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Michele Pagano - One of the best experts on this subject based on the ideXlab platform.
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scF cyclin F controls centrosome homeostasis and mitotic Fidelity through cp110 degradation
Nature, 2010Co-Authors: Vincenzo Dangiolella, Michele Pagano, Valerio Donato, Sangeetha Vijayakumar, Anita Saraf, Laurence Florens, Michael P Washburn, Brian David DynlachtAbstract:Cyclin F is the Founding member oF the F-Box protein Family but its Functions are unknown. In contrast to most cyclins, it does not bind or activate cyclin-dependent kinases (CDKs). Here, a protein essential For centrosome duplication, CP110, is identiFied as a substrate oF Cyclin F. CP110 and Cyclin F associate on centrioles during the cell cycle, and Cyclin F is proposed to limit centrosome duplication by targeting CP110 For degradation. Cyclin F is the Founding member oF the F-Box protein Family but its Functions are unknown; unlike most cyclins, it does not bind or activate cyclin-dependent kinases. Here the authors identiFy CP110, a protein essential For centrosome duplication, as a substrate oF Cyclin F. CP110 and Cyclin F associate on centrioles during the cell cycle, and Cyclin F is proposed to limit centrosome duplication by targeting CP110 For degradation. Generally, F-Box proteins are the substrate recognition subunits oF SCF (Skp1–Cul1–F-Box protein) ubiquitin ligase complexes, which mediate the timely proteolysis oF important eukaryotic regulatory proteins1,2. Mammalian genomes encode roughly 70 F-Box proteins, but only a handFul have established Functions3,4. The F-Box protein Family obtained its name From Cyclin F (also called Fbxo1), in which the F-Box motiF (the ∼40-amino-acid domain required For binding to Skp1) was First described5. Cyclin F, which is encoded by an essential gene, also contains a cyclin Box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs)5,6,7. However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the Founding member oF the F-Box protein Family, Cyclin F remains an orphan protein, whose Functions are unknown. Starting From an unbiased screen, we identiFied CP110, a protein that is essential For centrosome duplication, as an interactor and substrate oF Cyclin F. Using a mode oF substrate binding distinct From other F-Box protein–substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase oF the cell cycle, and CP110 is ubiquitylated by the SCFCyclin F ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion oF Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant oF CP110 that cannot bind Cyclin F. Finally, expression oF a stable CP110 mutant in cultured cells also promotes the Formation oF micronuclei, a hallmark oF chromosome instability. We propose that SCFCyclin F-mediated degradation oF CP110 is required For the Fidelity oF mitosis and genome integrity.
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jhdm1b Fbxl10 is a nucleolar protein that represses transcription oF ribosomal rna genes
Nature, 2007Co-Authors: David Frescas, Daniele Guardavaccaro, Floria Asserma, Ryo Koyamanasu, Michele PaganoAbstract:JHDM1B is an evolutionarily conserved and ubiquitously expressed member oF the JHDM (JmjC-domain-containing histone demethylase) Family. Because it contains an F-Box motiF, this protein is also known as FBXL10 (reF. 4). With the use oF a genome-wide RNAi screen, the JHDM1B worm orthologue (T26A5.5) was identiFied as a gene that regulates growth. In the mouse, Four independent screens have identiFied JHDM1B as a putative tumour suppressor by retroviral insertion analysis. Here we identiFy human JHDM1B as a nucleolar protein and show that JHDM1B preFerentially binds the transcribed region oF ribosomal DNA to repress the transcription oF ribosomal RNA genes. We also show that repression oF ribosomal RNA genes by JHDM1B is dependent on its JmjC domain, which is necessary For the speciFic demethylation oF trimethylated lysine 4 on histone H3 in the nucleolus. In agreement with the notion that ribosomal RNA synthesis and cell growth are coupled processes, we show a JmjC-domain-dependent negative eFFect oF JHDM1B on cell size and cell proliFeration. Because aberrant ribosome biogenesis and the disruption oF epigenetic control mechanisms contribute to cellular transFormation, these results, together with the low levels oF JHDM1B expression Found in aggressive brain tumours, suggest a role For JHDM1B in cancer development.
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JHDM1B/FBXL10 is a nucleolar protein that represses transcription oF ribosomal RNA genes
Nature, 2007Co-Authors: David Frescas, Daniele Guardavaccaro, Florian Bassermann, Ryo Koyama-nasu, Michele PaganoAbstract:JHDM1B is an evolutionarily conserved and ubiquitously expressed member oF the JHDM (JmjC-domain-containing histone demethylase) Family. Because it contains an F-Box motiF, this protein is also known as FBXL10 (reF. 4). With the use oF a genome-wide RNAi screen, the JHDM1B worm orthologue (T26A5.5) was identiFied as a gene that regulates growth. In the mouse, Four independent screens have identiFied JHDM1B as a putative tumour suppressor by retroviral insertion analysis. Here we identiFy human JHDM1B as a nucleolar protein and show that JHDM1B preFerentially binds the transcribed region oF ribosomal DNA to repress the transcription oF ribosomal RNA genes. We also show that repression oF ribosomal RNA genes by JHDM1B is dependent on its JmjC domain, which is necessary For the speciFic demethylation oF trimethylated lysine 4 on histone H3 in the nucleolus. In agreement with the notion that ribosomal RNA synthesis and cell growth are coupled processes, we show a JmjC-domain-dependent negative eFFect oF JHDM1B on cell size and cell proliFeration. Because aberrant ribosome biogenesis and the disruption oF epigenetic control mechanisms contribute to cellular transFormation, these results, together with the low levels oF JHDM1B expression Found in aggressive brain tumours, suggest a role For JHDM1B in cancer development.
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Systematic analysis and nomenclature oF mammalian F-Box proteins
Genes & development, 2004Co-Authors: Jianping Jin, Michele Pagano, Stephen J Elledge, Timothy Cardozo, Ruth C. Lovering, J. Wade HarperAbstract:Much oF the targeted protein ubiquitylation that occurs in eukaryotes is perFormed by cullin-based E3 ubiquitin ligases, which Form a superFamily oF modular E3s. The best understood cullin-based E3 is the SCF ubiquitin ligase (Feldman et al. 1997; Skowyra et al. 1997), which is composed oF a modular E3 core containing CUL1 and RBX1 (also called ROC1), and a substrate speciFicity module composed oF SKP1 and a member oF the F-Box Family oF proteins (Cardozo and Pagano 2004). The CUL1/RBX1 complex Functions as a scaFFold to assemble the E2 ubiquitin conjugating enzyme with the substrate speciFicity module (Zheng et al. 2002). CUL1 interacts with RBX1 through its C terminus and with SKP1 through its N terminus. The interaction oF F-Box proteins with SKP1 occurs through the F-Box motiF, an ∼40-amino acid motiF First identiFied in budding yeast Cdc4p and human cyclin F, the latter giving the name to the entire Family (Bai et al. 1996). F-Box proteins contain additional protein interaction domains that bind ubiquitylation targets. The overall architecture oF SCF complexes is conserved in the superFamily oF SCF-like ubiquitin ligases that use cullin proteins as a scaFFold. All cullins characterized to date (CUL1-5) are known to interact with RBX1 or RBX2 but use distinct speciFicity modules, which generally display structural and Functional similarities with the SKP1/F-Box protein module. For example, CUL2 and CUL5 are known to interact with the SKP1-like protein elongin C, which, in turn, interacts with F-Box protein-like speciFicity Factors called BC/SOCS-Box proteins (Deshaies 1999; Guardavaccaro and Pagano 2003). In addition, CUL3 interacts with the BTB/POZ Family oF proteins, which appear to merge the Functions oF SKP1 and the F-Box protein into a single polypeptide (Furukawa et al. 2003; Geyer et al. 2003; Pintard et al. 2003; Xu et al. 2003), with the BTB domain displaying structural relationships with SKP1 (Schulman et al. 2000; Xu et al. 2003). Cul4 Forms a complex wherein DDB1/DDB2 and CSA proteins appear to Function as substrate speciFicity modules (Groisman et al. 2003). Thus, the current expectation is that all cullin-containing ligases will share the modular nature oF the original SCF Family oF ligases.
