Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and ß, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

RNA-Seq Analysis of Trans-Differentiated ARPE-19 Cells Transduced by AAV9-AIPL1 Vectors.

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes' transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4.

The ubiquitin-like modifier FAT10 inhibits retinal PDE6 activity and mediates its proteasomal degradation.

The retina-specific chaperone aryl hydrocarbon interacting protein-like 1 (AIPL1) is essential for the correct assembly of phosphodiesterase 6 (PDE6), which is a pivotal effector enzyme for phototransduction and vision because it hydrolyzes cGMP. AIPL1 interacts with the cytokine-inducible ubiquitin-like modifier FAT10, which gets covalently conjugated to hundreds of proteins and targets its conjugation substrates for proteasomal degradation, but whether FAT10 affects PDE6 function or turnover is unknown. Here, we show that FAT10 mRNA is expressed in human retina and identify rod PDE6 as a retina-specific substrate of FAT10 conjugation. We found that AIPL1 stabilizes the FAT10 monomer and the PDE6-FAT10 conjugate. Additionally, we elucidated the functional consequences of PDE6 FAT10ylation. On the one hand, we demonstrate that FAT10 targets PDE6 for proteasomal degradation by formation of a covalent isopeptide linkage. On the other hand, FAT10 inhibits PDE6 cGMP hydrolyzing activity by noncovalently interacting with the PDE6 GAFa and catalytic domains. Therefore, FAT10 may contribute to loss of PDE6 and, as a consequence, degeneration of retinal cells in eye diseases linked to inflammation and inherited blindness-causing mutations in AIPL1.

Unique structural features of the AIPL1-FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness.

FKBP-domain proteins (FKBPs) are pivotal modulators of cellular signaling, protein folding, and gene transcription. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a distinctive member of the FKBP superfamily in terms of its biochemical properties, and it plays an important biological role as a chaperone of phosphodiesterase 6 (PDE6), an effector enzyme of the visual transduction cascade. Malfunction of mutant AIPL1 proteins triggers a severe form of Leber congenital amaurosis and leads to blindness. The mechanism underlying the chaperone activity of AIPL1 is largely unknown, but involves the binding of isoprenyl groups on PDE6 to the FKBP domain of AIPL1. We solved the crystal structures of the AIPL1-FKBP domain and its pathogenic mutant V71F, both in the apo form and in complex with isoprenyl moieties. These structures reveal a module for lipid binding that is unparalleled within the FKBP superfamily. The prenyl binding is enabled by a unique "loop-out" conformation of the ß4-α1 loop and a conformational "flip-out" switch of the key W72 residue. A second major conformation of apo AIPL1-FKBP was identified by NMR studies. This conformation, wherein W72 flips into the ligand-binding pocket and renders the protein incapable of prenyl binding, is supported by molecular dynamics simulations and appears to underlie the pathogenicity of the V71F mutant. Our findings offer critical insights into the mechanisms that underlie AIPL1 function in health and disease, and highlight the structural and functional diversity of the FKBPs.

A Novel AIPL1 Nonsense Mutation: Case Report of Three Siblings Diagnosed with Leber Congenital Amaurosis.

Background: Leber congenital amaurosis (LCA) is a subgroup of early onset retinal dystrophy, manifesting with early or congenital visual loss, wandering nystagmus, amaurotic pupils, oculodigital sign, reduced retinal thickness on optical coherence tomography and abnormal electroretinogram. Today, mutations of about 25 genes account for 80% of individuals with LCA. The AIPL1 mutations causing LCA type 4 account for about 5-10% of this group. Case Report: Three affected siblings with vision loss, nystagmus, cataracts, stage 4 keratoconus, retinal abnormalities (black spots), lack of glaucoma, and dysmorphic features from a consanguineous marriage had LCA type 4 with a novel homozygous missense mutations of AIPL1(c.862 C > T). Conclusion: Cortical cataracts, stage 4 keratoconus, retinal black spots, and lack of glaucoma along with mutations of AIPL1 (c.862 C > T) can be present in LCA type 4.

