Distinct expression patterns of the E3 ligase SIAH-1 and its partner Kid/KIF22 in normal tissues and in the breast tumoral processes

SIAH proteins are the human members of an highly conserved family of E3 ubiquitin ligases. Several data suggest that SIAH proteins may have a role in tumor suppression and apoptosis. Previously, we reported that SIAH-1 induces the degradation of Kid (KIF22), a chromokinesin protein implicated in the normal progression of mitosis and meiosis, by the ubiquitin proteasome pathway. In human breast cancer cells stably transfected with SIAH-1, Kid/KIF22 protein level was markedly reduced whereas, the Kid/KIF22 mRNA level was increased. This interaction has been further elucidated through analyzing SIAH and Kid/KIF22 expression in both paired normal and tumor tissues and cell lines. It was observed that SIAH-1 protein is widely expressed in different normal tissues, and in cells lines but showing some differences in western blotting profiles. Immunofluorescence microscopy shows that the intracellular distribution of SIAH-1 and Kid/KIF22 appears to be modified in human tumor tissues compared to normal controls. When mRNA expression of SIAH-1 and Kid/KIF22 was analyzed by real-time PCR in normal and cancer breast tissues from the same patient, a large variation in the number of mRNA copies was detected between the different samples. In most cases, SIAH-1 mRNA is decreased in tumor tissues compared to their normal counterparts. Interestingly, in all breast tumor tissues analyzed, variations in the Kid/KIF22 mRNA levels mirrored those seen with SIAH-1 mRNAs. This concerted variation of SIAH-1 and Kid/KIF22 messengers suggests the existence of an additional level of control than the previously described protein-protein interaction and protein stability regulation. Our observations also underline the need to re-evaluate the results of gene expression obtained by qRT-PCR and relate it to the protein expression and cellular localization when matched normal and tumoral tissues are analyzed

The Coiled-coil Domain Is the Structural Determinant for Mammalian Homologues of Drosophila Sina-mediated Degradation of Promyelocytic Leukemia Protein and Other Tripartite Motif Proteins by the Proteasome

Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation

Funktionelle Relevanz der Ubiquitin-Ligasen seven in absentia homologue (SIAH)-1 und SIAH-2 in der humanen Hepatokarzinogenese

Das hepatozelluläre Karzinom (HCC) ist der häufigste primäre maligne Tumor der Leber; es geht mit einer sehr schlechten Prognose einher. Neben der Dysregulation von Syntheseprozessen stellt auch der alterierte proteasomale Abbau von Proteinen durch Ubiquitin-Ligasen einen potentiellen protumorigenen Mechanismus dar. Der wichtigste Prozess zur Degradation von Zielstrukturen in eukaryontischen Zellen ist die Ubiquitin-26S-Proteasom-vermittelte Degradation durch die mehr als 80% aller zellulären Proteine spezifisch und energie-abhängig abgebaut werden. Für die E3-Ubiquitin-Ligasen seven in absentia homologue (SIAH)-1 und SIAH-2 wird der durch Polyubiquitylierung vermittelte Abbau verschiedener funktioneller und struktureller Klassen von Proteinen, wie β-Catenin und Synaptophysin, bewirkt. In unabhängigen Studien konnte eine Reduktion SIAH-1 Expression in HCCs nachgewiesen werden, was einen Einfluss auf die Bioverfügbarkeit dieser Bindepartner im Rahmen der Lebertumorentstehung oder Progression vermuten lässt. Ziel dieser Studie war daher die vergleichende Analyse von SIAH-1 und SIAH-2 hinsichtlich Expression und Funktion in der humanen Hepatokarzinogenese. In einem HCC Kollektiv wurde für SIAH-1 eine Reduktion der Expression bestätigt, wohingegen keine signifikante Expressionsänderung für SIAH-2 detektiert wurde. Während in Hepatozyten eine zytoplasmatische Expression für beide E3-Ligasen nachgewiesen wurde, war eine signifikante und progressive nukleäre Akkumulation in Vorläuferläsionen und HCC Zellen zu beobachten. In HCC-Zelllinien führte die Inhibierung beider Ubiquitin-Ligasen zu einer Reduktion der Tumorzellproliferation und Migration, als auch zu einer Induktion von Apoptose. Die gleichzeitige Inhibierung beider Ligasen verstärkte die beobachteten biologischen Effekte nur geringfügig, was für nicht redundante Effekte beider Ligasen spricht. Eine Inhibierung der Ubiquitin-Ligasen führte darüber hinaus zu einer signifikanten Sensibilisierung der HCC Zellen gegenüber der Behandlung mit solchen Chemotherapeutika, welche sowohl die Stabilität des Mikrotubulusnetzwerkes verändern als auch in DNA interkalieren. Im Gegensatz zu bereits beschriebenen in vitro-Modellen konnte in den HCC Zelllinien keine Regulation von SIAH-1 durch das Tumorsuppressorgen p53 nachgewiesen werden. Überraschenderweise induzierte die Inhibierung von SIAH-1 und SIAH-2 in HCC Zellen nicht die Bioverfügbarkeit von bekannten Bindungspartnern (z.B. T-Star und β-Catenin), sondern führte zu einer verringerten Expression von far upstream sequence element (FUSE) binding protein (FBP)-Familienmitgliedern, welche zentrale Mediatoren der Tumorzellproliferation und Migration darstellen. Zusammenfassend demonstrieren diese Daten, dass das protumorigene Potential von SIAH-1 und SIAH-2 nicht durch eine veränderte (reduzierte) Expression, sondern durch nukleäre Akkumulation und durch die Aktivierung distinkter molekulare Mechanismen vermittelt wird. Aufgrund der Tumorzell-assoziierten nukleären Expression unterscheidet sich die Funktion von SIAH-1 und SIAH-2 als auch die molekularen Effektormechanismen in HCC Zellen deutlich von anderen Zelltypen. Darüber hinaus stellt die Inhibierung der subzellulären Translokation bzw. die Reduktion der nukleären Bioaktivität der SIAH-Ligasen eine neue Möglichkeit zur Sensibilisierung von Tumorzellen mit zytotoxischen Substanzen dar

