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Titus J. Boggon - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.
Methods in Molecular Biology, 2020Co-Authors: Oriana S. Fisher, Rong Zhang, Weizhi Liu, Titus J. BoggonAbstract:Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.
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The cerebral cavernous malformations proteins.
Oncotarget, 2015Co-Authors: Oriana S. Fisher, Titus J. BoggonAbstract:Inherited mutations in three genes lead to the familial form of Cerebral Cavernous Malformations (CCM). These vascular dysplasias most commonly occur in the brain, and manifest as dilated, mulberry-shaped lesions with a single endothelial layer. The consequences of these lesions can be leakage and sequelae such as focal neurological deficits, epilepsy, or hemorrhagic stroke. Until recently, however, the molecular basis for the acquisition of CCM disease was unknown. The three genes associated with CCM disease encode the proteins KRIT1/CCM1 (Krev interaction trapped 1/cerebral cavernous malformations 1), CCM2/malcavernin/OSM (cerebral cavernous malformations 2, osmosensing scaffold for MEKK3), and CCM3/PDCD10 (cerebral cavernous malformations 3/programmed cell death 10). Maintenance of a normal vasculature requires expression of all three proteins. Almost all of the discovered mutations in these genes result in truncations of their protein products, although some rare missense mutations have been found to encode misfolded protein [1]. The three proteins implicated in the disease were predicted to be distinct from one another in structure, but their molecular level architecture and many details of their normal function and protein-protein interaction networks were unknown. Therefore, to better understand the signaling processes that are affected by CCM disease it was necessary to address these questions from the ground up, starting at the atomic level [2]. The formation of a ‘CCM complex’ between KRIT1, CCM2 and CCM3 was previously suggested, but without targeted disruption of the interactions and selective probing of the functional consequences of disruption, the specific role(s) of heterotrimerization have been hard to define. We tackled these questions using a structure-directed approach. Our studies revealed the molecular basis of a preferred interaction site between KRIT1 and CCM2 [1] and the molecular basis for the interaction of CCM2 with CCM3 [3], thus providing the atomic-level framework for the CCM complex. When we crystallized CCM3, we found that it is of an unexpected fold encompassing two domains [4], the C-terminal of which directly interacts with a conserved motif in CCM2 [3]. Targeted disruption of the interaction between CCM2 and CCM3 has a number of functional consequences. CCM2 and CCM3 reciprocally stabilize one another; knockdown of either CCM2 or CCM3 results in severely reduced stability of the other protein. Importantly, the decreased stability can be rescued by re-expression of the wild-type protein but not protein that has been mutated at the binding site. Loss of either expressed protein also deleteriously impacts proliferation and network formation in endothelial cells. Interestingly, CCM3 expression can rescue proliferation in CCM2 depleted cells, but CCM2 cannot rescue expression in CCM3 depleted cells and the interaction surface between CCM2 and CCM3 must be preserved to rescue proliferation. Conversely, CCM2 is better able to rescue endothelial network formation than CCM3. An important role of the CCM complex in endothelial cells therefore seems to be stabilization of the CCM proteins, allowing them to achieve their overlapping but distinct roles. The CCM proteins also interact with other signaling proteins. We, and others, have shown the molecular basis for interactions of KRIT1 with the Rap1 small GTPase [5] and with the suppressor of integrin activation, ICAP1 [6]. We have also very recently discovered how CCM2 interacts with the MAP kinase kinase kinase, MEKK3. A previously uncharacterized N-terminal helical region of MEKK3 directly binds the C-terminal HHD (harmonin homology domain) of CCM2 [7]. Targeted disruption of this interaction was not observed to have an impact on MEKK3 catalytic activity, but did alter MEKK3 sub-cellular localization. Disruption of the CCM2:MEKK3 interaction also increases Rho/ROCK signaling, potentially implying that this upregulation may be related to the ability of CCM2 and MEKK3 to interact. In vivo, targeted disruption of the CCM2:MEKK3 interaction increased the permeability of the neurovasculature. This study therefore provides a link between CCM2, MEKK3, and the dysregulation of Rho/ROCK signaling that has previously been observed in CCM disease. Taken together, our recent studies, and those from other groups not mentioned here due to space and formatting constraints, suggest that the proteins of the CCM complex not only require one another for reciprocal stabilization, but also act as a platform for signal transduction. A more intricate understanding of how the CCM complex signaling platform is formed, how its formation is regulated, and how it interacts with binding partners are clearly required. Nonetheless, the molecular level reasons why CCM disease is so distinctly associated with loss of KRIT1, CCM2 and CCM3 are now becoming clearer.
