Dissertations / Theses on the topic 'XIAP inhibitors'

Depuis leur découverte, les interactions protéine-protéine (IPPs) sont au centre de nombreux processus biologiques. Ces IPPs peuvent ainsi être la cible de médicaments, soit en mimant une interaction défectueuse ou en inhibant une interaction inadéquate. Parmi les IPPs les plus impliquées dans les pathologies, on trouve les protéines inhibitrices de l’apoptose (IAPs) Ces membres sont considérés comme des régulateurs-clés de la mort cellulaire programmée et au sein des voies de signalisation de la cellule, XIAP, membre de la famille des IAPs est parmi les protéines les plus ciblées actuellement. Son implication dans les cancers et les maladies inflammatoires rares en fait une cible thérapeutique de choix. Les données récentes de la littérature ont soulevé l’importance de la conception de perturbateurs sélectifs de cette protéine pour écarter les effets indésirables graves. De ce fait, il est indispensable de bien connaître les éléments structuraux essentiels associés aux différents domaines de cette protéine, qui la distinguent des autres membres similaires de la famille IAP. Il est également essentiel de connaitre les mécanismes d’interaction dans lesquels XIAP est engagée avec ses partenaires. Les outils de CADD, notamment l’approche Structure Based Drug Design, ainsi que les techniques d’évaluation expérimentale utilisant l’anisotropie de fluorescence (FPA) ou la technologie AlphaScreen® ont été mises en œuvre dans le cadre de ce travail de thèse. Les résultats de ces travaux in silico et in vitro ont conduit à proposer la conception rationnelle de petites molécules sélectives potentiellement perturbateurs des IPPs médiées par XIAP
Since their discovery, protein-protein interactions (PPIs) have been at the heart of many biological processes. These PPIs can be drug targets, either by mimicking a defective interaction or by inhibiting an inadequate interaction. Among the most implicated PPIs in pathologies, we found the Inhibitor Apoptosis Proteins (AAPs). These members are considered as key regulators of programmed cell death and, within the cell signalling pathways, XIAP, a member of the IAP family, is one of the most targeted proteins actually. Its involvement in cancers and rare inflammatory diseases makes it a therapeutic target of choice. Recent data in the literature have highlighted the importance of designing selective disruptors for this protein in order to avoid serious adverse effects. For this reason, it is essential to know the essential structural elements associated with the different domains of this protein, which distinguish it from other similar members of the IAP family. It is also essential to know the interaction mechanisms in which XIAP is involved with its partners. CADD tools, in particular the Structure-based drug design approach, as well as experimental evaluation techniques using fluorescence anisotropy (FPA) or AlphaScreen® technology were used as part of this thesis work. The results of in silico and in vitro work led to rational design proposals of selective small molecules that could potentially disrupt XIAP-mediated PPIs

Two methods of XIAP down regulation were considered; transient knock down using antisense (AS) oligonucleotides (OGNs) and stable down regulation by generating short hairpin RNA expression cells. AEG35156 (Aegera Inc), a 2^nd generation mixed backbone AS OGN, binds specifically to the sense XIAP and mRNA strand, stimulating its breakdown and preventing translation into protein. A clinical phase I trial of AEG35156 was undertaken in patients with advanced cancer, in Manchester and Edinburgh. The maximum tolerated dose was 96 mg/m²/day of AEG35156, using a 7 day continuous infusion regimen. Dose limiting toxicities were all abnormal laboratory values, reported at higher dose levels (125mg/m²/day and 160mg/m²/day). In laboratory studies, a panel of colorectal cell lines (Colo205, HT29, SW620, HCT15, HCT116) have been characterised according to p53, MLH1 and XIAP status. The HCT116 cell line is mismatch repair deficient (MLH1-) but has a normal functioning p53. It expresses XIAP at levels similar to other members of the cell line panel and is up regulated when compared to normal tissue. A method of transient transfection of XIAP AS (AEG35156) in vitro was developed which achieved 81% down regulation of XIAP mRNA using the AEG35156 compound. Down regulation of XIAP mRNA was also seen with the missense (MS) OGN, AEG35187. Cytotoxicity experiments showed no significant therapeutic benefit of AS over MS. Using short hairpin (shRNA) against XIAP, stably expressed in a parent HCT116 human colon cancer cell line, a series of clones were developed. XIAP mRNA levels were established by RT-PCR, the 4 X (XIAP knockdown) clonal cell lines showing 82-92% reduction in XIAP mRNA when compared to the 4 L (luciferase control) cell lines. Immunoblot analysis showed a 63-89% reduction in XIAP protein in X cell lines compared to L. A colorectal cancer model has been developed using isogenic cell lines which differ only in their XIAP status. The XIAP deficient cell lines show a 2-fold increase in sensitivity to rhTRAIL and 20% increase in sensitivity to radiotherapy. There was a >2-fold increase in sensitivity to paclitaxel and docetaxel. All 8 shRNA cell lines can be established in vivo, the L8 and X23 cell lines showing a similar growth pattern. XIAP knockdown is maintained at the mRNA level for 26 days after implantation, though the down regulation is less than that seen in vitro. However, treatment of X23 and L8 xenografts with docetaxel showed no significant different effect on growth.

