Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB.

PubMed

Burles, Kristin; van Buuren, Nicholas; Barry, Michele

2014-11-01

A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. Copyright © 2014 Elsevier Inc. All rights reserved.

Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burles, Kristin, E-mail: burles@ualberta.ca; Buuren, Nicholas van; Barry, Michele

2014-11-15

A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and thismore » inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. - Highlights: • Ectromelia virus encodes four Ank/F-box proteins, EVM002, EVM005, EVM154 and EVM165. • The Ank/F-box proteins inhibit NFκB nuclear translocation, dependent on the F-box. • The Ank/F-box proteins prevent IκBα degradation, suggesting they target the SCF. • Deletion of a single Ank/F-box gene from ECTV does not prevent viral NFκB inhibition. • This study identifies a new mechanism by which ectromelia virus inhibits NFκB.« less

JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation

PubMed Central

Sun, Luyang; Shi, Lei; Li, Wenqian; Yu, Wenhua; Liang, Jing; Zhang, Hua; Yang, Xiaohan; Wang, Yan; Li, Ruifang; Yao, Xingrong; Yi, Xia; Shang, Yongfeng

2009-01-01

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types—MDM2, Pirh2, and COP1—and the HECT-domain type—ARF-BP1—have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We showed that JFK promotes ubiquitination and degradation of p53. But unlike MDM2, Pirh2, COP1, and ARF-BP1, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Significantly, JFK inhibits p53-dependent transcription, and depletion of JFK stabilizes p53, promotes cell apoptosis, arrests cells in the G1 phase, and sensitizes cells to ionizing radiation-induced cell death. These data indicate that JFK is a critical negative regulator of p53 and represents a pathway for the maintenance of p53 levels in unstressed cells. Our experiments link the Skp1-Cul1-F-box system to p53 regulation. PMID:19509332

JFK, a Kelch domain-containing F-box protein, links the SCF complex to p53 regulation.

PubMed

Sun, Luyang; Shi, Lei; Li, Wenqian; Yu, Wenhua; Liang, Jing; Zhang, Hua; Yang, Xiaohan; Wang, Yan; Li, Ruifang; Yao, Xingrong; Yi, Xia; Shang, Yongfeng

2009-06-23

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Currently, several ubiquitin ligases, including the single-subunit RING-finger types--MDM2, Pirh2, and COP1--and the HECT-domain type--ARF-BP1--have been reported to target p53 for degradation. Here, we report the identification of a human Kelch domain-containing F-box protein, JFK. We showed that JFK promotes ubiquitination and degradation of p53. But unlike MDM2, Pirh2, COP1, and ARF-BP1, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Significantly, JFK inhibits p53-dependent transcription, and depletion of JFK stabilizes p53, promotes cell apoptosis, arrests cells in the G(1) phase, and sensitizes cells to ionizing radiation-induced cell death. These data indicate that JFK is a critical negative regulator of p53 and represents a pathway for the maintenance of p53 levels in unstressed cells. Our experiments link the Skp1-Cul1-F-box system to p53 regulation.

Novel regulation of Skp1 by the Dictyostelium AgtA α-galactosyltransferase involves the Skp1-binding activity of its WD40 repeat domain.

PubMed

Schafer, Christopher M; Sheikh, M Osman; Zhang, Dongmei; West, Christopher M

2014-03-28

The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-α type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in α-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40 repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40 repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function.

Novel Regulation of Skp1 by the Dictyostelium AgtA α-Galactosyltransferase Involves the Skp1-binding Activity of Its WD40 Repeat Domain*

PubMed Central

Schafer, Christopher M.; Sheikh, M. Osman; Zhang, Dongmei; West, Christopher M.

2014-01-01

The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-α type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in α-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40 repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40 repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function. PMID:24550398

The stomatin-like protein SLP-1 and Cdk2 interact with the F-Box protein Fbw7-γ.

PubMed

Zhang, Wei; MacDonald, Elizabeth M; Koepp, Deanna M

2012-01-01

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-γ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-γ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-γ. Overexpression of SLP-1 inhibits the degradation of Fbw7-γ, suggesting that this interaction can happen in vivo. When Fbw7-γ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF(Fbw7-γ) complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-γ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-γ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

PubMed

Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

2016-07-01

Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

PubMed Central

Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

2016-01-01

ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

PubMed

Cui, Hao-Ran; Zhang, Zheng-Rong; Lv, Wei; Xu, Jia-Ning; Wang, Xiao-Yun

2015-08-01

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

Identification of a Skp1-like protein interacting with SFB, the pollen S determinant of the gametophytic self-incompatibility in Prunus.

PubMed

Matsumoto, Daiki; Yamane, Hisayo; Abe, Kazuyuki; Tao, Ryutaro

2012-07-01

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCF(SLF) in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus.

The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

PubMed

Samach, A; Klenz, J E; Kohalmi, S E; Risseeuw, E; Haughn, G W; Crosby, W L

1999-11-01

Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors

PubMed Central

Li, Xin-Chun; Zhang, Jian; Wen, Zhe-Sheng; Huang, Zhi-Liang; Gao, Qin-Lei; Yang, Li-Na; Cheng, Yong-Xian; Tao, Sheng-Ce; Liu, Jinsong; Zhou, Guang-Biao

2015-01-01

Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and β-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials. PMID:26474281

Identification of a Skp1-Like Protein Interacting with SFB, the Pollen S Determinant of the Gametophytic Self-Incompatibility in Prunus1[W

PubMed Central

Matsumoto, Daiki; Yamane, Hisayo; Abe, Kazuyuki; Tao, Ryutaro

2012-01-01

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCFSLF in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus. PMID:22548785

F-box proteins Pof3 and Pof1 regulate Wee1 degradation and mitotic entry in fission yeast.

PubMed

Qiu, Cui; Yi, Yuan-Yuan; Lucena, Rafael; Wu, Meng-Juan; Sun, Jia-Hao; Wang, Xi; Jin, Quan-Wen; Wang, Yamei

2018-02-02

The key cyclin-dependent kinase Cdk1 (Cdc2) promotes irreversible mitotic entry, mainly by activating the phosphatase Cdc25 while suppressing the tyrosine kinase Wee1. Wee1 needs to be downregulated at the onset of mitosis to ensure rapid activation of Cdk1. In human somatic cells, one mechanism of suppressing Wee1 activity is mediated by ubiquitylation-dependent proteolysis through the Skp1/Cul1/F-box protein (SCF) ubiquitin E3 ligase complex. This mechanism is believed to be conserved from yeasts to humans. So far, the best-characterized human F-box proteins involved in recognition of Wee1 are β-TrCP (BTRCP) and Tome-1 (CDCA3). Although fission yeast Wee1 was the first identified member of its conserved kinase family, the F-box proteins involved in recognition and ubiquitylation of Wee1 have not been identified in this organism. In this study, our screen using Wee1- Renilla luciferase as the reporter revealed that two F-box proteins, Pof1 and Pof3, are required for downregulating Wee1 and are possibly responsible for recruiting Wee1 to SCF. Our genetic analyses supported a functional relevance between Pof1 and Pof3 and the rate of mitotic entry, and Pof3 might play a major role in this process. © 2018. Published by The Company of Biologists Ltd.

Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy

NASA Technical Reports Server (NTRS)

Gomes, M. D.; Lecker, S. H.; Jagoe, R. T.; Navon, A.; Goldberg, A. L.

2001-01-01

Muscle wasting is a debilitating consequence of fasting, inactivity, cancer, and other systemic diseases that results primarily from accelerated protein degradation by the ubiquitin-proteasome pathway. To identify key factors in this process, we have used cDNA microarrays to compare normal and atrophying muscles and found a unique gene fragment that is induced more than ninefold in muscles of fasted mice. We cloned this gene, which is expressed specifically in striated muscles. Because this mRNA also markedly increases in muscles atrophying because of diabetes, cancer, and renal failure, we named it atrogin-1. It contains a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the other components of SCF-type Ub-protein ligases (E3s), as well as a nuclear localization sequence and PDZ-binding domain. On fasting, atrogin-1 mRNA levels increase specifically in skeletal muscle and before atrophy occurs. Atrogin-1 is one of the few examples of an F-box protein or Ub-protein ligase (E3) expressed in a tissue-specific manner and appears to be a critical component in the enhanced proteolysis leading to muscle atrophy in diverse diseases.

Linking F-box protein 7 and parkin to neuronal degeneration in Parkinson's disease (PD).

PubMed

Zhou, Zhi Dong; Sathiyamoorthy, Sushmitha; Angeles, Dario C; Tan, Eng King

2016-04-18

Mutations of F-box protein 7 (FBXO7) and Parkin, two proteins in ubiquitin-proteasome system (UPS), are both implicated in pathogenesis of dopamine (DA) neuron degeneration in Parkinson's disease (PD). Parkin is a HECT/RING hybrid ligase that physically receives ubiquitin on its catalytic centre and passes ubiquitin onto its substrates, whereas FBXO7 is an adaptor protein in Skp-Cullin-F-box (SCF) SCF(FBXO7) ubiquitin E3 ligase complex to recognize substrates and mediate substrates ubiquitination by SCF(FBXO7) E3 ligase. Here, we discuss the overlapping pathophysiologic mechanisms and clinical features linking Parkin and FBXO7 with autosomal recessive PD. Both proteins play an important role in neuroprotective mitophagy to clear away impaired mitochondria. Parkin can be recruited to impaired mitochondria whereas cellular stress can promote FBXO7 mitochondrial translocation. PD-linked FBXO7 can recruit Parkin into damaged mitochondria and facilitate its aggregation. WT FBXO7, but not PD-linked FBXO7 mutants can rescue DA neuron degeneration in Parkin null Drosophila. A better understanding of the common pathophysiologic mechanisms of these two proteins could unravel specific pathways for targeted therapy in PD.

A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

PubMed

Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

2008-10-01

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

Unilateral incompatibility gene ui1.1 encodes an S-locus F-box protein expressed in pollen of Solanum species.

PubMed

Li, Wentao; Chetelat, Roger T

2015-04-07

Unilateral interspecific incompatibility (UI) is a postpollination, prezygotic reproductive barrier that prevents hybridization between related species when the female parent is self-incompatible (SI) and the male parent is self-compatible (SC). In tomato and related Solanum species, two genes, ui1.1 and ui6.1, are required for pollen compatibility on pistils of SI species or hybrids. We previously showed that ui6.1 encodes a Cullin1 (CUL1) protein. Here we report that ui1.1 encodes an S-locus F-box (SLF) protein. The ui1.1 gene was mapped to a 0.43-cM, 43.2-Mbp interval at the S-locus on chromosome 1, but positional cloning was hampered by low recombination frequency. We hypothesized that ui1.1 encodes an SLF protein(s) that interacts with CUL1 and Skp1 proteins to form an SCF-type (Skp1, Cullin1, F-box) ubiquitin E3 ligase complex. We identified 23 SLF genes in the S. pennellii genome, of which 19 were also represented in cultivated tomato (S. lycopersicum). Data from recombination events, expression analysis, and sequence annotation highlighted 11 S. pennellii genes as candidates. Genetic transformations demonstrated that one of these, SpSLF-23, is sufficient for ui1.1 function. A survey of cultivated and wild tomato species identified SLF-23 orthologs in each of the SI species, but not in the SC species S. lycopersicum, S. cheesmaniae, and S. galapagense, pollen of which lacks ui1.1 function. These results demonstrate that pollen compatibility in UI is mediated by protein degradation through the ubiquitin-proteasome pathway, a mechanism related to that which controls pollen recognition in SI.

The F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula.

PubMed

Song, Jian Bo; Wang, Yan Xiang; Li, Hai Bo; Li, Bo Wen; Zhou, Zhao Sheng; Gao, Shuai; Yang, Zhi Min

2015-07-01

F-box protein is a subunit of Skp1-Rbx1-Cul1-F-box protein (SCF) complex with typically conserved F-box motifs of approximately 40 amino acids and is one of the largest protein families in eukaryotes. F-box proteins play critical roles in selective and specific protein degradation through the 26S proteasome. In this study, we bioinformatically identified 972 putative F-box proteins from Medicago truncatula genome. Our analysis showed that in addition to the conserved motif, the F-box proteins have several other functional domains in their C-terminal regions (e.g., LRRs, Kelch, FBA, and PP2), some of which were found to be M. truncatula species-specific. By phylogenetic analysis of the F-box motifs, these proteins can be classified into three major families, and each family can be further grouped into more subgroups. Analysis of the genomic distribution of F-box genes on M. truncatula chromosomes revealed that the evolutional expansion of F-box genes in M. truncatula was probably due to localized gene duplications. To investigate the possible response of the F-box genes to abiotic stresses, both publicly available and customer-prepared microarrays were analyzed. Most of the F-box protein genes can be responding to salt and heavy metal stresses. Real-time PCR analysis confirmed that some of the F-box protein genes containing heat, drought, salicylic acid, and abscisic acid responsive cis-elements were able to respond to the abiotic stresses.

F-box protein interactions with the hallmark pathways in cancer.

PubMed

Randle, Suzanne J; Laman, Heike

2016-02-01

F-box proteins (FBP) are the substrate specifying subunit of Skp1-Cul1-FBP (SCF)-type E3 ubiquitin ligases and are responsible for directing the ubiquitination of numerous proteins essential for cellular function. Due to their ability to regulate the expression and activity of oncogenes and tumour suppressor genes, FBPs themselves play important roles in cancer development and progression. In this review, we provide a comprehensive overview of FBPs and their targets in relation to their interaction with the hallmarks of cancer cell biology, including the regulation of proliferation, epigenetics, migration and invasion, metabolism, angiogenesis, cell death and DNA damage responses. Each cancer hallmark is revealed to have multiple FBPs which converge on common signalling hubs or response pathways. We also highlight the complex regulatory interplay between SCF-type ligases and other ubiquitin ligases. We suggest six highly interconnected FBPs affecting multiple cancer hallmarks, which may prove sensible candidates for therapeutic intervention. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Polyubiquitination of the B-cell translocation gene 1 and 2 proteins is promoted by the SCF ubiquitin ligase complex containing βTrCP.

PubMed

Sasajima, Hitoshi; Nakagawa, Koji; Kashiwayanagi, Makoto; Yokosawa, Hideyoshi

2012-01-01

B-cell translocation gene 1 and 2 (BTG1 and BTG2) are members of the BTG/Tob antiproliferative protein family, which is able to regulate the cell cycle and cell proliferation. We previously reported that BTG1, BTG2, Tob, and Tob2 are degraded via the ubiquitin-proteasome pathway. In this study, we investigated the mechanism of polyubiquitination of BTG1 and BTG2. Since the Skp1-Cdc53/Cullin 1-F-box protein (SCF) complex functions as one of the major ubiquitin ligases for cell cycle regulation, we first examined interactions between BTG proteins and components of the SCF complex, and found that BTG1 and BTG2 were capable of interacting with the SCF complex containing Cullin-1 (a scaffold protein) and Skp1 (a linker protein). As the SCF complex can ubiquitinate various target proteins by substituting different F-box proteins as subunits that recognize different target proteins, we next examined which F-box proteins could bind the two BTG proteins, and found that Skp2, β-transducin repeat-containing protein 1 (βTrCP1), and βTrCP2 were able to associate with both BTG1 and BTG2. Furthermore, we obtained evidence showing that βTrCP1 enhanced the polyubiquitination of both BTG1 and BTG2 more efficiently than Skp2 did, and that an F-box truncated mutant of βTrCP1 had a dominant negative effect on this polyubiquitination. Thus, we propose that BTG1 and BTG2 are subjected to polyubiquitination, more efficiently when it is mediated by SCFβTrCP than by SCFSkp2.

Evolution of the F-Box Gene Family in Euarchontoglires: Gene Number Variation and Selection Patterns

PubMed Central

Wang, Ailan; Fu, Mingchuan; Jiang, Xiaoqian; Mao, Yuanhui; Li, Xiangchen; Tao, Shiheng

2014-01-01

F-box proteins are substrate adaptors used by the SKP1–CUL1–F-box protein (SCF) complex, a type of E3 ubiquitin ligase complex in the ubiquitin proteasome system (UPS). SCF-mediated ubiquitylation regulates proteolysis of hundreds of cellular proteins involved in key signaling and disease systems. However, our knowledge of the evolution of the F-box gene family in Euarchontoglires is limited. In the present study, 559 F-box genes and nine related pseudogenes were identified in eight genomes. Lineage-specific gene gain and loss events occurred during the evolution of Euarchontoglires, resulting in varying F-box gene numbers ranging from 66 to 81 among the eight species. Both tandem duplication and retrotransposition were found to have contributed to the increase of F-box gene number, whereas mutation in the F-box domain was the main mechanism responsible for reduction in the number of F-box genes, resulting in a balance of expansion and contraction in the F-box gene family. Thus, the Euarchontoglire F-box gene family evolved under a birth-and-death model. Signatures of positive selection were detected in substrate-recognizing domains of multiple F-box proteins, and adaptive changes played a role in evolution of the Euarchontoglire F-box gene family. In addition, single nucleotide polymorphism (SNP) distributions were found to be highly non-random among different regions of F-box genes in 1092 human individuals, with domain regions having a significantly lower number of non-synonymous SNPs. PMID:24727786

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

PubMed Central

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; Kota, Krishna P.; Whitehouse, Chris A.; Bavari, Sina

2016-01-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection. PMID

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase.

PubMed

Mudhasani, Rajini; Tran, Julie P; Retterer, Cary; Kota, Krishna P; Whitehouse, Chris A; Bavari, Sina

2016-02-01

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)(FBXW11) E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF(FBXW11) E3 ligase. We further show that disrupting the assembly of the SCF(FBXW11-NSs) E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF(FBXW11-NSs) E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF(FBXW11) complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

DOE PAGES

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary; ...

2016-02-02

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.« less

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mudhasani, Rajini; Tran, Julie P.; Retterer, Cary

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through anmore » alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.« less

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

PubMed

Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

2013-06-01

Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis.

PubMed

Wang, Jie; Yao, Wenkong; Wang, Lei; Ma, Fuli; Tong, Weihuo; Wang, Chen; Bao, Rui; Jiang, Changyue; Yang, Yazhou; Zhang, Jianxia; Xu, Yan; Wang, Xiping; Zhang, Chaohong; Wang, Yuejin

2017-10-01

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H 2 O 2 ) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCF VpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H 2 O 2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system. Copyright © 2017 Elsevier B.V. All rights reserved.

Stringent Control of NFE2L3 (Nuclear Factor, Erythroid 2-Like 3; NRF3) Protein Degradation by FBW7 (F-box/WD Repeat-containing Protein 7) and Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Kannan, Meenakshi B.; Dodard-Friedman, Isadore; Blank, Volker

2015-01-01

The NFE2L3 transcription factor has been implicated in various cellular processes, including carcinogenesis, stress response, differentiation, and inflammation. Previously it has been shown that NFE2L3 has a rapid turnover and is stabilized by proteasomal inhibitors. The mechanisms regulating the degradation of this protein have not been investigated. Here we report ubiquitination of NFE2L3 and demonstrate that F-box/WD repeat-containing protein 7 (FBW7 or FBWX7), a component of Skp1, Cullin 1, F-box containing complex (SCF)-type E3 ligase, is the E3 ligase mediating the degradation of NFE2L3. We showed that FBW7 interacts with NFE2L3 and that dimerization of FBW7 is required for the degradation of the transcription factor. We also demonstrate that the kinase glycogen synthase kinase 3 (GSK3) mediates the FBW7-dependent ubiquitination of NFE2L3. We show phosphorylation of NFE2L3 by GSK3 and its significance in the regulation of NFE2L3 by the tumor suppressor FBW7. FBW7 abrogated NFE2L3-mediated repression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene antioxidant response element (ARE). Our findings reveal FBW7 and GSK3 as novel regulators of the NFE2L3 transcription factor and a potential mechanism by which FBW7 might regulate detoxification and the cellular response to stress. PMID:26306035

Small molecule therapeutics targeting F-box proteins in cancer.

PubMed

Liu, Yuan; Mallampalli, Rama K

2016-02-01

The ubiquitin proteasome system (UPS) plays vital roles in maintaining protein equilibrium mainly through proteolytic degradation of targeted substrates. The archetypical SCF ubiquitin E3 ligase complex contains a substrate recognition subunit F-box protein that recruits substrates to the catalytic ligase core for its polyubiquitylation and subsequent proteasomal degradation. Several well-characterized F-box proteins have been demonstrated that are tightly linked to neoplasia. There is mounting information characterizing F-box protein-substrate interactions with the rationale to develop unique therapeutics for cancer treatment. Here we review that how F-box proteins function in cancer and summarize potential small molecule inhibitors for cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

MEIOTIC F-BOX Is Essential for Male Meiotic DNA Double-Strand Break Repair in Rice[OPEN

PubMed Central

Wang, Chong; Yu, Junping; Zong, Jie; Lu, Pingli

2016-01-01

F-box proteins constitute a large superfamily in plants and play important roles in controlling many biological processes, but the roles of F-box proteins in male meiosis in plants remain unclear. Here, we identify the rice (Oryza sativa) F-box gene MEIOTIC F-BOX (MOF), which is essential for male meiotic progression. MOF belongs to the FBX subfamily and is predominantly active during leptotene to pachytene of prophase I. mof meiocytes display disrupted telomere bouquet formation, impaired pairing and synapsis of homologous chromosomes, and arrested meiocytes at late prophase I, followed by apoptosis. Although normal, programmed double-stranded DNA breaks (DSBs) form in mof mutants, foci of the phosphorylated histone variant γH2AX, a marker for DSBs, persist in the mutant, indicating that many of the DSBs remained unrepaired. The recruitment of Completion of meiosis I (COM1) and Radiation sensitive51C (RAD51C) to DSBs is severely compromised in mutant meiocytes, indicating that MOF is crucial for DSB end-processing and repair. Further analyses showed that MOF could physically interact with the rice SKP1-like Protein1 (OSK1), indicating that MOF functions as a component of the SCF E3 ligase to regulate meiotic progression in rice. Thus, this study reveals the essential role of an F-box protein in plant meiosis and provides helpful information for elucidating the roles of the ubiquitin proteasome system in plant meiotic progression. PMID:27436711

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2014-01-07

The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

PubMed

Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

2016-02-26

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

PubMed Central

Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

2012-01-01

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

Sulforaphane down-regulates SKP2 to stabilize p27(KIP1) for inducing antiproliferation in human colon adenocarcinoma cells.

PubMed

Chung, Yuan-Kai; Chi-Hung Or, Richard; Lu, Chien-Hsing; Ouyang, Wei-Ting; Yang, Shu-Yi; Chang, Chia-Che

2015-01-01

Sulforaphane is a cruciferous vegetable-derived isothiocyanate with promising chemopreventive and therapeutic activities. Induction of proliferation arrest and apoptosis principally contribute to sulforaphane's anticancer activity, but the precise molecular mechanisms remain elusive. The oncoprotein SKP2 is a key component of the SKP1-CULLIN1-F-box (SCF) E3 ligase complex and is responsible for directing SCF-mediated degradation of cyclin-dependent kinase inhibitor p27(KIP1) to promote cell proliferation. We herein provide the first evidence supporting the critical involvement of the SKP2-p27(KIP1) axis in sulforaphane-induced antiproliferation in various human colon adenocarcinoma cell lines. Specifically, sulforaphane markedly suppressed the levels of bromodeoxyuridine (BrdU) incorporation and clonogenicity in all tested cell lines, illustrating the antiproliferative effect of sulforaphane. Of note, sulforaphane-induced antiproliferation was accompanied with down-regulation of SKP2, leading to the stabilization and thus up-regulation of p27(KIP1). Additionally, sulforaphane was found to down-regulate SKP2 mainly through transcriptional repression, as sulforaphane lowered SKP2 mRNA expression and the SKP2 promoter activity. Furthermore, sulforaphane treatment led to the activation of both AKT and ERK, thus ruling out the possibility that sulforaphane down-regulates SKP2 by inhibiting AKT or ERK. Notably, sulforaphane-elicited suppression of BrdU incorporation and clonogenicity were significantly rescued in the context of SKP2 overexpression or p27(KIP1) depletion, therefore highlighting the important role of SKP2 down-regulation and the ensuing stabilization of p27(KIP1) in sulforaphane-induced antiproliferation. Collectively, these data expand our molecular understanding about how sulforaphane elicits proliferation arrest, but also implicate the application of sulforaphane in therapeutic modalities targeting SKP2. Copyright © 2014 The Society for Biotechnology

Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures.

PubMed

Stefanowicz, Karolina; Lannoo, Nausicaä; Proost, Paul; Van Damme, Els J M

2012-01-01

The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.

Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

PubMed

Saritas-Yildirim, Banu; Pliner, Hannah A; Ochoa, Angelica; Silva, Elena M

2015-01-01

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.

CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata.

PubMed

Sun, Linhan; Kao, Teh-Hui

2018-06-01

Function of Petunia PiSSK1. Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S 2 -haplotype and S 3 -haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCF SLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1 and examined the SI behavior of a T 0 plant (S 2 S 3 ) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2 ) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatible S-genotypes, but fully compatible with pistils of an S 3 S 3 transgenic plant in which production of S 3 -RNase was completely suppressed by an antisense S 3 -RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.

Pollen S-locus F-box proteins of Petunia involved in S-RNase-based self-incompatibility are themselves subject to ubiquitin-mediated degradation.

PubMed

Sun, Penglin; Li, Shu; Lu, Dihong; Williams, Justin S; Kao, Teh-Hui

2015-07-01

Many flowering plants show self-incompatibility, an intra-specific reproductive barrier by which pistils reject self-pollen to prevent inbreeding and accept non-self pollen to promote out-crossing. In Petunia, the polymorphic S-locus determines self/non-self recognition. The locus contains a gene encoding an S-RNase, which controls pistil specificity, and multiple S-locus F-box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F-box) complex that is responsible for mediating degradation of non-self S-RNase(s), with which the SLF interacts, via the ubiquitin-26S proteasome pathway. A complete set of SLFs is required to detoxify all non-self S-RNases to allow cross-compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin-26S proteasome pathway, and identify an 18 amino acid sequence in the C-terminal region of S2 -SLF1 (SLF1 of S2 haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S2 haplotype and S3 haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S2 -SLF1 stabilized the protein but abolished its function in self-incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self-incompatibility. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.

PubMed

Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M; West, Christopher M

2012-01-13

Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.

Skp1: Implications in cancer and SCF-oriented anti-cancer drug discovery.

PubMed

Hussain, Muzammal; Lu, Yongzhi; Liu, Yong-Qiang; Su, Kai; Zhang, Jiancun; Liu, Jinsong; Zhou, Guang-Biao

2016-09-01

In the last decade, the ubiquitin proteasome system (UPS), in general, and E3 ubiquitin ligases, in particular, have emerged as valid drug targets for the development of novel anti-cancer therapeutics. Cullin RING Ligases (CRLs), which can be classified into eight groups (CRL1-8) and comprise approximately 200 members, represent the largest family of E3 ubiquitin ligases which facilitate the ubiquitination-derived proteasomal degradation of a myriad of functionally and structurally diverse substrates. S phase kinase-associated protein 1 (Skp1)-Cullin1-F-Box protein (SCF) complexes are the best characterized among CRLs, which play crucial roles in numerous cellular processes and physiological dysfunctions, such as in cancer biology. Currently, there is growing interest in developing SCF-targeting anti-cancer therapies for clinical application. Indeed, the research in this field has seen some progress in the form of cullin neddylation- and Skp2-inhibitors. However, it still remains an underdeveloped area and needs to design new strategies for developing improved form of therapy. In this review, we venture a novel strategy that rational pharmacological targeting of Skp1, a central regulator of SCF complexes, may provide a novel avenue for SCF-oriented anti-cancer therapy, expected: (i) to simultaneously address the critical roles that multiple SCF oncogenic complexes play in cancer biology, (ii) to selectively target cancer cells with minimal normal cell toxicity, and (iii) to offer multiple chemical series, via therapeutic interventions at the Skp1 binding interfaces in SCF complex, thereby maximizing chances of success for drug discovery. In addition, we also discuss the challenges that might be posed regarding rational pharmacological interventions against Skp1. Copyright © 2016 Elsevier Ltd. All rights reserved.

Functional redundancy and/or ongoing pseudogenization among F-box protein genes expressed in Arabidopsis male gametophyte.

PubMed

Ikram, Sobia; Durandet, Monique; Vesa, Simona; Pereira, Serge; Guerche, Philippe; Bonhomme, Sandrine

2014-06-01

F-box protein genes family is one of the largest gene families in plants, with almost 700 predicted genes in the model plant Arabidopsis. F-box proteins are key components of the ubiquitin proteasome system that allows targeted protein degradation. Transcriptome analyses indicate that half of these F-box protein genes are found expressed in microspore and/or pollen, i.e., during male gametogenesis. To assess the role of F-box protein genes during this crucial developmental step, we selected 34 F-box protein genes recorded as highly and specifically expressed in pollen and isolated corresponding insertion mutants. We checked the expression level of each selected gene by RT-PCR and confirmed pollen expression for 25 genes, but specific expression for only 10 of the 34 F-box protein genes. In addition, we tested the expression level of selected F-box protein genes in 24 mutant lines and showed that 11 of them were null mutants. Transmission analysis of the mutations to the progeny showed that none of the single mutations was gametophytic lethal. These unaffected transmission efficiencies suggested leaky mutations or functional redundancy among F-box protein genes. Cytological observation of the gametophytes in the mutants confirmed these results. Combinations of mutations in F-box protein genes from the same subfamily did not lead to transmission defect either, further highlighting functional redundancy and/or a high proportion of pseudogenes among these F-box protein genes.

