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Johnson, C. A.

Publications and source records attributed to Johnson, C. A..

25 records · Page 2Linked to original sources

Drug and siRNA screens identify ROCK2 as a therapeutic target for ciliopathies.

Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need. We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

cell biology↗

Regulation of canonical Wnt signalling by ciliary protein MKS1 and Ubiquitin Proteasome System component UBE2E1.

A functional primary cilium is essential for normal and regulated signalling. Primary ciliary defects cause a group of developmental conditions known as ciliopathies, but the precise mechanisms of signal regulation by the cilium remain unclear. Previous studies have implicated the ubiquitin proteasome system (UPS) in regulation of Wnt signalling at the ciliary basal body. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for a ciliopathy mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitizes cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. To substantiate a direct link between MKS1 and the UPS, we identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates {beta}-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalized, particularly during conditions of ciliary resorption, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. Furthermore, we demonstrate that processing of phosphorylated {beta}-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct {beta}-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1), confirming the fundamental role of UPS defects in the molecular pathogenesis of ciliopathies.

molecular biology↗

Cardiac contraction velocity has evolved to match heart rate with body size through variation in β-cardiac myosin sequence.

Heart rate and the maximum velocity of contraction of striated muscle are inversely related to species size. As mammals evolve to different sizes, adaptations are required such as slower contracting heart and skeletal muscles. Analysis of the motor domain of {beta}-myosin from 67 mammals from two clades identifies 14 sites, out of 800, strongly associated with body mass (p<0.01) but not with the clade (p>0.05). Nine of these sites were mutated in the human {beta}-myosin to make it resemble the rat sequence. Biochemical analysis revealed that the rat-human {beta}-myosin chimera functioned like the native rat myosin with a two fold increase in both motility and in the rate of ADP release from the actin.myosin cross-bridge (the step that limits contraction velocity). Both clades use the same small set of amino acids to adjust contraction velocity, suggesting a limited number of ways in which velocity can be manipulated.

biochemistry↗

The ATPase cycle of Human Muscle Myosin II Isoforms: Adaptation of a single mechanochemical cycle for different physiological roles.

Striated muscle myosins are encoded by a large gene family in all mammals, including human. These isoforms define several of the key characteristics of the different striated muscle fiber types including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of eight different human myosin isoforms to define the actin.myosin cross-bridge cycle in solution. Here, we use a recently developed modeling approach MUSICO to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction including duty ratio, shortening velocity, ATP economy and the load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.

biochemistry↗

Pediatric and adult-onset HCM mutations in the myosin motor domain have similar properties

Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and cardiac hypercontractility. Mutations in {beta} cardiac myosin heavy chain ({beta}-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to the disease remain incompletely understood. Predicting the severity of any single {beta}-MyHC mutation is hindered by a lack of detailed evaluation at the molecular level. In addition, since the cardiomyopathy can take 20 or more years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in early onset disease may show more severe molecular changes in function compared to later onset mutations. In this work, we performed steady-state and transient kinetic analyses of myosins carrying 1 of 7 missense mutations in the motor domain. Of these 7, 4 have been identified in early onset cardiomyopathy screens. The derived parameters were used to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results show no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related Duty Ratio (DR; fraction of myosin in strongly attached force-holding states) and the measured ATPases all change in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the 7 HCM mutations are clearly distinct from a set of DCM mutations previously characterized.

biophysics↗

Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.\n\neTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.\n\nHighlightsO_LISuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patients\nC_LIO_LIRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype protein\nC_LIO_LIPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoids\nC_LIO_LIPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retina\nC_LIO_LIRPE cells display loss of polarity, reduced barrier function and phagocytosis\nC_LIO_LIPhotoreceptors display shorter and fewer cilia and degenerative features\nC_LIO_LIRPE cells display most abnormalities suggesting they might be the primary site of pathogenesis\nC_LIO_LIIn situ gene editing corrects the mutation and rescues key phenotypes\nC_LI

genetics↗

CiliaCarta: An Integrated And Validated Compendium Of Ciliary Genes

The cilium is an essential organelle at the surface of most 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 found experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins. In addition, we show that OSCP1, which has previously been implicated in two distinct non-ciliary functions, causes a cilium dysfunction phenotype when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 836 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

bioinformatics↗