Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence

Kristen A. Kelps; Jadwiga Turchan-Cholewo; Erin R. Hascup; Tiffany L. Taylor; Don M. Gash; Greg A. Gerhardt; Luke H. Bradley

doi:10.1016/j.npep.2011.03.003

Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence

Kristen A. Kelps, Jadwiga Turchan-Cholewo, Erin R. Hascup, Tiffany L. Taylor, Don M. Gash, Greg A. Gerhardt, Luke H. Bradley

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively. Far-UV circular dichroism spectra show that the three DNSPs are soluble and act independently in vitro. Reverse phase HPLC and mass spectrometry analysis show that the three peptides are stable for one month at a variety of storage and experimental conditions. To gain insight into their biodistribution properties in the brain, we used affinity chromatography to show that DNSP-17 binds heparin equally as tight as GDNF, whereas DNSP-5 and DNSP-11 do not bind heparin, which should facilitate their delivery in vivo. Finally, we present data showing that DNSP-11 provides dose-dependent protection of HEK-293 cells from staurosporine and 3-nitropropionate (3-NP) cytotoxicity, thereby supporting its broad mitochondrial-protective properties.

Original language	English
Pages (from-to)	213-218
Number of pages	6
Journal	Neuropeptides
Volume	45
Issue number	3
DOIs	https://doi.org/10.1016/j.npep.2011.03.003
State	Published - Jun 2011

Bibliographical note

Funding Information:
The authors thank Raymond Bartus for his helpful discussion. The authors acknowledge Stewart Surgener, Jack Schmidt, Martin Chow and Louis Hersh for their discussion and use of HPLC instrumentation. FPLC and CD spectroscopy experiments were performed in the University of Kentucky Center of Structural Biology. Mass spectrometry was performed at the University of Kentucky Mass Spectrometry Facility. Peptide amino acid sequencing analysis was performed at the University of Nebraska – Lincoln Center of Biotechnology. T.L.T. conducted research as part of her University of Kentucky Undergraduate Honors Thesis project. This research was supported by training grants to K.A.K. (T32 DA022738), J.T-C. (T32 AG000242) and funds from NIH COBRE Pilot ( P20RR20171 – L.H.B.), PhRMA Foundation (L.H.B.), NINDS ( NS039787 – L.H.B., D.M.G., G.A.G.), Columbus Foundation (L.H.B.), and University of Kentucky College of Medicine Startup Funds (L.H.B.).

Funding

The authors thank Raymond Bartus for his helpful discussion. The authors acknowledge Stewart Surgener, Jack Schmidt, Martin Chow and Louis Hersh for their discussion and use of HPLC instrumentation. FPLC and CD spectroscopy experiments were performed in the University of Kentucky Center of Structural Biology. Mass spectrometry was performed at the University of Kentucky Mass Spectrometry Facility. Peptide amino acid sequencing analysis was performed at the University of Nebraska – Lincoln Center of Biotechnology. T.L.T. conducted research as part of her University of Kentucky Undergraduate Honors Thesis project. This research was supported by training grants to K.A.K. (T32 DA022738), J.T-C. (T32 AG000242) and funds from NIH COBRE Pilot ( P20RR20171 – L.H.B.), PhRMA Foundation (L.H.B.), NINDS ( NS039787 – L.H.B., D.M.G., G.A.G.), Columbus Foundation (L.H.B.), and University of Kentucky College of Medicine Startup Funds (L.H.B.).

Funders	Funder number
University of Kentucky College of Medicine
National Institutes of Health (NIH)	P20RR20171
National Institute of Neurological Disorders and Stroke	P50NS039787
Pharmaceutical Research and Manufacturers of America Foundation
Columbus Foundation

Keywords

Biotherapeutic
GDNF
Heparin
Neuroprotection
Propeptides

ASJC Scopus subject areas

Endocrinology
Neurology
Endocrine and Autonomic Systems
Cellular and Molecular Neuroscience