Gene and Cell Therapy for AIPL1-Associated Leber Congenital Amaurosis: Challenges and Prospects.

Leber congenital amaurosis (LCA) caused by AIPL1 mutations is one of the most severe forms of inherited retinal degeneration (IRD). The rapid and extensive photoreceptor degeneration challenges the development of potential treatments. Nevertheless, preclinical studies show that both gene augmentation and photoreceptor transplantation can regenerate and restore retinal function in animal models of AIPL1-associated LCA. However, questions regarding long-term benefit and safety still remain as these therapies advance towards clinical application. Ground-breaking advances in stem cell technology and genome editing are examples of alternative therapeutic approaches and address some of the limitations associated with previous methods. The continuous development of these cutting-edge biotechnologies paves the way towards a bright future not only for AIPL1-associated LCA patients but also other forms of IRD.

How Excessive cGMP Impacts Metabolic Proteins in Retinas at the Onset of Degeneration.

Aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1) is essential to stabilize cGMP phosphodiesterase 6 (PDE6) in rod photoreceptors. Mutation of AIPL1 leads to loss of PDE6, accumulation of intracellular cGMP, and rapid degeneration of rods. To understand the metabolic basis for the photoreceptor degeneration caused by excessive cGMP, we performed proteomics and phosphoproteomics analyses on retinas from AIPL1-/- mice at the onset of rod cell death. AIPL1-/- retinas have about 18 times less than normal PDE6a and no detectable PDE6b. We identified twelve other proteins and thirty-nine phosphorylated proteins related to cell metabolism that are significantly altered preceding the massive degeneration of rods. They include transporters, kinases, phosphatases, transferases, and proteins involved in mitochondrial bioenergetics and metabolism of glucose, lipids, amino acids, nucleotides, and RNA. In AIPLI-/- retinas mTOR and proteins involved in mitochondrial energy production and lipid synthesis are more dephosphorylated, but glycolysis proteins and proteins involved in leucine catabolism are more phosphorylated than in normal retinas. Our findings indicate that elevating cGMP rewires cellular metabolism prior to photoreceptor degeneration and that targeting metabolism may be a productive strategy to prevent or slow retinal degeneration.

The Leber Congenital Amaurosis-Linked Protein AIPL1 and Its Critical Role in Photoreceptors.

Mutations in the photoreceptor/pineal-expressed gene, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), are mainly associated with autosomal recessive Leber congenital amaurosis (LCA), the most severe form of inherited retinopathy that occurs in early childhood. AIPL1 functions as a photoreceptor-specific molecular co-chaperone that interacts specifically with the molecular chaperones HSP90 and HSP70 to facilitate the correct folding and assembly of the retinal cGMP phosphodiesterase (PDE6) holoenzyme. The absence of AIPL1 leads to a dramatic degeneration of rod and cone cells and a complete loss of any light-dependent electrical response. Here we review the important role of AIPL1 in photoreceptor functionality.

Neurotoxicity of cGMP in the vertebrate retina: from the initial research on rd mutant mice to zebrafish genetic approaches.

Zebrafish are an excellent animal model for research on vertebrate development and human diseases. Sophisticated genetic tools including large-scale mutagenesis methodology make zebrafish useful for studying neuronal degenerative diseases. Here, we review zebrafish models of inherited ophthalmic diseases, focusing on cGMP metabolism in photoreceptors. cGMP is the second messenger of phototransduction, and abnormal cGMP levels are associated with photoreceptor death. cGMP concentration represents a balance between cGMP phosphodiesterase 6 (PDE6) and guanylate cyclase (GC) activities in photoreceptors. Various zebrafish cGMP metabolism mutants were used to clarify molecular mechanisms by which dysfunctions in this pathway trigger photoreceptor degeneration. Here, we review the history of research on the retinal degeneration (rd) mutant mouse, which carries a genetic mutation of PDE6b, and we also highlight recent research in photoreceptor degeneration using zebrafish models. Several recent discoveries that provide insight into cGMP toxicity in photoreceptors are discussed.

