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R T Hay

Publications and source records attributed to R T Hay.

At least 19 recordsLinked to original sources
PubMed2006

Apoptin is modified by SUMO conjugation and targeted to promyelocytic leukemia protein nuclear bodies.

Apoptin, a protein of the chicken anemia virus (CAV), represents a novel potential anticancer therapeutic, because it induces apoptotic death specifically in tumor but not normal cells. The cellular localization appears to be crucial for apoptin's selective toxicity. In normal cells apoptin remains in the cytoplasm, whereas in transformed cells it migrates into the nucleus and kills the cell. However, the manner by which apoptin is able to distinguish between tumor and normal cells is unknown. Here, we report for the first time that apoptin interacts directly with the promyelocytic leukemia protein (PML) in tumor cells and accumulates in PML nuclear bodies (NBs), which are involved in apoptosis induction and viral replication. We also demonstrate that apoptin is sumoylated and that a sumoylation-deficient apoptin mutant is no longer recruited to PML-NBs, but localizes in the nuclear matrix. This mutant fails to bind PML, but can still induce apoptosis as efficiently as wild-type apoptin. Moreover, apoptin kills also PML-/- cells and promyelocytic leukemia cells with defective PML expression. Our results therefore suggest that apoptin kills tumor cells independently of PML and sumoylation, however, the interaction of apoptin with PML and small ubiquitin-like modifier (SUMO) proteins might be relevant for CAV replication.

Capsid Proteins↗
PubMed2006

Repression of SOX6 transcriptional activity by SUMO modification.

SOX6 plays key functions in several developmental processes, including neurogenesis and skeleton formation. In this report, we show that SOX6 is modified in vitro and in vivo by small ubiquitin-related modifier (SUMO) on two distinct sites. Mutation of both sites abolished SOX6 sumoylation and increased SOX6 transcriptional activity. SUMO dependent repression of SOX6 transcription was promoted by UBC9 whereas siRNA to UBC9, cotransfection of inactive UBC9 or a SUMO protease increased SOX6 transcriptional activity. Furthermore, co-expression of SOX6 with SUMO2 results in the appearance of SOX6 in a punctate nuclear pattern that colocalized with promyelocytic leukemia protein, which was partially abolished by mutations in SOX6 sumoylation sites.

Animals↗
PubMed2006

Role of ubiquitin-like proteins in transcriptional regulation.

Conjugation of ubiquitin-like proteins (Ubls) to components of the transcriptional machinery represents an important mechanism to allow switching between different activity states. While ubiquitin modification of transcription factors is associated with transcriptional activation, SUMO modification of transcription factors is most often associated with transcriptional repression. Recent experiments indicate that another Ubl, NEDD8, can also influence transcription. One of the characteristics of Ubl modification is that the biological consequences of conjugation do not appear proportionate to the small fraction of substrate that is modified. The low steady state levels of Ubl-modified substrates can be attributed to a highly dynamic situation in which proteins are conjugated to a particular Ubl only for the modification to be removed by Ubl-specific proteases. It therefore appears that an unmodified protein with a history of Ubl modification may have different properties from a protein that never has been modified. Here the diverse effects of Ubl modification are discussed and models proposed to explain Ubl actions.

Animals↗
PubMed2004

Dual role of the adenovirus pVI C terminus as a nuclear localization signal and activator of the viral protease.

Adenain, the protease produced by adenovirus, is regulated by formation of a heterodimer with an 11 aa peptide derived from the C terminus of another adenoviral protein, pVI. Here, the role of the basic motif KRRR, which is conserved in pVI sequences from human adenovirus serotypes, was investigated. It was shown that this motif is less important than the N- or C-terminal regions in the formation of the adenain-peptide heterodimer and in the activity of the subsequent complex. This motif, however, acted as a nuclear localization signal that was capable of targeting heterologous proteins to the nucleus, resulting in a distinctive intranuclear distribution consisting of discrete foci, which is similar to that found for pVI during adenovirus infection.

Adenoviruses, Human↗
PubMed2003

Adenovirus DNA replication.

Replication of the adenovirus genome is catalysed by adenovirus DNA polymerase in which the adenovirus preterminal protein acts as a protein primer. DNA polymerase and preterminal protein form a heterodimer which, in the presence of the cellular transcription factors NFI/CTFI and NFIII/Oct-1, binds to the origin of DNA replication. DNA replication is initiated by DNA polymerase mediated transfer of dCMP onto preterminal protein. Further DNA synthesis is catalysed by DNA polymerase in a strand displacement mechanism which also requires adenovirus DNA binding protein. Here, we discuss the role of individual proteins in this process as revealed by biochemical analysis, mutagenesis and molecular modelling.