Skp2 regulates androgen receptor through ubiquitin-mediated degradation independent of Akt/mTOR pathways in prostate cancer.

PubMed

Li, Bo; Lu, Wenfu; Yang, Qing; Yu, Xiuping; Matusik, Robert J; Chen, Zhenbang

2014-04-01

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling. © 2013 Wiley Periodicals, Inc.

Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

PubMed

Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas; Weber, Friedemann

2014-03-01

The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

Virulence Factor NSs of Rift Valley Fever Virus Recruits the F-Box Protein FBXO3 To Degrade Subunit p62 of General Transcription Factor TFIIH

PubMed Central

Kainulainen, Markus; Habjan, Matthias; Hubel, Philipp; Busch, Laura; Lau, Simone; Colinge, Jacques; Superti-Furga, Giulio; Pichlmair, Andreas

2014-01-01

ABSTRACT The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity. PMID:24403578

Overexpression of SKP2 Inhibits the Radiation-Induced Bystander Effects of Esophageal Carcinoma.

PubMed

Wang, Xiao-Chun; Zhang, Tie-Jun; Guo, Zi-Jian; Xiao, Chang-Yan; Ding, Xiao-Wen; Fang, Fang; Sheng, Wen-Tao; Shu, Xu; Li, Jue

2017-02-06

To investigate the effects of S-phase kinase protein 2 (SKP2) expression on the radiation induced bystander effect (RIBE) in esophageal cancer (EC) cells. Western blot was used to detect the levels of SKP2, Rad51, and Ku70 in EC cells. Positive transfection, RNAi, micronucleus (MN), and γ-H2AX focus formation assay were used to investigate the effects of SKP2 on RIBE induced by irradiated cells. We found a significant negative correlation between SKP2 expression and MN frequency ( p < 0.05) induced by RIBE. The results were further confirmed by positive transfection, RNAi, and rescue experiments.γ-H2AX focus formation assay results indicated that overexpression of SKP2 in the irradiated cells inhibited the DNA damage of RIBE cells. However, when SKP2 expression decreased in irradiated cells, the DNA damage of RIBE cells increased. Increased or decreased expression levels of SKP2 had effects on Rad51 expression under the conditions of RIBE. These results showed, for the first time, that SKP2 expression can inhibit RIBE of EC cells. The mechanism may function, at least partly, through the regulation of Rad51 in the ability to repair DNA damage.

Hsp27 and F-box protein β-TrCP promote degradation of mRNA decay factor AUF1.

PubMed

Li, Mei-Ling; Defren, Jennifer; Brewer, Gary

2013-06-01

Activation of the mitogen-activated protein (MAP) pathway kinases p38 and MK2 induces phosphorylation of the chaperone Hsp27 and stabilization of mRNAs containing AU-rich elements (AREs) (ARE-mRNAs). Likewise, expression of phosphomimetic mutant forms of Hsp27 also stabilizes ARE-mRNAs. It appears to perform this function by promoting degradation of the ARE-mRNA decay factor AUF1 by proteasomes. In this study, we examined the molecular mechanism linking Hsp27 phosphorylation to AUF1 degradation by proteasomes. AUF1 is a target of β-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase Skp1-cullin-F-box protein complex, SCF(β-TrCP). Depletion of β-TrCP stabilized AUF1. In contrast, overexpression of β-TrCP enhanced ubiquitination and degradation of AUF1 and led to stabilization of reporter mRNAs containing cytokine AREs. Enhanced AUF1 degradation required expression of phosphomimetic mutant forms of both Hsp27 and AUF1. Our results suggest that a signaling axis composed of p38 MAP kinase-MK2-Hsp27-β-TrCP may promote AUF1 degradation by proteasomes and stabilization of cytokine ARE-mRNAs.

Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins.

PubMed

Nelson, David E; Randle, Suzanne J; Laman, Heike

2013-10-09

F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases. To date, 69 FBPs have been identified in humans, but ubiquitinated substrates have only been identified for a few, with the majority of FBPs remaining 'orphans'. In recent years, a growing body of work has identified non-canonical, SCF-independent roles for about 12% of the human FBPs. These atypical FBPs affect processes as diverse as transcription, cell cycle regulation, mitochondrial dynamics and intracellular trafficking. Here, we provide a general review of FBPs, with a particular emphasis on these expanded functions. We review Fbxo7 as an exemplar of this special group as it has well-defined roles in both SCF and non-SCF complexes. We review its function as a cell cycle regulator, via its ability to stabilize p27 protein and Cdk6 complexes, and as a proteasome regulator, owing to its high affinity binding to PI31. We also highlight recent advances in our understanding of Fbxo7 function in Parkinson's disease, where it functions in the regulation of mitophagy with PINK1 and Parkin. We postulate that a few extraordinary FBPs act as platforms that seamlessly segue their canonical and non-canonical functions to integrate different cellular pathways and link their regulation.

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

2011-03-08

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding an F-box protein that interacts with a EIN3 involved in an ethylene response of plants, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding an F-box protein. The inventions also relates to methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein.

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana

PubMed Central

Baute, Joke; Polyn, Stefanie; De Block, Jolien; Blomme, Jonas; Van Lijsebettens, Mieke

2017-01-01

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates. PMID:28340173

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana.

PubMed

Baute, Joke; Polyn, Stefanie; De Block, Jolien; Blomme, Jonas; Van Lijsebettens, Mieke; Inzé, Dirk

2017-05-01

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Regulating the ethylene response of a plant by modulation of F-box proteins

DOEpatents

Guo, Hongwei; Ecker, Joseph R.

2010-02-02

The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding a F-box protein, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding a F-box protein.

BAG3 promotes proliferation of ovarian cancer cells via post-transcriptional regulation of Skp2 expression.

PubMed

Yan, Jing; Liu, Chuan; Jiang, Jing-Yi; Liu, Hans; Li, Chao; Li, Xin-Yu; Yuan, Ye; Zong, Zhi-Hong; Wang, Hua-Qin

2017-10-01

Bcl-2 associated athanogene 3 (BAG3) contains a modular structure, through which BAG3 interacts with a wide range of proteins, thereby affording its capacity to regulate multifaceted biological processes. BAG3 is often highly expressed and functions as a pro-survival factor in many cancers. However, the oncogenic potential of BAG3 remains not fully understood. The cell cycle regulator, S-phase kinase associated protein 2 (Skp2) is increased in various cancers and plays an important role in tumorigenesis. The current study demonstrated that BAG3 promoted proliferation of ovarian cancer cells via upregulation of Skp2. BAG3 stabilized Skp2 mRNA via its 3'-untranslated region (UTR). The current study demonstrated that BAG3 interacted with Skp2 mRNA. In addition, miR-21-5p suppressed Skp2 expression, which was compromised by forced BAG3 expression. These results indicated that at least some oncogenic functions of BAG3 were mediated through posttranscriptional regulation of Skp2 via antagonizing suppressive action of miR-21-5p in ovarian cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcriptional Regulation of S Phase Kinase-associated Protein 2 by NR4A Orphan Nuclear Receptor NOR1 in Vascular Smooth Muscle Cells*

PubMed Central

Gizard, Florence; Zhao, Yue; Findeisen, Hannes M.; Qing, Hua; Cohn, Dianne; Heywood, Elizabeth B.; Jones, Karrie L.; Nomiyama, Takashi; Bruemmer, Dennis

2011-01-01

Members of the NR4A subgroup of the nuclear hormone receptor superfamily have emerged as key transcriptional regulators of proliferation and inflammation. NOR1 constitutes a ligand-independent transcription factor of this subgroup and induces cell proliferation; however, the transcriptional mechanisms underlying this mitogenic role remain to be defined. Here, we demonstrate that the F-box protein SKP2 (S phase kinase-associated protein 2), the substrate-specific receptor of the ubiquitin ligase responsible for the degradation of p27KIP1 through the proteasome pathway, constitutes a direct transcriptional target for NOR1. Mitogen-induced Skp2 expression is silenced in vascular smooth muscle cells (VSMC) isolated from Nor1-deficient mice or transfected with Nor1 siRNA. Conversely, adenovirus-mediated overexpression of NOR1 induces Skp2 expression in VSMC and decreases protein abundance of its target p27. Transient transfection experiments establish that NOR1 transactivates the Skp2 promoter through a nerve growth factor-induced clone B response element (NBRE). Electrophoretic mobility shift and chromatin immunoprecipitation assays further revealed that NOR1 is recruited to this NBRE site in the Skp2 promoter in response to mitogenic stimulation. In vivo Skp2 expression is increased during the proliferative response underlying neointima formation, and this transcriptional induction depends on the expression of NOR1. Finally, we demonstrate that overexpression of Skp2 rescues the proliferative arrest of Nor1-deficient VSMC. Collectively, these results characterize Skp2 as a novel NOR1-regulated target gene and detail a previously unrecognized transcriptional cascade regulating mitogen-induced VSMC proliferation. PMID:21868379

A novel F-box protein CaF-box is involved in responses to plant hormones and abiotic stress in pepper (Capsicum annuum L.).

PubMed

Chen, Rugang; Guo, Weili; Yin, Yanxu; Gong, Zhen-Hui

2014-02-10

The F-box protein family is characterized by an F-box motif that has been shown to play an important role in regulating various developmental processes and stress responses. In this study, a novel F-box-containing gene was isolated from leaves of pepper cultivar P70 (Capsicum annuum L.) and designated CaF-box. The full-length cDNA is 2088 bp and contains an open reading frame of 1914 bp encoding a putative polypeptide of 638 amino acids with a mass of 67.8 kDa. CaF-box was expressed predominantly in stems and seeds, and the transcript was markedly upregulated in response to cold stress, abscisic acid (ABA) and salicylic acid (SA) treatment, and downregulated under osmotic and heavy metal stress. CaF-box expression was dramatically affected by salt stress, and was rapidly increased for the first hour, then sharply decreased thereafter. In order to further assess the role of CaF-box in the defense response to abiotic stress, a loss-of-function experiment in pepper plants was performed using a virus-induced gene silencing (VIGS) technique. Measurement of thiobarbituric acid reactive substances (TBARS) and electrolyte leakage revealed stronger lipid peroxidation and cell death in the CaF-box-silenced plants than in control plants, suggesting CaF-box plays an important role in regulating the defense response to abiotic stress resistance in pepper plants.

Rottlerin exerts its anti-tumor activity through inhibition of Skp2 in breast cancer cells.

PubMed

Yin, Xuyuan; Zhang, Yu; Su, Jingna; Hou, Yingying; Wang, Lixia; Ye, Xiantao; Zhao, Zhe; Zhou, Xiuxia; Li, Yali; Wang, Zhiwei

2016-10-11

Studies have investigated the tumor suppressive role of rottlerin in carcinogenesis. However, the molecular mechanisms of rottlerin-induced anti-tumor activity are largely unclear. Skp2 (S-phase kinase associated protein 2) has been validated to play an oncogenic role in a variety of human malignancies. Therefore, inactivation of Skp2 could be helpful for the treatment of human cancers. In the current study, we explore whether rottlerin could inhibit Skp2 expression, leading to inhibition of cell growth, migration and invasion in breast cancer cells. We found that rottlerin treatment inhibited cell growth, induced apoptosis and cell cycle arrest. We also revealed that rottlerin suppressed cell migration and invasion in breast cancer cells. Mechanically, we observed that rottlerin significantly down-regulated the expression of Skp2 in breast cancer cells. Importantly, overexpression of Skp2 abrogated rottlerin-mediated tumor suppressive activity, whereas down-regulation of Skp2 enhanced rottlerin-triggered anti-tumor function. Strikingly, we identified that rottlerin exhibited its anti-tumor potential partly through inactivation of Skp2 in breast cancer. Our findings indicate that rottlerin could be a potential safe agent for the treatment of breast cancer.

A Novel F-Box Protein CaF-Box Is Involved in Responses to Plant Hormones and Abiotic Stress in Pepper (Capsicum annuum L.)

PubMed Central

Chen, Rugang; Guo, Weili; Yin, Yanxu; Gong, Zhen-Hui

2014-01-01

The F-box protein family is characterized by an F-box motif that has been shown to play an important role in regulating various developmental processes and stress responses. In this study, a novel F-box-containing gene was isolated from leaves of pepper cultivar P70 (Capsicum annuum L.) and designated CaF-box. The full-length cDNA is 2088 bp and contains an open reading frame of 1914 bp encoding a putative polypeptide of 638 amino acids with a mass of 67.8 kDa. CaF-box was expressed predominantly in stems and seeds, and the transcript was markedly upregulated in response to cold stress, abscisic acid (ABA) and salicylic acid (SA) treatment, and downregulated under osmotic and heavy metal stress. CaF-box expression was dramatically affected by salt stress, and was rapidly increased for the first hour, then sharply decreased thereafter. In order to further assess the role of CaF-box in the defense response to abiotic stress, a loss-of-function experiment in pepper plants was performed using a virus-induced gene silencing (VIGS) technique. Measurement of thiobarbituric acid reactive substances (TBARS) and electrolyte leakage revealed stronger lipid peroxidation and cell death in the CaF-box-silenced plants than in control plants, suggesting CaF-box plays an important role in regulating the defense response to abiotic stress resistance in pepper plants. PMID:24518684

All 17 S-locus F-box proteins of the S2 - and S3 -haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility.

PubMed

Li, Shu; Williams, Justin S; Sun, Penglin; Kao, Teh-Hui

2016-09-01

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S2 -SLF1 (SLF1 with an S2 -haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCF(S) (2-) (SLF) (1) complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCF(SLF) complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S2 and S3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S2 and S3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCF(SLF) complexes have evolved specifically to function in self-incompatibility. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis.

PubMed

Durfee, Tim; Roe, Judith L; Sessions, R Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A; Weigel, Detlef; Zambryski, Patricia C

2003-07-08

The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity.

Rational redesign of a cation···π···π stacking at cardiovascular Fbw7-Skp1 complex interface and its application for deriving self-inhibitory peptides to disrupt the complex interaction.

PubMed

Zhou, Jing; Wang, Yao-Sheng

2017-09-26

The Fbw7-Skp1 complex is an essential component in the formation and development of the mammalian cardiovascular system; the complex interaction is mediated through binding of Skp1 C-terminal peptide (qGlu-peptide) to the F-box domain of Fbw7. By visually examining the crystal structure, we identified a typical cation ···π···π stacking system at the complex interface, which is formed by the Trp1159 residue of qGlu-peptide with the Lys2299 and His2359 residues of Fbw7 F-box domain. Both hybrid quantum mechanics/molecular mechanics (QM/MM) analysis of the real domain-peptide complex and electron-correlation ab initio calculation of the stacking system model suggested that the cation···π···π plays an important role in stabilizing the complex; substitution of peptide Trp1159 residue with aromatic Phe and Tyr would not cause a considerable effect on the configuration and energetics of cation···π···π stacking system, whereas His substitution seems to largely destabilize the system. Subsequently, the qGlu-peptide was stripped from the full-length Skp1 protein to define a so-called self-inhibitory peptide, which may rebind to the domain-peptide complex interface and thus disrupt the complex interaction. Fluorescence polarization (FP) assays revealed that the Trp1159Phe and Trp1159Tyr variants have a comparable or higher affinity (K d  = 41 and 62 μM) than the wild-type qGlu-peptide (K d  = 56 μM), while the Trp1159His mutation would largely impair the binding potency of qGlu-peptide to Fbw7 F-box domain (K d  = 280 μM), confirming that the cation···π···π confers both affinity and specificity to the domain-peptide recognition, which can be reshaped by rational molecular design of the nonbonded interaction system. Graphical abstract Stereoview of the complex structure of Fbw7 with Skp1 (PDB: 2ovp), where the Trp1159 residue of Skp1 qGlu-peptide can form a cation···π···π stacking system with the Lys2299 and His2359 residues

F-box only protein 2 (Fbxo2) regulates amyloid precursor protein levels and processing.

PubMed

Atkin, Graham; Hunt, Jack; Minakawa, Eiko; Sharkey, Lisa; Tipper, Nathan; Tennant, William; Paulson, Henry L

2014-03-07

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

F-BOX proteins in cancer cachexia and muscle wasting: emerging regulators and therapeutic opportunities

PubMed Central

Sukari, Ammar; Muqbil, Irfana; Mohammad, Ramzi M.; Philip, Philip A.; Azmi, Asfar S.

2016-01-01

Cancer cachexia is a debilitating metabolic syndrome accounting for fatigue, an impairment of normal activities, loss of muscle mass associated with body weight loss eventually leading to death in majority of patients with advanced disease. Cachexia patients undergoing skeletal muscle atrophy show consistent activation of the SCF ubiquitin ligase (F-BOX) family member Atrogin-1 (also known as MAFBx/FBXO32) alongside the activation of the muscle ring finger protein1 (MuRF1). Other lesser known F-BOX family members are also emerging as key players supporting muscle wasting pathways. Recent work highlights a spectrum of different cancer signaling mechanisms impacting F-BOX family members that feed forward muscle atrophy related genes during cachexia. These novel players provide unique opportunities to block cachexia induced skeletal muscle atrophy by therapeutically targeting the SCF protein ligases. Conversely, strategies that induce the production of proteins may be helpful to counter the effects of these F-BOX proteins. Through this review, we bring forward some novel targets that promote atrogin-1 signaling in cachexia and muscle wasting and highlight newer therapeutic opportunities that can help in the better management of patients with this devastating and fatal disorder. PMID:26804424

Hippo Signaling Suppresses Cell Ploidy and Tumorigenesis through Skp2.

PubMed

Zhang, Shihao; Chen, Qinghua; Liu, Qingxu; Li, Yuxi; Sun, Xiufeng; Hong, Lixin; Ji, Suyuan; Liu, Chengyan; Geng, Jing; Zhang, Weiji; Lu, Zhonglei; Yin, Zhen-Yu; Zeng, Yuanyuan; Lin, Kwang-Huei; Wu, Qiao; Li, Qiyuan; Nakayama, Keiko; Nakayama, Keiich I; Deng, Xianming; Johnson, Randy L; Zhu, Liang; Gao, Daming; Chen, Lanfen; Zhou, Dawang

2017-05-08

Polyploidy can lead to aneuploidy and tumorigenesis. Here, we report that the Hippo pathway effector Yap promotes the diploid-polyploid conversion and polyploid cell growth through the Akt-Skp2 axis. Yap strongly induces the acetyltransferase p300-mediated acetylation of the E3 ligase Skp2 via Akt signaling. Acetylated Skp2 is exclusively localized to the cytosol, which causes hyper-accumulation of the cyclin-dependent kinase inhibitor p27, leading to mitotic arrest and subsequently cell polyploidy. In addition, the pro-apoptotic factors FoxO1/3 are overly degraded by acetylated Skp2, resulting in polyploid cell division, genomic instability, and oncogenesis. Importantly, the depletion or inactivation of Akt or Skp2 abrogated Hippo signal deficiency-induced liver tumorigenesis, indicating their epistatic interaction. Thus, we conclude that Hippo-Yap signaling suppresses cell polyploidy and oncogenesis through Skp2. Copyright © 2017 Elsevier Inc. All rights reserved.

Overexpression of SKP2 promotes the radiation resistance of esophageal squamous cell carcinoma.

PubMed

Wang, Xiao-Chun; Tian, Li-Li; Tian, Jing; Jiang, Xiao-Yan

2012-01-01

SKP2 is the substrate recognition subunit of the SCF(SKP2) ubiquitin ligase complex. It is implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase (CDK) inhibitor p27(KIP1) and positively regulates the G(1)/S transition. Overexpression of SKP2 has been found in many kinds of tumors. In the present study, we found that SKP2 expression levels increased in esophageal squamous cell carcinoma tissues. Elevated expression of SKP2 correlated significantly with tumor stage and positive lymph node metastasis (P < 0.05). Moreover, a significantly negative correlation was found between SKP2 expression and the survival of patients who received radiotherapy (P < 0.05). At the molecular level, induced expression of SKP2 promoted the radioresistance of EC9706 cells. Knockdown of SKP2 expression sensitized cancer cells to radiation, and a wobble mutant of SKP2 that was resistant to SKP2 siRNA was able to rescue this effect. Increased or decreased expression levels of SKP2 had effects on Rad51 expression after irradiation. These results demonstrate for the first time that overexpression of SKP2 was correlated with the increased radioresistance of esophageal squamous cell carcinoma. Elevated expression of SKP2 promoted the radioresistance of cancer cells, and this effect was mediated at least in part by the Rad51 pathway.

F-BOX proteins in cancer cachexia and muscle wasting: Emerging regulators and therapeutic opportunities.

PubMed

Sukari, Ammar; Muqbil, Irfana; Mohammad, Ramzi M; Philip, Philip A; Azmi, Asfar S

2016-02-01

Cancer cachexia is a debilitating metabolic syndrome accounting for fatigue, an impairment of normal activities, loss of muscle mass associated with body weight loss eventually leading to death in majority of patients with advanced disease. Cachexia patients undergoing skeletal muscle atrophy show consistent activation of the SCF ubiquitin ligase (F-BOX) family member Atrogin-1 (also known as MAFBx/FBXO32) alongside the activation of the muscle ring finger protein1 (MuRF1). Other lesser known F-BOX family members are also emerging as key players supporting muscle wasting pathways. Recent work highlights a spectrum of different cancer signaling mechanisms impacting F-BOX family members that feed forward muscle atrophy related genes during cachexia. These novel players provide unique opportunities to block cachexia induced skeletal muscle atrophy by therapeutically targeting the SCF protein ligases. Conversely, strategies that induce the production of proteins may be helpful to counter the effects of these F-BOX proteins. Through this review, we bring forward some novel targets that promote atrogin-1 signaling in cachexia and muscle wasting and highlight newer therapeutic opportunities that can help in the better management of patients with this devastating and fatal disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

Expression of p27 and its ubiquitin ligase subunit Skp2 in upper urinary tract transitional cell carcinoma.

PubMed

Langner, Cord; von Wasielewski, Reinhard; Ratschek, Manfred; Rehak, Peter; Zigeuner, Richard

2004-09-01

To analyze p27 and S-phase kinase-associated protein 2 (Skp2) expression in upper urinary tract transitional cell carcinoma (TCC) with respect to biologic significance. p27 (p27/kip1) is involved in cell cycle control, and loss of p27 protein expression may result in tumor development and/or progression. The association of p27 with the ubiquitin ligase subunit Skp2 targets p27 for degradation. A total of 53 upper urinary tract TCC specimens were investigated immunohistochemically using a tissue microarray technique. The immunoreactivity of p27 and Skp2 was analyzed with respect to associations with pT stage, grade, and prognosis. Non-neoplastic renal tissue showed p27 immunoreactivity in tubule epithelium and pelvic urothelium, but lacked immunoreactivity for Skp2. In the TCC specimens, p27 immunoreactivity was noted in 47 (89%) of 53 cases. High p27 expression (50% or greater of tumor cell nuclei) tended to decrease with rising tumor stage (14 [45%] of 31 with pT1-pT2 versus 4 [18%] of 22 with pT3; P = 0.076), but was independent of tumor grade (11 [39%] of 28 grade 2 versus 7 [28%] of 25 grade 3-4; P = 0.56). Skp2 immunoreactivity was noted in 32 (60%) of 53 tumors. Skp2 expression increased with rising tumor stage (9 [41%] of 22 pT1 versus 23 [74%] of 31 pT2-pT3; P = 0.023) and tumor grade (12 [43%] of 28 grade 2 versus 20 [80%] of 25 grade 3; P = 0.043) and was associated with angioinvasion (P = 0.017). In multivariate analysis, tumor stage proved to be the only independent prognostic factor regarding disease-free survival. p27 and Skp2 are additional biomarkers in urogenital pathologic findings. The statistically significant association of Skp2 expression with high-grade TCC, as well as the lack of expression in non-neoplastic tissue, suggests that Skp2 could be a promising target for future cancer therapy strategies.

SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

PubMed

Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

2009-08-01

S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

Erbin loss promotes cancer cell proliferation through feedback activation of Akt-Skp2-p27 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Hao; Laboratory of Cellular and Molecular Immunology, Medical School of Henan University, Kaifeng 475004; Song, Yuhua

2015-07-31

Erbin localizes at the basolateral membrane to regulate cell junctions and polarity in epithelial cells. Dysregulation of Erbin has been implicated in tumorigenesis, and yet it is still unclear if and how disrupted Erbin regulates the biological behavior of cancer cells. We report here that depletion of Erbin leads to cancer cell excessive proliferation in vitro and in vivo. Erbin deficiency accelerates S-phase entry by down-regulating CDK inhibitors p21 and p27 via two independent mechanisms. Mechanistically, Erbin loss promotes p27 degradation by enhancing E3 ligase Skp2 activity though augmenting Akt signaling. Interestingly, we also show that Erbin is an unstable protein whenmore » the Akt-Skp2 signaling is aberrantly activated, which can be specifically destructed by SCF-Skp2 ligase. Erbin loss facilitates cell proliferation and migration in Skp2-dependent manner. Thus, our finding illustrates a novel negative feedback loop between Erbin and Akt-Skp2 signaling. It suggests disrupted Erbin links polarity loss, hyperproliferation and tumorigenesis. - Highlights: • Erbin loss leads to cancer cell excessive proliferation in vitro and in vivo. • Erbin loss accelerates cell cycle though down-regulating p21 and p27 expression. • Erbin is a novel negative modulator of Akt1-Skp2-p27 signaling pathway. • Our study suggests that Erbin loss contributes to Skp2 oncogenic function.« less

Deuterium Labeling Together with Contrast Variation Small-angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins

PubMed Central

Zaccai, Nathan R.; Sandlin, Clifford W.; Hoopes, James T.; Curtis, Joseph E.; Fleming, Patrick J.; Fleming, Karen G.; Krueger, Susan

2016-01-01

In gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archeal prefoldins and the mitochondrial Tim chaperones, that is α-helical ‘tentacles’ extend from a β-strand ‘body’ to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis and structure modeling. PMID:26791979

Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27Kip1 Signaling Pathway

PubMed Central

Ke, Rui; Liu, Lu; Zhu, Yanting; Li, Shaojun; Xie, Xinming; Li, Fangwei; Song, Yang; Yang, Lan; Gao, Li; Li, Manxiang

2016-01-01

It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27Kip1. Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension. PMID:27258250

Apple F-Box Protein MdMAX2 Regulates Plant Photomorphogenesis and Stress Response.

PubMed

An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2016-01-01

MAX2 (MORE AXILLARY GROWTH2) is involved in diverse physiological processes, including photomorphogenesis, the abiotic stress response, as well as karrikin and strigolactone signaling-mediated shoot branching. In this study, MdMAX2, an F-box protein that is a homolog of Arabidopsis MAX2, was identified and characterized. Overexpression of MdMAX2 in apple calli enhanced the accumulation of anthocyanin. Ectopic expression of MdMAX2 in Arabidopsis exhibited photomorphogenesis phenotypes, including increased anthocyanin content and decreased hypocotyl length. Further study indicated that MdMAX2 might promote plant photomorphogenesis by affecting the auxin signaling as well as other plant hormones. Transcripts of MdMAX2 were noticeably up-regulated in response to NaCl and Mannitol treatments. Moreover, compared with the wild-type, the MdMAX2 -overexpressing apple calli and Arabidopsis exhibited increased tolerance to salt and drought stresses. Taken together, these results suggest that MdMAX2 plays a positive regulatory role in plant photomorphogenesis and stress response.

Deuterium Labeling Together with Contrast Variation Small-Angle Neutron Scattering Suggests How Skp Captures and Releases Unfolded Outer Membrane Proteins.

PubMed

Zaccai, Nathan R; Sandlin, Clifford W; Hoopes, James T; Curtis, Joseph E; Fleming, Patrick J; Fleming, Karen G; Krueger, Susan

2016-01-01

In Gram-negative bacteria, the chaperone protein Skp forms specific and stable complexes with membrane proteins while they are transported across the periplasm to the outer membrane. The jellyfish-like architecture of Skp is similar to the eukaryotic and archaeal prefoldins and the mitochondrial Tim chaperones, that is the α-helical "tentacles" extend from a β-strand "body" to create an internal cavity. Contrast variation small-angle neutron scattering (SANS) experiments on Skp alone in solution and bound in two different complexes to unfolded outer membrane proteins (uOMPs), OmpA and OmpW, demonstrate that the helical tentacles of Skp bind their substrate in a clamp-like mechanism in a conformation similar to that previously observed in the apo crystal structure of Skp. Deuteration of the uOMP component combined with contrast variation analysis allowed the shapes of Skp and uOMP as well as the location of uOMP with respect to Skp to be determined in both complexes. This represents unique information that could not be obtained without deuterium labeling of the uOMPs. The data yield the first direct structural evidence that the α-helical Skp tentacles move closer together on binding its substrate and that the structure of Skp is different when binding different uOMPs. This work presents, by example, a tutorial on performing SANS experiments using both deuterium labeling and contrast variation, including SANS theory, sample preparation, data collection, sample quality validation, data analysis, and structure modeling. © 2016 Elsevier Inc. All rights reserved.

The Skp1 Protein from Toxoplasma Is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium*

PubMed Central

Xu, Yuechi; Brown, Kevin M.; Wang, Zhuo A.; van der Wel, Hanke; Teygong, Crystal; Zhang, Dongmei; Blader, Ira J.; West, Christopher M.

2012-01-01

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O2 and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3SCF ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O2 sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O2 levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O2 requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O2. The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O2 by detecting the levels of O2-regulated metabolites such as α-ketoglutarate. PMID:22648409

Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.

PubMed

Brenner, Wolfram G; Leuendorf, Jan Erik; Cortleven, Anne; Martin, Laetitia B B; Schaller, Hubert; Schmülling, Thomas

2017-05-17

Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic cycloartenol synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

The retinoid X receptor agonist, 9-cis UAB30, inhibits cutaneous T-cell lymphoma proliferation through the SKP2-p27kip1 axis.

PubMed

Chou, Chu-Fang; Hsieh, Yu-Hua; Grubbs, Clinton J; Atigadda, Venkatram R; Mobley, James A; Dummer, Reinhard; Muccio, Donald D; Eto, Isao; Elmets, Craig A; Garvey, W Timothy; Chang, Pi-Ling

2018-06-01

Bexarotene (Targretin ® ) is currently the only FDA approved retinoid X receptor (RXR) -selective agonist for the treatment of cutaneous T-cell lymphomas (CTCLs). The main side effects of bexarotene are hypothyroidism and elevation of serum triglycerides (TGs). The novel RXR ligand, 9-cis UAB30 (UAB30) does not elevate serum TGs or induce hypothyroidism in normal subjects. To assess preclinical efficacy and mechanism of action of UAB30 in the treatment of CTCLs and compare its action with bexarotene. With patient-derived CTCL cell lines, we evaluated UAB30 function in regulating growth, apoptosis, cell cycle check points, and cell cycle-related markers. Compared to bexarotene, UAB30 had lower half maximal inhibitory concentration (IC 50 ) values and was more effective in inhibiting the G1 cell cycle checkpoint. Both rexinoids increased the stability of the cell cycle inhibitor, p27kip1 protein, in part, through targeting components involved in the ubiquitination-proteasome system: 1) decreasing SKP2, a F-box protein that binds and targets p27kip1 for degradation by 26S proteasome and 2) suppressing 20S proteasome activity (cell line-dependent) through downregulation of PSMA7, a component of the 20S proteolytic complex in 26S proteasome. UAB30 and bexarotene induce both early cell apoptosis and suppress cell proliferation. Inhibition of the G1 to S cell cycle transition by rexinoids is mediated, in part, through downregulation of SKP2 and/or 20S proteasome activity, leading to increased p27kip1 protein stability. Because UAB30 has minimal effect in elevating serum TGs and inducing hypothyroidism, it is potentially a better alternative to bexarotene for the treatment of CTCLs. Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Curcumin suppresses cell growth and invasion and induces apoptosis by down-regulation of Skp2 pathway in glioma cells

PubMed Central

Su, Jingna; Ma, Renqiang; Yin, Xuyuan; Zhou, Xiuxia; Li, Huabin; Wang, Zhiwei

2015-01-01

Studies have demonstrated that curcumin exerts its tumor suppressor function in a variety of human cancers including glioma. However, the exact underlying molecular mechanisms remain obscure. Emerging evidence has revealed that Skp2 (S-phase kinase associated protein 2) plays an oncogenic role in tumorigenesis. Therefore, we aim to determine whether curcumin suppresses the Skp2 expression, leading to the inhibition of cell growth, invasion, induction of apoptosis, and cell cycle arrest. To this end, we conducted multiple methods such as MTT assay, Flow cytometry, Wound healing assay, invasion assay, RT-PCR, Western blotting, and transfection to explore the functions and molecular insights of curcumin in glioma cells. We found that curcumin significantly inhibited cell growth, suppressed cell migration and invasion, induced apoptosis and cell cycle arrest in glioma cells. Furthermore, we observed that overexpression of Skp2 promoted cell growth, migration, and invasion, whereas depletion of Skp2 suppressed cell growth, migration, and invasion and triggered apoptosis in glioma cells. Mechanistically, we defined that curcumin markedly down-regulated Skp2 expression and subsequently up-regulated p57 expression. Moreover, our results demonstrated that curcumin exerts its antitumor activity through inhibition of Skp2 pathway. Collectively, our findings suggest that targeting Skp2 by curcumin could be a promising therapeutic approach for glioma prevention and therapy. PMID:26046466

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE PAGES

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.; ...

2016-03-03

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Dihong; Ni, Weimin; Stanley, Bruce A.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown. Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type,more » but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development. In conclusion, our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.« less

Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction.

PubMed

Singh, Rajinder; Sran, Arvinder; Carroll, David C; Huang, Jianing; Tsvetkov, Lyuben; Zhou, Xiulan; Sheung, Julie; McLaughlin, John; Issakani, Sarkiz D; Payan, Donald G; Shaw, Simon J

2015-11-15

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Genome-wide identification and characterisation of F-box family in maize.

PubMed

Jia, Fengjuan; Wu, Bingjiang; Li, Hui; Huang, Jinguang; Zheng, Chengchao

2013-11-01

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.

S locus-linked F-box genes expressed in anthers of Hordeum bulbosum.

PubMed

Kakeda, Katsuyuki

2009-09-01

Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S (3) haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S (3)) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.

Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells

PubMed Central

Li, Fanni; Dong, Xiwen; Lin, Peng; Jiang, Jianli

2018-01-01

The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21Cip1 and p27Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21Cip1 and p27Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR. PMID:29360760

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

PubMed

Tao, Tao; Zhou, Cui-Ji; Wang, Qian; Chen, Xiang-Ru; Sun, Qian; Zhao, Tian-Yu; Ye, Jian-Chun; Wang, Ying; Zhang, Zong-Ying; Zhang, Yong-Liang; Guo, Ze-Jian; Wang, Xian-Bing; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2017-01-01

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway

PubMed Central

Wang, Qian; Chen, Xiang-Ru; Sun, Qian; Zhao, Tian-Yu; Ye, Jian-Chun; Wang, Ying; Zhang, Zong-Ying; Zhang, Yong-Liang; Guo, Ze-Jian; Wang, Xian-Bing; Li, Da-Wei; Yu, Jia-Lin

2017-01-01

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection. PMID:28494021

Genome-wide survey and expression analysis of F-box genes in chickpea.

PubMed

Gupta, Shefali; Garg, Vanika; Kant, Chandra; Bhatia, Sabhyata

2015-02-13

The F-box genes constitute one of the largest gene families in plants involved in degradation of cellular proteins. F-box proteins can recognize a wide array of substrates and regulate many important biological processes such as embryogenesis, floral development, plant growth and development, biotic and abiotic stress, hormonal responses and senescence, among others. However, little is known about the F-box genes in the important legume crop, chickpea. The available draft genome sequence of chickpea allowed us to conduct a genome-wide survey of the F-box gene family in chickpea. A total of 285 F-box genes were identified in chickpea which were classified based on their C-terminal domain structures into 10 subfamilies. Thirteen putative novel motifs were also identified in F-box proteins with no known functional domain at their C-termini. The F-box genes were physically mapped on the 8 chickpea chromosomes and duplication events were investigated which revealed that the F-box gene family expanded largely due to tandem duplications. Phylogenetic analysis classified the chickpea F-box genes into 9 clusters. Also, maximum syntenic relationship was observed with soybean followed by Medicago truncatula, Lotus japonicus and Arabidopsis. Digital expression analysis of F-box genes in various chickpea tissues as well as under abiotic stress conditions utilizing the available chickpea transcriptome data revealed differential expression patterns with several F-box genes specifically expressing in each tissue, few of which were validated by using quantitative real-time PCR. The genome-wide analysis of chickpea F-box genes provides new opportunities for characterization of candidate F-box genes and elucidation of their function in growth, development and stress responses for utilization in chickpea improvement.

Apple EIN3 BINDING F-box 1 inhibits the activity of three apple EIN3-like transcription factors

PubMed Central

Tacken, Emma J.; Ireland, Hilary S.; Wang, Yen-Yi; Putterill, Jo; Schaffer, Robert J.

2012-01-01

Background and aims Fruit ripening in Malus× domestica (apple) is controlled by ethylene. Work in model species has shown that following the detection of ethylene, the ETHYLENE INSENSITIVE 3 (EIN3) transcription factor is stabilized, leading to an increase in transcript accumulation of ethylene-responsive genes, such as POLYGALACTURONASE1 (PG1). In the absence of ethylene, the EIN3 BINDING F-box (EBF) proteins rapidly degrade EIN3 via the ubiquitination/SCF (Skp, Cullin, F-Box) proteasome pathway. In this study, we aim to identify and characterize the apple EBF genes, and test their activity against apple EIN3-like proteins (EILs). Methodology The apple genome sequence was mined for EBF-like genes. The expression of EBF-like genes was measured during fruit development. Using a transient assay in Nicotiana benthamiana leaves, the activity of three apple EILs was tested against the PG1 promoter, with and without ethylene and EBF1. Principal results Four EBF-like genes in apple were identified and grouped into two sub-clades. Sub-clade I genes had constant expression over fruit development while sub-clade II genes increased in expression at ripening. EBF1 was shown to reduce the transactivation of the apple PG1 promoter by the EIL1, EIL2 and EIL3 transcription factors in the presence of ethylene. Conclusions The apple EBF1 gene identified here is likely to be a functionally conserved EBF orthologue, modulating EIL activity in apples. The activity of EBF1 suggests that it is not specific to a single EIL, instead acting as a global regulator of apple EIL transcription factors. PMID:23585922

Identification and Characterization of Components of a Putative Petunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility[W

PubMed Central

Hua, Zhihua; Kao, Teh-hui

2006-01-01

Petunia inflata S-locus F-box (Pi SLF) is thought to function as a typical F-box protein in ubiquitin-mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S-RNase but not self S-RNase. We isolated three P. inflata Skp1s (Pi SK1, -2, and -3), two Cullin-1s (Pi CUL1-C and -G), and an Rbx1 (Pi RBX1) cDNAs and found that Pi CUL1-G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF2. We also isolated a RING-HC protein, S-RNase Binding Protein1 (Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S-RNases, Pi CUL1-G, and an E2 ubiquitin-conjugating enzyme, suggesting that Pi CUL1-G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S-RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S-RNases than with self S-RNases. Bacterially expressed S1-, S2-, and S3-RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S-allele–specific manner. Native glycosylated S3-RNase is not degraded to any significant extent; however, deglycosylated S3-RNase is degraded as efficiently as the bacterially expressed S-RNases. Finally, S-RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF–containing E3 complex is unknown. PMID:17028207

Inhibiting the role of Skp2 suppresses cell proliferation and tumorigenesis of human gastric cancer cells via the upregulation of p27kip1.

PubMed

Wen, Yanguang; Wang, Kuansong; Yang, Kaiyan

2016-10-01

Gastric cancer is a malignant disease of the digestive system with high rates of incidence and mortality. S‑phase kinase‑associated protein 2 (Skp2) is a novel oncogene, which has been identified to be important in tumor progression and metastasis. In order to clarify the role of Skp2 in human gastric cancer, the present study detected the expression of Skp2 in human gastric cancer tissues, and investigated the molecular mechanism of Skp2 in the progression of gastric carcinoma. The results of the initial bioinformatics analysis showed that Skp2 was significantly upregulated in 31 specimens of primary gastric cancer from a UK patient cohort, and in 10 gastric cancer lines of a side population, compared with normal gastric tissues (P<0.01). Specimens from 47 patients with gastric cancer and 19 normal gastric tissue specimens were obtained and analyzed using western blot analysis. The positive rate of expression of Skp2 was 87.2%, indicating that the expression of Skp2 was observed in 41 specimens of the detected gastric cancer samples, whereas the positive rate of the expression of Skp2 was 5.6% in the normal gastric samples (P<0.01). In the human gastric cancer cell lines, the defective regulation of Skp2 or presence of an Skp2 inhibitor inhibited the proliferation of BGC‑823 and MKN‑45 cells. In addition, the Skp2 inhibitor suppressed the proliferation of gastric cancer cells in a time‑ and dose‑dependent manner. Furthermore, transfection with Skp2 short hairpin (sh)RNA or treatment with SKP inhibitor C1 for 48 and 72 h led to the accumulation of p27kip1 in Hela cells. Tumorigenicity experiments involving nude mice showed that interference of the expression of Skp2 inhibited the growth of the human gastric tumor cells in the nude mice, and the tumor weights and volumes in the Skp2 shRNA group were significantly lower, compared with those in the negative control shRNA group (P<0.01) and untreated group (P<0.01). Taken together, these data

Photoactivatable protein labeling by singlet oxygen mediated reactions.

PubMed

To, Tsz-Leung; Medzihradszky, Katalin F; Burlingame, Alma L; DeGrado, William F; Jo, Hyunil; Shu, Xiaokun

2016-07-15

Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover

PubMed Central

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J.; Shi, Yang; Harper, J. Wade

2014-01-01

Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

PubMed

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J; Shi, Yang; Harper, J Wade

2013-10-10

Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

A novel gene, MdSSK1, as a component of the SCF complex rather than MdSBP1 can mediate the ubiquitination of S-RNase in apple.

PubMed

Yuan, Hui; Meng, Dong; Gu, Zhaoyu; Li, Wei; Wang, Aide; Yang, Qing; Zhu, Yuandi; Li, Tianzhong

2014-07-01

As a core factor in S-RNase-based gametophytic self-incompatibility (GSI), the SCF (SKP1-Cullin1-F-box-Rbx1) complex (including pollen determinant SLF, S-locus-F-box) functions as an E3 ubiquitin ligase on non-self S-RNase. The SCF complex is formed by SKP1 bridging between SLF, CUL1, and Rbx1; however, it is not known whether an SCF complex lacking SKP1 can mediate the ubiquitination of S-RNase. Three SKP1-like genes from pollen were cloned based on the structural features of the SLF-interacting-SKP1-like (SSK) gene and the 'Golden Delicious' apple genome. These genes have a motif of five amino acids following the standard 'WAFE' at the C terminal and, in addition, contain eight sheets and two helices. All three genes were expressed exclusively in pollen. In the yeast two-hybrid and pull-down assays only one was found to interact with MdSFBB and MdCUL1, suggesting it is the SLF-interacting SKP1-like gene in apple which was named MdSSK1. In vitro experiments using MdSSK1, S2-MdSFBB1 (S2-Malus domestica S-locus-F-box brother) and MdCUL1 proteins incubated with S 2-RNase and ubiquitin revealed that the SCF complex ubiquitinylates S-RNase in vitro, while MdSBP1 (Malus domestica S-RNase binding protein 1) could not functionally replace MdSSK1 in the SCF complex in ubiquitinylating S-RNase. According to the above experiments, MdSBP1 is probably the only factor responsible for recognition with S-RNase, while not a component of the SCF complex, and an SCF complex containing MdSSK1 is required for mediating the ubiquitination of S-RNase. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Vaccinia Virus C9 Ankyrin Repeat/F-Box Protein Is a Newly Identified Antagonist of the Type I Interferon-Induced Antiviral State.

PubMed

Liu, Ruikang; Moss, Bernard

2018-05-01

Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells coevolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pretreated with beta interferon (IFN-β), suggesting that one or more of the missing 17 open reading frames (ORFs) encode an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN resistance. Replication of the C9L deletion mutant (vΔC9) was impaired in human cells that had been pretreated with IFN-β. Expression of viral early genes occurred, but subsequent events, including genome uncoating, genome replication, and postreplicative gene expression, were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 contains six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that copurified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins. IMPORTANCE Poxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds, and mammals, all

Roles of F-box proteins in human digestive system tumors (Review).

PubMed

Gong, Jian; Lv, Liang; Huo, Jirong

2014-12-01

F-box proteins (FBPs), the substrate-recognition subunit of E3 ubiquitin (Ub) ligase, are the important components of Ub proteasome system (UPS). FBPs are involved in multiple cellular processes through ubiquitylation and subsequent degradation of their target proteins. Many studies have described the roles of FBPs in human cancers. Digestive system tumors account for a large proportion of all the tumors, and their mortality is very high. This review summarizes for the first time the roles of FBPs in digestive system tumorige-nesis and tumor progression, aiming at finding new routes for the rational design of targeted anticancer therapies in digestive system tumors.

The rice F-box protein KISS ME DEADLY2 functions as a negative regulator of cytokinin signalling.

PubMed

Kim, Hyo Jung; Kieber, Joseph J; Schaller, G Eric

2013-01-01

Cytokinins are plant hormones that play critical roles in growth and development. We recently determined that the transcriptional response to cytokinin of Arabidopsis is modulated by the KISS ME DEADLY (KMD) family of F-box proteins. Here we demonstrate a conserved function for a member of the rice KMD family. Ectopic overexpression of OsKMD2 in Arabidopsis results in decreased cytokinin sensitivity based on a hypocotyl growth response assay, the decrease in sensitivity correlating with a decrease in the levels of the transcriptional regulator AtARR12. Furthermore, OsKMD2 directly interacts with AtARR12 based on yeast two-hybrid and co-immunoprecipitation assays. These results indicate that both monocots and dicots employ a similar KMD-dependent mechanism to regulate the transcriptional response to cytokinin.

KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1

USDA-ARS?s Scientific Manuscript database

Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here, we show that the Arabidopsis F-Box protein Cold Temperature-Germinating (CTG)-10, identified by activation tagging, is a positive regulator during this p...

F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana

PubMed Central

Nelson, David C.; Scaffidi, Adrian; Dun, Elizabeth A.; Waters, Mark T.; Flematti, Gavin R.; Dixon, Kingsley W.; Beveridge, Christine A.; Ghisalberti, Emilio L.; Smith, Steven M.

2011-01-01

Smoke is an important abiotic cue for plant regeneration in postfire landscapes. Karrikins are a class of compounds discovered in smoke that promote seed germination and influence early development of many plants by an unknown mechanism. A genetic screen for karrikin-insensitive mutants in Arabidopsis thaliana revealed that karrikin signaling requires the F-box protein MAX2, which also mediates responses to the structurally-related strigolactone family of phytohormones. Karrikins and the synthetic strigolactone GR24 trigger similar effects on seed germination, seedling photomorphogenesis, and expression of a small set of genes during these developmental stages. Karrikins also repress MAX4 and IAA1 transcripts, which show negative feedback regulation by strigolactone. We demonstrate that all of these common responses are abolished in max2 mutants. Unlike strigolactones, however, karrikins do not inhibit shoot branching in Arabidopsis or pea, indicating that plants can distinguish between these signals. These results suggest that a MAX2-dependent signal transduction mechanism was adapted to mediate responses to two chemical cues with distinct roles in plant ecology and development. PMID:21555559

F-box protein MAX2 has dual roles in karrikin and strigolactone signaling in Arabidopsis thaliana.

PubMed

Nelson, David C; Scaffidi, Adrian; Dun, Elizabeth A; Waters, Mark T; Flematti, Gavin R; Dixon, Kingsley W; Beveridge, Christine A; Ghisalberti, Emilio L; Smith, Steven M

2011-05-24

Smoke is an important abiotic cue for plant regeneration in postfire landscapes. Karrikins are a class of compounds discovered in smoke that promote seed germination and influence early development of many plants by an unknown mechanism. A genetic screen for karrikin-insensitive mutants in Arabidopsis thaliana revealed that karrikin signaling requires the F-box protein MAX2, which also mediates responses to the structurally-related strigolactone family of phytohormones. Karrikins and the synthetic strigolactone GR24 trigger similar effects on seed germination, seedling photomorphogenesis, and expression of a small set of genes during these developmental stages. Karrikins also repress MAX4 and IAA1 transcripts, which show negative feedback regulation by strigolactone. We demonstrate that all of these common responses are abolished in max2 mutants. Unlike strigolactones, however, karrikins do not inhibit shoot branching in Arabidopsis or pea, indicating that plants can distinguish between these signals. These results suggest that a MAX2-dependent signal transduction mechanism was adapted to mediate responses to two chemical cues with distinct roles in plant ecology and development.

An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development.

PubMed

Li, Li; Li, Yixing; Song, Shufeng; Deng, Huafeng; Li, Na; Fu, Xiqin; Chen, Guanghui; Yuan, Longping

2015-01-01

In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

USDA-ARS?s Scientific Manuscript database

Ubiquitylation, which regulates most biological pathways, occurs through an enzymatic cascade involving a ubiquitin (ub) activating enzyme (E1), a ub conjugating enzyme (E2) and a ub ligase (E3). UbcH3 is the E2 that interacts with SCF (Skp1/Cul1/F-box protein) complex and ubiquitylates many protein...

Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

PubMed

Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

2004-06-18

Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

The differential susceptibilities of MCF-7 and MDA-MB-231 cells to the cytotoxic effects of curcumin are associated with the PI3K/Akt-SKP2-Cip/Kips pathway.

PubMed

Jia, Tao; Zhang, Li; Duan, Yale; Zhang, Min; Wang, Gang; Zhang, Jun; Zhao, Zheng

2014-01-01

The mechanism underlying the differential cytotoxicity of curcumin in various cancer types, however, remains largely unclear. The aims of this study is to examine the concentration- and time-related effects of curcumin on two different breast cancer cells, MCF-7 and MDA-MB-231, and investigated the functional changes induced by curcumin treatment, as well as their relationship to the PI3K/Akt-SKP2-Cip/Kips pathway. First, WST-1 and clonogenic assay were performed to determine the cytotoxicity of curcumin in MCF-7 and MDA-MB-231 cells. Then, the expression of CDK interacting protein/Kinase inhibitory protein (Cip/Kips) members (p27, p21 and p57) and S-phase kinase-associated protein-2 (SKP2) was investigated by QRT PCR and Western Blotting. Curcumin's effect on PI3K (phosphatidylinositol 3-kinase) /Akt and its substrates Foxo1 and Foxo3a were then studied by Western Blotting. Small interfering RNAs (siRNAs) targeting SKP2 was used to explore the relationship between SKP2 and Cip/Kips members. Finally, WST-1 assay was tested to explore the concomitant treatment with curcumin and the inhibition of PKB or SKP2 signaling on curcumin sensitivity in MCF-7 and MDA-MB-231 cells. We demonstrated MCF-7 and MDA-MB-231 cells exhibited differential responses to curcumin by WST-1 and clonogenic assay (MDA-MB-231 cells was sensitive, and MCF-7 cells was resistant), which were found to be related to the differential curcumin-mediated regulation of SKP2-Cip/Kips (p21 and p27 but not p57) signaling. The differential cellular responses were further linked to the converse effects of curcumin on PI3K/Akt and its substrates Foxo1 and Foxo3a. Importantly, PI3K inhibitor wortmannin could counteract both curcumin-induced phosphorylation of Akt and up-regulation of SKP2 in MCF-7 cells. Subsequent WST-1 assay demonstrated concomitant treatment with curcumin and wortmannin or SKP2 siRNA not only further augmented curcumin sensitivity in MDA-MB-231 cells but also overcame curcumin resistance in

FBXL5 interacts with p150 {sup Glued} and regulates its ubiquitination

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang Ning; Liu Jing; Ding Xia

2007-07-20

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. p150 {sup Glued} is the dynactin subunit responsible for binding to dynein and microtubules. The F-box proteins constitute one of the four subunits of ubiquitin protein ligase complex called SCFs (SKP1-cullin-F-box), which governs phosphorylation-dependent ubiquitination and subsequent proteolysis. Our recent study showed that the proteolysis of mitotic kinesin CENP-E is mediated by SCF via a direct Skp1 link [D. Liu, N. Zhang, J. Du, X. Cai, M. Zhu, C. Jin, Z. Dou, C. Feng, Y. Yang, L. Liu, K. Takeyasu, W. Xie, X. Yao,more » Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis, Biochem. Biophys. Res. Commun. 345 (2006) 394-402]. Here we show that F-box protein FBXL5 interacts with p150 {sup Glued} and orchestrates its turnover via ubiquitination. FBXL5 binds to p150 {sup Glued} in vitro and in vivo. FBXL5 and p150 {sup Glued} co-localize primarily in the cytoplasm with peri-nuclear enrichment in HeLa cells. Overexpression of FBXL5 promotes poly-ubiquitination of p150 {sup Glued} and protein turnover of p150 {sup Glued} . Our findings provide a potential mechanism by which p150 {sup Glued} protein function is regulated by SCFs.« less

Retinoic acid downregulates Rae1 leading to APC(Cdh1) activation and neuroblastoma SH-SY5Y differentiation.

PubMed

Cuende, J; Moreno, S; Bolaños, J P; Almeida, A

2008-05-22

In neuroblastoma cells, retinoic acid induces cell cycle arrest and differentiation through degradation of the F-box protein, Skp2, and stabilization of cyclin-dependent kinase inhibitor, p27. However, the mechanism responsible for retinoic acid-mediated Skp2 destabilization is unknown. Since Skp2 is degraded by anaphase-promoting complex (APC)(Cdh1), here we studied whether retinoic acid promotes differentiation of human SH-SY5Y neuroblastoma cells by modulating Cdh1. We found that retinoic acid induced the nuclear accumulation of Cdh1 that paralleled Skp2 destabilization and p27 accumulation. The mRNA and protein abundance of Rae1-a nuclear export factor that limits APC(Cdh1) activity in mitosis-decreased upon retinoic acid-induced inhibition of neuroblastoma cell proliferation. Furthermore, either Rae1 overexpression or Cdh1 inhibition promoted Skp2 accumulation, p27 destabilization and prevented retinoic acid-induced cell cycle arrest and differentiation. Conversely, inhibition of Rae1 accelerated retinoic acid-induced differentiation. Thus, retinoic acid downregulates Rae1, hence facilitating APC(Cdh1)-mediated Skp2 degradation leading to the arrest of cell cycle progression and neuroblastoma differentiation.

A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

PubMed

Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R

2014-02-01

Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

PubMed

Miguel-Rojas, Cristina; Hera, Concepcion

2016-01-01

F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development.

PubMed

Chae, Eunyoung; Tan, Queenie K-G; Hill, Theresa A; Irish, Vivian F

2008-04-01

Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation, indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species.

Identification of FBXO25-interacting Proteins Using an Integrated Proteomics Approach

PubMed Central

Teixeira, Felipe R.; Yokoo, Sami; Gartner, Carlos G.; Manfiolli, Adriana O.; Baqui, Munira M. A.; Assmann, Eliana M.; Maragno, Ana Leticia G. C.; Yu, Huijun; de Lanerolle, Primal; Kobarg, Jörg; Gygi, Steven P.; Gomes, Marcelo D.

2011-01-01

FBXO25 is one of 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of Skp1, Rbx1, Cullin1 and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FANDs). Combining two-step affinity purification followed by mass spectrometry with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, β-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FANDs, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions. PMID:20473970

SCF(SAP) controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana.

PubMed

Wang, Zhibiao; Li, Na; Jiang, Shan; Gonzalez, Nathalie; Huang, Xiahe; Wang, Yingchun; Inzé, Dirk; Li, Yunhai

2016-04-06

Control of organ size by cell proliferation and growth is a fundamental process, but the mechanisms that determine the final size of organs are largely elusive in plants. We have previously revealed that the ubiquitin receptor DA1 regulates organ size by repressing cell proliferation in Arabidopsis. Here we report that a mutant allele of STERILE APETALA (SAP) suppresses the da1-1 mutant phenotype. We show that SAP is an F-box protein that forms part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex and controls organ size by promoting the proliferation of meristemoid cells. Genetic analyses suggest that SAP may act in the same pathway with PEAPOD1 and PEAPOD2, which are negative regulators of meristemoid proliferation, to control organ size, but does so independently of DA1. Further results reveal that SAP physically associates with PEAPOD1 and PEAPOD2, and targets them for degradation. These findings define a molecular mechanism by which SAP and PEAPOD control organ size.

Measuring Helicase Inhibition of the DEAD-box Protein Dbp2 by Yra1

PubMed Central

Ma, Wai Kit; Tran, Elizabeth J.

