Identification of mammalian proteins inhibiting apoptosis downstream of cytochrome c release in a yeast survival screen

One of the known apoptotic pathways in mammalian cells involves release of mitochondrial Cytochrome c into the cytosol. Cyt c then together with ATP or dATP induces a conformational change in the adaptator protein Apaf-1 (a homologue of the C. elegans CED4 protein) (Zou, Henzel et al. 1997), leading to its oligomerization and the recruitment of several pro-Casp-9 molecules. This protein complex assembly called "apoptosome" leads to the activation of Casp-9 which then initiates or amplifies the caspase cascade. The cell death program can be stalled at several points and we were interested in identifying new proteins inhibiting cell death downstream of Cyt c release. This thesis describes how I have screened a cDNA library derived from a pool of human breast carcinomas in a yeast-based survival screen, using the S. pombe yeast strain HC4 containing an inducible CED4 construct(James, Gschmeissner et al. 1997). The screen resulted in the identification of six proteins displaying cell death-inhibiting activity in S. pombe as well as anti-apoptotic potential in mammalian cells. Those six molecules were RoRet (Ruddy, Kronmal et al. 1997), Aven (Chau, Cheng et al. 2000), Fte-1/S3a (Kho, Wang et al. 1996), PGC2 (Padilla, Kaur et al. 2000; Goetze, Eilers et al. 2002), SAA1-2ß (Moriguchi, Terai et al. 2001) and FBP (Brockstedt, Rickers et al. 1998) of which I selected RoRet, Aven and Fte-1/S3a for further analysis. RoRet is a new anti-apoptotic molecule that can inhibit the mitochondrial pathway via its PRY-SPRY domain. RoRet does not seem to bind to Apaf-1, and does not co-localize with the activated Apaf-1/Caspase-9 complex. Aven was published to act as an anti-apoptotic protein and suggested to function via the recruitment of Bcl-XL to Apaf-1. This work shows that its C-terminal domain can bind to Apaf-1 and has a strong anti-apoptotic activity by itself. Moreover, Aven co-localizes with the activated Apaf-1/Caspase-9 complex suggesting that it is a component of the apoptosome. Furthermore, the expression of Aven is regulated in mammary glands during the pregnancy cycle. Fte-1/S3a has been already implicated in increased transformation capacity of v-Fos in fibroblasts (Kho and Zarbl 1992; Kho, Wang et al. 1996). This work shows that it has anti-apoptotic activity and can protect against Bak- and Apaf-1-induced apoptosis. It can bind directly to activated Apaf-1 at the linker domain between the WD40 repeats and the CED4-like domain, suggesting that it may protect by sequestering the activated Apaf-1 to some organelles whose nature remains to be determined. Moreover, expression studies on mRNA and protein level showed upregulation of Fte-1/S3a in colon, lung and kidney carcinoma. Hmgb1 (Flohr, Rogalla et al. 2001; Pasheva, Ugrinova et al. 2002; Stros, Ozaki et al. 2002) was identified during a survival screen performed with a NIH 3T3 mouse fibroblast cDNA library in a Bak-expressing yeast S. pombe strain. HMGB1 can protect against Bak-, UV-, FasL- and TRAIL-induced apoptosis. Significant overexpression of HMGB1 was found in breast and colon carcinoma, and elevated mRNA amounts were detected in uterus, colon and stomach carcinoma, suggesting that it may be a tumour marker (Brezniceanu et al., 2003).Die Fehlsteuerung apoptotischen Zelltodes spielt eine große Rolle bei Krankheiten wie Krebs, AIDS oder bei neurodegenerativen Erkrankungen. Daher ist die Identifizierung weiterer apoptoseregulierender Proteine als potentielle Targets für molekulare Therapien ein wichtiges Ziel der Apoptoseforschung. Die Freisetzung von mitochondrialem Cytochrom c in das Zytosol ist ein zentraler Bestandteil der apoptotischen Signalübertragung in Säugetierzellen. Cytochrom c induziert in Gegenwart von ATP oder dATP die Bildung eines Multiproteinkomplexes, der als Apoptosom bezeichnet wird und aus jeweils mehreren Apaf-1- (homologes Adaptorprotein zu CED4 in C. elegans) und pro-Caspase-9-Molekülen besteht. Die Rekrutierung von Caspase-9 zum Apaf-1-Heptamer führt zu ihrer autokatalytischen Spaltung und Aktivierung. Dadurch wird die Kaskade weiterer Caspasen in Gang gesetzt. Apoptose kann an verschiedenen Punkten vor und nach der Freisetzung von Cytochrom c