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

Prognostic Role of Liver Biopsy in Patients With Severe Indeterminate Acute Hepatitis

Background and aims: Severe indeterminate acute hepatitis (sIAH) is a poorly understood rare disease with no specific therapy. This study aims to define the clinicopathological characteristics of sIAH and the role of liver biopsy in determining prognosis. Methods: Patients with sIAH admitted to a single center between 2010 and 2019 were included. Histopathological patterns of liver biopsies were reviewed by 2 histopathologists, and key findings further were specified by multiplex immunofluorescence. Patients that died or underwent liver transplantation were analyzed as nonsurvivors. Results: Of 294 patients with acute hepatitis, 43 with sIAH were included. Seventeen (39.5%) underwent liver transplantation and 7 (16.2%) died within 3 months. Multilobular necrosis was the predominant histopathological feature, being significantly more frequent in nonsurvivors (62.5% vs 21.1%; P = .016). Necrotic areas showed low HNF4α and Ki67 expression but high expression of CK19 and cell death markers identifying areas of severe tissue injury and inadequate regenerative response. Patients with multilobular necrosis had higher international normalized ratio, Model for End-Stage Liver Disease, and Model for End-Stage Liver Disease-Sodium scores compared with those without (P values for all markers <.05). Multivariate Cox analysis revealed that multilobular necrosis (hazard ratio, 3.675; 95% confidence interval, 1.322-10.211) and lower body mass index (hazard ratio, 0.916; 95% confidence interval, 0.848-0.991) independently predicted death or transplantation. Conclusions: The results of this study provide novel insights into the important role of liver biopsy in sIAH patients, suggesting that the presence of multilobular necrosis is an early indicator of poor prognosis.info:eu-repo/semantics/publishedVersio

Regulation of Synaptophysin Degradation by Mammalian Homologues of Seven in Absentia

Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin

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

Prognostic Role of Liver Biopsy in Patients With Severe Indeterminate Acute Hepatitis

Background and Aims: Severe indeterminate acute hepatitis (sIAH) is a poorly understood rare disease with no specific therapy. This study aims to define the clinicopathological characteristics of sIAH and the role of liver biopsy in determining prognosis. / Methods: Patients with sIAH admitted to a single center between 2010 and 2019 were included. Histopathological patterns of liver biopsies were reviewed by 2 histopathologists, and key findings further were specified by multiplex immunofluorescence. Patients that died or underwent liver transplantation were analyzed as nonsurvivors. / Results: Of 294 patients with acute hepatitis, 43 with sIAH were included. Seventeen (39.5%) underwent liver transplantation and 7 (16.2%) died within 3 months. Multilobular necrosis was the predominant histopathological feature, being significantly more frequent in nonsurvivors (62.5% vs 21.1%; P = .016). Necrotic areas showed low HNF4α and Ki67 expression but high expression of CK19 and cell death markers identifying areas of severe tissue injury and inadequate regenerative response. Patients with multilobular necrosis had higher international normalized ratio, Model for End-Stage Liver Disease, and Model for End-Stage Liver Disease-Sodium scores compared with those without (P values for all markers <.05). Multivariate Cox analysis revealed that multilobular necrosis (hazard ratio, 3.675; 95% confidence interval, 1.322–10.211) and lower body mass index (hazard ratio, 0.916; 95% confidence interval, 0.848–0.991) independently predicted death or transplantation. / Conclusions: The results of this study provide novel insights into the important role of liver biopsy in sIAH patients, suggesting that the presence of multilobular necrosis is an early indicator of poor prognosis

Alterations in ubiquitin ligase SIAH-2 and its corepressor N-COR after P-mapa immunotherapy and anti-androgen therapy: new therapeutic opportunities for non-muscle invasive bladder cancer

The present study describes the role of the ubiquitin ligase Siah-2 and corepressor N-CoR in controlling androgen receptor (AR) and estrogen receptors (ERa and ER beta) signaling in an appropriate animal model (Fischer 344 female rats) of non-muscle invasive bladder cancer (NMIBC), especially under conditions of anti-androgen therapy with flutamide. Furthermore, this study describes the mechanisms of a promising therapeutic alternative for NMIBC based on Protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) intravesical immunotherapy combined with flutamide, involving the interaction among steroid hormone receptors, their regulators and Toll-like receptors (TLRs). Our results demonstrated that increased Siah-2 and AR protein levels and decreased N-CoR, cytochrome P450 (CYP450) and estrogen receptors levels played a critical role in the urothelial carcinogenesis, probably leading to escape of urothelial cancer cells from immune system attack. P-MAPA immunotherapy led to distinct activation of innate immune system TLRs 2 and 4-mediated, resulting in increase of interferon signaling pathway, which was more effective in recovering the immunosuppressive tumor immune microenvironment and in recovering the bladder histology features than BCG (Bacillus Calmette-Guerin) treatments. The AR blockade therapy was important in the modulating of downstream molecules of TLR2 and TLR4 signaling pathway, decreasing the inflammatory cytokines signaling and enhancing the interferon signaling pathway when associated with P-MAPA. Taken together, the data obtained suggest that interferon signaling pathway activation and targeting AR and Siah-2 signals by P-MAPA intravesical immunotherapy alone and/or in combination with AR blockade may provide novel therapeutic approaches for NMIBC8544274443CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP490519/2011-32012/20706-2; 2012/13585-

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