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Structure and vascular function of MEKK3–cerebral cavernous malformations 2 complex
Nature communications, 2015Co-Authors: Oriana S. Fisher, Ya Zhang, Angeliki Louvi, Hanqiang Deng, Dou Liu, Rong Wei, Yong Deng, Fan Zhang, Benjamin E. Turk, Titus J. BoggonAbstract:Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 A cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho–ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease. The protein kinase MEKK3 interacts with CCM2, which is associated with the predominantly cerebrovascular CCM disease. Here the authors use structural, biochemical, cell biology and in vivotechniques to show that regulation of Rho signalling by the CCM2:MEKK3 complex is needed to maintain neurovascular integrity.
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CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation
Journal of Cell Biology, 2015Co-Authors: Kyle M. Draheim, Rong Zhang, Titus J. Boggon, Oriana S. Fisher, Giulia Villari, David A. CalderwoodAbstract:Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
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CCM2 ccm3 interaction stabilizes their protein expression and permits endothelial network formation
Journal of Cell Biology, 2015Co-Authors: Kyle M. Draheim, Rong Zhang, Titus J. Boggon, Oriana S. Fisher, Giulia Villari, David A. CalderwoodAbstract:Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
Oriana S. Fisher - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.
Methods in Molecular Biology, 2020Co-Authors: Oriana S. Fisher, Rong Zhang, Weizhi Liu, Titus J. BoggonAbstract:Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.
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The cerebral cavernous malformations proteins.
Oncotarget, 2015Co-Authors: Oriana S. Fisher, Titus J. BoggonAbstract:Inherited mutations in three genes lead to the familial form of Cerebral Cavernous Malformations (CCM). These vascular dysplasias most commonly occur in the brain, and manifest as dilated, mulberry-shaped lesions with a single endothelial layer. The consequences of these lesions can be leakage and sequelae such as focal neurological deficits, epilepsy, or hemorrhagic stroke. Until recently, however, the molecular basis for the acquisition of CCM disease was unknown. The three genes associated with CCM disease encode the proteins KRIT1/CCM1 (Krev interaction trapped 1/cerebral cavernous malformations 1), CCM2/malcavernin/OSM (cerebral cavernous malformations 2, osmosensing scaffold for MEKK3), and CCM3/PDCD10 (cerebral cavernous malformations 3/programmed cell death 10). Maintenance of a normal vasculature requires expression of all three proteins. Almost all of the discovered mutations in these genes result in truncations of their protein products, although some rare missense mutations have been found to encode misfolded protein [1]. The three proteins implicated in the disease were predicted to be distinct from one another in structure, but their molecular level architecture and many details of their normal function and protein-protein interaction networks were unknown. Therefore, to better understand the signaling processes that are affected by CCM disease it was necessary to address these questions from the ground up, starting at the atomic level [2]. The formation of a ‘CCM complex’ between KRIT1, CCM2 and CCM3 was previously suggested, but without targeted disruption of the interactions and selective probing of the functional consequences of disruption, the specific role(s) of heterotrimerization have been hard to define. We tackled these questions using a structure-directed approach. Our studies revealed the molecular basis of a preferred interaction site between KRIT1 and CCM2 [1] and the molecular basis for the interaction of CCM2 with CCM3 [3], thus providing the atomic-level framework for the CCM complex. When we crystallized CCM3, we found that it is of an unexpected fold encompassing two domains [4], the C-terminal of which directly interacts with a conserved motif in CCM2 [3]. Targeted disruption of the interaction between CCM2 and CCM3 has a number of functional consequences. CCM2 and CCM3 reciprocally stabilize one another; knockdown of either CCM2 or CCM3 results in severely reduced stability of the other protein. Importantly, the decreased stability can be rescued by re-expression of the wild-type protein but not protein that has been mutated at the binding site. Loss of either expressed protein also deleteriously impacts proliferation and network formation in endothelial cells. Interestingly, CCM3 expression can rescue proliferation in