MAML1-induced HPV E6 oncoprotein stability is required for cellular proliferation and migration of cervical tumor-derived cells.

While a small proportion of high-risk (HR) alpha (α) human papillomaviruses (HPVs) is associated with numerous human malignancies, of which cervical cancer is the most prevalent, beta (ß) HPVs predominantly act as co-factors in skin carcinogenesis. A characteristic feature of both α- and ß-E6 oncoproteins is the presence of the LXXLL binding motif, which α-E6s utilize to form a complex with E6AP and which enables ß-E6s to interact with MAML1. Here we show that multiple α-E6 oncoproteins bind to MAML1 via the LXXLL binding motif and that this results in increased protein stability. Moreover, ß-E6 oncoprotein stability is also dependent on the interaction with MAML1. Additionally, in the absence of MAML1, endogenous HPV-8 E6 and HPV-18 E6 are rapidly degraded at the proteasome. Ablation of both E6AP and MAML1 leads to an even more profound downregulation of α-E6 protein expression, whereas this is not observed with ß-E6. This highly suggests that there is one cellular pool for most of ß-E6 that interacts solely with MAML1, whereas there are two cellular pools of HR α-E6, one forming a complex with MAML1 and the other interacting with E6AP. Furthermore, MAML1 induces HPV-8 E6 shuttling from the nucleus to the cytosolic fraction, while MAML1 interaction with HR E6 induces a drastic nuclear and membrane upregulation of E6. Interestingly, the HR α-E6/MAML1 complex does not affect targeting of some of the known HR E6 cellular substrates such as p53 and DLG1. However, MAML1 and E6AP joint co-expression with HR α-E6 leads to a significant increase in cellular proliferation, whereas silencing MAML1 decreases wound closure in HeLa cells. These results demonstrate that HR α-E6 interaction with MAML1 results in a stable form of E6, which likely modulates MAML1's normal cellular activities, one consequence of which being an increased proliferative capacity of HPV-transformed cancer cells. Thus, this study shows a novel function of the α-E6 oncoprotein and how it's activity might affect HPV-induced pathogenesis.

Identification of SH2 Domain-Containing Protein 3C as a Novel, Putative Interactor of Dipeptidyl Peptidase 3.

Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent exopeptidase with broad specificity for four to eight amino acid residue substrates. It has a role in the regulation of oxidative stress response NRF2-KEAP1 pathway through the interaction with KEAP1. We have conducted stable isotope labeling by amino acids in a cell culture coupled to mass spectrometry (SILAC-MS) interactome analysis of TRex HEK293T cells using DPP3 as bait and identified SH2 Domain-Containing Protein 3C (SH2D3C) as prey. SH2D3C is one of three members of a family of proteins that contain both the SH2 domain and a domain similar to guanine nucleotide exchange factor domains of Ras family GTPases (Ras GEF-like domain), named novel SH2-containing proteins (NSP). NSPs, including SH2D3C (NSP3), are adaptor proteins involved in the regulation of adhesion, migration, tissue organization, and immune response. We have shown that SH2D3C binds to DPP3 through its C-terminal Ras GEF-like domain, detected the colocalization of the proteins in living cells, and confirmed direct interaction in the cytosol and membrane ruffles. Computational analysis also confirmed the binding of the C-terminal domain of SH2D3C to DPP3, but the exact model could not be discerned. This is the first indication that DPP3 and SH2D3C are interacting partners, and further studies to elucidate the physiological significance of this interaction are on the way.

IQGAP-related protein IqgC suppresses Ras signaling during large-scale endocytosis.

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC- and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.

PDZ Domain-Containing Protein NHERF-2 Is a Novel Target of Human Papillomavirus 16 (HPV-16) and HPV-18.

