The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Justin A. Macdonald - One of the best experts on this subject based on the ideXlab platform.
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Intrinsically Disordered N-Terminus of Calponin Homology-Associated Smooth Muscle Protein (CHASM) Interacts with the Calponin Homology Domain to Enable Tropomyosin Binding
Biochemistry, 2012Co-Authors: Justin A. Macdonald, Annegret Ulke-lemée, Hiroaki Ishida, Eric Ian Butler, Mona Chappellaz, Sarah E. Tulk, John K. Chik, Hans J. VogelAbstract:The Calponin Homology-associated smooth muscle (CHASM) protein plays an important adaptive role in smooth and skeletal muscle contraction. CHASM is associated with increased muscle contractility and can be localized to the contractile thin filament via its binding interaction with tropomyosin. We sought to define the structural basis for the interaction of CHASM with smooth muscle tropomyosin as a first step to understanding the contribution of CHASM to the contractile capacity of smooth muscle. Herein, we provide a structure-based model for the tropomyosin-binding Domain of CHASM using a combination of hydrogen/deuterium exchange mass spectrometry (HDX-MS) and NMR analyses. Our studies provide evidence that a portion of the N-terminal intrinsically disordered region forms intramolecular contacts with the globular C-terminal Calponin Homology (CH) Domain. Ultimately, cooperativeness between these structurally dissimilar regions is required for CHASM binding to smooth muscle tropomyosin. Furthermore, it ap...
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tropomyosin binding properties of the chasm protein are dependent upon its Calponin Homology Domain
FEBS Letters, 2010Co-Authors: Annegret Ulkelemee, Hiroaki Ishida, Meredith A. Borman, Alexandra Valderrama, Hans J. Vogel, Justin A. MacdonaldAbstract:Abstract The Calponin Homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the Calponin Homology (CH) Domain or expression of the CH Domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0 × 10−6 M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)–CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH Domain. Structured summary MINT- 7966126 : CHASM (uniprotkb: Q99LM3 ), Tropomyosin alpha (uniprotkb: P04268 ) and Tropomyosin beta (uniprotkb: P19352 ) physically interact (MI: 0915 ) by isothermal titration calorimetry (MI: 0065 ) MINT- 7966073 : CHASM (uniprotkb: Q99LM3 ) physically interacts (MI: 0914 ) with Tropomyosin beta (uniprotkb: P58776 ) and Tropomyosin alpha (uniprotkb: P58772 ) by pull down (MI: 0096 ) MINT- 7966187 : Tropomyosin alpha (uniprotkb: P04268 ) and Tropomyosin beta (uniprotkb: P19352 ) physically interact (MI: 0915 ) with CHASM (uniprotkb: Q99LM3 ) by pull down (MI: 0096 ) MINT- 7966090 : CHASM (uniprotkb: Q99LM3 ) binds (MI: 0407 ) to Tropomyosin alpha (uniprotkb: P04268 ) by pull down (MI: 0096 )
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Tropomyosin‐binding properties of the CHASM protein are dependent upon its Calponin Homology Domain
FEBS letters, 2010Co-Authors: Annegret Ulke-lemée, Hiroaki Ishida, Meredith A. Borman, Alexandra Valderrama, Hans J. Vogel, Justin A. MacdonaldAbstract:The Calponin Homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the Calponin Homology (CH) Domain or expression of the CH Domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0x10(-6) M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)-CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH Domain.
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The role of the Calponin Homology Domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction.
Molecular and cellular biochemistry, 2009Co-Authors: Meredith A. Borman, Tiffany A Freed, Timothy A J Haystead, Justin A. MacdonaldAbstract:In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) Calponin Homology (CH)-Domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of beta-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t(1/2)) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the Calponin Homology (CH)-Domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess "substrate trapping" properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC(20) in vitro, truncated SMTNL1 lacking the CH-Domain was ineffective. In summary, our findings suggest an important role for the CH-Domain in mediating the effects of SMTNL1 on smooth muscle contraction.
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the role of the Calponin Homology Domain of smoothelin like 1 smtnl1 in myosin phosphatase inhibition and smooth muscle contraction
Molecular and Cellular Biochemistry, 2009Co-Authors: Meredith A. Borman, Tiffany A Freed, Timothy A J Haystead, Justin A. MacdonaldAbstract:In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) Calponin Homology (CH)-Domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of β-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t1/2) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the Calponin Homology (CH)-Domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess “substrate trapping” properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC20 in vitro, truncated SMTNL1 lacking the CH-Domain was ineffective. In summary, our findings suggest an important role for the CH-Domain in mediating the effects of SMTNL1 on smooth muscle contraction.