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insights into scF ubiquitin ligases From the structure oF the skp1 skp2 complex
Nature, 2000Co-Authors: Brenda A Schulman, Michele Pagano, Wade J Harper, Stephen J Elledge, Andrea C Carrano, Philip D Jeffrey, Zachary Bowen, E Kinnucan, Michael S Finnin, Nikola P PavletichAbstract:F-Box proteins are members oF a large Family that regulates the cell cycle, the immune response, signalling cascades and developmental programmes by targeting proteins, such as cyclins, cyclin-dependent kinase inhibitors, IκBα and β-catenin, For ubiquitination (reviewed in reFs 1,2,3). F-Box proteins are the substrate-recognition components oF SCF (Skp1–Cullin–F-Box protein) ubiquitin-protein ligases4,5. They bind the SCF constant catalytic core by means oF the F-Box motiF interacting with Skp1, and they bind substrates through their variable protein–protein interaction domains6. The large number oF F-Box proteins is thought to allow ubiquitination oF numerous, diverse substrates6. Most organisms have several Skp1 Family members, but the Function oF these Skp1 homologues and the rules oF recognition between diFFerent F-Box and Skp1 proteins remain unknown. Here we describe the crystal structure oF the human F-Box protein Skp2 bound to Skp1. Skp1 recruits the F-Box protein through a bipartite interFace involving both the F-Box and the substrate-recognition domain. The structure raises the possibility that diFFerent Skp1 Family members evolved to Function with diFFerent subsets oF F-Box proteins, and suggests that the F-Box protein may not only recruit substrate, but may also position it optimally For the ubiquitination reaction.
Pascal Genschik - One of the best experts on this subject based on the ideXlab platform.
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The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing
Current biology : CB, 2007Co-Authors: Diane Bortolamiol, Maghsoud Pazhouhandeh, Katia Marrocco, Pascal Genschik, Véronique Ziegler-graffAbstract:Plants employ post-transcriptional gene silencing (PTGS) as an antiviral deFense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation oF the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component oF RISC: it carries the RNA slicer activity. As a counter-deFense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F Box motiF required to Form an SCF-like complex, which is also essential For P0's silencing suppressor Function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not aFFect the biogenesis oF primary siRNAs in an inverted repeat-PTGS assay, but it does aFFect their activity. Moreover, P0's expression in transFormed Arabidopsis plants leads to various developmental abnormalities reminiscent oF mutants aFFected in miRNA pathways, which is accompanied by enhanced levels oF several miRNA-target transcripts, suggesting that P0 acts at the level oF RISC. Interestingly, ectopic expression oF P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.
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F-Box-like domain in the polerovirus protein P0 is required For silencing suppressor Function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Maghsoud Pazhouhandeh, Katia Marrocco, Esther Lechner, Bassam Berry, Thomas Kretsch, Odile Hemmer, Kenneth Richards, Monika Dieterle, Veronique Brault, Pascal GenschikAbstract:Plants employ small RNA-mediated posttranscriptional gene silencing as a virus deFense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode oF action oF most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 oF two Arabidopsis-inFecting poleroviruses interacts by means oF a conserved minimal F-Box motiF with Arabidopsis thaliana orthologs oF S-phase kinase-related protein 1 (SKP1), a component oF the SCF Family oF ubiquitin E3 ligases. Point mutations in the F-Box-like motiF abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity oF P0. Knockdown oF expression oF a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus inFection. Together, the results support a model in which P0 acts as an F-Box protein that targets an essential component oF the host posttranscriptional gene silencing machinery.
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F-Box-like domain in the polerovirus protein PO is required For silencing suppressor Function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Maghsoud Pazhouhandeh, Katia Marrocco, Esther Lechner, Bassam Berry, Thomas Kretsch, Odile Hemmer, Kenneth Richards, Monika Dieterle, Veronique Brault, Pascal GenschikAbstract:Plants employ small RNA-mediated posttranscriptional gene silencing as a virus deFense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode oF action oF most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 oF two Arabidopsis-inFecting poleroviruses interacts by means oF a conserved minimal F-Box motiF with Arabidopsis thaliana orthologs oF S-phase kinase-related protein 1 (SKP1), a component oF the SCF Family oF ubiquitin E3 ligases. Point mutations in the F-Box-like motiF abolished the P0-SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity oF P0. Knockdown oF expression oF a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus inFection. Together, the results support a model in which P0 acts as an F-Box protein that targets an essential component oF the host posttranscriptional gene silencing machinery.