Adenoviridae↗
PubMed2001

Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence psiKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.

Amino Acid Sequence↗
PubMed2001

Common properties of nuclear body protein SP100 and TIF1alpha chromatin factor: role of SUMO modification.

The SP100 protein, together with PML, represents a major constituent of the PML-SP100 nuclear bodies (NBs). The function of these ubiquitous subnuclear structures, whose integrity is compromised in pathological situations such as acute promyelocytic leukemia (APL) or DNA virus infection, remains poorly understood. There is little evidence for the occurrence of actual physiological processes within NBs. The two NB proteins PML and SP100 are covalently modified by the ubiquitin-related SUMO-1 modifier, and recent work indicates that this modification is critical for the regulation of NB dynamics. In exploring the functional relationships between NBs and chromatin, we have shown previously that SP100 interacts with members of the HP1 family of nonhistone chromosomal proteins and that a variant SP100 cDNA encodes a high-mobility group (HMG1/2) protein. Here we report the isolation of a further cDNA, encoding the SP100C protein, that contains the PHD-bromodomain motif characteristic of chromatin proteins. We further show that TIF1alpha, a chromatin-associated factor with homology to both PML and SP100C, is also modified by SUMO-1. Finally, in vitro experiments indicate that SUMO modification of SP100 enhances the stability of SP100-HP1 complexes. Taken together, our results suggest an association of SP100 and its variants with the chromatin compartment and, further, indicate that SUMO modification may play a regulatory role in the functional interplay between the nuclear bodies and chromatin.

Amino Acid Sequence↗
PubMed2001

Interaction between hnRNPA1 and IkappaBalpha is required for maximal activation of NF-kappaB-dependent transcription.

Transcriptional activation of NF-kappaB is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. NF-kappaB activation induces a rapid resynthesis of IkappaBalpha which is responsible for postinduction repression of transcription. Following resynthesis, IkappaBalpha translocates to the nucleus, removes template bound NF-kappaB, and exports NF-kappaB to the cytoplasm in a transcriptionally inactive form. Here we demonstrate that IkappaBalpha interacts directly with another nucleocytoplasmic shuttling protein, hnRNPA1, both in vivo and in vitro. This interaction requires one of the N-terminal RNA binding domains of hnRNPA1 and the C-terminal region of IkappaBalpha. Cells lacking hnRNPA1 are defective in NF-kappaB-dependent transcriptional activation, but the defect in these cells is complemented by ectopic expression of hnRNPA1. hnRNPA1 expression in these cells increased the amount of IkappaBalpha degradation, compared to that of the control cells, in response to activation by Epstein-Barr virus latent membrane protein 1. Thus in addition to regulating mRNA processing and transport, hnRNPA1 also contributes to the control of NF-kappaB-dependent transcription.

Animals↗
PubMed2000

SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting.

SUMO-1 is a small ubiquitin-related modifier that is covalently linked to many cellular protein targets. Proteins modified by SUMO-1 and the SUMO-1-activating and -conjugating enzymes are located predominantly in the nucleus. Here we define a transferable sequence containing the PsiKXE motif, where Psi represents a large hydrophobic amino acid, that confers the ability to be SUMO-1-modified on proteins to which it is linked. Whereas addition of short sequences from p53 and IkappaBalpha, containing the PsiKXE motif, to a carrier protein is sufficient for modification in vitro, modification in vivo requires the additional presence of a nuclear localization signal. Thus, protein substrates must be targeted to the nucleus to undergo SUMO-1 conjugation.

Amino Acid Sequence↗
PubMed2000

Activation of NF-kappaB by PM(10) occurs via an iron-mediated mechanism in the absence of IkappaB degradation.

Exposure to particulate air pollution (PM(10)) is associated with exacerbations of respiratory diseases and increased cardiopulmonary mortality. PM(10) induces lung inflammation in rats, which has been attributed to many factors, including the ultrafine components of PM(10), endotoxins, and transition metals. In this study, we investigated in alveolar epithelial (A549) cells whether PM(10) could activate nuclear factor-kappa B (NF-kappaB), a transcription factor stimulated in response to many proinflammatory agents. Our results show that PM(10) samples from various sites within the United Kingdom cause nuclear translocation, DNA-binding, and transcriptional activation of NF-kappaB in A549 cells. Furthermore, increased NF-kappaB activity was observed in the absence of IkappaB degradation. To evaluate the role of iron, A549 cells were exposed to PM(10) previously treated with phosphate-buffered saline (PBS), deferoxamine mesylate, or deferoxamine plus ferrozine. PBS-treated and, to a lesser extent, deferoxamine-treated PM(10) were able to activate NF-kappaB, whereas this response was completely abrogated in cells exposed to PM(10) treated with both deferoxamine and ferrozine. Moreover, we studied the effects of soluble components of PM(10) on NF-kappaB activation by exposing alveolar epithelial cells to soluble fractions from PM(10) treated with PBS or the metal chelators. We found that, compared with fractions from PBS-treated PM(10) which activated NF-kappaB, fractions from PM(10) treated with deferoxamine and ferrozine did not stimulate NF-kappaB activity above background levels. Coincubation of polymixin B, an endotoxin-binding compound, and PM(10) did not inhibit NF-kappaB. In summary, PM(10) activates NF-kappaB in A549 cells by an iron-mediated mechanism in the absence of IkappaB degradation.

Adenocarcinoma↗
PubMed2000

An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo.

The p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.

Amino Acid Sequence↗
PubMed2000

NF-kappaB inhibits apoptosis in murine mammary epithelia.

The transcription factor NF-kappaB is a key modulator of apoptosis in a variety of cell types, but to date this specific function of NF-kappaB has not been demonstrated in epithelia. Here, we describe the activation of NF-kappaB during post-lactational involution of the mouse mammary gland, a period of extensive apoptosis of luminal epithelial cells. Significantly, active NF-kappaB localized exclusively to nonapoptotic epithelial cells both in vivo and in the mammary epithelial cell line, KIM-2, transduced with an NF-kappaB-dependent green fluorescent protein reporter. Activation of NF-kappaB in vitro coincided with a decrease in the cytosolic repressor, IkappaBalpha. Furthermore, induction of NF-kappaB either by extracellular ligands or, more specifically, by inhibition of the IkappaB repressor with adenoviral constructs expressing antisense mRNA, resulted in enhanced survival of KIM-2 cells. Therefore, although coincident with induction of apoptosis both in vivo and in vitro, NF-kappaB appeared to exert a selective survival function in epithelial cells. This study highlights for the first time a role for NF-kappaB in modulating apoptosis in epithelium.

Adenoviridae↗
PubMed2000

Cytokine-induced nuclear factor kappa B activation promotes the survival of developing neurons.

Ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and interleukin 6 (IL-6) comprise a group of structurally related cytokines that promote the survival of subsets of neurons in the developing peripheral nervous system, but the signaling pathways activated by these cytokines that prevent neuronal apoptosis are unclear. Here, we show that these cytokines activate NF-kappaB in cytokine-dependent developing sensory neurons. Preventing NF-kappaB activation with a super-repressor IkappaB-alpha protein markedly reduces the number of neurons that survive in the presence of cytokines, but has no effect on the survival response of the same neurons to brain-derived neurotrophic factors (BDNF), an unrelated neurotrophic factor that binds to a different class of receptors. Cytokine-dependent sensory neurons cultured from embryos that lack p65, a transcriptionally active subunit of NF-kappaB, have a markedly impaired ability to survive in response to cytokines, but respond normally to BDNF. There is increased apoptosis of cytokine- dependent neurons in p65(-/)- embryos in vivo, resulting in a reduction in the total number of these neurons compared with their numbers in wild-type embryos. These results demonstrate that NF-kappaB plays a key role in mediating the survival response of developing neurons to cytokines.

Apoptosis↗
PubMed2000

Regulation of transcription factors by protein degradation.

The level of transcription factors is tightly controlled by their rates of synthesis and degradation. Many critical factors are maintained at an appropriate level by targeted addition of ubiquitin and degradation via the proteasome. Whereas ubiquitination targets modified proteins for degradation, modification of substrates by the family of ubiquitin-like proteins does not target the proteins for degradation but can alter the stability and other properties of the modified proteins. Here we discuss the elaborate mechanisms that have evolved to allow specific recognition of substrates targeted for modification. Specific examples are discussed to illustrate the different mechanisms involved and the importance of regulated degradation in diseases such as cancer.

Animals↗
PubMed2000

Identification of conserved residues contributing to the activities of adenovirus DNA polymerase.

Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase alpha family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.

Adenoviridae↗
PubMed2000

Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation.

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.

Amino Acid Substitution↗
PubMed1999

SUMO-1 modification activates the transcriptional response of p53.

The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wild-type p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.

Binding Sites↗