CCM2 depleted cells, but CCM2 cannot rescue expression in CCM3 depleted cells and the interaction surface between CCM2 and CCM3 must be preserved to rescue proliferation. Conversely, CCM2 is better able to rescue endothelial network formation than CCM3. An important role of the CCM complex in endothelial cells therefore seems to be stabilization of the CCM proteins, allowing them to achieve their overlapping but distinct roles. The CCM proteins also interact with other signaling proteins. We, and others, have shown the molecular basis for interactions of KRIT1 with the Rap1 small GTPase [5] and with the suppressor of integrin activation, ICAP1 [6]. We have also very recently discovered how CCM2 interacts with the MAP kinase kinase kinase, MEKK3. A previously uncharacterized N-terminal helical region of MEKK3 directly binds the C-terminal HHD (harmonin homology domain) of CCM2 [7]. Targeted disruption of this interaction was not observed to have an impact on MEKK3 catalytic activity, but did alter MEKK3 sub-cellular localization. Disruption of the CCM2:MEKK3 interaction also increases Rho/ROCK signaling, potentially implying that this upregulation may be related to the ability of CCM2 and MEKK3 to interact. In vivo, targeted disruption of the CCM2:MEKK3 interaction increased the permeability of the neurovasculature. This study therefore provides a link between CCM2, MEKK3, and the dysregulation of Rho/ROCK signaling that has previously been observed in CCM disease. Taken together, our recent studies, and those from other groups not mentioned here due to space and formatting constraints, suggest that the proteins of the CCM complex not only require one another for reciprocal stabilization, but also act as a platform for signal transduction. A more intricate understanding of how the CCM complex signaling platform is formed, how its formation is regulated, and how it interacts with binding partners are clearly required. Nonetheless, the molecular level reasons why CCM disease is so distinctly associated with loss of KRIT1, CCM2 and CCM3 are now becoming clearer.
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Structure and vascular function of MEKK3–cerebral cavernous malformations 2 complex
Nature communications, 2015Co-Authors: Oriana S. Fisher, Ya Zhang, Angeliki Louvi, Hanqiang Deng, Dou Liu, Rong Wei, Yong Deng, Fan Zhang, Benjamin E. Turk, Titus J. BoggonAbstract:Cerebral cavernous malformations 2 (CCM2) loss is associated with the familial form of CCM disease. The protein kinase MEKK3 (MAP3K3) is essential for embryonic angiogenesis in mice and interacts physically with CCM2, but how this interaction is mediated and its relevance to cerebral vasculature are unknown. Here we report that Mekk3 plays an intrinsic role in embryonic vascular development. Inducible endothelial Mekk3 knockout in neonatal mice is lethal due to multiple intracranial haemorrhages and brain blood vessels leakage. We discover direct interaction between CCM2 harmonin homology domain (HHD) and the N terminus of MEKK3, and determine a 2.35 A cocrystal structure. We find Mekk3 deficiency impairs neurovascular integrity, which is partially dependent on Rho–ROCK signalling, and that disruption of MEKK3:CCM2 interaction leads to similar neurovascular leakage. We conclude that CCM2:MEKK3-mediated regulation of Rho signalling is required for maintenance of neurovascular integrity, unravelling a mechanism by which CCM2 loss leads to disease. The protein kinase MEKK3 interacts with CCM2, which is associated with the predominantly cerebrovascular CCM disease. Here the authors use structural, biochemical, cell biology and in vivotechniques to show that regulation of Rho signalling by the CCM2:MEKK3 complex is needed to maintain neurovascular integrity.
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CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation
Journal of Cell Biology, 2015Co-Authors: Kyle M. Draheim, Rong Zhang, Titus J. Boggon, Oriana S. Fisher, Giulia Villari, David A. CalderwoodAbstract:Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
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CCM2 ccm3 interaction stabilizes their protein expression and permits endothelial network formation
Journal of Cell Biology, 2015Co-Authors: Kyle M. Draheim, Rong Zhang, Titus J. Boggon, Oriana S. Fisher, Giulia Villari, David A. CalderwoodAbstract:Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
Ute Felbor - One of the best experts on this subject based on the ideXlab platform.
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Fibronectin rescues aberrant phenotype of endothelial cells lacking either CCM1, CCM2 or CCM3.
The FASEB Journal, 2020Co-Authors: Konrad Schwefel, Stefanie Spiegler, Bettina C. Kirchmaier, Patricia K. E. Dellweg, Christiane D. Much, Jan Pané-farré, Tim M. Strom, Katharina Riedel, Ute Felbor, Matthias RathAbstract:Loss-of-function variants in CCM1/KRIT1, CCM2, and CCM3/PDCD10 are associated with autosomal dominant cerebral cavernous malformations (CCMs). CRISPR/Cas9-mediated CCM3 inactivation in human endothelial cells (ECs) has been shown to induce profound defects in cell-cell interaction as well as actin cytoskeleton organization. We here show that CCM3 inactivation impairs fibronectin expression and consequently leads to reduced fibers in the extracellular matrix. Despite the complexity and high molecular weight of fibronectin fibrils, our in vitro model allowed us to reveal that fibronectin supplementation restored aberrant spheroid formation as well as altered EC morphology, and suppressed actin stress fiber formation. Yet, fibronectin replacement neither enhanced the stability of tube-like structures nor inhibited the survival advantage of CCM3-/- ECs. Importantly, CRISPR/Cas9-mediated introduction of biallelic loss-of-function variants into either CCM1 or CCM2 demonstrated that the impaired production of a functional fibronectin matrix is a common feature of CCM1-, CCM2-, and CCM3-deficient ECs.
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First interchromosomal insertion in a patient with cerebral and spinal cavernous malformations.
Scientific Reports, 2020Co-Authors: Robin A Pilz, Konrad Schwefel, Stefanie Spiegler, Anja Weise, Thomas Liehr, Philipp Demmer, Andreas Spuler, Eberhard Gilberg, Christian A. Hübner, Ute FelborAbstract:Autosomal dominant cerebral cavernous malformations (CCM) are leaky vascular lesions that can cause epileptic seizures and stroke-like symptoms. Germline mutations in either CCM1, CCM2 or CCM3 are found in the majority of patients with multiple CCMs or a positive family history. Recently, the first copy number neutral inversion in CCM2 has been identified by whole genome sequencing in an apparently mutation-negative CCM family. We here asked the question whether further structural genomic rearrangements can be detected within NGS gene panel data of unsolved CCM cases. Hybrid capture NGS data of eight index patients without a pathogenic single nucleotide, indel or copy number variant were analyzed using two bioinformatics pipelines. In a 58-year-old male with multiple CCMs in his brain and spinal cord, we identified a 294 kb insertion within the coding sequence of CCM2. Fine mapping of the breakpoints, molecular cytogenetic studies, and multiplex ligation-dependent probe amplification verified that the structural variation was an inverted unbalanced insertion that originated from 1p12-p11.2. As this rearrangement disrupts exon 6 of CCM2 on 7p13, it was classified as pathogenic. Our study demonstrates that efforts to detect structural variations in known disease genes increase the diagnostic sensitivity of genetic analyses for well-defined Mendelian disorders.
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First large genomic inversion in familial cerebral cavernous malformation identified by whole genome sequencing.
neurogenetics, 2017Co-Authors: Stefanie Spiegler, Tim M. Strom, Matthias Rath, Axel Pagenstecher, Ulrich Sure, Sabine Hoffjan, Philipp Dammann, Ute FelborAbstract:Familial cerebral cavernous malformations (CCMs) predispose to seizures and hemorrhagic stroke. Molecular genetic analyses of CCM1, CCM2, and CCM3 result in a mutation detection rate of up to 98%. However, only whole genome sequencing (WGS) in combination with the Manta algorithm for analyses of structural variants revealed a heterozygous 24 kB inversion including exon 1 of CCM2 in a 12-year-old boy with familial CCMs. Its breakpoints were fine-mapped, and quantitative analysis on RNA confirmed reduced CCM2 expression. Our data expand the spectrum of CCM mutations and indicate that the existence of a fourth CCM disease gene is rather unlikely.
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high throughput sequencing of the entire genomic regions of ccm1 krit1 CCM2 and ccm3 pdcd10 to search for pathogenic deep intronic splice mutations in cerebral cavernous malformations
European Journal of Medical Genetics, 2017Co-Authors: Matthias Rath, Konrad Schwefel, Stefanie Spiegler, Sönke E. Jenssen, Dana Kleimeier, Christian Sperling, Lars Kaderali, Ute FelborAbstract:Abstract Cerebral cavernous malformations (CCM) are vascular lesions of the central nervous system that can cause headaches, seizures and hemorrhagic stroke. Disease-associated mutations have been identified in three genes: CCM1/KRIT1 , CCM2 and CCM3/PDCD10 . The precise proportion of deep-intronic variants in these genes and their clinical relevance is yet unknown. Here, a long-range PCR (LR-PCR) approach for target enrichment of the entire genomic regions of the three genes was combined with next generation sequencing (NGS) to screen for coding and non-coding variants. NGS detected all six CCM1/KRIT1 , two CCM2 and four CCM3/PDCD10 mutations that had previously been identified by Sanger sequencing. Two of the pathogenic variants presented here are novel. Additionally, 20 stringently selected CCM index cases that had remained mutation-negative after conventional sequencing and exclusion of copy number variations were screened for deep-intronic mutations. The combination of bioinformatics filtering and transcript analyses did not reveal any deep-intronic splice mutations in these cases. Our results demonstrate that target enrichment by LR-PCR combined with NGS can be used for a comprehensive analysis of the entire genomic regions of the CCM genes in a research context. However, its clinical utility is limited as deep-intronic splice mutations in CCM1/KRIT1 , CCM2 and CCM3/PDCD10 seem to be rather rare.
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High-throughput sequencing of the entire genomic regions of CCM1/KRIT1, CCM2 and CCM3/PDCD10 to search for pathogenic deep-intronic splice mutations in cerebral cavernous malformations.
European Journal of Medical Genetics, 2017Co-Authors: Matthias Rath, Konrad Schwefel, Stefanie Spiegler, Sönke E. Jenssen, Dana Kleimeier, Christian Sperling, Lars Kaderali, Ute FelborAbstract:Abstract Cerebral cavernous malformations (CCM) are vascular lesions of the central nervous system that can cause headaches, seizures and hemorrhagic stroke. Disease-associated mutations have been identified in three genes: CCM1/KRIT1 , CCM2 and CCM3/PDCD10 . The precise proportion of deep-intronic variants in these genes and their clinical relevance is yet unknown. Here, a long-range PCR (LR-PCR) approach for target enrichment of the entire genomic regions of the three genes was combined with next generation sequencing (NGS) to screen for coding and non-coding variants. NGS detected all six CCM1/KRIT1 , two CCM2 and four CCM3/PDCD10 mutations that had previously been identified by Sanger sequencing. Two of the pathogenic variants presented here are novel. Additionally, 20 stringently selected CCM index cases that had remained mutation-negative after conventional sequencing and exclusion of copy number variations were screened for deep-intronic mutations. The combination of bioinformatics filtering and transcript analyses did not reveal any deep-intronic splice mutations in these cases. Our results demonstrate that target enrichment by LR-PCR combined with NGS can be used for a comprehensive analysis of the entire genomic regions of the CCM genes in a research context. However, its clinical utility is limited as deep-intronic splice mutations in CCM1/KRIT1 , CCM2 and CCM3/PDCD10 seem to be rather rare.
Bruce P Briegleb - One of the best experts on this subject based on the ideXlab platform.
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Polar Radiation Budgets of the NCAR CCM3
Journal of Climate, 1998Co-Authors: Bruce P Briegleb, David H. BromwichAbstract:Present-day Arctic and Antarctic radiation budgets of the National Center for Atmospheric Research Community Climate Model version 3 (CCM3) are presented. The CCM3 simulation is from a prescribed and interannually varying sea surface temperature integration from January 1979 through August 1993. Earth Radiation Budget Experiment (ERBE) data from 1985 through 1989 are used for validation of top-of-atmosphere (TOA) absorbed shortwave radiation (ASR) and outgoing longwave radiation (OLR). Summer ASR in both polar regions is less than the observations by about 20 W m22. While the annual mean OLR in both polar regions is only 2‐ 3Wm 22 less than the ERBE data, the seasonal amplitude in OLR of 40 W m22 is smaller than the observed of 55‐60 W m22. The annual polar TOA radiation balance is smaller than observations by 5‐10 W m22. Compared to selected model and observational surface data, downward shortwave (SW) is too small by 50‐70 W m22 and downward longwave (LW) too large by 10‐30 W m 22. Surface downward LW in clear atmospheres is too small by 10‐20 W m22. The absence of sea-ice melt ponds results in 10‐20 W m22 too much SW absorption during early summer and from 20 to 40 W m22 too little during late summer. Summer cloud covers are reasonably well simulated, but winter low cloud cover is too high by 0.5‐0.7 compared to surface cloud observations. Comparison with limited satellite and in situ observations indicates cloud water path (CWP) is too high by about a factor of 2. While cloud particle sizes are approximately in the range of observed values, regional variation between maritime and continental droplet sizes is too strong over coastlines. Despite several improvements in CCM3 radiation physics, the accuracy of polar TOA annual radiation balance is degraded against the ERBE data compared to CCM2. Improvement in CCM3 polar radiation budgets will require improved simulation of CWP, clear sky LW, and sea ice albedo.