Internal ribosome entry site (IRES)-mediated translation is critical for the cell’s ability to respond to stress. Understanding how RNA binding proteins (IRES trans-acting factors; ITAFs) regulate IRESes is crucial to elucidating the mechanism of alternative translation initiation. Furthermore, determining how these ITAFs are regulated is central to understanding their functions in diseased states. I have identified the tumour suppressor programmed cell death 4 (PDCD4) as a novel ITAF of the XIAP and Bcl-xL IRES elements. I demonstrate that under normal conditions, PDCD4 acts to inhibit translation from these IRES elements by preventing formation of the 48S translation initiation complex. Furthermore, I show that in response to treatment with the pro-survival fibroblast growthfactor-2 (FGF-2), S6 kinase 2 (S6K2) phosphorylates PDCD4 leading to its degradation and the subsequent de-repression of XIAP and Bcl-xL translation. Importantly, I demonstrate the clinical significance of this regulation in glioblastoma multiforme (GBM) tumours where the loss of PDCD4 expression correlates with an increase in Bcl-xL protein and poor patient outcome. Additionally, re-expression of PDCD4 down-regulates Bcl-xL and decreases cell viability, and direct inhibition of Bcl-xL by a small molecule antagonist ABT-737 sensitizes GBM cells to the chemotherapeutic doxorubicin. Finally, I demonstrate that PDCD4 can be regulated at multiple levels. Importantly, I identify the RNA binding protein HuR as a regulator of microRNA (miR) -21 induced silencing of PDCD4. I show that HuR can bind the PDCD4 3'UTR and prevent miR-21 binding, and that a loss of PDCD4 expression following H2O2 treatment is mediated via miR-21. These results provide novel insight into the role of PDCD4 as a tumour suppressor and highlight the importance of ITAFs in cancer progression.

Les mutations du gène codant pour la protéine XIAP (X-Linked Inhibitor of Apoptosis Protein) sont à l’origine du syndrome lymphoprolifératif lié à l’X de type 2 (XLP-2). Il s’agit d’un déficit immunitaire rare caractérisé par une susceptibilité anormale à l’infection par le virus d’Epstein Barr (EBV). De plus, certains patients déficients en XIAP peuvent souffrir d’une pathologie intestinale parfois sévère. XIAP est molécule anti-apoptique qui a aussi été impliquée dans la signalisation et les fonctions de récepteurs de l’immunité innée, les récepteurs NOD1 et NOD2. Mon travail de thèse a eu pour objectif de caractériser cette pathologie intestinale et ses mécanismes physiopathologiques. Pour cela, nous avons étudié une cohorte de patients déficients en XIAP présentant une pathologie inflammatoire intestinale. Nous avons également recherché des mutations de XIAP dans une cohorte d’enfants ayant présenté comme unique signe clinique une pathologie intestinale précoce. Sur 83 patients testés, 3 patients porteurs de mutations de XIAP ont été identifiés. Nous avons ensuite montré que cette pathologie intestinale est très proche sur les plans clinique et histologique de la maladie de Crohn, qui est une des principales affections inflammatoires de l’intestin chez l’adulte. La maladie de Crohn est associée à des facteurs environnementaux et une susceptibilité génétique, dont les polymorphismes dans le gène NOD2 qui représentent le facteur plus important identifié à ce jour. Nous avons ensuite montré que les monocytes des patients déficients en XIAP ont un défaut de production d’IL-8, de MCP-1 et d’IL-10 en réponse à la stimulation de la voie NOD2. Par contre, nous n’avons pas mis en évidence d’excès d’apoptose des cellules épithéliales digestives chez les patients. En revanche, ils présentaient un nombre diminué de leur lymphocytes T innés circulants, Enfin, au cours de cette étude, nous avons identifié pour la première fois des femmes vectrices d’une mutation de XIAP à l’état hétérozygote, ayant développé des manifestations inflammatoires intestinales. Chez ces patientes, l’inactivation du chromosome X, qui normalement est biaisée en faveur de l’allèle sain chez les vectrices asymptomatiques, est de façon inhabituelle biaisée vers l’allèle muté contribuant à une diminution de l’expression de XIAP dans les monocytes et une altération de la voie NOD2. Ce travail a permis de montrer que le déficit en XIAP est responsable d’une forme monogénique de la maladie de Crohn. Nos résultats suggèrent que le défaut d’activation des monocytes par NOD2 est un mécanisme important de la pathogénèse de la maladie. Sur le plan thérapeutique, la greffe de moelle osseuse semble indiquée dans les formes sévères, puisque le principal défaut identifié est une anomalie du compartiment hématopoïétique, et chez deux de nos patients, elle a permis en effet une amélioration franche de la pathologie digestive qui était très sévère
Mutations in the gene encoding for XIAP (X-Linked Inhibitor of Apoptosis Protein) are causing the X-linked lymphoproliferative syndrome type 2 (XLP-2). It is a rare immunodeficiency characterized by an abnormal susceptibility to infection with Epstein Barr virus (EBV). In addition, some XIAP-deficient patients may suffer from an intestinal disease that can be severe. XIAP is an anti-apoptotic molecule which has also been involved in the signaling and the functions of receptors of the innate immunity, NOD1 and NOD2. My thesis work aimed to characterize this intestinal pathology and its pathophysiology. For this, we studied a cohort of known XIAP-deficient patients with inflammatory bowel disease. We also looked for mutations of XIAP in a cohort of children who presented as the only clinical sign an early intestinal pathology. In 83 patients tested, three were identified as carrier of a XIAP mutation. We then showed that this intestinal pathology is clinically and histologically very close to Crohn’s disease, which is a major inflammatory bowel disease in adults. Crohn's disease is associated with environmental factors and genetic susceptibility, including polymorphisms in the NOD2 gene that represent the most important factor identified to date. We then showed that the monocytes from XIAP-deficient patients have a defect in production of IL-8, MCP-1 and IL-10 in response to stimulation of the NOD2 pathway. However, we did not reveal any excess of apoptosis in intestinal epithelial cells from XIAP-deficient patients. On the other hand, they showed a decreased number of their circulating innate T cells. Finally, during this study, we identified for the first time, female carriers of a mutation of XIAP in the heterozygous state, who developed intestinal inflammatory manifestations. In these patients, the inactivation of the X chromosome, which is normally biased toward the healthy allele in asymptomatic vectors, is biased to the unusually mutated allele contributing to a decrease of the expression of XIAP in monocytes and an alteration of the NOD2 pathway. This work showed that XIAP deficiency is responsible for a monogenic form of Crohn's disease. Our results suggest that the lack of monocyte activation by NOD2 is an important mechanism in the pathogenesis of the disease. Therapeutically, the bone marrow transplant seems indicated in severe cases, since the main identified defect is an abnormality of the hematopoietic compartment and in two of our patients, it allowed a clear improvement of the digestive pathology that was very severe

