Roger J. A. Grand

DNA viruses and the cellular DNA-damage response.

It is clear that a number of host-cell factors facilitate virus replication and, conversely, a number of other factors possess inherent antiviral activity. Research, particularly over the last decade or so, has revealed that there is a complex inter-relationship between viral infection and the host-cell DNA-damage response and repair pathways. There is now a realization that viruses can selectively activate and/or repress specific components of these host-cell pathways in a temporally coordinated manner, in order to promote virus replication. Thus, some viruses, such as simian virus 40, require active DNA-repair pathways for optimal virus replication, whereas others, such as adenovirus, go to considerable lengths to inactivate some pathways. Although there is ever-increasing molecular insight into how viruses interact with host-cell damage pathways, the precise molecular roles of these pathways in virus life cycles is not well understood. The object of this review is to consider how DNA viruses have evolved to manage the function of three principal DNA damage-response pathways controlled by the three phosphoinositide 3-kinase (PI3K)-related protein kinases ATM, ATR and DNA-PK and to explore further how virus interactions with these pathways promote virus replication.

The APC/C and CBP/p300 cooperate to regulate transcription and cell-cycle progression

The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein–protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C–CBP/p300 complexes in growth regulation.

Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair

DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.

Regulation of the 26S proteasome by adenovirus E1A

We have identified the N‐terminus of adenovirus early region 1A (AdE1A) as a region that can regulate the 26S proteasome. Specifically, in vitro and in vivo co‐precipitation studies have revealed that the 19S regulatory components of the proteasome, Sug1 (S8) and S4, bind through amino acids (aa) 4–25 of Ad5 E1A. In vivo expression of wild‐type (wt) AdE1A, in contrast to the N‐terminal AdE1A mutant that does not bind the proteasome, reduces ATPase activity associated with anti‐S4 immunoprecipitates relative to mock‐infected cells. This reduction in ATPase activity correlates positively with the ability of wt AdE1A, but not the N‐terminal deletion mutant, to significantly reduce the ability of HPV16 E6 to target p53 for ubiquitin‐mediated proteasomal degradation. AdE1A/proteasomal complexes are present in both the cytoplasm and the nucleus, suggesting that AdE1A interferes with both nuclear and cytoplasmic proteasomal degradation. We have also demonstrated that wt AdE1A and the N‐terminal AdE1A deletion mutant are substrates for proteasomal‐mediated degradation. AdE1A degradation is not, however, mediated through ubiquitylation, but is regulated through phosphorylation of residues within a C‐terminal PEST region (aa 224–238).

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

Adenovirus E1B 55-Kilodalton Protein : Multiple Roles in Viral Infection and Cell Transformation

Viruses are intracellular parasites that require host cell functions to reproduce. They must modify the host cell environment to maximize their replication and avoid the activation of antiviral responses, while keeping the cell alive long enough to produce viral progeny. Accordingly, many of the

THE AMINO ACID SEQUENCE OF THE TROPONIN C-LIKE PROTEIN (MODULATOR PROTEIN) FROM BOVINE UTERUS

Recent studies have shown that a troponin C-like protein is present in vertebrate smooth muscle [l-5] . This protein can form Ca”-dependent complexes with troponin I from fast skeletal muscle in a similar manner to those obtained with skeletal muscle troponin C [2,3] . It can be isolated from rabbit uterus using an affinity chromatographic procedure which depends on this property of complexing with skeletal muscle troponin I [l] but amore convenient, relatively simple, method of isolating the protein from smooth muscle and other tissues in good yields has recently been described [3]. The troponin C-like protein from smooth muscle possesses other properties similar to those of skeletal muscle troponin C. These are the abilities to inhibit the phosphorylation of troponin I by 3’,5’-cyclic AMP-dependent protein kinase and to neutralise the inhibitory activity of skeletal troponin I on the Mg2+-stimulated ATPase of desensitized skeletal actomyosin [2] . Unlike skeletal muscle troponin C, however, the smooth muscle protein activates cyclic nucleotide phosphodiesterase and contains the unusual amino acid, trimethyl lysine [3] . These latter properties are a feature of the Ca2+binding protein of bovine brain, the brain modulator protein, which Amphlett et al. [6] have demonstrated can substitute in the skeletal troponin complex in the place of troponin C and will restore full calcium sensitivity to desensitized actomyosin in the presence of troponin I and tropomyosin alone. Cheung et al. [7] have shown that a protein similar to the brain modulator protein is present in many of the tissues of mammals, and Waisman et al. [8] have

The fate of E- and P-cadherin during the early stages of apoptosis.

Caspases are responsible for the proteolysis of many cytoskeletal proteins in apoptotic cells. It has been demonstrated here that during cisplatin-induced apoptosis of human embryo retinoblasts both E- and P-cadherin were degraded by caspases, giving initially major polypeptide products of apparent molecular weights 48 K and 104 K respectively. This proteolysis occurred over a similar time-scale to the observed degradation of PARP and to the onset of DNA fragmentation but appreciably later than p53 induction and cleavage of Mdm2 and p21. Addition of caspase inhibitors such as Z-VAD-FMK inhibited apoptosis and cadherin degradation. Co-immunoprecipitation studies carried out on viable cells confirmed previously observed complexes between cadherins and α and β catenin and between the catenins themselves. These interactions were sustained in apoptotic cells as long as the protein components remained intact. Using confocal microscopy it has been shown that cytoskeletal changes associated with apoptosis precede degradation of catenins and cadherins by several hours. In particular, after addition of cisplatin relatively rapid (within 3 h) re-localization of adherens junction proteins from the cell periphery to the cytoplasm was observed whereas little cadherin or catenin degradation occurred until 10 h. We conclude that neither caspase-mediated degradation of cytoskeletal components nor disruption of adherens junction protein-protein interactions is required for morphological change.

Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation

Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIβ–binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.