Cancer-causing human papillomavirus (HPV) E6 oncoproteins have a class I PDZ-binding motif (PBM) on their C termini, which play critical roles that are related to the HPV life cycle and HPV-induced malignancies. E6 oncoproteins use these PBMs to interact with, to target for proteasome-mediated degradation, a plethora of cellular substrates that contain PDZ domains and that are involved in the regulation of various cellular pathways. In this study, we show that both HPV-16 and HPV-18 E6 oncoproteins can interact with Na+/H+ exchange regulatory factor 2 (NHERF-2), a PDZ domain-containing protein, which among other cellular functions also behaves as a tumor suppressor regulating endothelial proliferation. The interaction between the E6 oncoproteins and NHERF-2 is PBM dependent and results in proteasome-mediated degradation of NHERF-2. We further confirmed this effect in cells derived from HPV-16- and HPV-18-positive cervical tumors, where we show that NHERF-2 protein turnover is increased in the presence of E6. Finally, our data indicate that E6-mediated NHERF-2 degradation results in p27 downregulation and cyclin D1 upregulation, leading to accelerated cellular proliferation. To our knowledge, this is the first report to demonstrate that E6 oncoproteins can stimulate cell proliferation by indirectly regulating p27 through targeting a PDZ domain-containing protein.IMPORTANCE This study links HPV-16 and HPV-18 E6 oncoproteins to the modulation of cellular proliferation. The PDZ domain-containing protein NHERF-2 is a tumor suppressor that has been shown to regulate endothelial proliferation; here, we demonstrate that NHERF-2 is targeted by HPV E6 for proteasome-mediated degradation. Interestingly, this indirectly affects p27, cyclin D1, and CDK4 protein levels and, consequently, affects cell proliferation. Hence, this study provides information that will improve our understanding of the molecular basis for HPV E6 function, and it also highlights the importance of the PDZ domain-containing protein NHERF-2 and its tumor-suppressive role in regulating cell proliferation.

A Diaphanous-related formin links Ras signaling directly to actin assembly in macropinocytosis and phagocytosis.

Phagocytosis and macropinocytosis are Ras-regulated and actin-driven processes that depend on the dynamic rearrangements of the plasma membrane that protrudes and internalizes extracellular material by cup-shaped structures. However, the regulatory mechanisms underlying actin assembly in large-scale endocytosis remain elusive. Here, we show that the Diaphanous-related formin G (ForG) from the professional phagocyte Dictyostelium discoideum localizes to endocytic cups. Biochemical analyses revealed that ForG is a rather weak nucleator but efficiently elongates actin filaments in the presence of profilin. Notably, genetic inactivation of ForG is associated with a strongly impaired endocytosis and a markedly diminished F-actin content at the base of the cups. By contrast, ablation of the Arp2/3 (actin-related protein-2/3) complex activator SCAR (suppressor of cAMP receptor) diminishes F-actin mainly at the cup rim, being consistent with its known localization. These data therefore suggest that ForG acts as an actin polymerase of Arp2/3-nucleated filaments to allow for efficient membrane expansion and engulfment of extracellular material. Finally, we show that ForG is directly regulated in large-scale endocytosis by RasB and RasG, which are highly related to the human proto-oncogene KRas.

Modulation of small GTPase activity by NME proteins.

NME proteins are reported to influence signal transduction activity of small GTPases from the Ras superfamily by diverse mechanisms in addition to their generic NDP kinase activity, which replenishes the cytoplasmic pool of GTP. Comprehensive evidence shows that NME proteins modulate the activity of Ras GTPases, in particular members of the Rho family, via binding to their major activators GEFs. Direct interaction between several NMEs and Ras GTPases were also indicated in vitro and in vivo. These modes of regulation are mainly independent of the NME's kinase activity. NMEs also modulate the Ras-mediated signal transduction by interfering with the formation of a Ras signaling complex at the plasma membrane. In several examples, NMEs were proposed to perform the role of GAP proteins by promoting hydrolysis of the bound GTP, but this activity still requires additional verification. Early suggestions that NMEs can activate small GTPases by direct phosphorylation of the bound GDP, or by high-rate loading of GTP onto a closely apposed GTPase, were largely dismissed. In this review article, we survey and put into perspective published examples of identified and hypothetical mechanisms of Ras signaling modulation by NME proteins. We also point out involvement of NMEs in the transcriptional regulation of components of Ras GTPases-mediated signal transduction pathways, and reciprocal regulation of NME function by small GTPases, particularly related to NME's binding to membranes.

