Constantinos Demonacos

Acetylation control of the retinoblastoma tumour-suppressor protein

The retinoblastoma tumour-suppressor protein (pRb) and p300/CBP co-activator proteins are important for control of proliferation and in tumour cells these are sequestered by viral oncoproteins such as E1A. pRb is involved in negatively regulating growth, and p300/CBP proteins have histone acetyltransferase (HAT) activity, which influences gene expression. Although it is known that phosphorylation by G1 cyclin-dependent kinases (CDKs) regulates pRb activity, the nature and role of other post-translational modifications is not understood. Here we identify acetylation as a new type of modification and level of control in pRb function. Adenovirus E1A, which binds p300/CBP through an amino-terminal transformation-sensitive domain, stimulates the acetylation of pRb by recruiting p300 and pRb into a multimeric-protein complex. Furthermore, pRb acetylation is under cell-cycle control, and acetylation hinders the phosphorylation of pRb by cyclin-dependent kinases. pRb binds more strongly when acetylated to the MDM2 oncoprotein, which indicates that acetylation may regulate protein–protein interactions in the pRb pathway. The acetylation of pRb defines a new level of cell-cycle control mediated by HAT. Furthermore, our results establish a relationship between p300, pRb and acetylation in which E1A acts to recruit and target a cellular HAT activity to pRb.

Sirtuins: Molecular Traffic Lights in the Crossroad of Oxidative Stress, Chromatin Remodeling, and Transcription

Transcription is regulated by acetylation/deacetylation reactions of histone and nonhistone proteins mediated by enzymes called KATs and HDACs, respectively. As a major mechanism of transcriptional regulation, protein acetylation is a key controller of physiological processes such as cell cycle, DNA damage response, metabolism, apoptosis, and autophagy. The deacetylase activity of class III histone deacetylases or sirtuins depends on the presence of NAD+ (nicotinamide adenine dinucleotide), and therefore, their function is closely linked to cellular energy consumption. This activity of sirtuins connects the modulation of chromatin dynamics and transcriptional regulation under oxidative stress to cellular lifespan, glucose homeostasis, inflammation, and multiple aging-related diseases including cancer. Here we provide an overview of the recent developments in relation to the diverse biological activities associated with sirtuin enzymes and stress responsive transcription factors, DNA damage, and oxidative stress and relate the involvement of sirtuins in the regulation of these processes to oncogenesis. Since the majority of the molecular mechanisms implicated in these pathways have been described for Sirt1, this sirtuin family member is more extensively presented in this paper.

PCAF is an HIF-1alpha cofactor that regulates p53 transcriptional activity in hypoxia.

The p53 tumour suppressor is involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 stabilization occurs under hypoxic and DNA damage conditions. However, only in the latter scenario is stabilized p53 capable of inducing the expression of its pro-apoptotic targets. Here we present evidence that under hypoxia-mimicking conditions p53 acetylation is reduced to a greater extent at K320 site targeted by P300/CBP-associated factor (PCAF) than at K382 site targeted by p300/CBP. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the p21WAF-1/CIP-1 gene, which appears to be unaffected by hypoxia, but are not recruited to the BID promoter and hence p53 is incapable of upregulating pro-apoptotic BID in hypoxic conditions. As the K320 p53 acetylation is the site predominantly affected in hypoxia, the PCAF histone acetyltransferase activity is the key regulator of the cellular fate modulated by p53 under these conditions. In addition, we provide evidence that PCAF acetylates hypoxia-inducible factor-1α (HIF-1α) in hypoxic conditions and that the acetylated HIF-1α is recruited to a particular subset of its targets. In conclusion, PCAF regulates the balance between cell-cycle arrest and apoptosis in hypoxia by modulating the activity and protein stability of both p53 and HIF-1α.

Cross talk of signaling pathways in the regulation of the glucocorticoid receptor function.

