☆ 4.4 Article

Interaction and Localization of the Retinitis Pigmentosa Protein RP2 and NSF in Retinal Photoreceptor Cells

BIOCHEMISTRY (2010)

Journal

BIOCHEMISTRY

Volume 49, Issue 35, Pages 7439-7447

Publisher

AMER CHEMICAL SOC

DOI: 10.1021/bi1005249

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Funding

National Institutes of Health [EY 02422]
Canadian Institutes for Health Research (CIHR) [RMF-92101, MT 5822]
Macula Vision Research Foundation
Osk. Huttunen Foundation
Sigrid Juselius Foundation
NSERC
Foundation Fighting Blindness
Arthur and June Willms Postdoctoral Fellowship

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Abstract

RP2 is a ubiquitously expressed protein encoded by a gene associated with X-linked retinitis pigmentosa (XLRP), a retinal degenerative disease that causes severe vision loss. Previous in vitro studies have shown that RP2 binds to ADP ribosylation factor-like 3 (Ar13) and activates its intrinsic GTPase activity, but the function of RP2 in the retina, and in particular photoreceptor cells, remains unclear. To begin to define the role of RP2 in the retina and XLRP, we have conducted biochemical studies to identify proteins in retinal cell extracts that interact with RP2. Here, we show that RP2 interacts with N-ethylmaleimide sensitive factor (NSF) in retinal cells as well as cultured embryonic kidney (HEK293) cells by mass spectrometry-based proteomics and biochemical analysis. This interaction is mediated by the N-terminal domain of NSF. The E138G and Delta I137 mutations of RP2 known to cause XLRP abolished the interaction of RP2 with the N-terminal domain of NSF. Immunofluorescence labeling studies further showed that RP2 colocalized with NSF in photoreceptors and other cells of the retina. Intense punctate staining of RP2 was observed close to the junction between the inner and outer segments beneath the connecting cilium, as well as within the synaptic region of rod and cone photoreceptors. Our studies indicate that RP2, in addition to serving as a regulator of Ar13, interacts with NSF, and this complex may play an important role in membrane protein trafficking in photoreceptors and other cells of the retina.

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Inherited Retinal Degeneration: Towards the Development of a Combination Therapy Targeting Histone Deacetylase, Poly (ADP-Ribose) Polymerase, and Calpain

Yujie Dong, Jie Yan, Ming Yang, Wenrong Xu, Zhulin Hu, Francois Paquet-Durand, Kangwei Jiao

Summary: Inherited retinal degeneration (IRD) is a group of blinding diseases caused by gene mutations. Excessive activation of HDAC, PARP, and calpain is linked to photoreceptor loss in IRD. Inhibition of HDAC, PARP, or calpain has shown promise in preventing photoreceptor cell death, but their relationship is still unclear. In this study, organotypic retinal explant cultures from wild-type and rd1 mice were treated with specific inhibitors for HDAC, PARP, and calpain. The results showed that inhibition of HDAC, PARP, or calpain reduced rd1 mouse photoreceptor degeneration, with HDAC inhibitor Vorinostat being the most effective. HDAC inhibition reduced PARP activity but both HDAC and PARP inhibition were needed to reduce calpain activity. Unexpectedly, combined treatment with inhibitors for PARP and calpain or HDAC and calpain did not produce synergistic photoreceptor rescue. These findings suggest that in rd1 photoreceptors, HDAC, PARP, and calpain are part of the same degenerative pathway, activated in a sequence starting with HDAC and ending with calpain.

BIOMOLECULES (2023)