Krishna M. G. Mallela - One of the best experts on this subject based on the ideXlab platform.
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The N-Terminal Flanking Region Modulates the Actin Binding Affinity of the Utrophin Tandem Calponin-Homology Domain.
Biochemistry, 2017Co-Authors: Surinder M. Singh, Swati Bandi, Krishna M. G. MallelaAbstract:Despite sharing a high degree of sequence similarity, the tandem Calponin-Homology (CH) Domain of utrophin binds to actin 30 times stronger than that of dystrophin. We have previously shown that this difference in actin binding affinity could not be ascribed to the differences in inter-CH-Domain linkers [Bandi, S., et al. (2015) Biochemistry 54, 5480–5488]. Here, we examined the role of the N-terminal flanking region. The utrophin tandem CH Domain contains a 27-residue flanking region before its CH1 Domain. We examined its effect by comparing the structure and function of full-length utrophin tandem CH Domain Utr(1–261) and its truncated Utr(28–261) construct. Both full-length and truncated constructs are monomers in solution, with no significant differences in their secondary or tertiary structures. Truncated construct Utr(28–261) binds to actin 30 times weaker than that of the full-length Utr(1–261), similar to that of the dystrophin tandem CH Domain with a much shorter flanking region. Deletion of the ...
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The C-terminal Domain of the utrophin tandem Calponin-Homology Domain appears to be thermodynamically and kinetically more stable than the full-length protein.
Biochemistry, 2014Co-Authors: Swati Bandi, Surinder M. Singh, Krishna M. G. MallelaAbstract:Domains are in general less stable than the corresponding full-length proteins. Human utrophin tandem Calponin-Homology (CH) Domain seems to be an exception. Reversible, equilibrium denaturant melts indicate that the isolated C-terminal Domain (CH2) is thermodynamically more stable than the tandem CH Domain. Thermal melts show that CH2 unfolds at a temperature higher than that at which the full-length protein unfolds. Stopped-flow kinetics indicates that CH2 unfolds slower than the full-length protein, indicating its higher kinetic stability. Thus, the utrophin tandem CH Domain may be one of the few proteins in which an isolated Domain is more stable than the corresponding full-length protein.
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The actin binding affinity of the utrophin tandem Calponin-Homology Domain is primarily determined by its N-terminal Domain.
Biochemistry, 2014Co-Authors: Surinder M. Singh, Steve J. Winder, Swati Bandi, Krishna M. G. MallelaAbstract:The structural determinants of the actin binding function of tandem Calponin-Homology (CH) Domains are poorly understood, particularly the role of individual Domains. We determined the actin binding affinity of isolated CH Domains from human utrophin and compared them with the affinity of the full-length tandem CH Domain. Traditional cosedimentation assays indicate that the C-terminal CH2 Domain binds to F-actin much weaker than the full-length tandem CH Domain. The N-terminal CH1 Domain is less stable and undergoes severe protein aggregation; therefore, traditional actin cosedimentation assays could not be used. To address this, we have developed a folding-upon-binding method. We refolded the CH1 Domain from its unfolded state in the presence of F-actin. This results in a competition between actin binding and aggregation. A differential centrifugation technique was used to distinguish actin binding from aggregation. Low-speed centrifugation pelleted CH1 aggregates, but not F-actin or its bound protein. Subsequent high-speed centrifugation resulted in the cosedimentation of bound CH1 along with F-actin. The CH1 Domain binds to F-actin with an affinity similar to that of the full-length tandem CH Domain, unlike the CH2 Domain. The actin binding cooperativity between the two Domains was quantitatively calculated from the association constants of the full-length tandem CH Domain and its CH Domains, and found to be much smaller than the association constant of the CH1 Domain alone. These results indicate that the actin binding affinity of the utrophin tandem CH Domain is primarily determined by its CH1 Domain, when compared to that of its CH2 Domain or the cooperativity between the two CH Domains.
Meredith A. Borman - One of the best experts on this subject based on the ideXlab platform.