2016-01-01

Despite the highly conserved helicase core, individual DEAD-box proteins are specialized in diverse RNA metabolic processes. One mechanism that determines DEAD-box protein specificity is enzymatic regulation by other protein cofactors. In this chapter, we describe a protocol for purifying the Saccharomyces cerevisiae DEAD-box RNA helicase Dbp2 and RNA-binding protein Yra1 and subsequent analysis of helicase regulation. The experiments described here can be adapted to RNA helicase and purified co-factor. PMID:25579587

Evaluation of genetic and metabolic role of SKIP11 in Arabidopsis thaliana

NASA Astrophysics Data System (ADS)

Hassan, Muhammad Naeem ul; Ismail, Ismanizan

2015-09-01

Most of the regulatory proteins are degraded by 26S proteasome complex, only when they are tagged by Ubiquitin. A complex of four proteins, SKP1-Cullin-Ring box-F box (SCF) catalyses the final step to link the Ubiquitin tag with the target proteins. SCF complex interacts with the target proteins through F-box proteins, which confer the overall substrate specificity to the complex. F-box proteins, one of the largest family of proteins in plants have an N-terminal F-box domain and variable C-terminal domains, like leucine-rich repeat, WD-40 repeat and the kelch-repeat domains. In this study, we analysed the role of SKIP11, a kelch containing F-box protein (KFB) from Arabidopsis thaliana, by using reverse genetics strategy. The results show that SKIP11 is involved in the down-regulation of oxylipin pathway, possibly through the degradation of enzymes or/ and the regulatory factors of the pathway.

F-box genes: Genome-wide expansion, evolution and their contribution to pollen growth in pear (Pyrus bretschneideri).

PubMed

Wang, Guo-Ming; Yin, Hao; Qiao, Xin; Tan, Xu; Gu, Chao; Wang, Bao-Hua; Cheng, Rui; Wang, Ying-Zhen; Zhang, Shao-Ling

2016-12-01

F-box gene family, as one of the largest gene families in plants, plays crucial roles in regulating plant development, reproduction, cellular protein degradation and responses to biotic and abiotic stresses. However, comprehensive analysis of the F-box gene family in pear (Pyrus bretschneideri Rehd.) and other Rosaceae species has not been reported yet. Herein, we identified a total of 226 full-length F-box genes in pear for the first time. And these genes were further divided into various subgroups based on specific domains and phylogenetic analysis. Intriguingly, we observed that whole-genome duplication and dispersed duplication have a major contribution to F-box family expansion. Furthermore, the dynamic evolution for different modes of gene duplication was dissected. Interestingly, we found that dispersed and tandem duplicate have been evolving at a high rate. In addition, we found that F-box genes exhibited functional specificity based on GO analysis, and most of the F-box genes were significantly enriched in the protein binding (GO: 0005515) term, supporting that F-box genes might play a critical role for gene regulation in pear. Transcriptome and digital expression profiles revealed that F-box genes are involved in the development of multiple pear tissues. Overall, these results will set stage for elaborating the biological role of F-box genes in pear and other plants. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The involvement of wheat F-box protein gene TaFBA1 in the oxidative stress tolerance of plants.

PubMed

Zhou, Shu-Mei; Kong, Xiang-Zhu; Kang, Han-Han; Sun, Xiu-Dong; Wang, Wei

2015-01-01

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants' tolerance to multiple stress conditions.

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

PubMed Central

Zhou, Shu-Mei; Kong, Xiang-Zhu; Kang, Han-Han; Sun, Xiu-Dong; Wang, Wei

2015-01-01

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions. PMID:25906259

SKP2- and OTUD1-regulated non-proteolytic ubiquitination of YAP promotes YAP nuclear localization and activity.

PubMed

Yao, Fan; Zhou, Zhicheng; Kim, Jongchan; Hang, Qinglei; Xiao, Zhenna; Ton, Baochau N; Chang, Liang; Liu, Na; Zeng, Liyong; Wang, Wenqi; Wang, Yumeng; Zhang, Peijing; Hu, Xiaoyu; Su, Xiaohua; Liang, Han; Sun, Yutong; Ma, Li

2018-06-11

Dysregulation of YAP localization and activity is associated with pathological conditions such as cancer. Although activation of the Hippo phosphorylation cascade is known to cause cytoplasmic retention and inactivation of YAP, emerging evidence suggests that YAP can be regulated in a Hippo-independent manner. Here, we report that YAP is subject to non-proteolytic, K63-linked polyubiquitination by the SCF SKP2 E3 ligase complex (SKP2), which is reversed by the deubiquitinase OTUD1. The non-proteolytic ubiquitination of YAP enhances its interaction with its nuclear binding partner TEAD, thereby inducing YAP's nuclear localization, transcriptional activity, and growth-promoting function. Independently of Hippo signaling, mutation of YAP's K63-linkage specific ubiquitination sites K321 and K497, depletion of SKP2, or overexpression of OTUD1 retains YAP in the cytoplasm and inhibits its activity. Conversely, overexpression of SKP2 or loss of OTUD1 leads to nuclear localization and activation of YAP. Altogether, our study sheds light on the ubiquitination-mediated, Hippo-independent regulation of YAP.

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCFVBF ubiquitin–ligase complex pathway

PubMed Central

Zaltsman, Adi; Lacroix, Benoît; Gafni, Yedidya; Citovsky, Vitaly

2013-01-01

One the most intriguing, yet least studied, aspects of the bacterium–host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA—as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins—into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity. PMID:23248273

Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress

PubMed Central

Jia, Qi; Xiao, Zhi-Xia; Wong, Fuk-Ling; Sun, Song; Liang, Kang-Jing; Lam, Hon-Ming

2017-01-01

The F-box family is one of the largest gene families in plants that regulate diverse life processes, including salt responses. However, the knowledge of the soybean F-box genes and their roles in salt tolerance remains limited. Here, we conducted a genome-wide survey of the soybean F-box family, and their expression analysis in response to salinity via in silico analysis of online RNA-sequencing (RNA-seq) data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to predict their potential functions. A total of 725 potential F-box proteins encoded by 509 genes were identified and classified into 9 subfamilies. The gene structures, conserved domains and chromosomal distributions were characterized. There are 76 pairs of duplicate genes identified, including genome-wide segmental and tandem duplication events, which lead to the expansion of the number of F-box genes. The in silico expression analysis showed that these genes would be involved in diverse developmental functions and play an important role in salt response. Our qRT-PCR analysis confirmed 12 salt-responding F-box genes. Overall, our results provide useful information on soybean F-box genes, especially their potential roles in salt tolerance. PMID:28417911

Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress.

PubMed

Jia, Qi; Xiao, Zhi-Xia; Wong, Fuk-Ling; Sun, Song; Liang, Kang-Jing; Lam, Hon-Ming

2017-04-12

The F-box family is one of the largest gene families in plants that regulate diverse life processes, including salt responses. However, the knowledge of the soybean F-box genes and their roles in salt tolerance remains limited. Here, we conducted a genome-wide survey of the soybean F-box family, and their expression analysis in response to salinity via in silico analysis of online RNA-sequencing (RNA-seq) data and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to predict their potential functions. A total of 725 potential F-box proteins encoded by 509 genes were identified and classified into 9 subfamilies. The gene structures, conserved domains and chromosomal distributions were characterized. There are 76 pairs of duplicate genes identified, including genome-wide segmental and tandem duplication events, which lead to the expansion of the number of F-box genes. The in silico expression analysis showed that these genes would be involved in diverse developmental functions and play an important role in salt response. Our qRT-PCR analysis confirmed 12 salt-responding F-box genes. Overall, our results provide useful information on soybean F-box genes, especially their potential roles in salt tolerance.

SCFSAP controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana

PubMed Central

Wang, Zhibiao; Li, Na; Jiang, Shan; Gonzalez, Nathalie; Huang, Xiahe; Wang, Yingchun; Inzé, Dirk; Li, Yunhai

2016-01-01

Control of organ size by cell proliferation and growth is a fundamental process, but the mechanisms that determine the final size of organs are largely elusive in plants. We have previously revealed that the ubiquitin receptor DA1 regulates organ size by repressing cell proliferation in Arabidopsis. Here we report that a mutant allele of STERILE APETALA (SAP) suppresses the da1-1 mutant phenotype. We show that SAP is an F-box protein that forms part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex and controls organ size by promoting the proliferation of meristemoid cells. Genetic analyses suggest that SAP may act in the same pathway with PEAPOD1 and PEAPOD2, which are negative regulators of meristemoid proliferation, to control organ size, but does so independently of DA1. Further results reveal that SAP physically associates with PEAPOD1 and PEAPOD2, and targets them for degradation. These findings define a molecular mechanism by which SAP and PEAPOD control organ size. PMID:27048938

SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis

PubMed Central

Jia, Fengjuan; Wang, Chunyan; Huang, Jinguang; Yang, Guodong; Wu, Changai; Zheng, Chengchao

2015-01-01

Skp1–Cullin–F-box (SCF) E3 ligases are essential to the post-translational regulation of many important factors involved in cellular signal transduction. In this study, we identified an F-box protein from Arabidopsis thaliana, AtPP2-B11, which was remarkably induced with increased duration of salt treatment in terms of both transcript and protein levels. Transgenic Arabidopsis plants overexpressing AtPP2-B11 exhibited obvious tolerance to high salinity, whereas the RNA interference line was more sensitive to salt stress than wild-type plants. Isobaric tag for relative and absolute quantification analysis revealed that 4311 differentially expressed proteins were regulated by AtPP2-B11 under salt stress. AtPP2-B11 could upregulate the expression of annexin1 (AnnAt1) and function as a molecular link between salt stress and reactive oxygen species accumulation in Arabidopsis. Moreover, AtPP2-B11 influenced the expression of Na+ homeostasis genes under salt stress, and the AtPP2-B11 overexpressing lines exhibited lower Na+ accumulation. These results suggest that AtPP2-B11 functions as a positive regulator in response to salt stress in Arabidopsis. PMID:26041321

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

PubMed Central

Yoshida, Yukiko; Yasuda, Sayaka; Fujita, Toshiharu; Hamasaki, Maho; Murakami, Arisa; Kawawaki, Junko; Iwai, Kazuhiro; Saeki, Yasushi; Yoshimori, Tamotsu; Matsuda, Noriyuki; Tanaka, Keiji

2017-01-01

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy. PMID:28743755

Assaying Auxin Receptor Activity Using SPR Assays with F-Box Proteins and Aux/IAA Degrons.

PubMed

Quareshy, Mussa; Uzunova, Veselina; Prusinska, Justyna M; Napier, Richard M

2017-01-01

The identification of TIR1 as an auxin receptor combined with advanced biophysical instrumentation has led to the development of real-time activity assays for auxins. Traditionally, molecules have been assessed for auxinic activity using bioassays, and agrochemical compound discovery continues to be based on "spray and pray" technologies. Here, we describe the methodology behind an SPR-based assay that uses TIR1 and related F-box proteins with surface plasmon resonance spectrometry for rapid compound screening. In addition, methods for collecting kinetic binding data and data processing are given so that they may support programs for rational design of novel auxin ligands.

Strigolactone- and Karrikin-Independent SMXL Proteins Are Central Regulators of Phloem Formation.

PubMed

Wallner, Eva-Sophie; López-Salmerón, Vadir; Belevich, Ilya; Poschet, Gernot; Jung, Ilona; Grünwald, Karin; Sevilem, Iris; Jokitalo, Eija; Hell, Rüdiger; Helariutta, Yrjö; Agustí, Javier; Lebovka, Ivan; Greb, Thomas

2017-04-24

Plant stem cell niches, the meristems, require long-distance transport of energy metabolites and signaling molecules along the phloem tissue. However, currently it is unclear how specification of phloem cells is controlled. Here we show that the genes SUPPRESSOR OF MAX2 1-LIKE3 (SMXL3), SMXL4, and SMXL5 act as cell-autonomous key regulators of phloem formation in Arabidopsis thaliana. The three genes form an uncharacterized subclade of the SMXL gene family that mediates hormonal strigolactone and karrikin signaling. Strigolactones are endogenous signaling molecules regulating shoot and root branching [1] whereas exogenous karrikin molecules induce germination after wildfires [2]. Both activities depend on the F-box protein and SCF (Skp, Cullin, F-box) complex component MORE AXILLARY GROWTH2 (MAX2) [3-5]. Strigolactone and karrikin perception leads to MAX2-dependent degradation of distinct SMXL protein family members, which is key for mediating hormonal effects [6-12]. However, the nature of events immediately downstream of SMXL protein degradation and whether all SMXL proteins mediate strigolactone or karrikin signaling is unknown. In this study we demonstrate that, within the SMXL gene family, specifically SMXL3/4/5 deficiency results in strong defects in phloem formation, altered sugar accumulation, and seedling lethality. By comparing protein stabilities, we show that SMXL3/4/5 proteins function differently to canonical strigolactone and karrikin signaling mediators, although being functionally interchangeable with those under low strigolactone/karrikin signaling conditions. Our observations reveal a fundamental mechanism of phloem formation and indicate that diversity of SMXL protein functions is essential for a steady fuelling of plant meristems. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner

PubMed Central

Yumimoto, Kanae; Akiyoshi, Sayuri; Ueo, Hiroki; Sagara, Yasuaki; Onoyama, Ichiro; Ueo, Hiroaki; Ohno, Shinji; Mori, Masaki; Mimori, Koshi; Nakayama, Keiichi I.

2015-01-01

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis. PMID:25555218

SCFAtPP2-B11 modulates ABA signaling by facilitating SnRK2.3 degradation in Arabidopsis thaliana

PubMed Central

Ren, Ziyin; Zhi, Liya; Yao, Bin; Su, Chao; Liu, Liu; Li, Xia

2017-01-01

The phytohormone abscisic acid (ABA) is an essential part of the plant response to abiotic stressors such as drought. Upon the perception of ABA, pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) proteins interact with co-receptor protein phosphatase type 2Cs to permit activation Snf1-related protein kinase2 (SnRK2) kinases, which switch on ABA signaling by phosphorylating various target proteins. Thus, SnRK2 kinases are central regulators of ABA signaling. However, the mechanisms that regulate SnRK2 degradation remain elusive. Here, we show that SnRK2.3 is degradated by 26S proteasome system and ABA promotes its degradation. We found that SnRK2.3 interacts with AtPP2-B11 directly. AtPP2-B11 is an F-box protein that is part of a SKP1/Cullin/F-box E3 ubiquitin ligase complex that negatively regulates plant responses to ABA by specifically promoting the degradation of SnRK2.3. AtPP2-B11 was induced by ABA, and the knockdown of AtPP2-B11 expression markedly increased the ABA sensitivity of plants during seed germination and postgerminative development. Overexpression of AtPP2-B11 does not affect ABA sensitivity, but inhibits the ABA hypersensitive phenotypes of SnRK2.3 overexpression lines. These results reveal a novel mechanism through which AtPP2-B11 specifically degrades SnRK2.3 to attenuate ABA signaling and the abiotic stress response in Arabidopsis. PMID:28787436

F2Dock: Fast Fourier Protein-Protein Docking

PubMed Central

Bajaj, Chandrajit; Chowdhury, Rezaul; Siddavanahalli, Vinay

2009-01-01

The functions of proteins is often realized through their mutual interactions. Determining a relative transformation for a pair of proteins and their conformations which form a stable complex, reproducible in nature, is known as docking. It is an important step in drug design, structure determination and understanding function and structure relationships. In this paper we extend our non-uniform fast Fourier transform docking algorithm to include an adaptive search phase (both translational and rotational) and thereby speed up its execution. We have also implemented a multithreaded version of the adaptive docking algorithm for even faster execution on multicore machines. We call this protein-protein docking code F2Dock (F2 = Fast Fourier). We have calibrated F2Dock based on an extensive experimental study on a list of benchmark complexes and conclude that F2Dock works very well in practice. Though all docking results reported in this paper use shape complementarity and Coulombic potential based scores only, F2Dock is structured to incorporate Lennard-Jones potential and re-ranking docking solutions based on desolvation energy. PMID:21071796

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

PubMed

Lopez-Obando, Mauricio; de Villiers, Ruan; Hoffmann, Beate; Ma, Linnan; de Saint Germain, Alexandre; Kossmann, Jens; Coudert, Yoan; Harrison, C Jill; Rameau, Catherine; Hills, Paul; Bonhomme, Sandrine

2018-05-21

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors. © 2018 INRA New Phytologist © 2018 New Phytologist Trust.

Hepatic F-Box Protein FBXW7 Maintains Glucose Homeostasis Through Degradation of Fetuin-A.

PubMed

Zhao, Jiejie; Xiong, Xuelian; Li, Yao; Liu, Xing; Wang, Tao; Zhang, Hong; Jiao, Yang; Jiang, Jingjing; Zhang, Huijie; Tang, Qiqun; Gao, Xin; Li, Xuejun; Lu, Yan; Liu, Bin; Hu, Cheng; Li, Xiaoying

2018-05-01

Type 2 diabetes mellitus (T2DM) has become one of the most serious and long-term threats to human health. However, the molecular mechanism that links obesity to insulin resistance remains largely unknown. Here, we show that F-box and WD repeat domain-containing 7 (FBXW7), an E3 ubiquitin protein ligase, is markedly downregulated in the liver of two obese mouse models and obese human subjects. We further identify a functional low-frequency human FBXW7 coding variant (p.Ala204Thr) in the Chinese population, which is associated with elevated blood glucose and T2DM risk. Notably, mice with liver-specific knockout of FBXW7 develop hyperglycemia, glucose intolerance, and insulin resistance even on a normal chow diet. Conversely, overexpression of FBXW7 in the liver not only prevents the development of high-fat diet-induced insulin resistance but also attenuates the disease signature of obese mice. Mechanistically, FBXW7 directly binds to hepatokine fetuin-A to induce its ubiquitination and subsequent proteasomal degradation, comprising an important mechanism maintaining glucose homeostasis. Thus, we provide evidence showing a beneficial role of FBXW7 in glucose homeostasis. © 2018 by the American Diabetes Association.

Functional roles and efficiencies of the thioredoxin boxes of calcium-binding proteins 1 and 2 in protein folding.

PubMed Central

Kramer, B; Ferrari, D M; Klappa, P; Pöhlmann, N; Söling, H D

2001-01-01

The rat luminal endoplasmic-recticulum calcium-binding proteins 1 and 2 (CaBP1 and CaBP2 respectively) are members of the protein disulphide-isomerase (PDI) family. They contain two and three thioredoxin boxes (Cys-Gly-His-Cys) respectively and, like PDI, may be involved in the folding of nascent proteins. We demonstrate here that CaBP1, similar to PDI and CaBP2, can complement the lethal phenotype of the disrupted Saccharomyces cerevisiae PDI gene, provided that the natural C-terminal Lys-Asp-Glu-Leu sequence is replaced by His-Asp-Glu-Leu. Both the in vitro RNase AIII-re-activation assays and in vivo pro-(carboxypeptidase Y) processing assays using CaBP1 and CaBP2 thioredoxin (trx)-box mutants revealed that, whereas the three trx boxes in CaBP2 seem to be functionally equivalent, the first trx box of CaBP1 is significantly more active than the second trx box. Furthermore, only about 65% re-activation of denatured reduced RNase AIII could be obtained with CaBP1 or CaBP2 compared with PDI, and the yield of PDI-catalysed reactions was significantly reduced in the presence of either CaBP1 or CaBP2. In contrast with PDI, neither CaBP1 nor CaBP2 could catalyse the renaturation of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a redox-independent process, and neither protein had any effect on the PDI-catalysed refolding of GAPDH. Furthermore, although PDI can bind peptides via its b' domain, a property it shares with PDIp, the pancreas-specific PDI homologue, and although PDI can bind malfolded proteins such as 'scrambled' ribonuclease, no such interactions could be detected for CaBP2. We conclude that: (1) both CaBP2 and CaBP1 lack peptide-binding activity for GAPDH attributed to the C-terminal region of the a' domain of PDI; (2) CaBP2 lacks the general peptide-binding activity attributed to the b' domain of PDI; (3) interaction of CaBP2 with substrate (RNase AIII) is different from that of PDI and substrate; and (4) both CaBP2 and CaBP1 may

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

PubMed

Franciosini, Anna; Lombardi, Benedetta; Iafrate, Silvia; Pecce, Valeria; Mele, Giovanni; Lupacchini, Leonardo; Rinaldi, Gianmarco; Kondou, Youichi; Gusmaroli, Giuliana; Aki, Shiori; Tsuge, Tomohiko; Deng, Xing-Wang; Matsui, Minami; Vittorioso, Paola; Costantino, Paolo; Serino, Giovanna

2013-09-01

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations

PubMed Central

Hernández-Ramírez, Laura C.; Martucci, Federico; Morgan, Rhodri M. L.; Trivellin, Giampaolo; Tilley, Daniel; Ramos-Guajardo, Nancy; Iacovazzo, Donato; D'Acquisto, Fulvio; Prodromou, Chrisostomos

2016-01-01

Context: The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. Objective: This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. Design and Setting: Half-life and protein–protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. Patients: Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. Interventions: Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. Main Outcome Measures: Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. Results: Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7–92.9 h), and those with short (median, 27 h; IQR, 21.6–28.7 h) or very short (median, 7.7 h; IQR, 5.6–10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1–CUL1–F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. Conclusions: AIP is a stable protein, driven to ubiquitination by the SKP1–CUL1–F-box protein complex

Investigating the Role of Akt1 in Prostate Cancer Development Through Phosphorylation-Dependent Regulation of Skp2 Stability and Oncogenic Function

DTIC Science & Technology

2010-09-01

ubiquitin ligase. Science 2001, 294(5540):173-177. 7. Strohmaier H, Spruck CH, Kaiser P, Won KA , Sangfelt O, Reed SI: Human F-box protein hCdc4 targets...Anders H. Berg,1 Alan Tseng,1 Bo Zhai,2 Shavali Shaik,1 Eric Bennett,3 Adriana E. Tron,4 Jessica A. Gasser,1 Alan Lau,1 Steven P. Gygi,2 J. Wade Harper

Rice ABERRANT PANICLE ORGANIZATION 1, encoding an F-box protein, regulates meristem fate.

PubMed

Ikeda, Kyoko; Ito, Momoyo; Nagasawa, Nobuhiro; Kyozuka, Junko; Nagato, Yasuo

2007-09-01

Inflorescence architecture is one of the most important agronomical traits. Characterization of rice aberrant panicle organization 1 (apo1) mutants revealed that APO1 positively controls spikelet number by suppressing the precocious conversion of inflorescence meristems to spikelet meristems. In addition, APO1 is associated with the regulation of the plastchron, floral organ identity, and floral determinacy. Phenotypic analyses of apo1 and floral homeotic double mutants demonstrate that APO1 positively regulates class-C floral homeotic genes, but not class-B genes. Molecular studies revealed that APO1 encodes an F-box protein, an ortholog of Arabidopsis UNUSUAL FLORAL ORGAN (UFO), which is a positive regulator of class-B genes. Overexpression of APO1 caused an increase in inflorescence branches and spikelets. As the mutant inflorescences and flowers differed considerably between apo1 and ufo, the functions of APO1 and UFO appear to have diverged during evolution.

Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution

PubMed Central

Halling, D. Brent; Kenrick, Sophia A.; Riggs, Austen F.

2014-01-01

Ca2+ activates SK Ca2+-activated K+ channels through the protein Ca2+ sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca2+ regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca2+ concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca2+, SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca2+, 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca2+, the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca2+ and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca2+ or with CaM in molar excess. In low Ca2+ both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca2+. These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating. PMID:24420768

Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution.

PubMed

Halling, D Brent; Kenrick, Sophia A; Riggs, Austen F; Aldrich, Richard W

2014-02-01

Ca(2+) activates SK Ca(2+)-activated K(+) channels through the protein Ca(2+) sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca(2+) regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca(2+) concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca(2+), SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca(2+), 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca(2+), the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca(2+) and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca(2+) or with CaM in molar excess. In low Ca(2+) both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca(2+). These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.

Bimolecular fluorescence complementation studies support an in vivo interaction between the F-BOX protein COLD TEMPERATURE GERMINATING10 and PHYTOCHROME INTERACTING FACTOR1

USDA-ARS?s Scientific Manuscript database

The Arabidopsis thaliana F-BOX protein COLD TEMPERATURE GERMINATING10 (CTG10) was identified from an activation tagged mutant screen as causing seeds to complete germination faster than wild type at 10°C when its expression is increased (Salaita et al. 2005. J. Exp. Bot. 56: 2059). Our unpublished d...

Tubulin chaperone E binds microtubules and proteasomes and protects against misfolded protein stress.

PubMed

Voloshin, Olga; Gocheva, Yana; Gutnick, Marina; Movshovich, Natalia; Bakhrat, Anya; Baranes-Bachar, Keren; Bar-Zvi, Dudy; Parvari, Ruti; Gheber, Larisa; Raveh, Dina

2010-06-01

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.

The DEAD-box Protein Dbp2 Functions with the RNA-binding Protein Yra1 to Promote mRNP Assembly

PubMed Central

Ma, Wai Kit; Cloutier, Sara C.; Tran, Elizabeth J.

2013-01-01

Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoprotein (RNP) complex formation. The DEAD-box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-box proteins, our laboratory recently demonstrated that the DEAD-box protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding proteins Yra1, Nab2 and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby mRNP assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus. PMID:23721653

Recognition of a wide-range of S-RNases by S locus F-box like 2, a general-inhibitor candidate in the Prunus-specific S-RNase-based self-incompatibility system.

PubMed

Matsumoto, Daiki; Tao, Ryutaro

2016-07-01

Many species in the Rosaceae, the Solanaceae, and the Plantaginaceae exhibit S-RNase-based gametophytic self-incompatibility (GSI). This system comprises S-ribonucleases (S-RNases) as the pistil S determinant and a single or multiple F-box proteins as the pollen S determinants. In Prunus, pollen specificity is determined by a single S haplotype-specific F-box protein (SFB). The results of several studies suggested that SFB exerts cognate S-RNase cytotoxicity, and a hypothetical general inhibitor (GI) is assumed to detoxify S-RNases in non-specific manner unless it is affected by SFB. Although the identity of the GI is unknown, phylogenetic and evolutionary analyses have indicated that S locus F-box like 1-3 (or S locus F-box with low allelic sequence polymorphism 1-3; SLFL1-3), which are encoded by a region of the Prunus genome linked to the S locus, are good GI candidates. Here, we examined the biochemical characteristics of SLFL1-3 to determine whether they have appropriate GI characteristics. Pull-down assays and quantitative expression analyses indicated that Prunus avium SLFL1-3 mainly formed a canonical SCF complex with PavSSK1 and PavCul1A. Binding assays with PavS(1,3,4,6)-RNases showed that PavSLFL1, PavSLFL2, and PavSLFL3 bound to PavS(3)-RNase, all PavS-RNases tested, and none of the PavS-RNases tested, respectively. Together, these results suggested that SLFL2 has the appropriate characteristics to be the GI in sweet cherry pollen, while SLFL1 may redundantly work with SLFL2 to detoxify all S-RNases. We discuss the possible roles of SLFL1-3 as the GI in the Prunus-specific S-RNase-based GSI mechanism.

The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

PubMed Central

Bakó, László; Umeda, Masaaki; Tiburcio, Antonio F.; Schell, Jeff; Koncz, Csaba

2003-01-01

The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant, animal, and human cells. Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases. CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein. VirD2 is found in tight association with the TATA box-binding protein in vivo. These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes. PMID:12900506

Regulation of Aspergillus nidulans CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47.

PubMed

de Assis, Leandro José; Ulas, Mevlut; Ries, Laure Nicolas Annick; El Ramli, Nadia Ali Mohamed; Sarikaya-Bayram, Ozlem; Braus, Gerhard H; Bayram, Ozgur; Goldman, Gustavo Henrique

2018-06-19

The attachment of one or more ubiquitin molecules by SCF ( S kp- C ullin- F -box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans , CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δ fbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications. IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally

The Polerovirus F box protein P0 targets ARGONAUTE1 to suppress RNA silencing.

PubMed

Bortolamiol, Diane; Pazhouhandeh, Maghsoud; Marrocco, Katia; Genschik, Pascal; Ziegler-Graff, Véronique

2007-09-18

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.

Cofactor-dependent specificity of a DEAD-box protein.

PubMed

Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin

2013-07-16

DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.

Modified release terbutaline (SKP1052) for hypoglycaemia prevention: a proof-of-concept study in people with type 1 diabetes mellitus.