X&barbelow;IAP a&barbelow;ssociated f&barbelow;actor 1&barbelow; (XAF1) was initially isolated as novel 34 kDa protein which bound XIAP in a two-hybrid screening. The XAF1A protein consists of 301 a.a. and contains seven potential zinc finger domains. Two alternatively splice variants of XAF1 were later isolated. One isoform (XAF1B) was formed by the removal of a 57 bp exon, which leads to an in-frame deletion of the third zinc finger and the creation of a shorter 32.5 kDa protein. The other splice variant (XAF1C) contains a 154 bp exon insertion, which truncates the sixth and seventh zinc fingers to produce an 18.7 kDa protein. XAF1A and XAF1B, but not XAF1C, bound XIAP in in vitro pull down assays. Northern blot analysis showed at least four distinct sizes of xaf1 mRNA ranging between 3.9 and 7.0 kb, which may indicate other XAF1 isoforms yet to be discovered. Though the possible role of these zinc fingers on the XAF1/XIAP interaction has yet to be determined, recent experiments indicate that XAF1A can block the ability of XIAP to inhibit caspase-3 in vitro. Furthermore, overexpression of XAF1A in HEL299 cells triggered a G1 cell cycle arrest. This G1 arrest coincides with an increase in p21, but not p53. The ability of XAF1 to block XIAP function and induce cell cycle arrest suggests a role for XAF1 in the control of both apoptosis and cell growth. The coding regions of XAF1A, B and C are encoded on a total of 9 exons within a span of 20 kb. The single copy xaf1 gene has been mapped, using FISH analysis, distal to the TP53 gene on 17p13.2. Southern blot analysis of YACS within this region further localizes the xaf1 gene on YAC 746 C 10, which contains the markers D17S1831, D17S796, and D17S1881. These markers are located approximately 3 cM telomeric to the TP53 gene. Since the xaf1 gene is located in a region commonly deleted in numerous types of cancers, this may suggest a tumour suppressor role for XAF1 in cancer. To test this theory, a 60 cell line panel from the NCl was analyzed for xaf1 RNA expression by Taqman and heterozygosity status of markers proximal to xaf1. Taqman analysis indicated that the majority of cell lines expressed little or no xaf1 RNA while xiap levels were relatively high. A PCR study of markers near xaf1 showed significant loss of heterozygosity (LOH) in this region. The loss of xaf1 expression and significant LOH near the xaf1 gene indicate that the down-regulation of XAF1 may be important in the development of the transformed phenotype.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death, or apoptosis, has during the last decade received a lot of attention due to its involvement in a large number of pathological conditions. Since death is always irreversible, it is important for cells to fully control the initiation and execution of this process. One of many apoptosis-regulatory proteins is XIAP, which blocks the action of caspases, a family of proteases that are important during apoptosis. However, apoptosis inhibitors have to be tightly controlled since too little cell death can lead to the development of tumors and other diseases. This thesis is the result of an aspiration to fully understand the function and regulation of XIAP.

By using the yeast-2-hybrid system, we identified two novel binding partners of XIAP. The first, GPS2, was found to bind XIAP and inhibit its ability to block caspase-activity. In addition, GPS2 induced caspase-mediated cell death in two different tumour cell lines and XIAP inhibited this effect.

The second binding partner, Nulp1, preferentially bound XIAP in the presence of the apoptosis-inducer staurosporine. Nulp1 induced or sensitized cell lines to cell death when overexpressed, but this was not blocked by caspase-inhibitors or XIAP, suggesting a different reason for binding than apoptosis regulation. With the aim to understand the Nulp1-XIAP interaction, we continued to study Nulp1 in vivo and in vitro. We studied three different splice variants of Nulp1 and found that they were regulated by poly-ubiquitination and nuclear shuttling. Also, Nulp1 was expressed in embryonic mice, especially in the cortical plate, hippocampal neurons and cerebellar granular neurons. Expression of Nulp1 decreased with age but was still present in cerebellar deep nuclei and Purkinje cells of adult mice.

To summarize, we have identified GPS2 as an apoptosis-inducing factor and an inhibitor of XIAP in vitro, and Nulp1 as a XIAP-interacting protein during staurosporine-induced apoptosis.

The X-linked Inhibitor of Apoptosis Protein (XIAP) counters diverse apoptotic pathways through inhibition of caspases. The levels of XIAP protein can be increased translationally in response to pathophysiological stress, elevating the apoptotic threshold of cells. Here, I have characterized two XIAP mRNA isoforms, which differ only in their 5' untranslated regions. When global translation is attenuated during pathophysiological stress, the translation of one XIAP mRNA isoform is dramatically increased through the action of an Internal Ribosome Entry Site (IRES). In contrast, the second XIAP mRNA isoform supports cap-dependent translation of Xiap, but does not contain a functional IRES element. The physiological relevance and contribution of the XIAP mRNA variants to XIAP protein levels have been examined using RNA Interference, in conjunction with a cellular stress model. The distinct translational activities of these two transcripts under cellular stress suggest a model for a dual mode of XIAP regulation, which may be a common mechanism.

Thrombosis is one of the leading causes of mortality and morbidity that is associated with myocardial infarction, stroke and pulmonary embolism. Anti-thrombotic agents which intend to reduce the occurrence and severity of thrombosis usually target the enzymes of the coagulation cascade. FXIa, a 160 kDa homodimer is gaining popularity of late as a potential target for anti-thrombotic agents due to its relative safety. A number of inhibitors which target the active site of FXIa have been reported but to our knowledge there have been no inhibitors which act via an allosteric mechanism. The aim of this project was to screen for allosteric inhibitors of FXIa from of pool of sulfated small-molecules.These molecules were primarily designed to act as heparin mimetics; heparin being a natural anti-coagulant. These compounds were then analyzed to determine whether inhibition was via an allosteric mechanism.

