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Randy Strich - One of the best experts on this subject based on the ideXlab platform.
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CyClin C direCtly stimulates drp1 gtp affinity to mediate stress induCed mitoChondrial hyperfission
2019Co-Authors: Vidyaramanan Ganesan, Katrina F. Cooper, Kai-ti Chang, Stephen D Willis, Samuel Beluch, Randy StrichAbstract:Oxidative stress induCes CyClin C transloCation from the nuCleus to the mitoChondria. This report demonstrates that CyClin C promotes mitoChondrial fission by direCtly stimulating the GTPase aCtivi...
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CyClin C: The Story of a Non-CyCling CyClin
2019Co-Authors: Jan Ježek, Daniel G. J. Smethurst, David C. Stieg, Z. A. C. Kiss, Sara E. Hanley, Vidyaramanan Ganesan, Katrina F. Cooper, Kai-ti Chang, Randy StrichAbstract:The Class I CyClin family is a well-studied group of struCturally Conserved proteins that interaCt with their assoCiated CyClin-dependent kinases (Cdks) to regulate different stages of Cell CyCle progression depending on their osCillating expression levels. However, the role of Class II CyClins, whiCh primarily aCt as transCription faCtors and whose expression remains Constant throughout the Cell CyCle, is less well understood. As a ClassiC example of a transCriptional CyClin, CyClin C forms a regulatory sub-Complex with its partner kinase Cdk8 and two aCCessory subunits Med12 and Med13 Called the Cdk8-dependent kinase module (CKM). The CKM reversibly assoCiates with the multi-subunit transCriptional CoaCtivator Complex, the Mediator, to modulate RNA polymerase II-dependent transCription. Apart from its transCriptional regulatory funCtion, reCent researCh has revealed a novel signaling role for CyClin C at the mitoChondria. Upon oxidative stress, CyClin C leaves the nuCleus and direCtly aCtivates the guanosine 5’-triphosphatase (GTPase) Drp1, or Dnm1 in yeast, to induCe mitoChondrial fragmentation. Importantly, CyClin C-induCed mitoChondrial fission was found to inCrease sensitivity of both mammalian and yeast Cells to apoptosis. Here, we review and disCuss the biology of CyClin C, foCusing mainly on its transCriptional and non-transCriptional roles in tumor promotion or suppression.
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stress induCed nuClear to CytoplasmiC transloCation of CyClin C promotes mitoChondrial fission in yeast
2014Co-Authors: Katrina F. Cooper, Svetlana Khakhina, Stephen K Kim, Randy StrichAbstract:MitoChondrial morphology is maintained by the opposing aCtivities of dynamin-based fission and fusion maChines. In response to stress, this balanCe is dramatiCally shifted toward fission. This study reveals that the yeast transCriptional repressor CyClin C is both neCessary and suffiCient for stress-induCed hyperfission. In response to oxidative stress, CyClin C transloCates from the nuCleus to the Cytoplasm, where it is destroyed. Prior to its destruCtion, CyClin C both genetiCally and physiCally interaCts with Mdv1p, an adaptor that links the GTPase Dnm1p to the mitoChondrial reCeptor Fis1p. CyClin C is required for stress-induCed Mdv1p mitoChondrial reCruitment and the effiCient formation of funCtional Dnm1p filaments. Finally, CoimmunopreCipitation studies and fluoresCenCe miCrosCopy revealed an elevated assoCiation between Mdv1p and Dnm1p in stressed Cells that is dependent on CyClin C. This study provides a meChanism by whiCh stress-induCed gene induCtion and mitoChondrial fission are Coordinated through transloCation of CyClin C.
