agonist activity, we tested the hypothesis that dimeric peptidomimetics corresponding to NGF loop 4 can function as NGF agonists to mimic NGF biological activity and activate well established NGF signaling intermediates.…”

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confidence: 99%

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

Xie

Tisi

Yeo

et al. 2000

Journal of Biological Chemistry

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Previous work indicating that nerve growth factor (NGF) protein loops 2 and 4 interact with TrkA receptors raise the possibility that small molecule mimetics corresponding to TrkA-interacting domains that have NGF agonist activity can be developed. We applied our previously developed strategy of dimeric peptidomimetics to address the hypothesis that loop 4 small molecule dimeric mimetics would activate TrkA-related signal transduction and mimic NGF neurotrophic effects in a structure-specific manner. A loop 4 cyclized peptide dimer demonstrated NGF-like neurotrophic activity, whereas peptides with scrambled sequence, added or substituted residues, or cyclized in monomeric form were inactive. Activity was blocked by the TrkA inhibitors K252a and AG879 but not by NGF p75 receptor blocking antibody. Dimeric, but not monomeric, peptides partially blocked NGF activity. This profile was consistent with that of a NGF partial agonist. ERK and AKT phosphorylation was stimulated only by biologically active peptides and was blocked by K252a. The ERK inhibitor U0126 blocked the neurite-but not the survival-promoting activity of both NGF and active peptide. These studies support the proof of concept that small molecule NGF loop 4 mimetics can activate NGF signaling pathways and can mimic death-preventing and neurite-promoting effects of NGF. This finding will guide the rational design of NGF single-domain mimetics and contribute to elucidating NGF signal transduction mechanisms.Nerve growth factor (NGF) 1 acts via TrkA and p75 receptors to regulate neuronal survival, promote neurite outgrowth, and up-regulate certain neuronal functions such as mediation of pain and inflammation (1-5). These actions suggest that NGF agonists or antagonists might be useful in regulating these processes (6 -8). Factors limiting therapeutic applications of the NGF protein include restricted penetration of the central nervous system and the poor medicinal properties characteristic of most proteins (9, 10). The development of small molecule mimetics with favorable chemical properties that function as agonists or antagonists that mimic or inhibit NGF functions in the appropriate biological context will be critical in advancing potential in vivo applications of NGF. Moreover, in settings in which NGF might contribute to neuronal death, pain, or inflammatory mechanisms, NGF antagonists may be particularly relevant. Creation of single domain NGF mimetics will also constitute a powerful approach for linking specific NGF domains with specific patterns of intracellular signal transduction.A NGF mimetic (agonist or antagonist) would be expected to contain structural determinants of one or more NGF active sites that interact with NGF receptors. Multiple techniques have been used to deduce which domains of the NGF protein interact with NGF receptors (11). A peptide mapping approach in which synthetic peptides with sequences corresponding to specific NGF regions were tested for their ability to inhibit NGF activity pointed to residues 29 -35 as a key active ...

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“…Recently, a series of synthetic peptides [EPO-mimetic peptides (EMPs)] were discovered to mimic EPO activity (38)(39)(40). A small peptide screened from random phage display peptide library inhibits the binding of 125 I-labeled EPO to the extracellular binding domain of EPOR (hEPOsR, 1-225 residues of EPOR) with an IC 50 of 10 M. EMPs with higher binding affinities to hEPOsR were then designed based on the sequence of this peptide.…”

Section: Discussionmentioning

confidence: 99%

Nonnatural protein–protein interaction-pair design by key residues grafting

Liu

Zhu

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLC␦ 1-PH (pleckstrin homology domain of phospholipase C-␦1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLC␦ 1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLC␦ 1-PH (ERPH1) was found to bind EPOR with high affinity (K D of 24 nM and an IC50 of 5.7 M) both in vitro and in a cell-based assay, respectively, although the WT PLC␦ 1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLC␦ 1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting proteinprotein interfaces.de novo design of protein-protein interaction pair ͉ erythropoietin ͉ functional site grafting ͉ key residue at interface P rotein-protein interaction is critical in many biological processes ranging from cell differentiation to apoptosis; however, little is known about the general principles governing the specificity and binding affinity of protein-protein interactions. Recent developments in structural bioinformatics have contributed greatly to our understanding of protein-protein interactions (1-6). Several groups have addressed the feasibility of computational redesign of protein-protein interactions by using native or homologous protein-protein interfaces (7-10). Because different protein backbones can perform similar functions (11), in the current study we explored the feasibility of designing nonnatural protein-protein interaction pairs using known protein scaffolds.To reconstruct the function of a protein, one of the common strategies is grafting, i.e., transferring the functional epitopes from one protein to another. The critical step for protein grafting is to find suitable sites for functional epitope transfer. To this purpose, several computational methods have been developed including a geometric hashing paradigm (12), FITSITE (13), GRAFTER (14), and DEZYMER (15, 16). We have developed a strategy for protein-protein interface redesign by grafting discontinuous interaction epitopes to nonhomologous proteins (17-19). The erythropoietin (EPO)-EPO receptor (EPOR) system was used as an example for nonnatural protein-protein interaction-...

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Identification of a 13 Amino Acid Peptide Mimetic of Erythropoietin and Description of Amino Acids Critical for the Mimetic Activity of EMP1

Cited by 91 publications

References 24 publications

Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library

Selection of novel peptide mimics of the GD2 ganglioside from a constrained phage-displayed peptide library

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

Nonnatural protein–protein interaction-pair design by key residues grafting

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