inhibiert werden. Ziel dieser Arbeit war es, solche Proteine zu identifizieren, die nach Cytochrom c-Freisetzung bzw. nach Apoptosombildung den Zelltod verhindern. Ich habe einen Hefe-Survival-Screen mit einer cDNA-Bibliothek, die aus humanem Mammakarzinomgewebe hergestellt wurde, durchgeführt. Die Expression verschiedener pro-apoptotischer Säugerproteine in Hefen führt zu schnellem und effizienten Hefezelltod. Dieses System läßt sich aber für das Durchsuchen von Säugerzellbibliotheken nach hefezelltodinhibierenden cDNAs verwenden, die dann darauf hin untersucht werden können, ob sie auch Säugerapoptose verhindern. Ein induzierbares CED4-Expressionskonstrukt wurde in die Hefe S. pombe eingebracht. In den resultierenden Hefestamm HC4 wurde dann die Mammakarzinom-cDNA-Bibliothek transformiert und anschließend CED4-Expression induziert, was normalerweise Zelltod in den Hefen auslöst. Die cDNAs der Tumorbibliothek in überlebenden Hefekolonien wurden anschließend isoliert und auf ihre anti-apoptotische Wirkung in Säugerzellen hin analysiert. Ich habe sechs neue Proteine entdeckt, die Apoptose hemmen können. Dabei handelt es sich um RoRet, Aven, Fte-l, PGC2, SAAlß und FBP. RoRet, Aven und Fte-1 wurden dann näher untersucht. Die Expression von RoRet, Aven und Fte-1 kann solche apoptotische Signale in Säugerzellen hemmen, die den intrinsischen mitochondrialen Apoptose-Signalübertragungsweg auslösen z.B. Bak- oder Apaf-1/Caspase-9-Überexpression). RoRet ist ein neues Protein, das durch seine C-terminale PRY-SPRY-Domäne Apoptose hemmen kann. Es bindet nicht an Apaf-l und ko-lokalisiert auch nicht mit dem Apoptosom, sondern nur mit Caspase-9 (aber nicht mit Apaf-1). Überexprimiertes RoRet-GFP befindet sich offensichtlich in vesikulären zytoplasmatischen Strukturen, die noch charakterisiert und identifiziert werden müssen. Eine C-terminale Deletionsmutante, die die PRY-SPRY-Domäne enthält und anti-apoptotisch wirkt, wird nicht nur im Zytoplasma, sondern auch im Zellkern detektiert. Außerdem konnte eine Hochregulation von roret mRNA in Eierstock-, Schilddrüsen- und Magentumoren festgestellt werden. Aven wurde bereits als anti-apoptotisches Protein publiziert, das vermutlich das anti-apoptotische Bcl-2-Familienmitglied Bcl-XL zum Adaptorprotein Apaf-1 rekrutiert. In meiner Arbeit konnte ich zeigen, dass das C-terminale Ende an Apaf-1 binden kann. Diese Domäne weist auch ein starkes anti-apoptotisches Potential auf. Der C-Terminus von Aven ko-lokalisiert mit dem aktivierten Apaf-1/Caspase-9 Komplex. Es ist möglich, dass Aven sich im Apoptosomkomplex befindet. Ich konnte auch nachweisen, dass die Expression von Aven im Brustdrüsengewebe der Maus auf RNA-Ebene deutlich reguliert wird. Eine Erhöhung der aven mRNA konnte in Darm-, Lungen- und Nierentumoren festgestellt werden. Das ribosomale Protein Fte-1/S3a ist als ein Faktor isoliert worden, der für die Transformation von Mausfibroblasten durch c-Fos wichtig ist. Meine Arbeit zeigt, dass es anti-apoptotische Aktivität aufweist und Säugerzellen gegen Bak- und Apaf-1/Caspase-9-induzierte Apoptose schützt. Ich konnte auch feststellen, dass Fte-1 an die Linker-Region von Apaf-l (zwischen der CED4-homologen Domäne und den WD40-repeats) binden kann. Fte-1 scheint mit Apaf-1 in noch näher zu identifizierenden Organellen zu ko-lokalisieren. Es ist möglich, dass Fte-1 durch seine Bindung an Apaf-1 anti-apoptotisch wirkt. Untersuchungen auf RNA- und Proteineben zeigen, dass Fte-1 in Lungen-, Darm- und Nierenkarzinomen hochreguliert wird. HMGB 1 wurde in einem Hefe-Survival-Screen gefunden, bei dem die Hefen durch Bak-Überexpression getötet wurden. HMGB 1 kann auch Bak-, UV-, FasL- und TRAIL-induzierte Apoptose in Säugerzellen hemmen. Ich habe erhöhte HMBG1-Proteinmengen in Brust- und Darmkarzinomen nachgewiesen. Eine Hochregulation von HMGB1 mRNA konnte zudem in Darin-, Uterus- und Magentumoren festgestellt werden. Über welchen Wirkmechanismus HMGB 1 Apoptose inhibiert, ist unklar. Ausgeschlossen werden können die direkte Bindung von HMGB 1 an Bak und die transkriptionelle Herrunterregulation von Bak. Diese Arbeit hat über ihre konkreten Ergebnisse hinaus als "Proof of principle" gezeigt, dass ein Hefe-Survival-Screen zur Identifizierung antiapoptotischer Säugeproteine, die in Tumoren überexprimiert werden, geeignet ist

Upregulation of intrarenal angiotensinogen in diabetes

Universidade Federal de São Paulo, Dept Med, Div Nephrol, BR-04023040 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, BR-04023040 São Paulo, BrazilWeb of Scienc

Bcl-2–Modifying Factor Induces Renal Proximal Tubular Cell Apoptosis in Diabetic Mice

This study investigated the mechanisms underlying tubular apoptosis in diabetes by identifying proapoptotic genes that are differentially upregulated by reactive oxygen species in renal proximal tubular cells (RPTCs) in models of diabetes. Total RNAs isolated from renal proximal tubules (RPTs) of 20-week-old heterozygous db/m+, db/db, and db/db catalase (CAT)-transgenic (Tg) mice were used for DNA chip microarray analysis. Real-time quantitative PCR assays, immunohistochemistry, and mice rendered diabetic with streptozotocin were used to validate the proapoptotic gene expression in RPTs. Cultured rat RPTCs were used to confirm the apoptotic activity and regulation of proapoptotic gene expression. Additionally, studies in kidney tissues from patients with and without diabetes were used to confirm enhanced proapoptotic gene expression in RPTs. Bcl-2–modifying factor (Bmf) was differentially upregulated (P < 0.01) in RPTs of db/db mice compared with db/m+ and db/db CAT-Tg mice and in RPTs of streptozotocin-induced diabetic mice in which insulin reversed this finding. In vitro, Bmf cDNA overexpression in rat RPTCs coimmunoprecipated with Bcl-2, enhanced caspase-3 activity, and promoted apoptosis. High glucose (25 mmol/L) induced Bmf mRNA expression in RPTCs, whereas rotenone, catalase, diphenylene iodinium, and apocynin decreased it. Knockdown of Bmf with small interfering RNA reduced high glucose–induced apoptosis in RPTCs. More important, enhanced Bmf expression was detected in RPTs of kidneys from patients with diabetes. These data demonstrate differential upregulation of Bmf in diabetic RPTs and suggest a potential role for Bmf in regulating RPTC apoptosis and tubular atrophy in diabetes

