Association of SAP130/SF3b-3 with Cullin-RING ubiquitin ligase complexes and its regulation by the COP9 signalosome

<p>Abstract</p> <p>Background</p> <p>Cullin-RING ubiquitin E3 ligases (CRLs) are regulated by modification of an ubiquitin-like protein, Nedd8 (also known as Rub1) on the cullin subunit. Neddylation is shown to facilitate E3 complex assembly; while un-neddylated cullins are bound by CAND1 that prevents recruitment of the substrates. The level of Nedd8 modification is critically dependent on the COP9 signalosome (CSN), an eight-subunit protein complex containing Nedd8 isopeptidase activity.</p> <p>Results</p> <p>We report isolation of SAP130 (SF3b-3) as a CSN1 interacting protein. SAP130 is homologous to DDB1, and is a component of SF3b RNA splicing complex and STAGA/TFTC transcription complexes, but its specific function within these complexes is unknown. We show that SAP130 can interact with a variety of cullin proteins. It forms tertiary complexes with fully assembled CRL E3 complexes such as SCF^Skp2, Elongin B/C -Cul2- VHL and Cul4-DDB complex by binding to both N-terminal and C-terminal domain of cullins. SAP130 preferentially associates with neddylated cullins <it>in vivo</it>. However knock-down of CAND1 abolished this preference and increased association of SAP130 with Cul2. Furthermore, we provide evidence that CSN regulates SAP130-Cul2 interaction and SAP130-associated polyubiquitinating activity.</p> <p>Conclusion</p> <p>SAP130 is a cullin binding protein that is likely involved in the Nedd8 pathway. The association of SAP130 with various cullin member proteins such as Cul1, Cul2 and Cul4A is modulated by CAND1 and CSN. As an established component of transcription and RNA processing complexes, we hypothesis that SAP130 may link CRL mediated ubiquitination to gene expression.</p

Hepatitis C Virus Infection Suppresses the Interferon Response in the Liver of the Human Hepatocyte Chimeric Mouse

BACKGROUND AND AIMS: Recent studies indicate that hepatitis C virus (HCV) can modulate the expression of various genes including those involved in interferon signaling, and up-regulation of interferon-stimulated genes by HCV was reported to be strongly associated with treatment outcome. To expand our understanding of the molecular mechanism underlying treatment resistance, we analyzed the direct effects of interferon and/or HCV infection under immunodeficient conditions using cDNA microarray analysis of human hepatocyte chimeric mice. METHODS: Human serum containing HCV genotype 1b was injected into human hepatocyte chimeric mice. IFN-α was administered 8 weeks after inoculation, and 6 hours later human hepatocytes in the mouse livers were collected for microarray analysis. RESULTS: HCV infection induced a more than 3-fold change in the expression of 181 genes, especially genes related to Organismal Injury and Abnormalities, such as fibrosis or injury of the liver (P = 5.90E-16∼3.66E-03). IFN administration induced more than 3-fold up-regulation in the expression of 152 genes. Marked induction was observed in the anti-fibrotic chemokines such as CXCL9, suggesting that IFN treatment might lead not only to HCV eradication but also prevention and repair of liver fibrosis. HCV infection appeared to suppress interferon signaling via significant reduction in interferon-induced gene expression in several genes of the IFN signaling pathway, including Mx1, STAT1, and several members of the CXCL and IFI families (P = 6.0E-12). Genes associated with Antimicrobial Response and Inflammatory Response were also significantly repressed (P = 5.22×10(-10)∼1.95×10(-2)). CONCLUSIONS: These results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy

Involvement of Arabidopsis thaliana phospholipase Dzeta2 in root hydrotropism through the suppression of root gravitropism.