PubMed

Nosek, L; Cardot, J-M; Owens, D R; Ibarra, P; Bagate, K; Vergnault, G; Kaiser, K; Fischer, A; Heise, T

2012-12-01

In this randomized, single blind, cross-over study 2.5 mg and 5 mg of the modified-release terbutaline formulation (SKP-1052) were compared with conventional immediate-release terbutaline (IRT, 5 mg) and placebo on overnight blood glucose (BG) and hypoglycaemia in 30 subjects with type 1 diabetes mellitus. Subjects received subcutaneous injections of insulin glargine (individualized doses) before dinner. SKP-1052, IRT or placebo was administered around 21:00 hours. BG and terbutaline concentrations were monitored overnight for 10 h post-dosing. Endpoints comprised of the nadir BG (BGn 0-10 h, primary endpoint), mean overnight BG (BGmean), morning BG (BGmorning) and hypoglycaemia rates as well as pharmacokinetic (PK) endpoints. SKP-1052 delayed release of terbutaline by 2 h [PK-tmax (mean ± SD) 5.0 ± 2.1 h (2.5 mg) and 4.7 ± 1.7 h (5 mg) vs. 2.6 ± 1.3 h with IRT, p < 0.01, respectively]. Compared with placebo, no significant differences were observed for BGn 0-10 h across treatments, but both 5 mg formulations showed less hypoglycaemic events [10 (IRT), 16 (SKP-1052) vs. 33], higher BGmean (120, 114 and 95 mg/dl) and BGmorning (126, 126 and 101 mg/dl, all comparisons p < 0.05 vs. placebo). Numerically higher BG-levels between 3 and 8 h post-dosing were observed with 2.5 mg SKP-1052 vs. placebo. Compared with IRT SKP-1052 delays release of terbutaline. 2.5 mg SKP-1052 led to numerically higher BG 3 to 8 h post-dose without fasting hyperglycaemia while 5 mg SKP-1052 resulted in fasting hyperglycaemia vs. placebo. Future studies will investigate optimized doses of SKP-1052 for nocturnal hypoglycaemia prevention. © 2012 Blackwell Publishing Ltd.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

PubMed

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates

PubMed Central

Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory

2009-01-01

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541

High mobility group box (HMGB) proteins of Plasmodium falciparum: DNA binding proteins with pro-inflammatory activity.

PubMed

Kumar, Krishan; Singal, Ankita; Rizvi, M Moshahid A; Chauhan, Virander S

2008-06-01

High mobility group box chromosomal protein 1 (HMGB1), known as an abundant, non-histone architectural chromosomal protein, is highly conserved across different species. Homologues of HMGB1 were identified and cloned from malaria parasite, Plasmodium falciparum. Sequence analyses showed that the P. falciparum HMGB1 (PfHMGB1) exhibits 45, 23 and 18%, while PfHMGB2 shares 42, 21 and 17% homology with Saccharomyces cerevisiae, human and mouse HMG box proteins respectively. Parasite PfHMGB1and PfHMGB2 proteins contain one HMG Box domain similar to B-Box of mammalian HMGB1. Electrophoretic Mobility Shift Assay (EMSA) showed that recombinant PfHMGB1 and PfHMGB2 bind to DNA. Immunofluorescence Assay using specific antibodies revealed that these proteins are expressed abundantly in the ring stage nuclei. Significant levels of PfHMGB1 and PfHMGB2 were also present in the parasite cytosol at trophozoite and schizont stages. Both, PfHMGB1 and PfHMGB2 were found to be potent inducers of pro-inflammatory cytokines such as TNFalpha from mouse peritoneal macrophages as analyzed by both reverse transcription PCR and by ELISA. These results suggest that secreted PfHMGB1 and PfHMGB2 may be responsible for eliciting/ triggering host inflammatory immune responses associated with malaria infection.

Substrate phosphorylation and feedback regulation in JFK-promoted p53 destabilization.

PubMed

Sun, Luyang; Shi, Lei; Wang, Feng; Huangyang, Peiwei; Si, Wenzhe; Yang, Jie; Yao, Zhi; Shang, Yongfeng

2011-02-11

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Previously, we reported that JFK, the only Kelch domain-containing F-box protein in human, promotes ubiquitination and degradation of p53 and that unlike the other E3 ligases for p53, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Here, we report that the substrate recognition by JFK requires phosphorylation of p53 in its central core region by CSN (COP9 signalosome)-associated kinase. Significantly, inhibition of CSN-associated kinase activity or knockdown of CSN5 impairs JFK-promoted p53 degradation, enhances p53-dependent transcription, and promotes cell growth suppression, G(1) arrest, and apoptosis. Moreover, we showed that JFK is transcriptionally regulated by p53 and forms an auto-regulatory negative feedback loop with p53. These data may shed new light on the functional connection between CSN, Skp1-Cul1-F-box ubiquitin ligase, and p53 and provide a molecular mechanism for the regulation of JFK-promoted p53 degradation.

Substrate Phosphorylation and Feedback Regulation in JFK-promoted p53 Destabilization*

PubMed Central

Sun, Luyang; Shi, Lei; Wang, Feng; Huangyang, Peiwei; Si, Wenzhe; Yang, Jie; Yao, Zhi; Shang, Yongfeng

2011-01-01

The p53 tumor suppressor plays a central role in integrating cellular responses to various stresses. Tight regulation of p53 is thus essential for the maintenance of genome integrity and normal cell proliferation. Previously, we reported that JFK, the only Kelch domain-containing F-box protein in human, promotes ubiquitination and degradation of p53 and that unlike the other E3 ligases for p53, all of which possess an intrinsic ubiquitin ligase activity, JFK destabilizes p53 through the assembly of a Skp1-Cul1-F-box complex. Here, we report that the substrate recognition by JFK requires phosphorylation of p53 in its central core region by CSN (COP9 signalosome)-associated kinase. Significantly, inhibition of CSN-associated kinase activity or knockdown of CSN5 impairs JFK-promoted p53 degradation, enhances p53-dependent transcription, and promotes cell growth suppression, G1 arrest, and apoptosis. Moreover, we showed that JFK is transcriptionally regulated by p53 and forms an auto-regulatory negative feedback loop with p53. These data may shed new light on the functional connection between CSN, Skp1-Cul1-F-box ubiquitin ligase, and p53 and provide a molecular mechanism for the regulation of JFK-promoted p53 degradation. PMID:21127074

The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis.

PubMed

Zhao, D; Yu, Q; Chen, M; Ma, H

2001-07-01

The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

The role of two F-box proteins, SLEEPY1 and SNEEZY, in arabidopsis GA signaling

USDA-ARS?s Scientific Manuscript database

The F-box gene SLY1 is a positive regulator of gibberellin (GA) signaling and loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes can be partially rescued by overexpression of the SLY1 homolog...

F-box protein DOR functions as a novel inhibitory factor for abscisic acid-induced stomatal closure under drought stress in Arabidopsis,.

PubMed

Zhang, Yu'e; Xu, Wenying; Li, Zhonghui; Deng, Xing Wang; Wu, Weihua; Xue, Yongbiao

2008-12-01

Guard cells, which form stoma in leaf epidermis, sense and integrate environmental signals to modulate stomatal aperture in response to diverse conditions. Under drought stress, plants synthesize abscisic acid (ABA), which in turn induces a rapid closing of stoma, to prevent water loss by transpiration. However, many aspects of the molecular mechanism for ABA-mediated stomatal closure are still not understood. Here, we report a novel negative regulator of guard cell ABA signaling, DOR, in Arabidopsis (Arabidopsis thaliana). The DOR gene encodes a putative F-box protein, a member of the S-locus F-box-like family related to AhSLF-S(2) and specifically interacting with ASK14 and CUL1. A null mutation in DOR resulted in a hypersensitive ABA response of stomatal closing and a substantial increase of drought tolerance; in contrast, the transgenic plants overexpressing DOR were more susceptible to the drought stress. DOR is strongly expressed in guard cells and suppressed by ABA treatment, suggesting a negative feedback loop of DOR in ABA responses. Double-mutant analyses of dor with ABA-insensitive mutant abi1-1 showed that abi1-1 is epistatic to dor, but no apparent change of phospholipase Dalpha1 was detected between the wild type and dor. Affymetrix GeneChip analysis showed that DOR likely regulates ABA biosynthesis under drought stress. Taken together, our results demonstrate that DOR acts independent of phospholipase Dalpha1 in an ABA signaling pathway to inhibit the ABA-induced stomatal closure under drought stress.

SCF(KMD) controls cytokinin signaling by regulating the degradation of type-B response regulators.

PubMed

Kim, Hyo Jung; Chiang, Yi-Hsuan; Kieber, Joseph J; Schaller, G Eric

2013-06-11

Cytokinins are plant hormones that play critical roles in growth and development. In Arabidopsis, the transcriptional response to cytokinin is regulated by action of type-B Arabidopsis response regulators (ARRs). Although central elements in the cytokinin signal transduction pathway have been identified, mechanisms controlling output remain to be elucidated. Here we demonstrate that a family of F-box proteins, called the kiss me deadly (KMD) family, targets type-B ARR proteins for degradation. KMD proteins form an S-phase kinase-associated PROTEIN1 (SKP1)/Cullin/F-box protein (SCF) E3 ubiquitin ligase complex and directly interact with type-B ARR proteins. Loss-of-function KMD mutants stabilize type-B ARRs and exhibit an enhanced cytokinin response. In contrast, plants with elevated KMD expression destabilize type-B ARR proteins leading to cytokinin insensitivity. Our results support a model in which an SCF(KMD) complex negatively regulates cytokinin responses by controlling levels of a key family of transcription factors.

Identification of SFBB-containing canonical and noncanonical SCF complexes in pollen of apple (Malus × domestica).

PubMed

Minamikawa, Mai F; Koyano, Ruriko; Kikuchi, Shinji; Koba, Takato; Sassa, Hidenori

2014-01-01

Gametophytic self-incompatibility (GSI) of Rosaceae, Solanaceae and Plantaginaceae is controlled by a single polymorphic S locus. The S locus contains at least two genes, S-RNase and F-box protein encoding gene SLF/SFB/SFBB that control pistil and pollen specificity, respectively. Generally, the F-box protein forms an E3 ligase complex, SCF complex with Skp1, Cullin1 (CUL1) and Rbx1, however, in Petunia inflata, SBP1 (S-RNase binding protein1) was reported to play the role of Skp1 and Rbx1, and form an SCFSLF-like complex for ubiquitination of non-self S-RNases. On the other hand, in Petunia hybrida and Petunia inflata of Solanaceae, Prunus avium and Pyrus bretschneideri of Rosaceae, SSK1 (SLF-interacting Skp1-like protein1) is considered to form the SCFSLF/SFB complex. Here, we isolated pollen-expressed apple homologs of SSK1 and CUL1, and named MdSSK1, MdCUL1A and MdCUL1B. MdSSK1 was preferentially expressed in pollen, but weakly in other organs analyzed, while, MdCUL1A and MdCUL1B were almost equally expressed in all the organs analyzed. MdSSK1 transcript abundance was significantly (>100 times) higher than that of MdSBP1. In vitro binding assays showed that MdSSK1 and MdSBP1 interacted with MdSFBB1-S9 and MdCUL1, and MdSFBB1-S9 interacted more strongly with MdSSK1 than with MdSBP1. The results suggest that both MdSSK1-containing SCFSFBB1 and MdSBP1-containing SCFSFBB1-like complexes function in pollen of apple, and the former plays a major role.

Protein-Protein Docking with F2Dock 2.0 and GB-Rerank

PubMed Central

Chowdhury, Rezaul; Rasheed, Muhibur; Keidel, Donald; Moussalem, Maysam; Olson, Arthur; Sanner, Michel; Bajaj, Chandrajit

2013-01-01

Motivation Computational simulation of protein-protein docking can expedite the process of molecular modeling and drug discovery. This paper reports on our new F2 Dock protocol which improves the state of the art in initial stage rigid body exhaustive docking search, scoring and ranking by introducing improvements in the shape-complementarity and electrostatics affinity functions, a new knowledge-based interface propensity term with FFT formulation, a set of novel knowledge-based filters and finally a solvation energy (GBSA) based reranking technique. Our algorithms are based on highly efficient data structures including the dynamic packing grids and octrees which significantly speed up the computations and also provide guaranteed bounds on approximation error. Results The improved affinity functions show superior performance compared to their traditional counterparts in finding correct docking poses at higher ranks. We found that the new filters and the GBSA based reranking individually and in combination significantly improve the accuracy of docking predictions with only minor increase in computation time. We compared F2 Dock 2.0 with ZDock 3.0.2 and found improvements over it, specifically among 176 complexes in ZLab Benchmark 4.0, F2 Dock 2.0 finds a near-native solution as the top prediction for 22 complexes; where ZDock 3.0.2 does so for 13 complexes. F2 Dock 2.0 finds a near-native solution within the top 1000 predictions for 106 complexes as opposed to 104 complexes for ZDock 3.0.2. However, there are 17 and 15 complexes where F2 Dock 2.0 finds a solution but ZDock 3.0.2 does not and vice versa; which indicates that the two docking protocols can also complement each other. Availability The docking protocol has been implemented as a server with a graphical client (TexMol) which allows the user to manage multiple docking jobs, and visualize the docked poses and interfaces. Both the server and client are available for download. Server: http://www.cs.utexas.edu/~bajaj/cvc/software/f

SVP-like MADS-box protein from Carya cathayensis forms higher-order complexes.

PubMed

Wang, Jingjing; Hou, Chuanming; Huang, Jianqin; Wang, Zhengjia; Xu, Yingwu

2015-03-01

To properly regulate plant flowering time and construct floral pattern, MADS-domain containing transcription factors must form multimers including homo- and hetero-dimers. They are also active in forming hetero-higher-order complexes with three to five different molecules. However, it is not well known if a MADS-box protein can also form homo-higher-order complex. In this study a biochemical approach is utilized to provide insight into the complex formation for an SVP-like MADS-box protein cloned from hickory. The results indicated that the protein is a heterogeneous higher-order complex with the peak population containing over 20 monomers. Y2H verified the protein to form homo-complex in yeast cells. Western blot of the hickory floral bud sample revealed that the protein exists in higher-order polymers in native. Deletion assays indicated that the flexible C-terminal residues are mainly responsible for the higher-order polymer formation and the heterogeneity. Current results provide direct biochemical evidences for an active MADS-box protein to be a high order complex, much higher than a quartermeric polymer. Analysis suggests that a MADS-box subset may be able to self-assemble into large complexes, and thereby differentiate one subfamily from the other in a higher-order structural manner. Present result is a valuable supplement to the action of mechanism for MADS-box proteins in plant development. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

PubMed Central

Sharma, Manju; Dearth, Andrea; Smith, Benjamin; Braun, Robert E.

2015-01-01

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets. PMID:26646932

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family†

PubMed Central

Meyer, Damien; Cunnac, Sébastien; Guéneron, Mareva; Declercq, Céline; Van Gijsegem, Frédérique; Lauber, Emmanuelle; Boucher, Christian; Arlat, Matthieu

2006-01-01

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum. PMID:16788199

Ubc13 and COOH Terminus of Hsp70-interacting Protein (CHIP) Are Required for Growth Hormone Receptor Endocytosis*

PubMed Central

Slotman, Johan A.; da Silva Almeida, Ana C.; Hassink, Gerco C.; van de Ven, Robert H. A.; van Kerkhof, Peter; Kuiken, Hendrik J.; Strous, Ger J.

2012-01-01

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCFβTrCP (Skp Cullin F-box). Despite a specific interaction between β-transducin repeat-containing protein (βTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCFβTrCP-directed Lys48 polyubiquitination. We now show that also Lys63-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin-conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition to βTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR. PMID:22433856

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation.

PubMed

Ramachandran, Shyam; Osterhaus, Samantha R; Parekh, Kalpaj R; Jacobi, Ashley M; Behlke, Mark A; McCray, Paul B

2016-12-02

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl - conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl - transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl - conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

PubMed

Strand, Nicholas S; Allen, John M; Ghulam, Mahjoobah; Taylor, Matthew R; Munday, Roma K; Carrillo, Melissa; Movsesyan, Artem; Zayas, Ricardo M

2018-01-15

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of She3 as an SCFGrr1 Substrate in Budding Yeast

PubMed Central

Wang, Ruiwen; Solomon, Mark J.

2012-01-01

The highly orchestrated progression of the cell cycle depends on the degradation of many regulatory proteins at different cell cycle stages. One of the key cell cycle ubiquitin ligases is the Skp1-cullin-F-box (SCF) complex. Acting in concert with the substrate-binding F-box protein Grr1, SCFGrr1 promotes the degradation of cell cycle regulators as well as various metabolic enzymes. Using a yeast two-hybrid assay with a Grr1 derivative as the bait, we identified She3, which is an adaptor protein in the asymmetric mRNA transport system, as a novel Grr1 substrate. We generated stabilized She3 mutants, which no longer bound to Grr1, and found that the degradation of She3 is not required for regulating asymmetric mRNA transport. However, She3 stabilization leads to slower growth compared to wild-type cells in a co-culture assay, demonstrating that the degradation of She3 by Grr1 is required for optimal cell growth. PMID:23144720

The B-Box Domain Protein BBX21 Promotes Photomorphogenesis.

PubMed

Xu, Dongqing; Jiang, Yan; Li, Jian; Holm, Magnus; Deng, Xing Wang

2018-03-01

B-box-containing (BBX) proteins play critical roles in a variety of cellular and developmental processes in plants. BBX21 (also known as SALT TOLERANCE HOMOLOG2), which contains two B-box domains in tandem at the N terminus, has been previously demonstrated as a key component involved in the COP1-HY5 signaling hub. However, the exact molecular and physiological roles of B-box domains in BBX21 are largely unclear. Here, we found that structurally disruption of the second B-box domain, but not the first one, in BBX21 completely abolishes its biological and physiological activity in conferring hyperphotomorphogenetic phenotype in Arabidopsis ( Arabidopsis thaliana ). Intact B-box domains in BBX21 are not required for interaction with COP1 and its degradation by COP1 via the 26S proteasome system. However, disruption of the second B-box of BBX21 nearly impairs its ability for binding of T/G-box within the HY5 promoter both in vitro and in vivo, as well as controlling HY5 and HY5-regulated gene expression in Arabidopsis seedlings. Taken together, this study provides a mechanistic framework in which BBX21 directly binds to the T/G-box present in the HY5 promoter possibly through its second B-box domain, which in turn controls HY5 and HY5-regulated gene expression to promote photomorphogenesis. © 2018 American Society of Plant Biologists. All Rights Reserved.

Molecular characterization of atrogin-1/F-box protein-32 (FBXO32) and F-box protein 25 (FBXO25) in rainbow trout (Oncorhynchus mykiss); expression across tissues in response to feed deprivation

USDA-ARS?s Scientific Manuscript database

The characteristic increase in protein catabolism during muscle atrophy is largely the result of an increase in E3 ubiquitin ligase expression, specifically that of atrogin-1, or FBXO32, which functions to polyubiquitinate proteins. In rainbow trout, the cDNA sequences of two E3 ubiquitin ligase F-...

Mutations in the F-box gene SNEEZY result in decreased arabidopsis GA signaling

USDA-ARS?s Scientific Manuscript database

We previously reported that the SLEEPY1 (SLY1) homolog, F-box gene SNEEZY/SLEEPY2 (SNE/SLY2), can partly replace SLY1 in gibberellin (GA) hormone signaling through interaction with DELLAs RGA and GAI. To determine whether SNE normally functions in GA signaling, we characterized the phenotypes of tw...

Self-incompatibility in Petunia inflata: the relationship between a self-incompatibility locus F-box protein and its non-self S-RNases.

PubMed

Sun, Penglin; Kao, Teh-hui

2013-02-01

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S2-SLF1 (for S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF.

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Zimmer, J; Takahashi, T; Hofmann, A D; Puri, Prem

2016-12-01

High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n = 11) and control group (n = 11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047 ± 0.108, FoxF1 Ctrl 1.419 ± 0.01, p = 0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74 ± 8.925, FoxC2 Ctrl 27.408 ± 7.487, p = 0.776; FoxO1 CDH 0.011 ± 0.002, FoxO1 Ctrl 0.011 ± 0.001, p = 0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression

Strigolactone and karrikin signal perception: receptors, enzymes, or both?

PubMed Central

Janssen, Bart J.; Snowden, Kimberley C.

2012-01-01

The signaling molecules strigolactone (SL) and karrikin are involved in seed germination, development of axillary meristems, senescence of leaves, and interactions with arbuscular mycorrhizal fungi. The signal transduction pathways for both SLs and karrikins require the same F-box protein (MAX2) and closely related α/β hydrolase fold proteins (DAD2 and KAI2). The crystal structure of DAD2 has been solved revealing an α/β hydrolase fold protein with an internal cavity capable of accommodating SLs. DAD2 responds to the SL analog GR24 by changing conformation and binding to MAX2 in a GR24 concentration-dependent manner. DAD2 can also catalyze hydrolysis of GR24. Structure activity relationships of analogs indicate that the butenolide ring common to both SLs and karrikins is essential for biological activity, but the remainder of the molecules can be significantly modified without loss of activity. The combination of data from the study of DAD2, KAI2, and chemical analogs of SLs and karrikins suggests a model for binding that requires nucleophilic attack by the active site serine of the hydrolase at the carbonyl atom of the butenolide ring. A conformational change occurs in the hydrolase that results in interaction with the F-box protein MAX2. Downstream signal transduction is then likely to occur via SCF (Skp-Cullin-F-box) complex-mediated ubiquitination of target proteins and their subsequent degradation. The role of the catalytic activity of the hydrolase is unclear but it may be integral in binding as well as possibly allowing the signal to be cleared from the receptor. The α/β hydrolase fold family consists mostly of active enzymes, with a few notable exceptions. We suggest that DAD2 and KAI2 represent an intermediate stage where some catalytic activity is retained at the same time as a receptor role has evolved. PMID:23293648

Strigolactone and karrikin signal perception: receptors, enzymes, or both?

PubMed

Janssen, Bart J; Snowden, Kimberley C

2012-01-01

The signaling molecules strigolactone (SL) and karrikin are involved in seed germination, development of axillary meristems, senescence of leaves, and interactions with arbuscular mycorrhizal fungi. The signal transduction pathways for both SLs and karrikins require the same F-box protein (MAX2) and closely related α/β hydrolase fold proteins (DAD2 and KAI2). The crystal structure of DAD2 has been solved revealing an α/β hydrolase fold protein with an internal cavity capable of accommodating SLs. DAD2 responds to the SL analog GR24 by changing conformation and binding to MAX2 in a GR24 concentration-dependent manner. DAD2 can also catalyze hydrolysis of GR24. Structure activity relationships of analogs indicate that the butenolide ring common to both SLs and karrikins is essential for biological activity, but the remainder of the molecules can be significantly modified without loss of activity. The combination of data from the study of DAD2, KAI2, and chemical analogs of SLs and karrikins suggests a model for binding that requires nucleophilic attack by the active site serine of the hydrolase at the carbonyl atom of the butenolide ring. A conformational change occurs in the hydrolase that results in interaction with the F-box protein MAX2. Downstream signal transduction is then likely to occur via SCF (Skp-Cullin-F-box) complex-mediated ubiquitination of target proteins and their subsequent degradation. The role of the catalytic activity of the hydrolase is unclear but it may be integral in binding as well as possibly allowing the signal to be cleared from the receptor. The α/β hydrolase fold family consists mostly of active enzymes, with a few notable exceptions. We suggest that DAD2 and KAI2 represent an intermediate stage where some catalytic activity is retained at the same time as a receptor role has evolved.

Identification of Drosophila melanogaster yellow-f and yellow-f2 proteins as dopachrome-conversion enzymes.

PubMed Central

Han, Qian; Fang, Jianmin; Ding, Haizhen; Johnson, Jody K; Christensen, Bruce M; Li, Jianyong

2002-01-01

This study describes the identification of Drosophila yellow-f and yellow-f2 as dopachrome-conversion enzymes responsible for catalysing the conversion of dopachrome into 5,6-dihydroxyindole in the melanization pathway. Drosophila yellow -y gene and yellow -b, -c, -f and -f2 genes were expressed in an insect cell/baculovirus expression system and their corresponding recombinant proteins were screened for dopachrome-conversion enzyme activity. Among the yellow and yellow -related genes, the yellow -f and yellow -f2 genes were identified as the genes coding for Drosophila dopachrome-conversion enzyme based on the high activity of their recombinant proteins in catalysing the production of 5,6-dihydroxyindole from dopachrome. Both yellow-f and yellow-f2 are capable of mediating a decarboxylative structural rearrangement of dopachrome, as well as an isomerization/tautomerization of dopamine chrome and dopa methyl ester chrome. Northern hybridization revealed the transcription of yellow -f in larvae and pupae, but a high abundance of mRNA was observed in later larval and early pupal stages. In contrast, yellow-f2 transcripts were present at all stages, but high abundance of its mRNA was observed in later-stage pupae and adults. These data indicate that yellow-f and yellow-f2 complement each other during Drosophila development and that the yellow-f is involved in larval and pupal melanization, and yellow-f2 plays a major role in melanization reactions in Drosophila during later pupal and adult development. Results from this study provide the groundwork towards a better understanding of the physiological roles of the Drosophila yellow gene family. PMID:12164780

Self-Incompatibility in Petunia inflata: The Relationship between a Self-Incompatibility Locus F-Box Protein and Its Non-Self S-RNases[W

PubMed Central

Sun, Penglin; Kao, Teh-hui

2013-01-01

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S2-SLF1 (for S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF. PMID:23444333

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis.

PubMed

Bragantini, Benoit; Tiotiu, Decebal; Rothé, Benjamin; Saliou, Jean-Michel; Marty, Hélène; Cianférani, Sarah; Charpentier, Bruno; Quinternet, Marc; Manival, Xavier

2016-06-05

Zf–HIT family members share the zf–HIT domain (ZHD), which is characterized by a fold in “treble-clef” through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf–HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf–HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf–HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.

Tethering of SCFDia2 to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase

PubMed Central

Maculins, Timurs; Nkosi, Pedro Junior; Nishikawa, Hiroko; Labib, Karim

2015-01-01

Summary Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood [1, 2]. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase [3]. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex [4, 5], which assembles around the CMG helicase at replication forks [6]. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 [4] or else tethering SCFDia2 (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks [5]. Here, we show that SCFDia2 does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCFDia2 to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned. PMID:26255844

Role of indirect readout mechanism in TATA box binding protein-DNA interaction.

PubMed

Mondal, Manas; Choudhury, Devapriya; Chakrabarti, Jaydeb; Bhattacharyya, Dhananjay

2015-03-01

Gene expression generally initiates from recognition of TATA-box binding protein (TBP) to the minor groove of DNA of TATA box sequence where the DNA structure is significantly different from B-DNA. We have carried out molecular dynamics simulation studies of TBP-DNA system to understand how the DNA structure alters for efficient binding. We observed rigid nature of the protein while the DNA of TATA box sequence has an inherent flexibility in terms of bending and minor groove widening. The bending analysis of the free DNA and the TBP bound DNA systems indicate presence of some similar structures. Principal coordinate ordination analysis also indicates some structural features of the protein bound and free DNA are similar. Thus we suggest that the DNA of TATA box sequence regularly oscillates between several alternate structures and the one suitable for TBP binding is induced further by the protein for proper complex formation.

Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

PubMed

Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

2015-01-01

TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes

PubMed Central

Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

2015-01-01

TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

DEAD-box Helicases as Integrators of RNA, Nucleotide and Protein Binding

PubMed Central

Putnam, Andrea A.

2013-01-01

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. PMID:23416748

Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

PubMed

Li, Junhui; Zhang, Yue; Song, Yanzhai; Zhang, Hui; Fan, Jiangbo; Li, Qun; Zhang, Dongfen; Xue, Yongbiao

2017-01-01

Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression.

PubMed

Gonzalez, Lauren E; Keller, Kristen; Chan, Karen X; Gessel, Megan M; Thines, Bryan C

2017-07-17

The ubiquitin 26S proteasome system (UPS) selectively degrades cellular proteins, which results in physiological changes to eukaryotic cells. F-box proteins are substrate adaptors within the UPS and are responsible for the diversity of potential protein targets. Plant genomes are enriched in F-box genes, but the vast majority of these have unknown roles. This work investigated the Arabidopsis F-box gene F-BOX STRESS INDUCED 1 (FBS1) for its effects on gene expression in order elucidate its previously unknown biological function. Using publically available Affymetrix ATH1 microarray data, we show that FBS1 is significantly co-expressed in abiotic stresses with other well-characterized stress response genes, including important stress-related transcriptional regulators. This gene suite is most highly expressed in roots under cold and salt stresses. Transcriptome analysis of fbs1-1 knock-out plants grown at a chilling temperature shows that hundreds of genes require FBS1 for appropriate expression, and that these genes are enriched in those having roles in both abiotic and biotic stress responses. Based on both this genome-wide expression data set and quantitative real-time PCR (qPCR) analysis, it is apparent that FBS1 is required for elevated expression of many jasmonic acid (JA) genes that have established roles in combatting environmental stresses, and that it also controls a subset of JA biosynthesis genes. FBS1 also significantly impacts abscisic acid (ABA) regulated genes, but this interaction is more complex, as FBS1 has both positive and negative effects on ABA-inducible and ABA-repressible gene modules. One noteworthy effect of FBS1 on ABA-related stress processes, however, is the restraint it imposes on the expression of multiple class I LIPID TRANSFER PROTEIN (LTP) gene family members that have demonstrated protective effects in water deficit-related stresses. FBS1 impacts plant stress responses by regulating hundreds of genes that respond to the plant

Isolation and characterization of multiple F-box genes linked to the S9- and S10-RNase in apple (Malus × domestica Borkh.).

PubMed

Okada, Kazuma; Moriya, Shigeki; Haji, Takashi; Abe, Kazuyuki

2013-06-01

Using 11 consensus primer pairs designed from S-linked F-box genes of apple and Japanese pear, 10 new F-box genes (MdFBX21 to 30) were isolated from the apple cultivar 'Spartan' (S(9)S(10)). MdFBX21 to 23 and MdFBX24 to 30 were completely linked to the S(9) -RNase and S(10-)RNase, respectively, and showed pollen-specific expression and S-haplotype-specific polymorphisms. Therefore, these 10 F-box genes are good candidates for the pollen determinant of self-incompatibility in apple. Phylogenetic analysis and comparison of deduced amino acid sequences of MdFBX21 to 30 with those of 25 S-linked F-box genes previously isolated from apple showed that a deduced amino acid identity of greater than 88.0 % can be used as the tentative criterion to classify F-box genes into one type. Using this criterion, 31 of 35 F-box genes of apple were classified into 11 types (SFBB1-11). All types included F-box genes derived from S(3-) and S(9-)haplotypes, and seven types included F-box genes derived from S(3-), S(9-), and S(10-)haplotypes. Moreover, comparison of nucleotide sequences of S-RNases and multiple F-box genes among S(3-), S(9-), and S(10-)haplotypes suggested that F-box genes within each type showed high nucleotide identity regardless of the identity of the S-RNase. The large number of F-box genes as candidates for the pollen determinant and the high degree of conservation within each type are consistent with the collaborative non-self-recognition model reported for Petunia. These findings support that the collaborative non-self-recognition system also exists in apple.

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

PubMed Central

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-01-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence that CRL4Cdt2-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4Cdt2 as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage. PMID:24423875

PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage.

PubMed

Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

2014-04-01

Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4(Cdt2). Here we provide evidence that CRL4(Cdt2)-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4(Cdt2) as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4(Cdt2) pathway in the switch of PCNA partners on DNA damage.

[NMR structure and dynamics of the chimeric protein SH3-F2].

PubMed

Kutyshenko, V P; Gushchina, L V; Khristoforov, V S; Prokhorov, D A; Timchenko, M A; Kudrevatykh, Iu A; Fediukina, D V; Filimonov, V V

2010-01-01

For the further elucidation of structural and dynamic principles of protein self-organization and protein-ligand interactions the design of new chimeric protein SH3-F2 was made and genetically engineered construct was created. The SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamics properties of the protein were studied by high-resolution NMR. According to NMR data the tertiary structure of the chimeric protein SH3-F2 has the topology which is typical of SH3 domains in the complex with the ligand, forming polyproline type II helix, located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However the interaction of ligand and the part of globule relative to SH3 domain is not too large because the analysis of protein dynamic characteristics points to the low amplitude, high-frequency ligand tumbling in relation to the slow intramolecular motions of the main globule. The constructed chimera permits to carry out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant.

PubMed

Ueguchi-Tanaka, Miyako; Hirano, Ko; Hasegawa, Yasuko; Kitano, Hidemi; Matsuoka, Makoto

2008-09-01

The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.

Stability: Conservation laws, Painlevé analysis and exact solutions for S-KP equation in coupled dusty plasma

NASA Astrophysics Data System (ADS)

EL-Kalaawy, O. H.; Moawad, S. M.; Wael, Shrouk

The propagation of nonlinear waves in unmagnetized strongly coupled dusty plasma with Boltzmann distributed electrons, iso-nonthermal distributed ions and negatively charged dust grains is considered. The basic set of fluid equations is reduced to the Schamel Kadomtsev-Petviashvili (S-KP) equation by using the reductive perturbation method. The variational principle and conservation laws of S-KP equation are obtained. It is shown that the S-KP equation is non-integrable using Painlevé analysis. A set of new exact solutions are obtained by auto-Bäcklund transformations. The stability analysis is discussed for the existence of dust acoustic solitary waves (DASWs) and it is found that the physical parameters have strong effects on the stability criterion. In additional to, the electric field and the true Mach number of this solution are investigated. Finally, we will study the physical meanings of solutions.

Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of potato leafroll virus from Inner Mongolia.

PubMed

Zhuo, Tao; Li, Yuan-Yuan; Xiang, Hai-Ying; Wu, Zhan-Yu; Wang, Xian-Bin; Wang, Ying; Zhang, Yong-Liang; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2014-06-01

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors

PubMed Central

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios

2017-01-01

Abstract Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. PMID:28973457

Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

PubMed Central

Roy Choudhury, Swarup; Roy, Sujit; Nag, Anish; Singh, Sanjay Kumar; Sengupta, Dibyendu N.

2012-01-01

The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development. PMID:22984496

Characterization of an AGAMOUS-like MADS box protein, a probable constituent of flowering and fruit ripening regulatory system in banana.

PubMed

Roy Choudhury, Swarup; Roy, Sujit; Nag, Anish; Singh, Sanjay Kumar; Sengupta, Dibyendu N

2012-01-01

The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor (Musa sp, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ~95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation in vitro using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. In vivo and in vitro assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

PubMed

Truttmann, Matthias C; Guye, Patrick; Dehio, Christoph

2011-01-01

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

PubMed

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Msx1 and Msx2 are functional interacting partners of T-box factors in the regulation of Connexin43.

PubMed

Boogerd, Kees-Jan; Wong, L Y Elaine; Christoffels, Vincent M; Klarenbeek, Meinke; Ruijter, Jan M; Moorman, Antoon F M; Barnett, Phil

2008-06-01

T-box factors Tbx2 and Tbx3 play key roles in the development of the cardiac conduction system, atrioventricular canal, and outflow tract of the heart. They regulate the gap-junction-encoding gene Connexin43 (Cx43) and other genes critical for heart development and function. Discovering protein partners of Tbx2 and Tbx3 will shed light on the mechanisms by which these factors regulate these gene programs. Employing an yeast 2-hybrid screen and subsequent in vitro pull-down experiments we demonstrate that muscle segment homeobox genes Msx1 and Msx2 are able to bind the cardiac T-box proteins Tbx2, Tbx3, and Tbx5. This interaction, as that of the related Nkx2.5 protein, is supported by the T-box and homeodomain alone. Overlapping spatiotemporal expression patterns of Msx1 and Msx2 together with the T-box genes during cardiac development in mouse and chicken underscore the biological significance of this interaction. We demonstrate that Msx proteins together with Tbx2 and Tbx3 suppress Cx43 promoter activity and down regulate Cx43 gene activity in a rat heart-derived cell line. Using chromatin immunoprecipitation analysis we demonstrate that Msx1 can bind the Cx43 promoter at a conserved binding site located in close proximity to a previously defined T-box binding site, and that the activity of Msx proteins on this promoter appears dependent in the presence of Tbx3. Msx1 and Msx2 can function in concert with the T-box proteins to suppress Cx43 and other working myocardial genes.

BID-F1 and BID-F2 Domains of Bartonella henselae Effector Protein BepF Trigger Together with BepC the Formation of Invasome Structures

PubMed Central

Truttmann, Matthias C.; Guye, Patrick; Dehio, Christoph

2011-01-01

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation. PMID:22043280

Vero/BC-F: an efficient packaging cell line stably expressing F protein to generate single round-infectious human parainfluenza virus type 2 vector.

PubMed

Ohtsuka, J; Fukumura, M; Tsurudome, M; Hara, K; Nishio, M; Kawano, M; Nosaka, T

2014-08-01

A stable packaging cell line (Vero/BC-F) constitutively expressing fusion (F) protein of the human parainfluenza virus type 2 (hPIV2) was established for production of the F-defective and single round-infectious hPIV2 vector in a strategy for recombinant vaccine development. The F gene expression has not evoked cytostatic or cytotoxic effects on the Vero/BC-F cells and the F protein was physiologically active to induce syncytial formation with giant polykaryocytes when transfected with a plasmid expressing hPIV2 hemagglutinin-neuraminidase (HN). Transduction of the F-defective replicon RNA into the Vero/BC-F cells led to the release of the infectious particles that packaged the replicon RNA (named as hPIV2ΔF) without detectable mutations, limiting the infectivity to a single round. The maximal titer of the hPIV2ΔF was 6.0 × 10(8) median tissue culture infections dose per ml. The influenza A virus M2 gene was inserted into hPIV2ΔF, and the M2 protein was found to be highly expressed in a human lung cancer cell line after transduction. Furthermore, in vivo airway infection experiments revealed that the hPIV2ΔF was capable of delivering transgenes to hamster tracheal cells. Thus, non-transmissible or single round-infectious hPIV2 vector will be potentially applicable to human gene therapy or recombinant vaccine development.

Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y.

PubMed

Matuoka, Koozi; Chen, Kuang Yu

2002-09-01

Cellular ageing is a systematic process affecting the entirety of cell structure and function. Since changes in gene expression are extensive and global during ageing, involvement of general transcription regulators in the phenomenon is likely. Here, we focus on NF-Y, the major CCAAT box-binding factor, which exerts differential regulation on a wide variety of genes through its interaction with the CCAAT box present in as many as 25% of the eukaryotic genes. When a cell ages, senescing signals arise, typically through DNA damage due to oxidative stress or telomere shortening, and are transduced to proteins such as p53, retinoblastoma protein, and phosphatidylinositol 3-kinase. Among them, activated p53 family proteins suppress the function of NF-Y and thereby downregulate a set of cell cycle-related genes, including E2F1, which further leads to downregulation of E2F-regulated genes and cell cycle arrest. The p53 family also induces other ageing phenotypes such as morphological alterations and senescence-associated beta-galactosidase (SA-gal) presumably by upregulation of some genes through NF-Y suppression. In fact, the activities of NF-Y and E2F decrease during ageing and a dominant negative NF-YA induces SA-gal. Based on these observations, NF-Y appears to play an important role in the process of cellular ageing.

A truncated F-box protein confers the dwarfism in cucumber

USDA-ARS?s Scientific Manuscript database

Dwarfism is an important plant architecture trait for cucumber breeding. In the present study, we identified a dwarf mutant 406M in cucumber which showed a shorter internode length as compared with its wild type. In a BC1F2 population from the cross of 406M with its wild type parental line 406, the ...

49 CFR 178.515 - Standards for reconstituted wood boxes.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 2 2010-10-01 2010-10-01 false Standards for reconstituted wood boxes. 178.515... wood boxes. (a) The identification code for a reconstituted wood box is 4F. (b) Construction requirements for reconstituted wood boxes are as follows: (1) The walls of boxes must be made of water...

Transcriptional Regulation of Fruit Ripening by Tomato FRUITFULL Homologs and Associated MADS Box Proteins[W

PubMed Central

Fujisawa, Masaki; Shima, Yoko; Nakagawa, Hiroyuki; Kitagawa, Mamiko; Kimbara, Junji; Nakano, Toshitsugu; Kasumi, Takafumi; Ito, Yasuhiro

2014-01-01

The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening. PMID:24415769

Both Ubiquitin Ligases FBXW8 and PARK2 Are Sequestrated into Insolubility by ATXN2 PolyQ Expansions, but Only FBXW8 Expression Is Dysregulated

PubMed Central

Halbach, Melanie Vanessa; Stehning, Tanja; Damrath, Ewa; Jendrach, Marina; Şen, Nesli Ece; Başak, A. Nazlı; Auburger, Georg

2015-01-01

The involvement of the ubiquitin-proteasome system (UPS) in the course of various age-associated neurodegenerative diseases is well established. The single RING finger type E3 ubiquitin-protein ligase PARK2 is mutated in a Parkinson’s disease (PD) variant and was found to interact with ATXN2, a protein where polyglutamine expansions cause Spinocerebellar ataxia type 2 (SCA2) or increase the risk for Levodopa-responsive PD and for the motor neuron disease Amyotrophic lateral sclerosis (ALS). We previously reported evidence for a transcriptional induction of the multi-subunit RING finger Skp1/Cul/F-box (SCF) type E3 ubiquitin-protein ligase complex component FBXW8 in global microarray profiling of ATXN2-expansion mouse cerebellum and demonstrated its role for ATXN2 degradation in vitro. Now, we documented co-localization in vitro and co-immunoprecipitations both in vitro and in vivo, which indicate associations of FBXW8 with ATXN2 and PARK2. Both FBXW8 and PARK2 proteins are driven into insolubility by expanded ATXN2. Whereas the FBXW8 transcript upregulation by ATXN2- expansion was confirmed also in qPCR of skin fibroblasts and blood samples of SCA2 patients, a FBXW8 expression dysregulation was not observed in ATXN2-deficient mice, nor was a PARK2 transcript dysregulation observed in any samples. Jointly, all available data suggest that the degradation of wildtype and mutant ATXN2 is dependent on FBXW8, and that ATXN2 accumulation selectively modulates FBXW8 levels, while PARK2 might act indirectly through FBXW8. The effects of ATXN2-expansions on FBXW8 expression in peripheral tissues like blood may become useful for clinical diagnostics. PMID:25790475

Influenza A virus protein PB1-F2 exacerbates IFN-beta expression of human respiratory epithelial cells.

PubMed

Le Goffic, Ronan; Bouguyon, Edwige; Chevalier, Christophe; Vidic, Jasmina; Da Costa, Bruno; Leymarie, Olivier; Bourdieu, Christiane; Decamps, Laure; Dhorne-Pollet, Sophie; Delmas, Bernard

2010-10-15

The PB1-F2 protein of the influenza A virus (IAV) contributes to viral pathogenesis by a mechanism that is not well understood. PB1-F2 was shown to modulate apoptosis and to be targeted by the CD8(+) T cell response. In this study, we examined the downstream effects of PB1-F2 protein during IAV infection by measuring expression of the cellular genes in response to infection with wild-type WSN/33 and PB1-F2 knockout viruses in human lung epithelial cells. Wild-type virus infection resulted in a significant induction of genes involved in innate immunity. Knocking out the PB1-F2 gene strongly decreased the magnitude of expression of cellular genes implicated in antiviral response and MHC class I Ag presentation, suggesting that PB1-F2 exacerbates innate immune response. Biological network analysis revealed the IFN pathway as a link between PB1-F2 and deregulated genes. Using quantitative RT-PCR and IFN-β gene reporter assay, we determined that PB1-F2 mediates an upregulation of IFN-β expression that is dependent on NF-κB but not on AP-1 and IFN regulatory factor-3 transcription factors. Recombinant viruses knocked out for the PB1-F2 and/or the nonstructural viral protein 1 (the viral antagonist of the IFN response) genes provide further evidence that PB1-F2 increases IFN-β expression and that nonstructural viral protein 1 strongly antagonizes the effect of PB1-F2 on the innate response. Finally, we compared the effect of PB1-F2 variants taken from several IAV strains on IFN-β expression and found that PB1-F2-mediated IFN-β induction is significantly influenced by its amino acid sequence, demonstrating its importance in the host cell response triggered by IAV infection.

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells.

PubMed

Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

2016-11-15

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCF β-TrCP ) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine's potential as an anti-tumor agent for clinical cancer therapy.

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells

PubMed Central

Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

2016-01-01

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCFβ-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine′s potential as an anti-tumor agent for clinical cancer therapy. PMID:27854312

Molecular and functional characterization of two drought-induced zinc finger proteins, ZmZnF1 and ZmZnF2 from maize kernels

USDA-ARS?s Scientific Manuscript database

We have isolated two cDNA clones encoding Zinc Finger proteins, designated as ZmZnF1 and ZmZnF2, from water-stressed maize kernels. Sequence analyses indicates that ZmZnF1 is homologous to the A20/AN1-type zinc finger protein and contains the zinc finger motif of Cx2–Cx10–CxCx4Cx2Hx5HxC. Whereas ZmZ...

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family.

PubMed

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L; Salt, Jennifer N; Goring, Daphne R

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation▿

PubMed Central

Gardner, Amanda E.; Dutch, Rebecca E.

2007-01-01

Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion. PMID:17507474

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

PubMed Central

Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

2013-01-01

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

The role of the F-box gene TaFBA1 from wheat (Triticum aestivum L.) in drought tolerance.

PubMed

Zhou, Shumei; Sun, Xiudong; Yin, Suhong; Kong, Xiangzhu; Zhou, Shan; Xu, Ying; Luo, Yin; Wang, Wei

2014-11-01

Drought is one of the most important factors limiting plant growth and development. We identified a gene in wheat (Triticum aestivum L.) under drought stress named TaFBA1. TaFBA1 encodes a putative 325-amino-acid F-box protein with a conserved N-terminal F-box domain and a C-terminal AMN1 domain. Real-time RT-PCR analysis revealed that TaFBA1 transcript accumulation was upregulated by high-salinity, water stress, and abscisic acid (ABA) treatment. To evaluate the functions of TaFBA1 in the regulation of drought stress responses, we produced transgenic tobacco lines overexpressing TaFBA1. Under water stress conditions, the transgenic tobacco plants had a higher germination rate, higher relative water content, net photosynthesis rate (Pn), less chlorophyll loss, and less growth inhibition than WT. These results demonstrate the high tolerance of the transgenic plants to drought stress compared to the WT. The enhanced oxidative stress tolerance of these plants, which may be involved in their drought tolerance, was indicated by their lower levels of reactive oxygen species (ROS) accumulation, MDA content, and cell membrane damage under drought stress compared to WT. The antioxidant enzyme activities were higher in the transgenic plants than in WT, which may be related to the upregulated expression of some antioxidant genes via overexpression of TaFBA1. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

PubMed

Saliou, Jean-Michel; Manival, Xavier; Tillault, Anne-Sophie; Atmanene, Cédric; Bobo, Claude; Branlant, Christiane; Van Dorsselaer, Alain; Charpentier, Bruno; Cianférani, Sarah

2015-08-01

Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase.

PubMed

Villa, Fabrizio; Simon, Aline C; Ortiz Bazan, Maria Angeles; Kilkenny, Mairi L; Wirthensohn, David; Wightman, Mel; Matak-Vinkovíc, Dijana; Pellegrini, Luca; Labib, Karim

2016-08-04

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Titration of DnaA protein by oriC DnaA-boxes increases dnaA gene expression in Escherichia coli.

PubMed Central

Hansen, F G; Koefoed, S; Sørensen, L; Atlung, T

1987-01-01

Binding of the DnaA protein to its binding sites, the DnaA-boxes (TTATCCACA), was measured by a simple physiological approach. The presence of extra DnaA-boxes in growing cells leads to a derepression of dnaA gene expression, measured as beta-galactosidase activity of a dnaA-lacZ fusion polypeptide. Different DnaA-boxes caused different degrees of derepression indicating that the DnaA protein requires sequences in addition to the DnaA-box for efficient binding. The DnaA-boxes in oriC might act cooperatively in binding of the DnaA protein. The derepressed levels of DnaA protein obtained in a strain carrying an oriC+-pBR322 chimera were very high and sufficient to activate oriC on the chimeric plasmid, which was maintained at a copy number more than three times that of pBR322. PMID:3034578

Cullin1-P is an Essential Component of Non-Self Recognition System in Self-Incompatibility in Petunia.

PubMed

Kubo, Ken-Ichi; Tsukahara, Mai; Fujii, Sota; Murase, Kohji; Wada, Yuko; Entani, Tetsuyuki; Iwano, Megumi; Takayama, Seiji

2016-11-01

Self-incompatibility (SI) in flowering plants is a genetic reproductive barrier to distinguish self- and non-self pollen to promote outbreeding. In Solanaceae, self-pollen is rejected by the ribonucleases expressed in the styles (S-RNases), via its cytotoxic function. On the other side, the male-determinant is the S-locus F-box proteins (SLFs) expressed in pollen. Multiple SLFs collaboratively detoxify non-self S-RNases, therefore, non-self recognition is the mode of self-/non-self discrimination in Solanaceae. It is considered that SLFs function as a substrate-recognition module of the Skp1-Cullin1-F-box (SCF) complex that inactivates non-self S-RNases via their polyubiquitination, which leads to degradation by 26S proteasome. In fact, PhSSK1 (Petunia hybrida SLF-interacting Skp1-like1) was identified as a specific component of SCF SLF and was shown to be essential for detoxification of S-RNase in Petunia However, different molecules are proposed as the candidate Cullin1, another component of SCF SLF , and there is as yet no definite conclusion. Here, we identified five Cullin1s from the expressed sequence tags (ESTs) derived from the male reproductive organ in Petunia Among them, only PhCUL1-P was co-immunoprecipitated with S 7 -SLF2. In vitro protein-binding assay suggested that PhSSK1 specifically forms a complex with PhCUL1-P in an SLF-dependent manner. Knockdown of PhCUL1-P suppressed fertility of transgenic pollen in cross-compatible pollination in the functional S-RNase-dependent manner. These results suggested that SCF SLF selectively uses PhCUL1-P. Phylogeny of Cullin1s indicates that CUL1-P is recruited into the SI machinery during the evolution of Solanaceae, suggesting that the SI components have evolved differently among species in Solanaceae and Rosaceae, despite both families sharing the S-RNase-based SI. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For

Expression analysis of genes encoding double B-box zinc finger proteins in maize.

PubMed

Li, Wenlan; Wang, Jingchao; Sun, Qi; Li, Wencai; Yu, Yanli; Zhao, Meng; Meng, Zhaodong

2017-11-01

The B-box proteins play key roles in plant development. The double B-box (DBB) family is one of the subfamily of the B-box family, with two B-box domains and without a CCT domain. In this study, 12 maize double B-box genes (ZmDBBs) were identified through a genome-wide survey. Phylogenetic analysis of DBB proteins from maize, rice, Sorghum bicolor, Arabidopsis, and poplar classified them into five major clades. Gene duplication analysis indicated that segmental duplications made a large contribution to the expansion of ZmDBBs. Furthermore, a large number of cis-acting regulatory elements related to plant development, response to light and phytohormone were identified in the promoter regions of the ZmDBB genes. The expression patterns of the ZmDBB genes in various tissues and different developmental stages demonstrated that ZmDBBs might play essential roles in plant development, and some ZmDBB genes might have unique function in specific developmental stages. In addition, several ZmDBB genes showed diurnal expression pattern. The expression levels of some ZmDBB genes changed significantly under light/dark treatment conditions and phytohormone treatments, implying that they might participate in light signaling pathway and hormone signaling. Our results will provide new information to better understand the complexity of the DBB gene family in maize.

DWARF 53 acts as a repressor of strigolactone signalling in rice

NASA Astrophysics Data System (ADS)

Jiang, Liang; Liu, Xue; Xiong, Guosheng; Liu, Huihui; Chen, Fulu; Wang, Lei; Meng, Xiangbing; Liu, Guifu; Yu, Hong; Yuan, Yundong; Yi, Wei; Zhao, Lihua; Ma, Honglei; He, Yuanzheng; Wu, Zhongshan; Melcher, Karsten; Qian, Qian; Xu, H. Eric; Wang, Yonghong; Li, Jiayang

2013-12-01

Strigolactones (SLs) are a group of newly identified plant hormones that control plant shoot branching. SL signalling requires the hormone-dependent interaction of DWARF 14 (D14), a probable candidate SL receptor, with DWARF 3 (D3), an F-box component of the Skp-Cullin-F-box (SCF) E3 ubiquitin ligase complex. Here we report the characterization of a dominant SL-insensitive rice (Oryza sativa) mutant dwarf 53 (d53) and the cloning of D53, which encodes a substrate of the SCFD3 ubiquitination complex and functions as a repressor of SL signalling. Treatments with GR24, a synthetic SL analogue, cause D53 degradation via the proteasome in a manner that requires D14 and the SCFD3 ubiquitin ligase, whereas the dominant form of D53 is resistant to SL-mediated degradation. Moreover, D53 can interact with transcriptional co-repressors known as TOPLESS-RELATED PROTEINS. Our results suggest a model of SL signalling that involves SL-dependent degradation of the D53 repressor mediated by the D14-D3 complex.

Crystal structure of a DEAD box protein from the hyperthermophile Methanococcus jannaschii

PubMed Central

Story, Randall M.; Li, Hong; Abelson, John N.

2001-01-01

We have determined the structure of a DEAD box putative RNA helicase from the hyperthermophile Methanococcus jannaschii. Like other helicases, the protein contains two α/β domains, each with a recA-like topology. Unlike other helicases, the protein exists as a dimer in the crystal. Through an interaction that resembles the dimer interface of insulin, the amino-terminal domain's 7-strand β-sheet is extended to 14 strands across the two molecules. Motifs conserved in the DEAD box family cluster in the cleft between domains, and many of their functions can be deduced by mutational data and by comparison with other helicase structures. Several lines of evidence suggest that motif III Ser-Ala-Thr may be involved in binding RNA. PMID:11171974

Neurochemical aftermath of amateur boxing.

PubMed

Zetterberg, Henrik; Hietala, M Albert; Jonsson, Michael; Andreasen, Niels; Styrud, Ewa; Karlsson, Ingvar; Edman, Ake; Popa, Cornel; Rasulzada, Abdullah; Wahlund, Lars-Olof; Mehta, Pankaj D; Rosengren, Lars; Blennow, Kaj; Wallin, Anders

2006-09-01

Little solid information is available on the possible risks for neuronal injury in amateur boxing. To determine whether amateur boxing and severity of hits are associated with elevated levels of biochemical markers for neuronal injury in cerebrospinal fluid. Longitudinal study. Referral center specializing in evaluation of neurodegenerative disorders. Fourteen amateur boxers (11 men and 3 women) and 10 healthy male nonathletic control subjects. The boxers underwent lumbar puncture 7 to 10 days and 3 months after a bout. The control subjects underwent LP once. Neurofilament light protein, total tau, glial fibrillary acidic protein, phosphorylated tau, and beta-amyloid protein 1-40 (Abeta([1-40])) and 1-42 (Abeta([1-42])) concentrations in cerebrospinal fluid were measured. Increased levels after a bout compared with after 3 months of rest from boxing were found for 2 markers for neuronal and axonal injury, neurofilament light protein (mean +/- SD, 845 +/- 1140 ng/L vs 208 +/- 108 ng/L; P = .008) and total tau (mean +/- SD, 449 +/- 176 ng/L vs 306 +/- 78 ng/L; P = .006), and for the astroglial injury marker glial fibrillary acidic protein (mean +/- SD, 541 +/- 199 ng/L vs 405 +/- 138 ng/L; P = .003). The increase was significantly higher among boxers who had received many hits (>15) or high-impact hits to the head compared with boxers who reported few hits. In the boxers, concentrations of neurofilament light protein and glial fibrillary acidic protein, but not total tau, were significantly elevated after a bout compared with the nonathletic control subjects. With the exception of neurofilament light protein, there were no significant differences between boxers after 3 months of rest from boxing and the nonathletic control subjects. Amateur boxing is associated with acute neuronal and astroglial injury. If verified in longitudinal studies with extensive follow-up regarding the clinical outcome, analyses of cerebrospinal fluid may provide a scientific basis for

Glycogen Synthase Kinase-3β (GSK3β) Negatively Regulates PTTG1/Human Securin Protein Stability, and GSK3β Inactivation Correlates with Securin Accumulation in Breast Tumors*

PubMed Central

Mora-Santos, Mar; Limón-Mortés, M. Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

2011-01-01

PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCFβTrCP E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers. PMID:21757741

Glycogen synthase kinase-3beta (GSK3beta) negatively regulates PTTG1/human securin protein stability, and GSK3beta inactivation correlates with securin accumulation in breast tumors.

PubMed

Mora-Santos, Mar; Limón-Mortés, M Cristina; Giráldez, Servando; Herrero-Ruiz, Joaquín; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2011-08-26

PTTG1, also known as securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for securin in tumor progression. Stability of securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting securin for proteolysis have not been identified, and mechanistic insight into the cause of securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates securin to promote its proteolysis via SCF(βTrCP) E3 ubiquitin ligase. Importantly, a strong correlation between securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for securin accumulation in breast cancers.

A Large Complement of the Predicted Arabidopsis ARM Repeat Proteins Are Members of the U-Box E3 Ubiquitin Ligase Family1[w

PubMed Central

Mudgil, Yashwanti; Shiu, Shin-Han; Stone, Sophia L.; Salt, Jennifer N.; Goring, Daphne R.

2004-01-01

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis. PMID:14657406

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

PubMed

Zhao, D; Yang, M; Solava, J; Ma, H

1999-09-01

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo. Copyright 1999 Wiley-Liss, Inc.

The Crystal Structure of the Escherichia coli Autoinducer-2 Processing Protein LsrF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diaz, Z.; Xavier, K; Miller, S

2009-01-01

Many bacteria produce and respond to the quorum sensing signal autoinducer-2 (AI-2). Escherichia coli and Salmonella typhimurium are among the species with the lsr operon, an operon containing AI-2 transport and processing genes that are up regulated in response to AI-2. One of the Lsr proteins, LsrF, has been implicated in processing the phosphorylated form of AI-2. Here, we present the structure of LsrF, unliganded and in complex with two phospho-AI-2 analogues, ribose-5-phosphate and ribulose-5-phosphate. The crystal structure shows that LsrF is a decamer of (??)8-barrels that exhibit a previously unseen N-terminal domain swap and have high structural homology withmore » aldolases that process phosphorylated sugars. Ligand binding sites and key catalytic residues are structurally conserved, strongly implicating LsrF as a class I aldolase.« less

Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development

PubMed Central

Jin, Lian; Qin, Qingqing; Wang, Yu; Pu, Yingying; Liu, Lifang; Wen, Xing; Ji, Shaoyi; Wu, Jianguo; Wei, Chunhong; Li, Yi

2016-01-01

The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis. PMID:27606959

lncRNA-HIT promotes cell proliferation of non-small cell lung cancer by association with E2F1.

PubMed

Yu, L; Fang, F; Lu, S; Li, X; Yang, Y; Wang, Z

2017-05-01

Lung cancer is the leading cause of cancer-related death around the world. Long noncoding RNA (lncRNA) has pivotal roles in cancer occurrence and development. However, only a few lncRNAs have been functionally characterized. In the present study, we investigated the effects of lncRNA-HIT (HOXA transcript induced by TGFβ) expression on non-small cell lung cancer (NSCLC) cell phenotype with the gain-of-function and loss-of-function assays. We found that ectopic expression or knockdown of lncRNA-HIT markedly increased or decreased NSCLC cell proliferation, respectively. Moreover, we also showed that lncRNA-HIT interacted with E2F1 to regulate its target genes, such as Survivin, FOXM1, SKP2, NELL2 and DOK1. Collectively, our findings indicated that lncRNA-HIT affected the proliferation of NSCLC cells at least in part via regulating the occupancy of E2F1 in the promoter regions of its target genes. The lncRNA-HIT-E2F1 complex may be a potential target for NSCLC treatment.

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window.

PubMed

James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K; Barclay, Wendy S; Shelton, Holly

2016-10-01

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness - infectivity, spread and pathogenesis - is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.

Nucleolar proteins Bfr2 and Enp2 interact with DEAD-box RNA helicase Dbp4 in two different complexes

PubMed Central

Soltanieh, Sahar; Lapensée, Martin; Dragon, François

2014-01-01

Different pre-ribosomal complexes are formed during ribosome biogenesis, and the composition of these complexes is highly dynamic. Dbp4, a conserved DEAD-box RNA helicase implicated in ribosome biogenesis, interacts with nucleolar proteins Bfr2 and Enp2. We show that, like Dbp4, Bfr2 and Enp2 are required for the early processing steps leading to the production of 18S ribosomal RNA. We also found that Bfr2 and Enp2 associate with the U3 small nucleolar RNA (snoRNA), the U3-specific protein Mpp10 and various pre-18S ribosomal RNA species. Thus, we propose that Bfr2, Dbp4 and Enp2 are components of the small subunit (SSU) processome, a large complex of ∼80S. Sucrose gradient sedimentation analyses indicated that Dbp4, Bfr2 and Enp2 sediment in a peak of ∼50S and in a peak of ∼80S. Bfr2, Dbp4 and Enp2 associate together in the 50S complex, which does not include the U3 snoRNA; however, they associate with U3 snoRNA in the 80S complex (SSU processome). Immunoprecipitation experiments revealed that U14 snoRNA associates with Dbp4 in the 50S complex, but not with Bfr2 or Enp2. The assembly factor Tsr1 is not part of the ‘50S’ complex, indicating this complex is not a pre-40S ribosome. A combination of experiments leads us to propose that Bfr2, Enp2 and Dbp4 are recruited at late steps during assembly of the SSU processome. PMID:24357410

A MADS Box Protein Interacts with a Mating-Type Protein and Is Required for Fruiting Body Development in the Homothallic Ascomycete Sordaria macrospora

PubMed Central

Nolting, Nicole; Pöggeler, Stefanie

2006-01-01

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Δmcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development. PMID:16835449

A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora.

PubMed

Nolting, Nicole; Pöggeler, Stefanie

2006-07-01

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window

PubMed Central

James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K.; Barclay, Wendy S.

2016-01-01

Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness – infectivity, spread and pathogenesis – is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field. PMID:27558742

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

PubMed

de Abreu da Silva, Isabel Caetano; Vicentino, Amanda Roberta Revoredo; Dos Santos, Renata Coutinho; da Fonseca, Rodrigo Nunes; de Mendonça Amarante, Anderson; Carneiro, Vitor Coutinho; de Amorim Pinto, Marcia; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Bisch, Paulo Mascarello; da Silva-Neto, Mario Alberto Cardoso; Fantappié, Marcelo Rosado

2018-05-30

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis. Copyright © 2017. Published by Elsevier B.V.

A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility

PubMed Central

2014-01-01

Background The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean. Result Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins. Conclusion In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral

A soybean MADS-box protein modulates floral organ numbers, petal identity and sterility.

PubMed

Huang, Fang; Xu, Guangli; Chi, Yingjun; Liu, Haicui; Xue, Qian; Zhao, Tuanjie; Gai, Junyi; Yu, Deyue

2014-04-02

The MADS-box transcription factors play fundamental roles in reproductive developmental control. Although the roles of many plant MADS-box proteins have been extensively studied, there are almost no functional studies of them in soybean, an important protein and oil crop in the world. In addition, the MADS-box protein orthologs may have species-specific functions. Controlling male fertility is an important goal in plant hybrid breeding but is difficult in some crops like soybean. The morphological structure of soybean flowers prevents the cross-pollination. Understanding the molecular mechanisms for floral development will aid in engineering new sterile materials that could be applied in hybrid breeding programs in soybean. Through microarray analysis, a flower-enriched gene in soybean was selected and designated as GmMADS28. GmMADS28 belongs to AGL9/SEP subfamily of MADS-box proteins, localized in nucleus and showed specific expression patterns in floral meristems as well as stamen and petal primordia. Expression of GmMADS28 in the stamens and petals of a soybean mutant NJS-10Hfs whose stamens are converted into petals was higher than in those of wild-type plants. Constitutive expression of GmMADS28 in tobacco promoted early flowering and converted stamens and sepals to petals. Interestingly, transgenic plants increased the numbers of sepal, petal and stamen from five to six and exhibited male sterility due to the shortened and curly filaments and the failure of pollen release from the anthers. The ectopic expression of GmMADS28 was found to be sufficient to activate expression of tobacco homologs of SOC1, LEAFY, AGL8/FUL, and DEF. In addition, we observed the interactions of GmMADS28 with soybean homologs of SOC1, AP1, and AGL8/FUL proteins. In this study, we observed the roles of GmMADS28 in the regulation of floral organ number and petal identity. Compared to other plant AGL9/SEP proteins, GmMADS28 specifically regulates floral organ number, filament length and

A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans.

PubMed

Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y

1997-02-01

Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.

Remaining Sites Verification Package for the 100-F-44:2, Discovery Pipeline Near 108-F Building, Waste Site Reclassification Form 2007-006

DOE Office of Scientific and Technical Information (OSTI.GOV)

J. M. Capron

2008-05-30

The 100-F-44:2 waste site is a steel pipeline that was discovered in a junction box during confirmatory sampling of the 100-F-26:4 pipeline from December 2004 through January 2005. The 100-F-44:2 pipeline feeds into the 100-F-26:4 subsite vitrified clay pipe (VCP) process sewer pipeline from the 108-F Biology Laboratory at the junction box. In accordance with this evaluation, the confirmatory sampling results support a reclassification of this site to No Action. The current site conditions achieve the remedial action objectives and the corresponding remedial action goals established in the Remaining Sites ROD. The results of confirmatory sampling show that residual contaminantmore » concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also demonstrate that residual contaminant concentrations are protective of groundwater and the Columbia River.« less

The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc

PubMed Central

Sun, Xiao-Xin; He, Xia; Yin, Li; Komada, Masayuki; Sears, Rosalie C.; Dai, Mu-Shui

2015-01-01

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCFFbw7 (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc’s nucleolar degradation pathway. PMID:25775507

Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

PubMed

Chang, Wing Y; Andrews, Joseph; Carter, David E; Dagnino, Lina

2006-08-01

E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes.

The Emerging Roles of Forkhead Box (FOX) Proteins in Osteosarcoma

PubMed Central

Zhang, Wentao; Duan, Ning; Song, Tao; Li, Zhong; Zhang, Caiguo; Chen, Xun

2017-01-01

Osteosarcoma is the most common bone cancer primarily occurring in children and young adults. Over the past few years, the deregulation of a superfamily transcription factors, known as forkhead box (FOX) proteins, has been demonstrated to contribute to the pathogenesis of osteosarcoma. Molecular mechanism studies have demonstrated that FOX family proteins participate in a variety of signaling pathways and that their expression can be regulated by multiple factors. The dysfunction of FOX genes can alter osteosarcoma cell differentiation, metastasis and progression. In this review, we summarized the evidence that FOX genes play direct or indirect roles in the development and progression of osteosarcoma, and evaluated the emerging role of FOX proteins as targets for therapeutic intervention. PMID:28775781

Down-regulation of kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly)phenols and tolerance to ultraviolet radiation

DOE PAGES

Zhang, Xuebin; Liu, Chang -Jun; Gou, Mingyue; ...

2014-12-01

Phenylpropanoid biosynthesis in plants engenders myriad phenolics with diverse biological functions. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway, directing primary metabolic flux into a phenylpropanoid branch. Previously, we demonstrated that the Arabidopsis Kelch-domain containing F-box proteins, AtKFB01, -20, and -50, function as the negative regulators controlling phenylpropanoid biosynthesis via mediating PAL’s ubiquitination and subsequent degradation. Here, we reveal that Arabidopsis KFB39, a close homolog of AtKFB50, also interacts physically with PAL isozymes and modulates PALs' stability and activity. Disturbing the expression of KFB39 reciprocally affects the accumulation/deposition of a set of phenylpropanoid end products, suggesting thatmore » KFB39 is an additional post-translational regulator responsible for the turnover of PAL and negatively controlling phenylpropanoid biosynthesis. Furthermore, we discover that exposure of Arabidopsis to UV-B radiation suppresses the expression of all four KFB genes while inducing the transcription of PAL isogenes; these data suggest that Arabidopsis consolidates both transcriptional and post-translational regulation mechanisms to maximize its responses to UV stress. Simultaneous down-regulation of all four identified KFBs significantly enhances the production of (poly)phenols and the plant’s tolerance to UV irradiation. This study offers a biotechnological approach for engineering the production of useful phenolic chemicals and for increasing a plant’s resistance to environmental stress.« less

Max-E47, a Designed Minimalist Protein that Targets the E-Box DNA Site In Vivo and In Vitro

PubMed Central

Xu, Jing; Chen, Gang; De Jong, Antonia T.; Shahravan, S. Hesam; Shin, Jumi A.

2009-01-01

Max-E47 is a designed hybrid protein comprising the Max DNA-binding basic region and E47 HLH dimerization subdomain. In the yeast one-hybrid system (Y1H), Max-E47 shows strong transcriptional activation from the E-box site, 5'-CACGTG, targeted by the Myc/Max/Mad network of transcription factors; two mutants, Max-E47Y and Max-E47YF, activate more weakly from the E-box in the Y1H. Quantitative fluorescence anisotropy titrations to gain free energies of protein:DNA binding gave low nM Kd values for the native MaxbHLHZ, Max-E47, and the Y and YF mutants binding to the E-box site (14 nM, 15 nM, 9 nM, and 6 nM, respectively), with no detectable binding to a nonspecific control duplex. Because these minimalist, E-box-binding hybrids have no activation domain and no interactions with the c-MycbHLHZ, as shown by the yeast two-hybrid assay, they can potentially serve as dominant-negative inhibitors that suppress activation of E-box-responsive genes targeted by transcription factors including the c-Myc/Max complex. As proof-of-principle, we used our modified Y1H, which allows direct competition between two proteins vying for a DNA target, to show that Max-E47 effectively outcompetes the native MaxbHLHZ for the E-box; weaker competition is observed from the two mutants, consistent with Y1H results. These hybrids provide a minimalist scaffold for further exploration of the relationship between protein structure and DNA-binding function and may have applications as protein therapeutics or biochemical probes capable of targeting the E-box site. PMID:19449889

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Gene transfer of high-mobility group box 1 box-A domain in a rat acute liver failure model.

PubMed

Tanaka, Masayuki; Shinoda, Masahiro; Takayanagi, Atsushi; Oshima, Go; Nishiyama, Ryo; Fukuda, Kazumasa; Yagi, Hiroshi; Hayashida, Tetsu; Masugi, Yohei; Suda, Koichi; Yamada, Shingo; Miyasho, Taku; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Obara, Hideaki; Itano, Osamu; Takeuchi, Hiroya; Sakamoto, Michiie; Tanabe, Minoru; Maruyama, Ikuro; Kitagawa, Yuko

2015-04-01

High-mobility group box 1 (HMGB1) has recently been identified as an important mediator of various kinds of acute and chronic inflammation. The protein encoded by the box-A domain of the HMGB1 gene is known to act as a competitive inhibitor of HMGB1. In this study, we investigated whether box-A gene transfer results in box-A protein production in rats and assessed therapeutic efficacy in vivo using an acute liver failure (ALF) model. Three types of adenovirus vectors were constructed-a wild type and two mutants-and a mutant vector was then selected based on the secretion from HeLa cells. The secreted protein was subjected to a tumor necrosis factor (TNF) production inhibition test in vitro. The vector was injected via the portal vein in healthy Wistar rats to confirm box-A protein production in the liver. The vector was then injected via the portal vein in rats with ALF. Western blot analysis showed enhanced expression of box-A protein in HeLa cells transfected with one of the mutant vectors. The culture supernatant from HeLa cells transfected with the vector inhibited TNF-α production from macrophages. Expression of box-A protein was confirmed in the transfected liver at 72 h after transfection. Transfected rats showed decreased hepatic enzymes, plasma HMGB1, and hepatic TNF-α messenger RNA levels, and histologic findings and survival were significantly improved. HMGB1 box-A gene transfer results in box-A protein production in the liver and appears to have a beneficial effect on ALF in rats. Copyright © 2015 Elsevier Inc. All rights reserved.

A potent transrepression domain in the retinoblastoma protein induces a cell cycle arrest when bound to E2F sites.

PubMed Central

Sellers, W R; Rodgers, J W; Kaelin, W G

1995-01-01

An intact T/E1A-binding domain (the pocket) is necessary, but not sufficient, for the retinoblastoma protein (RB) to bind to DNA-protein complexes containing E2F and for RB to induce a G1/S block. Indirect evidence suggests that the binding of RB to E2F may, in addition to inhibiting E2F transactivation function, generate a complex capable of functioning as a transrepressor. Here we show that a chimera in which the E2F1 transactivation domain was replaced with the RB pocket could, in a DNA-binding and pocket-dependent manner, mimic the ability of RB to repress transcription and induce a cell cycle arrest. In contrast, a transdominant negative E2F1 mutant that is capable of blocking E2F-dependent transactivation did not. Fusion of the RB pocket to a heterologous DNA-binding domain unrelated to E2F likewise generated a transrepressor protein when scored against a suitable reporter. These results suggest that growth suppression by RB is due, at least in part, to transrepression mediated by the pocket domain bound to certain promoters via E2F. Images Fig. 4 Fig. 5 PMID:8524800

Characterization of the mammalian DEAD-box protein DDX5 reveals functional conservation with S. cerevisiae ortholog Dbp2 in transcriptional control and glucose metabolism.

PubMed

Xing, Zheng; Wang, Siwen; Tran, Elizabeth J

2017-07-01

DEAD-box proteins are a class of nonprocessive RNA helicases that dynamically modulate the structure of RNA and ribonucleoprotein complexes (RNPs). However, the precise roles of individual members are not well understood. Work from our laboratory revealed that the DEAD-box protein Dbp2 in Saccharomyces cerevisiae is an active RNA helicase in vitro that functions in transcription by promoting mRNP assembly, repressing cryptic transcription initiation, and regulating long noncoding RNA activity. Interestingly, Dbp2 is also linked to glucose sensing and hexose transporter gene expression. DDX5 is the mammalian ortholog of Dbp2 that has been implicated in cancer and metabolic syndrome, suggesting that the role of Dbp2 and DDX5 in glucose metabolic regulation is conserved. Herein, we present a refined biochemical and biological comparison of yeast Dbp2 and human DDX5 enzymes. We find that human DDX5 possesses a 10-fold higher unwinding activity than Dbp2, which is partially due to the presence of a mammalian/avian specific C-terminal extension. Interestingly, ectopic expression of DDX5 rescues the cold sensitivity, cryptic initiation defects, and impaired glucose import in dbp2 Δ cells, suggesting functional conservation. Consistently, we show that DDX5 promotes glucose uptake and glycolysis in mouse AML12 hepatocyte cells, suggesting that mammalian DDX5 and S. cerevisiae Dbp2 share conserved roles in cellular metabolism. © 2017 Xing et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

GPS-ARM: Computational Analysis of the APC/C Recognition Motif by Predicting D-Boxes and KEN-Boxes

PubMed Central

Ren, Jian; Cao, Jun; Zhou, Yanhong; Yang, Qing; Xue, Yu

2012-01-01

Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase incorporated with Cdh1 and/or Cdc20 recognizes and interacts with specific substrates, and faithfully orchestrates the proper cell cycle events by targeting proteins for proteasomal degradation. Experimental identification of APC/C substrates is largely dependent on the discovery of APC/C recognition motifs, e.g., the D-box and KEN-box. Although a number of either stringent or loosely defined motifs proposed, these motif patterns are only of limited use due to their insufficient powers of prediction. We report the development of a novel GPS-ARM software package which is useful for the prediction of D-boxes and KEN-boxes in proteins. Using experimentally identified D-boxes and KEN-boxes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted. By extensive evaluation and comparison, the GPS-ARM performance was found to be much better than the one using simple motifs. With this powerful tool, we predicted 4,841 potential D-boxes in 3,832 proteins and 1,632 potential KEN-boxes in 1,403 proteins from H. sapiens, while further statistical analysis suggested that both the D-box and KEN-box proteins are involved in a broad spectrum of biological processes beyond the cell cycle. In addition, with the co-localization information, we predicted hundreds of mitosis-specific APC/C substrates with high confidence. As the first computational tool for the prediction of APC/C-mediated degradation, GPS-ARM is a useful tool for information to be used in further experimental investigations. The GPS-ARM is freely accessible for academic researchers at: http://arm.biocuckoo.org. PMID:22479614

Fauna using nest boxes in four timber types in eastern Texas

Treesearch

Richard N. Conner; Daniel Saenz; D. Craig Rudolph

1995-01-01

Occupancy of 240 nest boxes in pure pine, pine-hardwood, upland hardwood, and bottomland hardwood forests (60 boxes in each forest type) were monitored for six years on the Stephen F. Austin Experimental Forest, Nacogdoches County in eastern Texas. Three boxes were placed at twenty sites in each forest type. Initially, each site had a box with 3.2, 4.7, or 5.7 cm...

Regulation of flower development in Arabidopsis by SCF complexes.

PubMed

Ni, Weimin; Xie, Daoxin; Hobbie, Lawrence; Feng, Baomin; Zhao, Dazhong; Akkara, Joseph; Ma, Hong

2004-04-01

SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.

Protein arginine methyltransferase 1 regulates herpes simplex virus replication through ICP27 RGG-box methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Jungeun; Shin, Bongjin; Park, Eui-Soon

2010-01-01

Protein arginine methylation is involved in viral infection and replication through the modulation of diverse cellular processes including RNA metabolism, cytokine signaling, and subcellular localization. It has been suggested previously that the protein arginine methylation of the RGG-box of ICP27 is required for herpes simplex virus type-1 (HSV-1) viral replication and gene expression in vivo. However, a cellular mediator for this process has not yet been identified. In our current study, we show that the protein arginine methyltransferase 1 (PRMT1) is a cellular mediator of the arginine methylation of ICP27 RGG-box. We generated arginine substitution mutants in this domain andmore » examined which arginine residues are required for methylation by PRMT1. R138, R148 and R150 were found to be the major sites of this methylation but additional arginine residues serving as minor methylation sites are still required to sustain the fully methylated form of ICP27 RGG. We also demonstrate that the nuclear foci-like structure formation, SRPK interactions, and RNA-binding activity of ICP27 are modulated by the arginine methylation of the ICP27 RGG-box. Furthermore, HSV-1 replication is inhibited by hypomethylation of this domain resulting from the use of general PRMT inhibitors or arginine mutations. Our data thus suggest that the PRMT1 plays a key role as a cellular regulator of HSV-1 replication through ICP27 RGG-box methylation.« less

M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP

PubMed Central

Watanabe, Nobumoto; Arai, Harumi; Nishihara, Yoshifumi; Taniguchi, Makoto; Watanabe, Naoko; Hunter, Tony; Osada, Hiroyuki

2004-01-01

Wee1, the Cdc2 inhibitory kinase, needs to be down-regulated at the onset of mitosis to ensure rapid activation of Cdc2. Previously, we have shown that human somatic Wee1 (Wee1A) is down-regulated both by protein phosphorylation and degradation, but the underlying mechanisms had not been elucidated. In the present study, we have identified the β-transducin repeat-containing protein 1/2 (β-TrCP1/2) F-box protein-containing SKP1/Cul1/F-box protein (SCF) complex (SCFβ-TrCP1/2) as an E3 ubiquitin ligase for Wee1A ubiquitination. Although Wee1A lacks a consensus DS(p)GXXS(p) phospho-dependent binding motif for β-TrCP, recognition of Wee1A by β-TrCP depended on phosphorylation, and two serine residues in Wee1A, S53 and S123, were found to be the most important phosphorylation sites for β-TrCP recognition. We have found also that the major M-phase kinases polo-like kinase 1 (Plk1) and Cdc2 are responsible for the phosphorylation of S53 and S123, respectively, and that in each case phosphorylation generates an unconventional phospho-degron (signal for degradation) that can be recognized by β-TrCP. Phosphorylation of Wee1A by these kinases cooperatively stimulated the recognition and ubiquitination of Wee1A by SCFβ-TrCP1/2 in vitro. Mutation of these residues or depletion of β-TrCP by small-interfering RNA treatment increased the stability of Wee1A in HeLa cells. Moreover, our analysis indicates that β-TrCP-dependent degradation of Wee1A is important for the normal onset of M-phase in vivo. These results also establish the existence of a feedback loop between Cdc2 and Wee1A in somatic cells that depends on ubiquitination and protein degradation and ensures the rapid activation of Cdc2 when cells are ready to divide. PMID:15070733

UV and arsenate toxicity: a specific and sensitive yeast bioluminescence assay.

PubMed

Bakhrat, Anya; Eltzov, Evgeni; Finkelstein, Yishay; Marks, Robert S; Raveh, Dina

2011-06-01

We describe a Saccharomyces cerevisiae bioluminescence assay for UV and arsenate in which bacterial luciferase genes are regulated by the promoter of the yeast gene, UFO1. UFO1 encodes the F-box subunit of the Skp1–Cdc53–F-box protein ubiquitin ligase complex and is induced by DNA damage and by arsenate. We engineered the UFO1 promoter into an existing yeast bioreporter that employs human genes for detection of steroid hormone-disrupting compounds in water bodies. Our analysis indicates that use of an endogenous yeast promoter in different mutant backgrounds allows discrimination between different environmental signals. The UFO1-engineered yeast give a robust bioluminescence response to UVB and can be used for evaluating UV protective sunscreens. They are also effective in detecting extremely low concentrations of arsenate, particularly in pdr5Δ mutants that lack a mechanism to extrude toxic chemicals; however, they do not respond to cadmium or mercury. Combined use of endogenous yeast promoter elements and mutants of stress response pathways may facilitate development of high-specificity yeast bioreporters able to discriminate between closely related chemicals present together in the environment.

Inhibition of E2F1 activity and cell cycle progression by arsenic via retinoblastoma protein.

PubMed

Sheldon, Lynn A

2017-01-01

The regulation of cell cycle progression by steroid hormones and growth factors is important for maintaining normal cellular processes including development and cell proliferation. Deregulated progression through the G1/S and G2/M cell cycle transitions can lead to uncontrolled cell proliferation and cancer. The transcription factor E2F1, a key cell cycle regulator, targets genes encoding proteins that regulate cell cycle progression through the G1/S transition as well as proteins important in DNA repair and apoptosis. E2F1 expression and activity is inhibited by inorganic arsenic (iAs) that has a dual role as a cancer therapeutic and as a toxin that leads to diseases including cancer. An understanding of what underlies this dichotomy will contribute to understanding how to use iAs as a more effective therapeutic and also how to treat cancers that iAs promotes. Here, we show that quiescent breast adenocarcinoma MCF-7 cells treated with 17-β estradiol (E2) progress through the cell cycle, but few cells treated with E2 + iAs progress from G1 into S-phase due to a block in cell cycle progression. Our data support a model in which iAs inhibits the dissociation of E2F1 from the tumor suppressor, retinoblastoma protein (pRB) due to changes in pRB phosphorylation which leads to decreased E2F1 transcriptional activity. These findings present an explanation for how iAs can disrupt cell cycle progression through E2F1-pRB and has implications for how iAs acts as a cancer therapeutic as well as how it may promote tumorigenesis through decreased DNA repair.

Inhibition of master transcription factors in pluripotent cells induces early stage differentiation

PubMed Central

De, Debojyoti; Jeong, Myong-Ho; Leem, Young-Eun; Svergun, Dmitri I.; Wemmer, David E.; Kang, Jong-Sun; Kim, Kyeong Kyu; Kim, Sung-Hou

2014-01-01

The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein–protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation. PMID:24434556

Purification, crystallization and preliminary X-ray analysis of the inverse F-BAR domain of the human srGAP2 protein.

PubMed

Wang, Hongpeng; Zhang, Yan; Zhang, Zhenyi; Jin, Wei Lin; Wu, Geng

2014-01-01

Bin-Amphiphysin-Rvs (BAR) domain proteins play essential roles in diverse cellular processes by inducing membrane invaginations or membrane protrusions. Among the BAR superfamily, the `classical' BAR and Fes/CIP4 homology BAR (F-BAR) subfamilies of proteins usually promote membrane invaginations, whereas the inverse BAR (I-BAR) subfamily generally incur membrane protrusions. Despite possessing an N-terminal F-BAR domain, the srGAP2 protein regulates neurite outgrowth and neuronal migration by causing membrane protrusions reminiscent of the activity of I-BAR domain proteins. In this study, the inverse F-BAR (IF-BAR) domain of human srGAP2 was overexpressed, purified and crystallized. The crystals of the srGAP2 IF-BAR domain protein diffracted to 3.50 Å resolution and belonged to space group P2(1). These results will facilitate further structural determination of the srGAP2 IF-BAR domain and the ultimate elucidation of its peculiar behaviour of inducing membrane protrusions rather than membrane invaginations.

Coilin association with Box C/D scaRNA suggests a direct role for the Cajal body marker protein in scaRNP biogenesis

PubMed Central

Enwerem, Isioma I.; Velma, Venkatramreddy; Broome, Hanna J.; Kuna, Marija; Begum, Rowshan A.; Hebert, Michael D.

2014-01-01

ABSTRACT Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase. PMID:24659245

Non-avian animal reservoirs present a source of influenza A PB1-F2 proteins with novel virulence-enhancing markers.

PubMed

Alymova, Irina V; York, Ian A; McCullers, Jonathan A

2014-01-01

PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

PubMed

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

High Mobility Group Box 1 Protein as an Auxiliary Biomarker for Dengue Diagnosis

PubMed Central

Allonso, Diego; Vázquez, Susana; Guzmán, Maria G.; Mohana-Borges, Ronaldo

2013-01-01

Despite the availability of many methods for rapid and early diagnosis of dengue, there is still a need to develop new approaches that not only combine low cost, specificity, and sensitivity, but also are capable of accurately detecting secondary infection in the early stages of the disease. We report the potential of the high mobility group box 1 protein as an auxiliary biomarker for early dengue diagnosis. We tested a 205-sample serum panel that included negative and positive samples from primary and secondary dengue cases, as well as samples from patients with dengue-like symptoms. We observed that high mobility group box 1 protein was generally detected only in dengue-positive samples for persons with primary and secondary infections. These results highlight the possibility of using this endogenous molecule as an auxiliary biomarker to aid in dengue detection and improve current methods for early diagnosis of dengue. PMID:23269659

SCFSLF-mediated cytosolic degradation of S-RNase is required for cross-pollen compatibility in S-RNase-based self-incompatibility in Petunia hybrida

PubMed Central

Liu, Wei; Fan, Jiangbo; Li, Junhui; Song, Yanzhai; Li, Qun; Zhang, Yu'e; Xue, Yongbiao

2014-01-01

Many flowering plants adopt self-incompatibility (SI) to maintain their genetic diversity. In species of Solanaceae, Plantaginaceae, and Rosaceae, SI is genetically controlled by a single S-locus with multiple haplotypes. The S-locus has been shown to encode S-RNases expressed in pistil and multiple SLF (S-locus F-box) proteins in pollen controlling the female and male specificity of SI, respectively. S-RNases appear to function as a cytotoxin to reject self-pollen. In addition, SLFs have been shown to form SCF (SKP1/Cullin1/F-box) complexes to serve as putative E3 ubiquitin ligase to interact with S-RNases. Previously, two different mechanisms, the S-RNase degradation and the S-RNase compartmentalization, have been proposed as the restriction mechanisms of S-RNase cytotoxicity allowing compatible pollination. In this study, we have provided several lines of evidence in support of the S-RNase degradation mechanism by a combination of cellular, biochemical and molecular biology approaches. First, both immunogold labeling and subcellular fractionation assays showed that two key pollen SI factors, PhS3L-SLF1 and PhSSK1 (SLF-interacting SKP1-like1) from Petunia hybrida, a Solanaceous species, are co-localized in cytosols of both pollen grains and tubes. Second, PhS3L-RNases are mainly detected in the cytosols of both self and non-self-pollen tubes after pollination. Third, we found that PhS-RNases selectively interact with PhSLFs by yeast two-hybrid and co-immunoprecipitation assays. Fourth, S-RNases are specifically degraded in compatible pollen tubes by non-self SLF action. Taken together, our results demonstrate that SCFSLF-mediated non-self S-RNase degradation occurs in the cytosol of pollen tube through the ubiquitin/26S proteasome system serving as the major mechanism to neutralize S-RNase cytotoxicity during compatible pollination in P. hybrida. PMID:25101113

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

PubMed

Gao, William N D; Carpentier, David C J; Ewles, Helen A; Lee, Stacey-Ann; Smith, Geoffrey L

2017-08-01

Vaccinia virus (VACV) utilizes microtubule-mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin-1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin-1, yet in its absence VACV egress still occurs on microtubules. During a co-immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C-terminal tail of KLC2, to a region that overlaps the binding site of cellular 14-3-3 proteins. F12/E2 displaces 14-3-3 from KLC and, unlike 14-3-3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N-terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co-operatively enhance A36 association with KLC, particularly when using a KLC1-KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co-operatively associate with kinesin-1. © 2017 The Authors. Traffic published by John Wiley & Sons Ltd.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

PubMed

Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

2015-07-01

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparative structural analysis of human DEAD-box RNA helicases.

PubMed

Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

2010-09-30

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.

Comparative Structural Analysis of Human DEAD-Box RNA Helicases

PubMed Central

Schütz, Patrick; Karlberg, Tobias; van den Berg, Susanne; Collins, Ruairi; Lehtiö, Lari; Högbom, Martin; Holmberg-Schiavone, Lovisa; Tempel, Wolfram; Park, Hee-Won; Hammarström, Martin; Moche, Martin; Thorsell, Ann-Gerd; Schüler, Herwig

2010-01-01

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members. PMID:20941364

FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation.

PubMed

Babaei-Jadidi, Roya; Li, Ningning; Saadeddin, Anas; Spencer-Dene, Bradley; Jandke, Anett; Muhammad, Belal; Ibrahim, ElSayed E; Muraleedharan, Ranjithmenon; Abuzinadah, Mohammed; Davis, Hayley; Lewis, Annabelle; Watson, Susan; Behrens, Axel; Tomlinson, Ian; Nateri, Abdolrahman Shams

2011-02-14

The Fbxw7 (F-box/WD repeat-containing protein 7; also called CDC4, Sel10, Ago, and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase complex acts as a tumor suppressor in several tissues and targets multiple transcriptional activators and protooncogenes for ubiquitin-mediated degradation. To understand Fbxw7 function in the murine intestine, in this study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre (Fbxw7(ΔG)). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and induced development of adenomas at 9-10 mo of age. In the context of APC (adenomatous polyposis coli) deficiency (Apc(Min/+) mice), loss of Fbxw7 accelerated intestinal tumorigenesis and death and promoted accumulation of β-catenin in adenomas at late but not early time points. At early time points, Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may also influence cell proliferation. DEK accumulation and altered TPM RNA splicing were also detected in FBXW7 mutant human colorectal tumor tissues. Given their reduced lifespan and increased incidence of intestinal tumors, Apc(Min/+)Fbxw7(ΔG) mice may be used for testing carcinogenicity and drug screening.

Multiple interactions amongst floral homeotic MADS box proteins.

PubMed Central

Davies, B; Egea-Cortines, M; de Andrade Silva, E; Saedler, H; Sommer, H

1996-01-01

Most known floral homeotic genes belong to the MADS box family and their products act in combination to specify floral organ identity by an unknown mechanism. We have used a yeast two-hybrid system to investigate the network of interactions between the Antirrhinum organ identity gene products. Selective heterodimerization is observed between MADS box factors. Exclusive interactions are detected between two factors, DEFICIENS (DEF) and GLOBOSA (GLO), previously known to heterodimerize and control development of petals and stamens. In contrast, a third factor, PLENA (PLE), which is required for reproductive organ development, can interact with the products of MADS box genes expressed at early, intermediate and late stages. We also demonstrate that heterodimerization of DEF and GLO requires the K box, a domain not found in non-plant MADS box factors, indicating that the plant MADS box factors may have different criteria for interaction. The association of PLENA and the temporally intermediate MADS box factors suggests that part of their function in mediating between the meristem and organ identity genes is accomplished through direct interaction. These data reveal an unexpectedly complex network of interactions between the factors controlling flower development and have implications for the determination of organ identity. Images PMID:8861961

NLRX1 prevents mitochondrial induced apoptosis and enhances macrophage antiviral immunity by interacting with influenza virus PB1-F2 protein

PubMed Central

Jaworska, Joanna; Coulombe, François; Downey, Jeffrey; Tzelepis, Fanny; Shalaby, Karim; Tattoli, Ivan; Berube, Julie; Rousseau, Simon; Martin, James G.; Girardin, Stephen E.; McCullers, Jonathan A.; Divangahi, Maziar

2014-01-01

To subvert host immunity, influenza A virus (IAV) induces early apoptosis in innate immune cells by disrupting mitochondria membrane potential via its polymerase basic protein 1-frame 2 (PB1-F2) accessory protein. Whether immune cells have mechanisms to counteract PB1-F2–mediated apoptosis is currently unknown. Herein, we define that the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 binds to viral protein PB1-F2, preventing IAV-induced macrophage apoptosis and promoting both macrophage survival and type I IFN signaling. We initially observed that Nlrx1-deficient mice infected with IAV exhibited increased pulmonary viral replication, as well as enhanced inflammatory-associated pulmonary dysfunction and morbidity. Analysis of the lungs of IAV-infected mice revealed markedly enhanced leukocyte recruitment but impaired production of type I IFN in Nlrx1−/− mice. Impaired type I IFN production and enhanced viral replication was recapitulated in Nlrx1−/− macrophages and was associated with increased mitochondrial mediated apoptosis. Through gain- and loss-of-function strategies for protein interaction, we identified that NLRX1 directly bound PB1-F2 in the mitochondria of macrophages. Using a recombinant virus lacking PB1-F2, we confirmed that deletion of PB1-F2 abrogated NLRX1-dependent macrophage type I IFN production and apoptosis. Thus, our results demonstrate that NLRX1 acts as a mitochondrial sentinel protecting macrophages from PB1-F2–induced apoptosis and preserving their antiviral function. We further propose that NLRX1 is critical for macrophage immunity against IAV infection by sensing the extent of viral replication and maintaining a protective balance between antiviral immunity and excessive inflammation within the lungs. PMID:24799673

Antrodia cinnamomea extract inhibits the proliferation of tamoxifen-resistant breast cancer cells through apoptosis and skp2/microRNAs pathway.

PubMed

Lin, Yu-Shih; Lin, Yin-Yin; Yang, Yao-Hsu; Lin, Chun-Liang; Kuan, Feng-Che; Lu, Cheng-Nan; Chang, Geng-He; Tsai, Ming-Shao; Hsu, Cheng-Ming; Yeh, Reming-Albert; Yang, Pei-Rung; Lee, I-Yun; Shu, Li-Hsin; Cheng, Yu-Ching; Liu, Hung-Te; Lee, Kuan-Der; Chang, De-Ching; Wu, Ching-Yuan

2018-05-09

Breast cancer is the most common cancer in women and affects 1.38 million women worldwide per year. Antiestrogens such as tamoxifen, a selective estrogen receptor (ER) modulator, are widely used in clinics to treat ER-positive breast tumors. However, remissions of breast cancer are often followed by resistance to tamoxifen and disease relapse. Despite the increasing understanding of the resistance mechanisms, effective regimens for treating tamoxifen-resistant breast cancer are limited. Antrodia cinnamomea is a traditional medicinal mushroom native only to Taiwan. In this study, we aimed to examine in vitro effect of antrodia cinnamomea in the tamoxifen-resistant cancer. Antrodia cinnamomea was studied for its biological activity against proliferation of tamoxifen-resistant breast cancer by XTT assay. Next, the underlying mechanism was studied by flow cytometry, qPCR and Western's blotting assay. Our results revealed that the ethanol extract of antrodia cinnamomea (AC) can inhibit the growth of breast cancer cells, including MCF-7 cell and tamoxifen-resistant MCF-7 cell lines. Combination treatment with AC and 10 - 6  M tamoxifen have the better inhibitory effect on the proliferation of tamoxifen-resistant MCF-7 cells than only AC did. AC can induce apoptosis in these breast cancer cells. Moreover, it can suppress the mRNA expression of skp2 (S-phase kinase-associated protein 2) by increasing the expressions of miR-21-5p, miR-26-5p, and miR-30-5p in MCF-7 and tamoxifen-resistant MCF-7 cells. These results suggest that the ethanol extract of antrodia cinnamomea could be a novel anticancer agent in the armamentarium of tamoxifen-resistant breast cancer management. Moreover, we hope to identify additional pure compounds that could serve as promising anti-breast cancer candidates for further clinical trials.

Role of Forkhead Box Class O proteins in cancer progression and metastasis.

PubMed

Kim, Chang Geun; Lee, Hyemin; Gupta, Nehal; Ramachandran, Sharavan; Kaushik, Itishree; Srivastava, Sangeeta; Kim, Sung-Hoon; Srivastava, Sanjay K

2018-06-01

It is now widely accepted that several gene alterations including transcription factors are critically involved in cancer progression and metastasis. Forkhead Box Class O proteins (FoxOs) including FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX and FoxO6 transcription factors are known to play key roles in proliferation, apoptosis, metastasis, cell metabolism, aging and cancer biology through their phosphorylation, ubiquitination, acetylation and methylation. Though FoxOs are proved to be mainly regulated by upstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt signaling pathway, the role of FoxOs in cancer progression and metastasis still remains unclear so far. Thus, with previous experimental evidences, the present review discussed the role of FoxOs in association with metastasis related molecules including cannabinoid receptor 1 (CNR1), Cdc25A/Cdk2, Src, serum and glucocorticoid inducible kinases (SGKs), CXCR4, E-cadherin, annexin A8 (ANXA8), Zinc finger E-box-binding homeobox 2 (ZEB2), human epidermal growth factor receptor 2 (HER2) and mRNAs such as miR-182, miR-135b, miR-499-5p, miR-1274a, miR-150, miR-34b/c and miR-622, subsequently analyzed the molecular mechanism of some natural compounds targeting FoxOs and finally suggested future research directions in cancer progression and metastasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

PubMed

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells

PubMed Central

Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

2015-01-01

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells. PMID:25892555

Down-Regulation of Kelch Domain-Containing F-Box Protein in Arabidopsis Enhances the Production of (Poly)phenols and Tolerance to Ultraviolet Radiation1[OPEN

PubMed Central

Zhang, Xuebin; Gou, Mingyue; Guo, Chunrong; Yang, Huijun; Liu, Chang-Jun

2015-01-01

Phenylpropanoid biosynthesis in plants engenders myriad phenolics with diverse biological functions. Phenylalanine ammonia-lyase (PAL) is the first committed enzyme in the pathway, directing primary metabolic flux into a phenylpropanoid branch. Previously, we demonstrated that the Arabidopsis (Arabidopsis thaliana) Kelch domain-containing F-box proteins, AtKFB01, AtKFB20, and AtKFB50, function as the negative regulators controlling phenylpropanoid biosynthesis via mediating PAL’s ubiquitination and subsequent degradation. Here, we reveal that Arabidopsis KFB39, a close homolog of AtKFB50, also interacts physically with PAL isozymes and modulates PAL stability and activity. Disturbing the expression of KFB39 reciprocally affects the accumulation/deposition of a set of phenylpropanoid end products, suggesting that KFB39 is an additional posttranslational regulator responsible for the turnover of PAL and negatively controlling phenylpropanoid biosynthesis. Furthermore, we discover that exposure of Arabidopsis to ultraviolet (UV)-B radiation suppresses the expression of all four KFB genes while inducing the transcription of PAL isogenes; these data suggest that Arabidopsis consolidates both transcriptional and posttranslational regulation mechanisms to maximize its responses to UV light stress. Simultaneous down-regulation of all four identified KFBs significantly enhances the production of (poly)phenols and the plant’s tolerance to UV irradiation. This study offers a biotechnological approach for engineering the production of useful phenolic chemicals and for increasing a plant’s resistance to environmental stress. PMID:25502410

A STE12 homologue of the homothallic ascomycete Sordaria macrospora interacts with the MADS box protein MCM1 and is required for ascosporogenesis.

PubMed

Nolting, Nicole; Pöggeler, Stefanie

2006-11-01

The MADS box protein MCM1 controls diverse developmental processes and is essential for fruiting body formation in the homothallic ascomycete Sordaria macrospora. MADS box proteins derive their regulatory specificity from a wide range of different protein interactions. We have recently shown that the S. macrospora MCM1 is able to interact with the alpha-domain mating-type protein SMTA-1. To further evaluate the functional roles of MCM1, we used the yeast two-hybrid approach to identify MCM1-interacting proteins. From this screen, we isolated a protein with a putative N-terminal homeodomain and C-terminal C2/H2-Zn2+ finger domains. The protein is a member of the highly conserved fungal STE12 transcription factor family of proteins and was therefore termed STE12. Furthermore, we demonstrate by means of two-hybrid and far western analysis that in addition to MCM1, the S. macrospora STE12 protein is able to interact with the mating-type protein SMTA-1. Unlike the situation in the closely related heterothallic ascomycete Neurospora crassa, deletion (Delta) of the ste12 gene in S. macrospora neither affects vegetative growth nor fruiting body formation. However, ascus and ascospore development are highly impaired by the Deltaste12 mutation. Our data provide another example of the functional divergence within the fungal STE12 transcription factor family.

The controversial role of forkhead box F2 (FOXF2) transcription factor in breast cancer.

PubMed

Lo, Pang-Kuo

2017-01-01

Deregulating the subcellular localization, functions and expression of Forkhead box (FOX) transcription factors that are critically involved in embryonic development and multiple biological processes is known to result in the development and progression of diseases, in particular cancer. Human FOXF transcription factors, including FOXF1 and FOXF2, are a subfamily of the FOX gene family. The recent findings from ours and others have linked FOXF2 to breast cancer development and progression. Our studies have shown that FOXF2 acts as a tumor-suppressive inhibitor of DNA replication in luminal and HER2-positive breast cancers and as an oncogenic activator of the epithelial-mesenchymal transition (EMT) in triple-negative/basal-like breast cancers (TN/BLBC), suggesting that FOXF2 plays a dual role in breast cancer. However, studies from Feng's research group have pointed out an opposite role of FOXF2 in TN/BLBC, which acts as an inhibitor of the EMT and as a promoter of cell proliferation in TN/BLBC. These discrepancies between our and Feng's studies have caused controversy in the role of FOXF2 in breast cancer. This article reviews both studies and discusses what causes might have led to these inconsistencies as well as what future experiments are needed to solve this debate.

Activation of the NLRP3 inflammasome by IAV virulence protein PB1-F2 contributes to severe pathophysiology and disease.

PubMed

McAuley, Julie L; Tate, Michelle D; MacKenzie-Kludas, Charley J; Pinar, Anita; Zeng, Weiguang; Stutz, Andrea; Latz, Eicke; Brown, Lorena E; Mansell, Ashley

2013-01-01

The ability for a host to recognize infection is critical for virus clearance and often begins with induction of inflammation. The PB1-F2 of pathogenic influenza A viruses (IAV) contributes to the pathophysiology of infection, although the mechanism for this is unclear. The NLRP3-inflammasome has been implicated in IAV pathogenesis, but whether IAV virulence proteins can be activators of the complex is unknown. We investigated whether PB1-F2-mediated activation of the NLRP3-inflammasome is a mechanism contributing to overt inflammatory responses to IAV infection. We show PB1-F2 induces secretion of pyrogenic cytokine IL-1β by activating the NLRP3-inflammasome, contributing to inflammation triggered by pathogenic IAV. Compared to infection with wild-type virus, mice infected with reverse engineered PB1-F2-deficient IAV resulted in decreased IL-1β secretion and cellular recruitment to the airways. Moreover, mice exposed to PB1-F2 peptide derived from pathogenic IAV had enhanced IL-1β secretion compared to mice exposed to peptide derived from seasonal IAV. Implicating the NLRP3-inflammasome complex specifically, we show PB1-F2 derived from pathogenic IAV induced IL-1β secretion was Caspase-1-dependent in human PBMCs and NLRP3-dependent in mice. Importantly, we demonstrate PB1-F2 is incorporated into the phagolysosomal compartment, and upon acidification, induces ASC speck formation. We also show that high molecular weight aggregated PB1-F2, rather than soluble PB1-F2, induces IL-1β secretion. Furthermore, NLRP3-deficient mice exposed to PB1-F2 peptide or infected with PB1-F2 expressing IAV were unable to efficiently induce the robust inflammatory response as observed in wild-type mice. In addition to viral pore forming toxins, ion channel proteins and RNA, we demonstrate inducers of NLRP3-inflammasome activation may include disordered viral proteins, as exemplified by PB1-F2, acting as host pathogen 'danger' signals. Elucidating immunostimulatory PB1-F2 mediation

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF

PubMed Central

García-Cano, Elena; Magori, Shimpei; Sun, Qi; Ding, Zehong; Lazarowitz, Sondra G.; Citovsky, Vitaly

2015-01-01

Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis. PMID:26571494

Effects of Quercetin Supplementation on Lipid and Protein Metabolism after Classic Boxing Training

ERIC Educational Resources Information Center

Demirci, Nevzat

2017-01-01

The metabolic fitness (MF) is a component of athletes' physical conditioning. This study aims to investigate the effects of quercetin supplementation on Turkish Junior athletes' lipid and protein metabolism relating to MF after one month classic boxing training. Totally 20 voluntary junior male athletes were separated into two equal groups as the…

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

PubMed Central

Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

2013-01-01

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation. PMID:23797863

Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

NASA Astrophysics Data System (ADS)

Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

2013-09-01

Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

Two high-mobility group box domains act together to underwind and kink DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.

The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A ismore » thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.« less

Identification and expression analysis of the SQUAMOSA promoter-binding protein (SBP)-box gene family in Prunus mume.

PubMed

Xu, Zongda; Sun, Lidan; Zhou, Yuzhen; Yang, Weiru; Cheng, Tangren; Wang, Jia; Zhang, Qixiang

2015-10-01

SQUAMOSA promoter-binding protein (SBP)-box family genes encode plant-specific transcription factors that play crucial roles in plant development, especially flower and fruit development. However, little information on this gene family is available for Prunus mume, an ornamental and fruit tree widely cultivated in East Asia. To explore the evolution of SBP-box genes in Prunus and explore their functions in flower and fruit development, we performed a genome-wide analysis of the SBP-box gene family in P. mume. Fifteen SBP-box genes were identified, and 11 of them contained an miR156 target site. Phylogenetic and comprehensive bioinformatics analyses revealed that different groups of SBP-box genes have undergone different evolutionary processes and varied in their length, structure, and motif composition. Purifying selection has been the main selective constraint on both paralogous and orthologous SBP-box genes. In addition, the sequences of orthologous SBP-box genes did not diverge widely after the split of P. mume and Prunus persica. Expression analysis of P. mume SBP-box genes revealed their diverse spatiotemporal expression patterns. Three duplicated SBP-box genes may have undergone subfunctionalization in Prunus. Most of the SBP-box genes showed high transcript levels in flower buds and young fruit. The four miR156-nontargeted genes were upregulated during fruit ripening. Together, these results provide information about the evolution of SBP-box genes in Prunus. The expression analysis lays the foundation for further research on the functions of SBP-box genes in P. mume and other Prunus species, especially during flower and fruit development.

Distinct domains of the AVRPM3A2/F2 avirulence protein from wheat powdery mildew are involved in immune receptor recognition and putative effector function

USDA-ARS?s Scientific Manuscript database

Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after coexpression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity is unknown....

Composition-dependent Membrane Disruption by the Proapoptotic Protein PB1F2 from HK97 Influenza A Virus.

PubMed

Wang, Yujuan; Yang, Jing; Wang, Jiarong; Zhu, Lei; Wang, Junfeng

2018-06-22

PB1F2 is a proapoptotic protein encoded by an alternative reading frame in the influenza A virus. Its accumulation accelerates mitochondrial fragmentation by decreasing the mitochondrial membrane potential following translocation into the mitochondrial inner membrane space, but the mechanistic underpinnings remain unclear. Herein, the PB1F2 from HK97 was expressed and purified in soluble form. The interaction between PB1F2 and the mitochondrial membrane were investigated using three membrane mimics, liposomes, bicelles and nanodiscs. We show that the interactions between PB1F2 and membrane mimics depend on lipid type and are time- and dose-dependent. The primary membrane target of PB1F2 is phosphatidylcholine, the lipid that forms the major component of mitochondrial inner membranes. PB1F2 disrupts the integrity of lipid membranes by forming micelle-like PB1F2-lipid assemblies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A respiratory syncytial virus (RSV) anti-G protein F(ab')2 monoclonal antibody suppresses mucous production and breathing effort in RSV rA2-line19F-infected BALB/c mice.

PubMed

Boyoglu-Barnum, Seyhan; Gaston, Kelsey A; Todd, Sean O; Boyoglu, Cemil; Chirkova, Tatiana; Barnum, Thomas R; Jorquera, Patricia; Haynes, Lia M; Tripp, Ralph A; Moore, Martin L; Anderson, Larry J

2013-10-01

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Increased airway resistance and increased airway mucin production are two manifestations of RSV infection in children. RSV rA2-line19F infection induces pulmonary mucous production and increased breathing effort in BALB/c mice and provides a way to assess these manifestations of RSV disease in an animal model. In the present study, we investigated the effect of prophylactic treatment with the F(ab')2 form of the anti-G protein monoclonal antibody (MAb) 131-2G on disease in RSV rA2-line19F-challenged mice. F(ab')2 131-2G does not affect virus replication. It and the intact form that does decrease virus replication prevented increased breathing effort and airway mucin production, as well as weight loss, pulmonary inflammatory-cell infiltration, and the pulmonary substance P and pulmonary Th2 cytokine levels that occur in mice challenged with this virus. These data suggest that the RSV G protein contributes to prominent manifestations of RSV disease and that MAb 131-2G can prevent these manifestations of RSV disease without inhibiting virus infection.

Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F.

PubMed

Loftus, Kyle M; Cui, Heying; Coutavas, Elias; King, David S; Ceravolo, Amanda; Pereiras, Dylan; Solmaz, Sozanne R

2017-08-03

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development.

PubMed

Litholdo, Celso G; Parker, Benjamin L; Eamens, Andrew L; Larsen, Martin R; Cordwell, Stuart J; Waterhouse, Peter M

2016-06-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic Identification of Putative MicroRNA394 Target Genes in Arabidopsis thaliana Identifies Major Latex Protein Family Members Critical for Normal Development*

PubMed Central

Litholdo, Celso G.; Parker, Benjamin L.; Eamens, Andrew L.; Larsen, Martin R.; Cordwell, Stuart J.; Waterhouse, Peter M.

2016-01-01

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem organization. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development. PMID:27067051

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

PubMed

Kamencayová, M; Košík, I; Hunková, J; Subr, Z W

2014-01-01

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

PubMed

Hattori, Yoshikazu; Heidenreich, David; Ono, Yuki; Sugiki, Toshihiko; Yokoyama, Kei-Ichi; Suzuki, Ei-Ichiro; Fujiwara, Toshimichi; Kojima, Chojiro

2017-08-01

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15 N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19 F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19 F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19 F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19 F-labeling method was 3.5-fold more sensitive than 15 N-labeling, and could be combined with other chemical modification techniques such as lysine 13 C-methylation. 13 C-dimethylated- 19 F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

Olympic boxing is associated with elevated levels of the neuronal protein tau in plasma.

PubMed

Neselius, Sanna; Zetterberg, Henrik; Blennow, Kaj; Randall, Jeffrey; Wilson, David; Marcusson, Jan; Brisby, Helena

2013-01-01

The aim of this study was to investigate if olympic (amateur) boxing is associated with elevation of brain injury biomarkers in peripheral blood compared to controls. Thirty olympic boxers competing in at least 47 bouts were compared to 25 controls. Blood was collected from the controls at one occasion and from the boxers within 1-6 days after a bout and after a rest period of at least 14 days. Tau concentration in plasma was determined using a novel single molecule ELISA assay and S-100B, glial fibrillary acidic protein, brain-derived neurotrophic factor and amyloid β 1-42 were determined using standard immunoassays. None of the boxers had been knocked-out during the bout. Plasma-tau was significantly increased in the boxers after a bout compared to controls (mean ± SD, 2.46 ± 5.10 vs. 0.79 ± 0.961 ng L(-1), p = 0.038). The other brain injury markers did not differ between the groups. Plasma-tau decreased significantly in the boxers after a resting period compared to after a bout (p = 0.030). Olympic boxing is associated with elevation of tau in plasma. The repetitive minimal head injury in boxing may lead to axonal injuries that can be diagnosed with a blood test.

Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model.

PubMed

Shin, Heon-Sub; Park, Sang-Yong; Hwang, Eun-Son; Lee, Don-Gil; Mavlonov, Gafurjon Turdalievich; Yi, Tae-Hoo

2014-01-01

This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

Respiratory syncytial virus prolifically infects N2a neuronal cells, leading to TLR4 and nucleolin protein modulations and RSV F protein co-localization with TLR4 and nucleolin.

PubMed

Yuan, Xiaoling; Hu, Tao; He, Hanwen; Qiu, Huan; Wu, Xuan; Chen, Jingxian; Wang, Minmin; Chen, Cheng; Huang, Shenghai

2018-02-10

Respiratory syncytial virus (RSV) infects the central nervous system, resulting in neurological symptoms. However, the precise underlying pathogenic mechanisms have not been elucidated. In the present study, the infectivity of RSV on N2a neuronal cells and the possible roles of Toll-like receptor 4 (TLR4) and nucleolin (C23) during RSV infection were investigated. We compared two experimental groups (infected and non-infected) and monitored the RSV viral titers in the culture supernatant by a viral plaque assay. We also inspected the morphology of the nucleus in infected N2a cells. We measured the level of RSV F protein and studied its co-localization with TLR4 and nucleolin using immunofluorescence assays and laser confocal microscopy. The potential interaction of RSV F protein with TLR4 and nucleolin was examined by coimmunoprecipitation. The expression changes of TLR4, nucleolin, TLR3 and TLR7 proteins in N2a cells and IL-6 and TNF-α in the culture supernatant were investigated by Western Blot analysis and ELISA assay. Changes in neuronal cell apoptosis status was examined by flow cytometry. The results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, F protein expression, RSV viral titers, and late apoptotic levels of N2a cells. RSV infection induced co-localization of RSV F protein with TLR4 and nucleolin, which could potentially lead to a direct interaction. Furthermore, it was found that TLR4 and nucleolin levels increased early after infection and decreased subsequently, whereas TLR3 and TLR7 expression increased throughout RSV infection. The RSV Long strain can prolifically infect N2a neuronal cells, modulating the expression of TLR4 and nucleolin, as well as TLR3, TLR7 and their downstream inflammatory factors, and inducing the co-localization of the RSV F protein with TLR4 and nucleolin.

FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation

PubMed Central

Babaei-Jadidi, Roya; Li, Ningning; Saadeddin, Anas; Spencer-Dene, Bradley; Jandke, Anett; Muhammad, Belal; Ibrahim, ElSayed E.; Muraleedharan, Ranjithmenon; Abuzinadah, Mohammed; Davis, Hayley; Lewis, Annabelle; Watson, Susan; Behrens, Axel; Tomlinson, Ian

2011-01-01

The Fbxw7 (F-box/WD repeat–containing protein 7; also called CDC4, Sel10, Ago, and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase complex acts as a tumor suppressor in several tissues and targets multiple transcriptional activators and protooncogenes for ubiquitin-mediated degradation. To understand Fbxw7 function in the murine intestine, in this study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre (Fbxw7ΔG). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and induced development of adenomas at 9–10 mo of age. In the context of APC (adenomatous polyposis coli) deficiency (ApcMin/+ mice), loss of Fbxw7 accelerated intestinal tumorigenesis and death and promoted accumulation of β-catenin in adenomas at late but not early time points. At early time points, Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may also influence cell proliferation. DEK accumulation and altered TPM RNA splicing were also detected in FBXW7 mutant human colorectal tumor tissues. Given their reduced lifespan and increased incidence of intestinal tumors, ApcMin/+Fbxw7ΔG mice may be used for testing carcinogenicity and drug screening. PMID:21282377

Sexually Dimorphic Expression of Foxl2 and Ftz-F1 in Chinese Giant Salamander Andrias Davidianus.