Several posttranslational modifications including phosphorylation have been detected on the glucocorticoid receptor (GR). However, the interdependence and combinatorial regulation of these modifications and their role in GR functions are poorly understood. We studied the effects of c-Jun N-terminal kinase (JNK)-dependent phosphorylation of GR on its sumoylation status and the impact that these modifications have on GR transcriptional activity. GR is targeted for phosphorylation at serine 246 (S246) by the JNK protein family in a rapid and transient manner. The levels of S246 phosphorylation of endogenous GR increased significantly in cells treated with UV radiation that activates JNK. S246 GR phosphorylation by JNK facilitated subsequent GR sumoylation at lysines 297 and 313. GR sumoylation increased with JNK activation and was inhibited in cells treated with JNK inhibitor. GR sumoylation in cells with activated JNK was mediated preferentially by small ubiquitin-like modifier (SUMO)2 rather than SUMO1. An increase in GR transcriptional activity was observed after inhibition of JNK or SUMO pathways and suppression of GR transcriptional activity after activation of both pathways in cells transfected with GR-responsive reporter genes. Endogenous GR transcriptional activity was inhibited on endogenous target genes IGF binding protein (IGFBP) and glucocorticoid-induced leucine zipper (GILZ) when JNK and SUMO pathways were induced individually or simultaneously. Activation of both of these signals inhibited GR-mediated regulation of human inhibitor of apoptosis gene (hIAP), whereas simultaneous activation had no effect. We conclude that phosphorylation aids GR sumoylation and that cross talk of JNK and SUMO pathways fine tune GR transcriptional activity in a target gene-specific manner, thereby modulating the hormonal response of cells exposed to stress.

Acute or chronic stress induce cell compartment-specific phosphorylation of glucocorticoid receptor and alter its transcriptional activity in Wistar rat brain

Chronic stress and impaired glucocorticoid receptor (GR) feedback are important factors for the compromised hypothalamic–pituitary–adrenal (HPA) axis activity. We investigated the effects of chronic 21 day isolation of Wistar rats on the extrinsic negative feedback part of HPA axis: hippocampus (HIPPO) and prefrontal cortex (PFC). In addition to serum corticosterone (CORT), we followed GR subcellular localization, GR phosphorylation at serine 232 and serine 246, expression of GR regulated genes: GR, CRF and brain-derived neurotropic factor (BDNF), and activity of c-Jun N-terminal kinase (JNK) and Cdk5 kinases that phosphorylate GR. These parameters were also determined in animals subjected to acute 30 min immobilization, which was taken as ‘normal’ adaptive response to stress. In isolated animals, we found decreased CORT, whereas in animals exposed to acute immobilization, CORT was markedly increased. Even though the GR was predominantly localized in the nucleus of HIPPO and PFC in acute, but not in chronic stress, the expression of GR, CRF, and BDNF genes was similarly regulated under both acute and chronic stresses. Thus, the transcriptional activity of GR under chronic isolation did not seem to be exclusively dependent on high serum CORT levels nor on the subcellular location of the GR protein. Rather, it resulted from the increased Cdk5 activation and phosphorylation of the nuclear GR at serine 232 and the decreased JNK activity reflected in decreased phosphorylation of the nuclear GR at serine 246. Our study suggests that this nuclear isoform of hippocampal and cortical GR may be related to hypocorticism i.e. HPA axis hypoactivity under chronic isolation stress.

Delivery of therapeutic shRNA and siRNA by Tat fusion peptide targeting bcr-abl fusion gene in Chronic Myeloid Leukemia cells

Gene silencing by RNA interference (RNAi) is a promising therapeutic approach for a wide variety of diseases for which the biological cause is known. The main challenge remains the ineffective RNAi delivery inside the cells. Non-viral gene delivery vectors have low immunogenicity compared to viral vectors, but are constrained by their reduced transfection efficiency. Silencing of the bcr-abl gene expression by RNAi confers therapeutic potential in Chronic Myeloid Leukemia (CML), but is limited by the cytotoxicity of the existing delivery methods. Here, we present evidence that the fusion between the cell penetrating peptide (CPP) HIV-Tat (49-57) and the membrane lytic peptide (LK15), Tat-LK15, mediates high transfection efficiency in delivering short hairpin RNA (shRNA) and small interfering RNA (siRNA) targeting the BCR-ABL oncoprotein in K562 CML cells. Our results show that shRNA complexes induce a more stable gene silencing of bcr-abl when compared to silencing mediated by siRNA complexes. In addition, silencing of the BCR-ABL oncoprotein by both shRNA and siRNA delivered by Tat-LK15 is more efficient and longer lasting than that achieved using Lipofectamine and more importantly without considerable cytotoxicity. In these terms Tat-LK15 can be an alternative to DNA/siRNA delivery in difficult-to-transfect leukemic cells.