Extended conformation of the proline-rich domain of human aryl hydrocarbon receptor-interacting protein-like 1: implications for retina disease.

Mutations in the primate-specific proline-rich domain (PRD) of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are thought to cause Leber congenital amaurosis or dominant cone-rod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL11-316 with PRD truncation, and the P351Δ12 and P376S mutants were examined by small angle X-ray scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506-binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecule's radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase-6A and HSP90. We also identified the γ-subunit of phosphodiesterase-6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351Δ12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants. Mutations in the proline-rich domain (PRD) of human AIPL1 cause severe retinal diseases, yet the role of PRD and the mechanisms of PRD mutations are unknown. Here, we describe a SAXS-derived solution structure of AIPL1 and functional properties of disease-linked AIPL1-PRD mutants. This structure and functional analyses provide a framework for understanding the mechanisms of PRD in disease.

Interaction of aryl hydrocarbon receptor-interacting protein-like 1 with the farnesyl moiety.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

Phenotyping and genotyping inherited retinal diseases: Molecular genetics, clinical and imaging features, and therapeutics of macular dystrophies, cone and cone-rod dystrophies, rod-cone dystrophies, Leber congenital amaurosis, and cone dysfunction syndromes.

Inherited retinal diseases (IRD) are a leading cause of blindness in the working age population and in children. The scope of this review is to familiarise clinicians and scientists with the current landscape of molecular genetics, clinical phenotype, retinal imaging and therapeutic prospects/completed trials in IRD. Herein we present in a comprehensive and concise manner: (i) macular dystrophies (Stargardt disease (ABCA4), X-linked retinoschisis (RS1), Best disease (BEST1), PRPH2-associated pattern dystrophy, Sorsby fundus dystrophy (TIMP3), and autosomal dominant drusen (EFEMP1)), (ii) cone and cone-rod dystrophies (GUCA1A, PRPH2, ABCA4, KCNV2 and RPGR), (iii) predominant rod or rod-cone dystrophies (retinitis pigmentosa, enhanced S-Cone syndrome (NR2E3), Bietti crystalline corneoretinal dystrophy (CYP4V2)), (iv) Leber congenital amaurosis/early-onset severe retinal dystrophy (GUCY2D, CEP290, CRB1, RDH12, RPE65, TULP1, AIPL1 and NMNAT1), (v) cone dysfunction syndromes (achromatopsia (CNGA3, CNGB3, PDE6C, PDE6H, GNAT2, ATF6), X-linked cone dysfunction with myopia and dichromacy (Bornholm Eye disease; OPN1LW/OPN1MW array), oligocone trichromacy, and blue-cone monochromatism (OPN1LW/OPN1MW array)). Whilst we use the aforementioned classical phenotypic groupings, a key feature of IRD is that it is characterised by tremendous heterogeneity and variable expressivity, with several of the above genes associated with a range of phenotypes.

Unique interface and dynamics of the complex of HSP90 with a specialized cochaperone AIPL1.

Photoreceptor phosphodiesterase PDE6 is central for visual signal transduction. Maturation of PDE6 depends on a specialized chaperone complex of HSP90 with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Disruption of PDE6 maturation underlies a severe form of retina degeneration. Here, we report a 3.9 Å cryoelectron microscopy (cryo-EM) structure of the complex of HSP90 with AIPL1. This structure reveals a unique interaction of the FK506-binding protein (FKBP)-like domain of AIPL1 with HSP90 at its dimer interface. Unusually, the N terminus AIPL1 inserts into the HSP90 lumen in a manner that was observed previously for HSP90 clients. Deletion of the 7 N-terminal residues of AIPL1 decreased its ability to cochaperone PDE6. Multi-body refinement of the cryo-EM data indicated large swing-like movements of AIPL1-FKBP. Modeling the complex of HSP90 with AIPL1 using crosslinking constraints indicated proximity of the mobile tetratricopeptide repeat (TPR) domain with the C-terminal domain of HSP90. Our study establishes a framework for future structural studies of PDE6 maturation.

NMR resonance assignments of the TPR domain of human aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1).

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor-specific chaperone of phosphodiesterase-6, a key effector enzyme in the phototransduction cascade. It contains an N-terminal FK506-binding protein (FKBP) domain and a C-terminal tetratricopeptide repeat (TPR) domain. Mutations in AIPL1, including many missense mutations in both FKBP and TPR domains, have been associated with Leber congenital amaurosis, a severe inherited retinopathy that causes blindness. TPR-domain containing proteins are known to interact with HSP90. However, the structure of AIPL1-TPR domain is presently not determined and little is known about the contribution of the TPR domain to the chaperone function of AIPL1. Here, we report the backbone and sidechain assignments of the TPR domain of AIPL1. These assignments reveal that AIPL1-TPR is an α-helical protein containing seven α-helices connected via short loops. Peak broadening or structural disorder is observed for a cluster of hydrophobic residues of W218, W222 and L223. Therefore, these assignments provide a framework for further structural determination of AIPL1-TPR domain and its interactions with various binding partners for elucidation of the mechanism of TPR contribution to the chaperone function of AIPL1.

TNFα Induces Müller Glia to Transition From Non-proliferative Gliosis to a Regenerative Response in Mutant Zebrafish Presenting Chronic Photoreceptor Degeneration.

Unlike mammals, zebrafish have the capacity to regenerate neurons in response to damage. Most zebrafish retinal injury models employ acute damage, which is unlike the chronic, gradual damage that occurs in human retinal diseases. Here, we studied the regenerative response in the zebrafish aipl1b mutant, gold rush (gosh). In gosh mutants, both cones and rods degenerate by 3 weeks post-fertilization (wpf). Müller glia do not exhibit a regenerative response by 3 wpf; however, they do present non-proliferative gliosis. Only at 5 wpf, is proliferation of Müller cells and rod precursor cells activated. Rods start to recover at 5 wpf and by 12 wpf they reach a level of recovery comparable to wild type, but cones remain absent in the adult stage. TNFα was detected in degenerating cones at 5-7 wpf and in Müller glia at 7 wpf in gosh mutants. At 5 wpf, proliferating Müller glia express Sox2, followed by Pax6 expression in neuronal progenitor cells (NPCs), confirming that the neuronal regeneration program is activated in gosh mutants after 5 wpf. Although acute light-induced damage did not activate proliferation of Müller glia, TNFα injection caused Müller glia to commence a proliferative response at 3 wpf in gosh mutants. These results suggest that Müller glia transition from non-proliferative gliosis to a regenerative state in gosh mutants, and that ectopic introduction of TNFα promotes this Müller cell transition even at 3 wpf. Thus, zebrafish gosh mutants provide a useful model to investigate mechanisms underlying retinal regeneration in a chronic photoreceptor degeneration model.

Chaperones and retinal disorders.

Defects in protein folding and trafficking are a common cause of photoreceptor degeneration, causing blindness. Photoreceptor cells present an unusual challenge to the protein folding and transport machinery due to the high rate of protein synthesis, trafficking and the renewal of the outer segment, a primary cilium that has been modified into a specialized light-sensing compartment. Phototransduction components, such as rhodopsin and cGMP-phosphodiesterase, and multimeric ciliary transport complexes, such as the BBSome, are hotspots for mutations that disrupt proteostasis and lead to the death of photoreceptors. In this chapter, we review recent studies that advance our understanding of the chaperone and transport machinery of phototransduction proteins.

FKBP Ligands-Where We Are and Where to Go?