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tropomyosin binding properties of the chasm protein are dependent upon its Calponin Homology Domain
FEBS Letters, 2010Co-Authors: Annegret Ulkelemee, Hiroaki Ishida, Meredith A. Borman, Alexandra Valderrama, Hans J. Vogel, Justin A. MacdonaldAbstract:Abstract The Calponin Homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the Calponin Homology (CH) Domain or expression of the CH Domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0 × 10−6 M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)–CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH Domain. Structured summary MINT- 7966126 : CHASM (uniprotkb: Q99LM3 ), Tropomyosin alpha (uniprotkb: P04268 ) and Tropomyosin beta (uniprotkb: P19352 ) physically interact (MI: 0915 ) by isothermal titration calorimetry (MI: 0065 ) MINT- 7966073 : CHASM (uniprotkb: Q99LM3 ) physically interacts (MI: 0914 ) with Tropomyosin beta (uniprotkb: P58776 ) and Tropomyosin alpha (uniprotkb: P58772 ) by pull down (MI: 0096 ) MINT- 7966187 : Tropomyosin alpha (uniprotkb: P04268 ) and Tropomyosin beta (uniprotkb: P19352 ) physically interact (MI: 0915 ) with CHASM (uniprotkb: Q99LM3 ) by pull down (MI: 0096 ) MINT- 7966090 : CHASM (uniprotkb: Q99LM3 ) binds (MI: 0407 ) to Tropomyosin alpha (uniprotkb: P04268 ) by pull down (MI: 0096 )
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Tropomyosin‐binding properties of the CHASM protein are dependent upon its Calponin Homology Domain
FEBS letters, 2010Co-Authors: Annegret Ulke-lemée, Hiroaki Ishida, Meredith A. Borman, Alexandra Valderrama, Hans J. Vogel, Justin A. MacdonaldAbstract:The Calponin Homology-associated smooth muscle protein (CHASM) can modulate muscle contractility, and its biological action may involve an interaction with the contractile filament. In this study, we demonstrate an interaction between CHASM and tropomyosin. Deletion constructs of CHASM were generated, and pull-down assays revealed a minimal deletion construct that could bind tropomyosin. Removal of the Calponin Homology (CH) Domain or expression of the CH Domain alone did not enable binding. The interaction was characterized by microcalorimetry with a dissociation constant of 2.0x10(-6) M. Confocal fluorescence microscopy also showed green fluorescent protein (GFP)-CHASM localization to filamentous structures within smooth muscle cells, and this targeting was dependent upon the CH Domain.
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The role of the Calponin Homology Domain of smoothelin-like 1 (SMTNL1) in myosin phosphatase inhibition and smooth muscle contraction.
Molecular and cellular biochemistry, 2009Co-Authors: Meredith A. Borman, Tiffany A Freed, Timothy A J Haystead, Justin A. MacdonaldAbstract:In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) Calponin Homology (CH)-Domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of beta-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t(1/2)) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the Calponin Homology (CH)-Domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess "substrate trapping" properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC(20) in vitro, truncated SMTNL1 lacking the CH-Domain was ineffective. In summary, our findings suggest an important role for the CH-Domain in mediating the effects of SMTNL1 on smooth muscle contraction.
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the role of the Calponin Homology Domain of smoothelin like 1 smtnl1 in myosin phosphatase inhibition and smooth muscle contraction
Molecular and Cellular Biochemistry, 2009Co-Authors: Meredith A. Borman, Tiffany A Freed, Timothy A J Haystead, Justin A. MacdonaldAbstract:In this study, we provide further insight into the contribution of the smoothelin-like 1 (SMTNL1) Calponin Homology (CH)-Domain on myosin light chain phosphatase (SMPP-1M) activity and smooth muscle contraction. SMTNL1 protein was shown to have inhibitory effects on SMPP-1M activity but not on myosin light chain kinase (MLCK) activity. Treatment of β-escin permeabilized rabbit, ileal smooth muscle with SMTNL1 had no effect on the time required to reach half-maximal force (t1/2) during stimulation with pCa6.3 solution. The addition of recombinant SMTNL1 protein to permeabilized, smooth muscle strips caused a significant decrease in contractile force. While the Calponin Homology (CH)-Domain was essential for maximal SMTNL1-associated relaxation, it alone did not cause significant changes in force. SMTNL1 was poorly dephosphorylated by PP-1C in the presence of the myosin targeting subunit (MYPT1), suggesting that phosphorylated SMTNL1 does not possess “substrate trapping” properties. Moreover, while full-length SMTNL1 could suppress SMPP-1M activity toward LC20 in vitro, truncated SMTNL1 lacking the CH-Domain was ineffective. In summary, our findings suggest an important role for the CH-Domain in mediating the effects of SMTNL1 on smooth muscle contraction.
Wen-ping Lin - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of leucine-rich repeats and Calponin Homology Domain containing 1 accelerates microglia-mediated neuroinflammation in a rat traumatic spinal cord injury model
Journal of neuroinflammation, 2020Co-Authors: Wen-kai Chen, Lin-juan Feng, Qiao-dan Liu, Pei-ya Cai, Pei-ru Zhang, Li-quan Cai, Nian-lai Huang, Wen-ping LinAbstract:Spinal cord injury (SCI) triggers the primary mechanical injury and secondary inflammation-mediated injury. Neuroinflammation-mediated insult causes secondary and extensive neurological damage after SCI. Microglia play a pivotal role in the initiation and progression of post-SCI neuroinflammation. To elucidate the significance of LRCH1 to microglial functions, we applied lentivirus-induced LRCH1 knockdown in primary microglia culture and tested the role of LRCH1 in microglia-mediated inflammatory reaction both in vitro and in a rat SCI model. We found that LRCH1 was downregulated in microglia after traumatic SCI. LRCH1 knockdown increased the production of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 after in vitro priming with lipopolysaccharide and adenosine triphosphate. Furthermore, LRCH1 knockdown promoted the priming-induced microglial polarization towards the pro-inflammatory inducible nitric oxide synthase (iNOS)-expressing microglia. LRCH1 knockdown also enhanced microglia-mediated N27 neuron death after priming. Further analysis revealed that LRCH1 knockdown increased priming-induced activation of p38 mitogen-activated protein kinase (MAPK) and Erk1/2 signaling, which are crucial to the inflammatory response of microglia. When LRCH1-knockdown microglia were adoptively injected into rat spinal cords, they enhanced post-SCI production of pro-inflammatory cytokines, increased SCI-induced recruitment of leukocytes, aggravated SCI-induced tissue damage and neuronal death, and worsened the locomotor function. Our study reveals for the first time that LRCH1 serves as a negative regulator of microglia-mediated neuroinflammation after SCI and provides clues for developing novel therapeutic approaches against SCI.
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Leucine rich repeats and Calponin Homology Domain containing 1 inhibits microglia-mediated neuroinflammation in a rat traumatic spinal cord injury model
2020Co-Authors: Wen-kai Chen, Lin-juan Feng, Qiao-dan Liu, Pei-ya Cai, Pei-ru Zhang, Li-quan Cai, Nian-lai Huang, Wen-ping LinAbstract:Abstract Background Spinal cord injury (SCI) triggers the primary mechanical injury and secondary inflammation-mediated injury. Neuroinflammation-mediated insult causes secondary and extensive neurological damage after SCI. Microglia play a pivotal role in the initiation and progression of post-SCI neuroinflammation. Methods To elucidate the significance of LRCH1 to microglial functions, we applied lentivirus-induced LRCH1 knockdown in primary microglia culture, and tested the role of LRCH1 in microglia-mediated inflammatory reaction both in vitro and in a rat SCI model. Results We found that LRCH1 was down-regulated in microglia after traumatic SCI. LRCH1 knockdown increased the production of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 after in vitro priming with lipopolysaccharide and adenosine triphosphate. Furthermore, LRCH1 knockdown promoted the priming-induced microglial polarization towards the pro-inflammatory inducible nitric oxide synthase (iNOS)-expressing microglia. LRCH1 knockdown also enhanced microglia-mediated N27 neuron death after priming. Further analysis revealed that LRCH1 knockdown increased priming-induced activation of p38 mitogen-activated protein kinase (MAPK) and Erk1/2 signaling, which are crucial to the inflammatory response of microglia. When LRCH1-knockdown microglia were adoptively injected into rat spinal cords, they enhanced post-SCI production of pro-inflammatory cytokines, increased SCI-induced recruitment of leukocytes, aggravated SCI-induced tissue damage and neuronal death, and worsened the locomotor function. Conclusion Our study reveals for the first time that LRCH1 serves as a negative regulator of microglia-mediated neuroinflammation after SCI, and provides clues for developing novel therapeutic approaches against SCI.