The suppression of programmed cell death is an essential alteration in the transformation from normal to neoplastic growth. The X-linked inhibitor of apoptosis (XIAP) is a potent suppressor of apoptosis. It is a member of the Inhibitor of Apoptosis (IAP) family and functions by inhibiting caspases 3, 7 and 9 critical proteases in the process of programmed cell death. XIAP expression levels are frequently elevated in cancer cell lines and tumors, yet the link between XIAP expression and tumorigenesis has not been demonstrated experimentally. The objective of this project is to determine whether XIAP over-expression predisposes mice to cancer. A XIAP transgenic mouse model has been created with expression of the transgene driven by the ubiquitin C promoter. The UbiC-6-myc XIAP transgenics demonstrate ubiquitous over-expression of the human homolog of XIAP. Transgene over-expression was detected by western blot in all tissues tested including brain, retina, thymus, lung, heart, liver, kidney, pancreas, and spleen. The mice develop normally and show no unusual phenotype. Homozygous mice have been bred that show a further two-fold over-expression relative to their heterozygous littermates. Suppression of apoptosis has been documented in in vitro studies of mouse embryo fibroblasts and hepatocyte cultures. Transgenic XIAP provides protection against injury-induced apoptosis in vivo in a high dose streptozotocin induced pancreatic beta cell damage model in adult mice. The mice have been bred with an inducible c-myc oncogene transgenic strain that expresses c-myc in the beta cells of the islets of Langerhans when activated by tamoxifen (obtained from Dr. Gerard Evan). The myc/XIAP double transgenics responded in the same manner as the single c-myc transgenics and the beta cells underwent apoptosis. These results suggest a model where XIAP suppresses injury or stress induced apoptosis but is unable to block genetically pre-determined or oncogene-activated apoptosis.

Retinitis Pigmentosa (RP) is the leading cause of hereditary blindness. The clinical manifestations of RP are night blindness, loss of peripheral vision leading to tunnel vision and a decreased electroretinogram (ERG) amplitude. There are numerous genes that when mutated cause RP. Regardless of the causative mutation, the end point is the same, photoreceptor cell death by apoptosis or programmed cell death (PCD). Interestingly, though all photoreceptors in an affected individual would have a mutation, disease progresses at a relatively slow rate indicating the affected cells can function. It is unclear as to why completely unrelated mutations would cause photoreceptors to die via the same mechanism. Somatic gene replacement studies using viral vectors in animal models have been successful in slowing down disease. However, attempting to cure all retinal degeneration mutations by this approach would be costly and difficult for two reasons. First, there are some mutations that comprise a small percentage of all RP cases and the cost/benefit ratio to develop a therapeutic agent for a few people would be enormous. Secondly, a number of genes that cause retinal degenerations have not been cloned. A more practical approach involves preventing apoptosis of the photoreceptor cell. The advantage of this approach is that the causative mutation does not need to be known. Developing a single therapeutic agent to target RP would also be economically more favourable than developing multiple therapeutic agents. The X-linked Inhibitor of Apoptosis (XIAP) can inhibit cell death from numerous triggers both in vitro and in vivo. XIAP prevents apoptosis by binding to and inhibiting caspase-3, -7 and -9. Work presented here shows that adeno-associated virus (AAV) encoding XIAP is neuroprotective when injected sub-retinally in an acute retinal degeneration model in Sprague Dawley rats. In addition, transgenic mice overexpressing XIAP throughout the neural retina have normal retinal morphology and their ERGs appear normal. These findings suggest that XIAP holds promise as a therapeutic agent in the treatment of hereditary retinal diseases such as retinitis pigmentosa.

Retinal detachments cause photoreceptor apoptosis. XIAP (X-linked inhibitor of apoptosis) inhibits caspases-3, -7, and -9, which prevents the apoptotic cascade. This study evaluates XIAP gene therapy as a means to provide photoreceptor neuroprotection following retinal detachment. Subretinal injections of virally-delivered XIAP or green fluorescent protein (GFP; injection control) were performed in rats. Two weeks later, retinal detachments were created at the viral injection site. Eyes were harvested 24 hours post-detachment to analyze caspase activity and at 3 days and 2 months for histological analysis. Caspase assays indicated rises in caspase-3 and -9 activities in detached GFP-treated retinas, whereas XIAP-treated retinas behaved comparably to attached controls. Three day TUNEL analysis showed less apoptosis in XIAP-treated detachments. Two month histology confirmed preservation of photoreceptors in XIAP-treated detachments, whereas GFP-treated detached retinas had deteriorated significantly. The results suggest that XIAP confers structural photoreceptor neuroprotection for at least two months following retinal detachment.

