0000000000322196
AUTHOR
Erwin Van Wijk
Usher syndrome: molecular links of pathogenesis, proteins and pathways.
Contains fulltext : 50437.pdf (Publisher’s version ) (Closed access) Usher syndrome is the most common form of deaf-blindness. The syndrome is both clinically and genetically heterogeneous, and to date, eight causative genes have been identified. The proteins encoded by these genes are part of a dynamic protein complex that is present in hair cells of the inner ear and in photoreceptor cells of the retina. The localization of the Usher proteins and the phenotype in animal models indicate that the Usher protein complex is essential in the morphogenesis of the stereocilia bundle in hair cells and in the calycal processes of photoreceptor cells. In addition, the Usher proteins are important in…
Usher syndrome and Leber congenital amaurosis are molecularly linked via a novel isoform of the centrosomal ninein-like protein.
Contains fulltext : 80984.pdf (Publisher’s version ) (Closed access) Usher syndrome (USH) and Leber congenital amaurosis (LCA) are autosomal recessive disorders resulting in syndromic and non-syndromic forms of blindness. In order to gain insight into the pathogenic mechanisms underlying retinal degeneration, we searched for interacting proteins of USH2A isoform B (USH2A(isoB)) and the LCA5-encoded protein lebercilin. We identified a novel isoform of the centrosomal ninein-like protein, hereby named Nlp isoform B (Nlp(isoB)), as a common interactor. Although we identified the capacity of this protein to bind calcium with one of its three EF-hand domains, the interacton with USH2A(isoB) did …
Direct interaction of the Usher syndrome 1G protein SANS and myomegalin in the retina
Contains fulltext : 96822.pdf (Publisher’s version ) (Closed access) The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically heterogeneous with at least 11 chromosomal loci assigned to 3 clinical types, USH1-3. We have previously demonstrated that all USH1 and 2 proteins in the eye and the inner ear are organized into protein networks by scaffold proteins. This has contributed essentially to our current understanding of the function of USH proteins and explains why defects in proteins of different families cause very similar phenotypes. We have previously shown that the USH1G protein SANS (scaffold protein containing ankyrin repeat…
Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis
Contains fulltext : 53618.pdf (Publisher’s version ) (Closed access) Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photore…
CiliaCarta: An integrated and validated compendium of ciliary genes
The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse…
KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Background Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. Results Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556-/- null mice possess…
MPP1 links the Usher protein network and the Crumbs protein complex in the retina.
Contains fulltext : 53571.pdf (Publisher’s version ) (Closed access) The highly ordered distribution of neurons is an essential feature of a functional mammalian retina. Disruptions in the apico-basal polarity complexes at the outer limiting membrane (OLM) of the retina are associated with retinal patterning defects in vertebrates. We have analyzed the binding repertoire of MPP5/Pals1, a key member of the apico-basal Crumbs polarity complex, that has functionally conserved counterparts in zebrafish (nagie oko) and Drosophila (Stardust). We show that MPP5 interacts with its MAGUK family member MPP1/p55 at the OLM. Mechanistically, this interaction involves heterodimerization of both MAGUK mo…
Association of Whirlin with Cav1.3 (α1D) Channels in Photoreceptors, Defining a Novel Member of the Usher Protein Network
Contains fulltext : 88383.pdf (Publisher’s version ) (Closed access) PURPOSE: Usher syndrome is the most common form of hereditary deaf-blindness. It is both clinically and genetically heterogeneous. The USH2D protein whirlin interacts via its PDZ domains with other Usher-associated proteins containing a C-terminal type I PDZ-binding motif. These proteins co-localize with whirlin at the region of the connecting cilium and at the synapse of photoreceptor cells. This study was undertaken to identify novel, Usher syndrome-associated, interacting partners of whirlin and thereby obtain more insights into the function of whirlin. METHODS: The database of ciliary proteins was searched for proteins…
An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.
Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub…
Usherin defects lead to early-onset retinal dysfunction in zebrafish
Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2a(rmc1): c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2a(b1245): c.15520_…
Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2.
Contains fulltext : 48386.pdf (Publisher’s version ) (Closed access) Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. USH is clinically and genetically heterogeneous with at least 11 chromosomal loci assigned to the three USH types (USH1A-G, USH2A-C, USH3A). Although the different USH types exhibit almost the same phenotype in human, the identified USH genes encode for proteins which belong to very different protein classes and families. We and others recently reported that the scaffold protein harmonin (USH1C-gene product) integrates all identified USH1 molecules in a USH1-protein network. Here, we investigated the relationship between the USH2 molecules a…
Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.
Item does not contain fulltext The human Usher syndrome (USH) is a complex ciliopathy with at least 12 chromosomal loci assigned to three clinical subtypes, USH1-3. The heterogeneous USH proteins are organized into protein networks. Here, we identified Magi2 (membrane-associated guanylate kinase inverted-2) as a new component of the USH protein interactome, binding to the multifunctional scaffold protein SANS (USH1G). We showed that the SANS-Magi2 complex assembly is regulated by the phosphorylation of an internal PDZ-binding motif in the sterile alpha motif domain of SANS by the protein kinase CK2. We affirmed Magi2's role in receptor-mediated, clathrin-dependent endocytosis and showed tha…
A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.
Contains fulltext : 69178.pdf (Publisher’s version ) (Closed access) The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families. The scaffold protein harmonin (USH1C) was shown to integrate all identified USH1 and USH2 molecules into protein networks. Here, we analyzed a protein network organized in the absence of harmonin by the scaffold proteins SANS (USH1G) and whirlin (USH2D). Immunoelectron microscopic anal…
Additional file 1: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
The phylogenetic distribution and sequence conservation of KIAA0556 orthologs in eukaryotes. Presence and sequence conservation of KIAA0556 are projected on the eukaryotic species tree to visualise the phylogenetic distribution of KIAA0556 orthologues as well as the distribution of the triple-repeat and quadruple-repeat configurations of the DUF4457 domains of unknown function. The black circles and white circles indicate which eukaryotic species contain or lack cilia/flagella. Recent KIAA0556 duplicates in Branchiostoma floridae and Paramecium tetraurelia are denoted by x2. *Dictyostelium discoideum protein sequence contains many â Nâ s (uncalled bases) in the N-terminal part of the sequen…
The mitotic spindle protein SPAG5/Astrin connects to the Usher protein network postmitotically
Abstract Background Mutations in the gene for Usher syndrome 2A (USH2A) are causative for non-syndromic retinitis pigmentosa and Usher syndrome, a condition that is the most common cause of combined deaf-blindness. To gain insight into the molecular pathology underlying USH2A-associated retinal degeneration, we aimed to identify interacting proteins of USH2A isoform B (USH2AisoB) in the retina. Results We identified the centrosomal and microtubule-associated protein sperm-associated antigen (SPAG)5 in the retina. SPAG5 was also found to interact with another previously described USH2AisoB interaction partner: the centrosomal ninein-like protein NINLisoB. Using In situ hybridization, we foun…
Additional file 1: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
The phylogenetic distribution and sequence conservation of KIAA0556 orthologs in eukaryotes. Presence and sequence conservation of KIAA0556 are projected on the eukaryotic species tree to visualise the phylogenetic distribution of KIAA0556 orthologues as well as the distribution of the triple-repeat and quadruple-repeat configurations of the DUF4457 domains of unknown function. The black circles and white circles indicate which eukaryotic species contain or lack cilia/flagella. Recent KIAA0556 duplicates in Branchiostoma floridae and Paramecium tetraurelia are denoted by x2. *Dictyostelium discoideum protein sequence contains many â Nâ s (uncalled bases) in the N-terminal part of the sequen…
Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa
Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactl…
Additional file 6: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Results of the SF-TAP analysis with over-expressed N-terminally SF-TAP-tagged KIAA0556 in HEK293T cells. Shown is the number of unique identified peptides as well as the sequence coverage for each protein detected by mass spectrometry. Proteins identified in >1 out 17 SF-TAP control experiments (empty vector) were removed. (XLSX 28 kb)
Additional file 4: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-type and K04F10.2(tm1830) mutant worms. Shown are the anterograde and retrograde velocities (μm.s-1/standard deviation (SD)) of GFP-tagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). t-test pairwise comparison with wild-type controls, n number of particles, N measured number of amphids and phasmids. OSM-3 is the worm orthologue of KIF17; CHE-11 is the worm orthologue of IFT140; OSM-6 is the worm orthologue of IFT52. b Representative fluorescence images of phasmid cilia showing normal IFT protein localisations and distributions in …
Additional file 8: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are fluorescence images of worms expressing a transcriptional GFP reporter under the control of the indicated geneâ s promoter, which stains the entire cell in which it is expressed. DiI (red) co-stain identifies six pairs of ciliated amphid neurons and both pairs of ciliated phasmid neurons. Arrowheads denote cells with both red and green signals. Other ciliated head cells are identifiable by long dendritic processes (arrows) extending to the anterior end of the worm. Scale bars, 20Â Îźm (all images similarly scaled). (JPG 611 kb)
Additional file 2: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Alignment of IFT25 with permutated KIAA0556 repeat sequences. When aligned using HHpred, a significant part of the Chlamydomonas IFT25 N-terminus was unmatched with human KIAA0556 and significant sequence remained at the C-terminus of the repeats, suggesting a circular permutation relationship between the repeats and IFT25. Shown is a HHpred alignment of IFT25 orthologues with permutated repeat sequences (r1–4) from KIAA0556 orthologues, which results in improved sequence matches. In each permutated repeat sequence, 30–40 amino acids from the beginning of each repeat have been added to the end of the same repeat (denoted by red box) using manual editing. The precise number of amino acids tr…
Additional file 3: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Ciliary phenotypes that are unaffected in C. elegans K04F10.2( tm1830 ) mutants. a K04F10.2 mutants possess normal fluorescent dye (DiI) filling in amphid (head) and phasmid (tail) neurons. Scale bars, 15 μm. b The lengths and morphologies of various sensory neuronal cilia are normal in K04F10.2 mutants. Shown are fluorescence images of cilia from worms expressing str-1p::GFP (AWB neuron), gcy-5p::GFP (ASER neuron) and OSM-6::GFP (PHA/B neurons) transgenes. Numbers (± standard error of the mean) refer to cilium lengths. Scale bars, 2 μm. c–e K04F10.2 mutants possess normal sensory benzaldehyde chemoattraction (n = 10), osmotic avoidance (n = 10), and foraging/roaming (n = 34) behaviours. ch…
Additional file 7: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Supplementary information to the data in Fig. 8 . a Schematic representation of all the different KIAA0556 fragments used to screen our selection of 200+ ciliary proteins. The predicted protein repeat domains, shown in Additional files 1 and 2, are depicted as d1 to d4. Constructs were generated containing isolated domains as well as a combination of domains. b Single transfections of PalMyr-KIAA0556 and mRFP-KATNBL1, showing that membrane localisation of the mRFP tagged protein is indeed dependent on the interaction with the PalMyr-tagged protein. (JPG 491 kb)
Additional file 5: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Data supplementary to the nocodazole destabilization assay shown in Fig. 7 . a, b Replicate images of DMSO or nocodazole-treated hTERT-RPE1 cells. Cells were transfected with SF-TAP-tagged KIAA0556 (detected with anti-FLAG immunostaining; green) or GFP-KIAA0556 and counterstained with anti-acetylated tubulin (red) and DAPI (blue). Cells with high KIAA0556 expression are characterised by a filamentous staining pattern and spots of accumulated KIAA0556 signal. In non-transfected cells, 10 minute nocodazole treatment resulted in the loss of a stabilised MT network (see especially the high exposure images), as judged by loss of (almost) all cytoplasmic acetylated tubulin staining and/or the abs…