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10.1016/j.npep.2011.03.003

Cite this

@article{139ce6f6e43246ff827b065eeefaabde,

title = "Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence",

abstract = "Recently, a small 11-amino acid amidated peptide, dopamine neuron stimulating peptide-11 (DNSP-11), was shown to exert neurotrophic-like actions on primary dopaminergic neurons and in parkinsonian rat models. This suggests smaller neurotrophic-like molecules may be deliverable and modifiable for therapeutic use. Here we evaluate the molecular and cellular protection properties of DNSP-11 and two other amidated-peptides, a 5-mer (DNSP-5) and a 17-mer (DNSP-17), hypothesized to be endoproteolytically processed from the pro- and mature glial cell line-derived neurotrophic factor (GDNF) protein sequence, respectively. Far-UV circular dichroism spectra show that the three DNSPs are soluble and act independently in vitro. Reverse phase HPLC and mass spectrometry analysis show that the three peptides are stable for one month at a variety of storage and experimental conditions. To gain insight into their biodistribution properties in the brain, we used affinity chromatography to show that DNSP-17 binds heparin equally as tight as GDNF, whereas DNSP-5 and DNSP-11 do not bind heparin, which should facilitate their delivery in vivo. Finally, we present data showing that DNSP-11 provides dose-dependent protection of HEK-293 cells from staurosporine and 3-nitropropionate (3-NP) cytotoxicity, thereby supporting its broad mitochondrial-protective properties.",

keywords = "Biotherapeutic, GDNF, Heparin, Neuroprotection, Propeptides",

author = "Kelps, {Kristen A.} and Jadwiga Turchan-Cholewo and Hascup, {Erin R.} and Taylor, {Tiffany L.} and Gash, {Don M.} and Gerhardt, {Greg A.} and Bradley, {Luke H.}",

note = "Funding Information: The authors thank Raymond Bartus for his helpful discussion. The authors acknowledge Stewart Surgener, Jack Schmidt, Martin Chow and Louis Hersh for their discussion and use of HPLC instrumentation. FPLC and CD spectroscopy experiments were performed in the University of Kentucky Center of Structural Biology. Mass spectrometry was performed at the University of Kentucky Mass Spectrometry Facility. Peptide amino acid sequencing analysis was performed at the University of Nebraska – Lincoln Center of Biotechnology. T.L.T. conducted research as part of her University of Kentucky Undergraduate Honors Thesis project. This research was supported by training grants to K.A.K. (T32 DA022738), J.T-C. (T32 AG000242) and funds from NIH COBRE Pilot ( P20RR20171 – L.H.B.), PhRMA Foundation (L.H.B.), NINDS ( NS039787 – L.H.B., D.M.G., G.A.G.), Columbus Foundation (L.H.B.), and University of Kentucky College of Medicine Startup Funds (L.H.B.). ",

year = "2011",

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doi = "10.1016/j.npep.2011.03.003",

language = "English",

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AU - Kelps, Kristen A.

AU - Turchan-Cholewo, Jadwiga

AU - Hascup, Erin R.

AU - Taylor, Tiffany L.

AU - Gash, Don M.

AU - Gerhardt, Greg A.

AU - Bradley, Luke H.

N1 - Funding Information: The authors thank Raymond Bartus for his helpful discussion. The authors acknowledge Stewart Surgener, Jack Schmidt, Martin Chow and Louis Hersh for their discussion and use of HPLC instrumentation. FPLC and CD spectroscopy experiments were performed in the University of Kentucky Center of Structural Biology. Mass spectrometry was performed at the University of Kentucky Mass Spectrometry Facility. Peptide amino acid sequencing analysis was performed at the University of Nebraska – Lincoln Center of Biotechnology. T.L.T. conducted research as part of her University of Kentucky Undergraduate Honors Thesis project. This research was supported by training grants to K.A.K. (T32 DA022738), J.T-C. (T32 AG000242) and funds from NIH COBRE Pilot ( P20RR20171 – L.H.B.), PhRMA Foundation (L.H.B.), NINDS ( NS039787 – L.H.B., D.M.G., G.A.G.), Columbus Foundation (L.H.B.), and University of Kentucky College of Medicine Startup Funds (L.H.B.).

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KW - Propeptides

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Evaluation of the physical and in vitro protective activity of three synthetic peptides derived from the pro- and mature GDNF sequence

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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