170,043 research outputs found
WC-1 and the proximal GATA sequence mediate a cis-/trans-acting repressive regulation of light-dependent gene transcription in the dark
No full textLight influences a wide range of physiological processes from prokaryotes to mammals. Neurospora crassa represents an important model system used for studying this signal pathway. At molecular levels, the WHITE COLLAR Complex (WCC), a heterodimer formed by WC-1 (the blue light photo-sensor) and WC-2 (the transcriptional activator), is the critical positive regulator of light-dependent gene expression. GATN (N indicates any other nucleotide) repeats are consensus sequences within the promoters of light-dependent genes recognized by the WCC. The distal GATN is also known as C-box since it is involved in the circadian clock. However, we know very little about the role of the proximal GATN, and the molecular mechanism that controls the transcription of light-induced genes during the dark/light transition it is still unclear. Here we showed a first indication that mutagenesis of the proximal GATA sequence within the target promoter of the albino-3 gene or deletion of the WC-1 zinc finger domain led to a rise in expression of light-dependent genes already in the dark, effectively decoupling light stimuli and transcriptional activation. This is the first observation of cis-/trans-acting repressive machinery, which is not consistent with the light-dependent regulatory mechanism observed in the eukaryotic world so far
Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins.
No full textThe genes coding for white collar-1 and white collar-2 (wc-1 and wc-2) have been isolated previously, and their products characterized as Zn-finger transcription factors involved in the control of blue light-induced genes. Here, we show that the PAS dimerization domains present in both proteins enable the WC-1 and WC-2 proteins to dimerize in vitro. Homodimers and heterodimers are formed between the white collar (WC) proteins. A computer analysis of WC-1 reveals a second domain, called LOV, also identified in NPH1, a putative blue light photoreceptor in plants and conserved in redox-sensitive proteins and in the phytochromes. The WC-1 LOV domain does not dimerize with canonical PAS domains, but it is able to self-dimerize. The isolation of three blind wc-1 strains, each with a single amino acid substitution only in the LOV domain, reveals that this region is essential for blue light responses in Neurospora. The demonstration that the WC-1 proteins in these LOV mutants are still able to self-dimerize suggests that this domain plays an additional role, essential in blue light signal transduction
SOD1, a new Kluyveromyces lactis helper gene for heterologous protein secretion
No full textBottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O(2)(-)) into oxygen and hydrogen peroxide. It is a Cu(2+)/Zn(2+) metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses
Specific down-modulation of Notch1 signaling in cervical cancer cells is required for sustained HPV-E6/E7 expression and late steps of malignant transformation
No full textThe Notch family of cell surface receptors plays a key role in cell-fate determination and differentiation, functioning in a cell- and context-specific manner. In mammalian cells, Notch activation is generally thought to maintain stem cell potential and inhibit differentiation, thereby promoting carcinogenesis. However, in other contexts such as primary epithelial cells (keratinocytes), increased Notch activity causes exit from the cell cycle and/or commitment to differentiation. We now report that expression of the endogenous Notch1 gene is markedly reduced in a panel of cervical carcinoma cells whereas expression of Notch2 remains elevated, and Notch1 expression is similarly reduced or absent in invasive cervical cancers. Conversely, expression of activated Notch1 causes strong growth inhibition of HPV-positive, but not HPV-negative, cervical carcinoma cells, but exerts no such effects on other epithelial tumor cells. Increased Notch1 signaling, but not Notch2, causes a dramatic down-modulation of HPV-driven transcription of the E6/E7 viral genes, through suppression of AP-1 activity by up-regulation of the Fra-1 family member and decreased c-Fos expression. Thus, Notch1 exerts specific protective effects against HPV-induced transformation through suppression of E6/E7 expression, and down-modulation of Notch1 expression is likely to play an important role in late stages of HPV-induced carcinogenesis
The impact of the ubiquitin system in the pathogenesis of squamous cell carcinomas
No full textThe ubiquitin system is a dynamic regulatory pathway controlling the activity, subcellular localization and stability of a myriad of cellular proteins, which in turn affects cellular homeostasis through the regulation of a variety of signaling cascades. Aberrant activity of key components of the ubiquitin system has been functionally linked with numerous human diseases including the initiation and progression of human tumors. In this review, we will contextualize the importance of the two main components of the ubiquitin system, the E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs), in the etiology of squamous cell carcinomas (SCCs). We will discuss the signaling pathways regulated by these enzymes, emphasizing the genetic and molecular determinants underlying their deregulation in SCCs