A TPR Motif Cofactor Contributes to p300 Activity in the p53 Response

The transcription of p53 target genes involves p300/CBP coactivators, which are multiprotein complexes that interact with the p53 activation domain. We report a cofactor in the p300 coactivator complex, Strap, which has an unusual structure, being composed almost entirely of a tandem series of six tetratricopeptide repeat (TPR) motifs. The TPR motif functions as a protein interaction domain, and it is consistent with this property that Strap harbors distinct and dedicated domains that allow it to bind and augment the interaction between different components of the p300 complex. Strap facilitates p53 activity in response to stress, in part through the stress-responsive accumulation of Strap protein and interfering with the MDM2-dependent downregulation of p53.

A new effector pathway links ATM kinase with the DNA damage response

The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.

The role of phosphorylated glucocorticoid receptor in mitochondrial functions and apoptotic signalling in brain tissue of stressed Wistar rats.

Mitochondrial dysfunction is increasingly recognized as a key component in compromised neuroendocrine stress response and, among other etiological causes, it may also involve action of glucocorticoid hormones. In the current study we followed glucocorticoid receptor and identified its mitochondrial phosphoisophorms in hippocampus and prefrontal brain cortex of Wistar male rats subjected to acute, chronic and combined neuroendocrine stresses. In both brain structures chronic social isolation caused marked increase in mitochondrial glucocorticoid receptor that was preferentially phosphorylated at serine 232 compared to serine 246 or serine 171. This increase corresponded with the decreased expression of mitochondrially encoded cytochrome oxidase subunits 1 and 3 in hippocampus, and with their increased expression in prefrontal brain cortex. Prefrontal brain cortex appeared to be more sensitive to chronic stress, since it exibited higher levels of mitochondrial Bax and cytoplasmic Bcl2 compared to hippocampus. Chronic stress also altered the response of both brain structures to subsequent acute stress according to the studied parameters. Therefore, prolonged social isolation may cause susceptibility to mitochondria triggered proapototic signalling, which at least in part may be mediated by the glucocorticoid receptor dependent mechanism.

Cytochrome P450 2E1 (CYP2E1) regulates the response to oxidative stress and migration of breast cancer cells

IntroductionThe cytochrome P450 (CYP) enzymes are a class of heme-containing enzymes involved in phase I metabolism of a large number of xenobiotics. The CYP family member CYP2E1 metabolises many xenobiotics and pro-carcinogens, it is not just expressed in the liver but also in many other tissues such as the kidney, the lung, the brain, the gastrointestinal tract and the breast tissue. It is induced in several pathological conditions including cancer, obesity, and type II diabetes implying that this enzyme is implicated in other biological processes beyond its role in phase I metabolism. Despite the detailed description of the role of CYP2E1 in the liver, its functions in other tissues have not been extensively studied. In this study, we investigated the functional significance of CYP2E1 in breast carcinogenesis.MethodsCellular levels of reactive oxygen species (ROS) were measured by H2DCFDA (2 2.9.2 2′,7′-dichlorodihydrofluorescein diacetate) staining and autophagy was assessed by tracing the cellular levels of autophagy markers using western blot assays. The endoplasmic reticulum stress and the unfolded protein response (UPR) were detected by luciferase assays reflecting the splicing of mRNA encoding the X-box binding protein 1 (XBP1) transcription factor and cell migration was evaluated using the scratch wound assay. Gene expression was recorded with standard transcription assays including luciferase reporter and chromatin immunoprecipitation.ResultsEctopic expression of CYP2E1 induced ROS generation, affected autophagy, stimulated endoplasmic reticulum stress and inhibited migration in breast cancer cells with different metastatic potential and p53 status. Furthermore, evidence is presented indicating that CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor.ConclusionsThese results support the notion that CYP2E1 exerts an important role in mammary carcinogenesis, provide a potential link between ethanol metabolism and breast cancer and suggest that progression, and metastasis, of advanced stages of breast cancer can be modulated by induction of CYP2E1 activity.