The fundamental physiological process of apoptosis plays a crucial role in maintaining the homeostasis of the mammalian immune system. The major sites of apoptotic influence occur throughout lymphopoiesis, during the deletion of unwanted carcinogenic or virally infected cells and at the waning of an antigen-induced immune response. The c&barbelow;ellular i&barbelow;nhibitor of apoptosis 2&barbelow; (cIAP2) is a potent inhibitor of apoptotic death. In contrast to the other members of the inhibitor of apoptosis (IAP) family cIAP2 is NF-kappaB-inducible via pro-inflammatory cytokines, such as IL-1beta and TNFalpha, and via bacterial components, such as l&barbelow;ipop&barbelow;olys&barbelow;accharide (LPS). The following studies demonstrate that cIAP2 -/- mice exhibit significant resistance to LPS-induced endotoxic shock, specifically via an attenuated inflammatory response. We show that due to a lack of cIAP2, cIAP2-/- macrophage cells have a heightened susceptibility to apoptosis in a LPS-induced pro-inflammatory environment and hence are unable to maintain a normal inflammatory response. These results raise the intriguing possibility that pharmacological agents targeting and inhibiting cIAP2 expression may be sufficient to confer protection to the lethal onset of sepsis. The X&barbelow;-linked i&barbelow;nhibitor of apoptosis (XIAP) and other members of the i&barbelow;nhibitor of apoptosis (IAP) family can suppress apoptosis induced by a diverse variety of triggers. Functional studies done to date have focused upon tissue culture models and adenovirus over-expression of XIAP and other IAP proteins. These studies report the phenotype of the first engineered transgenic mouse over-expressing a human IAP, as well as assessing the long-term consequence of IAP over-expression. The relative protein expression levels of the endogenous murine homologue to XIAP within thymocyte and T cell sub-populations are also documented. The consequence of lymphoid-targeted over-expression of XIAP in transgenic mice suggests a physiological role for the endogenous murine XIAP. Xiap-transgenic mice accumulated thymocytes and/or T cells in primary and secondary lymphoid tissue, T cell maturation was perturbed, and transgenic thymocytes resisted a variety of apoptotic triggers both in vitro and in vivo. These observations imply a possible key function for the intrinsic cellular inhibitor XIAP in maintaining the homeostasis of the immune system.

In Canada alone, there were an estimated 800,000 visually impaired people in 2007, costing the federal government an annual amount of $15.8 billion in services, treatments and lost revenue. These costs are estimated to double by the year 2032, as the population ages. The leading causes of visual impairment and blindness is retinal degeneration, characterized by the progressive death of retinal cells. The research presented in this PhD thesis aimed to prevent retinal degeneration by over-expressing the X-linked Inhibitor of Apoptosis (XIAP) in retinal cells using plasmid and adeno-associated viral vectors. The work is divided into four sequential chapters targeted at developing an anti-apoptotic gene therapy strategy to prevent retinal cell death. The first chapter examines XIAP gene therapy in the treatment of Leber’s Hereditary Optic Neuropathy (LHON). In vitro studies using the 661W cone-photoreceptor cell line showed that XIAP over-expression significantly lowers cell death when 661W cells are exposed to a number of apoptotic stimuli. In a mouse model of Leber’s Hereditary Optic Neuropathy (LHON), XIAP expression in retinal ganglion cells (RGCs) protected the ultrastructure of the RGC axons within the optic nerve, in addition to providing evidence of functional protection. The second and third chapters further examine the potential for XIAP gene therapy in the treatment of retinal disease by developing an in vivo model of retinal detachment in cats, followed by evaluating the efficacy of XIAP gene therapy intervention. When XIAP was over-expressed in the photoreceptor cells, there was significant structural protection and trends in preservation of function in this model of degeneration. Finally, the fourth chapter explores an alternate method to viral gene therapy by evaluating the efficacy and toxicity of chitosan microparticles as a protein delivery system to the retina. Results show that chitosan microparticles are mucosal-adhesive and are non-toxic at low concentrations in vitro in 661W cells and in vivo in rats. This thesis work provides strong evidence that XIAP gene therapy is an effective method for preventing retinal degeneration, and works as a broad spectrum gene therapy strategy that can be applied to different forms of retinal degeneration.

Nous avons exprimé dans différents systèmes hétérologues l'ADNc de la xylanase (XYNB) du champignon filamenteux Penicillium funiculosum ainsi que celle du phytopathogène Botrytis cinerea (XynBc) appartenant toutes deux à la famille des GH11 des glycosylhydrolases. Les propriétés physico-chimiques des enzymes recombinantes ont été déterminées. XYNB et XynBc montrent les mêmes spectres d'inhibitions en présence d'inhibiteurs protéiques probablement impliqués dans des mécanismes de défenses des plantes et issus du blé ; les activités sont fortement inhibées par XIP-I et TAXI-I tandis que TAXI-II n'a aucun effet. Des essais de RT-PCR semi-quantitatives ont montré la présence des ARNm au cours des 72 premières heures de l'infection de feuilles de tabacs par B. Cinerea
The phytopathogen fungus Botrytis cinerea and the filamentous fungus Penicillium funiculosum produce various glycosidases with xylanase activity. In the present study, we report the heterologous expression, purification and characterization of two family 11 xylanase produced by these fungi. These enzymes present similar activities on different substrates and identical suceptibilities to different proteinaceous inhibitors from wheat. These xylanases were sensitive to XIP-I, TAXI-I but not to TAXI-II. The inhibition was competitive and the inhibition constant were in the nanomolar range indicting a great affinity between these xylanases and the wheat inhibitors. Moreover, we have shown that the presence of glycosylation or of a carbohydrate binding module in the xylanase, did not affect the interaction of the enzyme with the inhibitor

碩士
國立臺灣大學
生化科學研究所
105
Previously, we demonstrated that disruption of protein-protein interaction (PPI) of caspase-7 (CASP7) and X-linked inhibitor of apoptosis protein (XIAP) by using I-Lys with an iodomethyl ketone warhead to alkylate Cys246 of CASP7, selectively killed cancer cells with accumulation of CASP7:XIAP complexes, which are caspase-3 down-regulated (CASP3/DR) [1]. CASP3/DR frequently confers resistance to cancer therapy due to reduced apoptotic machinery. This represents an effective and safe strategy for chemotherapy because the CASP7:XIAP complexes are not accumulated in normal cells. Unlike the I-Lys irreversible PPI inhibitor, through compute virtual screening, a reversible PPI inhibitor, 643943, a compound from Sigma compound bank was discovered by Chen, S. H. et al. In this thesis, I aimed to determine the structure-activity relationship of the I-Lys and 643943 analogues and the induced apoptotic mechanisms. We found that in I-Lys analogues, the weaker leaving group reduced the activity, but C-terminal protecting groups did not affect so much. We also found synergistic effect of 643943 combining STS in killing 7-TR, a Taxol-resistant MCF-7 cells. The analogue 0909 adopts different mechanism to induce apoptosis in MCF-7 cells through MAPK family to activate various caspases, unlike 643943 that mainly activated CASP7. Our studies demonstrated a promising therapeutic strategy against CASP3/DR and multidrug resistant cancers.

以前的研究表明，BRE是一種能在內源和外源凋亡路徑中均發揮作用的抗凋亡蛋白。然而，我們卻完全不知道它發揮抗凋亡功能的分子機制和生物化學機制是怎樣的。在本論文中，我們報導了BRE通過保護細胞內的XIAP水準來發揮抗凋亡功能。XIAP是一種強大的內源性的半胱氨酸天冬氨酸蛋白酶的抑制劑，與BRE一樣，XIAP也在內源和外源凋亡路徑中均發揮作用。
我們使用鼠Lewis細胞系為母細胞系，產生了shRNA介導的BRE基因敲除穩定細胞系。我們發現這種BRE敲除細胞系使得細胞對於在即使沒有放線菌酮作用下的腫瘤壞死因數α介導的凋亡也異常敏感。放線菌酮通過抑制抗凋亡蛋白的合成以發揮重要的促凋亡作用，相反地，腫瘤壞死因數-α通過NF-κB通路可以上調上述抗凋亡蛋白。蛋白質印跡結果顯示，上述通過NF-κB通路被上調且對放線菌酮敏感的抗凋亡蛋白中，只有XIAP的水準，而不是cIAP-1, cIAP-2 或者cFLIP的水準, 在BRE敲除細胞中明顯被降低。用具有高度同源性的人BRE在上述鼠BRE敲除細胞中恢復BRE的水準，XIAP水準也隨之上升。該實驗也證實了BRE正向調節XIAP的作用。並且當在上述BRE敲除細胞中恢復人BRE或者XIAP時，這些細胞對腫瘤壞死因數-α介導的凋亡的敏感性也都得以降低。
當具有使蛋白中半胱氨酸殘基改變作用的N-乙基馬來醯亞胺被加入細胞裂解液中時，抗BRE抗體Mab489-7 對於BRE的識別作用會因為BRE蛋白中半胱氨酸殘基的改變而受到影響。與之對應地，裂解液中的XIAP水準也同時降低。該結果提示在細胞裂解液中，BRE保護XIAP的基團可能與半胱氨酸殘基有關。
我們發現在凋亡過程中XIAP不僅被半胱氨酸天冬氨酸蛋白酶分解，它也會被蛋白體所降解。這提示我們XIAP的泛素化對於保持其穩定性有重要作用。我們已經知道BRE具有泛素鏈結合域，也可以形成具有降泛素作用的複合物。因此我們想通過實驗，瞭解BRE是否能降解掉鏈結在XIAP上的泛素鏈。實驗表明XIAP至少可以鏈結三種類型的泛素鏈，分別為K48型，K63型和K0型。然而BRE卻只特異性地降解鏈結在XIAP上的K0型泛素鏈或抑制XIAP上的K0型泛素鏈的形成。
綜上所述，BRE特異性地降解鏈結在XIAP上的線型泛素鏈或者抑制該種泛素鏈的形成，並通過它影響XIAP的穩定性，從而在外源和內源凋亡通路中發揮抗凋亡的作用。BRE和XIAP之間的相互作用是間接的還是直接的仍有待進一步證實。
BRE is a broad-spectrum anti-apoptotic protein, which attenuates both extrinsic and intrinsic apoptosis. However, the molecular and biochemical mechanisms by which BRE inhibits apoptosis remain completely unknown. Here I provide evidence that BRE attenuates apoptosis through maintaining the cellular level of XIAP, which is a potent endogenous inhibitor of caspases functioning in intrinsic and extrinsic pathways.
Using a mouse Lewis lung carcinoma cell line D122, we found that shRNA-mediated stable depletion of BRE rendered the cells susceptible to TNF-α-induced apoptosis even in the absence of cycloheximide (CHX). CHX plays a critical pro-apoptotic role by inhibiting synthesis of anti-apoptotic proteins, which TNF-α also up-regulates through activation of NF-κB pathway. Western blot analysis of the NF-κB-driven and CHX-sensitive anti-apoptotic proteins revealed only XIAP, but not cIAP-1, cIAP-2, or cFLIP, was down-regulated in BRE-depleted cells. Reconstitution of the BRE-depleted mouse cells with highly homologous human BRE restored the XIAP protein level, confirming a positive regulatory role of BRE on XIAP protein expression. Furthermore, reconstitution of the BRE-depleted cells with human BRE or XIAP rendered the cells less sensitive to TNF-α-induced apoptosis.
Addition of NEM (N-Ethylmaleimide), which binds irreversibly to cysteine residues of proteins, to cell lysates, was found to abrogate, the recognition of BRE by our anti-BRE antibody (Mab489-7) in Western blot analysis. Correspondingly, XIAP level was also found reduced in cell lysates. This correlation provides in vitro evidence that BRE has a protective role for XIAP, and that this role is related to cysteine residues of BRE.
I have shown that that XIAP is not only cleaved by caspases during apoptosis, but also subjected to proteaosomal degradation, indicating that ubiquitination of XIAP is an important regulatory mechanism for the stability of this protein. As BRE is known to contain polyubiquitin-binding domains and forms complexes with deubiquitination activity, the issue of whether BRE could remove the ubiquitination of XIAP was investigated. I found that over-expressed XIAP underwent K48-linked, K63-linked, and K0-linked polyubiquitination, respectively. Overexpression of BRE led to the removal of or inhibited K0- but not K48- or K63-linked ubiquitination of XIAP.
Taken together, I have provided evidence that BRE exerts its anti-apoptotic function through maintaining the cellular level of XIAP, a potent endogenous inhibitor of apoptosis. Promoting removal of linear ubiquitin chain of XIAP, or inhibition of the chain formation to prevent linear polyubiquitin-mediated proteasomal degradation of XIAP may be the mechanism by which BRE stabilizes this protein. Whether such interaction between BRE and XIAP is direct or indirect needs further investigation.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Li, Wei.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 114-126).
Abstracts also in Chinese.
Abstract --- p.i
Acknowledgements --- p.iv
List of Figures --- p.v
List of Tables --- p.vii
Abbreviations --- p.viii
Chapter CHAPTER 1 --- : Introduction --- p.1
Chapter 1.1 --- Apoptosis --- p.1
Chapter 1.1.1 --- Overview of apoptosis --- p.1
Chapter 1.1.2 --- Caspases --- p.1
Chapter 1.1.3 --- Extrinsic and intrinsic apoptotic pathways --- p.2
Chapter 1.2 --- NF-κB --- p.3
Chapter 1.2.1 --- Overview of NF-κB --- p.3
Chapter 1.2.2 --- NF-κB and two signaling complexes --- p.5
Chapter 1.3 --- IAP --- p.7
Chapter 1.3.1 --- Structure --- p.7
Chapter 1.3.2 --- Function --- p.8
Chapter 1.4 --- XIAP --- p.9
Chapter 1.4.1 --- Discovery and function --- p.9
Chapter 1.4.2 --- BIR domain of XIAP and its function as direct caspase inhibitor --- p.9
Chapter 1.4.3 --- RING domain of XIAP and its function as E3 in ubiquitination --- p.10
Chapter 1.4.4 --- XIAP associates with apoptosome --- p.12
Chapter 1.4.5 --- Antagonists of XIAP --- p.13
Chapter 1.4.6 --- Clinical significance of XIAP --- p.13
Chapter 1.5 --- Ubiquitin and ubiquitination --- p.13
Chapter 1.5.1 --- Overview of ubiquitin --- p.13
Chapter 1.5.2 --- Ubiquitination process --- p.14
Chapter 1.5.3 --- Ubiquitin-activating enzymes, E1s --- p.15
Chapter 1.5.4 --- Ubiquitin-conjugating enzymes, E2s --- p.17
Chapter 1.5.5 --- Ubiquitin-protein ligases, E3s --- p.18
Chapter 1.5.6 --- Proteasomes --- p.21
Chapter 1.5.7 --- Deubiquitinating enzymes --- p.23
Chapter 1.6 --- Background of BRE --- p.27
Chapter 1.6.1 --- DNA and RNA of BRE --- p.27
Chapter 1.6.2 --- Protein --- p.28
Chapter 1.6.3 --- BRE in two complexes --- p.29
Chapter 1.6.4 --- The anti-apoptotic function of BRE --- p.31
Chapter 1.6.5 --- BRE and ubiquitin --- p.33
Chapter 1.7 --- RNA interference --- p.33
Chapter 1.7.1 --- Mechanism of RNA interference --- p.33
Chapter 1.7.2 --- Small hairpin RNA --- p.34
Chapter CHAPTER 2 --- : Materials and methods --- p.36
Chapter 2.1 --- Materials --- p.36
Chapter 2.1.1 --- Primers used for cloning --- p.36
Chapter 2.1.2 --- DNA clones used in the studies --- p.36
Chapter 2.1.3 --- Materials for DNA manipulation --- p.44
Chapter 2.1.4 --- Materials for protein manipulation --- p.45
Chapter 2.1.5 --- Materials for virus manipulation --- p.45
Chapter 2.1.6 --- Antibodies --- p.46
Chapter 2.1.7 --- Chemicals --- p.46
Chapter 2.1.8 --- Kits --- p.46
Chapter 2.1.9 --- Culture media and reagents --- p.47
Chapter 2.1.10 --- Bacterial strains used for transformation and cloning --- p.47
Chapter 2.1.11 --- Instrumentation --- p.47
Chapter 2.2 --- Methods --- p.48
Chapter 2.2.1 --- Construction of plasmids --- p.48
Chapter 2.2.2 --- Plasmids preparation --- p.53
Chapter 2.2.3 --- Cell culture --- p.53
Chapter 2.2.4 --- Cell transfection --- p.54
Chapter 2.2.5 --- Generation of stable transfectants --- p.55
Chapter 2.2.6 --- Western blot analysis --- p.55
Chapter 2.2.7 --- Chemical treatment --- p.56
Chapter 2.2.8 --- Apoptosis assays by the flow cytometry --- p.57
Chapter 2.2.9 --- Immunoprecipitation --- p.58
Chapter 2.2.10 --- BRE containing retrovirus generation and transduction --- p.58
Chapter 2.2.11 --- BRE containing adenovirus generation and transduction --- p.61
Chapter CHAPTER 3 --- : BRE attenuates apoptosis through maintaining the cellular level of an apoptotic inhibitor XIAP --- p.65
Chapter 3.1 --- Establishment of cell lines with BRE expression stably knocked down by shRNA --- p.65
Chapter 3.2 --- BRE-depleted cells are more sensitive to apoptosis --- p.67
Chapter 3.3 --- BRE-depleted cells are susceptible to TNF-α induced apoptosis in the absence of cycloheximide --- p.69
Chapter 3.4 --- Reduction of XIAP in BRE-depleted cells --- p.72
Chapter 3.5 --- Recovery of BRE restores XIAP in BRE-depleted cells --- p.74
Chapter 3.6 --- Recovery of XIAP or BRE to BRE-depleted cells renders the cellsless sensitive to TNF-α induced apoptosis --- p.76
Chapter 3.7 --- N-Ethylmaleimide (NEM) affects BRE and XIAP --- p.79
Chapter 3.7.1 --- NEM affects BRE staining by anti-BRE (Mab489-7) antibody --- p.79
Chapter 3.7.2 --- NEM affects XIAP --- p.81
Chapter 3.7.3 --- NEM reduces XIAP instead of antibody-binding failure --- p.83
Chapter 3.8 --- Ubiquitination of XIAP is important for its stability --- p.85
Chapter 3.8.1 --- VAD cannot preserve XIAP upon 16 hours of etoposide treatment --- p.85
Chapter 3.8.2 --- Degradation of XIAP in response to etoposide in the presence of VAD is due to proteasomal degradation --- p.87
Chapter 3.9 --- BRE leads to the removal of or inhibits ubiquitination of XIAP --- p.89
Chapter 3.9.1 --- Immunoprecipitation of ubiquitinated XIAP --- p.89
Chapter 3.9.2 --- BRE leads to the removal of or inhibits endogenous ubiquitination of XIAP --- p.92
Chapter 3.9.3 --- BRE does not affect exogenous K48-linked ubiquitination of XIAP --- p.94
Chapter 3.9.4 --- BRE does not affect exogenous K63-linked ubiquitination of XIAP --- p.96
Chapter 3.9.5 --- BRE leads to the removal of or inhibits exogenous K0-linked ubiquitination of XIAP --- p.98
Chapter CHAPTER 4 --- : Discussion --- p.100
Chapter 4.1 --- Summary --- p.100
Chapter 4.2 --- Shortcoming and improvement in future experiment --- p.101
Chapter 4.3 --- Significance of the research findings --- p.102
Chapter 4.4 --- Possibility of BRE to attenuate apoptosis through affecting other NF-κB-activated anti-apoptotic proteins --- p.103
Chapter 4.5 --- BRE may function with other deubiquitinase to mediate deubiquitination of XIAP --- p.104
Chapter 4.6 --- Comparison of methodologies --- p.109
Chapter 4.7 --- Lysine-linkage specificity of the ubiquitination of XIAP --- p.110
Chapter 4.8 --- Possibility of BRE to maintain XIAP level utilizing other mechanisms --- p.111
Chapter 4.9 --- Interpretation of NEM-related data --- p.112
Chapter 4.10 --- Conclusion --- p.112
Reference --- p.114
Supplementary result --- p.127

Inflammatory breast cancer (IBC) is an extremely rare but highly aggressive form of breast cancer characterized by the rapid development of therapeutic resistance leading to particularly poor survival. Our previous work focused on the elucidation of factors that mediate therapeutic resistance in IBC and identified increased expression of the anti-apoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), to correlate with the development of resistance to chemotherapeutics. Although XIAP is classically thought of as an inhibitor of caspase activation, multiple studies have revealed that XIAP can also function as a signaling intermediate in numerous pathways. Based on preliminary evidence revealing high expression of XIAP in pre-treatment IBC cells rather than only subsequent to the development of resistance, we hypothesized that XIAP could play an important signaling role in IBC pathobiology outside of its heavily published apoptotic inhibition function. Further, based on our discovery of inhibition of chemotherapeutic efficacy, we postulated that XIAP overexpression might also play a role in resistance to other forms of therapy, such as immunotherapy. Finally, we posited that targeting of specific redox adaptive mechanisms, which are observed to be a significant barrier to successful treatment of IBC, could overcome therapeutic resistance and enhance the efficacy of chemo-, radio-, and immuno- therapies. To address these hypotheses our objectives were: 1. to determine a role for XIAP in IBC pathobiology and to elucidate the upstream regulators and downstream effectors of XIAP; 2. to evaluate and describe a role for XIAP in the inhibition of immunotherapy; and 3. to develop and characterize novel redox modulatory strategies that target identified mechanisms to prevent or reverse therapeutic resistance.

Using various genomic and proteomic approaches, combined with analysis of cellular viability, proliferation, and growth parameters both in vitro and in vivo, we demonstrate that XIAP plays a central role in both IBC pathobiology in a manner mostly independent of its role as a caspase-binding protein. Modulation of XIAP expression in cells derived from patients prior to any therapeutic intervention significantly altered key aspects IBC biology including, but not limited to: IBC-specific gene signatures; the tumorigenic capacity of tumor cells; and the metastatic phenotype of IBC, all of which are revealed to functionally hinge on XIAP-mediated NFκB activation, a robust molecular determinant of IBC. Identification of the mechanism of XIAP-mediated NFκB activation led to the characterization of novel peptide-based antagonist which was further used to identify that increased NFκB activation was responsible for redox adaptation previously observed in therapy-resistant IBC cells. Lastly, we describe the targeting of this XIAP-NFκB-ROS axis using a novel redox modulatory strategy both in vitro and in vivo. Together, the data presented here characterize a novel and crucial role for XIAP both in therapeutic resistance and the pathobiology of IBC; these results confirm our previous work in acquired therapeutic resistance and establish the feasibility of targeting XIAP-NFκB and the redox adaptive phenotype of IBC as a means to enhance survival of patients.

Dissertation

碩士
國立陽明大學
藥理學研究所
94
Positive transcription elongation factor b (P-TEFb) composed of cyclin dependent kinase 9 (CDK9) and cyclin T1 is required for transcription elongation by phosphorylating serine 2 residues in the C-terminal domain (CTD) of RNA polymerase II. It is proposed that P-TEFb is a co-activator of NF-κB . X-linked inhibitor of apoptosis (XIAP) is a member of inhibitors of apoptosis. The anti-apoptotic function of XIAP is through the inhibition of caspase 3、caspase 7 and caspase 9 activities. It was reported that the expression of XIAP is regulated by NF-κB. This study investigated the possibility whether P-TEFb participates in the regulation of the expression of XIAP in hepatoma cells. The activation of NF-κB was stimulated by tumor necrosis factor-alpha (TNF-α) in Huh-7 cells. NF-κB-regulated XIAP protein level was also induced in this model system. Furthermore, a CDK9 pharmacological inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) and cyclin T1 siRNA abrogated NF-κB promoter activity and reduced the TNF-α-induced expression of XIAP. In addition, chromatin immunoprecipitation (CHIP) results revealed the binding of P-TEFb with the promoter and coding region of XIAP gene. Taken together, my results suggest that P-TEFb directly regulates XIAP expression mediated by NF-κB in Huh-7 cells.