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The SCF(COI1) ubiquitin-ligase complexes are required For jasmonate response in Arabidopsis.
The Plant cell, 2002Co-Authors: Fuquan Liu, Esther Lechner, Pascal Genschik, Wen Peng, William L. Crosby, Dafang Huang, Daoxin XieAbstract:Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required For response to jasmonates (JAs), which regulate root growth, pollen Fertility, wound healing, and deFense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either oF the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-Box motiF oF COI1, abolishes the Formation oF the SCF(COI1) complexes and results in loss oF the JA response. AtRbx1 double-stranded RNA-mediated genetic interFerence reduces AtRbx1 expression and aFFects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component oF SCF(COI1) complexes is modiFied in planta, where mutations in AXR1 decrease the abundance oF the modiFied AtCUL1 oF SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modiFication oF the AtCUL1 subunit oF SCF(COI1) complexes is important For JA signaling.
Maghsoud Pazhouhandeh - One of the best experts on this subject based on the ideXlab platform.
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Genetic analysis oF Iranian population oF Potato leaFroll virus based on ORF0
Virus genes, 2012Co-Authors: Shaheen Nourinejhad Zarghani, Masoud Shams-bakhsh, Neda Zand, Nemat Sokhandan-bashir, Maghsoud PazhouhandehAbstract:Potato leaFroll virus (PLRV) is a destructive virus oF potatoes and responsible For high yield losses wherever potatoes are grown. In this study, DNA Fragments containing ORF0 From each oF nine PLRV isolates was sequenced. Sequence analysis data using 36 isolates From 12 diFFerent countries including 14 Iranian isolates showed that the identities oF ORF0 at both nucleotide and amino acid levels between the Iranian isolates were 96–100 % and these isolates were more similar to the European PLRV isolates than to the other isolates. Furthermore, phylogenetic and population genetic analysis were carried out on the basis oF Full-length ORF0 and overlapping and non-overlapping regions oF ORF0 and ORF1 (ORF0/1) which revealed that PLRV isolates were not geographically resolved. Also, we identiFied negative selection with diFFerent ratios For each oF the mentioned genomic regions suggesting eFFects oF F-Box motiF and -1 FrameshiFt on ORF0 non-overlapping region and ORF0/1 in the selection pressure, respectively. Five recombination events were detected in the Iranian, Australian, and European isolates suggesting an important role For this phenomenon in inFluencing genetic diversity within this virus population.
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The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing
Current biology : CB, 2007Co-Authors: Diane Bortolamiol, Maghsoud Pazhouhandeh, Katia Marrocco, Pascal Genschik, Véronique Ziegler-graffAbstract:Plants employ post-transcriptional gene silencing (PTGS) as an antiviral deFense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation oF the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component oF RISC: it carries the RNA slicer activity. As a counter-deFense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F Box motiF required to Form an SCF-like complex, which is also essential For P0's silencing suppressor Function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not aFFect the biogenesis oF primary siRNAs in an inverted repeat-PTGS assay, but it does aFFect their activity. Moreover, P0's expression in transFormed Arabidopsis plants leads to various developmental abnormalities reminiscent oF mutants aFFected in miRNA pathways, which is accompanied by enhanced levels oF several miRNA-target transcripts, suggesting that P0 acts at the level oF RISC. Interestingly, ectopic expression oF P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.
