Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular CCAAT/enhancer binding protein alpha (C/EBPalpha) promotes cellular differentiation and has antimitotic activities involving cell cycle arrest at G(1)/S through stabilization of p21(CIP-1)/WAF1 and through transcriptional activation of the p21 promoter. The Epstein-Barr virus lytic-cycle transactivator protein ZTA is known to arrest the host cell cycle at G(1)/S via a p53-independent p21 pathway, but the detailed molecular mechanisms involved have not been defined. To further evaluate the role of ZTA in cell cycle arrest, we constructed a recombinant adenovirus vector expressing ZTA (Ad-ZTA), whose level of expression at a low multiplicity of infection in normal human diploid fibroblast (HF) cells was lower than or equal to the physiological level seen in Akata cells lytically induced by EBV (EBV-Akata cells). Fluorescence-activated cell sorting analysis of HF cells infected with Ad-ZTA confirmed that G(1)/S cell cycle arrest occurred in the majority of ZTA-positive cells, but not with an adenovirus vector expressing green fluorescent protein. Double-label immunofluorescence assays (IFA) performed with Ad-ZTA-infected HF cells revealed that only ZTA-positive cells induced the expression of both endogenous C/EBPalpha and p21 and blocked the progression into S phase, as detected by a lack of incorporation of bromodeoxyuridine. The stimulation of endogenous ZTA protein expression either through treatment with tetradecanoyl phorbol acetate in D98/HR1 cells or through B-cell receptor cross-linking with anti-immunoglobulin G antibody in EBV-Akata cells also coincided with the induction of both C/EBPalpha and p21 and their mRNAs, as assayed by Northern blot, Western blot, and IFA experiments. Mechanistically, the ZTA protein proved to directly interact with C/EBPalpha by coimmunoprecipitation in EBV-Akata cells and with DNA-bound C/EBPalpha in electrophoretic mobility shift assay experiments, and the in vitro interaction domain encompassed the basic leucine zipper domain of ZTA. ZTA also specifically protected C/EBPalpha from degradation in a protein stability assay with a non-EBV-induced Akata cell proteasome extract. Furthermore, both C/EBPalpha and ZTA were found to specifically associate with the C/EBPalpha promoter in chromatin immunoprecipitation assays, but the interaction with ZTA appeared to be mediated by C/EBPalpha because it was abolished by clearing with anti-C/EBPalpha antibody. ZTA did not bind to or activate the C/EBPalpha promoter directly but cooperatively enhanced the positive autoregulation of the C/EBPalpha promoter by cotransfected C/EBPalpha in transient luciferase reporter gene assays with Vero and HeLa cells as well as with DG75 B lymphocytes. Similarly, ZTA alone had little effect on the p21 promoter in transient reporter gene assays, but in the presence of cotransfected C/EBPalpha, ZTA enhanced the level of C/EBPalpha activation. This effect proved to require a previously unrecognized region in the proximal p21 promoter that contains three high-affinity C/EBPalpha binding sites. Finally, in C/EBPalpha-deficient mouse embryonic fibroblasts (MEF), Ad-ZTA was unable to induce either p21 or G(1) arrest, whereas it was able to induce both in wild-type MEF. Overall, we conclude that C/EBPalpha is essential for at least one pathway of ZTA-induced G(1) arrest during EBV lytic-cycle DNA replication and that this process involves a physical piggyback interaction between ZTA and C/EBPalpha leading to greatly enhanced C/EBPalpha and p21 levels through both transcriptional and posttranslational mechanisms.
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PMID:CCAAT/enhancer binding protein alpha interacts with ZTA and mediates ZTA-induced p21(CIP-1) accumulation and G(1) cell cycle arrest during the Epstein-Barr virus lytic cycle. 1250 63

CCAAT/enhancer-binding protein (C/EBP) family transcription factors are critical for transcription of several genes involved in tissue development and cellular function, proliferation, and differentiation. Here we show that inhibitory/regulatory C/EBP family proteins, Ig/EBP (C/EBPgamma) and CHOP (C/EBPzeta), but not positively functioning NF-IL6 (C/EBPbeta), are constitutively multiubiquitinated and subsequently degraded by the proteasome. In addition, ubiquitination and degradation of these proteins are suppressed by forming dimer through their leucine zipper domains. Deletion of leucine zipper domain in NF-IL6 caused the loss of its homodimerization activity and the degradation of protein by the ubiquitin-proteasome system. In addition, Ig/EBP with its leucine zipper domain substituted for that of NF-IL6 formed homodimer and was stabilized. These observations suggest that mammalian cells equip a novel regulatory system abrogating the excess C/EBP family transcription factors bereft of dimerizing partner.
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PMID:C/EBP family transcription factors are degraded by the proteasome but stabilized by forming dimer. 1261 52

c-fos gene is expressed constitutively in a number of tissues as well as in certain tumor cells and is inducible, in general rapidly and transiently, in virtually all other cell types by a variety of stimuli. Its protein product, c-Fos, is a short-lived transcription factor that heterodimerizes with various protein partners within the AP-1 transcription complex via leucine zipper/leucine zipper interactions for binding to specific DNA sequences. It is mostly, if not exclusively, degraded by the proteasome. To localize the determinant(s) responsible for its instability, we have conducted a genetic analysis in which the half-lives of c-Fos mutants and chimeras made with the stable EGFP reporter protein were compared under two experimental conditions taken as example of continous and inducible expression. Those were constitutive expression in asynchronously growing Balb/C 3T3 mouse embryo fibroblasts and transient induction in the same cells undergoing the G0/G1 phase transition upon stimulation by serum. Our work shows that c-Fos is degraded faster in synchronous- than in asynchronous cells. This difference in turnover is primarily accounted for by several mechanisms. First, in asynchronous cells, a unique C-terminal destabilizer is active whereas, in serum-stimulated cells two destabilizers located at both extremities of the protein are functional. Second, heterodimerization and/or binding to DNA accelerates protein degradation only during the G0/G1 phase transition. Adding another level of complexity to turnover control, phosphorylation at serines 362 and 374, which are c-Fos phosphorylation sites largely modified during the G0/G1 phase transition, stabilizes c-Fos much more efficiently in asynchronous than in serum-stimulated cells. In both cases, the reduced degradation rate is due to inhibition of the activity of the C-terminal destabilizer. However, in serum-stimulated cells, this effect is partially masked by the activation of the N-terminal destabilizer and basic domain/leucine zipper-dependent mechanisms. Taken together, our data show that multiple degradation mechanisms, differing according to the conditions of expression, may operate on c-Fos to ensure a proper level and/or timing of expression. Moreover, they also indicate that the half-life of c-Fos during the G0/G1 phase transition is determined by a delicate balance between opposing stabilizing and destabilizing mechanisms operating at the same time.
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PMID:The structural determinants responsible for c-Fos protein proteasomal degradation differ according to the conditions of expression. 1262 9

Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G(1) cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPalpha and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPalpha and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPalpha. Cotransfection of RAP with C/EBPalpha cooperatively stimulated both the C/EBPalpha and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPalpha, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPalpha in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPalpha sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPalpha promoter in vivo, but only in a C/EBPalpha-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPalpha. Expression of exogenous RAP (Ad-RAP) caused G(1)/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPalpha and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPalpha was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPalpha binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPalpha were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G(1) phase or inducing p21 in a C/EBPalpha-independent process.
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PMID:Cell cycle arrest by Kaposi's sarcoma-associated herpesvirus replication-associated protein is mediated at both the transcriptional and posttranslational levels by binding to CCAAT/enhancer-binding protein alpha and p21(CIP-1). 1288 7

The nuclear factor kappaB (NF-kappaB)/Rel family of transcription factors participates in a wide range of biological activities including inflammation, immunity, and apoptosis. NF-kappaB is kept inactive in the cytoplasm in unstimulated cells by virtue of the masking of its nuclear localization sequence by bound IkappaB protein. Cellular stimuli trigger the destruction of IkappaB proteins and the liberation of NF-kappaB to enter the nucleus and activate gene expression. A multisubunit IkappaB kinase complex (IKK) phosphorylates IkappaB proteins and mediates the activation of NF-kappaB by proinflammatory stimuli such as tumor necrosis factor alpha. Phosphorylation of IkappaB proteins triggers their polyubiquitination and their subsequent recognition and degradation by the proteasome. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, and a noncatalytic subunit, NF-kappaB essential modifier/IKKgamma. IKK activation depends upon the phosphorylation of residues in the activation loop of IKKbeta and the subsequent activation of IKKbeta kinase activity. However, the events contributing to IKKbeta phosphorylation are not well understood. Here, we present evidence that the activation of IKKbeta depends on its ability to form homotypic interactions and to transautophosphorylate. We find that an intact leucine zipper in IKKbeta is necessary for homotypic interactions, kinase activation, and phosphorylation on its activation loop. Enforced oligomerization of an IKKbeta mutant defective in forming homotypic interactions restores kinase activation. Homotypic interactions allow IKKbeta molecules to transautophosphorylate one another on their activation loops. Finally, the oligomerization of IKKbeta is stimulated by tumor necrosis factor alpha in cultured cells. Our findings support a model whereby ligand-induced homotypic interactions between IKKbeta molecules result in IKKbeta phosphorylation and consequently IKK activation.
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PMID:Roles for homotypic interactions and transautophosphorylation in IkappaB kinase beta IKKbeta) activation [corrected]. 1289 Jun 79

TCDD (2,3,7,8-tetrachlorodibenzo- p -dixoin) induces phase II drug-metabolizing enzyme NQO1 [NAD(P)H:quinone oxidoreductase; EC 1.6.99.2; DT-diaphorase] in a wide range of mammalian tissues and cells. Here, we analysed the molecular pathway mediating NQO1 induction by TCDD in mouse hepatoma cells. Inhibition of protein synthesis with CHX (cycloheximide) completely blocks induction of NQO1 by TCDD as well as the basal expression and induction by phenolic antioxidant tBHQ (2-t-butylbenzene-1,4-diol), implicating a labile factor in NQO1 mRNA expression. The inhibition is both time- and concentration-dependent, requires inhibition of protein synthesis, and occurs at a transcriptional level. Inhibition of NQO1 transcription by CHX correlates with a rapid reduction of the CNC bZip (cap 'n' collar basic leucine zipper) transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) through the 26 S proteasome pathway. Moreover, blocking Nrf2 degradation with proteasome inhibitor MG132 increases the amount of Nrf2 and superinduces NQO1 in the presence of TCDD or tBHQ. Finally, genetic experiments using AhR (aryl hydrocarbon receptor)-, Arnt (aryl hydrocarbon receptor nuclear translocator)- or Nrf2-deficient cells reveal that, while induction of NQO1 by TCDD depends on the presence of AhR and Arnt, the basal and inducible expression of NQO1 by either TCDD or tBHQ requires functional Nrf2. The findings demonstrate a novel role of Nrf2 in the induction of NQO1 by TCDD and provide new insights into the mechanism by which Nrf2 regulates the induction of phase II enzymes by both phenolic antioxidants and AhR ligands.
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PMID:Induction of murine NAD(P)H:quinone oxidoreductase by 2,3,7,8-tetrachlorodibenzo-p-dioxin requires the CNC (cap 'n' collar) basic leucine zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2): cross-interaction between AhR (aryl hydrocarbon receptor) and Nrf2 signal transduction. 1451 Jun 36

The role of the NH(2)-terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH(2)-terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and whole-cell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 +/- 1.3 min compared with 3.2 +/- 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH(2) terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27 degrees C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH(2) terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.
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PMID:Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1. 1475 84

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme involved in defence against reactive forms of oxygen and inhibition of neoplasia. Under conditions of oxidative stress, expression of NQO1 is induced, and the resulting increase in oxidoreductase protein provides the cell with multiple layers of protection against environmental insults. Firstly, the catalytic activity of NQO1 is directed towards the complete reduction and detoxication of highly reactive quinones. Secondly, the oxidoreductase maintains the endogenous lipid-soluble antioxidants, alpha-tocopherol-hydroquinone and ubiquinol in their reduced and active forms. Thirdly, NQO1 is required for the stabilisation of p53 protein in response to DNA-damaging stimuli, and it thereby influences cell fate decisions. In view of the anticarcinogenic actions of NQO1, an understanding of the mechanisms that govern its expression is desirable. The redox sensitivity of NQO1 transcription occurs through a cis-acting antioxidant response element (ARE) located within the regulatory region of the mouse, rat and human genes. This element recruits the positively acting basic leucine zipper (bZip) transcription factor NF-E2 p45-related factor 2 (Nrf2). Under normal constitutive conditions, Nrf2 associates with the cytoskeletal-binding protein Keap1, which regulates the subcellular distribution of the bZip factor and also targets it for proteasome-dependent degradation. Oxidative stress inhibits the Nrf2-Keap1 interaction, thus promoting nuclear accumulation of the transcription factor and transactivation of NQO1 and other ARE-driven genes. Mouse, rat and human NQO1 can also be induced by planar aromatic hydrocarbons through a cis-acting xenobiotic response element (XRE) located in their gene promoters. The XRE recruits the arylhydrocarbon receptor (AhR) and AhR nuclear translocator. Cross-talk may occur between Nrf2 and AhR, but the details of this process remain to be elucidated.
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PMID:Contribution of NAD(P)H:quinone oxidoreductase 1 to protection against carcinogenesis, and regulation of its gene by the Nrf2 basic-region leucine zipper and the arylhydrocarbon receptor basic helix-loop-helix transcription factors. 1547 58

The Arabidopsis HY5 protein is a basic leucine zipper (bZIP) transcription factor that promotes photomorphogenesis. HY5 binds directly to the promoters of light responsible element containing the G-box and thus regulates their transcriptional activity. The level and activity of HY5 are negatively regulated, in a light-dependent manner, by interaction with the COP1 protein, which targets HY5 for proteasome-mediated degradation in the nucleus. Despite its essential roles in plant development, no structural information exists for HY5. In this article, we report the first structural and biophysical characterization of HY5. Using limited proteolysis in combination with mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy, we have deduced that the N-terminal 77 amino acids of HY5 form a premolten globular structure, while amino acids 78-110, which constitute the basic region (BR) of the protein, exist in a molten globule state. Our studies also revealed that the overall structural features of full-length HY5 are dominated largely by the disordered N-terminal domain, despite the existence of a bZIP domain at its C-terminus. We propose that HY5 is a member of the intrinsically unstructured protein (IUP) family, and that HY5 functions as an unstructured protein and benefits from being the same, in vivo.
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PMID:Intrinsically unstructured N-terminal domain of bZIP transcription factor HY5. 1700 43

C/EBPdelta (CCAAT/enhancer-binding protein delta) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPdelta gene transcription is highly induced in G(0) growth-arrested mammary epithelial cells but the C/EBPdelta protein exhibits a t(1/2) of only approximately 120 min. The goal of the present study was to investigate the role of C/EBPdelta modification by ubiquitin and C/EBPdelta proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPdelta ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPdelta ubiquitination is not required for proteasome-mediated C/EBPdelta degradation and the presence of ubiquitin does not increase C/EBPdelta degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPdelta protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPdelta ubiquitination we treated G(0) growth-arrested mammary epithelial cells with DNA-damage- and oxidative-stress-inducing agents and found that C/EBPdelta ubiquitination is induced in response to H2O2. However, C/EBPdelta protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPdelta is ubiquitin-independent.
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PMID:Proteasome-mediated CCAAT/enhancer-binding protein delta (C/EBPdelta) degradation is ubiquitin-independent. 1737 9


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