The Experts below are selected from a list of 1023 Experts worldwide ranked by ideXlab platform
Colin A. Johnson - One of the best experts on this subject based on the ideXlab platform.
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Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1.
2020Co-Authors: Katarzyna Szymanska, Clare V. Logan, Gabrielle Wheway, Karsten Boldt, Matthiew Adams, Philip A. Robinson, Marius Ueffing, Elton Zeqiraj, Colin A. JohnsonAbstract:AbstractA 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 vivoloss 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 β-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 β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-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.
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regulation of canonical wnt signalling by ciliary protein MKS1 and ubiquitin proteasome system component ube2e1
bioRxiv, 2020Co-Authors: Katarzyna Szymanska, Clare V. Logan, Gabrielle Wheway, Karsten Boldt, Marius Ueffing, Elton Zeqiraj, Colin A. JohnsonAbstract:Abstract A functional primary cilium is a crucial cell appendage which is essential for normal, regulated signalling, and loss of the primary cilium is implicated in a suite of severe developmental conditions known as ciliopathies. The mechanisms of signal regulation by the cilium remain unclear. Previous studies have suggested links between the primary cilium/basal body, the ubiquitin proteasome system (UPS) and Wnt signalling. Here we provide further mechanistic insights into these processes in vivo by crossing the MKS1−/− knockout mouse with the UbG76V-GFP reporter line. We demonstrate in vivo that MKS1 is essential for normal proteasomal processing of ubiquitinated proteins, but that this only manifests as accumulation of ubiquitinated proteins when the proteasome is inhibited. We show that an increase in proteasomal enzymatic activity and Wnt signalling de-regulation in the absence of MKS1. Yeast 2-hybrid demonstrate that the UPS component UBE2E1, an E2 ubiquitin-conjugating enzyme which polyubiquitinates β-catenin, interacts with MKS1. Levels of UBE2E1 and MKS1 are co-dependent, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1, suggesting a functional association between the two proteins. We suggest that MKS1 regulates UBE2E1 and other UPS components at the base of the cilium, which leads to proteasomal and canonical Wnt signalling dysregulation. These findings provide further mechanistic detail of the interaction between the basal body and the UPS in regulating signal transduction through β-catenin, and confirm that the UPS plays a central role in the molecular pathogenesis of ciliopathies.
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Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome.
Developmental biology, 2013Co-Authors: Gabrielle Wheway, Zakia Abdelhamed, Subaashini Natarajan, Carmel Toomes, Chris F. Inglehearn, Colin A. JohnsonAbstract:Meckel-Gruber syndrome (MKS) is an embryonic lethal ciliopathy resulting from mutations in genes encoding proteins localising to the primary cilium. Mutations in the basal body protein MKS1 account for 7% of cases of MKS. The condition affects the development of multiple organs, including brain, kidney and skeleton. Here we present a novel MKS1(tm1a(EUCOMM)Wtsi) knockout mouse which accurately recapitulates the human condition, consistently developing pre-axial polydactyly, complex posterior fossa defects (including the Dandy-Walker malformation), and renal cystic dysplasia. TOPFlash Wnt reporter assays in mouse embryonic fibroblasts (MEFs) showed general de-regulated high levels of canonical Wnt/β-catenin signalling in MKS1(-/-) cells. In addition to these signalling defects, we also observed ectopic high proliferation in the brain and kidney of mutant animals at mid- to late-gestation. The specific role of MKS1 in regulating cell proliferation was confirmed in MKS1 siRNA knockdown experiments which showed increased levels of proliferation after knockdown, an effect not seen after knockdown of other ciliopathy genes. We suggest that this is a result of the de-regulation of multiple signalling pathways (Wnt, mTOR and Hh) in the absence of functional MKS1. This novel model system offers insights into the role of MKS1 in Wnt signalling and proliferation, and the impact of deregulation of these processes on brain and kidney development in MKS, as well as expanding our understanding of the role of MKS1 in multiple signalling pathways.