The role of reactive oxygen species in apoptosis of the diabetic kidney

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney

Analysis and verification of the HMGB1 signaling pathway

Background\ud Recent studies have found that overexpression of the High-mobility group box-1 (HMGB1) protein, in conjunction with its receptors for advanced glycation end products (RAGEs) and toll-like receptors (TLRs), is associated with proliferation of various cancer types, including that of the breast and pancreatic.\ud \ud Results\ud We have developed a rule-based model of crosstalk between the HMGB1 signaling pathway and other key cancer signaling pathways. The model has been simulated using both ordinary differential equations (ODEs) and discrete stochastic simulation. We have applied an automated verification technique, Statistical Model Checking, to validate interesting temporal properties of our model.\ud \ud Conclusions\ud Our simulations show that, if HMGB1 is overexpressed, then the oncoproteins CyclinD/E, which regulate cell proliferation, are overexpressed, while tumor suppressor proteins that regulate cell apoptosis (programmed cell death), such as p53, are repressed. Discrete, stochastic simulations show that p53 and MDM2 oscillations continue even after 10 hours, as observed by experiments. This property is not exhibited by the deterministic ODE simulation, for the chosen parameters. Moreover, the models also predict that mutations of RAS, ARF and P21 in the context of HMGB1 signaling can influence the cancer cell's fate - apoptosis or survival - through the crosstalk of different pathways

Increased expression of high mobility group box 1 (HMGB1) is associated with an elevated level of the antiapoptotic c‐IAP2 protein in human colon carcinomas

BACKGROUND: High mobility group box 1 (HMGB1) is a non‐histone chromosomal protein implicated in a variety of biologically important processes, including transcription, DNA repair, V(D)J recombination, differentiation, and development. Overexpression of HMGB1 inhibits apoptosis, arguing that the molecule may act as an antiapoptotic oncoprotein. Indeed, increased expression of HMGB1 has been reported for several different tumour types. In this study, we analysed human colon carcinoma for HMGB1 as well as for c‐IAP2 expression levels. c‐IAP2 is an antiapoptotic protein which may be upregulated as a consequence of nuclear factor κB (NFκB) activation via HMGB1. METHODS: A comparative genomic hybridisation (CGH) database comprising 1645 cases from different human tumour types was screened to detect cytogenetic changes at the HMGB1 locus. Immunohistochemical staining of human colon tissue microarrays and tumour biopsies, as well as western blot analysis of tumour lysates, were performed to detect elevated HMGB1 and c‐IAP2 expression in colon carcinomas. The antiapoptotic potential of HMGB1 was analysed by measuring caspase activities, and luciferase reporter assays and quantitative polymerase chain reaction analysis were employed to confirm NFκB activation and c‐IAP2 mRNA upregulation on HMGB1 overexpression. RESULTS: According to CGH analysis, the genomic locus containing the HMGB1 gene was overrepresented in one third (35/96) of colon cancers. Correspondingly, HMGB1 protein levels were significantly elevated in 90% of the 60 colon carcinomas tested compared with corresponding normal tissues evaluable from the same patients. HMGB1 increased NFκB activity and led to co‐overexpression of the antiapoptotic NFκB target gene product c‐IAP2 in vitro. Furthermore, increased HMGB1 levels correlated with enhanced amounts of c‐IAP2 in colon tumours analysed by us. Finally, we demonstrated that HMGB1 overexpression suppressed caspase‐9 and caspase‐3 activity, suggesting that HMGB1 interferes with the apoptotic machinery at the level of apoptosomal caspase‐9 activation. CONCLUSIONS: We identified in vitro a molecular pathway triggered by HMGB1 to inhibit apoptosis via c‐IAP2 induction. Our data indicate a strong correlation between upregulation of the apoptosis repressing HMGB1 and c‐IAP2 proteins in the pathogenesis of colon carcinoma