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Leucine rich repeats and Calponin Homology Domain containing 1 inhibits microglia-mediated neuroinflammation in a rat traumatic spinal cord injury model
2019Co-Authors: Wen-kai Chen, Lin-juan Feng, Qiao-dan Liu, Pei-ya Cai, Pei-ru Zhang, Li-quan Cai, Nian-lai Huang, Wen-ping LinAbstract:Abstract Background Spinal cord injury (SCI) triggers the primary mechanical injury and secondary inflammation-mediated injury. Neuroinflammation-mediated insult causes secondary and extensive neurological damage after SCI. Microglia play a pivotal role in the initiation and progression of post-SCI neuroinflammation.Methods To elucidate the significance of LRCH1 to microglial functions, we applied lentivirus-induced LRCH1 knockdown in primary microglia culture, and tested the role of LRCH1 in microglia-mediated inflammatory reaction both in vitro and in a rat SCI model.ResultsWe found that LRCH1 was down-regulated in microglia after traumatic SCI. LRCH1 knockdown increased the production of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 after in vitro priming with lipopolysaccharide and adenosine triphosphate. Furthermore, LRCH1 knockdown promoted the priming-induced microglial polarization towards the pro-inflammatory M1 type, as demonstrated by increased differentiation into inducible nitric oxide synthase (iNOS)+ microglia. LRCH1 knockdown also enhanced microglia-mediated N27 neuron death after priming. Further analysis revealed that LRCH1 knockdown increased priming-induced activation of p38 mitogen-activated protein kinase (MAPK) and Erk1/2 signaling, which are crucial for M1 polarization of microglia. When LRCH1-knockdown microglia were adoptively injected into rat spinal cords, they enhanced post-SCI production of pro-inflammatory cytokines, increased SCI-induced recruitment of leukocytes, aggravated SCI-induced tissue damage and neuronal death, and worsened the locomotor function.Conclusion Our study reveals for the first time that LRCH1 serves as a negative regulator of microglia-mediated neuroinflammation after SCI, and provides clues for developing novel therapeutic approaches against SCI.
Geert J. P. L. Kops - One of the best experts on this subject based on the ideXlab platform.
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Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling
Science (New York N.Y.), 2015Co-Authors: Yoshitaka Hiruma, Anastassis Perrakis, Eleonore Von Castelmur, Carlos Sacristan, Spyridon T. Pachis, Athanassios Adamopoulos, Timo E. F. Kuijt, Marcellus Ubbink, Geert J. P. L. KopsAbstract:Cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules through kinetochores and the spindle assembly checkpoint (SAC) is satisfied. We show that the amino-terminal localization module of the SAC protein kinase MPS1 (monopolar spindle 1) directly interacts with the HEC1 (highly expressed in cancer 1) Calponin Homology Domain in the NDC80 (nuclear division cycle 80) kinetochore complex in vitro, in a phosphorylation-dependent manner. Microtubule polymers disrupted this interaction. In cells, MPS1 binding to kinetochores or to ectopic NDC80 complexes was prevented by end-on microtubule attachment, independent of known kinetochore protein-removal mechanisms. Competition for kinetochore binding between SAC proteins and microtubules provides a direct and perhaps evolutionarily conserved way to detect a properly organized spindle ready for cell division.
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a tpr Domain containing n terminal module of mps1 is required for its kinetochore localization by aurora b
Journal of Cell Biology, 2013Co-Authors: Wilco Nijenhuis, Anastassis Perrakis, Eleonore Von Castelmur, Dene R. Littler, Valeria De Marco, Eelco Tromer, Mathijs Vleugel, Maria H.j. Van Osch, Berend Snel, Geert J. P. L. KopsAbstract:The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) Domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the Calponin Homology Domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR Domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.
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A TPR Domain–containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B
The Journal of cell biology, 2013Co-Authors: Wilco Nijenhuis, Anastassis Perrakis, Eleonore Von Castelmur, Dene R. Littler, Valeria De Marco, Eelco Tromer, Mathijs Vleugel, Maria H.j. Van Osch, Berend Snel, Geert J. P. L. KopsAbstract:The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) Domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the Calponin Homology Domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR Domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.