Inhibition of eukaryotic translation initiation factor 5a (Eif5a) hypusination suppress p53 translation and alters the association of eif5a to the ribosomes
No full textThe eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for the viability of the cells whose proposed function is to prevent the stalling of the ribosomes during translation elongation. eIF5A activity requires a unique and functionally essential post-translational modification, the change of a lysine to hypusine. eIF5A is recognized as a promoter of cell proliferation, but it has also been suggested to induce apoptosis. To date, the precise molecular mechanism through which eIF5A affects these processes remains elusive. In the present study, we explored whether eIF5A is involved in controlling the stress-induced expression of the key cellular regulator p53. Our results show that treatment of HCT-116 colon cancer cells with the deoxyhypusine (DHS) inhibitor N1-guanyl-1,7-diamineheptane (GC7) caused both inhibition of eIF5A hypusination and a significant reduction of p53 expression in UV-treated cells, and that eIF5A controls p53 expression at the level of protein synthesis. Furthermore, we show that treatment with GC7 followed by UV-induced stress counteracts the pro-apoptotic process triggered by p53 up-regulation. More in general, the importance of eIF5A in the cellular stress response is illustrated by the finding that exposure to UV light promotes the binding of eIF5A to the ribosomes, whereas UV treatment complemented by the presence of GC7 inhibits such binding, allowing a decrease of de novo synthesis of p53 protein
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Differential subcellular localization regulates c-Cbl E3 ligase activity upon Notch3 protein in T-cell leukemia
No full textNotch3 and pT alpha signaling events are essential for T-cell leukemogenesis and characterize murine and human T-cell acute lymphoblastic leukemia. Genetic ablation of pT alpha expression in Notch3 transgenic mice abrogates tumor development, indicating that pT alpha signaling is crucial to the Notch3-mediated leukemogenesis. Here we report a novel direct interaction between Notch3 and pT alpha. This interaction leads to the recruitment and persistence of the E3 ligase protein c-Cbl to the lipid rafts in Notch3-IC transgenic thymocytes. Conversely, deletion of pT alpha in Notch3 transgenic mice leads to cytoplasmic retention of c-Cbl that targets Notch3 protein to the proteasomal-degradative pathway. It appears that protein kinase C theta (PKC theta), by regulating tyrosine and serine phosphorylation of Cbl, is able to control its function. We report here that the increased Notch3-IC degradation correlates with higher levels of c-Cbl tyrosine phosphorylation in Notch3-IC/pT alpha(-/-) double-mutant thymocytes, which also display a decreased PKC theta activity. Our data indicate that pT alpha/pre-T-cell receptor is able to regulate the different subcellular localization of c-Cbl and, by regulating PKC theta activity, is also able to influence its ubiquitin ligase activity upon Notch3 protein. Oncogene (2010) 29, 1463-1474; doi:10.1038/onc.2009.446; published online 7 December 200
Cross talk among calcineurin, Sp1/Sp3, and NFAT in control of p21(WAF1/CIP1) expression in keratinocyte differentiation
No full textEfficacy of the melanocortin analogue Nle4-D-Phe7-α-melanocyte-stimulating hormone in the treatment of patients with Hailey-Hailey disease
No full textBackgroundHailey-Hailey disease (HHD) is a rare, chronic and recurrent blistering disorder, which is characterized clinically by erosions occurring primarily in intertriginous regions, and histologically by suprabasal acantholysis. Oxidative stress plays a specific role in the pathogenesis of HHD, by regulating the expression of factors playing an important role in keratinocyte proliferation and differentiation. AimGiven the significance of oxidative stress in HHD, we investigated the potential effects of the antioxidant properties of an -MSH analogue, Nle4-D-Phe7--MSH (afamelanotide), in HHD lesion-derived keratinocytes. ResultsTreatment of HHD-derived keratinocytes with afamelanotide contributed to upregulation of Nrf2 [nuclear factor (erythroid-derived 2)-like 2], a redox-sensitive transcription factor that plays a pivotal role in redox homeostasis during oxidative stress. Additionally, afamelanotide treatment restored the defective proliferative capability of lesion-derived keratinocytes. Our results show that Nrf2 is an important target of the afamelanotide signalling that reduces oxidative stress. Because afamelanotide possesses antioxidant effects, we also assessed the clinical potential of this -MSH analogue in the treatment of patients with HHD. In a phase II open-label pilot study, afamelanotide 16mg was administered subcutaneously as a sustained-release resorbable implant formulation to two patients with HHD, who had a number of long-standing skin lesions. For both patients, their scores on the Short Form-36 improved 30days after the first injection of afamelanotide, and both had 100% clearance of HHD lesions 60days after the first injection, independently of the lesion location. ConclusionsAfamelanotide is effective for the treatment of skin lesions in HHD
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