Quantitative imaging of Rac1 activity in Dictyostelium cells with a fluorescently labelled GTPase-binding domain from DPAKa kinase.

Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Sophisticated tools used to investigate their activity in living cells include probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation, and photoactivation. However, such methods are of limited use in quickly migrating cells due to a short time available for image acquisition leading to a low signal-to-noise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. Here we present design and characterization of a new fluorescent probe that selectively binds to active form of Rac1 GTPases, and demonstrate its superior properties for imaging in highly motile Dictyostelium cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase and was selected on the basis of yeast two-hybrid screen, GST pull-down assay and FRET measurements by fluorescence lifetime imaging microscopy. DPAKa(GBD) probe binds specifically to GTP-bound Rac1 at the cell membrane and features a low cytoplasmic background. The main advantage of DPAKa(GBD) in comparison with similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables quantitative measurements of the Rac1 activity in different parts of the membrane. Finally, expression of DPAKa(GBD) induces no adverse effects on cell growth, motility and cytokinesis.

The IQGAP-related protein DGAP1 mediates signaling to the actin cytoskeleton as an effector and a sequestrator of Rac1 GTPases.

Proteins are typically categorized into protein families based on their domain organization. Yet, evolutionarily unrelated proteins can also be grouped together according to their common functional roles. Sequestering proteins constitute one such functional class, acting as macromolecular buffers and serving as an intracellular reservoir ready to release large quantities of bound proteins or other molecules upon appropriate stimulation. Another functional protein class comprises effector proteins, which constitute essential components of many intracellular signal transduction pathways. For instance, effectors of small GTP-hydrolases are activated upon binding a GTP-bound GTPase and thereupon participate in downstream interactions. Here we describe a member of the IQGAP family of scaffolding proteins, DGAP1 from Dictyostelium, which unifies the roles of an effector and a sequestrator in regard to the small GTPase Rac1. Unlike classical effectors, which bind their activators transiently leading to short-lived signaling complexes, interaction between DGAP1 and Rac1-GTP is stable and induces formation of a complex with actin-bundling proteins cortexillins at the back end of the cell. An oppositely localized Rac1 effector, the Scar/WAVE complex, promotes actin polymerization at the cell front. Competition between DGAP1 and Scar/WAVE for the common activator Rac1-GTP might provide the basis for the oscillatory re-polarization typically seen in randomly migrating Dictyostelium cells. We discuss the consequences of the dual roles exerted by DGAP1 and Rac1 in the regulation of cell motility and polarity, and propose that similar signaling mechanisms may be of general importance in regulating spatiotemporal dynamics of the actin cytoskeleton by small GTPases.

Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress.

Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.

A dual role for Rac1 GTPases in the regulation of cell motility.

Rac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the GTP-bound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.

IqgC is a potent regulator of macropinocytosis in the presence of NF1 and its loading to macropinosomes is dependent on RasG.

RasG is a major regulator of macropinocytosis in Dictyostelium discoideum. Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo, the physiological significance of the Rab5A-IqgC interaction remains elusive.

Rho GTPases in Model Systems.

Since the discovery of their role in the regulation of actin cytoskeleton 30 years ago, Rho GTPases have taken center stage in cell motility research [...].

Regulation of the Actin Cytoskeleton via Rho GTPase Signalling in Dictyostelium and Mammalian Cells: A Parallel Slalom.

Both Dictyostelium amoebae and mammalian cells are endowed with an elaborate actin cytoskeleton that enables them to perform a multitude of tasks essential for survival. Although these organisms diverged more than a billion years ago, their cells share the capability of chemotactic migration, large-scale endocytosis, binary division effected by actomyosin contraction, and various types of adhesions to other cells and to the extracellular environment. The composition and dynamics of the transient actin-based structures that are engaged in these processes are also astonishingly similar in these evolutionary distant organisms. The question arises whether this remarkable resemblance in the cellular motility hardware is accompanied by a similar correspondence in matching software, the signalling networks that govern the assembly of the actin cytoskeleton. Small GTPases from the Rho family play pivotal roles in the control of the actin cytoskeleton dynamics. Indicatively, Dictyostelium matches mammals in the number of these proteins. We give an overview of the Rho signalling pathways that regulate the actin dynamics in Dictyostelium and compare them with similar signalling networks in mammals. We also provide a phylogeny of Rho GTPases in Amoebozoa, which shows a variability of the Rho inventories across different clades found also in Metazoa.

