PCARE requires coiled coil, RP62 kinase-binding and EVH1 domain-binding motifs for ciliary expansion

Abstract

Retinitis Pigmentosa (RP) is a genetically heterogeneous form of inherited retinal disease that leads to progressive visual impairment. One genetic subtype of RP, RP54, has been linked to mutations in PCARE (Photoreceptor Cilium Actin Regulator). We have recently shown that PCARE recruits WASF3 to the tip of a primary cilium, and thereby activates an Arp2/3 complex which results in the remodeling of actin filaments that drives the expansion of the ciliary tip membrane. Based on these findings, and the lack of proper photoreceptor development in mice lacking Pcare, we postulated that PCARE plays an important role in photoreceptor outer segment disk formation. In this study, we aimed to decipher the relationship between predicted structural and function amino acid motifs within PCARE and its function. Our results show that PCARE contains a predicted helical coiled coil domain together with evolutionary conserved binding sites for photoreceptor kinase MAK (type RP62), as well as EVH1 domain binding linear motifs. Upon deletion of the helical domain, PCARE failed to localize to the cilia. Furthermore, upon deletion of the EVH1 domain binding motifs separately or together, co-expression of mutant protein with WASF3 resulted in smaller ciliary tip membrane expansions. Finally, inactivation of the lipid modification on the cysteine residue at amino acid position 3 also caused a moderate decrease in the sizes of ciliary tip expansions. Taken together, our data illustrate the importance of amino acid motifs and domains within PCARE in fulfilling its physiological function.

Mutational Profile and Retinal Phenotypes of PCARE-Related Cone-Rod Dystrophies in a Mexican Cohort

“…Of note, five out of six variants identified here introduce premature termination codons (PTCs), and according to the canonical rules known for nonsense-mediated mRNA decay activation [ 26 ], all of them are predicted to activate the mechanism leading to transcript degradation. On the other hand, mutation modeling shows that the novel p.His184Arg variant affects a highly conserved residue of the PCARE protein ( Figure 2 ), located in a likely structured region corresponding to a helical coiled coil domain, which is predicted to have an important role in the function of the protein [ 18 , 19 ]. Previously characterized pathogenic PCARE missense variants as p.Ile201Phe [ 8 ] and p.Cys599Arg [ 27 ] also affect relatively conserved residues.…”

Cytoskeletal networks in primary cilia: Current knowledge and perspectives

“…Notably, our previous work revealed a critical role for tubulin deacetylase HDAC6 in photoreceptor‐connecting cilia, suggesting potential value for HDAC6‐targeting drugs for the prevention of retinopathy of prematurity (Ran et al, 2020; Ran et al, 2022). There is also increasing recognition of the critical involvement of many actin‐binding proteins in primary ciliogenesis, such as the Arp2/3 complex (Afanasyeva et al, 2022), neuronal Wiskott‐Aldrich syndrome protein (N‐WASP) (Drummond et al, 2018), formin (Copeland et al, 2018), and myosin‐VI (Magistrati et al, 2022). Fortunately, inhibitors are available for these targets, so it is therefore relevant to investigate whether their pharmacological inhibition could benefit ciliopathy patients (Figure 3).…”

Clinical and Molecular Aspects of C2orf71/PCARE in Retinal Diseases

“…However, after collecting all the identified variants, we found that 61% are located in the N-terminal (Figure 2). Afanasyeva et al [32] studied how the inactivation of the PCARE-relevant motifs affects their functions and localization. PCARE with an inactivated helix domain has been observed to change its localization from the cilia to the cytosol.…”

“…Unfortunately, the PCARE protein encoded by the C2orf71 gene shows no homology with other known human proteins. Therefore, there is a lack of information on the primary sequence and its function [ 1 , 4 , 12 , 31 , 32 ]. Yet, comparing the PCARE protein sequence between mammals, birds, and fish vertebrate classes presents some well-conserved areas undergoing post-translational lipid modifications [ 1 ].…”

“…PCARE-F1 also contains a likely structured domain, referring to the helical domain forming an antiparallel coiled coil from amino acids 171 to 388, relevant for ciliary trafficking. PCARE-F2 contains an actin-binding WH2 short-linear motif (SLiM), amino acids 597 to 615 [ 32 ], capable of binding to actin filaments. Moreover, this region contains two target sites (RP62a and RP62b) for the MAK/RP62 kinase, a photoreceptor cilium-associated protein crucial for ciliary length and retinal health [ 33 ].…”