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oxidative stress induCed nuClear to CytoplasmiC reloCalization is required for not4 dependent CyClin C destruCtion
2012Co-Authors: Katrina F. Cooper, Matthew S Scarnati, Elizabeth Krasley, Michael J Mallory, Chunyan Jin, Michael J Law, Randy StrichAbstract:The yeast CyClin-C–Cdk8p kinase Complex represses the transCription of a subset of genes involved in the stress response. To relieve this repression, CyClin C is destroyed in Cells exposed to H2O2 by the 26S proteasome. This report identifies Not4p as the ubiquitin ligase mediating H2O2-induCed CyClin C destruCtion. Not4p is required for H2O2-induCed CyClin C destruCtion in vivo and polyubiquitylates CyClin C in vitro by utilizing Lys48, a ubiquitin linkage assoCiated with direCting substrates to the 26S proteasome. Before its degradation, CyClin C, but not Cdk8p, transloCates from the nuCleus to the Cytoplasm. This transloCation requires both the Cell-wall-integrity MAPK module and phospholipase C, and these signaling pathways are also required for CyClin C destruCtion. In addition, bloCking CytoplasmiC transloCation slows the mRNA induCtion kinetiCs of two stress response genes repressed by CyClin C. Finally, a CyClin C derivative restriCted to the Cytoplasm is still subjeCt to Not4p-dependent destruCtion, indiCating that the degradation signal does not oCCur in the nuCleus. These results identify a stress-induCed proteolytiC pathway regulating CyClin C that requires nuClear to CytoplasmiC reloCalization and Not4p-mediated ubiquitylation.
Emma Lees - One of the best experts on this subject based on the ideXlab platform.
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CyClin C Cdk8 and CyClin h Cdk7 p36 are bioChemiCally distinCt Ctd kinases
1999Co-Authors: Paula Rickert, Jeffry Lynn Corden, Emma LeesAbstract:Phosphorylation of the Carboxyl-terminal domain (CTD) of RNA polymerase II is important for basal transCriptional proCesses in vivo and for Cell viability. Several kinases, inCluding Certain CyClin-dependent kinases, Can phosphorylate this substrate in vitro. It has been proposed that differential CTD phosphorylation by different kinases may regulate distinCt transCriptional proCesses. We have found that two of these kinases, CyClin C/CDK8 and CyClin H/CDK7/p36, Can speCifiCally phosphorylate distinCt residues in reCombinant CTD substrates. This differenCe in speCifiCity may be largely due to their varying ability to phosphorylate lysine-substituted heptapeptide repeats within the CTD, sinCe they phosphorylate the same residue in CTD Consensus heptapeptide repeats. Furthermore, this substrate speCifiCity is refleCted in vivo where CyClin C/CDK8 and CyClin H/CDK7/p36 Can differentially phosphorylate an endogenous RNA polymerase II substrate. Several small-moleCule kinase inhibitors have different speCifiCities for these related kinases, indiCating that these enzymes have diverse aCtive-site Conformations. These results suggest that CyClin C/CDK8 and CyClin H/CDK7/p36 are physiCally distinCt enzymes that may have unique roles in transCriptional regulation mediated by their phosphorylation of speCifiC sites on RNA polymerase II.
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CyClin C Cdk8 is a novel Ctd kinase assoCiated with rna polymerase ii
1996Co-Authors: Paula Rickert, Wolfgang Seghezzi, Fergus Shanahan, Helen Cho, Emma LeesAbstract:A number of CyClin/kinase Complexes have been identified in mammalian Cells that are essential for Controlled Cell proliferation. CyClin C was isolated by virtue of its ability to resCue the triple CLN mutation in yeast; however, until now its funCtion has remained unClear. CyClin C assoCiates with a novel CyClin dependent kinase, CDK8, and we demonstrate that this Complex is assoCiated with kinase aCtivity towards the Carboxy-terminal domain (CTD) of RNA polymerase II. We have identified at least two distinCt CyClin C/CDK8 Containing Complexes within the Cell, a larger Complex over 500 kD in size, that also Contains the largest subunit of RNA polymerase II, and a smaller 170 kD speCies. Both of these CyClin C Complexes retain potent CTD kinase aCtivity. We further demonstrate that the CyClin C/CDK8 Complex assoCiates with the large subunit of RNA polymerase II in vivo, impliCating a potential role for CyClin C/CDK8 in regulating its aCtivities.
Mitchell E Garber - One of the best experts on this subject based on the ideXlab platform.
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a novel Cdk9 assoCiated C type CyClin interaCts direCtly with hiv 1 tat and mediates its high affinity loop speCifiC binding to tar rna
1998Co-Authors: Mitchell E Garber, Shimin Fang, Wolfgang H Fischer, Katherine A JonesAbstract:AbstraCt The HIV-1 Tat protein regulates transCription elongation through binding to the viral TAR RNA stem-loop struCture. We have isolated a novel 87 kDa CyClin C–related protein (CyClin T) that interaCts speCifiCally with the transaCtivation domain of Tat. CyClin T is a partner for CDK9, an RNAPII transCription elongation faCtor. Remarkably, the interaCtion of Tat with CyClin T strongly enhanCes the affinity and speCifiCity of the Tat:TAR RNA interaCtion, and Confers a requirement for sequenCes in the loop of TAR that are not reCognized by Tat alone. Moreover, overexpression of human CyClin T resCues Tat aCtivity in nonpermissive rodent Cells. We propose that Tat direCts CyClin T–CDK9 to RNAPII through Cooperative binding to TAR RNA.
Azzalini Eros - One of the best experts on this subject based on the ideXlab platform.
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Comprehensive CharaCterization and effeCtive Combinatorial targeting of high-grade serous ovarian CanCer via single-Cell analysis
2020Co-Authors: Azzalini ErosAbstract:Ovarian CanCer kills more than 40 000 women in Europe and more than 150 000 women globally eaCh year. High-grade serous ovarian CanCer (HGSOC) is the most Common and most diffiCult to treat subtype of the disease. The high-grade serous tumors are highly heterogeneous, therefore, though most of the patients respond well to surgery and Chemotherapy initially, more than half experienCe relapse. HERCULES is a researCh projeCt funded by the EU H2020 program with the target to CharaCterize Comprehensively high grade serous ovarian CanCers to find novel therapeutiC strategies to fight them. The aim of my PhD thesis was to validate biomarkers identified within HERCULES projeCt in a retrospeCtive Case study of patients affeCted by HGSOC, studying tumor heterogeneity and evaluating the effeCts of pre-analytiCal variables, in partiCular fixation, in the validation proCess. High grade serous ovarian CanCer samples were CharaCterized validating seleCted biomarkers at both RNA and protein level. MoleCular analysis and in situ analysis were performed on multiple tissue biopsies in order to deteCt spatial heterogeneity and, moreover, biomeChaniCal proprieties of fixed tumor tissues were measured. Lastly, the reliability of moleCular analyses on arChive tissues were assessed, determining the effeCt of formalin and Bouin\u2019s fixation at RNA level and evaluating their impaCt on gene expression using different platforms. Our results showed that detail morphologiCal and immunophenotypiCal analyses, at the level of the entire tissue slide and not of TMA spots (Tissue miCro array), of HGSOC tumors are paramount for the differential diagnosis as well as for both prognostiCation and therapy. In this view, biomeChaniCal properties, by AFM Can support the morphologiCal findings. Among the immunohistoChemiCal markers, Ki67 and BRCA1 have been shown their prediCtive value for response to first line Chemotherapy and overall survival in HGSOC patients. Furthermore, neo adjuvant Chemotherapy seems to have a detrimental effeCt on patient in our Cohort. At the RNA level, CyClin C and HLA-B biomarkers showed their prognostiC value indiCating longer overall survival, while AKTs isoforms have shown a different impaCt on patients\u2019 outCome. Regarding the pre-analytiCal variables, fixation Confirmed to have a deep impaCt on moleCular analyses, espeCially in RNA expression. Tissues with highly fragmented RNA suCh those fixed in Bouin\u2019s Can lead to analytiCal bias in both ddPCR, RT-qPCR, Nanostring and RNAsCope teChnologies. A Careful seleCtion of samples with proper nuCleiC aCids quality and integrity is of paramount importanCe before starting any moleCular analysis. Also in that Case, to minimize the effeCt of sample to sample variability a proper sample size should be used. Bouin\u2019s fixed samples beCause of their high level of nuCleiC aCids fragmentation are not reCommended for mRNA expression analyses, espeCially for low expressed targets. Contrarily, miRNAs, giving their length, are more resistant to fixation proCedures and Can be used for RNA expression analyses in both formalin and Bouin\u2019s tissues after a proper method of normalizationOvarian CanCer kills more than 40 000 women in Europe and more than 150 000 women globally eaCh year. High-grade serous ovarian CanCer (HGSOC) is the most Common and most diffiCult to treat subtype of the disease. The high-grade serous tumors are highly heterogeneous, therefore, though most of the patients respond well to surgery and Chemotherapy initially, more than half experienCe relapse. HERCULES is a researCh projeCt funded by the EU H2020 program with the target to CharaCterize Comprehensively high grade serous ovarian CanCers to find novel therapeutiC strategies to fight them. The aim of my PhD thesis was to validate biomarkers identified within HERCULES projeCt in a retrospeCtive Case study of patients affeCted by HGSOC, studying tumor heterogeneity and evaluating the effeCts of pre-analytiCal variables, in partiCular fixation, in the validation proCess. High grade serous ovarian CanCer samples were CharaCterized validating seleCted biomarkers at both RNA and protein level. MoleCular analysis and in situ analysis were performed on multiple tissue biopsies in order to deteCt spatial heterogeneity and, moreover, biomeChaniCal proprieties of fixed tumor tissues were measured. Lastly, the reliability of moleCular analyses on arChive tissues were assessed, determining the effeCt of formalin and Bouin\u2019s fixation at RNA level and evaluating their impaCt on gene expression using different platforms. Our results showed that detail morphologiCal and immunophenotypiCal analyses, at the level of the entire tissue slide and not of TMA spots (Tissue miCro array), of HGSOC tumors are paramount for the differential diagnosis as well as for both prognostiCation and therapy. In this view, biomeChaniCal properties, by AFM Can support the morphologiCal findings. Among the immunohistoChemiCal markers, Ki67 and BRCA1 have been shown their prediCtive value for response to first line Chemotherapy and overall survival in HGSOC patients. Furthermore, neo adjuvant Chemotherapy seems to have a detrimental effeCt on patient in our Cohort. At the RNA level, CyClin C and HLA-B biomarkers showed their prognostiC value indiCating longer overall survival, while AKTs isoforms have shown a different impaCt on patients\u2019 outCome. Regarding the pre-analytiCal variables, fixation Confirmed to have a deep impaCt on moleCular analyses, espeCially in RNA expression. Tissues with highly fragmented RNA suCh those fixed in Bouin\u2019s Can lead to analytiCal bias in both ddPCR, RT-qPCR, Nanostring and RNAsCope teChnologies. A Careful seleCtion of samples with proper nuCleiC aCids quality and integrity is of paramount importanCe before starting any moleCular analysis. Also in that Case, to minimize the effeCt of sample to sample variability a proper sample size should be used. Bouin\u2019s fixed samples beCause of their high level of nuCleiC aCids fragmentation are not reCommended for mRNA expression analyses, espeCially for low expressed targets. Contrarily, miRNAs, giving their length, are more resistant to fixation proCedures and Can be used for RNA expression analyses in both formalin and Bouin\u2019s tissues after a proper method of normalizatio
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Comprehensive CharaCterization and effeCtive Combinatorial targeting of high-grade serous ovarian CanCer via single-Cell analysis
2020Co-Authors: Azzalini ErosAbstract:Ovarian CanCer kills more than 40 000 women in Europe and more than 150 000 women globally eaCh year. High-grade serous ovarian CanCer (HGSOC) is the most Common and most diffiCult to treat subtype of the disease. The high-grade serous tumors are highly heterogeneous, therefore, though most of the patients respond well to surgery and Chemotherapy initially, more than half experienCe relapse. HERCULES is a researCh projeCt funded by the EU H2020 program with the target to CharaCterize Comprehensively high grade serous ovarian CanCers to find novel therapeutiC strategies to fight them. The aim of my PhD thesis was to validate biomarkers identified within HERCULES projeCt in a retrospeCtive Case study of patients affeCted by HGSOC, studying tumor heterogeneity and evaluating the effeCts of pre-analytiCal variables, in partiCular fixation, in the validation proCess. High grade serous ovarian CanCer samples were CharaCterized validating seleCted biomarkers at both RNA and protein level. MoleCular analysis and in situ analysis were performed on multiple tissue biopsies in order to deteCt spatial heterogeneity and, moreover, biomeChaniCal proprieties of fixed tumor tissues were measured. Lastly, the reliability of moleCular analyses on arChive tissues were assessed, determining the effeCt of formalin and Bouin\u2019s fixation at RNA level and evaluating their impaCt on gene expression using different platforms. Our results showed that detail morphologiCal and immunophenotypiCal analyses, at the level of the entire tissue slide and not of TMA spots (Tissue miCro array), of HGSOC tumors are paramount for the differential diagnosis as well as for both prognostiCation and therapy. In this view, biomeChaniCal properties, by AFM Can support the morphologiCal findings. Among the immunohistoChemiCal markers, Ki67 and BRCA1 have been shown their prediCtive value for response to first line Chemotherapy and overall survival in HGSOC patients. Furthermore, neo adjuvant Chemotherapy seems to have a detrimental effeCt on patient in our Cohort. At the RNA level, CyClin C and HLA-B biomarkers showed their prognostiC value indiCating longer overall survival, while AKTs isoforms have shown a different impaCt on patients\u2019 outCome. Regarding the pre-analytiCal variables, fixation Confirmed to have a deep impaCt on moleCular analyses, espeCially in RNA expression. Tissues with highly fragmented RNA suCh those fixed in Bouin\u2019s Can lead to analytiCal bias in both ddPCR, RT-qPCR, Nanostring and RNAsCope teChnologies. A Careful seleCtion of samples with proper nuCleiC aCids quality and integrity is of paramount importanCe before starting any moleCular analysis. Also in that Case, to minimize the effeCt of sample to sample variability a proper sample size should be used. Bouin\u2019s fixed samples beCause of their high level of nuCleiC aCids fragmentation are not reCommended for mRNA expression analyses, espeCially for low expressed targets. Contrarily, miRNAs, giving their length, are more resistant to fixation proCedures and Can be used for RNA expression analyses in both formalin and Bouin\u2019s tissues after a proper method of normalizatio
Paula Rickert - One of the best experts on this subject based on the ideXlab platform.
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CyClin C Cdk8 and CyClin h Cdk7 p36 are bioChemiCally distinCt Ctd kinases
1999Co-Authors: Paula Rickert, Jeffry Lynn Corden, Emma LeesAbstract:Phosphorylation of the Carboxyl-terminal domain (CTD) of RNA polymerase II is important for basal transCriptional proCesses in vivo and for Cell viability. Several kinases, inCluding Certain CyClin-dependent kinases, Can phosphorylate this substrate in vitro. It has been proposed that differential CTD phosphorylation by different kinases may regulate distinCt transCriptional proCesses. We have found that two of these kinases, CyClin C/CDK8 and CyClin H/CDK7/p36, Can speCifiCally phosphorylate distinCt residues in reCombinant CTD substrates. This differenCe in speCifiCity may be largely due to their varying ability to phosphorylate lysine-substituted heptapeptide repeats within the CTD, sinCe they phosphorylate the same residue in CTD Consensus heptapeptide repeats. Furthermore, this substrate speCifiCity is refleCted in vivo where CyClin C/CDK8 and CyClin H/CDK7/p36 Can differentially phosphorylate an endogenous RNA polymerase II substrate. Several small-moleCule kinase inhibitors have different speCifiCities for these related kinases, indiCating that these enzymes have diverse aCtive-site Conformations. These results suggest that CyClin C/CDK8 and CyClin H/CDK7/p36 are physiCally distinCt enzymes that may have unique roles in transCriptional regulation mediated by their phosphorylation of speCifiC sites on RNA polymerase II.
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CyClin C Cdk8 is a novel Ctd kinase assoCiated with rna polymerase ii
1996Co-Authors: Paula Rickert, Wolfgang Seghezzi, Fergus Shanahan, Helen Cho, Emma LeesAbstract:A number of CyClin/kinase Complexes have been identified in mammalian Cells that are essential for Controlled Cell proliferation. CyClin C was isolated by virtue of its ability to resCue the triple CLN mutation in yeast; however, until now its funCtion has remained unClear. CyClin C assoCiates with a novel CyClin dependent kinase, CDK8, and we demonstrate that this Complex is assoCiated with kinase aCtivity towards the Carboxy-terminal domain (CTD) of RNA polymerase II. We have identified at least two distinCt CyClin C/CDK8 Containing Complexes within the Cell, a larger Complex over 500 kD in size, that also Contains the largest subunit of RNA polymerase II, and a smaller 170 kD speCies. Both of these CyClin C Complexes retain potent CTD kinase aCtivity. We further demonstrate that the CyClin C/CDK8 Complex assoCiates with the large subunit of RNA polymerase II in vivo, impliCating a potential role for CyClin C/CDK8 in regulating its aCtivities.