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the simulated earth radiation budget of the national center for atmospheric research community climate model CCM2 and comparisons with the earth radiation budget experiment erbe
Journal of Geophysical Research, 1994Co-Authors: J T Kiehl, James J. Hack, Bruce P BrieglebAbstract:This study documents the Earth radiation budget as simulated by the latest version of the National Center for Atmospheric Research community climate model (CCM2). The validation of the simulated Earth radiation budget is carried out through comparison with Earth Radiation Budget Experiment (ERBE) data. The study also documents the new cloud parameterization employed by CCM2. In general, the radiation budget of CCM2 is in better agreement with the ERBE data than previous versions of the CCM. In particular, the latitudinal structure of cloud radiative forcing is much improved over CCM1. The phase of the simulated seasonal cycle in top of atmosphere radiation quantities is well represented. In the tropics the magnitude is in good agreement with the observations from ERBE. In the northern hemisphere summer the model radiative properties contain a bias. In the shortwave spectral region the clouds reflect an insufficient amount of solar radiation, while in the longwave, too much radiation is emitted to space. These biases are associated with deficiencies in the cloud optical properties, namely, cloud liquid water path and cloud effective radius specification.
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Diurnal heating and cloudiness in the NCAR Community Climate Model (CCM2)
Journal of Climate, 1994Co-Authors: Ruth S. Lieberman, Byron A Boville, Conway B. Leovy, Bruce P BrieglebAbstract:Abstract In this paper, the authors assess the suitability of the heating fields in the latest version of the NCAR Community Climate Model (CCM2) for modeling the thermal forcing of atmospheric tides. Accordingly, diurnal variations of the surface pressure, outgoing longwave radiation, cloudiness, and precipitation are examined in the CCM2. The fields of radiative, sensible, and latent beating are similarly analyzed. These results are subjectively compared with available data. Equatorial diurnal surface pressure tides are fairly well simulated by CCM2. The model successfully reproduces the semidiurnal surface pressure tides; however, this may result in part from reflection of wave energy at the upper boundary. The CCM2 large-scale diurnal OLR is generally consistent with observations. The moist-convective scheme in the model is able to reproduce the diurnally varying cloudiness and precipitation patterns associated with land-sea contrasts; however, the amplitudes of CCM2 diurnal continental convective clo...
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Description of the NCAR community climate model (CCM2), June 1993. Technical note
1993Co-Authors: James J. Hack, Bruce P Briegleb, Byron A Boville, J T Kiehl, P.j. RaschAbstract:The report presents the details of the governing equations, physical parameterizations, and numerical algorithms defining the version of the NCAR Community Climate Model designated CCM2. The material provides an overview of the major model components, and the way in which they interact as the numerical integration proceeds. Details on the coding implementation, along with in-depth information on running the CCMe code, given in a separate technical report entitled User's Guide to NCAR CCMe (Bath et. a., 1992). As before, it is the authors objective that the model provide NCAR and the university research community with a reliable, well documented atmospheric general circulation model. In contrast with earlier versions of the CCM, however, the CCM2 represents a wholly new state-of-the-art atmospheric general circulation model, both functionally and in terms of its implementation. As such, the version of the CCM represents a major break from the evolutionary nature of earlier releases, and should provide the research community with a significantly improved atmospheric modeling capability.
Rong Zhang - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic Studies of the Cerebral Cavernous Malformations Proteins.
Methods in Molecular Biology, 2020Co-Authors: Oriana S. Fisher, Rong Zhang, Weizhi Liu, Titus J. BoggonAbstract:Cerebral cavernous malformations (CCM) are dysplasias that primarily occur in the neurovasculature, and are associated with mutations in three genes: KRIT1, CCM2, and PDCD10, the protein products of which are KRIT1 (Krev/Rap1 Interaction Trapped 1; CCM1, cerebral cavernous malformations 1), CCM2 (cerebral cavernous malformations 2; OSM, osmosensing scaffold for MEKK3), and CCM3 (cerebral cavernous malformations 3; PDCD10, programmed cell death 10). Until recently, these proteins were relatively understudied at the molecular level, and only three folded domains were documented. These were a band 4.1, ezrin, radixin, moesin (FERM), and an ankyrin repeat domain (ARD) in KRIT1, and a phosphotyrosine-binding (PTB) domain in CCM2. Over the past 10 years, a crystallographic approach has been used to discover a series of previously unidentified domains within the CCM proteins. These include a non-functional Nudix (or pseudonudix) domain in KRIT1, a harmonin homology domain (HHD) in CCM2, and dimerization and focal adhesion targeting (FAT)-homology domains within CCM3. Many of the roles of these domains have been revealed by structure-guided studies that show the CCM proteins can directly interact with one another to form a signaling scaffold, and that the "CCM complex" functions in signal transduction by interacting with other binding partners, including ICAP1, RAP1, and MEKK3. In this chapter, we describe the crystallization of CCM protein domains alone, and with their interaction partners.
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CCM2–CCM3 interaction stabilizes their protein expression and permits endothelial network formation
Journal of Cell Biology, 2015Co-Authors: Kyle M. Draheim, Rong Zhang, Titus J. Boggon, Oriana S. Fisher, Giulia Villari, David A. CalderwoodAbstract:Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
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CCM2 ccm3 interaction stabilizes their protein expression and permits endothelial network formation
Journal of Cell Biology, 2015Co-Authors: Kyle M. Draheim, Rong Zhang, Titus J. Boggon, Oriana S. Fisher, Giulia Villari, David A. CalderwoodAbstract:Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved “HP1” pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2–CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.
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Structural basis for the disruption of the Cerebral Cavernous Malformations 2 (CCM2) interaction with Krev Interaction Trapped 1 (KRIT1) by disease-associated mutations
Journal of Biological Chemistry, 2014Co-Authors: Oriana S. Fisher, Rong Zhang, Weizhi Liu, Amy L. Stiegler, Sondhya Ghedia, James L. Weber, Titus J. BoggonAbstract:Familial cerebral cavernous malformations (CCMs) are predominantly neurovascular lesions and are associated with mutations within the KRIT1, CCM2, and PDCD10 genes. The protein products of KRIT1 and CCM2 (Krev interaction trapped 1 (KRIT1) and cerebral cavernous malformations 2 (CCM2), respectively) directly interact with each other. Disease-associated mutations in KRIT1 and CCM2 mostly result in loss of their protein products, although rare missense point mutations can also occur. From gene sequencing of patients known or suspected to have one or more CCMs, we discover a series of missense point mutations in KRIT1 and CCM2 that result in missense mutations in the CCM2 and KRIT1 proteins. To place these mutations in the context of the molecular level interactions of CCM2 and KRIT1, we map the interaction of KRIT1 and CCM2 and find that the CCM2 phosphotyrosine binding (PTB) domain displays a preference toward the third of the three KRIT1 NPX(Y/F) motifs. We determine the 2.75 A co-crystal structure of the CCM2 PTB domain with a peptide corresponding to KRIT1(NPX(Y/F)3), revealing a Dab-like PTB fold for CCM2 and its interaction with KRIT1(NPX(Y/F)3). We find that several disease-associated missense mutations in CCM2 have the potential to interrupt the KRIT1-CCM2 interaction by destabilizing the CCM2 PTB domain and that a KRIT1 mutation also disrupts this interaction. We therefore provide new insights into the architecture of CCM2 and how the CCM complex is disrupted in CCM disease.
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Structural studies of cerebral cavernous malformations 2 (CCM2) reveal a folded helical domain at its C-terminus.
FEBS Letters, 2012Co-Authors: Oriana S. Fisher, Rong Zhang, James W. Murphy, Borries Demeler, Titus J. BoggonAbstract:Cerebral cavernous malformations (CCM) are neurovascular dysplasias affecting up to 0.5% of the population. Mutations in the CCM2 gene are associated with acquisition of CCM. We identify a previously uncharacterized domain at the C-terminus of CCM2 and determine its 1.9 A resolution crystal structure. Because this domain is structurally homologous to the N-terminal domain of harmonin, we name it the CCM2 harmonin-homology domain or HHD. CCM2 HHD is observed in two conformations, and we employ analytical ultracentrifugation to test its oligomerization. Additionally, CCM2 HHD contains an unusually long 13-residue 310 helix. This study provides the first structural characterization of CCM2.