Role of HIF-1, Siah-1 and SKN-1 in Inducing Adiposity for Caenorhabditis elegans under Hypoxic Conditions

BACKGROUND: Hypoxia has been shown to be able to induce adiposity. However, the mechanism and factors involved in this effect still remains unclear. Hence, we sought to investigate the role of oxygensensitive factors regarding hypoxia-induced adiposity in nematode Caenorhabditis elegans.METHODS: The C. elegans were grown on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 at 20°C. The ratio of width/body length was measured using the morphometry analysis. Fat accumulation was examined using Sudan Black methods. Protein levels of sterol binding protein (SBP)-1 were assessed by immunoblotting. Lifespan assay was performed at 20°C and was monitored every two days.RESULTS: The results showed that of all mutant used, only hif-1 mutant which did not experience an increase in the ratio of width/body length (p>0.05) and fat accumulation (p>0.05), indicating that hypoxia-inducible factors (HIF)-1 plays an important role in the pathogenesis of hypoxia-induced adiposity. Both siah-1 and skn-1 mutants experienced SBP-1 protein elevation (p0.05) further supporting that HIF-1 acts as an upstream regulator fromSBP-1.CONCLUSION: In general, the results of this study provide evidences of the involvement of the transcription factor HIF-1 in inducing adiposity under the hypoxic conditions. However, we did not find the involvement of seven in absentia homolog-1 (Siah-1) and skinhead-1 (SKN-1).KEYWORDS: hypoxia, adiposity, fat, HIF-1, Siah-1, SKN-1, C. elegan

The characterization of the human Siah-1 promoter11The nucleotide sequence of the Siah-1 gene promoter can be found in the DDBJ and GenBank databases with the following accession number: AB072970.

AbstractSiah-1, the human homologue of Drosophila seven in absentia, is related to apoptosis and tumor suppression. Although it was reported that the expression of Siah-1 is induced by p53 and p21/WAF1, little is known about the transcriptional regulation of the Siah-1 gene. To investigate the transcriptional regulation, we isolated and sequenced the genomic fragment of the Siah-1 promoter region. The Siah-1 promoter has no typical TATA box or CCAAT box. Transient transfection assays using reporter plasmids in which the promoter region of the Siah-1 gene was deleted or mutated showed that one Sp1 site was responsible for the basal promoter activity. In Northern blotting analysis, the expression of the Siah-1 gene was upregulated by p53, but activation of the reporter plasmid by the p53 co-transfection assay was not shown, suggesting that a p53 responsive element does not exist in the promoter region we examined in this study but might be present in another region

Membrane-bound β-catenin degradation is enhanced by ETS2-mediated Siah1 induction in Helicobacter pylori-infected gastric cancer cells.

β-catenin has two different cellular functions: intercellular adhesion and transcriptional activity. The E3 ubiquitin ligase Siah1 causes ubiquitin-mediated degradation of the cytosolic β-catenin and therefore, impairs nuclear translocation and oncogenic function of β-catenin. However, the effect of Siah1 on the cell membrane bound β-catenin has not been studied. In this study, we identified that the carcinogenic bacterium H. pylori increased ETS2 transcription factor-mediated Siah1 protein expression in gastric cancer cells (GCCs) MKN45, AGS and Kato III. Siah1 protein level was also noticeably higher in gastric adenocarcinoma biopsy samples as compared to non-cancerous gastric epithelia. Siah1 knockdown significantly decreased invasiveness and migration of H. pylori-infected GCCs. Although, Siah1 could not increase degradation of the cytosolic β-catenin and its nuclear translocation, it enhanced degradation of the membrane-bound β-catenin in the infected GCCs. This loss of membrane-bound pool of β-catenin was not associated with the proteasomal degradation of E-cadherin. Thus, this work delineated the role of Siah1 in increasing invasiveness of H. pylori-infected GCCs

Urban sign. The polis furnished

Just as the linguistic sign, defined by Saussure, is like a double-sided coin: significant + meaning, so the city can be defined by this dichotomy, that is, on the one hand is composed of matter and, second, to function or content. We travel the city as the baudelairean flâneur as untimely affiliates walkers to the Situationist International, and so we wear away the tread on the asphalt. Politics, associations and media are responsible for our public space is constantly bombarded by private interests with the consequent impact on the development of citizen relations in semi-privatized public space. The Art brings significant strategies of non-urban places and does so through the Public Art as artistic intervention appropriation of public space by the affirmation of ordinary citizens living there, i.e. by an art to the city and citizenship. Exploring the current state of the contemporary city gives us the framework to review part of urban artistic poetics emerged in recent decades.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech