The Experts below are selected from a list of 462 Experts worldwide ranked by ideXlab platform
Eckhard Jankowsky - One of the best experts on this subject based on the ideXlab platform.
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autoinhibitory interdomain interactions and subfamily specific extensions redefine the catalytic core of the human dead box protein ddx3
Journal of Biological Chemistry, 2016Co-Authors: Stephen N. Floor, Kendall J. Condon, Eckhard Jankowsky, Deepak Sharma, Jennifer A. DoudnaAbstract:DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 A resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.
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the ded1 ddx3 subfamily of dead box rna helicases
Critical Reviews in Biochemistry and Molecular Biology, 2014Co-Authors: Deepak Sharma, Eckhard JankowskyAbstract:AbstractIn eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.
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dead box protein assisted rna structure conversion towards and against thermodynamic equilibrium values
Journal of Molecular Biology, 2007Co-Authors: Quansheng Yang, Margaret E Fairman, Eckhard JankowskyAbstract:RNAs in biological processes often interconvert between defined structures. These RNA structure conversions are assisted by proteins and are frequently coupled to ATP hydrolysis. It is not well understood how proteins coordinate RNA structure conversions and which role ATP hydrolysis has in these processes. Here, we have investigated in vitro how the DEAD-box ATPase Ded1 facilitates RNA structure conversions in a simple model system. We find that Ded1 assists RNA structure conversions via two distinct pathways. One pathway requires ATP hydrolysis and involves the complete disassembly of the RNA strands. This pathway represents a kinetically controlled steady state between the RNA structures, which allows formation of less stable from more stable RNA conformations and thus RNA structure conversion against thermodynamic equilibrium values. The other pathway is ATP-independent and proceeds via multipartite intermediates that are stabilized by Ded1. Our results provide a basic mechanistic framework for protein-assisted RNA structure conversions that illuminates the role of ATP hydrolysis and reveal an unexpected diversity of pathways.
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the dead box protein ded1 unwinds rna duplexes by a mode distinct from translocating helicases
Nature Structural & Molecular Biology, 2006Co-Authors: Quansheng Yang, Eckhard JankowskyAbstract:Helicases unwind RNA or DNA duplexes and displace proteins from nucleic acids in an ATP-dependent fashion. To unwind duplexes, helicases typically load onto one of the two nucleic acid strands, usually at a single-stranded region, and then translocate on this strand in a unidirectional fashion, thereby displacing the complementary DNA or RNA. Here we show that the DEAD-box RNA helicase Ded1 unwinds duplexes in a different manner. Ded1 uses the single-stranded region to gain access to the duplex. Strand separation is directly initiated from the duplex region and no covalent connection between the single strand and the duplex region is required. This new type of helicase activity explains observations with other DEAD-box proteins and may be the prototype for duplex-unwinding reactions in RNA metabolism.
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discriminatory rnp remodeling by the dead box protein ded1
RNA, 2006Co-Authors: Heath A Bowers, Patricia A Maroney, Margaret E Fairman, Berthold Kastner, Reinhard Luhrmann, Timothy W Nilsen, Eckhard JankowskyAbstract:DExH/D proteins catalyze NTP-driven rearrangements of RNA and RNA-protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which a non-sequence specific DExH/D protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from a tightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the “inability” to actively displace other proteins from RNA can provide non-sequence specific DExH/D proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.
Josette Banroques - One of the best experts on this subject based on the ideXlab platform.
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the in silico identification of potential members of the ded1 ddx3 subfamily of dead box rna helicases from the protozoan parasite leishmania infantum and their analyses in yeast
Genes, 2021Co-Authors: Molka Mokdadi, Josette Banroques, Yosser Zina Abdelkrim, Emmeline Huvelle, Rafeh Oualha, Hilal Yeteralat, Ikram Guizani, Mourad Barhoumi, Kyle N TannerAbstract:DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.
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the dead box rna helicase ded1 from yeast is associated with the signal recognition particle and is regulated by srp21
bioRxiv, 2020Co-Authors: Kyle N Tanner, Molka Mokdadi, Emmeline Huvelle, Hilal Yeteralat, Naima Belgarehtouze, Josette BanroquesAbstract:The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation. It belongs to the DDX3 subfamily of proteins implicated in developmental and cell-cycle regulation. In vitro, the purified Ded1 protein is an ATP-dependent RNA binding protein and an RNA-dependent ATPase, but it lacks RNA substrate specificity and enzymatic regulation. Here we demonstrate by yeast genetics, in situ localization and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 with SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results open a new comprehension of the cellular role of Ded1 during translation.
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The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus
Nucleic Acids Research, 2014Co-Authors: Meriem Senissar, Josette Banroques, Agnès Le Saux, Naïma Belgareh-touzé, Céline Adam, N. Kyle TannerAbstract:The DEAD-box helicase Ded1 is an essential yeast protein that is closely related to mammalian DDX3 and to other DEAD-box proteins involved in developmental and cell cycle regulation. Ded1 is considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5 7-methylguanosine cap to the AUG start codon. We used IgG pull-down experiments, mass spectrom-etry analyses, genetic experiments, sucrose gradients , in situ localizations and enzymatic assays to show that Ded1 is a cap-associated protein that actively shuttles between the cytoplasm and the nucleus. NanoLC-MS/MS analyses of purified complexes show that Ded1 is present in both nuclear and cytoplasmic mRNPs. Ded1 physically interacts with purified components of the nuclear CBC and the cytoplasmic eIF4F complexes, and its enzymatic activity is stimulated by these factors. In addition, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes. We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.
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motif iii in superfamily 2 helicases helps convert the binding energy of atp into a high affinity rna binding site in the yeast dead box protein ded1
Journal of Molecular Biology, 2010Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Marc Dreyfus, Kyle N TannerAbstract:Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.
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a conserved phenylalanine of motif iv in superfamily 2 helicases is required for cooperative atp dependent binding of rna substrates in dead box proteins
Molecular and Cellular Biology, 2008Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Olivier Cordin, Kyle N TannerAbstract:We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-pi interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.
Kyle N Tanner - One of the best experts on this subject based on the ideXlab platform.
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the in silico identification of potential members of the ded1 ddx3 subfamily of dead box rna helicases from the protozoan parasite leishmania infantum and their analyses in yeast
Genes, 2021Co-Authors: Molka Mokdadi, Josette Banroques, Yosser Zina Abdelkrim, Emmeline Huvelle, Rafeh Oualha, Hilal Yeteralat, Ikram Guizani, Mourad Barhoumi, Kyle N TannerAbstract:DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.
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the dead box rna helicase ded1 from yeast is associated with the signal recognition particle and is regulated by srp21
bioRxiv, 2020Co-Authors: Kyle N Tanner, Molka Mokdadi, Emmeline Huvelle, Hilal Yeteralat, Naima Belgarehtouze, Josette BanroquesAbstract:The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation. It belongs to the DDX3 subfamily of proteins implicated in developmental and cell-cycle regulation. In vitro, the purified Ded1 protein is an ATP-dependent RNA binding protein and an RNA-dependent ATPase, but it lacks RNA substrate specificity and enzymatic regulation. Here we demonstrate by yeast genetics, in situ localization and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 with SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results open a new comprehension of the cellular role of Ded1 during translation.
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motif iii in superfamily 2 helicases helps convert the binding energy of atp into a high affinity rna binding site in the yeast dead box protein ded1
Journal of Molecular Biology, 2010Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Marc Dreyfus, Kyle N TannerAbstract:Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.
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a conserved phenylalanine of motif iv in superfamily 2 helicases is required for cooperative atp dependent binding of rna substrates in dead box proteins
Molecular and Cellular Biology, 2008Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Olivier Cordin, Kyle N TannerAbstract:We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-pi interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.
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the newly discovered q motif of dead box rna helicases regulates rna binding and helicase activity
The EMBO Journal, 2004Co-Authors: Olivier Cordin, Patrick Linder, Kyle N Tanner, Monique Doere, Josette BanroquesAbstract:DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs ∼17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group ∼17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis–Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
Toru Miyazaki - One of the best experts on this subject based on the ideXlab platform.
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death effector domain containing protein DEDD is required for uterine decidualization during early pregnancy in mice
Journal of Clinical Investigation, 2011Co-Authors: Mayumi Mori, Satoko Arai, Jun Kurokawa, Miwako Kitazume, Yutaka Osuga, Kaori Koga, Toru MiyazakiAbstract:During intrauterine life, the mammalian embryo survives via its physical connection to the mother. The uterine decidua, which differentiates from stromal cells after implantation in a process known as decidualization, plays essential roles in supporting embryonic growth before establishment of the placenta. Here we show that female mice lacking death effector domain–containing protein (DEDD) are infertile owing to unsuccessful decidualization. In uteri of DEDD–/– mice, development of the decidual zone and the surrounding edema after embryonic implantation was defective. This was subsequently accompanied by disintegration of implantation site structure, leading to embryonic death before placentation. Polyploidization, a hallmark of mature decidual cells, was attenuated in DEDD-deficient cells during decidualization. Such inefficient decidualization appeared to be caused by decreased Akt levels, since polyploidization was restored in DEDD-deficient decidual cells by overexpression of Akt. In addition, we showed that DEDD associates with and stabilizes cyclin D3, an important element in polyploidization, and that overexpression of cyclin D3 in DEDD-deficient cells improved polyploidization. These results indicate that DEDD is indispensable for the establishment of an adequate uterine environment to support early pregnancy in mice.
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Death effector domain–containing protein (DEDD) is required for uterine decidualization during early pregnancy in mice
Journal of Clinical Investigation, 2010Co-Authors: Mayumi Mori, Satoko Arai, Jun Kurokawa, Miwako Kitazume, Yutaka Osuga, Kaori Koga, Toru MiyazakiAbstract:During intrauterine life, the mammalian embryo survives via its physical connection to the mother. The uterine decidua, which differentiates from stromal cells after implantation in a process known as decidualization, plays essential roles in supporting embryonic growth before establishment of the placenta. Here we show that female mice lacking death effector domain–containing protein (DEDD) are infertile owing to unsuccessful decidualization. In uteri of DEDD–/– mice, development of the decidual zone and the surrounding edema after embryonic implantation was defective. This was subsequently accompanied by disintegration of implantation site structure, leading to embryonic death before placentation. Polyploidization, a hallmark of mature decidual cells, was attenuated in DEDD-deficient cells during decidualization. Such inefficient decidualization appeared to be caused by decreased Akt levels, since polyploidization was restored in DEDD-deficient decidual cells by overexpression of Akt. In addition, we showed that DEDD associates with and stabilizes cyclin D3, an important element in polyploidization, and that overexpression of cyclin D3 in DEDD-deficient cells improved polyploidization. These results indicate that DEDD is indispensable for the establishment of an adequate uterine environment to support early pregnancy in mice.
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death effector domain containing protein DEDD is an inhibitor of mitotic cdk1 cyclin b1
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Satoko Arai, Shino Nemoto, Toru Miyazaki, Edward K. Wakeland, Katsuhisa Miyake, Renate Voit, Ingrid GrummtAbstract:Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD−/−) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD−/− cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD−/− mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
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Death-effector domain-containing protein DEDD is an inhibitor of mitotic Cdk1/cyclin B1
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Satoko Arai, Shino Nemoto, Edward K. Wakeland, Katsuhisa Miyake, Renate Voit, Ingrid Grummt, Toru MiyazakiAbstract:Accumulating evidence has shown that many molecules, including some cyclin-dependent kinases (Cdks) and cyclins, as well as the death-effector domain (DED)-containing FADD, function for both apoptosis and cell cycle. Here we identified that DEDD, which also possesses the DED domain, acts as a novel inhibitor of the mitotic Cdk1/cyclin B1 complex. DEDD associates with mitotic Cdk1/cyclin B1 complexes via direct binding to cyclin B1 and reduces their function. In agreement, kinase activity of nuclear Cdk1/cyclin B1 in DEDD-null (DEDD−/−) embryonic fibroblasts is increased compared with that in DEDD+/+ cells, which results in accelerated mitotic progression, thus exhibiting a shortened G2/M stage. Interestingly, DEDD−/− cells also demonstrated decreased G1 duration, which perhaps enhanced the overall reduction in rRNA amounts and cell volume, primarily caused by the rapid termination of rRNA synthesis before cell division. Likewise, DEDD−/− mice show decreased body and organ weights relative to DEDD+/+ mice. Thus, DEDD is an impeder of cell mitosis, and its absence critically influences cell and body size via modulation of rRNA synthesis.
Patrick Linder - One of the best experts on this subject based on the ideXlab platform.
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motif iii in superfamily 2 helicases helps convert the binding energy of atp into a high affinity rna binding site in the yeast dead box protein ded1
Journal of Molecular Biology, 2010Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Marc Dreyfus, Kyle N TannerAbstract:Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.
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a conserved phenylalanine of motif iv in superfamily 2 helicases is required for cooperative atp dependent binding of rna substrates in dead box proteins
Molecular and Cellular Biology, 2008Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Olivier Cordin, Kyle N TannerAbstract:We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-pi interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.
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the newly discovered q motif of dead box rna helicases regulates rna binding and helicase activity
The EMBO Journal, 2004Co-Authors: Olivier Cordin, Patrick Linder, Kyle N Tanner, Monique Doere, Josette BanroquesAbstract:DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs ∼17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group ∼17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis–Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
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ded1p a dead box protein required for translation initiation in saccharomyces cerevisiae is an rna helicase
Journal of Biological Chemistry, 1999Co-Authors: Isabelle Iost, Marc Dreyfus, Patrick LinderAbstract:Abstract The Ded1 protein (Ded1p), a member of the DEAD-box family, has recently been shown to be essential for translation initiation in Saccharomyces cerevisiae. Here, we show that Ded1p purified from Escherichia coli has an ATPase activity, which is stimulated by various RNA substrates. Using an RNA strand-displacement assay, we show that Ded1p has also an ATP-dependent RNA unwinding activity. Hydrolysis of ATP is required for this activity: the replacement of ATP by a nonhydrolyzable analog or a mutation in the DEAD motif abolishing ATPase activity results in loss of RNA unwinding. We find that cells harboring a Ded1 protein with this mutated DEAD motif are nonviable, suggesting that the ATPase and RNA helicase activities of this protein are essential to the cell. Finally, RNA binding measurements indicate that the presence of ATP, but not ADP, increases the affinity of Ded1p for duplexversus single-stranded RNA; we discuss how this differential effect might drive the unwinding reaction.