In recent years, many members of the FK506-binding protein (FKBP) family were increasingly linked to various diseases. The binding domain of FKBPs differs only in a few amino acid residues, but their biological roles are versatile. High-affinity ligands with selectivity between close homologs are scarce. This review will give an overview of the most prominent ligands developed for FKBPs and highlight a perspective for future developments. More precisely, human FKBPs and correlated diseases will be discussed as well as microbial FKBPs in the context of anti-bacterial and anti-fungal therapeutics. The last section gives insights into high-affinity ligands as chemical tools and dimerizers.

NMR resonance assignments of the FKBP domain of human aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) in complex with a farnesyl ligand.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a specialized chaperone of phosphodiesterase 6, a key effector enzyme in the phototransduction cascade. The FKBP domain of AIPL1 is known to bind the farnesyl moiety of PDE6. Mutations in AIPL1, including many missense mutations in the FKBP domain, have been associated with Leber congenital amaurosis, a severe blinding disease. Here, we report the backbone and sidechain assignments of the N-terminal FKBPΔloop (with a loop deletion) of AIPL1 in complex with a farnesyl ligand. We also compare the predicted secondary structures of FKBPΔloop with those of a highly homologous AIP FKBP. These results show that the FKBP domains of AIP and AIPL1 have similar folds, but display subtle differences in structure and dynamics. Therefore, these assignments provide a framework for further elucidation of the mechanism of farnesyl binding and the function of AIPL1 FKBP.

Mechanisms of mutant PDE6 proteins underlying retinal diseases.

Mutations in PDE6 genes encoding the effector enzymes in rods and cones underlie severe retinal diseases including retinitis pigmentosa (RP), autosomal dominant congenital stationary night blindness (adCSNB), and achromatopsia (ACHM). Here we examined a spectrum of pathogenic missense mutations in PDE6 using the system based on co-expression of cone PDE6C with its specialized chaperone AIPL1 and the regulatory Pγ subunit as a potent co-chaperone. We uncovered two mechanisms of PDE6C mutations underlying ACHM: (a) folding defects leading to expression of catalytically inactive proteins and (b) markedly diminished ability of Pγ to co-chaperone mutant PDE6C proteins thereby dramatically reducing the levels of functional enzyme. The mechanism of the Rambusch adCSNB associated with the H258N substitution in PDE6B was probed through the analysis of the model mutant PDE6C-H262N. We identified two interrelated deficits of PDE6C-H262N: disruption of the inhibitory interaction of Pγ with mutant PDE6C that markedly reduced the ability of Pγ to augment the enzyme folding. Thus, we conclude that the Rambusch adCSNB is triggered by low levels of the constitutively active PDE6. Finally, we examined PDE6C-L858V, which models PDE6B-L854V, an RP-linked mutation that alters the protein isoprenyl modification. This analysis suggests that the type of prenyl modifications does not impact the folding of PDE6, but it modulates the enzyme affinity for its trafficking partner PDE6D. Hence, the pathogenicity of PDE6B-L854V likely arises from its trafficking deficiency. Taken together, our results demonstrate the effectiveness of the PDE6C expression system to evaluate pathogenicity and elucidate the mechanisms of PDE6 mutations in retinal diseases.

AIPL1: A specialized chaperone for the phototransduction effector.

Molecular chaperones play pivotal roles in protein folding, quality control, assembly of multimeric protein complexes, protein trafficking, stress responses, and other essential cellular processes. Retinal photoreceptor rod and cone cells have an unusually high demand for production, quality control, and trafficking of key phototransduction components, and thus, require a robust and specialized chaperone machinery to ensure the fidelity of sensing and transmission of visual signals. Misfolding and/or mistrafficking of photoreceptor proteins are known causes for debilitating blinding diseases. Phosphodiesterase 6, the effector enzyme of the phototransduction cascade, relies on a unique chaperone aryl hydrocarbon receptor (AhR)-interacting protein-like 1 (AIPL1) for its stability and function. The structure of AIPL1 and its relationship with the client remained obscure until recently. This review summarizes important recent advances in understanding the mechanisms underlying normal function of AIPL1 and the protein perturbations caused by pathogenic mutations.