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dzeta2 gene (PLDzeta2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldzeta2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldzeta2 mutant roots. In root hydrotropism experiments, pldzeta2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDzeta2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDzeta2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism

Functional Differentiation among the Arabidopsis Phosphatidylinositol 4-Phosphate 5-Kinase Genes PIP5K1, PIP5K2 and PIP5K3

International audienceAbstract Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is involved in regulating various cellular processes through the signaling function of its product, phosphatidylinositol (4,5)-bisphosphate. Higher plants encode a large number of PIP5Ks forming distinct clades in their molecular phylogenetic tree. Although biological functions of PIP5K genes have been analyzed intensively in Arabidopsis thaliana, it remains unclear how those functions differ across clades of paralogs. We performed comparative functional analysis of the Arabidopsis genes encoding PIP5K1, PIP5K2 and PIP5K3, of which the first two and the last belong to closely related but distinct clades, to clarify their conserved and/or differentiated functions. Genetic analysis with their single and multiple mutants revealed that PIP5K1 and PIP5K3 have non-overlapping functions, with the former in total plant growth and the latter in root hair elongation, whereas PIP5K2 redundantly functions in both phenomena. This pattern of functional redundancy is explainable in terms of the overlapping pattern of their promoter activities. In transformation rescue experiments, PIP5K3 promoter-directed PIP5K1-YFP completely rescued the short-root-hair phenotype of pip5k3. However, PIP5K3-YFP could substitute for PIP5K1-YFP only partially in rescuing the severe dwarfism of pip5k1pip5k2 when directed by the PIP5K1 promoter. Phylogenetic analysis of angiosperm PIP5Ks revealed that PIP5K3 orthologs have a faster rate of diversification in their amino-acid sequences compared with PIP5K1/2 orthologs after they arose through a eudicot-specific duplication event. These findings suggest that PIP5K3 specialized to promote root hair elongation and lost some of the protein-encoded functions retained by PIP5K1 and PIP5K2, whereas PIP5K1 differentiated from PIP5K2 only in its promoter-directed expression pattern

Characterization of the Last Subunit of the Arabidopsis COP9 Signalosome: Implications for the Overall Structure and Origin of the Complex[W]

The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests

The A-Type Cyclin CYCA2;3 Is a Key Regulator of Ploidy Levels in Arabidopsis Endoreduplication

Plant cells frequently undergo endoreduplication, a process in which chromosomal DNA is successively duplicated in the absence of mitosis. It has been proposed that endoreduplication is regulated at its entry by mitotic cyclin-dependent kinase activity. However, the regulatory mechanisms for its termination remain unclear, although plants tightly control the ploidy level in each cell type. In the process of searching for regulatory factors of endoreduplication, the promoter of an Arabidopsis thaliana cyclin A gene, CYCA2;3, was revealed to be active in developing trichomes during the termination period of endoreduplication as well as in proliferating tissues. Taking advantage of the situation that plants encode highly redundant cyclin A genes, we were able to perform functional dissection of CYCA2;3 using null mutant alleles. Null mutations of CYCA2;3 semidominantly promoted endocycles and increased the ploidy levels achieved in mature organs, but they did not significantly affect the proportion of cells that underwent endoreduplication. Consistent with this result, expression of the CYCA2;3–green fluorescent protein fusion protein restrained endocycles in a dose-dependent manner. Moreover, a mutation in the destruction box of CYCA2;3 stabilized the fusion protein in the nuclei and enhanced the restraint. We conclude that CYCA2;3 negatively regulates endocycles and acts as a key regulator of ploidy levels in Arabidopsis endoreduplication

The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex

The COP9 complex, genetically identified in Arabidopsis as a repressor of photomorphogenesis, is composed of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1-3]; unpublished observations). We have previously demonstrated the existence of the mammalian counterpart of the COP9 complex and purified the complex by conventional biochemical and immunoaffinity procedures [4]. Here, we report the molecular identities of all eight subunits of the mammalian COP9 complex. We show that the COP9 complex is highly conserved between mammals and higher plants, and probably among most multicellular eukaryotes. It is not present in the single-cell eukaryote Saccharomyces cerevisiae, however. All of the subunits of the COP9 complex contain structural features that are also present in the components of the proteasome regulatory complex and the translation initiation factor eIF3 complex. Six subunits of the COP9 complex have overall similarity with six distinct non-ATPase regulatory subunits of the 26S proteasome, suggesting that the COP9 complex and the proteasome regulatory complex are closely related in their evolutionary origin. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase (JNK) and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathway