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Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM)

Ilaria D'Atri, Emily-Rose Martin, Liming Yang, Elizabeth Sears, Emma Baple, Andrew H. Crosby, John K. Chilton, Asami Oguro-Ando | Neuroscience Letters | Vol 830 | May 2024 | doi.org/10.1016/j.neulet.2024.137778


Abstract

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins.


In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart.


To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.


Background

In eukaryotes the endoplasmic reticulum (ER) is the organelle where calcium homeostasis is maintained, and lipids or proteins are produced, modified, exported, and degraded [1], [2], [3]. The folding of proteins is a necessary step for export or membrane insertion, and failure of this process leads to accumulation of misfolded proteins [3], [4]. When the demand for the secretion of folded proteins and the accumulation of misfolded proteins occurs, the ER undergoes stress [4], [5]. Pathways of stress signaling, collectively called the unfolded protein response (UPR), are activated to restore homeostasis and survival of the cell or induce apoptosis, leading to cell death [5], [6].

Many proteins have been identified to be fundamental for retinal development and function (for example RHO, RP1, ATF6, CLCC1, etc.) [7], [8], [9], [10]. One of these, CLCC1, is an ER-resident chloride channel, and variants in it have been associated with neurodegeneration of the retina [11]. Although the variant (referred to as the Asp25Glu variant) results in pathological events, its expression is necessary for development [7]. We have previously shown that loss of function of CLCC1 during development in both mouse and zebrafish is lethal, while heterozygous knockout or knockdown negatively impact the development of the retina [7]. Specifically, cone and rod photoreceptors fail to develop, with fewer number of them being functional [7].


As mentioned above, CLCC1 localizes in the ER, and the loss of CLCC1 was linked with increase of UPR response and ER stress [7]. CLCC1 loss of function has also been reported to increase the levels of ER chaperone BiP (GRP78), consistent with the induction of ER stress [12]. Recently, Chu et al. [13] demonstrated that CLCC1 interacts with the microprotein PIGBOS at ER-mitochondria contact sites, where CLCC1 is necessary for PIGBOS to function as an UPR activator. In this work, we looked for binding partners of CLCC1 using mass spectrometry and validate novel CLCC1-interacting proteins by immunoprecipitation and microscopy. We observe that CLCC1 co-localizes and co-precipitates with Calnexin; as well as Sigma Non-Opioid Intracellular Receptor 1 (SigmaR1), an ER chaperone protein that localizes to mitochondria-associated endoplasmic reticulum membranes (MAMs). These results support the finding of CLCC1 interacting with MAM proteins at the interface between the ER and mitochondria.


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