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Renal Cystic Disease Proteins Play Critical Roles in the Organization of the Olfactory Epithelium
PloS one, 2011Co-Authors: Jennifer L. Pluznick, Diego J. Rodriguez-gil, Michael Hull, Kavita Mistry, Vincent H. Gattone, Colin A. Johnson, Scott D. Weatherbee, Charles A. Greer, Michael J. CaplanAbstract:It was reported that some proteins known to cause renal cystic disease (NPHP6; BBS1, and BBS4) also localize to the olfactory epithelium (OE), and that mutations in these proteins can cause anosmia in addition to renal cystic disease. We demonstrate here that a number of other proteins associated with renal cystic diseases – polycystin 1 and 2 (PC1, PC2), and Meckel-Gruber syndrome 1 and 3 (MKS1, MKS3) – localize to the murine OE. PC1, PC2, MKS1 and MKS3 are all detected in the OE by RT-PCR. We find that MKS3 localizes specifically to dendritic knobs of olfactory sensory neurons (OSNs), while PC1 localizes to both dendritic knobs and cilia of mature OSNs. In mice carrying mutations in MKS1, the expression of the olfactory adenylate cyclase (AC3) is substantially reduced. Moreover, in rats with renal cystic disease caused by a mutation in MKS3, the laminar organization of the OE is perturbed and there is a reduced expression of components of the odor transduction cascade (Golf, AC3) and α-acetylated tubulin. Furthermore, we show with electron microscopy that cilia in MKS3 mutant animals do not manifest the proper microtubule architecture. Both MKS1 and MKS3 mutant animals show no obvious alterations in odor receptor expression. These data show that multiple renal cystic proteins localize to the OE, where we speculate that they work together to regulate aspects of the development, maintenance or physiological activities of cilia.
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Molecular Genetics and Pathogenic Mechanisms for the Severe Ciliopathies: Insights into Neurodevelopment and Pathogenesis of Neural Tube Defects
Molecular Neurobiology, 2011Co-Authors: Clare V. Logan, Zakia Abdel-hamed, Colin A. JohnsonAbstract:Meckel–Gruber syndrome (MKS) is a severe autosomal recessively inherited disorder characterized by developmental defects of the central nervous system that comprise neural tube defects that most commonly present as occipital encephalocele. MKS is considered to be the most common syndromic form of neural tube defect. MKS is genetically heterogeneous with six known disease genes: MKS1 , MKS2/TMEM216 , MKS3/TMEM67 , RPGRIP1L , CEP290 , and CC2D2A with the encoded proteins all implicated in the correct function of primary cilia. Primary cilia are microtubule-based organelles that project from the apical surface of most epithelial cell types. Recent progress has implicated the involvement of cilia in the Wnt and Shh signaling pathways and has led to an understanding of their role in normal mammalian neurodevelopment. The aim of this review is to provide an overview of the molecular genetics of the human disorder, and to assess recent insights into the etiology and molecular cell biology of severe ciliopathies from mammalian animal models of MKS.
Gabrielle Wheway - One of the best experts on this subject based on the ideXlab platform.
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Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1.
2020Co-Authors: Katarzyna Szymanska, Clare V. Logan, Gabrielle Wheway, Karsten Boldt, Matthiew Adams, Philip A. Robinson, Marius Ueffing, Elton Zeqiraj, Colin A. JohnsonAbstract:AbstractA 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 vivoloss 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 β-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 β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-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.
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regulation of canonical wnt signalling by ciliary protein MKS1 and ubiquitin proteasome system component ube2e1
bioRxiv, 2020Co-Authors: Katarzyna Szymanska, Clare V. Logan, Gabrielle Wheway, Karsten Boldt, Marius Ueffing, Elton Zeqiraj, Colin A. JohnsonAbstract:Abstract A functional primary cilium is a crucial cell appendage which is essential for normal, regulated signalling, and loss of the primary cilium is implicated in a suite of severe developmental conditions known as ciliopathies. The mechanisms of signal regulation by the cilium remain unclear. Previous studies have suggested links between the primary cilium/basal body, the ubiquitin proteasome system (UPS) and Wnt signalling. Here we provide further mechanistic insights into these processes in vivo by crossing the MKS1−/− knockout mouse with the UbG76V-GFP reporter line. We demonstrate in vivo that MKS1 is essential for normal proteasomal processing of ubiquitinated proteins, but that this only manifests as accumulation of ubiquitinated proteins when the proteasome is inhibited. We show that an increase in proteasomal enzymatic activity and Wnt signalling de-regulation in the absence of MKS1. Yeast 2-hybrid demonstrate that the UPS component UBE2E1, an E2 ubiquitin-conjugating enzyme which polyubiquitinates β-catenin, interacts with MKS1. Levels of UBE2E1 and MKS1 are co-dependent, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1, suggesting a functional association between the two proteins. We suggest that MKS1 regulates UBE2E1 and other UPS components at the base of the cilium, which leads to proteasomal and canonical Wnt signalling dysregulation. These findings provide further mechanistic detail of the interaction between the basal body and the UPS in regulating signal transduction through β-catenin, and confirm that the UPS plays a central role in the molecular pathogenesis of ciliopathies.
