A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

Charest‐Morin, Xavier; Fortin, Jean‐Philippe; Lodge, Robert; Allaeys, Isabelle; Poubelle, Patrice E.; Marceau, François

doi:10.1016/j.peptides.2014.08.001

Cited by 5 publications

(17 citation statements)

References 22 publications

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“…PTH exerts its effects on osteoblasts through a G-protein-coupled transmembrane receptor, PTH1R [19], by which classical two G-protein signaling cascades, i.e., the cyclic AMP-(cAMP-) and phospholipase C (PLC)-pathways are dominantly transmitted [7]. Previous studies suggested that the important target genes from the cAMP-pathway might be the c-fos, Jun, and interleukin-11 for regulating osteoblast proliferation [15,20,21].…”

Section: Introductionmentioning

confidence: 99%

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression

et al. 2015

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Osteoporosis is a common disease that increases individual's fragility fracture risk. PTH is the only therapeutic agent for severe osteoporosis that requires anabolic action of bone formation. Although a part of the PTH actions is explained by increased proliferation of osteoblastic precursor cells, the mechanisms involved in the proliferation of osteoblastic cells by PTH have not been clarified yet. Therefore, in this study, we investigated the effects of PTH on gene expression in the cultured osteoblastic MC3T3-E1 cells, and found that the ubiquitin-specific peptidase 2 (Usp2) may be one of the direct target genes of PTHR signaling. Usp2 is a deubiquitination enzyme targeting various factors including CyclinD1 in cancer cells and PTH receptor 1 in osteoblasts. We confirmed that consistent induction of Usp2 expression peaked at 1 h by PTH1-34 (teriparatide) in MC3T3-E1 cells and primary calvarial osteoblasts. Among the three known splicing variants of the Usp2, we found the isoforms 1 and 2 are predominantly expressed in osteoblasts. Live-imaging analysis of the Fucci-transgenic mouse-derived primary osteoblasts indeed demonstrated that Usp2 is required for the PTH1-34-induced osteoblast proliferation. Western blotting analysis of the CyclinD1 indicated that Usp2 knock-down influences the paradoxical changes of CyclinD1 protein levels in this condition. Our data indicate that Usp2 is required for the PTH1-34-induced proliferation of osteoblasts.

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Section: Introductionmentioning

confidence: 99%

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression

et al. 2015

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“…The myc-tagged peptide f-Nle-Leu-Phe-Nle-Tyr-Lys-myc also competed with the fluorescent probe with a potency comparable with that of f-Met-Leu-Phe ( Fig. 9 3), but the prolonged design f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly) 5 -myc, a 26-mer, was ~300-fold less potent, suggesting that the receptor discriminates against long peptides.…”

Section: Resultsmentioning

confidence: 90%

“…4) due to either the excess of free agonist in the reaction or to some steric hindrance between the agonist-bound antibody and the receptor. The prolonged peptide f-NleLeu-Phe-Nle-Tyr-Lys-(Asn-Gly) 5 -myc, while presumably maintaining its affinity for the antibodies, did not bind well enough to the FPR 1 (Fig. 3) to directly address the issue of the role of steric hindrance in the system.…”

Section: Discussionmentioning

confidence: 98%

“…f-Nle-Leu-Phe-Nle-Tyr-Lysmyc and f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly) 5 -myc, the myc sequence representing the 10-residue epitope (EQKLISEEDL), were custom-synthesized by Peptide 2.0, Inc. (Chantilly, VA) and provided as % pure peptides (HPLC) with confirmed identity (mass spectrometry). The (Asn-Gly) n linker has been successfully used as a spacer compatible with the extracellular fluid in 6 recombinant protein constructions [10] and in the design of fluorescent bradykinin B 1 receptor ligands (X. Charest-Morin and F. Marceau, unpublished); it was used to increase the distance between the N-terminal FPR 1 pharmacophore and the C-terminal epitope.…”

Section: Drugsmentioning

confidence: 99%

“…This bifunctional agonist strategy applied particularly well to large recombinant myctagged peptides, CCL19 and parathyroid hormone, that formed complexes with the anti-myc antibody in the extracellular fluid; the complexes were internalized in endosomes by their cognate receptors, CCR7 and PTH 1 R, respectively [4,5]. A similar strategy applied to the bradykinin B 2 receptor concerned myc-KGP-B-9972, a synthetic tagged analog agonist that 4 carried some anti-myc antibody into receptor-expressing cells, but with a lesser efficacy [6].…”

Section: Introductionmentioning

confidence: 99%

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Pharmacological profile of a bifunctional ligand of the formyl peptide receptor1 fused to the myc epitope

Charest‐Morin

Roy

Fernandes

et al. 2015

International Immunopharmacology

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In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-binds to and activates formyl peptide receptor 1 (FPR 1 ) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, Cterminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10-1000 nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~7 nM (microscopy). The nonfluorescent version of the antibody (28 nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-NleLeu-Phe-Nle-Tyr-Lys-(Asn-Gly) 5 -myc, designed to decrease the possible steric hindrance between FPR 1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-NleLeu-Phe-Nle-Tyr-Lys-myc.

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Bifunctional ligands of the bradykinin B 2 and B 1 receptors: An exercise in peptide hormone plasticity

et al. 2018

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Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B receptor (BR) and to the inducible B receptor (BR). BRs and BRs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for BRs, Lys-des-Arg-BK for BR). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the BR antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at BR have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value.

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A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

Cited by 5 publications

References 22 publications

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression

PTH-Induced Osteoblast Proliferation Requires Upregulation of the Ubiquitin-Specific Peptidase 2 (Usp2) Expression

Pharmacological profile of a bifunctional ligand of the formyl peptide receptor1 fused to the myc epitope

Bifunctional ligands of the bradykinin B 2 and B 1 receptors: An exercise in peptide hormone plasticity

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