Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function

Sneha Singh; Dhiraj Srivastava; Kimberly Boyd; Nikolai O Artemyev

doi:10.1016/j.jbc.2023.105576

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Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function

Journal article

Open access

Peer reviewed

Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function

Sneha Singh, Dhiraj Srivastava, Kimberly Boyd and Nikolai O Artemyev

The Journal of biological chemistry, Vol.300(1), 105576

01/2024

DOI: 10.1016/j.jbc.2023.105576

PMCID: PMC10819763

PMID: 38110033

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Abstract

The 6 family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of the HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90 and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

Protein Folding

HSP90

chaperone

phosphodiesterase 6

photoreceptor

phototransduction

Details

Title: Subtitle: Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function
Creators: Sneha Singh - University of Iowa
Dhiraj Srivastava - University of Iowa
Kimberly Boyd - University of Iowa
Nikolai O Artemyev
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.300(1), 105576
DOI: 10.1016/j.jbc.2023.105576
PMID: 38110033
PMCID: PMC10819763
NLM abbreviation: J Biol Chem
eISSN: 1083-351X
Language: English
Electronic publication date: 12/16/2023
Date published: 01/2024
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9984532056002771

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