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Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome.
Developmental biology, 2013Co-Authors: Gabrielle Wheway, Zakia Abdelhamed, Subaashini Natarajan, Carmel Toomes, Chris F. Inglehearn, Colin A. JohnsonAbstract:Meckel-Gruber syndrome (MKS) is an embryonic lethal ciliopathy resulting from mutations in genes encoding proteins localising to the primary cilium. Mutations in the basal body protein MKS1 account for 7% of cases of MKS. The condition affects the development of multiple organs, including brain, kidney and skeleton. Here we present a novel MKS1(tm1a(EUCOMM)Wtsi) knockout mouse which accurately recapitulates the human condition, consistently developing pre-axial polydactyly, complex posterior fossa defects (including the Dandy-Walker malformation), and renal cystic dysplasia. TOPFlash Wnt reporter assays in mouse embryonic fibroblasts (MEFs) showed general de-regulated high levels of canonical Wnt/β-catenin signalling in MKS1(-/-) cells. In addition to these signalling defects, we also observed ectopic high proliferation in the brain and kidney of mutant animals at mid- to late-gestation. The specific role of MKS1 in regulating cell proliferation was confirmed in MKS1 siRNA knockdown experiments which showed increased levels of proliferation after knockdown, an effect not seen after knockdown of other ciliopathy genes. We suggest that this is a result of the de-regulation of multiple signalling pathways (Wnt, mTOR and Hh) in the absence of functional MKS1. This novel model system offers insights into the role of MKS1 in Wnt signalling and proliferation, and the impact of deregulation of these processes on brain and kidney development in MKS, as well as expanding our understanding of the role of MKS1 in multiple signalling pathways.
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MKS1 interacts with components of the ubiquitin proteasome pathway to regulate ciliogenesis and multiple signalling pathways
Cilia, 2012Co-Authors: Gabrielle Wheway, Zakia Abdelhamed, Subaashini Natarajan, Colin JohnsonAbstract:MKS1, a ciliary protein containing a B9 domain of unknown function, plays an important role in ciliogenesis. Mutation of the MKS1 gene causes the neonatal lethal multi-organ developmental condition Meckel-Gruber syndrome, characterized by severe ciliary defects and disruption of both Wnt and Shh signalling. We have performed a yeast two-hybrid screen for the MKS1 B9 domain and identified and validated interactions between MKS1, and both an E2 ubiquitin conjugating enzyme and an E3 ubiqutin ligase. Previous studies have shown the importance of the basal body in regulating Wnt signalling through selective proteolysis and the study of the MKS1 protein offers additional mechanistic insight into this process. We present evidence that the role of MKS1 in ciliogenesis and developmental signalling is mediated by targeted protein degradation. Work on a newly characterised MKS1 mutant mouse also provides further insight into the role of this particular ciliary protein normal processes of in vivo developmental signalling regulation and its disruption in Meckel-Gruber syndrome.
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Defects in non-canonical Wnt signalling and actin cytoskeleton remodelling as pathogenic mechanisms in Meckel–Gruber syndrome
International Journal of Developmental Neuroscience, 2010Co-Authors: Colin A. Johnson, Clare V. Logan, Gabrielle Wheway, Zakia Abdelhamed, Matthew AdamsAbstract:Primary cilia are mechano- and chemosensory organelles that have a fundamental role in regulating embryogenesis. Inherited disorders that involve aberrant ciliary structure or function are now known as “ciliopathies”, and they invariably present with cystic kidney dysplasia. Meckel–Gruber syndrome (MKS) is a pleiotropic ciliopathy characterized by severe defects in neurodevelopment that include occipital encephalocele, hydrocephaly and severe neural tube defects. Several MKS genes are now known, including MKS1 and MKS3/TMEM67, encoding the proteins MKS1 and meckelin, a novel receptor. Remarkably, MKS is allelic and overlaps in phenotype with the neurodevelopmental disorder Joubert syndrome (JS), with some of the causative genes implicated in regulation of the Hedgehog signalling pathway. However, our recent work has suggested a role for meckelin and some other MKS proteins in modulating non-canonical Wnt signalling and remodelling the actin cytoskeleton. Meckelin is localized at the apical cell surface, basal bodies and ciliary axoneme of ciliated cell lines and tissues, but also interacts with other MKS proteins and the actin-binding proteins nesprin-2 and filamin A. Loss of expression of MKS genes following RNAi-mediated knockdown or in MKS patient fibroblasts: (1) prevents the movement of the basal body to the apical cell surface prior to ciliogenesis; (2) causes hyperactivation of the small GTPase RhoA and Dishevelled, both implicated in the control of apical docking of basal bodies and planar polarization of epithelial cells; and (3) remodels the actin cytoskeleton. These findings are reiterated in the Mks3/Tmem67 knock-out mouse model of MKS/JS. In contrast, MKS1 is implicated in constraining canonical Wnt signalling. These findings therefore underline the critical role of MKS proteins in ciliogenesis and regulation of Wnt signalling, through interactions with apical cell surface proteins associated with the actin cytoskeleton and implicated in basal body docking.
Morten Petersen - One of the best experts on this subject based on the ideXlab platform.
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Constitutive expression of MKS1 confers susceptibility to Botrytis cinerea infection independent of PAD3 expression
Plant signaling & behavior, 2011Co-Authors: Berthe Katrine Fiil, Morten PetersenAbstract:Signal transduction through MAPK cascades is essential for eukaryotic cell response to various extracellular stimuli, such as the induction of innate immune responses. Arabidopsis thaliana relies in particular on three of its 20 MAPKs, MPK3,-4,-6, for a proper immune response. Recently we showed that one MPK4-substrate, MKS1, is required for basal resistance against the virulent Pseudomonas syringae and the oomycete Hyaloperonospora arabidopsidis. Overexpression of MKS1 (35S-MKS1) led to increased resistance to the same pathogens but also to an increased susceptibility towards the fungi Botrytis cinerea. MKS1 interacts with the transcription factor WRKY33, which in turn controls the regulation of PAD3 and CYP71A13, two genes, required for proper resistance to B. cinerea. Therefore, we tested if the increased susceptibility towards B. cinerea from 35S-MKS1 was due to deregulation of WRKY33 targets. PAD3 and CYP71A13 expression is similar in 35S-MKS1 and WT after B. cinerea treatment suggesting another mechanism controls 35S-MKS1 susceptibility.
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Arabidopsis MKS1 is involved in basal immunity and requires an intact N-terminal domain for proper function.
PloS one, 2010Co-Authors: Klaus Petersen, Jin-long Qiu, Berthe Katrine Fiil, John Mundy, Juri Lütje, Sidsel Hansen, Morten PetersenAbstract:Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. MAP kinase 4 (MPK4) functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins. We investigate the role of MKS1 in basal resistance and the importance of its N- and C-terminal domains for MKS1 function. We used the information that MKS1 loss-of-function partially suppresses the mpk4 loss-of-function phenotype, and that transgenic expression of functional MKS1 in mpk4/MKS1 double mutants reverted the mpk4 dwarf phenotype. Transformation of MKS1/mpk4 with mutant versions of MKS1 constructs showed that a single amino acid substitution in a putative MAP kinase docking domain, MKS1-L32A, or a truncated MKS1 version unable to interact with WRKY33, were deficient in reverting the double mutant to the mpk4 phenotype. These results demonstrate functional requirement in MKS1 for the interaction with MPK4 and WRKY33. In addition, nuclear localization of MKS1 was shown to depend on an intact N-terminal domain. Furthermore, loss-of-function MKS1 mutants exhibited increased susceptibility to strains of Pseudomonas syringae and Hyaloperonospora arabidopsidis, indicating that MKS1 plays a role in basal defense responses. Taken together, our results indicate that MKS1 function and subcellular location requires an intact N-terminus important for both MPK4 and WRKY33 interactions.
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arabidopsis MKS1 is involved in basal immunity and requires an intact n terminal domain for proper function
PLOS ONE, 2010Co-Authors: Klaus Petersen, Jin-long Qiu, Berthe Katrine Fiil, John Mundy, Juri Lütje, Sidsel Hansen, Morten PetersenAbstract:Background Innate immune signaling pathways in animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. MAP kinase 4 (MPK4) functions downstream of innate immune receptors via a nuclear substrate MKS1 to regulate the activity of the WRKY33 transcription factor, which in turn controls the production of anti-microbial phytoalexins.
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The MAP kinase substrate MKS1 is a regulator of plant defense responses
The EMBO journal, 2005Co-Authors: Erik Andreasson, Jin-long Qiu, Tom Jenkins, Peter Brodersen, Stephan Thorgrimsen, Nikolaj H.t. Petersen, Shijiang Zhu, Pernille Ollendorff Micheelsen, Anne Rocher, Morten PetersenAbstract:Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors.
Clare V. Logan - One of the best experts on this subject based on the ideXlab platform.
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Regulation of canonical Wnt signalling by the ciliopathy protein MKS1 and the E2 ubiquitin-conjugating enzyme UBE2E1.
2020Co-Authors: Katarzyna Szymanska, Clare V. Logan, Gabrielle Wheway, Karsten Boldt, Matthiew Adams, Philip A. Robinson, Marius Ueffing, Elton Zeqiraj, Colin A. JohnsonAbstract:AbstractA 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 vivoloss 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 β-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 β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-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.
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regulation of canonical wnt signalling by ciliary protein MKS1 and ubiquitin proteasome system component ube2e1
bioRxiv, 2020Co-Authors: Katarzyna Szymanska, Clare V. Logan, Gabrielle Wheway, Karsten Boldt, Marius Ueffing, Elton Zeqiraj, Colin A. JohnsonAbstract:Abstract A functional primary cilium is a crucial cell appendage which is essential for normal, regulated signalling, and loss of the primary cilium is implicated in a suite of severe developmental conditions known as ciliopathies. The mechanisms of signal regulation by the cilium remain unclear. Previous studies have suggested links between the primary cilium/basal body, the ubiquitin proteasome system (UPS) and Wnt signalling. Here we provide further mechanistic insights into these processes in vivo by crossing the MKS1−/− knockout mouse with the UbG76V-GFP reporter line. We demonstrate in vivo that MKS1 is essential for normal proteasomal processing of ubiquitinated proteins, but that this only manifests as accumulation of ubiquitinated proteins when the proteasome is inhibited. We show that an increase in proteasomal enzymatic activity and Wnt signalling de-regulation in the absence of MKS1. Yeast 2-hybrid demonstrate that the UPS component UBE2E1, an E2 ubiquitin-conjugating enzyme which polyubiquitinates β-catenin, interacts with MKS1. Levels of UBE2E1 and MKS1 are co-dependent, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1, suggesting a functional association between the two proteins. We suggest that MKS1 regulates UBE2E1 and other UPS components at the base of the cilium, which leads to proteasomal and canonical Wnt signalling dysregulation. These findings provide further mechanistic detail of the interaction between the basal body and the UPS in regulating signal transduction through β-catenin, and confirm that the UPS plays a central role in the molecular pathogenesis of ciliopathies.
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Founder mutations and genotype-phenotype correlations in Meckel-Gruber syndrome and associated ciliopathies
Cilia, 2012Co-Authors: Katarzyna Szymanska, Clare V. Logan, Ian R. Berry, Simon Rr Cousins, Helen Lindsay, Hussain Jafri, Yasmin Raashid, Saghira Malik-sharif, Bruce Castle, Mushtag AhmedAbstract:Meckel-Gruber syndrome (MKS) is an autosomal recessive lethal condition that is a ciliopathy. MKS has marked phenotypic variability and genetic heterogeneity, with mutations in nine genes identified as causative to date. Families diagnosed with Meckel-Gruber syndrome were recruited for research studies following informed consent. DNA samples were analyzed by microsatellite genotyping and direct Sanger sequencing. We now report the genetic analyses of 87 individuals from 49 consanguineous and 19 non-consanguineous families in an unselected cohort with reported MKS, or an associated severe ciliopathy in a kindred. Linkage and/or direct sequencing were prioritized for seven MKS genes (MKS1, TMEM216, TMEM67/MKS3, RPGRIP1L, CC2D2A, CEP290 and TMEM237) selected on the basis of reported frequency of mutations or ease of analysis. We have identified biallelic mutations in 39 individuals, of which 13 mutations are novel and previously unreported. We also confirm general genotype-phenotype correlations. TMEM67 was the most frequently mutated gene in this cohort, and we confirm two founder splice-site mutations (c.1546 + 1 G > A and c.870-2A > G) in families of Pakistani ethnic origin. In these families, we have also identified two separate founder mutations for RPGRIP1L (c. 1945 C > T p.R649X) and CC2D2A (c. 3540delA p.R1180SfsX6). Two missense mutations in TMEM67 (c. 755 T > C p.M252T, and c. 1392 C > T p.R441C) are also probable founder mutations. These findings will contribute to improved genetic diagnosis and carrier testing for affected families, and imply the existence of further genetic heterogeneity in this syndrome.
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Molecular Genetics and Pathogenic Mechanisms for the Severe Ciliopathies: Insights into Neurodevelopment and Pathogenesis of Neural Tube Defects
Molecular Neurobiology, 2011Co-Authors: Clare V. Logan, Zakia Abdel-hamed, Colin A. JohnsonAbstract:Meckel–Gruber syndrome (MKS) is a severe autosomal recessively inherited disorder characterized by developmental defects of the central nervous system that comprise neural tube defects that most commonly present as occipital encephalocele. MKS is considered to be the most common syndromic form of neural tube defect. MKS is genetically heterogeneous with six known disease genes: MKS1 , MKS2/TMEM216 , MKS3/TMEM67 , RPGRIP1L , CEP290 , and CC2D2A with the encoded proteins all implicated in the correct function of primary cilia. Primary cilia are microtubule-based organelles that project from the apical surface of most epithelial cell types. Recent progress has implicated the involvement of cilia in the Wnt and Shh signaling pathways and has led to an understanding of their role in normal mammalian neurodevelopment. The aim of this review is to provide an overview of the molecular genetics of the human disorder, and to assess recent insights into the etiology and molecular cell biology of severe ciliopathies from mammalian animal models of MKS.
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Defects in non-canonical Wnt signalling and actin cytoskeleton remodelling as pathogenic mechanisms in Meckel–Gruber syndrome
International Journal of Developmental Neuroscience, 2010Co-Authors: Colin A. Johnson, Clare V. Logan, Gabrielle Wheway, Zakia Abdelhamed, Matthew AdamsAbstract:Primary cilia are mechano- and chemosensory organelles that have a fundamental role in regulating embryogenesis. Inherited disorders that involve aberrant ciliary structure or function are now known as “ciliopathies”, and they invariably present with cystic kidney dysplasia. Meckel–Gruber syndrome (MKS) is a pleiotropic ciliopathy characterized by severe defects in neurodevelopment that include occipital encephalocele, hydrocephaly and severe neural tube defects. Several MKS genes are now known, including MKS1 and MKS3/TMEM67, encoding the proteins MKS1 and meckelin, a novel receptor. Remarkably, MKS is allelic and overlaps in phenotype with the neurodevelopmental disorder Joubert syndrome (JS), with some of the causative genes implicated in regulation of the Hedgehog signalling pathway. However, our recent work has suggested a role for meckelin and some other MKS proteins in modulating non-canonical Wnt signalling and remodelling the actin cytoskeleton. Meckelin is localized at the apical cell surface, basal bodies and ciliary axoneme of ciliated cell lines and tissues, but also interacts with other MKS proteins and the actin-binding proteins nesprin-2 and filamin A. Loss of expression of MKS genes following RNAi-mediated knockdown or in MKS patient fibroblasts: (1) prevents the movement of the basal body to the apical cell surface prior to ciliogenesis; (2) causes hyperactivation of the small GTPase RhoA and Dishevelled, both implicated in the control of apical docking of basal bodies and planar polarization of epithelial cells; and (3) remodels the actin cytoskeleton. These findings are reiterated in the Mks3/Tmem67 knock-out mouse model of MKS/JS. In contrast, MKS1 is implicated in constraining canonical Wnt signalling. These findings therefore underline the critical role of MKS proteins in ciliogenesis and regulation of Wnt signalling, through interactions with apical cell surface proteins associated with the actin cytoskeleton and implicated in basal body docking.
Ted Powers - One of the best experts on this subject based on the ideXlab platform.
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Accounting for strain-specific differences during RTG target gene regulation in Saccharomyces cerevisiae.
FEMS yeast research, 2006Co-Authors: Ivanka Dilova, Ted PowersAbstract:Mitochondrial dysfunction results in the expression, via the retrograde response pathway, of a concise set of genes (RTG target genes) that encode enzymes involved in the anapleurotic production of alpha-ketoglutarate. Inhibiting the rapamycin-sensitive TOR kinases, important regulators of cell growth, similarly results in RTG target gene expression under rich nutrient conditions. Retrograde and TOR-dependent regulation of RTG target genes requires a number of shared components, including the heterodimeric bZip/HLH transcription factors Rtg1p and Rtg3p, as well as their upstream regulator MKS1p. Two unresolved discrepancies exist with regard to the mechanism of RTG target gene control: (1) deletion of MKS1 results in constitutive expression of RTG target genes in most but not all strain backgrounds; and (2) RTG target gene expression has been correlated with both decreased as well as increased Rtg3p phosphorylation. Here we have addressed both of these issues. First, we demonstrate that the MKS1 deletion strain used in a previous study by Shamji and coworkers contains a nonsense mutation within codon Ser 231 in RTG3 that likely accounts for the inactivity of the RTG system in this strain. Second, we confirm results by Butow and coworkers that Rtg3p is dephosphorylated as a primary response to induction of the pathway. Hyper-phosphorylation of this protein appears to be a secondary consequence of rapamycin treatment and is influenced both by strain background as well as by specific supplied nutrients. That hyper-phosphorylation of Rtg3p is also caused by heat shock suggests that it may reflect a more generalized response to cell stress. Together these results contribute toward a uniform view of RTG target gene regulation.
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MKS1 in concert with tor signaling negatively regulates rtg target gene expression in s cerevisiae
Current Biology, 2002Co-Authors: Ivanka Dilova, Chingyi Chen, Ted PowersAbstract:The target of rapamycin (TOR) signaling pathway allows eukaryotic cells to regulate their growth in response to nutritional cues [1, 2]. In S. cerevisiae , TOR controls the expression of genes involved in several nutrient-responsive biosynthetic pathways [3–7]. In particular, we have demonstrated that TOR negatively regulates a concise cluster of genes (termed RTG target genes) that encode mitochondrial and peroxisomal enzymes required for de novo amino acid biosynthesis [7]. TOR acts in part by regulating the subcellular localization of the Rtg1/Rtg3 transcription factor complex. Nuclear entry of this complex requires the cytoplasmic protein Rtg2, whose precise function has remained ill defined. Here we establish that the likely role of Rtg2 is to antagonize the activity of another protein, MKS1, which we demonstrate is itself a negative regulator of RTG target gene activation. Results of epistasis analyses suggest that Rtg2 and MKS1 act downstream of TOR and upstream of Rtg1 and Rtg3. Moreover, we find that MKS1 phosphorylation responds to TOR as well as to each of the Rtg1-Rtg3 proteins, indicative of complex regulation within this branch of TOR signaling. In addition to RTG target genes, microarray analysis reveals robust expression of lysine biosynthetic genes in MKS1 Δ cells, which depends on a functional RTG pathway. This latter result provides a molecular explanation for the previous identification of MKS1 as LYS80 , a negative regulator of lysine biosynthesis [8].
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MKS1 in Concert with TOR Signaling Negatively Regulates RTG Target Gene Expression in S. cerevisiae
Current biology : CB, 2002Co-Authors: Ivanka Dilova, Chingyi Chen, Ted PowersAbstract:The target of rapamycin (TOR) signaling pathway allows eukaryotic cells to regulate their growth in response to nutritional cues. In S. cerevisiae, TOR controls the expression of genes involved in several nutrient-responsive biosynthetic pathways. In particular, we have demonstrated that TOR negatively regulates a concise cluster of genes (termed RTG target genes) that encode mitochondrial and peroxisomal enzymes required for de novo amino acid biosynthesis. TOR acts in part by regulating the subcellular localization of the Rtg1/Rtg3 transcription factor complex. Nuclear entry of this complex requires the cytoplasmic protein Rtg2, whose precise function has remained ill defined. Here we establish that the likely role of Rtg2 is to antagonize the activity of another protein, MKS1, which we demonstrate is itself a negative regulator of RTG target gene activation. Results of epistasis analyses suggest that Rtg2 and MKS1 act downstream of TOR and upstream of Rtg1 and Rtg3. Moreover, we find that MKS1 phosphorylation responds to TOR as well as to each of the Rtg1-Rtg3 proteins, indicative of complex regulation within this branch of TOR signaling. In addition to RTG target genes, microarray analysis reveals robust expression of lysine biosynthetic genes in MKS1Delta cells, which depends on a functional RTG pathway. This latter result provides a molecular explanation for the previous identification of MKS1 as LYS80, a negative regulator of lysine biosynthesis [8].