Role of LrrkA in the Control of Phagocytosis and Cell Motility in Dictyostelium discoideum.

LrrkA is a Dictyostelium discoideum kinase with leucine-rich repeats. LrrkA stimulates Kil2 and intra-phagosomal killing of ingested bacteria in response to folate. In this study, we show that genetic inactivation of lrrkA also causes a previously unnoticed phenotype: lrrkA KO cells exhibit enhanced phagocytosis and cell motility compared to parental cells. This phenotype is cell autonomous, is reversible upon re-expression of LrrkA, and is not due to an abnormal response to inhibitory quorum-sensing factors secreted by D. discoideum in its medium. In addition, folate increases motility in parental D. discoideum cells, but not in lrrkA KO cells, suggesting that LrrkA plays a pivotal role in the cellular response to folate. On the contrary, lrrkA KO cells regulate gene transcription in response to folate in a manner indistinguishable from parental cells. Overall, based on analysis of mutant phenotypes, we identify gene products that participate in the control of intracellular killing, cell motility, and gene transcription in response to folate. These observations reveal a mechanism by which D. discoideum encountering bacterially-secreted folate can migrate, engulf, and kill bacteria more efficiently.

Combination of sirtuin 3 and hyperoxia diminishes tumorigenic properties of MDA-MB-231 cells.

AIMS: Since the role of the major mitochondrial NAD+-dependent deacetylase, sirtuin 3 (Sirt3), is differential in cancer, opposite to the well-known tumor-suppressing effect of hyperoxia, this study aimed to investigate the role of Sirt3 in triple-negative breast cancer (TNBC) cell line MDA-MB-231 upon hyperoxic (95% O2) conditions. MAIN METHODS: MDA-MB-231 cells were stably transfected with Flag-tagged Sirt-3 or empty plasmid. Western blot and real-time PCR were used to monitor the expression of proteins or genes involved in mitochondrial biogenesis, metabolic regulation and antioxidant defense. Immunocytochemistry and confocal microscopy were used to confirm the cellular localization and abundance of proteins. Flow cytometry was used to analyze mitochondrial mass, potential and ROS production, and MTT test as a measure of metabolic activity. Mitotic index analysis, colony-forming unit assay, DNA damage and Annexin V-FITC analyses were used to assess the differences in the growth and apoptosis rate. KEY FINDINGS: Although Sirt3 seemed to improve mitochondrial properties by increasing mitochondrial mass and potential, metabolic activity (Warburg effect) and antioxidative defense (SOD2, Cat), it also increased mitochondrial ROS, induced DNA damage, timp-1 expression, formation of multinucleated cells and apoptosis, and finally markedly reduced the proliferation of MDA-MB-231 cells. All these effects were even more evident upon the hyperoxic treatment, thus pointing towards combined negative effect of Sirt3 and hyperoxia on MDA-MB-231 cells. SIGNIFICANCE: Both Sirt3 and hyperoxia, alone or in combination, have the potential to negatively affect the malignant properties of the MDA-MB-231 cells and should be further explored as a possible therapy for TNBC.

Sirt3 Exerts Its Tumor-Suppressive Role by Increasing p53 and Attenuating Response to Estrogen in MCF-7 Cells.

Estrogen (E2) is a major risk factor for the initiation and progression of malignancy in estrogen receptor (ER) positive breast cancers, whereas sirtuin 3 (Sirt3), a major mitochondrial NAD+-dependent deacetylase, has the inhibitory effect on the tumorigenic properties of ER positive MCF-7 breast cancer cells. Since it is unclear if this effect is mediated through the estrogen receptor alpha (ERα) signaling pathway, in this study, we aimed to determine if the tumor-suppressive function of Sirt3 in MCF-7 cells interferes with their response to E2. Although we found that Sirt3 improves the antioxidative response and mitochondrial fitness of the MCF-7 cells, it also increases DNA damage along with p53, AIF, and ERα expression. Moreover, Sirt3 desensitizes cells to the proliferative effect of E2, affects p53 by disruption of the ERα-p53 interaction, and decreases proliferation, colony formation, and migration of the cells. Our observations indicate that these tumor-suppressive effects of Sirt3 could be reversed by E2 treatment only to a limited extent which is not sufficient to recover the tumorigenic properties of the MCF-7 cells. This study provides new and interesting insights with respect to the functional role of Sirt3 in the E2-dependent breast cancers.

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli.

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB+ sbcC+ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3'-5' exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3' overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.

Prominent role of exopeptidase DPP III in estrogen-mediated protection against hyperoxia in vivo.

A number of age-related diseases have a low incidence in females, which is attributed to a protective effect of sex hormones. For instance, the female sex hormone estrogen (E2) has a well established cytoprotective effect against oxidative stress, which strongly contributes to ageing. However, the mechanism by which E2 exerts its protective activity remains elusive. In this study we address the question whether the E2-induced protective effect against hyperoxia is mediated by the Nrf-2/Keap-1 signaling pathway. In particular, we investigate the E2-induced expression and cellular distribution of DPP III monozinc exopeptidase, a member of the Nrf-2/Keap-1 pathway, upon hyperoxia treatment. We find that DPP III accumulates in the nucleus in response to hyperoxia. Further, we show that combined induction of hyperoxia and E2 administration have an additive effect on the nuclear accumulation of DPP III. The level of nuclear accumulation of DPP III is comparable to nuclear accumulation of Nrf-2 in healthy female mice exposed to hyperoxia. In ovariectomized females exposed to hyperoxia, supplementation of E2 induced upregulation of DPP III, Ho-1, Sirt-1 and downregulation of Ppar-γ. While other cytoprotective mechanisms cannot be excluded, these findings demonstrate a prominent role of DPP III, along with Sirt-1, in the E2-mediated protection against hyperoxia.

Mitochondrial dysfunction enhances Gal4-dependent transcription.

Mitochondrial dysfunction has been shown to elicit broad effects on nuclear gene expression. We show here that transcription dependent on the prototypical acidic activator Gal4 is responsive to mitochondrial dysfunction. In cells with no mitochondrial DNA, Gal4-dependent gene expression is elevated. A minimal Gal4 activator containing the DNA binding and activation domain is sufficient for this response. Transcription dependent on a fusion of Gal4 to a heterologous DNA binding domain is similarly elevated in a mitochondrial mutant. Analysis of different Gal4-dependent promoters and gel mobility shift assays suggest that the effect of mitochondrial dysfunction on Gal4 activity is related to increased DNA binding to the cognate Gal4 element. Given that fermentation is the only means to obtain energy in respiratory deficient cells, it is possible that higher Gal4 activity in cells with dysfunctional mitochondria works to promote more efficient fermentation of galactose.

Monoamine oxidases A and B gene polymorphisms in migraine patients.

Abnormal cortical activity and brainstem functioning are considered the possible etiopathogenetic factors of migraine. Monoamine oxidase A and B (MAO-A and -B) regulate the levels of monoamine neurotransmitters, so changes in their activity could participate in migraine pathogenesis. We have investigated the possible association of MAO-A and -B alleles and haplotypes with two common types of migraine, i.e. migraine without aura (MO) and migraine with aura (MA), on the sample of 110 migraineours (80 MO and 30 MA) and 150 controls. MAO-A promoter and MAO-B intron 13 polymorphisms were genotyped by the PCR-based methods. In addition, we have reevaluated the reported association between MAO-B intron 13 polymorphism and platelet MAO-B activity. The platelet MAO-B activity was determined fluorimetrically using kynuramine as a substrate. We have found a tendency toward association of the shorter variant of MAO-A gene promoter with migraine without aura in male subjects. Regarding investigated MAO-B polymorphism, no association with migraine or with platelet MAO-B activity was found. The suggestive association of the variant in MAO-A gene with migraine is considered worthy of independent replication. On the other hand, further studies on MAO-B polymorphism in migraine do not seem promising.