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F-Box-like domain in the polerovirus protein P0 is required For silencing suppressor Function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Maghsoud Pazhouhandeh, Katia Marrocco, Esther Lechner, Bassam Berry, Thomas Kretsch, Odile Hemmer, Kenneth Richards, Monika Dieterle, Veronique Brault, Pascal GenschikAbstract:Plants employ small RNA-mediated posttranscriptional gene silencing as a virus deFense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode oF action oF most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 oF two Arabidopsis-inFecting poleroviruses interacts by means oF a conserved minimal F-Box motiF with Arabidopsis thaliana orthologs oF S-phase kinase-related protein 1 (SKP1), a component oF the SCF Family oF ubiquitin E3 ligases. Point mutations in the F-Box-like motiF abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity oF P0. Knockdown oF expression oF a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus inFection. Together, the results support a model in which P0 acts as an F-Box protein that targets an essential component oF the host posttranscriptional gene silencing machinery.
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F-Box-like domain in the polerovirus protein PO is required For silencing suppressor Function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Maghsoud Pazhouhandeh, Katia Marrocco, Esther Lechner, Bassam Berry, Thomas Kretsch, Odile Hemmer, Kenneth Richards, Monika Dieterle, Veronique Brault, Pascal GenschikAbstract:Plants employ small RNA-mediated posttranscriptional gene silencing as a virus deFense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode oF action oF most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 oF two Arabidopsis-inFecting poleroviruses interacts by means oF a conserved minimal F-Box motiF with Arabidopsis thaliana orthologs oF S-phase kinase-related protein 1 (SKP1), a component oF the SCF Family oF ubiquitin E3 ligases. Point mutations in the F-Box-like motiF abolished the P0-SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity oF P0. Knockdown oF expression oF a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus inFection. Together, the results support a model in which P0 acts as an F-Box protein that targets an essential component oF the host posttranscriptional gene silencing machinery.
J. Wade Harper - One of the best experts on this subject based on the ideXlab platform.
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IdentiFication oF substrates For F-Box proteins
Methods in enzymology, 2005Co-Authors: Jianping Jin, Xiaolu L. Ang, Takahiro Shirogane, J. Wade HarperAbstract:Abstract F‐Box proteins serve as speciFicity Factors For a Family oF ubiquitin protein ligases composed oF Skp1, Cu11, and Rbx1. In SCF complexes, Cu11 serves as a scaFFold For assembly oF the catalytic components composed oF Rbx1 and a ubiquitin‐conjugating enzyme and the speciFicity module composed oF Skp1 and an F‐Box protein. F‐Box proteins interact with Skp1 through the F‐Box motiF and with ubiquitination substrates through C‐terminal protein interaction domains such as WD40 repeats. The human genome contains ∼68 F‐Box proteins, which Fall into three major classes: Fbws containing WD40 repeats, Fbls containing leucine‐rich repeats, and Fbxs containing other types oF domains. Most oFten, F‐Box proteins interact with their targets in a phosphorylation‐dependent manner. The interaction oF F‐Box proteins with substrates typically involves a phosphodegron, a small peptide motiF containing speciFic phosphorylation events whose sequence is complementary to the F‐Box protein. The identiFication oF substrates oF F‐Box proteins is Frequently a challenge because oF the relatively weak aFFinity oF substrates For the requisite F‐Box protein. Here we describe approaches For the identiFication oF substrates oF F‐Box proteins. Approaches include stabilization oF ubiquitination targets by Cu11‐dominant negatives, the use oF shRNA hairpins to disrupt F‐Box protein expression, and the use oF collections oF F‐Box proteins as biochemical reagents to identiFy interacting proteins that may be substrates. In addition, we describe approaches For the use oF immobilized phosphopeptides to identiFy F‐Box proteins that recognize particular phosphodegrons.
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Systematic analysis and nomenclature oF mammalian F-Box proteins
Genes & development, 2004Co-Authors: Jianping Jin, Michele Pagano, Stephen J Elledge, Timothy Cardozo, Ruth C. Lovering, J. Wade HarperAbstract:Much oF the targeted protein ubiquitylation that occurs in eukaryotes is perFormed by cullin-based E3 ubiquitin ligases, which Form a superFamily oF modular E3s. The best understood cullin-based E3 is the SCF ubiquitin ligase (Feldman et al. 1997; Skowyra et al. 1997), which is composed oF a modular E3 core containing CUL1 and RBX1 (also called ROC1), and a substrate speciFicity module composed oF SKP1 and a member oF the F-Box Family oF proteins (Cardozo and Pagano 2004). The CUL1/RBX1 complex Functions as a scaFFold to assemble the E2 ubiquitin conjugating enzyme with the substrate speciFicity module (Zheng et al. 2002). CUL1 interacts with RBX1 through its C terminus and with SKP1 through its N terminus. The interaction oF F-Box proteins with SKP1 occurs through the F-Box motiF, an ∼40-amino acid motiF First identiFied in budding yeast Cdc4p and human cyclin F, the latter giving the name to the entire Family (Bai et al. 1996). F-Box proteins contain additional protein interaction domains that bind ubiquitylation targets. The overall architecture oF SCF complexes is conserved in the superFamily oF SCF-like ubiquitin ligases that use cullin proteins as a scaFFold. All cullins characterized to date (CUL1-5) are known to interact with RBX1 or RBX2 but use distinct speciFicity modules, which generally display structural and Functional similarities with the SKP1/F-Box protein module. For example, CUL2 and CUL5 are known to interact with the SKP1-like protein elongin C, which, in turn, interacts with F-Box protein-like speciFicity Factors called BC/SOCS-Box proteins (Deshaies 1999; Guardavaccaro and Pagano 2003). In addition, CUL3 interacts with the BTB/POZ Family oF proteins, which appear to merge the Functions oF SKP1 and the F-Box protein into a single polypeptide (Furukawa et al. 2003; Geyer et al. 2003; Pintard et al. 2003; Xu et al. 2003), with the BTB domain displaying structural relationships with SKP1 (Schulman et al. 2000; Xu et al. 2003). Cul4 Forms a complex wherein DDB1/DDB2 and CSA proteins appear to Function as substrate speciFicity modules (Groisman et al. 2003). Thus, the current expectation is that all cullin-containing ligases will share the modular nature oF the original SCF Family oF ligases.
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BTB proteins are substrate-speciFic adaptors in an SCF-like modular ubiquitin ligase containing CUL-3
Nature, 2003Co-Authors: Yue Wei, Stephen J Elledge, Jérôme Reboul, Philippe Vaglio, Tae Ho Shin, Marc Vidal, J. Wade HarperAbstract:Programmed destruction oF regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism For controlling signalling pathways. Cullins are proteins that Function as scaFFolds For modular ubiquitin ligases typiFied by the SCF (Skp1-Cul1-F-Box) complex. The substrate selectivity oF these E3 ligases is dictated by a speciFicity module that binds cullins. In the SCF complex, this module is composed oF Skp1, which binds directly to Cul1, and a member oF the F-Box Family oF proteins. F-Box proteins bind Skp1 through the F-Box motiF, and substrates by means oF carBoxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-Box-containing speciFicity Factors through the Skp1-like protein elongin C. Cul3 is required For embryonic development in mammals and Caenorhabditis elegans but its speciFicity module is unknown. Here we report the identiFication oF a large Family oF BTB-domain proteins as substrate-speciFic adaptors For C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 and its genetic target MEI-1 (reFs 12, 13) indicate that BTB proteins merge the Functional properties oF Skp1 and F-Box proteins into a single polypeptide.
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A Family oF mammalian F-Box proteins.
Current biology : CB, 1999Co-Authors: Jeffrey T. Winston, Stephen J Elledge, Deanna M. Koepp, Cihui Zhu, J. Wade HarperAbstract:Abstract Ubiquitin-mediated destruction oF regulatory proteins is a Frequent means oF controlling progression through signaling pathways [1]. F-Box proteins [2] are components oF modular E3 ubiquitin protein ligases called SCFs, which Function in phosphorylation-dependent ubiquitination ([3–5], reviewed in [6,7]). F-Box proteins contain a carBoxy-terminal domain that interacts with substrates and a 42–48 amino-acid F-Box motiF which binds to the protein Skp1 [2–4]. Skp1 binding links the F-Box protein with a core ubiquitin ligase composed oF the proteins Cdc53/Cul1, Rbx1 (also called Hrt1 and Roc1) and the E2 ubiquitin-conjugating enzyme Cdc34 [8–11]. The genomes oF the budding yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans contain, respectively, 16 and more than 60 F-Box proteins [2,7]; in S. cerevisiae , the F-Box proteins Cdc4, Grr1 and Met30 target cyclin-dependent kinase inhibitors, G1 cyclins and transcriptional regulators For ubiquitination ([3–5,8,10], reviewed in [6,7]). Only Four mammalian F-Box proteins (Cyclin F, Skp1, β-TRCP and NFB42) have been identiFied so Far [2,12]. Here, we report the identiFication oF a Family oF 33 novel mammalian F-Box proteins. The large number oF these proteins in mammals suggests that the SCF system controls a correspondingly large number oF regulatory pathways in vertebrates. Four oF these proteins contain a novel conserved motiF, the F-Box-associated (FBA) domain, which may represent a new protein–protein interaction motiF. The identiFication oF these genes will help uncover pathways controlled by ubiquitin-mediated proteolysis in mammals.
Katia Marrocco - One of the best experts on this subject based on the ideXlab platform.
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The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing
Current biology : CB, 2007Co-Authors: Diane Bortolamiol, Maghsoud Pazhouhandeh, Katia Marrocco, Pascal Genschik, Véronique Ziegler-graffAbstract:Plants employ post-transcriptional gene silencing (PTGS) as an antiviral deFense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation oF the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component oF RISC: it carries the RNA slicer activity. As a counter-deFense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F Box motiF required to Form an SCF-like complex, which is also essential For P0's silencing suppressor Function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not aFFect the biogenesis oF primary siRNAs in an inverted repeat-PTGS assay, but it does aFFect their activity. Moreover, P0's expression in transFormed Arabidopsis plants leads to various developmental abnormalities reminiscent oF mutants aFFected in miRNA pathways, which is accompanied by enhanced levels oF several miRNA-target transcripts, suggesting that P0 acts at the level oF RISC. Interestingly, ectopic expression oF P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.
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F-Box-like domain in the polerovirus protein P0 is required For silencing suppressor Function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Maghsoud Pazhouhandeh, Katia Marrocco, Esther Lechner, Bassam Berry, Thomas Kretsch, Odile Hemmer, Kenneth Richards, Monika Dieterle, Veronique Brault, Pascal GenschikAbstract:Plants employ small RNA-mediated posttranscriptional gene silencing as a virus deFense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode oF action oF most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 oF two Arabidopsis-inFecting poleroviruses interacts by means oF a conserved minimal F-Box motiF with Arabidopsis thaliana orthologs oF S-phase kinase-related protein 1 (SKP1), a component oF the SCF Family oF ubiquitin E3 ligases. Point mutations in the F-Box-like motiF abolished the P0–SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity oF P0. Knockdown oF expression oF a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus inFection. Together, the results support a model in which P0 acts as an F-Box protein that targets an essential component oF the host posttranscriptional gene silencing machinery.
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F-Box-like domain in the polerovirus protein PO is required For silencing suppressor Function
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Maghsoud Pazhouhandeh, Katia Marrocco, Esther Lechner, Bassam Berry, Thomas Kretsch, Odile Hemmer, Kenneth Richards, Monika Dieterle, Veronique Brault, Pascal GenschikAbstract:Plants employ small RNA-mediated posttranscriptional gene silencing as a virus deFense mechanism. In response, plant viruses encode proteins that can suppress RNA silencing, but the mode oF action oF most such proteins is poorly understood. Here, we show that the silencing suppressor protein P0 oF two Arabidopsis-inFecting poleroviruses interacts by means oF a conserved minimal F-Box motiF with Arabidopsis thaliana orthologs oF S-phase kinase-related protein 1 (SKP1), a component oF the SCF Family oF ubiquitin E3 ligases. Point mutations in the F-Box-like motiF abolished the P0-SKP1 ortholog interaction, diminished virus pathogenicity, and inhibited the silencing suppressor activity oF P0. Knockdown oF expression oF a SKP1 ortholog in Nicotiana benthamiana rendered the plants resistant to polerovirus inFection. Together, the results support a model in which P0 acts as an F-Box protein that targets an essential component oF the host posttranscriptional gene silencing machinery.