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SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies

Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8 % of a patient population with azoospermia. Transfection of the mutant protein in COS-1 cell...

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Autores principales: Raju, Murugananth Kumar, Hassan, Sergio, Kavarthapu, Raghuveer, Anbazhagan, Rajakumar, Dufau, Maria
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Endocrine Society 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553102/
http://dx.doi.org/10.1210/js.2019-SUN-215
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author Raju, Murugananth Kumar
Hassan, Sergio
Kavarthapu, Raghuveer
Anbazhagan, Rajakumar
Dufau, Maria
author_facet Raju, Murugananth Kumar
Hassan, Sergio
Kavarthapu, Raghuveer
Anbazhagan, Rajakumar
Dufau, Maria
author_sort Raju, Murugananth Kumar
collection PubMed
description Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8 % of a patient population with azoospermia. Transfection of the mutant protein in COS-1 cells showed loss of the 61 kDa cytoplasmic phospho-species with preservation of the nuclear non-phospho form of GRTH (1). Mice with knock-in of the human GRTH mutation, lack the phospho-form of GRTH, are sterile due to lack of sperm, and have normal testosterone levels and mating behavior (2). These findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified T239, structurally adjacent to the patient’s mutant site, as the GRTH-phospho-site. Molecular modelling of the phospho-site based on the crystal structure of DDX19 (64% a.a. identity), provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These effects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA-interface, which leads to modest but consequential structural changes that can affect PKA binding or its catalytic efficiency. Inhibition of phosphorylation may be achieved by small molecules that bind to GRTH and reconfigure the GRTH/PKA-interface. These studies based on the abolition of the phospho-form of GRTH observed in vivo and in vitro provide the basis for drug design and virtual and throughput screening for the discovery of a non-toxic specific, reversible chemical inhibitor for use as a male contraceptive. (1) Tsai-Morris et al., Mol Hum Reprod. 2017; 13: 887-892. (2) Kavarthapu et al., Endo Reviews 2018 Vol 39, Issue 2 Suppl.
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spelling pubmed-65531022019-06-13 SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies Raju, Murugananth Kumar Hassan, Sergio Kavarthapu, Raghuveer Anbazhagan, Rajakumar Dufau, Maria J Endocr Soc Reproductive Endocrinology Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8 % of a patient population with azoospermia. Transfection of the mutant protein in COS-1 cells showed loss of the 61 kDa cytoplasmic phospho-species with preservation of the nuclear non-phospho form of GRTH (1). Mice with knock-in of the human GRTH mutation, lack the phospho-form of GRTH, are sterile due to lack of sperm, and have normal testosterone levels and mating behavior (2). These findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified T239, structurally adjacent to the patient’s mutant site, as the GRTH-phospho-site. Molecular modelling of the phospho-site based on the crystal structure of DDX19 (64% a.a. identity), provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These effects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA-interface, which leads to modest but consequential structural changes that can affect PKA binding or its catalytic efficiency. Inhibition of phosphorylation may be achieved by small molecules that bind to GRTH and reconfigure the GRTH/PKA-interface. These studies based on the abolition of the phospho-form of GRTH observed in vivo and in vitro provide the basis for drug design and virtual and throughput screening for the discovery of a non-toxic specific, reversible chemical inhibitor for use as a male contraceptive. (1) Tsai-Morris et al., Mol Hum Reprod. 2017; 13: 887-892. (2) Kavarthapu et al., Endo Reviews 2018 Vol 39, Issue 2 Suppl. Endocrine Society 2019-04-30 /pmc/articles/PMC6553102/ http://dx.doi.org/10.1210/js.2019-SUN-215 Text en Copyright © 2019 Endocrine Society https://creativecommons.org/licenses/by-nc-nd/4.0/ This article has been published under the terms of the Creative Commons Attribution Non-Commercial, No-Derivatives License (CC BY-NC-ND; https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Reproductive Endocrinology
Raju, Murugananth Kumar
Hassan, Sergio
Kavarthapu, Raghuveer
Anbazhagan, Rajakumar
Dufau, Maria
SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies
title SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies
title_full SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies
title_fullStr SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies
title_full_unstemmed SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies
title_short SUN-215 Phosphorylation Site of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) and GRTH/PKA Binding Interface: Biochemical and Structural Studies
title_sort sun-215 phosphorylation site of gonadotropin regulated testicular rna helicase (grth/ddx25) and grth/pka binding interface: biochemical and structural studies
topic Reproductive Endocrinology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553102/
http://dx.doi.org/10.1210/js.2019-SUN-215
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